Six-membered fused to five-membered heterocyclic compounds, pharmaceutical compositions thereof and uses thereof

Abstract

The application discloses a kind of six-membered and five-membered heterocyclic compounds and pharmaceutical composition and application thereof.The structure of the compound is as follows, also includes its pharmaceutically acceptable salt, can effectively inhibit FGFR kinase activity, especially the inhibitory activity of FGFR1 and FGFR4 kinase, also shows high selectivity to single kinase;It also has significant inhibitory effect on the proliferation of various tumor cells, FGFR-ERK signal pathway.The compound is widely used, and can be prepared as an antitumor drug, and shows significant inhibitory activity at the molecular level, cell level and signal pathway level, and has no obvious toxicity to normal cells.

Description

Technical Field

[0001] This invention relates to a six-membered pentagonal heterocyclic compound, pharmaceutical compositions thereof, and applications, and more particularly to a six-membered pentagonal heterocyclic compound that can be prepared as an antitumor drug, pharmaceutical compositions thereof, and applications. Background Technology

[0002] Fibroblast growth factor receptor (FGFR) is a member of the protein tyrosine kinase (PTK) family, comprising four conserved transmembrane tyrosine kinase receptors (FGFR1-4). It phosphorylates tyrosine residues by transferring the γ-phosphate group of ATP and participates in regulating cell proliferation, differentiation, migration, and metabolism. Studies have shown that when FGFR binds to its ligand, it induces FGFR dimerization, catalyzing its own phosphorylation and activating downstream signaling pathways, such as the RAS / RAF / MAPK signaling pathway, the PI3K / AKT signaling pathway, signal transducers and activators of transcription (STAT), and phospholipase Cγ (PLCγ). Furthermore, FGFR-mediated signaling pathways are involved in physiological processes such as angiogenesis, cell proliferation and migration, regulation of organ development, and wound healing. Mutations or overexpression of FGFR can lead to excessive activation of the FGFR signaling pathway and induce cancerous transformation in normal cells. Overactivation of RAS / RAF / MAPK can stimulate cell proliferation and differentiation; overactivation of PI3K / AKT inhibits apoptosis; STAT is closely related to promoting tumor invasion and metastasis and enhancing tumor immune escape ability; and the PLCγ signaling pathway is an important pathway for regulating tumor cell metastasis. Therefore, FGFRs are considered a very promising target for anticancer therapy.

[0003] Currently approved small molecule inhibitors targeting FGFR can be mainly divided into two categories: one is multi-target tyrosine kinase inhibitors, such as dovitinib, nintedanib, and lenvatinib, although FGFR is not their primary target; although they have shown good efficacy in clinical applications, their low selectivity inevitably leads to off-target toxicity and various adverse reactions. The other category is FGFR inhibitors, such as erdafitinib and pemigatinib, but these are pan-FGFR inhibitors and have no obvious selectivity for the four subtypes of FGFR; their main mechanism of action is to target the intracellular tyrosine kinase domain, non-covalently and reversibly binding to the FGFR protein, inhibiting the catalytic activity of FGFR or tyrosine autophosphorylation. Summary of the Invention

[0004] Purpose of the invention: In view of the shortcomings of existing FGFR inhibitor drugs, such as low selectivity and weak binding to the target, the present invention aims to provide a six-membered pentaneous heterocyclic compound with excellent selective inhibitory activity, its pharmaceutical composition and application.

[0005] Technical Solution: Covalent irreversible inhibitors are a class of small molecule inhibitors that can interact with specific target proteins and form covalent bonds, leading to irreversible conformational changes in the target proteins and thus inhibiting their activity. Compared with non-covalent reversible inhibitors, covalent irreversible inhibitors have significant advantages in terms of biological activity. Due to their unique pharmacokinetic properties, covalent irreversible inhibitors have lower effective concentrations and longer effective durations of action, and can effectively overcome resistance to non-covalent reversible inhibitors. It has been confirmed that many major diseases, such as malignant tumors, are regulated by kinases, and these enzymes have become attractive drug targets. Therefore, obtaining covalent irreversible inhibitors through rational design, screening, and structural optimization has become a hot topic in targeted drug development. Given that first-generation non-covalent reversible FGFR inhibitors used clinically have shown varying degrees of resistance, it is essential to develop a new generation of FGFR inhibitors that combine the advantages of covalent irreversible inhibitors to overcome the resistance and toxicity of first-generation inhibitors.

[0006] This invention designs a series of compounds with covalent irreversible FGFR inhibitory activity by coupling Michael receptors with p-aminobenzoic acid, 5-bromomethyl-2-fluoroaniline and p-aminobenzyl bromide as linkers on a six-membered and five-membered heterocyclic parent containing different numbers of nitrogen atoms.

[0007] As a first aspect of the present invention, the six-membered pentagonal heterocyclic compound of the present invention has the following structure and further comprises a pharmaceutically acceptable salt thereof.

[0008]

[0009] in:

[0010] And the ring system is selected from

[0011] U, V, W, X, Y, Z are selected from N or CH. It can contain at most 2 N. It can contain at most 3 N;

[0012] L is selected from carbonyl group or CH2;

[0013] M is selected from O and NH;

[0014] P and Q are selected from halogens. Preferably, the substituents are at the 3', 4', and 5' positions;

[0015] R1 and R2 are selected from H and halogens;

[0016] R3 is selected from C1-C4 alkyl, C1-C4 haloalkyl, C3-C7 cycloalkyl, and C3-C7 halocycloalkyl.

[0017] R 4 Selected from vinyl, 2-propenyl, 1-(2-methyl)propenyl, C1-C4 alkyl, C1-C4 haloalkyl, C3-C7 cycloalkyl, and C3-C7 halocycloalkyl;

[0018] R 5 The substituent is selected from halogen, phenyl, substituted phenyl, thienyl, benzothienyl, benzofuranyl, wherein the substituent is selected from at least one halogen, C1-C4 alkoxy, C1-C4 alkyl, or C1-C4 haloalkyl.

[0019] Preferably, the above-mentioned six-membered and five-membered heterocyclic compounds have any one of the structures of Formula I to Formula V:

[0020]

[0021] in:

[0022] R1 and R2 are selected from Cl and Br;

[0023] R3 is selected from C1-C2 alkyl, C1-C2 haloalkyl, C3-C5 cycloalkyl, and C3-C5 halocycloalkyl.

[0024] R4 is selected from vinyl, 2-propenyl, 1-(2-methyl)propenyl, C1-C3 alkyl, C1-C3 haloalkyl, C3-C5 cycloalkyl, and C3-C5 halocycloalkyl.

[0025] R5 is selected from phenyl, substituted phenyl, thienyl, benzothienyl, benzofuranyl, wherein the substituent is selected from at least one halogen, C1-C2 alkoxy, C1-C2 alkyl, or C1-C2 haloalkyl.

[0026] Further optimization, in the above structure:

[0027] R1 and R2 are selected from Cl and Br substituted at the 4-, 5-, and 6-positions, respectively;

[0028] R3 is selected from methyl, haloethyl, and halocyclobutyl;

[0029] R4 is selected from vinyl, 2-propenyl, 1-(2-methyl)propenyl, ethyl, haloethyl, isopropyl, and cyclopropyl.

[0030] R5 is selected from phenyl, substituted phenyl, thienyl, benzothienyl, benzofuranyl, wherein the substituent is selected from two halogens, C1-C2 alkoxy, C1-C2 alkyl, C1-C2 haloalkyl.

[0031] To further optimize, in the above structure:

[0032] R3 is selected from methyl, 2,2-difluoroethyl, and 3,3-difluorocyclobutyl;

[0033] R4 is selected from vinyl, 2-propenyl, 1-(2-methyl)propenyl, ethyl, 2-chloroethyl, 1-chloroethyl, isopropyl, and cyclopropyl.

[0034] R5 is selected from phenyl, substituted phenyl, thienyl, benzothienyl, benzofuranyl, wherein the substituent is selected from two fluorine or methoxy groups that are meta- or / and para-substituted.

[0035] Most preferably, the above-mentioned six-membered and five-membered heterocyclic compounds are selected from any of the following compounds:

[0036]

[0037]

[0038]

[0039]

[0040] The pharmaceutically acceptable salts of the aforementioned six-membered and five-membered heterocyclic compounds are those formed with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid.

[0041] The specific preparation methods for the above compounds are as follows:

[0042] The compound shown in I is obtained by a condensation reaction of 4-acrylamide benzoic acid and six-membered five-membered heterocyclic compounds I-M1 containing different numbers of nitrogen atoms, as shown in the following reaction formula:

[0043]

[0044] Reaction conditions: (i) HATU, DIPEA, DMF, 0℃~RT, 0.5h; wherein, the atoms represented by X, Y, Z and R1 in the structures of compounds I-M1 and I, and the position of R1 in the six-membered pentagonal heterocycle, are as defined above;

[0045] The reaction formulas for preparing 4-acrylamide benzoate and its acid are as follows:

[0046]

[0047] Reaction conditions: (ii) Acryloylchloride, TEA, DCM, 0℃~RT, 0.5h; (iii) TFA, DCM.

[0048] Compound II is derived from 4-fluoro-3-nitrobenzyl bromide and six-membered and five-membered heterocyclic compounds II-M1 containing different numbers of nitrogen atoms. The intermediate II-M2 is obtained through a substitution reaction, followed by reduction of the nitro group to obtain intermediate II-M3, which is then condensed with acryloyl chloride. The reaction equation is as follows:

[0049]

[0050] Reaction conditions: (i) K2CO3, MeCN, 80℃, 12h; (ii) Fe powder, NH4Cl, EtOH / H2O, 76℃, 6h; (iii) Acryloylchloride, TEA, DCM, 0℃~RT, 0.5h; wherein, the atoms represented by U, V, W, X, Y, Z and R2 in the structures of compounds II-M1, II-M2, II-M3 and II, and the position of R2 in the six-membered pentagonal heterocycle, are as defined above.

