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[Ahmed Nabil](https://orcid.org/0000-0002-5617-4726), Marwa Abdel-Motaal, Ayman Hassan, Mohamed M. Elshemy, Medhat Asem, Mariam Elwan, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350), Mohammed Abdelmageed, Gamal Shiha, Hassan M. E. Azzazy

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[Anti-hepatocellular carcinoma activities of novel hydrazone derivatives <i>via</i> downregulation of interleukin-6](https://mdr.nims.go.jp/datasets/bb2abfe7-4de0-4583-bca8-77e392a95076)

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Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6RSC AdvancesPAPEROpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueAnti-hepatocelluaResearch Center for Macromolecules anMaterials Science (NIMS), Tsukuba, 305-00nims.go.jp; Tel: +201000618349bBiotechnology and Life Sciences DepartmeAdvanced Sciences (PSAS), Beni-Suef UnivercEgyptian Liver Research Institute and HospitdDepartment of Chemistry, College of Scienc51452, Saudi Arabia. E-mail: ma.mohammeeFaculty of Science, Menoua University, MefDepartment of Civil Engineering, College ofOnaizah Colleges, Qassim, Saudi ArabiagEgyptian Ministry of Health, El Mansoura,hGraduate School of Pure and Applied SciencTsukuba, Ibaraki, 305-8577, JapaniGraduate School of Industrial Science and T3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585jDepartment of Pharmacology and ToxicColleges, Qassim, Saudi ArabiakHot Laboratory Center, Atomic Energy AuthlHepatology and Gastroenterology Unit, InMedicine, Mansoura University, EgyptmDepartment of Chemistry, School of Sciencein Cairo, New Cairo, 11835, Egypt.+201000565727† Electronic supplementary information (FTIR spectral analysis of differentphysicochemical properties and speccompounds (Table S1), caspase 3 assay (FSee DOI: https://doi.org/10.1039/d4ra0585Cite this: RSC Adv., 2024, 14, 37960Received 12th August 2024Accepted 17th November 2024DOI: 10.1039/d4ra05854brsc.li/rsc-advances37960 | RSC Adv., 2024, 14, 37960–lar carcinoma activities of novelhydrazone derivatives via downregulation ofinterleukin-6†Ahmed Nabil, *abc Marwa Abdel-Motaal,*d Ayman Hassan,c Mohamed M. Elshemy,eMedhat Asem,f Mariam Elwan,g Mitsuhiro Ebara, ahi Mohammed Abdelmageed,jkGamal Shihacl and Hassan M. E. Azzazy *mHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related morbidity worldwide.Sorafenib is a first-line drug for the treatment of HCC, however, it is reported to cause serious adverseeffects and may lead to resistance in many patients. In this study, 20 hydrazone derivativesincorporating triazoles, pyrazolone, pyrrole, pyrrolidine, imidazoline, quinazoline, and oxadiazinemoieties were designed, synthesized, and characterized. In addition to molecular docking and in silicoADME study, the cytotoxic activity of the synthesized compounds was evaluated against the humanhepatocellular cancer cell line (HepG2) and liver mesenchymal stem cells as a normal cell line. Theantitumor activities of the derivatives against sorafenib were compared. Of the 20 synthesizedcompounds, compound 16 demonstrated potential as a potent anti-HCC drug candidate throughdownregulation of interleukin 6 which reduces inflammation and tumorigenesis with a strong bindinginteraction and bioavailability.d Biomaterials, National Institute for44, Japan. E-mail: TOLBA.AhmedNabil@nt, Faculty of Postgraduate Studies forsity, Beni-Suef, Egyptal (ELRIAH), Sherbin, El Mansoura, Egypte, Qassim University, Qassim, Buraydah,d@qu.edu.sa; Tel: +966569909737noua, EgyptEngineering and Information Technology,Dakahlia, Egyptes, University of Tsukuba, 1-1-1 Tennodai,echnology, Tokyo University of Science, 6-, Japanology, Faculty of Pharmacy, Buraydahority, Cairo, Egyptternal Medicine Department, Faculty ofs & Engineering, The American UniversityE-mail: hazzazy@aucegypt.edu; Tel:ESI) available: 1H NMR, 13C NMR, andhydrazone derivatives (Fig. S1),troscopy data of the synthesizedig. S2), and cell cycle analysis (Fig. S3).4b379741. IntroductionHCC is considered the sixth most identied neoplasm and thesecond most common cause of cancer-related death world-wide.1,2 It usually occurs in patients with liver cirrhosis.3 Themost recognized risk factors for HCC include chronic viralinfections of hepatitis B and C, autoimmune hepatitis, alcohol,aatoxin B1, non-alcoholic steatohepatitis, obesity, and dia-betes mellitus.4Sorafenib is a rst-line drug for patients with advanced HCC.It reduces tumor cell growth and progression through inhibi-tion of multiple serine/threonine kinases involved in tumorprogression and angiogenesis.5 These include the vascularendothelial growth factor receptor (VEGFR-2/3), platelet-derivedgrowth factor receptor (PDGF-R), Flt3, c-Kit, and Raf kinase.5,6Although sorafenib has demonstrated clinical advantages inpatients with HCC, it is reported to exert multiple side effectsprimarily caused by its suppression of kinases in healthy cells.These include diarrhea and dermatological effects such ashand-foot skin reactions, alopecia, stomatitis, and multiformeerythema.7 Other side effects include fatigue, hypertension,cardiovascular problems, hemorrhage, renal toxicity, pancrea-titis, mouth ulcers, and weight loss.8 Furthermore, resistance tosorafenib represents a major challenge in the treatment ofadvanced/recurrent HCC.9 Therefore, there is a need to developother agents