A compound having btk inhibitory activity and preparation method and application thereof

Abstract

This invention discloses a compound with BTK inhibitory activity, its preparation method, and its application; the compound is shown in Formula I. The compound of this invention can effectively inhibit Bruton's tyrosine kinase and can be used as a targeted drug for the treatment of diseases related to the BTK signaling pathway, such as chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatoid arthritis, and chronic graft-versus-host disease.

Description

Technical Field

[0001] This invention relates to a compound with BTK inhibitory activity, as well as a method for preparing the compound and its applications. Background Technology

[0002] With the development of tumor biology and the advancement of molecular biology techniques, targeted therapy, a treatment method targeting specific molecular targets of tumors, has been proposed. Targeted drugs are drugs designed to target specific molecular targets of tumor cells, and can inhibit or block these targets to intervene in tumor growth and spread. These targets are usually proteins, signaling pathways, or other biomolecules closely related to the occurrence, growth, and metastasis of cancer. Currently, common targeted drugs include kinase inhibitors, anti-angiogenic drugs, immune checkpoint inhibitors, and hormone receptor modulators. Among them, research on kinase inhibitors has become a hot topic in the pharmaceutical field.

[0003] Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase selectively expressed in various immune cells and hematopoietic lineages. BTK plays a crucial role in B cell development and activation of mature B cells. It participates in regulating B cell growth, development, proliferation, differentiation, apoptosis, and cell migration. In downstream signaling of B cell receptors, BTK is an important component, activating phospholipase Cγ (PLCγ) through intermediate signaling from upstream Src family kinases and signals generated by phosphatidylinositol 3-kinase (PI3K). This leads to Ca2+ release and activation of the NF-κB and MAP kinase pathways, promoting B cell proliferation and survival. BTK also induces the production of pro-inflammatory cytokines IL-6 and IL-10 and controls B cell responses to integrin-mediated adhesion and chemokines such as SDF-1. Constitutive activation of this kinase also leads to the accumulation of autoreactive plasma cells, which ultimately causes immune dysregulations such as rheumatoid arthritis, systemic lupus erythematosus, and chronic urticaria. Furthermore, aberrant BTK activation is also associated with the pathogenesis of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Therefore, BTK inhibition may be a novel strategy for chemotherapy in various autoimmune diseases and hematologic malignancies.

[0004] Small molecule BTK inhibitors include those used for hematologic malignancies such as ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib, and orelabrutinib, as well as various BTK inhibitors with antitumor activity, such as poseltinib, TL-895, and spebrutinib. Despite the availability of numerous BTK inhibitors, emerging cases of resistance have become a major limitation to their clinical application. BTK point mutations (such as C481S / F / Y / R, T474I / S / M, L528W), mutations in downstream BTK genes (such as PLCγ2), and activation of alternative pathways maintaining B-cell receptor (BCR) signaling (such as kinases like PI3Kδ, JAK3, and FLT3) can all lead to resistance to BTK inhibitors, especially covalent inhibitors. Furthermore, existing covalent BTK inhibitors, especially ibrutinib, may nonspecifically inhibit other kinases (such as EGFR, ITK, and TEC) due to insufficient selectivity, leading to off-target effects and related adverse events, including arthralgia, atrial fibrillation, bleeding, and lymphocytosis. Therefore, there is an urgent need to develop novel non-covalent BTK inhibitors. Summary of the Invention

[0005] Purpose of the invention: To address the above-mentioned problems, this invention provides a compound with BTK inhibitory activity, as well as a method for preparing the compound and its applications.

[0006] The compound with BTK inhibitory activity of the present invention is a quinoline-4-amine compound containing a benzoylhydrazine structure, and its structure is shown in Formula I:

[0007]

[0008] 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;

[0009] 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).

[0010] Wherein, R1 or R2 is selected from H or OCH3.

[0011] Where R is selected from the following structures:

[0012] .

[0013] The compound comprises the following structure:

[0014] .

[0015] The compounds also include pharmaceutically acceptable salts, stereo and tautomers, metabolic precursors or products, solvates or prodrugs.

[0016] 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.

[0017] The method for preparing the above-mentioned compound with BTK inhibitory activity includes the following steps: reacting compound A with ethanol to prepare intermediate B, reacting B with hydrazine hydrate to prepare intermediate C, reacting compound D with p-aminobenzoic acid to obtain intermediate E, and reacting C with E to obtain the BTK inhibitor.

[0018] .

[0019] A pharmaceutical composition of the present invention comprises a pharmaceutically acceptable salt, stereo and tautomer, metabolic precursor or product, solvent compound or prodrug of the compound shown in Formula I above, and a pharmaceutically acceptable carrier.

[0020] The use of the above-mentioned compounds or pharmaceutical compositions with BTK inhibitory activity in the preparation of BTK inhibitors.

[0021] 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.