[0051] Compound III is prepared by reacting 1H-benzotriazole-7-carboxylic acid and a fatty alcohol or fatty amine compound via a condensation reaction to obtain intermediate III-M1, which then undergoes a substitution reaction with 4-fluoro-3-nitrobenzyl bromide to obtain intermediate III-M2. The nitro group in III-M2 is then reduced to obtain intermediate III-M3, which is finally condensed with acryloyl chloride to obtain the final product. The reaction formula is as follows:

[0052]

[0053] Reaction conditions: (i) HATU, DIPEA, DMF, 45℃, 6h; (ii) K2CO3, MeCN, 80℃, 12h; (iii) Fe powder, NH4Cl, EtOH / H2O, 76℃, 6h; (iv) Acryloylchloride, TEA, DCM, 0℃~RT; wherein, the atoms or groups represented by M and R3 in the structures of the compounds shown in III-M1, III-M2, III-M3 and III are as defined above.

[0054] Compound IV is derived from 6-chloropurine and p-nitrobenzyl bromide via a substitution reaction to obtain intermediate IV-M1. The nitro group in IV-M1 is then reduced to intermediate IV-M2, which is subsequently condensed with IV-M3 to yield the final product. The reaction equation is as follows:

[0055]

[0056] Reaction conditions: (i) K2CO3, MeCN, 80℃, 12h; (ii) Fe powder, NH4Cl, EtOH / H2O, 76℃, 6h; (iii) TEA, DCM, 0℃; wherein, the atoms or groups represented by R4 in the structures of compounds shown in IV-M1 and IV are as defined above.

[0057] Compound V is obtained by coupling IV-M2 and an aromatic compound V-M1 containing a boric acid group to obtain intermediate V-M2, which is then reacted with acryloyl chloride to produce the compound. The reaction formula is as follows:

[0058]

[0059] Reaction conditions: (i) Pd(PPh3)4, K2CO3, 1,4-Dioxane, 80℃, 8h; (b) Acryloylchloride, TEA, DCM, 0℃~RT, 0.5h; wherein, the atoms or groups represented by R5 in the structures of compounds shown in V-M1, V-M2 and V are as defined above.

[0060] In the reaction conditions for preparing the compound shown in IV, HATU represents the condensing agent 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, DIPEA represents N,N-diisopropylethylamine, TEA represents triethylamine, TFA represents trifluoroacetic acid, DCM represents dichloromethane, DMF represents N,N-dimethylformamide, and RT represents room temperature.

[0061] Salts are formed by reacting the corresponding acids with the six-membered and five-membered heterocyclic compounds prepared by the above methods to obtain pharmaceutically acceptable salts.

[0062] As a second aspect of this invention, the aforementioned six-membered and five-membered heterocyclic compounds and a pharmaceutically acceptable carrier constitute the pharmaceutical composition of this invention. Specifically, common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions, or injections, are prepared by adding a pharmaceutically acceptable carrier. The preparations may include commonly used pharmaceutical excipients such as flavorings, sweeteners, liquid / solid fillers, and diluents.

[0063] As a third aspect of the present invention, the above-mentioned hexa- and pentaneous heterocyclic compounds and their pharmaceutical compositions are used to prepare FGFR inhibitory drugs, preferably FGFR1 and FGFR4 inhibitory drugs, specifically antitumor drugs, and more specifically drugs for treating breast cancer, liver cancer and esophageal cancer.

[0064] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0065] 1. These compounds and their pharmaceutical compositions can effectively inhibit FGFR kinase activity, especially the inhibitory activity against FGFR1 and FGFR4 kinases, IC50. 50 The optimal concentration was below 0.5 μM; it also exhibited high selectivity for single kinases, with the optimal value reaching more than 40 times; at the same time, it had a significant inhibitory effect on the proliferation of various tumor cells and the FGFR-ERK signaling pathway.

[0066] 2. It has a wide range of applications and can be prepared as an anti-tumor drug. It exhibits significant inhibitory activity at the molecular, cellular, and signaling pathway levels, and has no obvious toxicity to normal cells. Attached Figure Description

[0067] Figure 1 The results of Western blot analysis of the effect of compound 30 on the EGFR-ERK signaling pathway. Detailed Implementation

[0068] The technical solution of the present invention will be further described below with reference to the embodiments.

[0069] All reagents used in the examples were of analytical grade. NMR data for compound structural characterization were obtained using a Bruker ARX-600 NMR spectrometer, with TMS as an internal standard; high-resolution mass spectrometry was performed using an Agilent 6224 TOF LC / MS instrument.

[0070] Example 1: Preparation of Compound 1

[0071] (a) Preparation of tert-butyl 4-acrylamide benzoate

[0072] 5.79 g (30 mmol) of tert-butyl 4-aminobenzoate was placed in a 500 mL round-bottom flask, and 200 mL of anhydrous dichloromethane was added. The mixture was stirred thoroughly at 0 °C. 4.55 g (45 mmol) of triethylamine and 2.26 g (36 mmol) of acryloyl chloride were slowly added dropwise, followed by stirring at room temperature. TLC was monitored until the reactants had completely reacted. The reaction was quenched with distilled water, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phase was collected, washed with 50 mL of saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and separated by column chromatography using a petroleum ether-ethyl acetate system (PE / EA = 6 / 1–5 / 1, v / v). A white solid (6.1 g) was finally obtained, with a yield of 67%. 1H NMR (600MHz, DMSO-d6) δ10.45(s,1H),7.87(d,J=8.8Hz,2H),7.78(d,J=8.8Hz,2H),6.46(dd,J= 17.0,10.1Hz,1H),6.30(dd,J=17.0,1.8Hz,1H),5.81(dd,J=10.1,1.8Hz,1H),1.54(s,9H)ppm. 13 C NMR(150MHz,DMSO-d6)δ167.15,164.15,143.53,141.02,132.95,132.03,130.80,12 8.30,127.76,127.08,125.40,123.93,123.48,119.38,119.10,118.65,118.17ppm.

[0073] (b) Preparation of 4-acrylamidobenzoic acid

[0074] 0.65 g (2.6 mmol) of tert-butyl 4-acrylamide benzoate was added to a 150 mL round-bottom flask, followed by 50 mL of anhydrous dichloromethane. The mixture was stirred thoroughly at room temperature, and then 7–8 mL of trifluoroacetic acid was slowly added dropwise while stirring continuously at room temperature. The reaction was monitored by TLC until the reactants were completely reacted. The solvent was removed by concentration under reduced pressure, and the pH was adjusted to alkaline with saturated sodium bicarbonate solution. Then, 3 M hydrochloric acid solution was slowly added dropwise until no more bubbles were produced. The mixture was filtered and dried under vacuum to give a white solid (0.40 g), with a yield of 62%. 1 H NMR (600MHz, DMSO-d6) δ12.73(s,1H),10.43(s,1H),7.91(d,J=8.7Hz,2H),7.78(d,J=8.7H z,2H),6.46(dd,J=17.0,10.2Hz,1H),6.30(dd,J=17.0,1.9Hz,1H),5.95-5.57(m,1H)ppm.

[0075] (c) Preparation of Compound 1

[0076] 0.19 g (1 mmol) of 4-acrylamide benzoic acid was placed in a 25 mL round-bottom flask, and 10 mL of DMF was slowly added. The mixture was stirred at 45 °C until homogeneous. 0.46 g (1.2 mmol) of HATU was added to the reaction mixture, and the mixture was stirred at 45 °C for 10 min. Then, 0.26 g (2 mmol) of DIPEA was added dropwise, and the mixture was stirred at this temperature for 15 min. Finally, 0.2 g (1 mmol) of 6-bromo-1H-indazole was added. The mixture was protected under nitrogen and reacted at 45 °C for 6 h. TLC was monitored until the reactants were completely reacted. The mixture was extracted three times with ethyl acetate (20 mL × 3), and the organic phase was collected. The organic phase was washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography. The eluent was a petroleum ether-ethyl acetate system (PE / EA = 5 / 1 to 1 / 1, v / v). A white solid (0.24 g) was finally obtained, with a yield of 60%. 1 H NMR (600MHz, DMSO-d6) δ10.58(s,1H),8.59(d,J=19.1Hz,2H),8.08(d,J=8.7Hz,2H),7.94(d,J=8.4Hz,1H),7. 87(d,J=8.7Hz,H),7.72-7.62(m,1H),6.50(dd,J=17.0,10.1Hz,1H),6.38-6.30(m,1H),5.85-5.83(m,1H)ppm. 13 C NMR(150MHz,DMSO-d6)δ167.15,164.15,143.53,141.02,132.95,132.03,130.80,128.30,127.7 6,127.08,125.40,123.93,123.48,119.38,119.10,118.65,118.17ppm.HR-MS(m / z)(ESI):calcd for C 17 H 12 BrN3O2[M+H] + 370.0185; found 370.0185.

[0077] Example 2: Preparation of Compound 2

[0078] Using 4-acrylamide benzoic acid and 5-chloro-1H-pyrrolo[2,3-b]pyridine as starting materials, a white solid (0.24 g) was obtained with a yield of 70%, following the synthesis method of compound 1. 1H NMR (600MHz, DMSO-d6) δ10.44(s,1H),8.77(dd,J=4.4,1.4Hz,2H),8.54(dd,J=8.4,1.4Hz,2H),7.96-7.85(m,1H),7.84-7.74(m ,1H),7.52(dd,J=8.4,4.4Hz,2H),6.46(dd,J=16.9,10.1Hz,1H),6.30(dd,J=17.0,1.8Hz,1H),5.81(dd,J=10.1,1.9Hz,1H)ppm. 13 C NMR(150MHz,DMSO-d6)δ167.37,164.02,151.55,143.48,140.07,135.08,132.0 1,130.86,129.27,128.15,125.86,121.16,119.15ppm.HR-MS(m / z)(ESI):calcd for C 17 H 12 ClN3O2[M+H] + 326.0690; found: 326.0688.

[0079] Example 3: Preparation of Compound 3

[0080] Using 4-acrylamide benzoic acid and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine as starting materials, a white solid (0.15 g) was obtained with a yield of 55%, following the synthetic method of compound 1. 1 H NMR (600MHz, DMSO-d6) δ10.59(s,1H),8.66(s,1H),8.09(d,J=4.0Hz,1H),7.91(d,J=8.7Hz,1H),7.84(s, 2H),7.78(d,J=8.7Hz,1H),6.96(d,J=4.0Hz,1H),6.57-6.43(m,1H),6.40-6.19(m,1H),5.83(m,1H)ppm. 13 C NMR(150MHz,DMSO-d6)δ160.99,154.73,153.04,149.80,140.68,134.98,130.34,128.31,122.17,102.25,97.66ppm.HR-MS(m / z)(ESI):calcd for C 16 H 11 ClN4O2[M+H] + :327.0643; found:327.0642.