to overcome the previous drawbacks.Interleukin 6 (IL-6) is a key molecule of the immuneresponse that is produced in response to infections and tissue© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://crossmark.crossref.org/dialog/?doi=10.1039/d4ra05854b&domain=pdf&date_stamp=2024-11-28http://orcid.org/0000-0002-5617-4726http://orcid.org/0000-0002-7906-0350http://orcid.org/0000-0003-2047-4222https://doi.org/10.1039/d4ra05854bhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bhttps://pubs.rsc.org/en/journals/journal/RAhttps://pubs.rsc.org/en/journals/journal/RA?issueid=RA014051Paper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinedamage.10 Elevated blood levels of IL-6, independent of otherrisk factors for HCC, have been associated with a higher risk ofdeveloping HCC.11 Furthermore, by promoting the repair andstimulation of countersignalling (antioxidant and anti-apoptotic/pro-survival) pathways, IL-6 protects tumor cellsagainst DNA damage, oxidative stress, and/or apoptosis causedby anticancer treatments. Therefore, a potential therapeuticapproach for cancer treatment could involve suppressing IL-6 orits signalling pathway alone or in conjunction with conven-tional anticancer strategies.12Hydrazone derivatives represent a class of compounds con-taining a biologically active pharmacophore (]N–NH–R) withvarious biological effects13–15 such as antibacterial,16–18 anti-inammatory,19 antimalarial,20 anticonvulsant,21 antidepres-sant, and antiproliferative22 activities. Different heterocycle-containing hydrazones have been reported to exert powerfulcytotoxic and antitumor actions.23,24 Importantly, variousclasses of hydrazones based on coumarin, triazoles, pyridine,indole, quinolone, caffeine, pyrimidine have been evaluated fortheir anticancer potential against various cell lines,25–27 asdepicted in Fig. 1. Additionally, hydrazone derivatives haveshown promising therapeutic potential for the treatment ofneurodegenerative diseases such as Alzheimer's disease.28Hydrazone links are also used as pH-responsive drug deliverysystems29 and in nifuroxazide, an antibiotic used for the treat-ment of colitis and dehydration. Consequently, these pharma-cophores were used as intermediates for the synthesis ofheterocycles with numerous biological activities.30–32Synthesis of broad-spectrum biologically active hydrazone-based heterocycles was reported.33 Some of these compoundsexhibited high in vitro anticancer activity against breast cancercell lines (MCF7). Therefore, in this study, 20 hydrazone deriv-atives were investigated using structure–activity relationships(SAR) and molecular docking to select those with better anti-cancer activity and higher selectivity. Furthermore, the cyto-toxicity of the hydrazone derivatives was assessed against thehuman hepatocellular carcinoma cell line (HepG2) and livermesenchymal stem cells using MTT assay and ow cytometry.Their effects on IL-6 and cytochrome C levels and tumor cellmigration were investigated.2. Materials and methods2.1 Synthesis and characterization of hydrazone derivativesAll chemicals, which are of the highest purity grade, wereacquired from Sigma-Aldrich Chemical Co. (St. Louis, MO,Fig. 1 Hydrazones based on heterocycles reported showing antitumor© 2024 The Author(s). Published by the Royal Society of ChemistryUSA). The synthesis of hydrazone derivatives was performed aspreviously reported.33 The melting points were measured usinga Gallenkamp electric melting point apparatus (Gemini Lab,Apeldoorn, The Netherlands), and the results were uncorrected.Using KBr discs and a FT-IR spectrophotometer, the IR spectraof different compounds were obtained and analyzed. 1H-NMRand 13C-NMR spectra were assessed in DMSO-d6 on a JNM-ECA500II at 500 MHz NMR spectrometer (JEOL Ltd., Peabody,MA) reported as d ppm and using tetramethyl silane (TMS) asinternal reference. With the Kratos MS apparatus (KratosAnalytical Ltd, Manchester, UK), mass spectra (EI) were recor-ded at 70 eV.2.2 Reagents and kitsDulbecco's modied Eagles culture medium (DMEM), fetalbovine serum (FBS), trypsin/EDTA 0.25%, MTT assay kit, trypanblue 0.4%, and penicillin–streptomycin were purchased from(Lonza, Belgium), while Kreps ringer bicarbonate buffer waspurchased from (sigma Aldrich, USA). Bovine serum albumin(BSA) and 1X phosphate buffer saline (PBS) were acquired from(Hyclone, USA). Sorafenib was purchased from (Bayer,Germany).The high-capacity cDNA reverse transcription kit, TRIzolreagent, and RT-PCR grade water were purchased from (ThermoFisher Scientic), and the Sso-Fast EvaGreen supermix was from(BIO-RAD). Propidium iodide was purchased from (MiltenyiBiotec), the cytochrome C ELISA kit was supplied by (Abcam,Cambridge, UK), and other chemicals and reagents used were ofthe highest purity grade.2.3 CytotoxicityThe HepG2 cell line was obtained from ATCC (Rockville, MD).Liver mesenchymal stem cells were harvested from liver biopsyaccording to previously published protocols.34 Cells were grownin DMEM media with 10% fetal bovine serum (FBS), 100 Uper mL penicillin, and 100 g per mL streptomycin at 37 °C, 5%CO2. The MTT assay kit, which relies on succinate dehydroge-nase in living cells' mitochondria to convert the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)dye to violet formazan crystals, was used to calculate the vitalityof individual cells. 