[0022] Invention Principle: The compound with BTK inhibitory activity of this invention is based on larotinib as a lead compound, which is structurally modified by introducing an acylhydrazine group into its side chain to obtain a novel BTK inhibitor. Larotinib is a multi-target kinase inhibitor that primarily targets the epidermal growth factor receptor EGFR and exhibits moderate inhibitory activity against BTK. Acylhydrazine is a bioactive group; drugs containing acylhydrazine groups were initially used to treat central nervous system diseases and infectious diseases, such as isocarboxazid, isoniazid, and nifurosine.

[0023] The compounds of this invention exhibit inhibitory activity against the proliferation of human histiocytic lymphoma cells (U-937). Structurally, the 6,7-dimethoxyquinoline core structure shows superior activity compared to the quinoline core. Regarding the relationship between the terminal benzene ring substituents connected to the benzoylhydrazine fragment and activity, the general trend is: dichloro substitution > monosubstituted; among monosubstituted groups, -Cl / -Br > -CH3 / -F; among dichloro substituted groups, 2,3-di-Cl / 2,4-di-Cl > 3,4-di-Cl / 2,6-di-Cl; for the substitution position on the monosubstituted ring, ortho / meta > para. Compounds F-30, F-42, and F-43 exhibit good inhibitory activity against Bruton's tyrosine kinase. Terminal benzene ring substituents can significantly modulate the effect of the target compounds; dichloro substituents may exhibit an activity-enhancing effect. In addition, the electronic effects of R1R2 substituents significantly affect activity, and electron-donating groups (such as methoxy groups or other aliphatic groups) can enhance the inhibitory effect.

[0024]

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The compounds of the present invention can effectively inhibit Bruton's tyrosine kinase and can be used as targeted drugs for the treatment of diseases related to the BTK signaling 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 IC50), the compounds can effectively inhibit Bruton's tyrosine kinase and can be used as targeted drugs for the treatment of diseases related to the BTK signaling pathway, such as chronic lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, systemic lupus erythematosus, rheumatoid arthritis, chronic graft-versus-host disease, etc.; 50 Compared to (2.03 μM), the compounds of this invention exhibit superior inhibitory activity against the proliferation of human histiocytic lymphoma cells (U-937), with F-43 showing the lowest IC50. 50 Value (0.89 μM); The compound F-43 of the present invention has the best selectivity for BTK (99.84), and has weak inhibitory activity against common off-target kinases of the existing BTK inhibitor ibrutinib, such as EGFR (-70.37), ITK (32.83), and TEC (74.80); (2) The preparation of the compound of the present invention does not require column chromatography, and the operation is simple and the conditions are mild. Attached Figure Description

[0026] Figure 1 This is a dendrogram showing the kinase activity distribution of compound F-43 against 76 tyrosine kinases.

[0027] Figure 2 This is a heatmap showing the kinase activity distribution of compound F-43 against 76 tyrosine kinases.

[0028] Figure 3 Western blot diagram of the inhibitory activity of compound F-43 on BTK protein phosphorylation;

[0029] Figure 4 Two-dimensional scatter plot of the apoptosis effect of compound F-43 on human histiocytic lymphoma cells (U-937);

[0030] Figure 5 A bar chart showing the apoptosis effect of compound F-43 on human histiocytic lymphoma cells (U-937). Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[0032] Example 1: N'-(2-(3-chlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-1)

[0033] Synthesis route:

[0034] Synthesis method:

[0035] 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 round-bottom flask. 2.5 ml of concentrated sulfuric acid (H2SO4) was slowly added along the flask wall with stirring. The mixture was then refluxed and stirred overnight at 78 °C. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), heating was stopped. After the mixture was allowed to return to room temperature, sodium bicarbonate solid was added in portions until no more bubbles were generated. At this point, the reaction system became a viscous paste. The mixture was filtered, and the filter cake was washed with a large amount of ethanol. The filtrates were combined and concentrated to obtain a white solid, which was then directly added to the next step.

[0036] Preparation of 2-(3-chlorophenyl)acetylhydrazine (C-1): 7 g of 85% hydrazine hydrate (NH2-NH2·H2O) and 30 ml of ethanol were added to the ethyl 2-(3-chlorophenyl) synthesized in the previous step. The mixture was stirred and refluxed overnight at 78 °C. After the reaction was confirmed to be complete by TLC, heating was stopped, and the ethanol in the reaction system was concentrated under reduced pressure to obtain an oily liquid. 30 ml of water was added to a round-bottom flask, and the mixture was extracted appropriately with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate for about 30 min, filtered, 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).

[0037] 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).

[0038] 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℃. 1 H 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, HRMS (ESI) + ): m / z calcd C 24 H 19 ClN4O2[M +H + ]=430.1197; found = 430.1196.

[0039] Example 2: N'-(2-(2-chlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-2)

[0040] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-2, with a yield of 34.11% and an mp of 270-271°C. 1H-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.

[0041] Example 3: N'-(2-(4-chlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-3)

[0042] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-3, with a yield of 63.67% and an mp of 268-270°C. 1 H-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.