[0081] Example 4: Preparation of Compound 4

[0082] Using 4-acrylamide benzoic acid and 1H-pyrazolo[3,4-b]pyridine as raw materials, a white solid (0.56 g) was obtained with a yield of 60%, following the synthetic method of compound 1. 1 H NMR (600MHz, DMSO-d6) δ10.55(s,1H),8.74(dd,J=4.6,1.6Hz,1H),8.55(s,1H),8.43(dd,J=8.0,1.6Hz,1H),7.96(d,J=8.8Hz,2H),7 .86(d,J=8.8Hz,2H),7.50(dd,J=8.0,4.6Hz,1H),6.49(dd,J=17.0,10.2Hz,1H),6.34(dd,J=17.0,1.8Hz,1H),6.02-5.66(m,1H)ppm. 13 C NMR(150MHz,DMSO-d6)δ165.84,164.18,152.39,150.87,143.62,138.83,132.79,13 2.00,131.74,128.34,127.74,120.64,118.72,118.21ppm.HR-MS(m / z)(ESI):calcd for C 16 H 12 N4O2[M+H] + :293.1033; found:293.1032.

[0083] Example 5: Preparation of Compound 5

[0084] Using 4-acrylamide benzoic acid and 5-bromo-7-azaindole pyridine as starting materials, a white solid (0.70 g) was obtained with a yield of 60%, following the synthetic method of compound 1. 1 H NMR (600MHz, DMSO-d6) δ10.54(s,1H),8.37(d,J=2.3Hz,1H),8.31(d,J=2.2Hz,1H),7.94(d,J=4.0Hz,1H),7.85-7.80(m,2H),7.79 -7.72(m,2H),6.80(d,J=4.0Hz,1H),6.49(dd,J=17.0,10.2Hz,1H),6.33(dd,J=17.0,1.8Hz,1H),5.84(dd,J=10.1,1.8Hz,1H)ppm. 13C NMR(150MHz,DMSO-d6)δ166.72,164.13,146.58,144.35,143.99,132.23,132.14,131.9 8,130.21,128.37,127.98,124.99,118.83,114.70,105.36ppm.HR-MS(m / z)(ESI):calcd for C 17 H 12 BrN3O2[M+H] + 370.0186; found 370.0183.

[0085] Example 6: Preparation of Compound 6

[0086] (a) Synthesis of intermediate II-M2

[0087] 0.98 g (5 mmol) of 6-bromoindole and 1.38 g (10 mmol) of anhydrous potassium carbonate were placed in a 250 mL round-bottom flask, and 150 mL of anhydrous acetonitrile was added. The mixture was refluxed and stirred at 80 °C for 30 min. Then, 1.17 g (5 mmol) of 4-fluoro-3-nitrobenzyl bromide was added, and the mixture was refluxed for 12 h. The reaction mixture was monitored by TLC until the reactants were completely reacted. The mixture was then extracted three times with ethyl acetate (50 mL × 3), and the organic phase was collected. The organic phase was washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography. The eluent was a petroleum ether-ethyl acetate system (PE / EA = 15 / 1, v / v). The final product was a yellow solid (0.96 g), with a yield of 55%. 1H NMR (600MHz, DMSO-d6) δ8.86 (s, 1H), 8.80 (s, 1H), 8.28 (dd, J = 7.1, 2.3Hz, 1H), 7 .83(ddd,J=8.6,4.2,2.4Hz,1H),7.59(dd,J=11.2,8.7Hz,1H),5.64(s,2H)ppm.

[0088] (b) Synthesis of intermediate II-M3

[0089] 0.56 g (10 mmol) of iron powder and 1.07 g (20 mmol) of ammonium chloride were placed in a 100 mL round-bottom flask. A mixed solution of 20 mL of ethanol and distilled water (EtOH / H2O = 5 / 1, v / v) was added. The mixture was reacted at 76 °C for 30 min. Then, 0.70 g (2 mmol) of II-M2 was added to the flask, and the mixture was stirred at 76 °C for 6 h. TLC monitoring was maintained until the reactants had completely reacted. The mixture was filtered hot through diatomaceous earth. The filtrate was concentrated under reduced pressure and separated by column chromatography. The eluent was a petroleum ether-ethyl acetate system (PE / EA = 5 / 1, v / v), yielding a gray solid (0.45 g) in 70% yield.1 H NMR (600MHz, DMSO-d6) δ8.79(s,2H),6.94(s,1H),6.65(s,1H),6.51(s,1H),5.38(s,2H),5.17(s,2H)ppm.

[0090] (c) Synthesis of Compound 6

[0091] Using intermediate II-M3 and acryloyl chloride as raw materials, and referring to the synthesis method of tert-butyl 4-acrylamide benzoate in Example 1, the final product was obtained as a white solid (0.48 g) with a yield of 61%. 1 H NMR (600MHz, DMSO-d6) δ9.91(s,1H),7.87(d,J=5.9Hz,1H),7.71(s,1H),7.54-7.49(m,2H),7.25-7.20(m,1H),7.14(d,J=8.2Hz,1H) ,6.97(s,1H),6.57(dd,J=16.9,10.4Hz,1H),6.52(d,J=2.7Hz,1H),6.24(d,J=17.0Hz,1H),5.75(d,J=10.1Hz,1H),5.42(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.90, 137.04, δ134.61 (d, J = 3.2Hz), 131.69, 130.53, 127.93, 127.77, 126.56, 126.52 (d, J = 12. 1Hz),124.43(d,J=7.8Hz),122.69,122.54,116.12,115.99,114.62,113.30,101.99,48.93ppm.HR-MS(m / z)(ESI):calcd for C 18 H 14 BrFN2O[M+H] + :332.0708; found:332.0706.

[0092] Example 7: Preparation of Compound 7

[0093] Using 6-bromoindazole and 4-fluoro-3-nitrobenzyl bromide as raw materials, and referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1, the first step intermediate product was obtained, a yellow solid (1.00 g), with a yield of 27%. 1¹H NMR (600MHz, DMSO-d⁶) δ 8.68 (s, 1H), 8.25 (dd, J = 7.2, 2.3Hz, 1H), 8.19 (d, J = 8.3Hz, 1H), 7.84–7.75 (m, 1H), 7.60 (dd, J = 11.3, 8.7Hz, 1H), 7.38 (d, J = 8.3Hz, 1H), 5.60 (s, 2H) ppm; Second step intermediate product, gray solid (0.93 g), yield 40%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.58 (s, 1H), 8.17 (d, J = 8.2Hz, 1H), 7.36 (d, J = 8.3Hz, 1H), 7.09–6.85 (m, 1H), 6.61 (d, J = 8.4Hz, 1H), 6.46 (s, 1H), 5.32 (s, 2H), 5.18 (s, 2H) ppm; final product, white solid (0.46 g), yield 30%. 1 H NMR (600MHz, DMSO-d6) δ9.90 (s, 1H), 8.15 (d, J = 0.8Hz, 1H), 8.09 (s, 1H), 7. 91(d,J=6.4Hz,1H),7.74(d,J=8.5Hz,1H),7.27(dd,J=8.5,1.5Hz,1H),7.22 (dd,J=10.8,8.5Hz,1H),7.06-7.02(m,1H),6.56(dd,J=17.0,10.2Hz,1H), 6.24(dd,J=17.0,1.9Hz,1H),5.75(dd,J=10.2,1.8Hz,1H),5.64(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.88, 140.55, 134.25, δ133.93 (d, J = 3.3Hz), 131.69, 127.93, 126.48 (d, J = 11.9Hz), 124.9 3(d,J=7.7Hz),124.26,123.62,123.25,123.13,120.41,116.08,115.94,112.98,51.60ppm.HR-MS(m / z)(ESI):calcd for C 17 H 13 BrFN3O[M+H] + :374.0304; found:374.0302.

[0094] Example 8: Preparation of Compound 8

[0095] Using 4-chloro-7-azaindole and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.10 g) with a yield of 56%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.21–8.04 (m, 1H), 7.89 (d, J = 7.0 Hz, 1H), 7.77–7.71 (m, 1H), 7.58 (dd, J = 11.2, 8.7 Hz, 1H), 7.39–7.07 (m, 2H), 6.64 (t, J = 5.8 Hz, 1H), 5.23 (s, 2H) ppm; Second step intermediate product, yellow solid (1.30 g), yield 90%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 7.87 (d, J = 7.0Hz, 1H), 7.21 (d, J = 5.0Hz, 2H), 6.92 (dd, J = 11.4, 8.3Hz, 1H), 6.67 (d, J = 7.3Hz, 1H), 6.62 (t, J = 6.1Hz, 1H), 6.50–6.40 (m, 1H), 5.15 (s, 2H), 4.93 (s, 2H) ppm; final product, white solid (0.73 g), yield 50%. 1 H NMR(600MHz, DMSO-d6)δ9.94(s,1H),7.96(d,J=6.5Hz,1H),7.88(d,J=7.1Hz,1H),7.28-7.24(m,1H),7.24-7.19(m,2H),7.14-7.09(m, 1H),6.63(ddd,J=8.4,5.0,2.5Hz,1H),6.61-6.55(m,1H),6.26(dd,J=17.0,1.9Hz,1H),5.77(dd,J=10.2,1.9Hz,1H),5.08(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.87, 148.08, 143.85, 134.88, δ 134.64 (d, J = 3.4Hz), 131.67, 130.77, 127.97, 126.45 (d, J =12.0Hz),124.81(d,J=7.5Hz),123.47,119.32,116.24,116.12,115.99,98.23,47.54ppm.HR-MS(m / z)(ESI):calcd for C 17 H 13 ClFN3O[M+H] +:330.0804; found:330.0802.