6000 cells per well were seeded in the 96-wellplate containing, which were then incubated for 24 h. Differentconcentrations of drug were dissolved in DMEM and thenadded to the wells to replace the culture medium. The mediumwas discarded aer incubation for 24 h under the sameactivity.RSC Adv., 2024, 14, 37960–37974 | 37961http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bRSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineconditions, and 100 mL of MTT (2 mg mL−1) were added. Aer3 h of incubation at 37 °C, the generated formazan crystalsgenerated were dissolved in 50 mL of DMSO. The optical densitywas then measured using a Stat Fax ELISA plate reader (RomerLabs, Getzersdorf, Austria) at 570 nm with a reference wave-length of 630 nm aer incubating the plate at 37 °C for 15 min.As a positive control, the tyrosine kinase inhibitor sorafenib(Bayer, Germany) was employed. DMSO was used as a solventand its nal concentration was less than 0.2%. SI and IC50 werecalculated. Triplicates of each in vitro test were performed.332.4 Flow cytometry analysisCells were harvested, aliquoted up to 1 × 106 cells/100 mL inFACS tubes, washed twice with 2 mL of PBS, centrifuged at 300× g for ve minutes, and the buffer was decanted. Reconstitutedcells in 100 mL of staining buffer for ow cytometry. HepG2 cellswere stained with propidium iodide according to the techniqueoffered via the kit and subsequently performed on the cytom-eter. The distribution of the cell cycles was estimated andanalyzed using a FACScan TM device (BD, Franklin Lakes, NJ).Furthermore, the caspase-3 apoptotic marker levels wereinvestigated by ow cytometry. During the ow cytometry assay,we investigated a population of HepG2 cells to obtain enoughcells for statistically signicant detection.FITC active caspase-3 apoptosis kit (BD Biosciences, FranklinLakes, NJ, USA) was used for detection of active caspase 3 fol-lowed by ow cytometry analysis according to the instructionsof the kit manufacturer.2.5 IL-6 gene expressionAer two rounds of PBS washing, total RNA was extracted usingthe TRIzol Reagent, and cDNA synthesis was performed usingthe high-capacity cDNA reverse transcription kit in accordancewith the manufacturer's instructions. The qPCR reactionmixture contained so-fast EvaGreen supermix (10 mL), cDNA (2mL), invitrogen RT-PCR grade water (6 mL), and the primer pair(500 nM; forward primer 50-CAAATTCGGTACATCCTC-30, reverseprimer 50-CTGGCTTGTTCCTCACTA-30). The amplication star-ted with heating 10 min at 95 °C, followed by 40 cycles of 15 s at95 °C, 20 s at 55 °C and 30 s at 72 °C. The amplication data wasthen analyzed as previously described previously with normal-ization to b-actin.352.6 Cell migration assayThe wound healing assay was used to investigate the anti-cellmigration/antiproliferation effects of synthesized hydrazonederivatives on the HepG2 cell line. In DMEM medium supple-mented with 10% fetal bovine serum (FBS), 100 U per mLpenicillin, and 100 g per mL streptomycin, cells were cultured at37 °C and 5% CO2. Aer reaching 80% density, the monolayercells were scratched using a sterile tip and then washed withphosphate buffered saline, pH 7.4. Different hydrazone deriva-tives were applied to cells, which were then incubated for 24 h at37 °C and 5% CO2. Finally, an Olympus microscope (OlympusEuropa, Hamburg, Germany) was used to take pictures of the37962 | RSC Adv., 2024, 14, 37960–37974wound areas at 0 and 24 h. The relative cell migration wascalculated using the following equation:Relative migration = (width0h − width24h)/width0h2.7 Cytochrome C assayCytochrome C was quantied using a sandwich ELISA kit(Abcam, Cambridge, UK) according to the manufacturer'sprocedures. Aer the addition of the TMB substrate and thedevelopment of the color, the optical density was measuredusing a Stat fax microplate reader, USA, at 450 nm.2.8 Docking studiesMolecular Operating Environment (MOE) 2015.10 soware wasused to perform the docking studies. The 3D structures andconformations of the broblast growth factor receptor 4(FGFR4) complex with N-(3,5-dichloro-2-((5-((2,6-dichloro-3,5-dimethoxybenzyl)oxy)pyrimidin-2-yl)amino)phenyl)acrylamidewere downloaded from the PDB website (http://www.rcsb.org/;ID: 6NVG). The structures of compound 16 and the standarddrug, 5-uorouracil (5-FU), were drawn using Chem DrawUltra 16.0 (ChemOffice). Before docking, preparatory stepswere performed for the ligand (including protonation,partial charge, and energy minimization in the database)and protein (including elimination of water molecules andrepeating chains, addition of hydrogens, calculation of thepartial charges, and determination of the active site). Theperformance of the docking method was assessed byredocking the co-crystalline ligand in the detected activepocket site of FGFR4. The validation of the docking processwas affirmed by determining the scoring energy (lowerbinding energy), the root means standard deviation (RMSD)values, and detecting amino acid interactions for the best poseby free rotation of the rotatable bonds into the rigid receptorbinding site. The validation process was carried out byredocking the co-crystal within its binding pocket. A validperformance was veried when an RMSD value less than 2 Åwas obtained.2.9 In silico ADME simulationThe prediction of the ADME properties of the synthesizedcompounds was determined from https://www.swissadme.ch/online toolkit according to Lipinski's molecular rules.Lipinski's ‘Rule of Five’ was used to establish the druglikenessof the synthesized hydrazone derivatives compared to thereference drug 5-uorouracil.2.10 Statistical analysisData were expressed as mean± standard error (SE) of the mean.Statistical signicance was determined by one-way ANOVA andTukey post hoc test using data analyzed using SPSS sowareversion 22 (SPSS Inc., Chicago, IL, United States). A level of p <0.05 was dened as statistically signicant.© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://www.rcsb.org/https://www.swissadme.ch/onlinehttps://www.swissadme.ch/onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bPaper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online3. Results and discussion3.1 Synthesis of hydrazone derivativesDicyclopropyl ketone 1 was condensed with hydrazine hydrateto generate dicyclopropylmethylene hydrazone (2). Compound 2was used as the lead compound for the synthesis of 3–20derivatives through the synthetic routes depicted inSchemes 1–3.Treatment of hydrazone 2 with suitable aromatic aldehydes(such as p-anisaldehyde, p-nitrobenzaldehyde, furfural, andsalicylaldehyde) in reuxed ethanol furnished the correspond-ing Schiff bases 3a–c and the bisazene derivative 5. Similarly,condensation of hydrazone 2 with isatin (1H-indole-2,3-dione)in EtOH and reux with a few drops of glacial acetic acid (gl.AcOH) gave a high yield of 3-(dicyclopropylmethylene)hydra-zone indolin-2-one (4). Triazoles 6a,b, 7a,b, and 8a,b weregenerated by cycloaddition on the azine derivatives 3a, 4, and 5with thiosemicarbazide or benzohydrazide (Scheme 1).Also, synthesis of imidazolones 10a,b by treating hydrazone 2with oxazolones 9a,b in acetic acid under reux. Furthermore,the reaction of hydrazone 2 with several anhydrides such asphthalic, succinic, maleic anhydrides proceeded by the samemethodology and generated amido and pyrrole derivatives 14–Scheme 1 Synthetic route to obtain triazoles 3–8. Reproduced from re© 2024 The Author(s). Published by the Royal Society of Chemistry16, respectively. Although 3-((dicyclopropylmethylene) amino)-2-methylquinazolin-4(3H)-one (11) was achieved by reaction ofhydrazone 2 with an equimolar amount of 2-methylbenzox-azinone in boiling acetic anhydride. Heating of hydrazone (2)(dicyclopropylmethylene) with 2,3-epoxy-1,4-naphthoquinone 12in CH3CN produced naphthoxadiazine-5,6-dione 13 (Scheme 2).Additionally, a mixture of cyano acetohydrazone and pyr-idazinone derivatives 17 and 18 was acquired by condensationof 1 with cyano acetic acid hydrazide in boiling ethanol and thetwo products were easily separated. Finally, dicyclopropylketone was treated with the thiosemicarbazide derivative 19 inreuxing ethyl alcohol, providing the corresponding quinazo-line derivative 20 (Scheme 3).The structures of these targets were identied and conrmedbased on IR, NMR, and MS spectral and analytical data aspreviously reported.33 FTIR, 1H-NMR, 13C-NMR, and MS spectraof most derivatives are provided in ESI (Fig. S1 and Table S1†).3.2. Cytotoxicity assayThe cytotoxic effects of the synthetic hydrazone derivatives werecompared to those of sorafenib using the HepG2 cell line usingtheMTT assay. Themean IC50 values ranged from 23.6 to 94.7 mMf. 33. Copyright (2019), Deuton-X Ltd.RSC Adv., 2024, 14, 37960–37974 | 37963http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bScheme 2 Synthesis of imidazole, cyclic imides, fused diazine, and oxadiazine targets. Reproduced from ref. 33. Copyright (2019), Deuton-X Ltd.Scheme 3 Condensation of dicyclopropyl ketone 1 with hydrazide,and thiosemicarbazide derivatives. Reproduced from ref. 33. Copyright(2019), Deuton-X Ltd.Table 1 IC50 and SI calculations for sorafenib and the preparedhydrazone derivativesa,bMTT assay IC50 24 h (mM)SICompounds HepG2 Liver mesenchymal stem cellsSorafenib 25.6 100 3.92 94.7 >100 1.583a 48.2 >100 —3b >100 90.2 —4 53.7 100 1.865 75.8 >100 —7a >100 >100 —7b >100 100 —8a 62.2 100 1.68b >100 75.5 —11 >100 >100 —13 61.4 85.2 1.3816 23.6 76.5 3.218 89.5 100 1.1119 >100 >100 —20 48.6 61.5 1.26a IC50: drug concentration that inhibits cell growth by 50%. b SI:calculated by dividing the IC50 value against liver mesenchymal stemRSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineand the SI values are presented in Table 1. The results demon-strated that sorafenib and compounds 2, 4, 8a, 13, 16, 18, and 20had excellent cytotoxic action against HepG2. Compound 16 hadIC50 and SI values similar to those of sorafenib, indicating a strongpotency and selectivity for activated HepG2 cell lines with littlecytotoxicity and the potential to be a successful anticancer agent.cells for each compound by the IC50 value of that compound againstthe cancer cell line HepG2.3.3 Cell migration assayThe ability of sorafenib and the synthesized hydrazone deriva-tives to attenuate the migration of HepG2 cells was evaluatedusing the wound healing assay. Cell migration increased aer24 h for untreated cells (control) but was substantially reducedin the presence of sorafenib and hydrazone derivativecompounds 2, 4, 8a, 13, 16, 18, and 20 (Fig. 2). These resultssuggest that compounds 2, 4, 8a, 13, 16, 18, and 20 delay wound37964 | RSC Adv., 2024, 14, 37960–37974closure and signicantly inhibit migration/invasion of HepG2cells similar to or better than sorafenib. Quantied relativemigration calculations showed that compounds 16, 13, and 20had a signicant antimigration and antiproliferative effects onHepG2 cells compared to the commercially available anticancerdrug sorafenib.© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bFig. 2 In vitro wound healing assay of HepG2 cells treated with sor-afenib and synthesized hydrazone derivatives (A) cell morphologybefore and after drug screening, (B) quantified relative migrationsummarized as a bar graph. Data are mean ±SD (n = 3). (a) significantwith DMEM Medium, (b) significant with sorafenib, (c) significant with16, (d) significant with 13, (e) significant with 20, (f) significant with 8a,(g) significant with 4, (h) significant with 18. Cell migration wasdramatically suppressed after treatment with compounds 2, 4, 8a, 13,16, 18, and 20 for 24 h. Wound healing assay of HepG2 cells treatedwith treatment with sorafenib and hydrazone derivatives. Compared tothe commercially available anticancer drug, sorafenib, the datashowed that compounds 16, 13, and 20 had a significant antimigrationeffect.Paper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online3.4 Apoptosis and cell cycle analysesThe effects of treatment with sorafenib and hydrazone deriva-tives on HepG2 cells were investigated. Apoptotic and necrotic© 2024 The Author(s). Published by the Royal Society of Chemistryeffects of the tested compounds on HepG2 cells were evaluatedusing caspase 3 assay kit which employs an FITC-antibody re-ported to specically recognize the active form of caspase 3 (notits proenzyme). On the other hand, cell cycle arrests at differentstages, caused by treatment with hydrazone derivatives wereassessed using propidium iodide staining (Fig. 3, ESI Fig. S2and S3†).Caspase 3 is synthesized as a proenzyme that is cleaved byother proteases into small and large subunits which thenassociate to form the active enzyme. Active caspase 3 proteo-lytically cleaves other caspases and cellular targets leading toexecution of apoptosis. Caspase 3 ow cytometry analysis ofHepG2 cells treated with the synthesized hydrazone derivativesor sorafenib was conducted and the results showed that,compared to the control group, the percentage of apoptotic cellsincreased signicantly aer treatment with sorafenib and thederivatives of hydrazone (2, 4, 8a, 13, 16, 18, and 20). Treatmentwith compound 16 showed a higher increase in the percentageof apoptotic cells (80%) than sorafenib (75%) (Fig. 3A and B).This explains the key effect, of the synthesized hydrazonederivatives on caspase-3 activation that leads to cellularapoptosis upregulation that supports the anticancer potentialof the synthesized compounds especially compound 16.Additionally, the anticancer potential of the most potenthydrazone derivatives and sorafenib on the cell cycle progres-sion, crucial for proliferation of cancer cells, was determined.Flow cytometry cell cycle analysis (Fig. 3C and D) was used toinvestigate the effect of compound 16 on HepG2 cell cyclestained with propidium iodide. Approximately 79.38% ofuntreated HepG2 cells were in the G0/G1 phase, 6.23% in the S-phase, and 3.5% in the G2/M phase. For cells treated with sor-afenib, a positive control, 74.93% of cells were in the G0/G1phase, 4.17% in S-phase, and 10.68% in G2/M. The mostpotent anticancer hydrazone derivative, compound 16, reducedthe number of cells in S-phase to 4.5% while 2.5% of the cellpopulation was in the G2/M phase which suggest a signicantinhibitory effect of compound 16 on the HepG2 cells. Thesedata conrm that compound 16 has a greater impact on HepG2cell cycle when compared to sorafenib. This cellular prolifera-tion inhibitory effect of the synthesized hydrazones, especiallycompound 16, could enable their application as anticanceragents following further assessments. Additional ow cytometrygraphs illustrating the effects of different treatments on caspase3 levels and cell cycle of HepG2 cells can be found in ESI.†3.5 Effects of sorafenib and compound 16 treatment on IL-6gene expression and cytochrome C concentrationIL-6 plays a key role in the regulation, inammation, andoncogenesis of the immune system.36 When IL-6 binds to itsreceptor, glycoprotein 130, JAK phosphorylates the receptor,activating the JAK/STAT3 signalling pathway. Through thereduction of oxidative damage and inhibition of the apoptoticcascade, these pathways play a crucial role in liver regenera-tion.37 However, continuing activation of the IL-6 signallingpathway is detrimental to the liver and can eventually lead toHCC progression or recurrence.12 High serum levels of IL-6 haveRSC Adv., 2024, 14, 37960–37974 | 37965http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bFig. 3 Effects of the selected derivatives of hydrazones on HepG2 cell apoptosis, necrosis, and cell cycle. The treated cells were stained usingpropidium iodide cell cycle staining and caspase 3 and analyzed by flow cytometry to determine percentages of necrotic and apoptotic cellscharacterizing cells in different cell cycle stages. (A) Caspase 3 flow cytometry histograms for all derivatives of hydrazone compared to Sorafinib.