[0043] Example 4: N'-(2-(2-bromophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-4)

[0044] The preparation method was the same as in Example 1, yielding a grayish-white solid, namely compound F-4, with a yield of 66.02% and an mp of 268-272°C.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.

[0045] Example 5: N'-(2-(3-bromophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-5)

[0046] The preparation method was the same as in Example 1, yielding a light yellow solid, namely compound F-6, with a yield of 53.64% and an mp of 264-266°C. 1 H-NMR(300 MHz, DMSO-d6): δ 0.28 (s, 2H), 9.24 (s, 1H), 8.57 (d, J = 5.1Hz, 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.

[0047] Example 6: N'-(2-(4-bromophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-6)

[0048] The preparation method was the same as in Example 1, yielding a grayish-white solid, namely compound F-6, with a yield of 37.14% and an mp of 268-270°C. 1 H-NMR (300 MHz, DMSO-d6): δ10.33 (s, 1H), 10.21 (s, 1H), 9.25 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.93 (t, J = 7.6 HRMS(ESI) + ): m / z calcd for C 24 H 19 BrN4O2[M+H + ]=474.0691; found=474.0694.

[0049] Example 7: N'-(2-(2-fluorophenyl)acetyl)-4-(quinoline-4-ylamino)benzoylhydrazine (F-7)

[0050] The preparation method was the same as in Example 1, yielding a light yellow solid, namely compound F-7, with a yield of 44.87% and an mp of 268-269°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.34 (s, 1H), 10.21 (s, 1H), 9.33 (d, J =43.7 Hz, 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), 7.62-7.11 (m, 8H), 3.62 (s, 2H).HRMS (ESI + ): m / z calcd for C 24 H 19 FN4O2[M+H + ]=414.1492; found=414.1490.

[0051] Example 8: N'-(2-(3-fluorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazide (F-8)

[0052] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-8, with a yield of 61.40% and an mp of 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 HRMS(ESI) + ): m / z calcd for C 24 H 19 FN4O2[M+H + ]=414.1492; found=414.1495.

[0053] Example 9: N'-(2-(4-fluorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-9)

[0054] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-9, with a yield of 33.06% and an mp of 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.

[0055] Example 10: 4-(quinoline-4-ylamino)-N'-(2-(o-tolyl)acetyl)benzoylhydrazide (F-10)

[0056] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-10, with a yield of 76.31% and an mp of 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.

[0057] Example 11: 4-(quinoline-4-ylamino)-N'-(2-(p-tolyl)acetyl)benzoylhydrazide (F-11)

[0058] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-11, with a yield of 38.15% and an mp of 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 forC 25 H 22 N4O2[M+H + ]=410.1743; found=410.1745.

[0059] Example 12: N'-(2-(2-methoxyphenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-12)

[0060] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-12, with a yield of 57.38% and an mp of 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.

[0061] Example 13: N'-(2-(3-methoxyphenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-13)

[0062] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-13, with a yield of 50.49% and an mp of 258-260°C. 1H-NMR (300MHz, 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 25 H 22 N4O3[M+H + ]=426.1692; found=426.1695.

[0063] Example 14: N'-(2-(4-nitrophenyl)acetyl)-4-(quinoline-4-ylamino)benzoylhydrazine (F-14)

[0064] The preparation method was the same as in Example 1, yielding a yellowish-brown solid, namely compound F-14, with a yield of 51.00% and an mp of 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 C 24 H 19 N5O4[M+H +]=441.1437; found=441.1439.

[0065] Example 15: 4-(quinoline-4-ylamino)-N'-(2-(4-(trifluoromethyl)phenyl)acetyl)benzoylhydrazine (F-15)

[0066] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-15, with a yield of 75.06% and an mp of 269-270°C. 1 H-NMR (300MHz, 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 forC 25 H 19 F3N4O2[M+H + ]=464.1460; found=464.1465.

[0067] Example 16: N'-(2-(3,4-dichlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-16)

[0068] The preparation method was the same as in Example 1, yielding a white solid, namely compound F-16, with a yield of 69.55% and an mp of 267°C. 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 18 Cl2N4O2[M+H+ ]=464.0807; found=464.0809.

[0069] Example 17: N'-(2-(2,3-dichlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-17)

[0070] The preparation method was the same as in Example 1, yielding a yellowish-brown solid, namely compound F-17, with a yield of 59.01% and an mp of 264-266℃. 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.

[0071] Example 18: N'-(2-(2,4-dichlorophenyl)acetyl)-4-(quinoline-4-ylamino)benzoylhydrazine (F-18)

[0072] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-18, with a yield of 73.77% and an mp of 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, HRMS (ESI) +):m / z calcd forC 24 H 18 Cl2N4O2[M+H + ]=464.0807; found=464.0805.