[0096] Example 9: Preparation of Compound 9

[0097] Using 5-bromo-7-azaindole and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (3.70 g) with a yield of 90%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.25 (d, J = 5.1 Hz, 1H), 8.13 (dd, J = 7.2, 2.2 Hz, 1H), 7.85 (d, J = 3.6 Hz, 1H), 7.67 (ddd, J = 8.6, 4.3, 2.3 Hz, 1H), 7.54 (dd, J = 11.3, 8.7 Hz, 1H), 7.28 (d, J = 5.1 Hz, 1H), 6.62 (d, J = 3.6 Hz, 1H), 5.60 (s, 2H) ppm; Second step intermediate product, gray solid (1.33 g), yield 75%. 1 ¹H NMR (600 MHz, DMSO-d⁶) δ 8.24 (s, 1H), 7.70 (s, 1H), 7.25 (s, 1H), 6.90 (s, 1H), 6.57 (s, 2H), 6.41 (s, 1H), 5.35 (s, 2H), 5.12 (s, 2H) ppm; final product, white solid (0.68 g), yield 45%. 1 H NMR (600MHz, DMSO-d6) δ9.91(s,1H),8.24(d,J=5.1Hz,1H),7.92(d,J=6.3Hz,1H),7.77(d,J=3.5Hz,1H),7.26(d,J=5.1Hz,1H),7.21(dd,J=10.8,8 .5Hz,1H),7.12-6.96(m,1H),6.59(d,J=3.5Hz,1H),6.58-6.52(m,1H),6. 24(dd,J=17.0,1.9Hz,1H),5.75(dd,J=10.2,1.9Hz,1H),5.48(s,2H)ppm. 13C NMR (150MHz, DMSO-d6) δ163.88,145.88,143.08,134.68(d,J=3.4Hz),131.69,131.44,131.12,127.93,126.46(d,J =12.1Hz),124.76(d,J=7.7Hz),123.45,122.34,116.10,115.96,111.57,99.95,47.29ppm.HR-MS(m / z)(ESI):calcd for C 17 H 13 BrFN3O[M+H] + :374.0299; found:374.0299.

[0098] Example 10: Preparation of Compound 10

[0099] Using 1H-pyrazolo[3,4-c]pyridine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.47 g) with a yield of 57%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.34 (s, 1H), 8.30–8.18 (m, 1H), 8.09 (dd, J = 7.2, 2.2 Hz, 1H), 7.78 (d, J = 3.5 Hz, 1H), 7.65 (ddd, J = 8.6, 4.2, 2.3 Hz, 1H), 7.59–7.44 (m, 1H), 6.55 (d, J = 3.5 Hz, 1H), 5.57 (s, 2H) ppm; Second step intermediate product, white solid (1.23 g), yield 88%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.32 (s, 1H), 8.23 ​​(s, 1H), 7.67–7.57 (m, 1H), 6.98–6.78 (m, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.50 (s, 1H), 6.39 (s, 1H), 5.30 (s, 2H), 5.09 (s, 2H) ppm; final product, white solid (0.62 g), yield 50%. 1H NMR(600MHz,DMSO-d6)δ9.90(s,1H),8.32(d,J=2.2Hz,1H),8.24(d,J=2.2Hz ,1H),7.90(d,J=6.6Hz,1H),7.70(d,J=3.5Hz,1H),7.20(dd,J=10.8,8.5Hz, 1H),7.05-7.00(m,1H),6.56(dd,J=17.1,10.3Hz,1H),6.52(d,J=3.5Hz,1H) ,6.24(dd,J=17.0,1.9Hz,1H),5.75(dd,J=10.2,1.9Hz,1H),5.45(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.87, 150.14, 149.45, δ134.10 (d, J = 3.3Hz), 133.20, 131.69, 131.12, 127.94, 126.46 ( d,J=12.0Hz),125.03(d,J=7.7Hz),123.61,117.63,116.06,115.92,115.59,49.82ppm.HR-MS(m / z)(ESI):calcd forC 16 H 13 FN4O[M+H] + :297.1146; found:297.1160.

[0100] Example 11: Preparation of Compound 11

[0101] Using 5-chloro-3H-imidazolium[4,5-b]pyridine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.4 g) with a yield of 50%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.06 (dd, J = 7.1, 1.9 Hz, 1H), 7.83 (s, 1H), 7.61–7.58 (m, H), 7.57–7.54 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.16 (dd, J = 8.4, 1.5 Hz, 1H), 6.56 (d, J = 3.1 Hz, 1H), 5.54 (s, 2H) ppm; Second step intermediate product, white solid (0.50 g), yield 71%. 1¹H NMR (600MHz, DMSO-d⁶) δ 7.65 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 3.1 Hz, 1H), 7.13 (dd, J = 8.4, 1.7 Hz, 1H), 6.91 (dd, J = 11.5, 8.3 Hz, 1H), 6.53 (dd, J = 8.7, 2.0 Hz, 1H), 6.50 (d, J = 3.1 Hz, 1H), 6.35 (ddd, J = 8.0, 4.2, 2.2 Hz, 1H), 5.26 (s, 2H), 5.12 (s, 2H) ppm; final product, white solid (0.45 g), yield 70%. 1 H NMR (600MHz, DMSO-d6) δ9.96(s,1H),8.64(s,1H),8.18(d,J=8.3Hz,1H),7.98(d,J=6.1Hz,1H),7.37(d,J=8.3Hz,1H),7.27(dd,J=10.8,8 .5Hz,1H),7.14-7.10(m,1H),6.58(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.9Hz,1H),5.77(dd,J=10.2,1.9Hz,1H),5.47(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.93, 146.52, 146.47, 144.83, 134.48, δ 133.30 (d, J = 3.2Hz), 131.65, 131.10, 128.04, 126.66(d,J=12.1Hz),124.95(d,J=7.3Hz),123.48,118.75,116.33,116.20,46.28ppm.HR-MS(m / z)(ESI):calcd for C 16 H 12 ClFN4O[M+H] + :331.0756; found:331.0757.

[0102] Example 12: Preparation of Compound 12

[0103] Using 4-chloropyrrolo[2,3-D]pyrimidine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.12 g) with a yield of 60%. 1¹H NMR (600MHz, DMSO-d⁶) δ 8.61 (s, ¹H), 8.31 (d, J = 7.0 Hz, ¹H), 8.27 (s, ¹H), 8.15–8.04 (m, ¹H), 7.67 (s, ¹H), 7.55 (t, J = 9.6 Hz, ¹H), 7.29 (s, ¹H), 5.82 (s, 2H) ppm; Second step intermediate product, gray solid (0.56 g), yield 70%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.58 (d, J = 3.5Hz, 1H), 8.27 (d, J = 7.8Hz, 1H), 8.19 (s, 1H), 7.24 (dd, J = 7.6, 4.4Hz, 1H), 6.94–6.79 (m, 1H), 6.64 (d, J = 8.2Hz, 1H), 6.43 (s, 1H), 5.51 (s, 2H), 5.13 (s, 2H) ppm; final product, white solid (0.57 g), yield 70%. 1 H NMR (600MHz, DMSO-d6) δ9.94(s,1H),8.65(s,1H),8.20(d,J=3.2Hz,1H),7.82(d,J=6.5Hz,1H),7.24(dd,J=10.8,8.6Hz,1H),6. 86(d,J=3.2Hz,2H),6.58(dd,J=17.0,10.2Hz,1H),6.24(dd,J=17.0,1.9Hz,1H),5.77(dd,J=10.1,1.9Hz,1H),5.76(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.91, 152.69, 149.93, 141.73, 139.82, δ 134.82 (d, J = 3.2Hz), 131.66, 128.03, 126.73 (d ,J=12.0Hz),123.45(d,J=7.7Hz),123.35,122.08,116.29,116.16,102.74,51.12ppm.HR-MS(m / z)(ESI):calcdfor C 16 H 12 ClFN4O[M+H] + :331.0756; found:331.0761.

[0104] Example 13: Preparation of Compound 13

[0105] Using 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.35 g) with a yield of 53%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.21 (d, J = 3.9Hz, 2H), 8.11 (dd, J = 7.2, 2.2Hz, 1H), 7.81–7.72 (m, 1H), 7.67 (ddd, J = 8.6, 4.2, 2.3Hz, 1H), 7.55 (dd, J = 11.2, 8.7Hz, 1H), 7.30 (d, J = 8.5Hz, 1H), 5.78 (s, 2H) ppm; Second step intermediate product, white solid (0.91 g), yield 87%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.12 (s, 1H), 8.00 (s, 1H), 7.73 (d, J = 8.5Hz, 1H), 7.26 (d, J = 8.4Hz, 1H), 6.90 (dd, J = 11.2, 8.4Hz, 1H), 6.58 (d, J = 8.0Hz, 1H), 6.50–6.35 (m, 1H), 5.49 (s, 2H), 5.12 (s, 2H) ppm; final product, white solid (0.65 g), yield 62%. 1 H NMR (600MHz, DMSO-d6) δ9.92(s,1H),8.67(s,1H),7.96(d,J=6.1Hz,1H),7.86(d,J=3.6Hz,1H),7.23(dd,J=10.8,8.5Hz,1H),7.08(ddd,J=8.3,4 .6,2.3Hz,1H),6.70(d,J=3.6Hz,1H),6.57(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.9Hz,1H),5.76(dd,J=10.2,1.9Hz,1H),5.50(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.91, 151.23, 150.99, δ133.82 (d, J = 3.3Hz), 131.87, 131.33, 130.42, 128.00, 126.60 (d,J=12.0Hz),124.95(d,J=7.9Hz),123.55,117.24,116.24,116.11,99.42,47.75ppm.HR-MS(m / z)(ESI):calcd forC 16H 12 ClFN4O[M+H] + :331.0756; found:331.0744.

[0106] Example 14: Preparation of Compound 14

[0107] Using 6-chloro-9H-purine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (2.10 g) with a yield of 56%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.83–8.59 (m, 1H), 8.18 (dd, J = 7.2, 2.2 Hz, 1H), 7.93 (d, J = 3.6 Hz, 1H), 7.72 (ddd, J = 8.6, 4.2, 2.3 Hz, 1H), 7.56 (t, J = 9.9 Hz, 1H), 6.73 (dd, J = 3.5, 1.7 Hz, 1H), 5.63 (s, 2H) ppm; Second step intermediate product, yellow solid (1.30 g), yield 90%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.66 (s, 1H), 7.78 (s, 1H), 7.05–6.82 (m, 1H), 6.69 (s, 1H), 6.57 (d, J = 7.3 Hz, 1H), 6.43 (s, 1H), 5.35 (s, 2H), 5.14 (s, 2H) ppm; final product, white solid (0.61 g), yield 40%. 1 H NMR(600MHz,DMSO-d6)δ9.95(s,1H),8.84(s,1H),8.80(s,1H),8.03(d,J=6.0Hz,1H),7.26(dd,J=10.8,8.5Hz,1H),7.20 -7.15(m,1H),6.58(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.9Hz,1H),5.76(dd,J=10.2,1.9Hz,1H),5.52(s,2H)ppm. 13C NMR (150MHz, DMSO-d6) δ163.95, 152.23, 149.65, 147.88, 132.67 (d, J = 3.4Hz), 131.65, 131.31, 130.43, 128. 05,126.68(d,J=12.1Hz),125.29(d,J=7.7Hz),123.83,116.37,116.24,46.98ppm.HR-MS(m / z)(ESI):calcd for C 15 H 11 ClFN5O[M+H] + 332.0709; found 332.0706.

