An egfr / her2 inhibitor based on hydrazone structure, preparation method and application thereof

Abstract

The application provides an EGFR / HER2 inhibitor based on a hydrazone structure, a preparation method and application thereof, and belongs to the field of medicines; specifically provides a hydrazone derivative, which has a chemical structural formula as shown in formula A, wherein R is a methyl group or a hydrogen group, and X is halogen, a hydroxyl group or a methoxyl group; and a preparation method of the hydrazone derivative is provided; the hydrazone derivative provided by the application has an antitumor effect as an EGFR and / or HER2 inhibitor, and can be used for preparing an antitumor drug. Formula A

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to an EGFR / HER2 inhibitor based on a hydrazone structure, its preparation method, and its application. Background Technology

[0002] Hepatoblastoma is a common malignant liver tumor in children, with a peak incidence in the first two years (Semin. Diagn. Pathol. 2017, 34(2), 192-200). Molecularly, it is characterized by gene mutations and alterations in key signaling pathways driving tumorigenesis, and its significant heterogeneity complicates the identification of the most aggressive subtypes and the development of targeted therapies (Genes 2024, 15(11), 1358). Survival rates for high-risk and metastatic cases of hepatoblastoma have been reported to be poor, and the exact etiology remains unclear (Asian J. Surg. 2024, 47(5), 2132-2137).

[0003] Breast cancer is one of the most common malignant tumors worldwide, and its global incidence rate is increasing year by year. The rapid development of targeted therapy has provided new hope for the treatment of breast cancer (Breast Cancer-Targets Ther. 2025, 17, 339-348). However, combined with factors such as social development, population growth, and population aging, the economic burden of breast cancer on women worldwide has been steadily increasing.

[0004] Colorectal cancer is a malignant tumor of the gastrointestinal tract originating in the colon or rectum. Treatment for colorectal cancer includes surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy (Ca-Cancer J. Clin. 2023, 73(3), 233-254). Although various drugs have been used to treat malignant tumors, their effectiveness is not as expected. This is mainly due to the lack of selectivity, poor solubility, and severe side effects of most drugs.

[0005] Epidermal growth factor receptor (EGFR) plays a crucial role in tumor growth and progression, including cell proliferation, inhibition of apoptosis, metastasis, and angiogenesis. Furthermore, human epidermal growth factor receptor 2 (HER2) serves as an important cancer biomarker.

[0006] The molecular mechanism and signaling pathways of EGFR / HER2 kinase inhibitors: Primarily, they compete with ATP molecules to occupy the ATP-binding pocket of the intracellular tyrosine kinase domain of EGFR / HER2, affecting the activity of intracellular tyrosine kinases and blocking kinase autophosphorylation induced by ligand binding to the extracellular region and cross-phosphorylation caused by EGFR family member dimerization. This inhibits downstream PI3K / AKT and MAPK signaling pathways, suppressing tumor cell proliferation and inducing tumor cell death. Dual-target protein kinase inhibitors can act on both EGFR and HER2 kinases simultaneously, inhibiting multiple downstream signaling pathways, resulting in better anti-tumor effects and reduced side effects.

[0007] Dual targeting of EGFR and HER2 is a well-established anti-cancer strategy, but existing small-molecule EGFR / HER2 dual inhibitors (such as lapatinib) often suffer from problems such as complex structures, difficult synthesis, and poor drug resistance (CancerChemother.Pharmacol.2011,68(5), 1315-1323). Therefore, researching and developing a small-molecule EGFR / HER2 dual inhibitor with a simple structure and easy synthesis could provide a potential new clinical treatment approach for patients with related malignant tumors. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an EGFR / HER2 inhibitor based on a hydrazone structure, its preparation method, and its application.

[0009] This invention has discovered seven hydrazone compounds that can bind to and inhibit the activity of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), thereby inhibiting the activity, proliferation, and invasion of hepatoblastoma cells, breast cancer cells, and colorectal cancer cells.

[0010] The technical solution of the present invention is as follows: A hydrazone derivative having the chemical structural formula shown in Formula A:

[0011] Formula A Where R is a methyl or hydrogen group; X is a halogen, hydroxyl or methoxy group.

[0012] Preferably, the halogen is fluorine, chlorine, bromine or iodine.

[0013] Preferably, the hydrazone derivative includes compound 1: C 16 H 14 F2N2, Compound 2: C 16 H 14 Cl2N2, Compound 3: C 16 H 14Br2N2, Compound 4: C 16 H 14 I₂N₂, Compound 5: C 16 H 20 N2O4, Compound 6: C 18 H 20 N2O2 or compound 7C 14 H 10 Cl2N2; The crystallographic parameters of compound 1 are: monoclinic system, space group 1. P twenty one / n The unit cell parameters are a =11.7001(5)±10 Å, b =7.3306(2)±10 Å, c =15.3389(6)±10 Å, α =90°, β =96.218(4)±5°, γ =90°, cell volume is 1307.86(58)±40 Å 3 ; The crystallographic parameters of compound 2 are: monoclinic system. P twenty one / c Space group, cell parameters are a =15.0756(5)±10 Å, b =9.1432(3)±10 Å, c =10.3122(3)±10 Å, α =90°, β =93.734(3)±5°, γ =90°, cell volume is 1418.41(8)±40 Å 3 ; The crystallographic parameters of compound 3 are: orthorhombic crystal system, space group 1. Pbca The unit cell parameters are a =9.3247(2)±10 Å, b =10.3535(3)±10 Å, c =30.3792(7)±10 Å, α =90°, β =90°, γ =90°, the cell volume is 2932.91(13)±40 Å 3 ; The crystallographic parameters of compound 4 are: orthorhombic crystal system, space group 4. Pcca The unit cell parameters are a =34.556(2)±10 Å, b=7.2485(4)±10 Å, c =6.3260(3)±10 Å, α =90°, β =90°, γ =90°, cell volume is 1584.53(15)±40 Å 3 ; The crystallographic parameters of compound 5 are: monoclinic system, space group 1. P twenty one / n The unit cell parameters are a =7.8538(5)±10 Å, b =5.5180(3)±10 Å, c =17.1022(8)±10 Å, α =90°, β =97.251(5)±5°, γ =90°, cell volume is 735.24(7)±40 Å 3 ; The crystallographic parameters of compound 6 are: monoclinic system, space group 6. P twenty one / n The unit cell parameters are a =6.2644(4)±10 Å, b =7.2188(5)±10 Å, c =16.8958(11)±10 Å, α =90°, β =96.87(1)±5°, γ =90°, cell volume is 758.56(9)±40 Å 3 ; The crystallographic parameters of compound 7 are: monoclinic system, space group 1. P twenty one / c The unit cell parameters are a =3.8553(2)±10 Å, b =6.9822(3)±10 Å, c =22.8906(10)±10 Å, α =90°, β =90.908(4)±5°, γ =90°, the volume of the unit cell is 616.10(5)±40 Å. 3 .