[0073] Example 19: N'-(2-(2,6-dichlorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-19)

[0074] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-19, with a yield of 21.08% and an mp of 266-267°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, HRMS (ESI) + ):m / z calcd forC 24 H 18 Cl2N4O2[M+H + ]=464.0807; found=464.0807.

[0075] Example 20: 4-(quinoline-4-ylamino)-N'-(2-(3,4,5-trimethoxyphenyl)acetyl)benzoylhydrazine (F-20)

[0076] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-20, with a yield of 32.19% and an mp of 216-218°C. 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, 3.7Hz, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.3Hz, 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.

[0077] Example 21: N'-(2-(4-(tert-butyl)phenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-21)

[0078] The preparation method was the same as in Example 1, yielding a light yellow solid, namely compound F-21, with a yield of 54.07% and an mp of 244-246℃. 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 = HRMS (ESI) + ):m / z calcd for C 28 H 28 N4O2[M+H + ]=452.2212; found=452.2217.

[0079] Example 22: N'-(2-(2-hydroxyphenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-22)

[0080] The preparation method was the same as in Example 1, yielding a yellowish-brown solid, namely compound F-22, with a yield of 21.36% and an mp of 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.

[0081] Example 23: N'-(2-(4-aminophenyl)acetyl)-4-(quinoline-4-ylamino)benzoylhydrazine (F-23)

[0082] The preparation method was the same as in Example 1, yielding a yellowish-brown solid, namely compound F-23, with a yield of 11.89% and an mp of 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 H21 N5O2[M+H + ]=411.1695; found=411.1693.

[0083] Example 24: N'-(2-(3,4-difluorophenyl)acetyl)-4-(quinolin-4-ylamino)benzoylhydrazine (F-24)

[0084] The preparation method was the same as in Example 1, yielding a grayish-white solid, namely compound F-24, with a yield of 67.90% and an mp of 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.

[0085] Example 25: N'-(2-phenylacetyl)-4-(quinolin-4-ylamino)benzoylhydrazide (F-25)

[0086] The preparation method was the same as in Example 1, yielding a yellow solid, namely compound F-25, with a yield of 76.54% and an mp of 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 + ]=396.1586; found=396.1588.

[0087] Example 26: N'-(2-(3-chlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-26)

[0088] Synthesis process: The preparation of intermediate 2-(3-chlorophenyl)acetylhydrazine is described in Example 1;

[0089] Preparation of 4-((6,7-dimethoxyquinoline-4-yl)amino)benzoic acid:

[0090]

[0091] 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) were placed in a 250 ml round-bottom flask, 100 ml of isopropanol was added, and 1.6 ml of trifluoroacetic acid was slowly added. The mixture was refluxed at 85 °C. During the reaction, a grayish-white solid gradually precipitated out. After the reaction was detected by thin-layer chromatography (TLC), the heating was stopped, the flask was sealed, and the mixture was placed in a refrigerator for 1 hour. The mixture was then removed, filtered, and rinsed alternately with methanol and ethyl acetate. The filter cake was dried under infrared light and collected to obtain 2.12 g of grayish-white solid, 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).

[0092] In a 25 ml single-necked flask, 0.33 g (1 mmol, 1 eq) of 4-((6,7-dimethoxyquinoline-4-yl)amino)benzoic acid, 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. 10 ml of N,N-dimethylformamide (DMF) was added as a solvent, and the mixture was stirred overnight at room temperature. After the reaction was complete as detected by TLC, the reaction mixture was added dropwise to 200 ml of water, resulting in the precipitation of a solid. The solid was slurried for 1 h, filtered, and the filter cake was dried. 0.31 g of a pale yellow solid, compound F-26, was collected after drying, with a yield of 62.12%. mp: 258-260℃; 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.

[0093] Example 27: N'-(2-(2-chlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-27)

[0094] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-27, with a yield of 54.10% and an mp of 266-268°C. 1H-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.

[0095] Example 28: N'-(2-(4-chlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-28)

[0096] 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.

[0097] Example 29: N'-(2-(2-bromophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-29)

[0098] 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.

[0099] Example 30: N'-(2-(3-bromophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-30)

[0100] The preparation method was the same as in Example 26, yielding a light yellow solid, namely compound F-30, with a yield of 62.52% and an mp of 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 = HRMS (ESI) + );m / z calcd for C 26 H 23 BrN4O4[M+H + ]=534.0903; found=534.0907.

[0101] Example 31: N'-(2-(4-bromophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-31)

[0102] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-31, with a yield of 84.59% and an mp of 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.

[0103] Example 32: N'-(2-(2-fluorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-32)

[0104] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-32, with a yield of 35.21% and an mp of 266-268°C. 1 H-NMR (300 MHz, DMSO-d6): δ10.21 (d, J = 27.1 Hz, 2H), 8.94 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.63 (s, 1H), 7.51-7.27(m, 5H), 7.23-7.12 (m, 2H), 7.08 (d, J = 5.2 Hz, 1H), 3.93 (d, J = 5.0 Hz,6H), 3.62 (s, 2H). HRMS (ESI + ):m / z calcd for C 26 H 23 FN4O4[M+H+ ]=474.1703; found=474.1707.