[0108] Example 15: Preparation of Compound 15

[0109] Using 2-chloro-9H-purine and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (1.35 g) with a yield of 62%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 9.13 (s, ¹H), 8.79 (s, ¹H), 8.27 (dd, J = 7.1, 2.3Hz, ¹H), 7.90–7.69 (m, ¹H), 7.61 (dd, J = 11.3, 8.7Hz, ¹H), 5.60 (s, 2H) ppm; Second step intermediate product, gray solid (0.22 g), yield 70%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 9.12 (s, 1H), 8.73 (s, 1H), 6.95 (dd, J = 11.5, 8.3Hz, 1H), 6.62 (dd, J = 8.6, 2.1Hz, 1H), 6.48 (ddd, J = 8.0, 4.1, 2.2Hz, 1H), 5.33 (s, 2H), 5.20 (s, 2H) ppm; final product, white solid (0.14 g), yield 62%. 1 HNMR(600MHz,DMSO-d6)δ9.98(s,1H),9.13(s,1H),8.78(s,1H),8.01(d,J=5.9Hz,1H),7.28(dd,J=10.8,8.5Hz,1H),7.19 -7.10(m,1H),6.59(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.9Hz,1H),5.77(dd,J=10.1,1.8Hz,1H),5.48(s,2H)ppm.13 C NMR (150MHz, DMSO-d6) δ163.95, 153.44, 150.53, 148.56, 133.51, δ 132.59 (d, J = 3.3Hz), 131.65, 131.33, 128 .07,126.73(d,J=12.1Hz),125.14(d,J=7.8Hz),123.64,116.43,116.29,46.43ppm.HR-MS(m / z)(ESI):calcd for C 15 H 11 ClFN5O[M+H] + :332.0709; found:332.0714.

[0110] Example 16: Preparation of Compound 16

[0111] Using methyl 1H-benzo[d][1,2,3]triazole-4-carboxylic acid and 4-fluoro-3-nitrobenzyl bromide as raw materials, the first-step intermediate product was obtained by referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1. The product was a yellow solid (0.96 g) with a yield of 44%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.69 (dd, J = 1.3, 0.8 Hz, 1H), 8.27 (dd, J = 7.2, 2.2 Hz, 1H), 8.20 (dd, J = 8.7, 0.8 Hz, 1H), 7.97 (dd, J = 8.7, 1.4 Hz, 1H), 7.79 (ddd, J = 8.6, 4.2, 2.4 Hz, 1H), 7.59 (dd, J = 11.3, 8.7 Hz, 1H), 6.22 (s, 2H), 3.93 (s, 3H) ppm; Second step intermediate product, yellow solid (0.65 g), yield 78%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.56 (d, J = 103.5 Hz, 1H), 8.13 (dd, J = 52.0, 7.6 Hz, 1H), 7.99–7.75 (m, 1H), 6.95 (s, 1H), 6.74–6.48 (m, 2H), 5.91 (d, J = 45.4 Hz, 2H), 5.20 (s, 2H), 3.91 (s, 3H) ppm; final product, yellow solid (0.36 g), yield 45%. 1H NMR (600MHz, DMSO-d6) δ9.95 (s, 1H), 8.62 (d, J = 50.3Hz, 1H), 8.20-8.09 (m, 1H) ,8.08-8.03(m,1H),8.02-7.94(m,1H),7.30-7.25(m,1H),7.18(ddd,J=10.6,7 .5,2.1Hz,1H),6.58(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.0Hz,1H),6. 10(s,1H),6.03(s,1H),5.76(dd,J=10.2,1.6Hz,1H),3.91(d,J=4.5Hz,3H)ppm. 13 C NMR (150MHz, DMSO-d6) δ166.31, 163.94, 147.73, 145.46, 135.42, 132.95, δ 132.37 (d, J = 3.4Hz), 131.63, 129.11, 128.08 (d,J=4.1Hz),126.81(d,J=10.8Hz),124.75,122.03,120.08,113.61,111.65,53.04,50.98ppm.HR-MS(m / z)(ESI):calcd for C 18 H 15 FN4O3[M+H] + :355.1201; found:355.1208.

[0112] Example 17: Preparation of Compound 17

[0113] (a) Synthesis of intermediate III-M1

[0114] Using 1H-benzotriazole-7-carboxylic acid and 2,2-difluoroethylamine as starting materials, and following the synthetic method of compound 1, the first-step intermediate product (III-M1) was obtained as a yellow solid (3.17 g) with a yield of 65%. 1 H NMR (600MHz, DMSO-d6) δ15.99(s,1H),9.04(s,1H),8.57(s,1H),7.97(s,2H),6.16(tt,J=56.0,4.1Hz,1H),3.73(tdd,J=15.6,5.7,4.2Hz,2H)ppm;

[0115] (b) Synthesis of Compound 17

[0116] Using III-M1 as raw material, and referring to the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1, the second intermediate product (III-M2) was obtained, a yellow solid (1.28 g), with a yield of 60%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 9.04 (t, J = 5.8 Hz, 1H), 8.58–8.39 (m, 1H), 8.31 (dd, J = 7.2, 2.2 Hz, 1H), 8.03 (dd, J = 9.0, 0.8 Hz, 1H), 7.92 (dd, J = 9.0, 1.5 Hz, 1H), 7.88 (ddd, J = 8.6, 4.2, 2.3 Hz, 1H), 7.63 (dd, J = 11.3, 8.7 Hz, 1H), 6.32–6.03 (m, 3H), 3.81–3.64 (m, 2H) ppm; Third step intermediate product (III-M₃), yellow solid (0.19 g), yield 95%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 9.03 (s, 1H), 8.49 (s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.06–6.89 (m, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.57 (s, 1H), 6.15 (t, J = 56.0 Hz, 1H), 5.84 (s, 2H), 5.22 (s, 2H), 3.72 (t, J = 14.9 Hz, 2H) ppm; final product, white solid (0.14 g), yield 58%. 1 H NMR(600MHz,DMSO-d6)δ9.97(s,1H),9.03(s,1H),8.50(s,1H),8.12(d,J=6 .5Hz,1H),8.02(d,J=8.9Hz,1H),7.91(d,J=8.7Hz,1H),7.32-7.27(m,1H), 7.24(s,1H),6.59(dd,J=16.8,10.2Hz,1H),6.25(d,J=16.6Hz,1H),6.11(d ,J=56.0Hz,1H),6.02(s,2H),5.77(d,J=10.1Hz,1H),3.76-3.68(m,2H)ppm. 13C NMR(150MHz,DMSO-d6)δ166.99,163.98,145.57,143.79,132.41,131.64,128.08,δ126.74(d,J=11.9Hz),125.98(d,J=11.5Hz),124.37 (d,J=1.6Hz),118.44(d,J=11.7Hz),116.59,116.33,116.20,115.00,113.40,59.73,42.24(t,J=26.5Hz)ppm.HR-MS(m / z)(ESI):calcd for C 19 H 16 F3N5O2[M+H] + 404.1328; found: 404.1327.

[0117] Example 18: Preparation of Compound 18

[0118] Using 1H-benzo[d][1,2,3]triazole-7-carboxylic acid and 3,3-difluorocyclobutylamine as raw materials, the first-step intermediate product was obtained by referring to the synthesis method of III-M1 in Example 17. The product was a gray solid (2.82 g) with a yield of 69%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 15.97 (s, 1H), 9.00 (d, J = 6.4Hz, 1H), 8.49 (s, 1H), 7.95 (s, 2H), 4.83–4.02 (m, 1H), 3.06–2.91 (m, 2H), 2.85–2.67 (m, 2H) ppm. Following the synthesis methods of II-M2 and II-M3 in Example 6 and tert-butyl 4-acrylamide benzoate in Example 1, the second intermediate product was obtained as a yellow solid (1.58 g), with a yield of 54%. 1 ¹H NMR (600MHz, DMSO-d⁶) δ 8.99 (d, J = 6.5 Hz, 1H), 8.49 (s, 1H), 8.30 (dd, J = 7.2, 2.2 Hz, 1H), 8.02 (dd, J = 9.0, 0.8 Hz, 1H), 7.92–7.88 (m, 1H), 7.87 (dq, J = 6.5, 2.1 Hz, 1H), 7.63 (dd, J = 11.3, 8.7 Hz, 1H), 6.18 (s, 2H), 4.30 (dt, J = 14.9, 6.8 Hz, 1H), 3.04–2.90 (m, 2H), 2.84–2.65 (m, 2H) ppm; Third step intermediate product, yellow solid (0.18 g), yield 90%. 1¹H NMR (600MHz, DMSO-d⁶) δ 8.96 (s, 1H), 8.44 (s, 1H), 7.92 (d, J = 61.9Hz, 2H), 7.19–6.33 (m, 3H), 5.84 (s, 2H), 5.22 (s, 2H), 4.31 (s, 1H), 2.99 (s, 2H), 2.79 (s, 2H) ppm; final product, white solid (0.11 g), yield 51%. 1 H NMR(600MHz,DMSO-d6)δ9.97(s,1H),8.98(d,J=6.5Hz,1H),8.64-8.37(m,1H),8.12(d,J =5.9Hz,1H),8.06-7.97(m,1H),7.88(dd,J=9.0,1.5Hz,1H),7.29(dd,J=10.7,8.5Hz,1H) ,7.26-7.20(m,1H),6.59(dd,J=17.0,10.2Hz,1H),6.25(dd,J=17.0,1.9Hz,1H),6.01(s, 2H),5.77(dd,J=10.2,1.9Hz,1H),4.30(s,1H),3.04-2.92(m,2H),2.82-2.75(m,2H)ppm. 13 C NMR(150MHz,DMSO-d6)δ166.37,163.99,145.53,143.81,132.80,δ131.67(d, J=3.7Hz),128.05,126.74(d,J=12.0Hz),125.96(d,J=9.4Hz),124.36,121.8 3,120.06,118.38,118.22,116.31,116.17,59.72,42.50(dd,J=23.0,21.6Hz ),42.35,35.05(d,J=5.6Hz),34.92(d,J=5.6Hz)ppm.HR-MS(m / z)(ESI):calcd for C 21 H 18 F3N5O2[M+H] + :430.1485; found:430.1499.