[0014] The preparation method of the above-mentioned hydrazone derivatives includes the following steps: In the presence of an acid catalyst, a para-substituted acetophenone compound or a para-substituted benzaldehyde compound is reacted with hydrazine hydrate in an organic solvent to obtain the hydrazone derivatives.

[0015] Preferably, in the preparation method, the para-substituted acetophenone compound or the para-substituted benzaldehyde compound has the chemical structural formula shown in Formula B:

[0016] Formula B Where R is a methyl or hydrogen group; X is a halogen, hydroxyl or methoxy group.

[0017] Preferably, in the preparation method, the para-substituted acetophenone compound includes p-fluoroacetophenone, p-chloroacetophenone, p-bromoacetophenone, p-iodoacetophenone, p-hydroxyacetophenone, or p-methoxyacetophenone; the para-substituted benzaldehyde compound includes p-chlorobenzaldehyde.

[0018] The synthesis route is as follows:

[0019] Where R is a methyl or hydrogen group; X is a halogen, hydroxyl or methoxy group.

[0020] Preferably, in the preparation method, the acid catalyst is acetic acid.

[0021] Preferably, in the preparation method, the organic solvent is ethanol.

[0022] Preferably, in the preparation method, the reaction conditions are 80-110℃ for 2-5 hours.

[0023] Preferably, in the preparation method, after the reaction is completed, the filter residue is collected by filtration, and the filter residue is washed and dried to obtain the product.

[0024] A further preferred method is to use ethanol at 0-35°C for washing.

[0025] In a further preferred embodiment, the product is recrystallized in ethanol to obtain crystals.

[0026] The above-mentioned hydrazone derivatives or hydrazone derivatives prepared by the above methods are used as EGFR and / or HER2 inhibitors in the preparation of drugs.

[0027] The application of the above-mentioned hydrazone derivatives or hydrazone derivatives prepared by the above method in the preparation of antitumor drugs.

[0028] Preferably, the above-mentioned hydrazone derivatives or hydrazone derivatives prepared by the above method are used as EGFR and / or HER2 inhibitors in the preparation of antitumor drugs.

[0029] Preferably, the tumor includes tumors associated with EGFR and / or HER2 activation.

[0030] Preferably, the tumor includes hepatoblastoma, breast cancer, or colorectal cancer.

[0031] A drug containing the above-mentioned hydrazone derivatives or hydrazone derivatives prepared by the above method.

[0032] Preferably, the drug is an anti-tumor drug.

[0033] Beneficial effects of the present invention 1. The hydrazone derivatives provided by this invention exhibit good binding interactions with EGFR and HER2, indicating their ability to inhibit the activity of both tyrosine kinases and demonstrating superior in vitro anticancer activity. In addition to altering related apoptosis factors, the hydrazone derivatives can also arrest the cell cycle at the G0-G1 phase, inducing early and late apoptosis. The hydrazone derivatives provided by this invention possess good anti-tumor cell proliferation activity and exhibit lower toxicity to normal cells. With excellent anticancer activity and good safety profile, they can be used to prepare drugs for treating tumors.

[0034] 2. The hydrazone derivatives provided by this invention have simple structures and are easy to synthesize, which is conducive to their application and promotion. Attached Figure Description

[0035] Figure 1 The figure shows the in vitro cytotoxicity test results of sunitinib and the hydrazone compounds provided by this invention.

[0036] Figure 2 The diagram shows the molecular docking results for compound 1.

[0037] Figure 3 This is a diagram showing the molecular docking results of compound 2.

[0038] Figure 4 The diagram shows the molecular docking results for compound 3.

[0039] Figure 5 The diagram shows the molecular docking results for compound 4.

[0040] Figure 6 This is a diagram showing the molecular docking results of compound 5.

[0041] Figure 7 This is a diagram showing the molecular docking results of compound 6.

[0042] Figure 8 The diagram shows the molecular docking results for compound 7.

[0043] Figure 9 This is a graph showing the molecular docking results of Sorafenib.

[0044] Figure 10 This is a diagram showing the molecular docking results of TAK-285.

[0045] Figure 11 Figure 1 shows the results of in vitro tyrosine kinase inhibition experiments on erlotinib, lapatinib, and compounds 4, 5, and 6 provided in this invention.

[0046] Figure 12 The zero-dimensional structure diagrams are for compounds 1-7. In the figure: (a) is compound 1, (b) is compound 2, (c) is compound 3, (d) is compound 4, (e) is compound 5, (f) is compound 6, and (g) is compound 7.

[0047] Figure 13 The ultraviolet spectra of seven hydrazone compounds provided for this invention.

[0048] Figure 14 Infrared spectra of compounds 1 and 2 provided for this invention.

[0049] Figure 15 Infrared spectra of compounds 3 and 4 provided for this invention.

[0050] Figure 16 Infrared spectra of compounds 5, 6 and 7 provided for this invention.

[0051] Figure 17 The DSC results of the seven hydrazone compounds provided by this invention are shown in the figure. Detailed Implementation

[0052] The technical solution of the present invention will be described in detail through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0053] Unless otherwise specified, the following implementation schemes generally follow standard testing conditions or the testing conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] Example 1: Preparation method of seven hydrazone compounds (i.e., hydrazone derivatives).

[0055] This invention employs a novel synthetic route to synthesize novel hydrazone compounds. The entire preparation process does not involve any special reaction types or conditions. The reaction conditions are mild, the reaction is a one-step process, the reaction yield is high, and the post-processing is simple, mostly using recrystallization instead of other complex purification methods.

