Compound with BTK inhibitory activity as well as preparation method and application thereof

Abstract

The invention discloses a compound with BTK inhibitory activity and a preparation method and application thereof. The compound is shown as a formula I. The compound can effectively inhibit Bruton's tyrosine kinase, and can be applied to targeted drugs for treating diseases related to BTK signal channels, such as chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatic arthritis, chronic graft versus host disease and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound having BTK inhibitory activity, and also relates to a preparation method and application of the compound. BACKGROUND

[0002] With the development of tumor biology and the progress of molecular biology technology, targeted therapy, a treatment method targeting specific molecular targets of tumors, is proposed. Targeted drugs are drugs designed for specific molecular targets of tumor cells, which can inhibit or block these targets to intervene in the growth and spread of tumors. These targets are usually proteins, signaling pathways or other biological molecules closely related to the occurrence, growth and metastasis of cancer. Common targeted drugs at present include kinase inhibitors, anti-angiogenic drugs, immune checkpoint inhibitors, hormone receptor modulators, etc. Among them, the research on kinase inhibitors has become a hot spot in the pharmaceutical industry.

[0003] Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that is selectively expressed in a variety of immune cells and hematopoietic system lineages. BTK plays a crucial role in the development of B cells and the activation of mature B cells. It is involved in the regulation of B cell growth and development, proliferation and differentiation, apoptosis and cell migration, etc. In the downstream signaling of B cell receptor, BTK is an important component, which activates phospholipase C gamma (PLC gamma) through the intermediate signaling of upstream Src family kinases and the signal generated by phosphatidylinositol 3-kinase (PI3K), leading to the release of Ca2+ and the activation of NF-kappa B and MAP kinase pathways, promoting the proliferation and survival of B cells. BTK also induces the production of pro-inflammatory cytokines IL-6 and IL-10, and controls B cell responses to integrin-mediated adhesion and to chemokines such as SDF-1. Constitutive activation of this kinase also leads to the accumulation of autoreactive plasma cells, which ultimately causes immune disorders such as rheumatoid arthritis, systemic lupus erythematosus and chronic urticaria, and abnormal activation of BTK is also related to the pathogenesis of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Therefore, BTK inhibition may be a new strategy for the chemotherapy of various autoimmune diseases and hematological malignancies.

[0004] Small molecule BTK inhibitors include BTK inhibitors Ibrutinib, Acalabrutinib, Zanubrutinib, Tirabrutinib and Orelabrutinib for hematological malignancies, and various BTK inhibitors with anti-tumor activity such as Poseltinib, TL-895 and Spebrutinib, etc. Despite the large number of BTK inhibitors, emerging drug-resistant cases have become a major limitation of their clinical application. BTK point mutations (such as C481S / F / Y / R, T474I / S / M, L528W), mutations in BTK downstream genes (such as PLCγ2) and the activation of alternative pathways (such as PI3Kδ, JAK3, FLT3, etc. kinases) that maintain B cell receptor (BCR) signaling can all lead to drug resistance to BTK inhibitors, especially covalent inhibitors. In addition, existing covalent BTK inhibitors, especially Ibrutinib, due to insufficient selectivity, may non-specifically inhibit other kinases (such as EGFR, ITK, TEC), thereby causing off-target effects and related adverse events, including joint pain, atrial fibrillation, bleeding and lymphocytosis. Therefore, there is an urgent need to develop new non-covalent BTK inhibitors. SUMMARY

[0005] The present application provides a compound with BTK inhibitory activity, and also provides a preparation method and application of the compound.

[0006] The compound with BTK inhibitory activity of the present application is a quinoline-4-amine compound containing a benzoyl hydrazine structure, and the structure is shown in formula I:

[0007] wherein R1 or R2 is selected from OCH3, H, CH3, OCH2F, OCHF2, OCF3 or , wherein X=CH2, NH or O, Y=CH3, , or , n=1~3; R is a single or double substitution on the benzene ring, and the substituent used for the substitution is one or more of F, Cl, Br, I, CH3, OCH3, CF3, OH, NH2, NO2 and C(CH3).

[0008] wherein R1 or R2 is selected from H or OCH3.

[0009] wherein R is selected from the following structures: .

[0010] The compound comprises the following structure: .

[0011] The compound further comprises pharmaceutically acceptable salts, stereoisomers and tautomers, metabolic precursors or products, solvates or prodrugs thereof.

[0012] The pharmaceutically acceptable salt is prepared by using an acid, and the acid includes inorganic acid, aliphatic monocarboxylic acid and dicarboxylic acid, phenyl-substituted alkanoic acid, hydroxyalkanoic acid and alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid, and the like organic acid, and the acid specifically includes hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, carbonic acid, formic acid, acetic acid, sebacic acid, beta-hydroxybutyric acid, oxalic acid, succinic acid, tartaric acid, mandelic acid, fumaric acid, maleic acid, lactic acid, citric acid, glutamic acid and aspartic acid.

[0013] The preparation method of the compound with BTK inhibitory activity comprises the following steps: preparing intermediate B by reacting compound A with ethanol, preparing intermediate C by reacting B with hydrazine hydrate, preparing intermediate E by reacting compound D with p-aminobenzoic acid, and preparing the BTK inhibitor by reacting C with E: .

[0014] The pharmaceutical composition comprises the compound of formula I, the pharmaceutically acceptable salt, stereoisomer and tautomer, metabolic precursor or product, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

[0015] The compound with BTK inhibitory activity or the pharmaceutical composition is used for preparing the BTK inhibitor.

[0016] The application is specifically used for preparing a drug for preventing and / or treating chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatoid arthritis or chronic graft-versus-host disease.

[0017] Principle: The compound with BTK inhibitory activity is prepared by modifying the structure of Larotinib as a lead compound, introducing a hydrazide group into the side chain to obtain a new BTK inhibitor. Larotinib is a multi-targeted kinase inhibitor mainly targeting epidermal growth factor receptor EGFR, and it shows moderate inhibitory activity on BTK. Hydrazide is a bioactive group, and drugs containing hydrazide groups are initially used for treating central nervous system diseases and infectious diseases, such as isocarboxazid, isoniazid and nialamide.

[0018] The compounds of the present application have inhibitory effect on the anti-proliferation of human histiocytic lymphoma cells (U-937), and from the structure, the 6,7-dimethoxyquinoline mother nucleus structure has better activity compared with the quinoline mother nucleus, and for the terminal benzene ring substituent group connected with the benzhydrazide fragment, the general trend is: di-chloro substitution > mono-substitution; among the mono-substituent, -Cl / -Br > -CH3 / -F; among the di-chloro substitution, 2,3-di-Cl / 2,4-di-Cl > 3,4-di-Cl / 2,6-di-Cl; for the substitution position on the mono-substituted ring, ortho / meta > para, and the compounds F-30, F-42, F-43 have better inhibitory activity on Bruton's tyrosine kinase. The terminal benzene ring substituent group can significantly adjust the effect of the target compound, the di-chloro substituent group can exhibit activity enhancement, in addition, the electronic effect of the R1R2 substituent group significantly affects the activity, and the electron-donating group (such as methoxy, or other aliphatic groups) can enhance the inhibitory effect.