[0105] Example 33: N'-(2-(3-fluorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-33)

[0106] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-33, with a yield of 33.14% and an mp of 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), HRMS (ESI) + ):m / z calcd for C 26 H 23 FN4O4[M+H + ]=474.1703; found=474.1701.

[0107] Example 34: N'-(2-(4-fluorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-34)

[0108] The preparation method is the same as in Example 26, yielding a grayish-white solid, namely compound F-34, with a yield of 43.50% and an mp of 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 C26 H 23 FN4O4[M+H + ]=474.1703; found=474.1708.

[0109] Example 35: 4-((6,7-dimethoxyquinoline-4-yl)amino)-N'-(2-(o-tolyl)acetyl)benzoylhydrazine (F-35)

[0110] The preparation method was the same as in Example 26, yielding a light yellow solid, namely compound F-35, with a yield of 52.22% and an mp of 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.

[0111] Example 36: 4-((6,7-dimethoxyquinoline-4-yl)amino)-N'-(2-(p-tolyl)acetyl)benzoylhydrazine (F-36)

[0112] The preparation method was the same as in Example 26, yielding a white solid, namely compound F-36, with a yield of 54.31% and an mp of 274-276 °C. 1H-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.

[0113] Example 37: N'-(2-(2-methoxyphenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-37)

[0114] The preparation method is the same as in Example 26, yielding a grayish-white solid, namely compound F-37, with a yield of 70.70% and an mp of 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.

[0115] Example 38: N'-(2-(3-methoxyphenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-38)

[0116] The preparation method is the same as in Example 26, yielding a grayish-white solid, namely compound F-38, with a yield of 66.66% and an mp of 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 / zcalcd for C 27 H 26 N4O5[M+H + ]=486.1903; found=486.1907.

[0117] Example 39: N'-(2-(4-nitrophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-39)

[0118] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-39, with a yield of 52.91% and an mp of 266-268°C. 1 H-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 forC 26 H 23 N5O6[M+H + ]=501.1648; found=501.1645.

[0119] Example 40: ((6,7-dimethoxyquinoline-4-yl)amino)-N'-(2-(4-(trifluoromethyl)phenyl)acetyl)benzoylhydrazine (F-40)

[0120] The preparation method was the same as in Example 26, yielding a yellow solid, namely compound F-40, with a yield of 62.48% and an mp of 262-264°C. 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.

[0121] Example 41: N'-(2-(3,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-41)

[0122] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-41, with a yield of 28.11% and an mp of 258-260°C. 1H-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.

[0123] Example 42: N'-(2-(2,3-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-42)

[0124] The preparation method is the same as in Example 26, yielding a grayish-white solid, namely compound F-42, with a yield of 74.96% and an mp of 268-272°C. 1 H-NMR (300 MHz, DMSO-d6): δ 10.32 (s, 1H), 10.19 (s, 1H), 8.96 (s,1H), 8.39 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.66-7.26 (m, 7H), 7.08 (d, J = 5.3 Hz, 1H), 3.93 (d, J = 5.3 Hz, 6H), 3.80 (s, 2H). HRMS (ESI + ):m / z calcd for C 26 H 22 Cl2N4O4[M+H + ]=524.1018; found=524.1019.

[0125] Example 43: N'-(2-(2,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-43)

[0126] 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. 1H-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.

[0127] Example 44: N'-(2-(2,6-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-44)

[0128] 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.

[0129] Example 45: 4-((6,7-dimethoxyquinoline-4-yl)amino)-N'-(2-(3,4,5-trimethoxyphenyl)acetyl)benzoylhydrazine (F-45)

[0130] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-45, with a yield of 50.35% and an mp of 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.

[0131] Example 46: N'-(2-(4-(tert-butyl)phenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-46)

[0132] The preparation method is the same as in Example 26, yielding a grayish-white solid, namely compound F-46, with a yield of 76.69% and an mp of 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 32 N4O4[M+H + ]=512.2424; found=512.2427.

[0133] Example 47: N'-(2-(2-hydroxyphenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-47)

[0134] The preparation method was the same as in Example 26, yielding a yellow solid, namely compound F-47, with a yield of 12.26% and an mp of 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.

[0135] Example 48: N'-(2-(4-aminophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-48)

[0136] The preparation method was the same as in Example 26, yielding a light yellow solid, namely compound F-48, with a yield of 45.86% and an mp of 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 forC 26 H 25 N5O4[M+H + ]=471.1907; found=471.1909.

[0137] Example 49: N'-(2-(3,4-difluorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-49)

[0138] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-49, with a yield of 69.85% and an mp of 266-268°C. 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.

[0139] Example 50: N'-(2-phenylacetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine (F-50)

[0140] The preparation method was the same as in Example 26, yielding a grayish-white solid, namely compound F-50, in 66.74% yield (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.