(B) The quantified percentages are summarized as a bar graph. Data are mean±SD (n = 3). (a) significant with DMEMMedium, (b) significant withSorafenib, (c) significant with 2, (d) significant with 4, (e) significant with 8a, (f) significant with 13, (g) significant with 16, (h) significant with 18.Treatment with sorafenib and hydrazone derivatives (compounds 2, 4, 8a, 13, 16, 18, and 20) significantly increased the apoptotic percentage ofHepG2 cells compared to the control group. Compound 16 caused a higher percentage of apoptosis compared to sorafenib and the otherderivatives. (C) Histograms showing different cell cycle stages for compound 16 compared to sorafenib and media. (D) The quantifiedpercentages are summarized as a bar graph.RSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinebeen suggested as a tumor marker of HCC. Furthermore, IL-6has been reported to protect cancer cells against DNAdamage, oxidative stress, and apoptosis caused by cancertherapy.38 Inhibition of IL-6 was proposed as a possible thera-peutic strategy to make HCC cells more susceptible to sor-afenib.39 Because the JAK/STAT3 cascade is critical for thedevelopment of HCC, and various intracellular signallingpathways are activated by the IL-6 cytokine, a potential thera-peutic approach for the treatment of malignancies could involveblocking IL-6 or interfering with its signalling pathways alone orin conjunction with traditional anticancer medicines. Cellulardeath is signicantly inuenced by cytochrome C.40 Theexpression of the mitochondrial respiratory chain proteinscytochrome C, cytochrome C oxidase subunits I and IV, and themanganese superoxide dismutase, which scavenges free37966 | RSC Adv., 2024, 14, 37960–37974radicals, increases in response to chemotherapy. An apoptoticsignal is produced when Bax encourages the release of cyto-chrome C from mitochondria, which in turn triggers the cas-pase 9 apoptotic pathway. Caspase 9 activates downstreamcaspases, including caspase 3, which execute apoptosis and celldeath.12,41 Cytochrome C is released from injured mitochondriainto plasma aer induction of apoptosis and its serum levelscould be used to assess the effect of chemotherapy. In thisstudy, the administration of sorafenib and compound 16caused a signicant (P < 0.001) decrease (P < 0.001) in theexpression of IL-6 (Fig. 4A) and a signicant increase in cyto-chrome C concentration (Fig. 4B) compared to the controlgroup. Treatment with compound 16 showed a signicantdecrease in the level of IL-6 level and an increase in cytochromeC greater than sorafenib. Therefore, the proposed anticancer© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bFig. 4 Sorafenib and compound 16 effects on (A) IL-6mRNA expression (RT-PCR) and (B) cytochromeC concentration (ELISA). All samples weremeasured separately in duplicate, and the data were provided as mean standard deviation. *Statistical significance compared to the controlgroup (p < 0.001); # statistical significance compared to the sorafenib group (p < 0.001). Treatment with compound 16 showed a significantdecrease in the level of IL-6 and an increase in cytochrome C greater than sorafenib. Therefore, the proposed anticancer mechanism ofcompound 16 may include a marked reduction in the expression of the IL-6 gene.Paper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinemechanism of compound 16 may include a marked reductionin IL-6 gene expression that causes suppression of the JAK/STAT3 pathway and ultimately decreases inammation andtumorigenesis, as shown in Fig. 5.3.6. Molecular docking studiesThe broblast growth factor receptor (FGFR) family of receptortyrosine kinases consists of 4 members (FGRF 1–4) encoded bydifferent genes (50–70% sequence homology). FGFRs are key toactivation of mitogen-activated protein kinase; phosphatidyli-nositol 3-kinase, phospholipase Cg, and signal transducer andFig. 5 Proposed mechanism by which compound 16 may exert its antic© 2024 The Author(s). Published by the Royal Society of Chemistryactivator of transcription (STAT) which lead to activation oftarget genes responsible for cellular proliferation, metastasis,angiogenesis, and development of HCC.42 The broblast growthfactor receptor 4 (FGFR4) is involved in cell proliferation,differentiation, and migration, and its abnormal signalling waslinked to development and progression of HCC. Similar to othertyrosine kinases, FGFR consists of an extracellular receptordomain and a transmembrane helix connected to a cytoplasmickinase domain.36,43–45 Therefore, compound 16 and a standarddrug, 5-uorouracil, were docked to the active site of the FGFR4kinase domain (PDB ID: 6NVG) to predict their ability to inhibitthe kinase function using Molecular Operating Environmentancer effect.RSC Adv., 2024, 14, 37960–37974 | 37967http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bRSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online2015.10 (MOE) soware to analyze all docking poses andbinding energies (Fig. 6 and 7). RMSD was employed to measurethe overall stability of the protein and ligand in relation to theinitial protein backbone structure. Therefore, validation of thedocking protocol indicates that the ligand is conrmed by theactive site pocket with a score = 4.6292 kcal mol−1 and rsmd =1.1392 Å. Compound 16 presented better binding interactionscompared to 5-uorouracil (score = −3.8501 kcal mol−1, rsmd =1.2706 Å). The imino and carbonyl groups of compound 16showed hydrogen bond interactions with His 704 and Arg 701amino acids. On the other hand, when the same pose wasdocked with a different pocket on the same protein, compound16 also formed a hydrogen bond with Lys 503. These interac-tions