[0019]

[0020] Advantages: Compared with the prior art, the present application has the following significant advantages: (1) The compounds of the present application can effectively inhibit Bruton's tyrosine kinase, and can be applied to targeted drugs for treating diseases related to the BTK signal pathway, such as chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatoid arthritis, chronic graft-versus-host disease, etc.; compared with Ibrutinib (half maximal inhibitory concentration IC 50 = 2.03 μM), the compounds of the present application have better inhibitory effect on the anti-proliferation of human histiocytic lymphoma cells (U-937), and F-43 has the lowest IC 50 value (0.89 μM); the compound F-43 of the present application has the best selectivity to BTK (99.84), and has weaker inhibitory activity to the off-target kinases EGFR (-70.37), ITK (32.83), and TEC (74.80) commonly found in the prior art BTK inhibitors; (2) The preparation of the compounds of the present application does not require the use of column chromatography, and the operation is simple and the conditions are mild. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A tree diagram of the kinase spectrum activity distribution of the compound F-43 to 76 tyrosine kinases; Figure 2 A heat map of the kinase spectrum activity distribution of the compound F-43 to 76 tyrosine kinases; Figure 3 A Western blot diagram of the inhibitory activity of the compound F-43 to BTK protein phosphorylation; Figure 4 Two-dimensional scatter plot of the apoptosis effect of compound F-43 on human histiocytic lymphoma cells (U-937); Figure 5 Bar chart of the apoptosis effect of compound F-43 on human histiocytic lymphoma cells (U-937). DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further described below in conjunction with examples. The test materials used in the examples can be purchased through conventional channels.

[0023] Example 1: N'- (2- (3-chlorophenyl) acetyl) -4- (quinoline-4-ylamino) benzohydrazide (F-1) Synthetic route:

[0024] Synthetic method: Preparation of ethyl 2- (3-chlorophenyl) acetate (B-1): 10 mmol of 3-chlorophenylacetic acid and 20 ml of ethanol (CH3CH2OH) were added to a 50 ml flask, 2.5 ml of concentrated sulfuric acid (H2SO4) was slowly added along the wall of the flask under stirring, and then the reaction was stirred under reflux at 78°C overnight. After the reaction was completed by TLC detection, the heating was stopped, and after the temperature returned to room temperature, sodium bicarbonate solid was added in batches until no bubbles were generated. At this time, the reaction system became a sticky paste, and the filter cake was washed with a large amount of ethanol. The combined filtrate was concentrated to obtain a white solid, which was directly used in the next step.

[0025] Preparation of 2- (3-chlorophenyl) acethydrazide (C-1): 7 g of 85% hydrazine hydrate (NH2-NH2·H2O) and 30 ml of ethanol were added to the ethyl 2- (3-chlorophenyl) acetate synthesized in the previous step, and the reaction was continued under stirring and reflux at 78°C overnight. After the reaction was completed by TLC detection, the heating was stopped, and the ethanol of the reaction system was concentrated under reduced pressure to obtain an oily liquid. 30 ml of water was added to the flask, and the organic layer was extracted with ethyl acetate. After drying with anhydrous sodium sulfate for about 30 min, the organic layer was combined and concentrated under reduced pressure to obtain a white cotton-like solid. 1 H NMR (300 MHz, Chloroform- d ) δ 7.34-7.31 (m, 3H), 7.23-7.18 (m, 1H), 3.57 (s, 2H), 3.39 (s, 2H). Preparation of 4-(quinoline-4-ylamino)benzoic acid (E-1): 1.64 g of p-aminobenzoic acid (12 mmol, 1 eq) and 1.96 g of 4-chloroquinoline (12 mmol, 1 eq) were placed in a 250 ml round-bottom flask. 120 ml of isopropanol [(CH3)2CHOH] was added, followed by the slow addition of 1.8 ml of trifluoroacetic acid (CF3COOH, 24 mmol, 2 eq) in portions. The mixture was refluxed at 85 °C. A pale yellow solid gradually precipitated during the reaction. After TLC detection showed the reaction was complete, heating was stopped, the flask was sealed, and the mixture was refrigerated for 1 hour. The mixture was then filtered, the filter cake was washed with isopropanol, dried, and then slurried in a mixture of methanol and ethanol for half an hour. The mixture was filtered again, and the filter cake was dried under an infrared lamp and collected to obtain 3.28 g of a pale yellow solid, with a yield of 98.29%. 1 H-NMR (300 MHz, DMSO-d6): δ 11.20 (s, 1H), 8.89 (d, J = 8.6 Hz, 1H), 8.60 (d, J = 7.0 Hz, 1H), 8.19-8.03 (m, 4H), 7.84(t, J = 7.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 6.9 Hz, 1H). Preparation of N'-(2-(3-chlorophenyl)acetyl)-4-(quinoline-4-ylamino)benzoylhydrazine (F-1): 0.27 g of 4-(quinoline-4-ylamino)benzoic acid (1 mmol, 1 eq), 0.482 g of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboronic acid (TBTU, 1.5 mmol, 1.5 eq), 0.31 g of triethylamine (Et3N, 3 mmol, 3 eq) and 0.28 g of 2-(3-chlorophenyl)acetylhydrazine (1.5 mmol, 1.5 eq) were added to a 25 ml single-necked flask. The mixture was dissolved in 10 ml of N,N-dimethylformamide (DMF) and stirred overnight at room temperature. After the reaction was completed as detected by TLC, the reaction solution was added dropwise to 200 ml of water, and a solid precipitated out. The mixture was then slurried for 1 hour and filtered. The filter cake was dried, and 0.18 g of a light yellow solid, namely compound F-1, was collected after drying, with a yield of 43.21%. mp: 264~268℃. 1H NMR (300 MHz, DMSO-d6) δ 10.34 (s, 1H), 10.24 (s,1H), 9.25 (s, 1H), 8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92(dd, J = 8.8, 3.1 Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz,1H), 7.50 – 7.17 (m, 7H), 3.58 (s, 2H). HRMS (ESI + ): m / z calcd C 24 H 19 ClN4O2[M +H + ]=430.1197;found = 430.1196。

[0026] Example 2: N'- (2- (2-chlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-2) Preparation method according to Example 1, white solid, namely compound F-2, yield 34.11%, mp: 270-271℃ was obtained. 1 H-NMR (300 MHz, DMSO- d6): δ 10.34 (s, 1H), 10.19 (s, 1H), 9.23 (s, 1H),8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 3H),7.73 (t, J = 7.6 Hz, 1H), 7.63-7.39 (m, 5H), 7.37-7.17 (m, 3H), 3.72 (s,2H).HRMS(ESI+):m / z calcd for C 24 H 19 ClN4O4[M + H + ] = 430.1197;found = 430.1199。

[0027] Example 3: N'- (2- (4-chlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-3) Preparation method according to Example 1, white solid, namely compound F-3, yield 63.67%, mp: 268-270℃ was obtained. 1H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.20 (s, 1H), 9.24 (s, 1H),8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 7.91 (t, J = 7.4 Hz, 3H),7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.48-7.33 (m, 6H), 7.21(d, J = 5.2 Hz, 1H), 3.55 (s, 2H). HRMS(ESI+):m / z calcd for C 24 H 19 ClN4O4[M + H + ]=430.1197;found = 430.1195。

[0028] Example 4: N'- (2- (2-bromophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-4) Preparation method according to example 1, to produce off-white solid, namely compound F-4, yield 66.02%, mp: 268-272℃. 1 H-NMR (300 MHz, DMSO-d6): δ 10.34 (s, 1H), 10.18 (s, 1H), 9.23 (s, 1H),8.57 (d, J = 5.1 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 3H),7.73 (t, J = 7.6 Hz, 1H), 7.65-7.28 (m, 6H), 7.22 (t, J = 5.9 Hz, 2H), 3.73(s, 2H). HRMS(ESI + ):m / z calcd for C 24 H 19 BrN4O2[M+H + ]=474.0691;found=474.0695。

[0029] Example 5: N'- (2- (3-bromophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-5) Preparation method according to example 1, to produce light yellow solid, namely compound F-6, yield 53.64%, mp: 264-266℃. 1H-NMR (300 MHz, DMSO-d6): δ 0.28 (s, 2H), 9.24 (s, 1H), 8.57 (d, J = 5.1 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.6, 5.7 Hz, 3H), 7.73 (t, J= 7.6 Hz, 1H), 7.62-7.52 (m, 2H), 7.50-7.26 (m, 5H), 7.21 (d, J = 5.3 Hz,1H), 3.57 (s, 2H). HRMS(ESI + ): m / z calcd for C 24 H 19 BrN4O2[M+ H + ]=474.0691;found=474.0690.