[0056] Synthesis of Compound 1: A mixture of p-fluoroacetophenone (121 μl, 1 mmol) and anhydrous ethanol (5 ml) was added with 50% hydrazine hydrate (49 μl, 0.5 mmol) and glacial acetic acid (5 drops). The reaction mixture was heated at 100 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature to obtain a solid-liquid mixture. The residue was filtered off and washed three times with cold ethanol (0 °C) (approximately 5 ml each time). The residue was dried in an oven at 80 °C for 1 hour to obtain a solid (76.3% yield, 0.1039 g). The solid was dissolved in ethanol, and after two days, the resulting brown filamentous crystals were collected. Melting point: 199–203 °C. 16 H 14 Elemental analysis calculations (%) of F₂N₂ (272.29): C, 70.58; H, 5.18; N, 10.29; Experimental values: C, 70.78; H, 5.17; N, 10.24. IR (KBr, cm⁻¹) -1 ): 3839(w), 3734(w), 3648(w), 3576(m), 3383(m), 3164(m), 2983(m), 1748(w), 1730(w), 1704(w), 1632(s), 1613(m), 1598( m), 1540(m), 1503(s), 1479(w), 1445(w), 1430(w), 1407(w), 1395(w), 1375(w), 1314(m), 1298(m), 1288(m), 1279(m), 12 31(m), 1214(s), 1202(s), 1159(m), 1131(w), 1103(w), 1050(w), 1025(w), 1012(w), 979(w), 966(w), 938(w), 918(m), 864 (m), 838(s), 819(m), 798(w), 768(w), 756(w), 684(w), 620(w), 600(w), 558(m), 518(w), 496(w), 481(m), 457(w), 435(w).

[0057] Synthesis of Compound 2: p-Chloroacetophenone (0.1546 g, 1 mmol) was mixed with anhydrous ethanol (5 ml) and dissolved by sonication. 50% hydrazine hydrate (49 μl, 0.5 mmol) and glacial acetic acid (5 drops) were added, and the mixture was heated at 100 °C for 3 hours. After cooling to room temperature, the mixture was filtered under reduced pressure to obtain a pale yellow powder. The powder was washed and dried (using the same method as Compound 1) to obtain a solid (yield 57.3%, 0.0875 g). Recrystallization (using the same method as Compound 1) yielded colorless blocky crystals. Melting point: 143-147 °C. 16 H 14Elemental analysis of Cl₂N₂ (305.19) calculated values ​​(%): C, 62.97; H, 4.62; N, 9.18; experimental values: C, 62.78; H, 4.61; N, 9.16. IR (KBr, cm⁻¹) -1 ): 3839(m), 3734(m), 3648(m), 3566(m), 3057(m), 2961(m), 2919(m), 1921(w), 1904(w), 1782(w), 1760(w), 1731(w), 1693(w), 1651(w), 1644(w), 1607(m), 1592(m), 1561(w), 1505(w), 1487(s), 1426(s), 1397(m), 1361(m), 1301(m), 1292(m), 1275(w), 1230(w), 1174(w), 1106(w), 1090(s), 1012(s), 976(w), 963(w), 94 3(w), 912(w), 830(s), 823(s), 789(w), 765(m), 717(m), 678(w), 630(w), 560(s), 514(w), 486(w), 468(w).

[0058] Synthesis of Compound 3: 50% hydrazine hydrate (49 μl, 0.5 mmol) and glacial acetic acid (5 drops) were dissolved in a mixture of p-bromoacetophenone (0.1990 g, 1 mmol) and anhydrous ethanol (5 ml). After thorough mixing, the mixture was heated at 100 °C for 3 hours, cooled to room temperature, filtered, washed, and dried (using the same method as Compound 1) to obtain an orange powdery solid (yield 68.8%, 0.1356 g). Recrystallization (using the same method as Compound 1) yielded colorless flaky crystals. Melting point: 212-218 °C. 16 H 14 Elemental analysis of Br₂N₂ (394.11) calculated values ​​(%): C, 48.76; H, 3.58; N, 7.11; experimental values: C, 48.59; H, 3.59; N, 7.12. IR (KBr, cm⁻¹) -1): 3746(m), 3713(m), 3673(m), 3647(m), 3611(m), 3565(m), 3257(m), 1899(w), 1730(w), 1637(w), 1607(m), 1 584(m), 1558(w), 1507(w), 1483(m), 1428(w), 1394(m), 1361(m), 1301(m), 1292(m), 1274(w), 1252(w), 12 24(w), 1180(w), 1140(w), 1105(w), 1077(m), 1008(s), 965(w), 944(w), 905(w), 889(w), 863(w), 824(s), 7 80(w), 760(m), 713(w), 676(w), 629(w), 560(m), 533(w), 512(w), 499(w), 486(w), 474(w), 463(w), 428(w).

[0059] Synthesis of Compound 4: A mixture of p-iodoacetophenone (0.2460 g, 1 mmol) and anhydrous ethanol (5 ml) was added to 50% hydrazine hydrate (49 μl, 0.5 mmol) and glacial acetic acid (5 drops). The reaction mixture was heated at 100 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, and dried (using the same method as Compound 1) to obtain a solid (yield 33%, 0.0807 g). The solid was dissolved in ethanol and recrystallized (using the same method as Compound 1). Yellow flaky crystals suitable for single-crystal X-ray diffraction analysis were isolated. Melting point: 200-203 °C. 16 H 14 Elemental analysis calculations (%) of I₂N₂ (488.09): C, 39.37; H, 2.89; N, 5.74; Experimental values: C, 39.45; H, 2.88; N, 5.73. IR (KBr, cm⁻¹) -1): 3726(m), 3705(m), 3623(m), 3599(m), 3073(m), 3049(m), 2970(m), 1951(w), 1925(w), 1895(w), 1 735(w), 1701(w), 1654(w), 1645(w), 1618(w), 1600(m), 1579(m), 1545(w), 1480(m), 1437(m), 13 89(s), 1358(m), 1301(w), 1288(m), 1273(w), 1182(w), 1120(w), 1107(w), 1074(m), 1003(s), 977 (w), 849(m), 823(s), 795(w), 751(m), 711(w), 659(w), 627(w), 560(s), 518(w), 482(w), 445(w).