[0119] Example 19: Preparation of Compound 19

[0120] (a) Synthesis of intermediate IV-M1

[0121] Using 6-chloro-9H-purine and p-nitrobenzyl bromide as raw materials, a yellow solid (3.90 g) was obtained by referring to the synthesis method of II-M2 in Example 6, with a yield of 52%.1 H NMR (600MHz, DMSO-d6) δ8.88(s,1H),8.79(s,1H),8.20(d,J=8.7Hz,2H),7.58(d,J=8.8Hz,2H),5.71(s,2H)ppm.

[0122] (b) Synthesis of intermediate IV-M2

[0123] Using intermediate IV-M1 as a raw material, a yellow solid (1.42 g) was obtained by referring to the synthesis method of II-M3 in Example 6, with a yield of 57%. 1 H NMR (600MHz, DMSO-d6) δ8.80(s,1H),8.76(s,1H),7.09(d,J=8.4Hz,2H),6.50(d,J=8.5Hz,2H),5.30(s,2H),5.14(s,2H)ppm.

[0124] (c) Synthesis of Compound 19

[0125] Using intermediate IV-M2 and acryloyl chloride as raw materials, and referring to the synthesis method of compound 6 in Example 6, the final product was obtained as a yellow solid (0.70 g) with a yield of 41%. 1 H NMR (600MHz, DMSO-d6) δ10.25(s,1H),8.82(d,J=22.5Hz,2H),7.64(d,J=8.5Hz,2H),7.34(d,J=8.5Hz,2H), 6.43(dd,J=17.0,10.2Hz,1H), 6.24(dd,J=17.0,1.8Hz,1H), 5.74(dd,J=10.2,1.8Hz,1H), 5.49(s,2H)ppm. 13 C NMR(150MHz,DMSO-d6)δ163.63,152.29,152.26,149.58,147.89,139.32,132.2 4,131.37,131.31,128.84,127.44,120.00,47.17ppm.HR-MS(m / z)(ESI):calcd for C 15 H 12 ClN5O[M+H] + 314.0803; found: 314.0808.

[0126] Example 20: Preparation of Compound 20

[0127] Using IV-M2 and methacryloyl chloride from Example 19 as raw materials, and referring to the synthesis method of compound 6 in Example 6, a white solid (0.31 g) was obtained with a yield of 56%. 1 H NMR(600MHz,DMSO-d6)δ9.85(s,1H),8.84(s,1H),8.80(s,1H),7.65(d,J=8.6Hz,2H),7.33(d ,J=8.6Hz,2H),5.79(s,1H),5.53-5.50(m,1H),5.48(s,2H),1.92(dd,J=1.6,1.2Hz,3H)ppm. 13 C NMR(150MHz,DMSO-d6)δ167.24,152.24,152.15,149.56,147.91,140.67,139.36,1 31.30,131.29,128.60,120.82,120.62,47.19,19.19ppm.HR-MS(m / z)(ESI):calcd for C 16 H 14 ClN5O[M+H] + :328.0959; found:328.0966.

[0128] Example 21: Preparation of compound 21

[0129] Using IV-M2 and 3-methylcrotonyl chloride from Example 19 as raw materials, and following the synthesis method of compound 6 in Example 6, a white solid (0.18 g) was obtained with a yield of 51%. 1 H NMR(600MHz,DMSO-d6)δ9.86(s,1H),8.83(s,1H),8.80(s,1H),7.58(d,J=8.3Hz,2 H),7.31(d,J=8.3Hz,2H),5.83(s,1H),5.46(s,2H),2.13(s,3H),1.85(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6) δ165.04,152.23,152.16,152.01,149.57,147.86,139.84,131. 30,130.69,128.78,119.63,119.49,47.21,27.50,19.95ppm.HR-MS(m / z)(ESI):calcd for C 17 H 16 ClN5O[M+H] + :342.1116; found:342.1119.

[0130] Example 22: Preparation of compound 22

[0131] Using IV-M2 and propionyl chloride from Example 19 as raw materials, and referring to the synthesis method of compound 6 in Example 6, a white solid (0.35 g) was obtained with a yield of 61%. 1 H NMR(600MHz,DMSO-d6)δ9.89(s,1H),8.82(s,1H),8.80(s,1H),7.55(d,J=8.5Hz,2H) ,7.30(d,J=8.6Hz,2H),5.46(s,2H),2.29(q,J=7.5Hz,2H),1.05(t,J=7.5Hz,3H)ppm. 13 C NMR (150MHz, DMSO-d6) δ172.47,152.23,152.16,149.57,147.87,139.66,131. 30,130.72,128.77,119.62,47.18,29.91,10.07ppm.HR-MS(m / z)(ESI):calcd for C 15 H 14 ClN5O[M+H] + 316.0959; found: 316.0968.

[0132] Example 23: Preparation of compound 23

[0133] Using IV-M2 and chloroacetyl chloride from Example 19 as raw materials, and following the synthesis method of compound 6 in Example 6, a white solid (0.22 g) was obtained with a yield of 61%. 1 H NMR (600MHz, DMSO-d6) δ10.69(s,1H),8.85(s,1H),8.79(s,1H),7.60(d,J=8.6Hz,2H),7.35(d,J=8.6Hz,2H),5.49(s,2H),4.28(s,2H)ppm. 13 CNMR(150MHz,DMSO-d6)δ165.19,152.24,152.16,149.56,147.91,138.87,131.67,131.30,128.88,120.00,47.15,43.94ppm.HR-MS(m / z)(ESI):calcd for C 14 H 11 Cl2N5O[M+H] + :336.0413; found:336.0409.

[0134] Example 24: Preparation of compound 24

[0135] Using IV-M2 and 2-chloropropionyl chloride from Example 19 as raw materials, and referring to the synthesis method of compound 6 in Example 6, a white solid (0.34 g) was obtained with a yield of 72%. 1 H NMR (600MHz, DMSO-d6) δ10.36(s,1H),8.81(d,J=19.7Hz,2H),7.57(d,J=8.3Hz,2H ),7.35(d,J=8.3Hz,2H),5.49(s,2H),4.93-4.30(m,1H),1.59(d,J=6.5Hz,3H)ppm. 13 C NMR(150MHz,DMSO-d6)δ167.77,152.25,152.17,149.58,147.88,138.74,131 .80,131.31,128.91,120.11,55.1847.14,21.43ppm.HR-MS(m / z)(ESI):calcd for:C 15 H 13 Cl2N5O[M+H] + :3,50.0569; found:350.0584.

[0136] Example 25: Preparation of Compound 25

[0137] Using IV-M2 and isobutyryl chloride from Example 19 as raw materials, and referring to the synthesis method of compound 6 in Example 6, a white solid (0.32 g) was obtained with a yield of 58%. 1 H NMR(600MHz,DMSO-d6)δ9.87(s,1H),8.82(s,1H),8.80(s,1H),7.60-7.54(m,2H) ,7.31(d,J=8.5Hz,2H),5.46(s,2H),2.60-2.52(m,1H),1.07(d,J=6.8Hz,6H)ppm. 13 C NMR(150MHz,DMSO-d6)δ175.71,152.22,152.15,149.56,147.85,139.75,131.3 0,130.74,128.77,119.74,47.20,35.33,19.92ppm.HR-MS(m / z)(ESI):calcdfor C 16 H 16 ClN5O[M+H] + :330.11161; found:330.11251.

[0138] Example 26: Preparation of Compound 26

[0139] Using IV-M2 and cyclopropylformyl chloride from Example 19 as raw materials, and referring to the synthesis method of compound 6 in Example 6, a white solid (0.25 g) was obtained with a yield of 63%. 1 H NMR (600MHz, DMSO-d6) δ10.23(s,1H),8.82(s,1H),8.80(s,1H),7.55(d,J=8.5Hz, 2H),7.31(d,J=8.6Hz,2H),5.47(s,2H),1.85-1.65(m,1H),0.90-0.57(m,4H)ppm. 13 C NMR(150MHz,DMSO-d6)δ172.12,152.23,152.16,149.57,147.87,139.63,131 .30,130.74,128.80,119.60,47.17,14.96,7.67ppm.HR-MS(m / z)(ESI):calcd for:C 16 H 14 ClN5O[M+H] + 328.0959; found 328.0971.

[0140] Example 27: Preparation of Compound 27

[0141] 0.10 g (0.39 mmol) of IV-M2 (see Example 19) and 0.11 g (0.77 mmol) of potassium carbonate were placed in a 150 mL double-necked round-bottom flask. 30 mL (v1 / v2 = 3 / 1) of a mixed solvent of 1,4-dioxane and water was added, followed by 0.044 g (0.039 mmol) of tetrakis(triphenylphosphine)palladium. The mixture was evacuated and protected with nitrogen. Then, 0.064 g (0.5 mmol) of thiophene-2-phenylboronic acid dissolved in a small amount of 1,4-dioxane was injected into the reaction flask. The reaction was carried out at 80 °C for 8 h. TLC was monitored until the reactants had completely reacted. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to remove the solvent. Column chromatography was used to separate the gray intermediate V-M2. Using V-M2 and acryloyl chloride as starting materials, and following the synthesis method of compound 6 in Example 6, a white solid (0.058 g) was obtained, with a yield of 61%. 1H NMR (600MHz, DMSO-d6) δ10.39(s,1H),8.96(s,1H),8.90(s,1H),8.74(d,J=3.6Hz,1H),8.02(d,J=5.0Hz,1H),7.76(d,J=8.4Hz,2 H),7.49-7.39(m,3H),6.54(dd,J=16.9,10.2Hz,1H),6.34(dd,J=17.0,1.6Hz,1H),5.84(dd,J=10.2,1.6Hz,1H),5.59(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.61,152.52,152.22,148.92,147.05,140.20,139.23,132.84,132.2 5,132.12,131.82,129.53,128.79,128.55,127.42,119.99,46.65ppm.HR-MS(m / z)(ESI):calcd for:C 19 H 15 N5OS[M+H] + :362.1070; found:362.1070.