[0030] Example 6: N'- (2- (4-bromophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-6) Preparation method according to example 1, to give off-white solid, compound F-6, yield 37.14%, mp: 268-270℃. 1 H-NMR (300 MHz, DMSO-d6): δ 0.28 (s, 2H), 9.24 (s, 1H), 8.57 (d, J = 5.1 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.6, 5.7 Hz, 3H), 7.73 (t, J= 7.6 Hz, 1H), 7.62-7.52 (m, 2H), 7.50-7.26 (m, 5H), 7.21 (d, J = 5.3 Hz,1H), 3.57 (s, 2H). HRMS(ESI + ): m / z calcd for C 24 H 19 BrN4O2[M+ H + ]=474.0691;found=474.0694.

[0031] Example 7: N'- (2- (2-fluorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-7) Preparation method according to example 1, to give compound F-7 as a light yellow solid in 44.87% yield, mp: 268-269 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.22 (s, 1H), 9.24 (s, 1H), 8.57(d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.97-7.86 (m, 3H), 7.73 (t, J= 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.49-7.32 (m, 3H), 7.27 – 7.02 (m,4H), 3.58 (s, 2H). HRMS(ESI + ): m / z calcd for C 24 H 19 FN4O2[M+H + ]=414.1492;found=414.1490.

[0032] Example 8: N'- (2- (3-fluorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-8) Preparation method according to example 1, to give compound F-8 as a white solid in 61.40% yield, mp: 256-260 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.22 (s, 1H), 9.24 (s, 1H), 8.57(d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.97-7.86 (m, 3H), 7.73 (t, J= 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.49-7.32 (m, 3H), 7.27 – 7.02 (m,4H), 3.58 (s, 2H). HRMS(ESI + ): m / z calcd for C 24 H 19 FN4O2[M+H + ]=414.1492;found=414.1495.

[0033] Example 9: N'- (2- (4-fluorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-9) Preparation method according to example 1, to give yellow solid, compound F-9, yield 33.06%, mp: 272-274°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.24 (d, J = 35.2 Hz, 2H), 9.23 (s, 1H), 8.57(d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.98-7.85 (m, 3H), 7.73 (t, J= 7.6 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.48-7.34 (m, 4H), 7.27-7.07 (m,3H), 3.54(s,2H). HRMS(ESI + ): m / z calcd for C 24 H 19 FN4O2 [M+H + ]=414.1492;found=414.1496。

[0034] Example 10: 4-(quinolin-4-ylamino)-N'-(2-(o-tolyl)acetyl)benzohydrazide (F-10) Preparation method according to example 1, to give white solid, compound F-10, yield 76.31%, mp: 272-274°C. 1 H-NMR (300 MHz, DMSO-d6): δ10.28 (s, 1H), 10.12 (s, 1H), 9.36-9.13 (m,1H), 8.57 (t, J = 4.5 Hz, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.91 (dd, J = 8.5,4.1 Hz, 3H), 7.80-7.05 (m, 9H), 3.56 (d, J = 3.3 Hz, 2H), 2.34 (d, J = 3.4Hz, 3H). HRMS(ESI + ):m / z calcd for C 25 H 22 N4O2[M+H + ]= 410.1743;found=410.1746。

[0035] Example 11: 4-(quinolin-4-ylamino)-N'-(2-(p-tolyl)acetyl)benzohydrazide (F-11) Preparation method according to example 1, to give yellow solid, compound F-11, yield 38.15%, mp: 264-271 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.29 (s, 1H), 10.15 (s, 1H), 9.24 (s, 1H),8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.98-7.85 (m, 3H), 7.80-7.38 (m, 4H), 7.29-7.05 (m, 5H), 2.28 (s, 3H). HRMS(ESI + ): m / z calcd for C 25 H 22 N4O2[M+H + ]= 410.1743; found = 410.1745.

[0036] Example 12: N'- (2- (2-methoxyphenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-12) Preparation method according to example 1, to give yellow solid, compound F-12, yield 57.38%, mp: 254-256 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.27 (s, 1H), 9.99 (s, 1H), 9.22 (s, 1H),8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.3 Hz, 3H),7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H),7.36-7.15 (m, 3H), 7.01-6.86 (m, 2H), 3.79 (s, 3H), 3.53 (s, 2H). HRMS(ESI + ):m / z calcd for C 25 H 22 N4O3[M+H + ]= 426.1692; found = 426.1696.

[0037] Example 13: N'- (2- (3-methoxyphenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-13) Preparation method according to example 1, to give white solid, compound F-13, yield 50.49%, mp: 258-260 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 10.18 (s, 1H), 9.25 (s, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 8.3, 4.8 Hz, 3H), 7.76 (t, J = 7.7 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.32-7.19 (m, 2H), 7.05-6.90 (m, 2H), 6.84 (d, J = 8.6 Hz, 1H), 3.78 (s, 3H), 3.54 (s, 2H). HRMS (ESI + ): m / z calcd for C 25 H 22 N4O3[M+H + ]= 426.1692; found = 426.1695.

[0038] Example 14: N'- (2- (4-nitrophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-14) Preparation method according to example 1, to give yellow-brown solid, compound F-14, yield 51.00%, mp: 252-254 °C. 1 H-NMR (300 MHz, DMSO- d 6): δ 10.33 (d, J = 18.7 Hz, 2H), 9.26 (s, 1H), 8.56 (d, J = 5.1 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.3 Hz, 2H), 7.92 (dd, J = 9.4, 4.0 Hz, 3H), 7.79-7.38 (m, 6H), 7.20 (d, J = 5.3 Hz, 1H), 3.74 (s, 2H).HRMS (ESI + ): m / z calcd for C24 H 19 N5O4[M+H + ]=441.1437;found=441.1439。

[0039] Example 15: 4-(quinolin-4-ylamino)-N'-(2-(4-(trifluoromethyl)phenyl)acetyl)benzohydrazide (F-15) Preparation method according to Example 1 to give yellow solid, compound F-15, yield 75.06%, mp: 269-270℃. 1 H-NMR (300 MHz, DMSO-d6): δ 10.33 (s, 1H), 10.26 (s, 1H), 9.23 (s, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.43-8.34 (m, 1H), 8.00-7.88 (m, 3H), 7.79-7.41 (m, 8H), 7.22 (d, J = 5.3 Hz, 1H), 3.69 (s, 2H). HRMS(ESI + ):m / z calcd for C 25 H 19 F3N4O2[M+H + ]=464.1460;found=464.1465。

[0040] Example 16: N'- (2-(3, 4-dichlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-16) Preparation method according to Example 1 to give white solid, compound F-16, yield 69.55%, mp: 267℃. 1 H-NMR (300 MHz, DMSO-d6): δ 10.34 (s, 1H), 10.23 (s, 1H), 9.25 (s, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H), 7.93 (t, J = 7.6 Hz, 3H), 7.75 (t, J = 7.6 Hz, 1H), 7.69-7.55 (m, 3H), 7.46 (d, J = 8.4 Hz, 2H), 7.30 (dd, J = 41.2, 6.7 Hz, 2H), 3.61 (s, 2H). HRMS(ESI + ):m / z calcd for C 24 H 18Cl2N4O2[M+H + ]=464.0807;found=464.0809.

[0041] Example 17: N'- (2- (2, 3-dichlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-17) Preparation method according to example 1, to give yellow brown solid, i.e. compound F-17, in 59.01% yield, mp: 264-266 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.20 (s, 1H), 9.22 (s, 1H), 8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.5 Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.63 - 7.27 (m, 6H), 7.21 (d, J = 5.2 Hz, 1H), 3.80 (s, 2H). HRMS (ESI + ): m / z calcd for C 24 H 18 Cl2N4O2[M+H + ]=464.0807;found=464.0810.