[0141] Example 51

[0142] 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.

[0143] (1) Test procedure: Prepare 1x kinase buffer: 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol (DTT), 0.01% Tween-20, 0.01% bovine serum albumin (BSA). (2) Prepare the compound: Serially dilute the compound and prepare the source plate: Dissolve the compound in 100% dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Dilute the compound with 100% DMSO to 100 times the final desired maximum inhibitor concentration. Add 100 μl of 100% DMSO to two empty wells, without a composite control and enzyme control in the same 96-well plate. Label this plate as the source plate. Transfer 40 μl of the compound to a new 384-well echo plate as an intermediate plate. Prepare the test plate: Transfer 200 nl of each well from the 384-well echo plate to the 384-well analytical plate, replicate. (3) Kinase reaction: Prepare 2x kinase solution: Prepare BTK solution in 1x kinase buffer, with a concentration twice the final concentration of each reagent in the experiment. Add 10 μl of kinase solution to each well except for the enzyme-free control well (add 10 μl of 1x kinase buffer instead). Incubate at room temperature for 10 minutes. Prepare 2x substrate solution. Prepare substrate solutions of Fluorescein-poly GT and ATP in 1x kinase reaction buffer, with a concentration twice the final concentration of each reagent required for the experiment. Add 10 μl of substrate solution to each well of the test plate to start the reaction. Incubate the kinase reaction at room temperature for a period of time. (4) Kinase detection: Prepare detection solution at 2x final concentration in antibody dilution buffer. Add 20 μl of detection solution to each well and stop the reaction. Mix briefly with a centrifuge, incubate at room temperature for 60 minutes, and then read the data on a fluorometer. (5) Data reading and curve fitting: Collect data at excitation wavelength of 340 nm and emission wavelengths of 520 nm and 495 nm on the Envision program. Calculate the ratio of relative fluorescence units (RFU) at 520 nm to 495 nm. Convert the calculated "ratio" value to a percentage inhibition value. Inhibition rate (%) = (maximum value - sample ratio) / (maximum value - minimum value) * 100. "min" represents the enzyme-free control; "max" represents the compound-free negative control.

[0144] The results of the compounds' BTK inhibitory activity are shown in Table 1. The experimental results show that compounds F-30, F-42, and F-43 in this invention exhibit good inhibitory activity against Bruton's tyrosine kinase. When the compounds in this invention have the same substituents, compounds F-26~F-36 and F-41~F-44 generally show higher BTK inhibitory activity than F-1~F-11 and F-16~F-19. This indicates that using the 6,7-dimethoxyquinoline core structure has superior activity compared to the quinoline core. Regarding the relationship between the terminal benzene ring substituents connected to the benzoylhydrazine fragment and the activity, the general trend is: dichloro substitution > monosubstituted; among monosubstituted groups, -Cl / -Br > -CH3 / -F; among dichloro substituted groups, 2,3-di-Cl / 2,4-di-Cl > 3,4-di-Cl / 2,6-di-Cl; for the substitution position on the monosubstituted ring, ortho / meta > para. In summary, terminal benzene ring substituents can significantly modulate the effects of target compounds, and dichloro substituents may exhibit an activity-enhancing effect.

[0145] Table 1 Results of the in vitro BTK inhibitory activity of the compounds

[0146]

[0147] Example 52

[0148] The inhibitory effect of the compound on the proliferation of human colon cancer cells (HCT116) was determined using the MTT assay [3-(4,5)-bismethyl-2-thiazolyl-(2,5)-phenyltetrazolyl bromide blue].

[0149] The inhibitory effect of the compound on the proliferation of human colon cancer cells (HCT116) was determined in vitro using the MTT assay [3-(4,5)-bismethyl-2-thiazolyl-(2,5)-phenyltetrazolium bromide blue]. 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: Prepare a 0.5% trypsin solution using D-Hanks buffer. Filter sterilize. Preparation of drug solution: Dissolve the compound in a small amount of triple-distilled water to prepare a stock solution, generally prepared at 10 times the highest experimental concentration. Depending on the solubility of the compound, it can be dissolved directly in triple-distilled water, or a small amount of DMSO can be used to aid dissolution. The concentration of DMSO in the culture medium should not be too high. The final concentration of DMSO in the cell suspension in each well after drug addition should generally not exceed 0.05-0.1%.