[0142] Example 28: Preparation of compound 28

[0143] Using intermediate IV-M2 (see Example 19) and phenylboronic acid as raw materials, a white solid (0.061 g) was obtained by referring to the synthesis methods of V-M2 in Example 27 and compound 6 in Example 6, with a yield of 55%. 1 H NMR (600MHz, DMSO-d6) δ10.24(s,1H),9.01(s,1H),8.88-8.78(m,2H),7.68-7.54(m,6H),7.37(d,J=8.5Hz,2H ), 6.42(dd,J=17.0,10.2Hz,1H), 6.24(dd,J=17.0,1.9Hz,1H), 5.74(dd,J=10.2,1.9Hz,1H), 5.51(s,2H)ppm. 13C NMR(150MHz,DMSO-d6)δ163.60,153.12,152.78,152.43,146.95,139.22,135.85,132.24,131.98,131.91,131.8 5,131.53,130.80,129.82,129.26,129.19,129.14,128.83,127.43,120.01,46.64ppm.HR-MS(m / z)(ESI):calcd for:C 21 H 17 N5O[M+H] + :356.1505; found:356.1500.

[0144] Example 29: Preparation of compound 29

[0145] Using intermediate IV-M2 (see Example 19) and 3,4-difluorophenylboronic acid as raw materials, a yellow solid (0.062 g) was obtained by referring to the synthesis methods of V-M2 in Example 27 and compound 6 in Example 6, with a yield of 53%. 1 H NMR (600MHz, DMSO-d6) δ10.23(s,1H),9.02(s,1H),8.87(s,1H),8.86-8.80(m,1H),8.79-8.74(m,1H),7.70(dt,J=10.4,8.6Hz,1H),7.64 (d,J=8.6Hz,2H),7.37(d,J=8.7Hz,2H),6.42(dd,J=17.0,10.2Hz,1H),6.24(dd,J=17.1,1.8Hz,1H),5.79-5.69(m,1H),5.51(s,2H)ppm. 13 C NMR (150MHz, DMSO-d6) δ163.60, 152.93, 152.54, 152.39, 150.50, 147.50, 139.23, δ 133.38 (dd, J = 5.8, 3.4Hz), 132.21, 131. 72,130.67,128.84,127.47,127.09(dd,J=6.5,3.4Hz),120.00,118.60,118.49,118.35,46.72ppm.HR-MS(m / z)(ESI):calcd for:C 21 H 15 F2N5O[M+H] + :392.1317; found:392.1323.

[0146] Example 30: Preparation of compound 30

[0147] Using intermediate IV-M2 (see Example 19) and 3,4-dimethoxyphenylboronic acid as raw materials, a gray solid (0.31 g) was obtained by referring to the synthesis methods of V-M2 in Example 27 and compound 6 in Example 6, with a yield of 60%. 1 HNMR (600MHz, DMSO-d6) δ10.18(s,1H),9.00(s,1H),8.82(s,1H),8.09(d,J=2.3Hz,2H),7.64(d,J=8.6Hz,2H),7.36(d,J=8.6Hz,2H),6.72 (t,J=2.3Hz,1H),6.40(dd,J=17.0,10.2Hz,1H),6.24(dd,J=16.8,1.8Hz,1H),5.74(dd,J=10.1,2.0Hz,1H),5.50(s,2H),3.85(s,6H)ppm. 13 C NMR(150MHz,DMSO-d6)δ163.59,161.01,152.84,152.59,152.27,147.06,139.18,137.68,132.19,1 31.84,130.86,128.82,127.49,120.00,107.68,103.57,55.85,46.65ppm.HR-MS(m / z)(ESI):calcd for:C 23 H 21 N5O3[M+H] + 416.1717; found: 416.1735.

[0148] Example 31: Preparation of compound 31

[0149] Using intermediate IV-M2 (see Example 19) and benzofuran-2-boronic acid as raw materials, a white solid (0.060 g) was obtained by referring to the synthesis methods of V-M2 in Example 27 and compound 6 in Example 6, with a yield of 58%. 1H NMR (600MHz, DMSO-d6) δ10.22(s,1H),9.01(s,1H),8.88(s,1H),8.35(s,1H),7.88(d,J=7.7Hz,1H),7.78(d,J=8.3Hz,1H),7.65(d,J=8.6Hz,2H), 7.56-7.47(m,1H),7.42-7.35(m,3H),6.42(dd,J=17.0,10.2Hz,1H),6.24(dd,J=17.0,1.9Hz,1H),5.74(dd,J=10.2,1.9Hz,1H),5.52(s,2H)ppm. 13 C NMR(150MHz,DMSO-d6)δ163.60,155.39,152.56,152.32,151.08,147.62,145.01,139.23,132.21,131.76,129.5 1,128.86,128.43,127.49,127.41,124.19,123.16,120.01,113.72,112.28,46.71ppm.HR-MS(m / z)(ESI):calcd for:C 23 H 17 N5O2[M+H] + :396.1455; found:396.1450.

[0150] Example 32: Preparation of compound 32

[0151] Using intermediate IV-M2 (see Example 19) and benzothiophene-2-boronic acid as raw materials, a yellow solid (0.050 g) was obtained by referring to the synthesis methods of V-M2 in Example 27 and compound 6 in Example 6, with a yield of 69%. 1 H NMR (600MHz, DMSO-d6) δ10.21(s,1H),9.03(s,1H),8.96(s,1H),8.88(s,1H),8.14-8.02(m,2H),7.65(d,J=8.6Hz,2H),7.47(p,J=8. 1,7.5Hz,2H),7.38(d,J=8.6Hz,2H),6.41(dd,J=17.0,10.2Hz,1H),6.24(dd,J=17.0,1.9Hz,1H),5.82-5.68(m,1H),5.52(s,2H)ppm. 13CNMR(150MHz,DMSO-d6)δ163.60,152.57,152.31,148.83,147.59,140.93,140.55,140.41,139.23,132.22,131.91,131. 77,129.99,129.49,129.19,128.83,127.47,126.93,125.80,125.46,123.25,120.01,46.74ppm.HR-MS(m / z)(ESI):calcd for:C 23 H 17 N5OS[M+H] + :412.1226; found:412.1225.

[0152] Example 33: Enzymatic activity of the compound

[0153] 1. Experimental methods: The inhibitory ability of the representative compounds obtained in this invention against FGFR1 and FGFR4 was tested using the commercially available FGFR1 and FGFR4 kinase (human) ELISA detection / inhibitor screening analysis kit provided by Shanghai Fusheng Industrial Co., Ltd., with known FGFR inhibitors AZD4547 and SSR128129E as positive controls.

[0154] 2. Experimental Procedure: Remove the 96-well plate from the kit and thaw it in a 37°C cell culture incubator. Then, divide the 96-well plate into three areas: the test group, the positive control group, and the blank group. Add 50 μL of 60 U / L FGFR1 and FGFR4 enzyme reaction solution to each well of the 96-well plate. Add five concentration gradients (500, 250, 50, 10, and 5 nM) of fresh test compound solution and AZD4547 and SSR128129E solution to each well of the test group and the positive control group, respectively. Add an equal volume of DMSO solvent to each well of the blank group. After sealing, incubate at 37°C for 2 hours. After the reaction, except for the blank group, 50.0 μL of FGFR1 or FGFR4 enzyme ligand was added to each well of the other groups, and incubated for 30 min. Then, 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B were added to each well, and the mixture was gently shaken and incubated at 37°C in the dark for 10 min. After the reaction, 50.0 μL of stop solution was added to each well, and the mixture was incubated at room temperature for 10 min to stop the reaction. At this point, the blue color immediately turned yellow. Finally, the blank well was used to zero the microscope, and the absorbance (OD value) was measured at 450 nm using a microplate reader. Each compound was tested in triplicate independently, and the results were taken as the average of the three experiments ± SD. The half-maximal inhibitory concentration (IC50) of each compound against FGFR1 and FGFR4 was calculated using SPSS 16.0 software. 50 ).

[0155] 3. Experimental Results

[0156] Table 1. Inhibitory activity of compounds against FGFR enzyme and antiproliferative activity against cells

[0157]

[0158]

[0159]