[0042] Example 18: N'- (2- (2, 4-dichlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-18) Preparation method according to example 1, to give yellow solid, i.e. compound F-18, in 73.77% yield, mp: 266-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.27 (d, J = 33.5 Hz, 2H), 9.24 (s, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.01 - 7.89 (m, 3H), 7.75 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.43 (dd, J = 18.7, 9.1 Hz, 4H), 7.23 (d, J = 5.3 Hz, 2H), 3.59 (s, 2H). HRMS (ESI + ): m / z calcd for C24 H 18 Cl2N4O2[M+H + ]=464.0807;found=464.0805.

[0043] Example 19: N'- (2- (2, 6-dichlorophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-19) Preparation method according to example 1, to produce yellow solid, namely compound F-19, yield 21.08%, mp: 266-267℃. 1 H-NMR (300 MHz, DMSO-d6): δ 10.27 (d, J = 33.5 Hz, 2H), 9.24 (s, 1H),8.59 (d, J = 5.2 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.01-7.89 (m, 3H), 7.75(t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.43 (dd, J = 18.7, 9.1 Hz,4H), 7.23 (d, J = 5.3 Hz, 2H), 3.59 (s, 2H). HRMS (ESI + ): m / z calcd for C 24 H 18 Cl2N4O2[M+H + ]=464.0807;found=464.0807.

[0044] Example 20: 4- (quinolin-4-ylamino) -N'- (2- (3, 4, 5-trimethoxyphenyl) acetyl) benzohydrazide (F-20) Preparation method according to example 1, to produce yellow solid, namely compound F-20, yield 32.19%, mp: 216-218℃. 1H-NMR (300 MHz, DMSO-d6): δ 10.29 (s, 1H), 10.13 (s, 1H), 9.22 (s, 1H), 8.57 (d, J = 5.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.5, 3.7 Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 5.1 Hz, 1H), 6.71 (s, 2H), 3.71 (dd, J = 44.6, 1.0 Hz, 9H), 3.48 (s, 2H). HRMS (ESI + ): m / z calcd for C 27 H 26 N4O5[M+ H + ]=486.1903; found = 486.1901.

[0045] Example 21: N'- (2- (4- (tert-butyl) phenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-21) Preparation method according to example 1, to give light yellow solid, compound F-21, yield 54.07%, mp: 244-246 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.28 (s, 1H), 10.15 (s, 1H), 9.23 (s, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H), 7.93 (t, J = 7.4 Hz, 3H), 7.75 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.49-7.18 (m, 7H), 3.51 (s, 2H), 1.30 (s, 9H). HRMS (ESI + ): m / z calcd for C 28 H 28 N4O2[M+ H + ]=452.2212; found = 452.2217.

[0046] Example 22: N'- (2- (2-hydroxyphenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-22) Preparation method according to example 1, to give brown yellow solid, compound F-22, in 21.36% yield, mp: 266-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 9.23 (s, 1H), 8.57 (s, 1H),8.37 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 3H), 7.73 (t, J = 7.7 Hz, 1H),7.64-7.02 (m, 7H), 6.88-6.70 (m, 1H), 3.54 (d, J = 20.5 Hz, 2H). HRMS(ESI + ):m / z calcd for C 24 H 20 N4O3[M+ H + ]=412.1535;found=412.1537.

[0047] Example 23: N'- (2- (4-aminophenyl) acetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-23) Preparation method according to example 1, to give brown yellow solid, compound F-23, in 11.89% yield, mp: 262-264 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 10.18 (s, 1H), 9.25 (s, 1H),8.59 (d, J = 5.1 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 8.3, 4.8Hz, 3H), 7.76 (t, J = 7.7 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.6Hz, 2H), 7.32-7.19 (m, 2H), 7.05-6.90 (m, 2H), 6.84 (d, J = 8.6 Hz, 1H), 3.78(s, 3H), 3.54 (s, 2H). HRMS(ESI + ):m / z calcd for C 24 H 21 N5O2[M+H +] = 411.1695; found = 411.1693.

[0048] Example 24: N'- (2- (3, 4-difluorophenyl) acetyl) -4- (quinolin-4- ylamino) benzohydrazide (F-24) Preparation method according to example 1, to give off-white solid, compound F-24, yield 67.90%, mp: 267-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.34 (s, 1H), 10.19 (s, 1H), 9.22 (s, 1H), 8.57 (d, J = 5.3 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.65-7.33 (m, 6H), 7.21 (d, J = 5.3 Hz, 1H), 3.73 (s, 2H). HRMS (ESI + ): m / z calcd for C 24 H 18 F2N4O2[M+ H + ]=432.1398; found=432.1399.

[0049] Example 25: N'- (2-phenylacetyl) -4- (quinolin-4-ylamino) benzohydrazide (F-25) Preparation method according to example 1, to give yellow solid, compound F-25, yield 76.54%, mp: 268-269 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 10.18 (s, 1H), 9.26 (d, J = 13.3 Hz, 1H), 8.57 (d, J = 4.7 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.1 Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.49-7.16 (m, 8H), 3.47 (s, 2H). HRMS (ESI + ): m / z calcd for C 24 H 20 N4O2[M+ H +] Found: 396.1586; found: 396.1588.

[0050] Example 26: N'- (2- (3-chlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-26) Synthetic procedure: Preparation of intermediate 2- (3-chlorophenyl) acethydrazide was done as per example 1; Preparation of 4- ( (6, 7-dimethoxyquinolin-4-yl) amino) benzoic acid:

[0051] A 250 ml flask was charged with 1.37 g of p-aminobenzoic acid (10 mmol, 1 eq) and 2.24 g of 4-chloro-6,7-dimethoxyquinoline (10 mmol, 1 eq), 100 ml of isopropyl alcohol was added, 1.6 ml of trifluoroacetic acid was slowly added, and heating was carried out under reflux at 85°C. During the reaction, white solid gradually precipitated. After TLC detection showed that the reaction was complete, heating was stopped, and the flask was placed in the refrigerator for 1 h. After being taken out, filtration was performed, and the filter cake was dried under infrared light. After being dried, 2.12 g of white solid was collected, with a yield of 64.73%. 1 H NMR (300 MHz, DMSO-d6) δ 14.51 (s, 1H), 10.86 (s,1H), 8.43 (d, J = 6.9 Hz, 1H), 8.19 (s, 1H), 8.10 (d, J = 8.2 Hz, 2H), 7.63(d, J = 8.3 Hz, 2H), 7.50 (s, 1H), 6.99 (d, J = 6.9 Hz, 1H), 4.02 (d, J =10.1 Hz, 6H)。

[0052] A 25 ml single-necked flask was charged with 0.33 g of 4- ( (6, 7-dimethoxyquinolin-4-yl) amino) benzoic acid (1 mmol, 1 eq), 0.482 g of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 1.5 mmol, 1.5 eq), 0.31 g of triethylamine (Et3N, 3 mmol, 3 eq), and 0.28 g of 2- (3-chlorophenyl) acethydrazide (1.5 mmol, 1.5 eq), 10 ml of N,N-dimethylformamide (DMF) was added as solvent, and the reaction was stirred at room temperature overnight. After TLC detection showed that the reaction was complete, the reaction solution was added dropwise into 200 ml of water, and the solid was precipitated. After being stirred for 1 h, filtration was performed, and the filter cake was dried. After drying was completed, 0.31 g of light yellow solid was collected, which was compound F-26, with a yield of 62.12%. mp: 258-260°C;1 H-NMR (300 MHz, DMSO- d 6): δ(ppm) 10.28 (s, 1H), 10.18 (s, 1H), 8.95 (s, 1H), 8.40 (d, J = 5.2 Hz, 1H),7.97-7.86 (m, 2H), 7.63 (s, 1H), 7.48-7.26 (m, 7H), 7.08 (d, J = 5.3 Hz, 1H),3.94 (d, J = 5.1 Hz, 6H), 3.59 (s, 2H). HRMS (ESI + ): m / z calcd C 26 H 23 ClN4O4 [M +H + ]=490.1408;found = 490.1410。