[0060] Synthesis of Compound 5: p-Hydroxyacetophenone (0.1089 g, 0.8 mmol) was mixed with anhydrous ethanol (5 ml) and dissolved by sonication. 23 μl (0.4 mmol) of 85% hydrazine hydrate and glacial acetic acid (5 drops) were added, and the mixture was heated at 100 °C for 3 hours. After cooling to room temperature, the mixture was filtered under reduced pressure, washed, and dried (using the same method as Compound 1) to obtain a yellow powdery solid (yield 61.2%, 0.0745 g). Recrystallization (using the same method as Compound 1) yielded yellow flaky crystals. Melting point: 204-207 °C. 16 H 20 Elemental analysis of N₂O₄ (304.34) calculated values ​​(%): C, 71.62; H, 6.01; N, 10.44; experimental values: C, 71.50; H, 6.02; N, 10.45. IR (KBr, cm⁻¹) -1): :3902(m), 3881(m), 3839(m), 3797(m), 3740(m), 3710(m), 3674(m), 3648(m), 3613(m), 3566(m), 3417(s), 3 242(s), 3064(s), 1747(w), 1703(w), 1649(s), 1624(s), 1603(s), 1573(m), 1558(m), 1536(m), 1511(s), 1440 (m), 1397(m), 1361(m), 1319(m), 1270(m), 1241(m), 1208(s), 1168(m), 1101(m), 1014(w), 912(w), 888(w), 859(w), 835(m), 751(w), 734(w), 698(w), 642(w), 622(w), 599(w), 565(w), 534(w), 519(w), 508(w), 468(w).

[0061] Synthesis of Compound 6: 50% hydrazine hydrate (49 μl, 0.5 mmol) and glacial acetic acid (5 drops) were dissolved in a mixture of p-methoxyacetophenone (0.1502 g, 1 mmol) and anhydrous ethanol (5 ml). After thorough mixing, the mixture was heated at 100 °C for 3 hours, cooled to room temperature, filtered, washed, and dried (using the same method as Compound 1) to obtain a yellow powdery solid (yield 67.6%, 0.1002 g). Recrystallization (using the same method as Compound 1) yielded yellow blocky crystals. Melting point: 194-208 °C. 18 H 20 Elemental analysis of N₂O₂ (296.36) calculated values ​​(%): C, 72.95; H, 6.80; N, 9.45; Actual values: C, 72.75; H, 6.81; N, 9.46. IR (KBr, cm⁻¹) -1):3839(m), 3740(m), 3714(m), 3647(m), 3613(m), 3565(m), 3446(m), 3011(m), 2964(m), 2928(m), 2837(m), 25 55(w), 2042(w), 1915(w), 1867(w), 1747(w), 1730(w), 1714(w), 1698(w), 1681(w), 1657(w), 1593(s), 1505(s) , 1455(m), 1442(m), 1411(w), 1366(m), 1303(s), 1252(s), 1181(m), 1173(s), 1116(m), 1026(s), 968(w), 912(w) ), 880(w), 835(s), 805(w), 760(w), 725(w), 697(w), 634(w), 605(m), 569(m), 525(w), 501(w), 456(w), 431(w).

[0062] Synthesis of Compound 7: p-Chlorobenzaldehyde (0.2811 g, 2 mmol) was dissolved in anhydrous ethanol (2 ml), then 85% hydrazine hydrate (57 μl, 1 mmol) and glacial acetic acid (5 drops) were added. The mixture was heated at 100 °C for 3 hours. After cooling to room temperature, the mixture was filtered, washed, and dried (using the same method as Compound 1) to obtain a yellow powdery solid (yield 73.3%, 0.2032 g). Recrystallization (using the same method as Compound 1) yielded yellow flaky crystals. Melting point: 205-208 °C. 14 H 10 Elemental analysis calculations (%) for Cl₂N₂ (277.14): C, 60.67; H, 3.64; N, 10.11; Actual values: C, 60.49; H, 3.65; N, 10.13. IR (KBr, cm⁻¹) -1 ): 3448(m), 3048(m), 2996(m), 2942(m), 2565(m), 1928(w), 1895(m), 1872 (m), 1785(m), 1653(m), 1625(s), 1593(m), 1568(m), 1485(s), 1401(s), 131 2(m), 1293(m), 1280(w), 1208(w), 1168(m), 1102(w), 1089(s), 1028(w), 1 012(s), 957(m), 933(m), 862(s), 825(s), 815(s), 717(w), 703(w), 629(w), 554(m), 498(s).

[0063] Example 2: Half-inhibitory concentration (IC50) of hydrazone compounds 50 and cytotoxicity testing The cytotoxicity of the seven hydrazone compounds obtained above (crystals obtained after recrystallization) against hepatoblastoma cells, breast cancer cells, colorectal cancer cells, and fibroblasts was determined using the CCK-8 assay. Experimental procedure: First, prepare the stock solution of the compound: Based on the weighed drug mass, determine the required molar concentration, and use the MCE molar calculator to calculate the volume of dimethyl sulfoxide (DMSO) to be added. Add the corresponding volume of DMSO according to the calculated volume. After adding the DMSO solution, vortex the solution until the drug is completely dissolved into a clear solution. The following experimental procedures are performed in a biosafety cabinet. Use tweezers to handle sterilized centrifuge tubes and label them. Add 250 μL of DMEM culture medium to each labeled centrifuge tube in a suspending position. Calculate the required stock solution using Formula 1. Seal the centrifuge tubes containing the stock solution and culture medium, and vortex for 1 min.

[0064] Formula 1 X : Volume of stock solution added; C: Final concentration; C0: Initial concentration; 0.25 mL: Volume of culture medium; 2: Number of cell wells to be added.

[0065] Post-drug incubation: Label the cell culture plates with the corresponding drug solutions. Before plating, vortex the centrifuge tubes containing the drug solutions again. Use a pipette to transfer 100 μL of the drug solution into one well of a 96-well plate, and add the same volume of drug solution to adjacent wells. After dispensing, place the cell culture plates in a CO2 incubator and incubate for 48 hours. After 48 hours, discard the culture medium, add 100 μL of a mixture of DMEM medium and CCK-8 reagent (DMEM medium: CCK-8 reagent = 9:1) to each well, and incubate at 37°C for approximately 1 hour.

[0066] Absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The inhibitory rate of the tested compound on tumor cell growth was calculated based on the absorbance values. The experimental data were compared with those of the multi-target tyrosine kinase inhibitor sunitinib, and the results are as follows: Figure 1 As shown in Table 1.