[0150] (1) Cell culture: HCT116 cells were routinely cultured in DMEM medium (containing 10% fetal bovine serum (FBS) and 100 U / mL of each of the two antibiotics) at 37 ℃ in a 5% CO2 incubator for 3-4 days. The cells were completely adherent and needed to be passaged once. When passaged, the original culture medium was discarded first, and then washed with D-Hanks buffer. Then, the cells were digested with trypsin for 30 seconds, and a small amount of fresh culture medium was added to stop the digestion. The cells were pipetted and transferred to a new culture flask, and then fresh culture medium was added to the original volume. (2) Cell incubation: When HCT116 cells reached the logarithmic growth phase, a cell suspension was prepared with a concentration of 1.5 × 10⁻⁶. 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well culture plate and incubate at 37 ℃ in a 5% CO2 incubator for 24 h. Then, add the drug solution according to the designed concentration. (3) Drug addition: Add the test drug solution to each well according to the concentration gradient of the final concentration, with 6 parallel wells for each concentration. The experiment is divided into 3 groups: drug test group A (containing different concentrations of test drug), control group B (containing culture medium and cells, without test drug) and blank group C (containing only culture medium, without cells and test drug). Place the 96-well plate after drug addition in a 37 ℃ incubator for 48 h. (4) Determination of viable cells: Add 40 μL of MTT (prepared with D-Hanks buffer to 4 mg / mL) to each well of the 96-well plate after 48 h of culture. After placing in a 37 ℃ incubator for 4 h, discard the supernatant. Add 150 μL of LDMSO to each well and shake for 5 min to completely dissolve the precipitate. Finally, the optical density (OD value) of each well was read using an automated microplate reader at a wavelength of 570 nm. (5) Calculation of cell inhibition rate: Growth inhibition rate = (1 - survival rate) × 100% = [1 - (OD value) × 100%] 实验 -OD 空白 ) / (OD 对照 -OD 空白 )]×100%; (OD 实验 The OD value represents the average optical density of test drug group A. 对照 The average optical density (OD) of control group B is represented by the value of OD. 空白 (This represents the average optical density of the blank group C). The obtained inhibition rate was fitted using GraphPad Prism 6 software to obtain the half-maximum inhibition concentration (IC50). 50 )value.

[0151] The in vitro proliferation results of the compounds against human colon cancer HCT116 cell line are shown in Table 2. The experimental results show that the IC50 of most compounds... 50Values ​​in the range of 1.33-10 μM showed better anti-tumor cell proliferation activity compared to positive control drugs. Among them, F-2, F-5, F-27, F-35, F-40, F-41, and F-43 exhibited IC50-90% anti-proliferation activity against HCT116 cells. 50 With a value less than 2μM, the F-43 has the lowest IC50. 50 The value indicates that it has the best activity among the compounds in this series.

[0152] Table 2. Results of the compounds' in vitro anti-HCT116 cell proliferation activity.

[0153]

[0154] Example 53

[0155] The inhibitory effect of the compound on the proliferation of human histiocytic lymphoma cells (U-937) was determined using the Cell Counting Kit-8 (CCK-8) method.

[0156] U-937 cells were prepared into a cell suspension of 50,000 cells / mL using complete culture medium and seeded into 96-well plates. Different concentration gradients of the compound (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM) and ibrutinib were added to the cells, and the cells were incubated at 37 °C, 5% CO2 for 48 h. After incubation, 10 μL of CCK-8 solution was added to each well under dark conditions, and the cells were incubated at 37 °C, 5% CO2 for 3 h. The OD value was measured at 450 nm, and the inhibition rate was calculated. Inhibition rate = (OD...) / (2000-2000-2000) = 10 μL / well. 对照组平均值 -OD 加药组 ) / OD control group × 100%, OD 对照组平均值 = (OD) 未加药 -OD 空白 () ÷ Number of wells without drug addition. The obtained inhibition rate was fitted using GraphPad Prism 8 software to obtain the IC50. 50 The in vitro antiproliferative activity of the compounds against human histiocytic lymphoma U-937 cell line is shown in Table 3. The results show that, compared with the positive control drug ibrutinib, compounds F-4, F-17, F-18, F-27, F-29, F-42, and F-43 exhibited superior inhibitory effects against the proliferation of human histiocytic lymphoma cells (U-937). F-43 showed the lowest IC50 value. 50 The value indicates that it has the best activity among the compounds in this series.

[0157] Table 3. Results of the compounds' in vitro anti-proliferation effects on U-937 cells.

[0158]

[0159] Example 54

[0160] Kinase selectivity test: The compound with the best activity, F-43 [N'-(2-(2,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine], was selected for kinase profile testing.