[0053] Example 27: N'- (2- (2-chlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-27) Preparation method according to example 26, to produce off-white solid, namely compound F-27, yield 54.10%, mp: 266-268℃. 1 H-NMR (300 MHz, DMSO-d6): δ 10.28 (s, 1H), 10.13 (s, 1H), 8.93 (s,1H), 8.39 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.54-7.27 (m, 7H), 7.07 (d, J = 5.2 Hz, 1H), 3.93 (d, J = 4.8 Hz, 6H), 3.72 (s,2H). HRMS(ESI+):m / z calcd for C 26 H 23 ClN4O4[M + H + ]=490.1408;found=490.1406。

[0054] Example 28: N'- (2- (4-chlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-28) The preparation method was the same as in Example 26, yielding a light yellow solid, namely compound F-28, with a yield of 60.11% and an mp of 266-268°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.25 (s, 1H), 10.14 (s, 1H), 8.93 (s,1H), 8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.61 (s, 1H), 7.43-7.34 (m, 6H), 7.29 (s, 1H), 7.07 (d, J = 5.3 Hz, 1H), 3.93 (d, J = 4.6 Hz, 6H), 3.55 (s, 2H). HRMS (ESI + ):m / z calcd for C 26 H 23 ClN4O4[M+H + ]=490.1408; found=490.1403.

[0055] Example 29: N'-(2-(2-bromophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-29) The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-29, with a yield of 68.04% and an mp of 258-260°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.31 (s, 1H), 10.15 (s, 1H), 9.08 (s,1H), 8.40 (d, J = 5.4 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.68-7.59 (m, 2H),7.52 (d, J = 7.7 Hz, 1H), 7.44-7.30 (m, 4H), 7.27 -7.18 (m, 1H), 7.07 (d, J =5.5 Hz, 1H), 3.94 (d, J = 4.4 Hz, 6H), 3.74 (s, 2H). HRMS (ESI + ):m / z calcdfor C 26 H 23 BrN4O4[M+H + ]= 534.0903; found=534.0905.

[0056] Example 30: N'-((2-(3-bromophenyl)acetyl)-4-((6,7-dimethoxyquinolin-4- yl)amino)benzohydrazide (F-30) Preparation method according to Example 26, to give light yellow solid, i.e. compound F-30, in 62.52% yield, mp: 256-258 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.27 (s, 1H), 10.17 (s, 1H), 8.95 (s, 1H),8.39 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 11.2 Hz,2H), 7.47 (d, J = 7.7 Hz, 1H), 7.42-7.28 (m, 5H), 7.08 (d, J = 5.3 Hz, 1H),3.93 (d, J = 4.7 Hz, 6H), 3.58 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 23 BrN4O4[M+H + ]=534.0903;found=534.0907.

[0057] Example 31: N'-((2-(4-bromophenyl)acetyl)-4-((6,7-dimethoxyquinolin-4- yl)amino)benzohydrazide (F-31) Preparation method according to Example 26, to give off-white solid, i.e. compound F-31, in 84.59% yield, mp: 266-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.36 (s, 1H), 10.20 (d, J = 1.3 Hz, 1H),8.41 (d, J = 6.0 Hz, 1H), 7.98 (d, J = 8.5 Hz, 2H), 7.82 (s, 1H), 7.58-7.44(m, 4H), 7.38-7.30 (m, 3H), 7.01 (d, J = 6.1 Hz, 1H), 3.97 (d, J = 3.8 Hz,6H), 3.55 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 23 BrN4O4[M+H += 534.0903; found = 534.0902.

[0058] Example 32: N'- (2- (2-fluorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-32) Preparation method according to example 26, to give off-white solid, compound F-32, yield 35.21%, mp: 266-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.26 (s, 1H), 10.16 (s, 1H), 8.93 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.44-7.33 (m, 3H), 7.30 (s, 1H), 7.25-7.16 (m, 2H), 7.14-7.03 (m, 2H), 3.93 (d, J = 4.8 Hz, 6H), 3.59 (s, 2H). HRMS (ESI + ): m / z calcd for C 26 H 23 FN4O4[M+H + ]= 474.1703; found = 474.1707.

[0059] Example 33: N'- (2- (3-fluorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-33) Preparation method according to example 26, to give off-white solid, compound F-33, yield 33.14%, mp: 268-270 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.26 (s, 1H), 10.16 (s, 1H), 8.93 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.44-7.33 (m, 3H), 7.30 (s, 1H), 7.25-7.16 (m, 2H), 7.14-7.03 (m, 2H), 3.93 (d, J = 4.8 Hz, 6H), 3.59 (s, 2H). HRMS (ESI + ): m / z calcd for C 26 H 23FN4O4[M+H + ]= 474.1703; found = 474.1701.

[0060] Example 34: N'- (2- (4-fluorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-34) Preparation method according to example 26, to give off-white solid, compound F-34, yield 43.50%, mp: 268-270 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.25 (s, 1H), 10.14 (s, 1H), 8.94 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.62 (s, 1H), 7.45-7.28 (m, 5H), 7.22-7.04 (m, 3H), 3.93 (d, J = 4.6 Hz, 6H), 3.55 (s, 2H). HRMS (ESI + ): m / z calcd for C 26 H 23 FN4O4[M+H + ]= 474.1703; found = 474.1708.

[0061] Example 35: 4- ( (6, 7-dimethoxyquinolin-4-yl) amino) -N'- (2- (o-tolyl) acetyl) benzohydrazide (F-35) Preparation method according to example 26, to give light yellow solid, compound F-35, yield 52.22%, mp: 262-264 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.23 (s, 1H), 10.07 (s, 1H), 8.94 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.97-7.88 (m, 2H), 7.63 (s, 1H), 7.44-7.28 (m, 4H), 7.20-7.06 (m, 4H), 3.93 (d, J = 5.0 Hz, 6H), 3.57 (s, 2H), 2.35 (s, 3H).HRMS (ESI + ): m / z calcd for C 27 H 26 N4O4[M+H +] = 470.1954; found = 470.1956.

[0062] Example 36: 4-((6,7-dimethoxyquinolin-4-yl)amino)-N'-(2-(p-tolyl)acetyl)benzohydrazide (F-36) Preparation method according to Example 26, to give compound F-36 as a white solid in 54.31% yield, mp: 274-276 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.23 (s, 1H), 10.08 (s, 1H), 8.94 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.30 (s, 1H), 7.24 (d, J = 7.8 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 7.07 (d, J = 5.3 Hz, 1H), 3.93 (d, J = 4.7 Hz, 6H), 3.50 (s, 2H), 2.29 (s, 3H). HRMS (ESI + ): m / z calcd for C 27 H 26 N4O4[M+H + ] = 470.1954; found = 470.1959.

[0063] Example 37: N'- (2-(2-methoxyphenyl)acetyl)-4-((6,7-dimethoxyquinolin-4-yl)amino)benzohydrazide (F-37) Preparation method according to Example 26, to give compound F-37 as an off-white solid in 70.70% yield, mp: 272-274 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.25 (s, 1H), 9.97 (s, 1H), 8.95 (s, 1H), 8.39 (d, J = 5.3 Hz, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.45-7.20 (m, 5H), 7.11-6.86 (m, 3H), 3.93 (d, J = 5.3 Hz, 6H), 3.79 (s, 3H), 3.52 (s, 2H). HRMS (ESI+ ): m / z calcd for C 27 H 26 N4O5[M+H + = 486.1903; found = 486.1906.

[0064] Example 38: N'- (2- (3-methoxyphenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-38) Preparation method according to example 26, to give off-white solid, compound F-38, yield 66.66%, mp: 272-274 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 10.18 (s, 1H), 9.00 (s, 1H), 8.41 (d, J = 5.3 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.34-7.21 (m, 2H), 7.10 (d, J = 5.3 Hz, 1H), 7.03-6.78 (m, 3H), 3.95 (d, J = 5.4 Hz, 6H), 3.78 (s, 3H), 3.54 (s, 2H). HRMS (ESI + ): m / z calcd for C 27 H 26 N4O5[M+H + = 486.1903; found = 486.1907.