[0067] Table 1. In vitro cytotoxicity of compounds 1-7 and sunitinib against normal and cancer cell lines.

[0068] The cytotoxicity of hydrazone compounds 1-7 to three cancer cell lines (HCT-116, HEPG-2, and MCF-7) and a normal fibroblast cell line (WI-38) was investigated using the CCK-8 assay. 50 The IC50 value represents the molecular concentration required to inhibit cell viability by 50%. 50 Lower values ​​indicate higher cytotoxicity. Table 1 shows that compounds 1-7 exhibited some anticancer activity against all tested cancer cell lines, while showing weak cytotoxicity against normal cell lines. Compound 4 (12.47±0.64-17.04±1.27 μM) showed good anticancer effects against all cancer cell lines and was stronger than other hydrazone compounds. Comparison of hydrazone compounds with the standard drug Sunitinib revealed that compounds 4 and 5 showed higher cytotoxicity than Sunitinib in the HCT-116 cell line. For the HepG-2 cell line, compounds 1-7 showed lower cytotoxicity than the standard drug. In the MCF-7 cell line, compounds 4, 5, and 6 showed higher cytotoxicity than Sunitinib. For the normal WI-38 cell line, the toxicity of the target compounds was lower than that of Sunitinib, with compound 4 (93.67±2.09 μM) showing the lowest toxicity. Based on the structure-activity relationship, it can be concluded that, except for compound 4, the other compounds with electron-withdrawing groups exhibit moderate activity, while compounds with electron-donating groups exhibit stronger activity and higher cytotoxicity. For HepG2 and MCF-7 cell lines, the hydrazone compounds provided in this invention all showed good anticancer activity.

[0069] Example 3: Flow Cytometry Analysis of the Cell Cycle Based on the in vitro cytotoxicity analysis results of the compounds in Table 1 against normal and cancer cell lines, compound 4 was selected for further cell cycle analysis. The effect of compound 4 on cell cycle progression was evaluated using the propidium iodide flow cytometry kit / BD (ab139418).

[0070] First, plate the cells according to the experimental requirements and collect them once they reach the appropriate density. The cell collection procedure is the same as for routine cell treatment. Note that when performing cell cycle experiments, the original culture medium should be collected in a centrifuge tube. After digestion, add an appropriate amount of the original culture medium to stop the digestion reaction, and then centrifuge the entire mixture. After centrifugation, discard the supernatant, add 1 ml of ice-cold PBS buffer to the centrifuge tube to resuspend the cells, and transfer to a 1.5 ml Eppendorf tube. Centrifuge at 4°C and 1000 rpm for 5 minutes, discard the supernatant (approximately 50 μl should remain), and gently tap the bottom of the centrifuge tube to separate the cells. Add the dispersed cell suspension to 1 ml of pre-chilled 70% ethanol, gently mix, and fix at 4°C for at least 4 hours. Remove the fixed cells, centrifuge at 4°C and 1000 rpm for 5 minutes, discard the supernatant, add 1 ml of pre-chilled PBS buffer to resuspend the cells, centrifuge again, discard the supernatant (approximately 50 μl of PBS buffer should remain), and gently tap the bottom of the tube to separate the cells. Prepare the dye according to the sample volume under light-protected conditions. Add 500 μl of propidium iodide staining solution to each sample tube, mix slowly with a pipette, incubate at 37°C in the dark for 30 min, and then perform flow cytometry analysis. Save the data for further processing. The final results are shown in Table 2.

[0071] Table 2. Effect of compound 4 on DNA content in HepG-2 cells

[0072] The effect of compound 4 on the cell cycle was assessed by measuring the DNA content at different stages of the cell cycle in treated HepG-2 cells. The results showed that the treated HepG-2 cells exhibited a significant increase in DNA content during the G0-G1 phase, altering the distribution of HepG-2 cells across different cell cycle stages. The percentage of cells in the G0-G1 phase increased from 50.97% to 68.26%, the percentage in the S phase decreased from 36.24% to 25.52%, and the percentage in the G2 / M phase decreased from 12.79% to 6.22%. The change in the cell cycle distribution index from 0.96 to 0.46 further clarified the effect of compound 4 on the HepG-2 cell cycle. This indicates that compound 4 can arrest HepG-2 cells in the G0-G1 phase and inhibit cancer cell proliferation.

[0073] Example 4: Annexin V / PI double staining method for analyzing cell apoptosis First, cell collection was performed. Suspended cells were collected directly into 10ml centrifuge tubes, with each sample containing 1×10⁶ cells. 4Centrifuge at 500-1000 rpm for 5 min, discarding the culture medium. Wash once with incubation buffer and centrifuge at 500-1000 rpm for 5 min. Resuspend cells in 100 μl of labeling solution (a mixture of 10 μl Annexin V-FITC stock solution (concentration 1 mg / mL) and 90 μl 1× Annexin V binding buffer. The 1× Annexin V binding buffer consists of 10 mmol / L HEPES (pH 7.4), 140 mmol / L NaCl, and 5 mmol / L CaCl2) and incubate at room temperature in the dark for 10-15 min. Centrifuge the incubated cells at 500-1000 rpm for 5 min, and wash the cell pellet once with incubation buffer. Add fluorescent (SA-FLOUS) solution to the cell pellet and incubate at 4°C for 20 min, in the dark, with occasional shaking. Cell viability was then analyzed using flow cytometry. The excitation wavelength of the flow cytometer was 488 nm, and FITC fluorescence was detected using a passband filter with a wavelength of 515 nm. PI was detected using another filter with a wavelength greater than 560 nm.

[0074] Results Interpretation: Apoptotic cells were resistant to all dyes used for cell viability assessment, such as PI, while necrotic cells were not. Cells with damaged cell membranes showed red fluorescence due to PI staining, while cells with intact cell membranes did not. Therefore, PI does not stain in the early stages of apoptosis, hence no red fluorescence signal. Normal living cells behave similarly. In the bivariate flow cytometry scatter plot, the lower left quadrant shows living cells (FITC- / PI-); the upper right quadrant shows non-living cells, i.e., necrotic cells (FITC+ / PI+); and the lower right quadrant shows apoptotic cells (FITC+ / PI-). The experimental study on compound 4-induced apoptosis in HepG-2 cells was evaluated, and the results are shown in Table 3.