[0160] As shown in Table 1, the IC50 values ​​of the positive control drugs AZD4547 and SSR128129E for exogenous FGFR1 enzyme are... 50 The values ​​were 0.95 μM and 0.66 μM, respectively. The IC50 values ​​of the representative compounds obtained in this invention for exogenous FGFR1 enzymes were... 50 Eleven compounds had inhibitory values ​​less than 0.66 μM, namely 1, 6, 7, 9, 11, 14, 19, 29, 30, 31, and 32. Compound 7 exhibited the strongest inhibitory activity, being 5.3 times and 3.7 times more potent than AZD4547 and SSR128129E, respectively. AZD4547 and SSR128129E showed relatively weak inhibitory activity against FGFR4 enzymes, with IC50 values ​​of [missing value]. 50 The values ​​were 11.48 μM and 1.64 μM, respectively. The IC50 values ​​of the obtained compound against the FGFR4 enzyme were... 50 Eleven compounds had inhibitory activity values ​​less than 1 μM: 4, 6, 8, 11, 14, 17, 19, 20, 24, 29, and 30. Compound 4 exhibited the highest inhibitory activity, being 35.9 times and 5.1 times more active than AZD4547 and SSR128129E, respectively. Structure-activity relationship analysis revealed that when p-aminobenzoic acid was used as the linker, only compounds 1 and 4 among the resulting compounds (1-5) showed good inhibitory activity against FGFR1 and FGFR4. Compound 4, in particular, demonstrated high selectivity for different FGFR isoforms, with an inhibitory activity of only 14.74 μM against FGFR1 and 0.32 μM against FGFR4, a difference of 46 times. When 4-fluoro-3-nitrobenzyl bromide was used as the linker, the resulting compounds (6-18) generally showed high inhibitory activity against FGFR1, with IC50 values ​​exceeding 1 μM. 50 Five compounds had values ​​less than 0.5 μM: compounds 6, 7, 9, 11, and 16. Among them, the compound with 6-bromoindole as the parent compound showed the best inhibitory activity against FGFR1, with an IC50 value of [missing value]. 50 The value was 0.18 μM; compounds based on 6-chloropurine did not show significant selectivity for FGFR1 and FGFR4 isoforms, but exhibited high inhibitory activity, with an IC50 value of 0.18 μM. 50The values ​​were 0.58 μM and 0.73 μM, respectively; when 2-chloropurine was used as the parent compound, the resulting compound showed moderate inhibitory activity against FGFR1, with an IC50 value of 0.58 μM and 0.73 μM. 50 The value was 6.42 μM. Based on this, 6-chloropurine was selected as the parent compound, and p-aminobenzyl bromide was used as the linker. Compounds 19-28 were obtained by reacting different Michael receptors with short-chain fatty acids. Compared with compound 14, compound 19, obtained by using p-aminobenzyl bromide as the linker, showed improved inhibitory activity against both FGFR1 and FGFR4. When the Michael receptor acrylamide was replaced with methacrylamide or 3-methylcrotonamide, the inhibitory activity against FGFR was not improved. When aliphatic chain amides such as propionamide, chloroacetamide, and 2-chloroacetamide were used to replace propionamide, except for compound 24, the inhibitory activity against FGFR of the other compounds was not effectively improved. Using acrylamide as the Michael receptor, 6-chloropurine as the parent compound and p-aminobenzyl bromide as the linker, aromatic compounds containing borate groups such as 2-thiopheneboronic acid, phenylboronic acid, 3,4-difluorophenylboronic acid, and 3,5-dimethoxyphenylboronic acid were used to modify the parent compound structure to obtain compounds 27-32. Compounds 29 and 30, obtained by modifying the 6-chloropurine core with 3,4-difluorophenylboronic acid and 3,5-dimethoxyphenylboronic acid, were found to effectively enhance their inhibitory activity against FGFR1 and FGFR4. Among them, compound 30 showed the best inhibitory activity, with an IC50 of 29% for both FGFR1 and FGFR4. 50 The values ​​were 0.20 μM and 0.40 μM, respectively.

[0161] Example 34: In vitro antiproliferative activity of the compound

[0162] 1. Experimental Methods

[0163] Esophageal squamous cell carcinoma cells (KYSE-150), breast cancer cells (MDA-MB-435), liver cancer cells (HepG2), triple-negative breast cancer cells (MDA-MB-231), and human umbilical vein endothelial cells (HUVEC) were selected to evaluate the antiproliferative activity of the compound. The inhibitory effect of the compound on the growth of different cell types at different concentrations was observed, and the inhibition rate and IC50 were calculated. 50 The values ​​were determined using AZD4547 and SSR128129E as positive controls.

[0164] 2. Experimental Procedure

[0165] (1) Preparation of compounds: All compounds were prepared into 2 mol / L solutions using DMSO as solvent, and then serially diluted with culture medium. The volume content of DMSO in the dilution solution must be less than 0.4%.

[0166] (2) Cell Culture: Cells were cultured in DMEM containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 μg / mL ampicillin sodium. During cell passage, the old culture medium was discarded, and the cells were slowly washed twice with PBS (5.0 mL × 2). 3.0 mL of 0.25% trypsin digestion solution (without EDTA) was added to digest the cells. After digestion, a certain amount of serum was added to stop the digestion. The cell solution was transferred to a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and an appropriate amount of culture medium was added to disperse the cells into a single-cell suspension. The single-cell suspension was then aliquoted into new cell culture flasks, and the culture medium was replenished. The cell culture flasks were incubated at 37°C in a cell culture incubator containing 95% air and 5% CO2 until the cell morphology stabilized before various cell experiments were performed.

[0167] (3) Cytotoxicity assay: The above single-cell suspension was seeded in 96-well plates, with a cell density of approximately 1 × 10⁶ cells per well. 4 The cells were incubated overnight in a cell culture incubator. The old culture medium was discarded, and 150.0 μL of fresh culture medium containing different concentrations of the target compound was added. The cells were then incubated for another 72 h. After incubation, the old culture medium was discarded, and 10.0 μL of 5 mg / mL MTT solution was added. The cells were incubated for another 4 h. The MTT solution in each well was discarded, and 100.0 μL of DMSO was added and gently shaken to dissolve the solution. The absorbance at 490 nm was measured using a microplate reader. Concentration-inhibition rate curves were plotted using SPSS 18 software, and the IC50 was calculated. 50 value.

[0168] 3. Experimental Results

[0169] IC 50 The values ​​are expressed as the mean ± SD of three independent experiments, and the results are shown in Table 1.

[0170] The antiproliferative activity data of the obtained compounds against cancer cells are shown in Table 1. The positive control drugs AZD4547 and SSR128129E showed only moderate cytotoxic activity against the four cancer cell types. Only AZD4547 showed strong inhibitory activity against HepG2 cells, with an IC50 value of [missing information]. 50 The value was 3.128 μM. Some compounds showed a positive correlation between their anti-proliferative activity against cancer cells and their enzyme inhibitory activity. For example, compound 1 exhibited high inhibitory activity against FGFR1 and also showed good inhibitory activity against KYSE-150 and MDA-MB-435 cells; compound 19 showed strong inhibitory activity against two FGFR enzymes and also demonstrated strong anti-proliferative activity against four types of cancer cells, particularly against the IC50 of KYSE-150, HepG2, and MDA-MB-231 cells. 50All values ​​were less than 3 μM, superior to positive control drugs. Compound 30 showed the strongest inhibitory activity against FGFR1 and FGFR4, and also exhibited strong inhibitory activity against KYSE-150 and MDA-MB-231 cells, with an IC50 value of less than 3 μM. 50 The concentrations were 1.965 μM and 1.893 μM, respectively, representing 4.5 times and 3.8 times the activity of AZD4547. Notably, the obtained compound exhibited low toxicity to normal HUVEC cells.

[0171] Example 35: Western blot analysis of the effects of compounds on the FGFR-ERK signaling pathway

[0172] 1. Experimental Methods

[0173] (1) Protein extraction: Add 2.0 mL of 1×10⁻⁶ protein solution to each well of a 6-well plate. 5 Cells were cultured in a suspension of MDA-MB-231 cells / mL at 37°C for 12 h. Then, 20.0 μL of 5.0 μM AZD4547 and different concentrations of compound 30 (2.5, 5.0, and 10.0 μM), along with an equal volume of DMSO, were added to each well, and the cells were incubated for another 24 h. After incubation, cells were collected in 15 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were slowly washed twice with 2.0 mL of PBS. The centrifuge tubes were then placed on crushed ice, and 80 μL of cell lysis buffer was added for lysis for 30 min. After lysis, the cells were transferred to 2 mL centrifuge tubes and centrifuged at 15000 rpm for 15 min in a refrigerated centrifuge. The supernatant was stored at -20°C.

[0174] (2) Protein quantification and sample preparation: First, the protein content was measured on a multi-mode microplate spectrophotometer. Then, Loading Buffer was added to the protein and stored at 100°C for 15 min.

[0175] (3) Gel preparation and sample loading: Add TEE to the pre-prepared separating gel and mix well. Then transfer it to the concave-convex glass plate of the electrophoresis apparatus and immediately seal with water. Remove the water, add 6% stacking gel, and insert the gouges. After the gel has completely solidified, slowly remove the gouges and add 10 μL of the above sample diluted with SDS to the gouges, using the markers as a reference. Adjust the voltage of the electrophoresis pool and stop the gel running when the markers are completely separated and the loading buffer reaches the bottom.

[0176] (4) Transfer: After wetting the PVDF membrane with methanol, immerse it and filter paper together in the transfer buffer. Completely immerse the target protein portion of the gel in the transfer buffer for a short time, then place it together with the PVDF membrane on a semi-dry transfer apparatus and treat it at 350 mA for 1 hour. After treatment, place the PVDF membrane in 5% skim milk powder and shake it on a shaker for 1 hour.

[0177] (5) Immunization and Exposure: The PVDF membrane treated above was washed with TBST. The membrane was incubated with primary antibody overnight at 4°C, and washed with TBST every 30 min on a shaker for a total of five washes. The membrane was then incubated with secondary antibody at 37°C for 1 h, followed by five more washes with TBST. Finally, the membrane was imaged using an Odyssey scanning system.

[0178] 2. Experimental Results

[0179] The effects of compound 30 at different concentrations on the expression of p-EGFR1, ERK, and p-ERK proteins were analyzed using Western blot technology. The results are as follows: Figure 1 As shown in the figure. Experimental results indicate that compound 30 exhibits dose-dependent inhibition of p-EGFR1 and p-ERK protein expression, and its inhibitory ability on p-EGFR1 and p-ERK protein expression at the same concentration is significantly better than that of AZD4547. This suggests that compound 30 has a strong inhibitory effect on endogenous FGFR1 protein activity and can effectively inhibit the EGFR-ERK signaling pathway.

[0180] In summary, by examining the inhibitory activity of the representative compounds obtained in this invention against FGFR1 and FGFR4 enzymes, it was found that some compounds exhibited strong inhibitory activity against both FGFR isoforms, with IC50 values ​​of [missing information]. 50 The concentration was less than 1 μM. Compounds 19 and 30 not only exhibited strong enzyme inhibitory activity but also showed strong anti-proliferative activity against cancer cells, particularly compound 30, which showed significant activity against the IC50 of KYSE-150 and MDA-MB-231 cells. 50 The values ​​were all less than 2 μM, which is superior to the positive control drugs AZD4547 and SSR128129E, and can be used as potential FGFR inhibitors for the preparation of anti-tumor drugs.

Claims

1. A six-membered pentagonal heterocyclic compound or a pharmaceutically acceptable salt thereof, characterized in that, Selected from any of the following compounds: 。 2. The six-membered pentagonal heterocyclic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound with any of the following acids: Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the six-membered and five-membered heterocyclic compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The use of a six-membered pentagonal heterocyclic compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of an FGFR inhibitor drug, wherein the FGFR inhibitor drug is selected from FGFR1 and FGFR4 inhibitor drugs.

5. The application according to claim 4, characterized in that, The FGFR inhibitor is an anti-tumor drug.

6. The application according to claim 5, characterized in that, The anti-tumor drugs mentioned are for the treatment of breast cancer, liver cancer, and esophageal cancer.

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