[0065] Example 39: N'- (2- (4-nitrophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-39) Preparation method according to example 26, to give off-white solid, compound F-39, yield 52.91%, mp: 266-268 °C. 1H-NMR (300 MHz, DMSO-d6): δ 10.27 (d, J = 12.1 Hz, 2H), 8.94 (s, 1H),8.39 (d, J = 5.2 Hz, 1H), 8.28- 8.18 (m, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.69-7.59 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.30 (s, 1H), 7.07 (d, J = 5.2 Hz,1H), 3.93 (d, J = 4.7 Hz, 6H), 3.74 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 23 N5O6[M+H + ]=501.1648;found=501.1645。

[0066] Example 40: ( (6, 7-dimethoxyquinolin-4-yl) amino) -N'- (2- (4- (trifluoromethyl) phenyl) acetyl) benzohydrazide (F-40) Preparation method according to example 26, to produce yellow solid, namely compound F-40, yield 62.48%, mp: 262-264℃. 1 H-NMR (300MHz, DMSO-d6): δ 10.25 (s, 1H), 10.12 (s, 1H), 8.95 (s, 1H),8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.43-7.24(m, 7H), 7.07 (d, J = 5.2 Hz, 1H), 3.93 (d, J = 5.2 Hz, 6H), 3.49 (s, 2H),1.28 (s, 9H). HRMS(ESI + ):m / z calcd for C 27 H 23 F3N4O4[M+H + ]=524.1671;found=524.1670.

[0067] Example 41: N'- (2- (3, 4-dichlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-41) Preparation method according to example 26, to give off-white solid, compound F-41, yield 28.11%, mp: 258-260°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.26-10.16 (m, 1H), 9.00(s, 1H), 8.41 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 18.3 Hz, 3H),7.47-7.25 (m, 4H), 7.09 (s, 1H), 3.95 (d, J = 5.1 Hz, 6H), 3.61 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 22 Cl2N4O4[M+H + ]=524.1018;found=524.1021.

[0068] Example 42: N'- (2- (2, 3-dichlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-42) Preparation method according to example 26, to give off-white solid, compound F-42, yield 74.96%, mp: 268-272°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.26-10.16 (m, 1H), 9.00(s, 1H), 8.41 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 18.3 Hz, 3H),7.47-7.25 (m, 4H), 7.09 (s, 1H), 3.95 (d, J = 5.1 Hz, 6H), 3.61 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 22 Cl2N4O4[M+H + ]=524.1018;found=524.1019.

[0069] Example 43: N'- (2- (2, 4-dichlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-43) The preparation method was the same as in Example 26, yielding a light green solid, namely compound F-43, with a yield of 82.46% and an mp of 258-260°C. 1 H-NMR (300MHz, DMSO-d6): δ 10.32 (s, 1H), 10.18 (s, 1H), 8.98 (s, 1H), 8.39 (d, J = 5.3 Hz, 1H), 7.97-7.87 (m, 2H), 7.62 (d, J = 2.4 Hz, 2H), 7.53(d, J = 8.3 Hz, 1H), 7.46-7.35 (m, 3H), 7.29 (s, 1H), 7.07 (d, J = 5.3 Hz,1H), 3.93 (d, J = 5.1 Hz, 6H), 3.72 (s, 2H). HRMS (ESI + ):m / z calcd forC 26 H 22 Cl2N4O4[M+H + ]=524.1018; found=524.1018.

[0070] Example 44: N'-(2-(2,6-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-44) The preparation method was the same as in Example 26, yielding a light yellow solid, namely compound F-44, with a yield of 33.73% and an mp of 266-268°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.35 (s, 1H), 10.21 (s, 1H), 9.02 (d, J =19.4 Hz, 1H), 8.40 (d, J = 5.8 Hz, 1H), 8.01-7.88 (m, 2H), 7.63 (d, J = 4.5Hz, 1H), 7.56-7.29 (m, 6H), 7.17-7.06 (m, 1H), 3.94 (d, J = 4.6 Hz, 8H). HRMS (ESI + ):m / z calcd for C 26 H 22 Cl2N4O4[M+H + ]=524.1018; found=524.1016.

[0071] Example 45: 4-((6,7-dimethoxyquinolin-4-yl)amino)-N'-(2-(3,4,5- trimethoxyphenyl)acetyl)benzohydrazide (F-45) Preparation method according to example 26, to give off-white solid, compound F-45, yield 50.35%, mp: 266-268 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.27 (s, 1H), 10.12 (s, 1H), 8.95 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.62 (s, 1H), 7.38 (d, J = 8.3 Hz, 2H), 7.29 (s, 1H), 7.07 (d, J = 5.3 Hz, 1H), 6.71 (s, 2H), 3.93 (d, J = 5.3 Hz, 6H), 3.71 (d, J = 44.7 Hz, 9H), 3.48 (s, 2H). HRMS (ESI + ): m / z calcd for C 29 H 30 N4O7[M+H + ]= 546.2114; found = 546.2117.

[0072] Example 46: N'- (2-(4-(tert-butyl)phenyl)acetyl)-4-((6,7-dimethoxyquinolin-4- yl)amino)benzohydrazide (F-46) Preparation method according to example 26, to give off-white solid, compound F-46, yield 76.69%, mp: 270-274 °C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.25 (s, 1H), 10.12 (s, 1H), 8.95 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.43-7.24 (m, 7H), 7.07 (d, J = 5.2 Hz, 1H), 3.93 (d, J = 5.2 Hz, 6H), 3.49 (s, 2H), 1.28 (s, 9H). HRMS (ESI + ): m / z calcd for C 30 H 32N4O4[M+H + ] = 512.2424; found = 512.2427.

[0073] Example 47: N'- (2- (2-hydroxyphenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-47) Preparation method according to example 26, to give yellow solid, compound F-47, yield 12.26%, mp: 224-226 °C. 1 H-NMR (300 MHz, DMSO- d 6): δ 10.26 (s, 1H), 8.94 (s, 1H), 8.37 (s, 1H),7.90 (s, 2H), 7.60 (s, 1H), 7.47- 6.95 (m, 6H), 6.76 (s, 1H), 3.91 (s, 6H),3.49 (d, J = 12.2 Hz, 2H). HRMS (ESI + ): m / z calcd for C 26 H 24 N4O5[M+H + ] = 472.1747; found = 472.1748.

[0074] Example 48: N'- (2- (4-aminophenyl) acetyl) -4- ( (6, 7-dimethoxyquinolin-4- yl) amino) benzohydrazide (F-48) Preparation method according to example 26, to give light yellow solid, compound F-48, yield 45.86%, mp: 252-254 °C. 1 H-NMR (300 MHz, DMSO- d 6): δ 10.20 (d, J = 23.4 Hz, 1H), 10.02 (s, 1H),9.00 (d, J = 10.5 Hz, 1H), 8.40 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.64(s, 1H), 7.48-7.27 (m, 3H), 7.15-6.96 (m, 2H), 6.53 (d, J = 8.2 Hz, 1H), 4.95(s, 1H), 3.94 (d, J= 5.4 Hz, 6H), 3.35 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 25 N5O4[M+H + ]=471.1907;found=471.1909.

[0075] Example 49: N'- (2- (3, 4-difluorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinoline-4- yl) amino) benzohydrazide (F-49) Preparation method according to example 26, to produce off-white solid, namely compound F-49, yield 69.85%, mp: 266-268℃. 1 H-NMR (300 MHz, DMSO- d 6): δ 10.29 (s, 1H), 10.18 (s, 1H), 8.96 (s, 1H),8.39 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.62 (s, 1H), 7.49-7.34 (m,4H), 7.29 (s, 1H), 7.19 (s, 1H), 7.07 (d, J = 5.2 Hz, 1H), 3.92 (d, J = 5.1 Hz,6H), 3.57 (s, 2H). HRMS(ESI + ):m / z calcd for C 26 H 22 F2N4O4[M+H + ]=492.1609;found=492.1608.