[0075] Table 3. Induction of apoptosis and necrosis in HepG-2 cells by compound 4

[0076] The data in the table demonstrate the induction of apoptosis and necrosis in HepG-2 cells by compound 4. Untreated HepG-2 cells exhibited early and late apoptosis rates of 0.81% and 0.23%, respectively. However, the apoptosis rate of HepG-2 cells treated with compound 4 significantly increased, with early apoptosis at 18.94% and late apoptosis at 12.65%. This indicates that compound 4 has a significant effect on promoting programmed cell death. Furthermore, compound 4 also affects cell necrosis; its addition led to a more than double increase in the percentage of necrosis in HepG-2 cells, reaching 2.23%. The survival rate of HepG-2 cells decreased after treatment, from 97.85% to 66.18%. These experimental results demonstrate the effective pro-apoptotic effect of compound 4 on HepG-2 cells, showcasing its potential as a targeted therapeutic agent.

[0077] Example 5: Molecular docking The target compound was docked to the active sites of EGFR and HER2 using Auto Dock software, and binding energy data were obtained. Image processing was performed using PyMOL software. Molecular docking studies were conducted to evaluate the binding affinity and hydrogen bonding interactions between the synthesized compound and EGFR and HER2. Results are as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown in Table 4.

[0078] Table 4. Molecular docking results of the target compound, Sorafenib, and TAK-285 with EGFR and HER2.

[0079] Using sorafenib, a tyrosine kinase inhibitor and a first-line treatment for hepatocellular carcinoma, and TAK-285, a dual EGFR / HER2 inhibitor, as reference standards, molecular docking analysis with the target protein revealed that the compounds exhibited high binding affinity to the target protein. Compound 4 showed the best anticancer activity, demonstrating a more significant therapeutic effect than both sorafenib and TAK-285. This demonstrates the potential of hydrazone-based EGFR / HER2 inhibitors as multi-target inhibitors.

[0080] Example 6: Inhibitory effect of compounds 4-6 provided by the present invention on the activity of EGFR and HER2 tyrosine kinases. The inhibitory effects of the target compound, erlotinib, and lapatinib on tyrosine kinases were evaluated using specific human ELISA (enzyme-linked immunosorbent assay) to measure EGFR and HER2. The results of the in vitro tyrosine kinase inhibition experiments are as follows: Figure 11 As shown in Table 5.

[0081] Table 5. Inhibitory effects of compounds 4-6 on EGFR and HER2 tyrosine kinase activity

[0082] *IC 50 ( μ The M value is the mean ± standard deviation of the three measurements.

[0083] The anticancer potential of hydrazone compounds was evaluated by testing the inhibitory activities of compounds 4-6 on tyrosine kinases EGFR and HER2. Table 5 shows that compound 4 exhibited stronger inhibitory effects on both enzymes than compounds 5 and 6. On the one hand, the inhibitory activities of compounds 5 (0.159±0.008) and 6 (0.265±0.014) on EGFR were lower than those of the reference compound Erlotinib (0.09±0.004). On the other hand, for HER2, compound 5 showed higher inhibitory activity than Lapatinib, while compound 6 showed lower inhibitory activity. Compound 4 demonstrated excellent inhibitory effects on both EGFR and HER2 tyrosine kinases, superior to the reference compounds Erlotinib and Lapatinib. In conclusion, compound 4 exhibits excellent dual inhibitory activity against both EGFR and HER2.

[0084] Example 7: Expression of relevant apoptosis factors The effects of compound 4 on gene expression alterations were evaluated using methods described in the Supplemental Information for cysteine ​​aspartate protease-3 (Caspase-3), BAX, and Bcl-2. Human Bax ELISA (EIA-4487), Bcl-2 ELISA kit, and human Caspase-3 (active) ELISA kit were employed. The experimental results are shown in Table 6.

[0085] Table 6. Gene expression levels of some apoptosis markers in HepG-2 cells treated with compound 4

[0086] Apoptosis is regulated by multiple proteins. Caspase-3 protein promotes apoptosis, leading to irreversible cell death. The ratio between apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) determines the incidence of apoptosis. As shown in the table, compound 4 induced a 5.98-fold increase in Caspase-3 expression, a 5.97-fold increase in Bax expression, and a 0.19-fold decrease in Bcl-2 expression. Staurosporine showed a significantly stronger induction of Caspase-3 than compound 4, with a similar effect on Bax expression, and a 0.21-fold decrease in Bcl-2 expression. These results indicate that both compound 4 and staurosporine induce apoptosis in HepG-2 cells by activating Caspase-3 and upregulating Bax, while downregulating Bcl-2. Although staurosporine showed higher Caspase-3 expression, compound 4 demonstrated a certain efficacy in promoting apoptosis.

[0087] Example 8: Relevant structural information of the compound The absolute configurations of seven hydrazone compounds were determined by single-crystal X-ray diffraction, and their crystallographic parameters were obtained. The seven hydrazone compounds include compound 1 (C... 16 H 14 F2N2), compound 2 (C 16 H 14 Cl2N2), compound 3 (C 16 H 14 Br2N2), compound 4 (C 16 H 14 I2N2), compound 5 (C 16 H 20 N2O4), compound 6 (C 18 H 20 N2O2) and compound 7 (C 14 H 10 Cl2N2). The zero-dimensional structures of compounds 1-7 are as follows: Figure 12 As shown in (a)-(g).

[0088] Compound 1 belongs to the monoclinic crystal system, space group 1. P twenty one / n Cell parameters a =11.7001(5)±10 Å, b =7.3306(2)±10 Å, c =15.3389(6)±10 Å, α =90°, β =96.218(4)±5°, γ=90°, cell volume is 1307.86(58)±40 Å 3 .

[0089] Compound 2 belongs to the monoclinic crystal system. P twenty one / c Space group. Cell parameters. a =15.0756(5)±10 Å, b =9.1432(3)±10 Å, c =10.3122(3)±10 Å, α =90°, β =93.734(3)±5°, γ =90°, cell volume is 1418.41(8)±40 Å 3 .

[0090] Compound 3 belongs to the orthorhombic crystal system, space group 3. Pbca Cell parameters a =9.3247(2)±10 Å, b =10.3535(3)±10 Å, c =30.3792(7)±10 Å, α =90°, β =90°, γ =90°, the cell volume is 2932.91(13)±40 Å 3 .