[0161] (1) Compound preparation: Dissolve compound F-43 in DMSO to obtain a 10 mM stock solution, and serially dilute to the specified experimental concentration 100 times (1 mM). (2) Compound screening: Homogeneous time-resolved fluorescence (HTRF) kinase assay: Prepare 2×ATP and substrate solutions and 2× kinase and metal solutions using HTRF buffer (5 mM MgCl2; 1 mM MnCl2; 12.5 nM staphylococcal enterotoxin B (SEB); 1 mM DTT; double-distilled water ddH2O). Transfer 50 nL of the compound to a 384-well plate using an Echo 655. After centrifugation, add 2.5 μL of 2× kinase-metal solution and incubate at 25 °C for 10 min. Add 2.5 μL of 2× substrate and ATP solution to each well and incubate at 25 °C for 60 min. Prepare 2× XL665 and antibody solutions using HTRF buffer. Add 5 μL of the solution prepared in the previous step to each well and incubate at 25 °C for 60 min. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665). ADP-Glo ​​kinase assay: Prepare 2×ATP and substrate solutions and 2× kinase and metal solutions 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). Transfer 40 nL of the compound to a 384-well plate using an Echo 655. Add 2 μL of 2× kinase and metal solution to each well and incubate at 25 °C for 10 min. Add 2 μL of 2×ATP and substrate solution to each well and incubate at 25 °C for 60 min. Add 4 μL of ADP-Glo ​​reagent to each well and incubate at 25 °C for 40 min. Add 8 μL of kinase detection reagent to each well and incubate at 25°C for 40 minutes. Read the fluorescence signal recorded on the plate reader. (3) Data analysis: Set the reading value of the negative control (1% DMSO) to 0% inhibition rate and the reading value of the 10µM positive control to 100% inhibition rate. Inhibition rate (%) = 100% - (compound - positive control) / (negative control - positive control) × 100%; where, positive control: 10µM positive drug; negative control: 1% DMSO.

[0162] The kinase activity results of compound F-43 against 76 tyrosine kinases are shown in Table 4. The results show that compound F-43 has the best selectivity for BTK (99.84%), and has weak inhibitory activity against common off-target kinases of the existing BTK inhibitor ibrutinib, such as EGFR (-70.37%), ITK (32.83%), and TEC (74.80%), which can reduce off-target effects and their side effects.

[0163] Table 4. Kinase profile test results of compound F-43

[0164]

[0165] Example 55

[0166] The activity of compound F-43 [N'-(2-(2,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine] in inhibiting BTK phosphorylation was detected by Western blot. Compound F-43 (final concentrations of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM) and the control compound (ibrutinib, final concentration of 1 μM) were added to six-well plates containing U-937 cells, with a blank control group included. After 48 h of culture, cells were collected by centrifugation. Cells were washed with 1xPBS buffer and lysed with RAPI lysis buffer. Cell lysates were separated by 4-20% gradient gel electrophoresis, transferred to membranes, blocked, incubated with primary antibodies (BTK, p-BTK, Beyotime), eluted, incubated with secondary antibodies, and finally analyzed by chemiluminescence imaging. Experimental results are shown below. Figure 3 As shown in the figure. The results indicate that compound F-43 inhibits BTK phosphorylation in a dose-dependent manner.

[0167] Example 56

[0168] The effect of compound F-43 [N'-(2-(2,4-dichlorophenyl)acetyl)-4-((6,7-dimethoxyquinoline-4-yl)amino)benzoylhydrazine] on apoptosis in human histiocytic lymphoma cells (U-937) was detected by flow cytometry. Compound F-43 (final concentrations of 2 μM, 4 μM, and 8 μM), and the control compound (ibrutinib, final concentration of 8 μM) were added to 6-well plates containing U-937 cells. After incubation for 48 h, cells were collected by centrifugation, stained with the Annexin V-FITC apoptosis detection kit (Beyotime), and the samples were analyzed by CytoFLEX flow cytometry. Data were analyzed using FlowJo software. Experimental results are shown below. Figure 4 and 5As shown in the figure. The results indicate that compound F-43 promotes apoptosis of human histiocytic lymphoma cells (U-937) in a dose-dependent manner, with an apoptosis rate of over 20% in U-937 cells at a concentration of 8 μM, and its pro-apoptotic ability is stronger than that of the positive control drug ibrutinib.

Claims

1. A compound having BTK inhibitory activity, characterized by, The compound is a quinoline-4-amine compound containing a benzohydrazide structure, and the structure is shown in formula I: ； Specifically selected from the following compounds: 。 2. The compound of claim 1, wherein The compound also includes pharmaceutically acceptable salts thereof.

3. The compound of claim 2, wherein The pharmaceutically acceptable salt, the acid used for salification is an inorganic acid or an organic acid, and the organic acid is aliphatic monocarboxylic acid and dicarboxylic acid, phenyl-substituted alkanoic acid, hydroxyalkanoic acid and alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid.

4. The compound of claim 3, wherein The pharmaceutically acceptable salt, the acid used for salification is 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 or aspartic acid.

5. A process for the preparation of a compound having BTK inhibitory activity according to claim 1, characterized by, The method comprises the following steps: Preparation of intermediate B by reacting compound A with ethanol, reaction of B with hydrazine hydrate to prepare intermediate C, reaction of compound D with p-aminobenzoic acid to obtain intermediate E, reaction of C with E to obtain a BTK inhibitor, wherein R1, R2 and R are as defined in claim 1. 。 6. A pharmaceutical composition comprising a compound of the formula: ###0002### or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. The method comprises the following steps:

7. Use of a compound having BTK inhibitory activity according to claim 1 or a pharmaceutical composition according to claim 6 for the manufacture of a BTK inhibitor.

8. Use according to claim 7, characterized in that, The application is specifically the use of 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.

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