[0076] Example 50: N'- (2-phenylacetyl) -4- ( (6, 7-dimethoxyquinoline-4- yl) amino) benzohydrazide (F-50) Preparation method according to example 26, to produce off-white solid, namely compound F-50, yield 66.74%, mp: 266-268 o C. 1 H-NMR (300 MHz, DMSO- d ): δ 10.27 (s, 1H), 10.15 (s, 1H), 8.97 (s, 1H),8.38 (d, J= 5.2 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.62 (s, 1H), 7.44-7.20 (m,8H), 7.07 (d, J = 5.4 Hz, 1H), 3.92 (d, J = 5.1 Hz, 6H), 3.54 (s, 2H). HRMS (ESI + ):m / z calcd for C 26 H 24 N4O4[M+H + ]=456.1798; found=456.1781.

[0077] Example 51 Kinase inhibition test: 30 compounds were selected for the experiment. The Bruton's tyrosine kinase (BTK) activity test method used in the experiment was the Lantha screening test. The positive drug was staurosporine. The concentration of adenine nucleoside triphosphate ATP was measured at the Michaelis constant Km.

[0078] (1) Test procedure: Prepare 1x kinase buffer: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol (DTT), 0.01% Tween-20, 0.01% bovine serum albumin (BSA). (2) Prepare compounds: Serially dilute and prepare source plate: Compound is dissolved in 100% dimethyl sulfoxide (DMSO) to 10 mM stock. Dilute compound to 100 times the final desired highest inhibitor concentration in 100% DMSO. Add 100 μl of 100% DMSO in two empty wells, no compound control and enzyme control in the same 96 well plate. Label this plate as the source plate. Take 40 μl of compound and transfer to a new 384 Echo plate as the intermediate plate. Prepare test plate: Transfer 200 nl per well from the 384 Echo plate to a 384 well assay plate, duplicate wells. (3) Kinase reaction: Prepare 2x kinase solution: Prepare BTK solution in 1x kinase buffer, solution concentration is 2 times the final concentration of each reagent in the assay. Add 10 μl of kinase solution per well except for no enzyme control well (add 10 μl of 1x kinase buffer instead). Incubate at room temperature for 10 minutes. Prepare 2x substrate solution: Prepare Fluorescein-poly GT and ATP substrate solution in 1x kinase reaction buffer, concentration is 2 times the final concentration of each reagent needed in the assay. Start the reaction by adding 10 μl of substrate solution per well in the test plate. Incubate the kinase reaction at room temperature for a period of time. (4) Kinase detection: Prepare 2 times final concentration of detection solution in antibody dilution buffer. Stop the reaction by adding 20 μl of detection solution per well. Mix briefly with a centrifuge and incubate at room temperature for 60 minutes, then read on a fluorescent plate reader. (5) Data collection and curve fitting: Collect data on Envision program with excitation wavelength at 340 nm and emission wavelength at 520 nm and 495 nm. Calculate the ratio of relative fluorescence units (RFU) 520 nm / 495 nm. Convert the "ratio" value calculated above to percent inhibition value. Inhibition (%) = (max - sample ratio) / (max - min)*100. "min" is the no enzyme control; "max" indicates no compound negative control.

[0079] The results of the BTK inhibitory activity of the compounds are shown in Table 1. From the experimental results, it can be seen that the compounds F-30, F-42, and F-43 in the present application have good inhibitory activity against Bruton's tyrosine kinase. When the compounds in the present application have the same substituent, the compounds F-26~F-36, and F-41~F-44 generally exhibit higher BTK inhibitory activity than F-1~F-11, and F-16~F-19. This indicates that the use of a 6,7-dimethoxyquinoline mother nucleus structure has better activity than a quinoline mother nucleus. For the relationship between the terminal benzene ring substituent connected to the benzhydrazide fragment and the activity, the general trend is: di-chloro substitution > mono-substitution; among the mono-substituents, -Cl / -Br > -CH3 / -F; among the di-chloro substitutions, 2,3-di-Cl / 2,4-di-Cl > 3,4-di-Cl / 2,6-di-Cl; and for the substitution position on the mono-substituted ring, ortho / meta > para. In general, the terminal benzene ring substituent can significantly modulate the effect of the target compound, and the di-chloro substituent can exhibit an activity enhancement effect.

[0080] Table 1. Results of the in vitro BTK inhibitory activity of the compounds

[0081] Example 52 The MTT [3-(4,5)-dimethyl-2-thiazole-(2,5)-phenyl tetrazolium bromide] method was used to determine the proliferation inhibitory effect of the compounds on human colon cancer cells (HCT116): The MTT [3-(4,5)-dimethyl-2-thiazole-(2,5)-phenyl tetrazolium bromide] method was used to determine the proliferation inhibitory effect of the compounds on human colon cancer cells (HCT116) in vitro, and the specific steps are as follows: Preparation of D-Hanks buffer (per liter): NaCl 8.00 g, KCl 0.40 g, Na2HPO4·12H2O 0.06 g, KH2PO4 0.06 g, NaHCO3 0.35 g, autoclaved. Preparation of trypsin solution: Use D-Hanks buffer to prepare a 0.5% trypsin solution. Filter sterilization. Preparation of drug solution: Dissolve the compound in a small amount of triple distilled water to prepare a stock solution, generally prepare a stock solution at 10 times the highest concentration for the experiment. Depending on the solubility of the compound, it can be directly dissolved with triple distilled water or a small amount of DMSO can be used for dissolution. The concentration of DMSO in the culture medium should not be too high, and the final concentration of DMSO in each well of the cell suspension after adding the drug is generally not more than 0.05-0.1%.

[0082] (1) Cell culture: HCT116 cells were routinely cultured in DMEM medium (containing 10% fetal bovine serum (FBS), 100 U / mL of two kinds of antibiotics) in a 37 °C, 5% CO2 incubator for 3-4 days, and the cells were completely adherent and needed to be passaged once. At the time of passage, the original culture solution was discarded, and then washed with D-Hanks buffer. Then, trypsin digestion was performed for 30 seconds, and a small amount of fresh culture solution was added to terminate the digestion. Blowing was performed, and an appropriate amount was transferred to a new culture bottle, and fresh culture solution was added to the original volume. 5 实验 空白 对照 空白 实验 对照 空白 50

[0083] The in vitro anti-human colon cancer HCT116 cell line proliferation results of the compounds are shown in Table 2. From the experimental results, it can be seen that most of the compounds have an IC 50 ​​​​​​​​​The IC50values were less than 2 μM, and F-43 had the lowest IC50value, which was the most active compound in this series. 50 The IC50values were less than 2 μM, and F-43 had the lowest IC50value, which was the most active compound in this series. 50 The IC50values were less than 2 μM, and F-43 had the lowest IC50value, which was the most active compound in this series.

[0084] Table 2, in vitro anti-HCT116 cell proliferation results of compounds

[0085] Example 53 The inhibitory effect of the compounds on the proliferation of human histiocytic lymphoma cells (U-937) was determined by using a cell counting kit-8 (CCK-8) method.