[0091] Compound 4 belongs to the orthorhombic crystal system, space group 4. Pcca Cell parameters a =34.556(2)±10 Å, b =7.2485(4)±10 Å, c =6.3260(3)±10 Å, α =90°, β =90°, γ =90°, cell volume is 1584.53(15)±40 Å 3 .

[0092] Compound 5 belongs to the monoclinic crystal system, space group 5. P twenty one / n Cell parameters a =7.8538(5)±10 Å, b =5.5180(3)±10 Å, c =17.1022(8)±10 Å, α =90°, β =97.251(5)±5°, γ=90°, cell volume is 735.24(7)±40 Å 3 .

[0093] Compound 6 is a monoclinic crystal system. P twenty one / n Space group, cell parameters a =6.2644(4)±10 Å, b =7.2188(5)±10 Å, c =16.8958(11)±10 Å, α =90°, β =96.87(1)±5°, γ =90°, cell volume is 758.56(9)±40Å 3 .

[0094] Compound 7 belongs to the monoclinic crystal system, space group 7. P twenty one / c Cell parameters a =3.8553(2)±10 Å, b =6.9822(3)±10 Å, c =22.8906(10)±10 Å, α =90°, β =90.908(4)±5°, γ =90°, the volume of the unit cell is 616.10(5)±40 Å. 3 .

[0095] The relevant information on hydrogen bonds and CX···π (X=H / Cl / Br) stacking interactions in compounds 1-7 is shown in Tables 7 and 8 below: Table 7. Hydrogen bond information in compounds 1-7

[0096] Symmetry codes: a x, y, 1+z for 1; a 1 / 2 - x, 1 / 2 + y, 1 / 2 - z; b 1-x, 2-y, -z; c 1 / 2 + x, 3 / 2 - y, -1 / 2 + z; d 1 + x, y, z for 5. Table 8. Information on CX···π (X = H / Cl / Br) stacking interactions in compounds 1-7

[0097] Symmetry codes: a 1-x, -y, 1-z; b 3 / 2 - x, 1 / 2 + y, 1 / 2 - z; c 1-x, 1-y, 1-z; d 3 / 2 - x, -1 / 2 + y, 3 / 2 - z for 1; a 1-x, 1-y, 1-z; b x, 1 / 2 - y, 1 / 2 + z; c 2-x, 1-y, 1-z for 2; a 3 / 2 - x, -1 / 2 + y, z; b 1-x, -1 / 2+y, 1 / 2-z for 3; a x, 1-y, 1 / 2+z; b x, 2-y, -1 / 2+z for 4; a 1 / 2 - x, -1 / 2 + y, 1 / 2 - z; b -1 / 2 - x, 1 / 2 + y, 1 / 2 - z for 5; a 1 / 2 - x, 1 / 2 + y, 1 / 2 - z; b -1 / 2-x, -1 / 2+y, 1 / 2-z for 6. Cg2=C9-C10-C11-C12-C13-C14; Cg1=C1-C2-C3-C4-C5-C6 for 1; Cg1=C1-C2-C3-C4-C5-C6; Cg2=C11-C12-C13-C14-C15-C16 for 2 and 3; Cg1= C1-C2-C3-C4-C5-C6 for 4, 5 and 6.

[0098] Example 9: Ultraviolet Spectroscopic Analysis of Compounds To further evaluate the structure of hydrazone compounds, ultraviolet-visible spectroscopy was performed in the 200-500 nm range using anhydrous ethanol as the solvent. Figure 13 As can be seen from the data, the UV spectra of compounds 1-7 show characteristic absorption peaks at 219 nm, 237 nm, 238 nm, 296 nm, 236 nm, 303 nm, and 307 nm, respectively, which can be attributed to π-π *Electronic transitions. The order of the absorption bands is: 1 < 5 < 2 < 3 < 4 < 6, which can be attributed to the difference in electronegativity of the substituents bonded to the aromatic ring. Due to the influence of the methyl group, the conjugation effect of the entire molecule is reduced, resulting in a large difference in the maximum absorption peaks of compounds 2 and 7. UV spectroscopy results show that the size of the absorption peak is inversely proportional to the electronegativity of the substituent.

[0099] Example 10: Infrared Spectroscopic Analysis of Compounds like Figure 14 , Figure 15 and Figure 16 As shown, the stretching vibration band of C=C / N can be observed in the FT-IR spectrum, specifically in the range of 1632-1479 cm⁻¹. -1 1644-1487cm -1 1637-1483cm -1 1645-1480cm -1 1649-1440cm -1 1657-1455cm -1 and 1653-1485cm -1 Typically, the strong band ranges from 1250 to 950 cm. -1 This indicates the presence of an aromatic bond between carbon and halogen. Located at 1202 cm⁻¹. -1 717cm -1 560cm -1 and 560cm -1 The strong peak values ​​are characteristic vibrations of the CX bonds in compounds 1-4 (where X represents F, Cl, Br, and I atoms). In compound 5, a peak value of 3417 cm⁻¹ was observed. -1 The peak at 1173 cm⁻¹ is attributed to the stretching vibration of the hydroxyl group. In compound 6, it is located at 1173 cm⁻¹. -1 The peak value should be attributed to the stretching vibration of COC. The subtle differences in the spectra of compounds 2 and 7 can be attributed to the effect of the methyl group. (979-676 cm⁻¹) -1 The peak positions can be attributed to the in-plane and out-of-plane bending vibrations of aromatic -CH. The current results can be further confirmed by single-crystal X-ray diffraction data.

[0100] Example 11: DSC analysis of the compound Differential scanning calorimetry (DSC) can obtain the heat capacity at high temperatures in a short time. The experiment was conducted under a nitrogen atmosphere with a heating rate of 10 °C / min. Figure 17The DSC curves show that compounds 1-4 exhibit endothermic peaks at 130℃, 153℃, 165℃, and 209℃, respectively, indicating the beginning of melting. The endothermic peak values ​​follow a pattern of 1 < 2 < 3 < 4, which corresponds well to the decreasing order of their total interaction energies. For compound 5, a narrow endothermic band was observed at 223℃, which can be attributed to the melting point of the organic structure of the sample, and its value is the largest among all compounds. The peaks of compounds 6 and 7 are located at 200℃ and 212℃, respectively, indicating the melting points of the samples.