[0086] The U-937 cells were configured into a cell suspension of 50000 cells / mL with complete culture medium, and different concentration gradients (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM and 5 μM) of the compounds and ibutinib were added to the cells, respectively, and incubated in a 37 °C, 5% CO2incubator for 48 h. After the incubation was completed, 10 μL of CCK-8 solution was added to each well under light shielding conditions, and the reaction was carried out in a 37 °C, 5% CO2incubator for 3 h, and the OD value was determined at 450 nm, and the inhibition rate was calculated. Inhibition rate = (OD 对照组平均值 -OD 加药组 ) / OD control group x 100%, OD 对照组平均值 = (OD 未加药 -OD 空白 ) ÷ no drug well. The obtained inhibition rate was fitted by GraphPad Prism 8 software to obtain the IC 50 value. The in vitro anti-proliferation activity results of the compounds on human histiocytic lymphoma U-937 cell lines are shown in Table 3: the experimental results showed that, compared with the positive drug ibutinib, the compounds F-4, F-17, F-18, F-27, F-29, F-42 and F-43 had better inhibitory effect on the anti-proliferation of human histiocytic lymphoma cells (U-937). F-43 had the lowest IC 50 value, which was the most active compound in this series.

[0087] Table 3, in vitro anti-U-937 cell proliferation results of compounds

[0088] Example 54 Kinase selectivity test: The best active compound F-43 [N'-((2-(2,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinolin-4-yl)amino)benzohydrazide] was subjected to kinase profiling test.

[0089] (1) Preparation of compound: Compound F-43 was dissolved in DMSO to obtain a 10 mM stock solution, and was serially diluted to 100 times (1 mM) of the prescribed test concentration. (2) Compound screening: Homogeneous time-resolved fluorescence (HTRF) method kinase assay: 2x ATP and substrate solution and 2x kinase and metal solution were prepared using HTRF buffer (5 mM MgCl2; 1 mM MnCl2; 12.5 nM Staphylococcal enterotoxin B (SEB); 1 mM DTT; double distilled water ddH2O). 50 nL of compound was transferred to a 384-well plate using Echo 655. After centrifugation, 2.5 μL of 2x kinase-metal solution was added, and incubated at 25°C for 10 minutes. 2.5 μL of 2x substrate and ATP solution was added to each well, and incubated at 25°C for 60 minutes. 2x XL665 and antibody solution was prepared with HTRF buffer. 5 μL of the solution prepared in the previous step was added to each well, and incubated at 25°C for 60 minutes. The fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were read. ADP-Glo method kinase test: 2x ATP and substrate solution and 2x kinase and metal solution were prepared using ADP-Glo buffer (50 mM Hepes; 10 mM MgCl2; 0.01% Brij35; 1 mM ethylene glycol tetraacetic acid (EGTA); 2 mM DTT; double distilled water ddH2O). 40 nL of compound was transferred to a 384-well plate using Echo 655. 2 μL of 2x kinase and metal solution was added to the 384-well plate, and incubated at 25°C for 10 minutes. 2 μL of 2x ATP and substrate solution was added to each well, and incubated at 25°C for 60 minutes. 4 μL of ADP-Glo reagent was added to each well, and incubated at 25°C for 40 minutes. 8 μL of kinase detection reagent was added to each well, and incubated at 25°C for 40 minutes. The fluorescence signal recorded on the plate reader was read. (3) Data analysis: The readout value of the negative control (1% DMSO) was set to 0% inhibition, and the readout value of the 10 µM positive control was set to 100% inhibition. Inhibition rate (%) = 100% - (compound - positive control) / (negative control - positive control) x 100%; wherein, positive control: 10 µM positive drug; negative control: 1% DMSO.

[0090] The results of the kinase profiling activity of compound F-43 on 76 tyrosine kinases are shown in Table 4. The results show that compound F-43 has the best selectivity on BTK (99.84%), and has weaker inhibitory activity on off-target kinases EGFR (-70.37%), ITK (32.83%), and TEC (74.80%) commonly found in existing BTK inhibitors ibutinib, which can reduce the off-target effect and the side reactions caused thereby.

[0091] Table 4. Results of the kinase profiling test of compound F-43

[0092] Example 55 The activity of compound F-43 [N'- (2- (2, 4-dichlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinoline-4-yl) amino) benzhydrazide] in inhibiting BTK phosphorylation was detected by Western blot. Compound F-43 (final concentration of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, respectively), and a control compound (ibutinib, final concentration of 1 μM) were added to a six-well plate containing U-937 cells, and a blank group was set up. After 48 h of culture, the cells were collected by centrifugation. The cells were washed with 1xPBS buffer and lysed with RAPI lysis buffer. The cell lysate was separated by 4-20% gradient gel electrophoresis, transferred to a membrane, blocked, incubated with a primary antibody (BTK, p-BTK, Biyun Tian), washed, and then incubated with a secondary antibody. Finally, chemiluminescence imaging analysis was performed. The experimental results are shown in Figure 3 The results show that compound F-43 inhibits the phosphorylation of BTK in a dose-dependent manner.

[0093] Example 56 The effect of compound F-43 [N'- (2- (2, 4-dichlorophenyl) acetyl) -4- ( (6, 7-dimethoxyquinoline-4-yl) amino) benzhydrazide] on the apoptosis of human histiocytic lymphoma cells (U-937) was detected by flow cytometry. Compound F-43 (final concentration of 2 μM, 4 μM, 8 μM, respectively), and a control compound (ibutinib, final concentration of 8 μM) were added to a six-well plate containing U-937 cells. After 48 h of incubation, the cells were collected by centrifugation, stained with an Annexin V-FITC apoptosis detection kit (Bi Yun Tian), and the sample was detected by flow cytometry CytoFLEX. The data were analyzed using FlowJo software. The experimental results are shown in Figure 4 and 5 The results show that compound F-43 promotes the apoptosis of human histiocytic lymphoma cells (U-937) in a dose-dependent manner, and the apoptosis rate of U-937 cells at a concentration of 8 μM is higher than 20%, and the pro-apoptotic ability is stronger than that of the positive drug ibutinib.

Claims

1. A compound having BTK inhibitory activity, characterized in that, The compound is a quinoline-4-amine compound containing a benzoylhydrazine structure, as shown in Formula I: ； Wherein, R1 or R2 is selected from OCH3, H, CH3, OCH2F, OCHF2, OCF3 or Where X = CH2, NH or O, Y = CH3, , or n=1~3; R is a mono- or di-substituted substance at any position on the benzene ring, and the substituent used is one or more of the following: F, Cl, Br, I, CH3, OCH3, CF3, OH, NH2, NO2, and C(CH3).

2. The compound according to claim 1, characterized in that, R1 or R2 is selected from H or OCH3.

3. The compound according to claim 1, characterized in that, R is selected from the following structures: 。 4. The compound according to claim 1, characterized in that, The compound comprises the following structure: 。 5. The compound according to claim 1, characterized in that, The compounds also include their pharmaceutically acceptable salts, stereo and tautomers, metabolic precursors or products, solvates or prodrugs.

6. The compound according to claim 5, characterized in that, The pharmaceutically acceptable salts include inorganic acids and aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes and alkanediacids, aromatic acids, aliphatic and aromatic sulfonic acids, and other organic acids, specifically hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, carbonic acid, formic acid, acetic acid, sebacic acid, β-hydroxybutyric acid, oxalic acid, succinic acid, tartaric acid, mandelic acid, fumaric acid, maleic acid, lactic acid, citric acid, glutamic acid, and aspartic acid.

7. A method for preparing the compound with BTK inhibitory activity as described in claim 1, characterized in that, Includes the following steps: Intermediate B is prepared by reacting compound A with ethanol; intermediate C is prepared by reacting B with hydrazine hydrate; intermediate E is prepared by reacting compound D with p-aminobenzoic acid; and BTK inhibitor is prepared by reacting C with E. 。 8. A pharmaceutical composition, characterized in that, The compound comprising a pharmaceutically acceptable salt, stereo and tautomer, metabolic precursor or product, solvate compound or prodrug of the compound of formula I of claim 1, and a pharmaceutically acceptable carrier.

9. The use of a compound having BTK inhibitory activity as described in claim 1 or a pharmaceutical composition as described in claim 8 in the preparation of a BTK inhibitor.

10. The application according to claim 9, characterized in that, The specific application is in the use of drugs for the prevention and / or treatment of chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatoid arthritis, or chronic graft-versus-host disease.

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