[0101] Applying the technical solution of this invention, the crystal configuration, differential thermal analysis (DSC), infrared absorption spectrum, and ultraviolet absorption spectrum of the sample are consistent with the structural characteristics of hydrazone compounds, and the elemental analysis results are consistent with the structural composition of hydrazone compounds.

[0102] Studies have revealed that EGFR and HER2 are the targets of the hydrazone compounds provided in this invention. Molecular docking results show that the compounds have high binding affinity to the target proteins, and EGFR and HER2 possess "pocket" structures that bind to the target compounds. In vitro pharmacological experiments show that the EGFR / HER2 inhibitors based on hydrazone structures (i.e., the hydrazone compounds provided in this invention) can effectively exert antitumor effects in colorectal cancer, breast cancer, and hepatoblastoma cells, with compound 4 showing superior anticancer efficacy.

Claims

1. A hydrazone derivative, characterized in that, It has the chemical structural formula shown in Formula A: Formula A Where R is a methyl or hydrogen group; X is a halogen, hydroxyl or methoxy group.

2. The hydrazone derivative as described in claim 1, characterized in that, The halogen is fluorine, chlorine, bromine or iodine.

3. The hydrazone derivative as described in claim 1, characterized in that, The hydrazone derivatives include compound 1: C 16 H 14 F2N2, Compound 2: C 16 H 14 Cl2N2, Compound 3: C 16 H 14 Br2N2, Compound 4: C 16 H 14 I₂N₂, Compound 5: C 16 H 20 N2O4, Compound 6: C 18 H 20 N2O2 or compound 7 C 14 H 10 Cl2N2; The crystallographic parameters of compound 1 are: monoclinic system, space group 1. P twenty one / n The unit cell parameters are a =11.7001(5)±10Å, b =7.3306(2)±10 Å, c =15.3389(6)±10 Å, α =90°, β =96.218(4)±5°, γ =90°, cell volume is 1307.86(58)±40 Å 3 ; The crystallographic parameters of compound 2 are: monoclinic system. P twenty one / c Space group, cell parameters are a =15.0756(5)±10 Å, b =9.1432(3)±10 Å, c =10.3122(3)±10 Å, α =90°, β =93.734(3)±5°, γ =90°, cell volume is 1418.41(8)±40 Å 3 ; The crystallographic parameters of compound 3 are: orthorhombic crystal system, space group 1. Pbca The unit cell parameters are a =9.3247(2)±10Å, b =10.3535(3)±10 Å, c =30.3792(7)±10 Å, α =90°, β =90°, γ =90°, the cell volume is 2932.91(13)±40 Å 3 ; The crystallographic parameters of compound 4 are: orthorhombic crystal system, space group 4. Pcca The unit cell parameters are a =34.556(2)±10Å, b =7.2485(4)±10 Å, c =6.3260(3)±10 Å, α =90°, β =90°, γ =90°, cell volume is 1584.53(15)±40 Å 3 ; The crystallographic parameters of compound 5 are: monoclinic system, space group 1. P twenty one / n The unit cell parameters are a =7.8538(5)±10Å, b =5.5180(3)±10 Å, c =17.1022(8)±10 Å, α =90°, β =97.251(5)±5°, γ =90°, cell volume is 735.24(7)±40 Å 3 ; The crystallographic parameters of compound 6 are: monoclinic system, space group 6. P twenty one / n The unit cell parameters are a =6.2644(4)±10 Å, b =7.2188(5)±10 Å, c =16.8958(11)±10 Å, α =90°, β =96.87(1)±5°, γ =90°, cell volume is 758.56(9)±40 Å 3 ; The crystallographic parameters of compound 7 are: monoclinic system, space group 1. P twenty one / c The unit cell parameters are a =3.8553(2)±10 Å, b =6.9822(3)±10 Å, c =22.8906(10)±10 Å, α =90°, β =90.908(4)±5°, γ =90°, the volume of the unit cell is 616.10(5)±40 Å. 3 .

4. The method for preparing the hydrazone derivative according to any one of claims 1-3, characterized in that, Includes the following steps: In the presence of an acid catalyst, a para-substituted acetophenone compound or a para-substituted benzaldehyde compound is reacted with hydrazine hydrate in an organic solvent to obtain the hydrazone derivatives.

5. The preparation method according to claim 4, characterized in that, In the preparation method, the para-substituted acetophenone compound or the para-substituted benzaldehyde compound has the chemical structural formula shown in Formula B: Formula B Wherein, R is methyl or hydrogen; X is halogen, hydroxyl or methoxy; Preferably, in the preparation method, the para-substituted acetophenone compound includes p-fluoroacetophenone, p-chloroacetophenone, p-bromoacetophenone, p-iodoacetophenone, p-hydroxyacetophenone, or p-methoxyacetophenone; the para-substituted benzaldehyde compound includes p-chlorobenzaldehyde.

6. The preparation method according to claim 4, characterized in that, In the preparation method, the acid catalyst is acetic acid; Preferably, in the preparation method, the organic solvent is ethanol; Preferably, in the preparation method, the reaction conditions are 80-110℃ for 2-5 hours.

7. The preparation method according to claim 4, characterized in that, In the preparation method, after the reaction is completed, the filter residue is collected by filtration, and the filter residue is washed and dried to obtain the product; Preferably, washing is performed using ethanol at 0-35°C; Preferably, the product is recrystallized in ethanol to obtain crystals.

8. The use of the hydrazone derivatives according to any one of claims 1-3 or the hydrazone derivatives prepared by the method according to any one of claims 4-7 as EGFR and / or HER2 inhibitors in the preparation of pharmaceuticals.

9. The use of the hydrazone derivatives according to any one of claims 1-3 or the hydrazone derivatives prepared by the method according to any one of claims 4-7 in the preparation of antitumor drugs; Preferably, the hydrazone derivatives according to any one of claims 1-3 or the hydrazone derivatives prepared by the method according to any one of claims 4-7 are used as EGFR and / or HER2 inhibitors in the preparation of antitumor drugs; Preferably, the tumor includes tumors associated with EGFR and / or HER2 activation; Preferably, the tumor includes hepatoblastoma, breast cancer, or colorectal cancer.

10. A drug, characterized in that, Contains a hydrazone derivative as described in any one of claims 1-3 or a hydrazone derivative prepared by the method described in any one of claims 4-7; Preferably, the drug is an anti-tumor drug.

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