suggest a strong polar connection between compound 16and the binding pocket of the protein, potentially stabilizing thecomplex (Table 2 and Fig. 7). In addition to hydrogen bonds,hydrophobic interactions (typically enable the molecule to tmore tightly into the binding site and increase its affinity for theprotein) are achieved by the orientation of compound 16 withinthe protein pocket which contribute to the binding stability ofcompound 16. The ability of compound 16 to display betterbinding interactions to EGFR4 as compared to 5-FU, a commonchemotherapeutic drug used for treatment of several cancers,supports its potential as an effective anticancer agent.3.7 In silico ADME predictionsThe preferable oral bioavailability of the compounds examinedwas conrmed by in silico ADME predictions (log P < 5, Mw <Fig. 6 2D and 3D interaction analysis of structural redocked with 5-fluoTable 2 Ligand interactions report for compound 16 and 5-fluoruracilLigand ReceptorCompound 16 N 5 N HIS 704O 15 NH1 ARG 701O 5 N Lys 5035-Fluorouracil O 5 NE ARG 714N 6 NE ARG 714O 5 NE ARG 69837968 | RSC Adv., 2024, 14, 37960–37974500, HBA 10 and HBD <5). The number of values of rotatablebonds (n-ROTB) (which should be <10), indicated molecularexibility. The topological polar surface area (TPSA) revealedthat most of the calculated values of the target compoundsindicated good permeability. According to ADME results (Table3), the compounds tested did not violate Lipinski's rules, whichprovides positive values of drug similarity. Skin permeation (logKp; cm s−1) showed that the more negative the log Kp, the lessskin permeant for each compound.46 The Lipinski qualitativemodel (mostly known as rule of ve) is a widely used approachfor prediction of the likelihood of a small compound beingorally bioavailable. It is used in early drug development to guidecompound selection by using criteria such as molecular weight,lipophilicity (log P), hydrogen bond donors, and acceptors. Ithelps to streamline the prioritize selection of compounds morelikely to be absorbed in the gastrointestinal tract and conse-quently and potentially succeed in clinical trials. Several studieshave proven a strong correlation between its criteria and thepharmacokinetic properties of successful drugs. Further, itassists in evaluating ADME parameters for drug candidates andmolecules and offers insights that help address uncertaintiesearly in the drug discovery process. Therefore, the predicted oralbioavailability was summarized using the bioavailability radar,in which the pink zone represents the ideal ranges of differentcharacteristics (lipophilicity: XLOGP3 0.7 to +5.0, size 150–500 gmol−1, polarity: TPSA between 20 and 130, solubility: loga-rithmic S with maximum value 6, saturation: the fraction ofhybridized carbons sp3 with minimum value 0.25, androuracil with FGFR4 showing clear active site interactions.Interaction Distance (oA) E (kcal mol−1)H-acceptor 3.49 −1.0H-acceptor 2.96 −4.8H-acceptor 3.28 −0.7H-acceptor 2.99 −5.5H-acceptor 3.43 −1.7Ionic 3.80 −0.9© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bFig. 7 2D and 3D interaction analysis of 16 newly redocked compounds with two different pockets of FGFR4 showing significant active siteinteractions.Paper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineexibility: rotatable bonds with maximum value 9). Accordingly,all of the evaluated hydrazones exhibit the probability of beingorally active drug-like candidates. Compared to the referencedrug (5-uorouracil), the hydrazone derivatives tested showedgreater bioavailability (Table 4). These In silico ADME predic-tions are matched with the in vitro investigations that showedthat compared to the control group, the percentage of apoptoticTable 3 In silico drug-likeness ADME predictions according to the LipinCompounds Mw (g mol−1) log P0/w (MLO GP) TPSA (Å) n-ROTB4 253.31 2.03 53.82 38a 297.32 2.03 53.82 313 296.33 1.43 67.43 216 206.25 1.76 49.74 318 193.25 1.69 63.81 320 326.43 3.66 91.37 55-Fluorouracil 130.08 −0.42 66.24 0a Molecular weight (Mw) < 500, partition coefficient between n-octanol andof rotatable bonds (n-RB) <10, hydrogen-bond donor (HBD) <5, and hydro© 2024 The Author(s). Published by the Royal Society of Chemistrycells increased signicantly aer treatment with sorafenib andthe derivatives of hydrazone (2, 4, 8a, 13, 16, 18, and 20) and thetreatment with compound 16 showed a higher increase in thepercentage of cell apoptosis than sorafenib (Fig. 3A and B). Also,these computational studies matched with cell cycle analysis asfor cells treated with sorafenib, 74.93% of cells were in the G0/G1 phase, 4.17% in S-phase, and 10.68% in G2/M while theski ruleaHBD HBA Drug likeness/violationlog Kp(skin permeation) (cm s−1)1 3 Yes/0 −6.351 3 Yes/0 −6.352 3 Yes/0 −6.420 3 Yes/0 −7.382 1 Yes/0 −6.801 3 Yes/0 −6.742 5 Yes/0 −6.91water (log P) < 5.0, topological polar surface area (TPSA) < 140 Å:, numbergen-bond acceptors (HBA) <10.RSC Adv., 2024, 14, 37960–37974 | 37969http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bTable 4 Bioavailability radar of different hydrazone derivativesaa The pink area represents the optimal range for each properties as follows: lipophilicity (LIPO): XLOGP3 between 0.7 and + 5.0, Size:Mw from 150 to500 g mol−1; polarity (POLAR): TPSA between 20 and 130 2, insolubility (INSOLU): log S not greater than 6; saturation (INSATU): fraction ofhybridized carbons of sp3 not less than 0.25; and exibility (FLEX): no more than 9 rotatable bonds.RSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinemost potent anticancer hydrazone derivative, compound 16,reduced the number of cells in S-phase to 4.5% while 1.2% ofthe cell population was in the G2/M phase which suggesta signicant inhibitory effect of compound 16 on the HepG2cells (Fig. 3C and D). Moreover, treatment with compound 1637970 | RSC Adv., 2024, 14, 37960–37974showed a signicant decrease in the level of IL-6 level and anincrease in cytochrome C greater than sorafenib. Therefore, theproposed anticancer mechanism of compound 16 may includea marked reduction in IL-6 gene expression that causes© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bFig. 8 Chemical structure of target compounds with important sites for the structure–activity relationship (SAR).Paper RSC AdvancesOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinesuppression of the JAK/STAT3 pathway and ultimately decreasesinammation and tumorigenesis, as shown in Fig. 5.3.8. Structure–activity relationship (SAR)The synthesized compounds were characterized and evaluatedagainst anticancer activities that support the described SAR(Fig. 8). In a previous investigation of lead cyclopropyl hydra-zone 2 with its structural modications, the presence of thecyclopropyl ring47,48 and the hydrazone fragment49,50 was pre-dicted to exert broad cytotoxic activities against cancer cells. Tofurther enhance its potential as an anticancer agent, importantmodications such as the addition of N-heterocycles wereintroduced to the lead compound.As evidenced by the effects of the synthesized compounds inthe MTT assay, compounds 2, 4, 8a, 13, 16, 18, and 20 possessedthe highest cytotoxic activities against HepG2 cells. Althoughthe starting material, compound 2, with the basic structure ofcyclopropyl rings, hydrazone, and primary amine exertedmoderate cytotoxicity against HepG2 cells, it was found thatcyclization of the amino group to a heterocycle moiety orintroduction of functional group such as formation of cyanoa-cetamide fragment (compound 18) increased its potency andselectivity. This may be essential to achieve good bindinginteractions between the tested compounds and the receptor,possibly through the formation of a hydrogen bond.Similarly, it is suggested that the incorporation of indoline-2-one (compound 4), aminotriazole moiety and imino group(compound 8a) and oxadiazine-5,6-dione (compound 13) isresponsible for their biological signicance. Introducinga thiourea fragment substituted with a 4-oxoquinazoline moietyremarkably improved the cytotoxic activity. However,compound 16 with the 2,5-pyrrolidinone moiety50 showed anexcellent effect compared to sorafenib and the othercompounds tested, possibly due to the formation of hydrogenbonds with the receptor (due to the presence of an imide group).Treatment with compound 16 showed a signicant increase incytochrome C greater than that observed upon treatment withsorafenib. Chemotherapeutic agents are reported to signi-cantly increase cytochrome C release from the mitochondrialmatrix into the cytosol in addition to the other mitochondrialrespiratory chain proteins cytochrome C oxidase subunits I andIV, and manganous superoxide dismutase (which scavengesfree radicals). The released cytochrome C indirectly activates© 2024 The Author(s). Published by the Royal Society of Chemistrycaspase-9 and caspase-3 which ultimately execute apoptosis.51–53This study showed that treatment of HepG2 cells withcompound 16 led to high increase in activation of cytochrome Cand activated caspase 3 which represent signicant markers ofapoptosis. This supports the potential role of the synthesizedhydrazone derivatives, especially compound 16, as anticanceragents mediated by their ability to trigger cellular apoptosis.To the best of our knowledge, this report is the rst toinvestigate the potential of the newly synthesized hydrazonederivatives for hepatocellular carcinoma treatment. However,further validations such as assessing the effect of hydrazonederivatives, namely compound 16, on the proteins involved incell proliferation, apoptosis, JAK/STAT3 pathway, as well asanimal studies to conrm the anticancer effects of hydrazonederivatives are required. A further computational moleculardynamics simulation and docking study compared with a co-crystallized ligand are required to better elucidate the interac-tion between the protein and the ligand.4. ConclusionsDespite recent improvements in the management of HCC, itremains one of the leading causes of cancer-related mortality.Various molecular targets involved in signaling pathways thatregulate tumor proliferation and angiogenesis that characterizeHCC were identied. The rst HCC medication approved by theFDA is the oral multikinase inhibitor sorafenib. This rst-linetreatment may not work for all patients for various causes,including drug resistance. Therefore, it is necessary to developadditional effective HCC drugs.In the current study, new hydrazone derivatives withimproved activities and selectivity were synthesized and evalu-ated in silico by molecular docking and structure–activitycorrelations (SAR) in addition to in vitro studies using HepG2human hepatocellular carcinoma cell line and liver mesen-chymal stem cells as a normal cell line. With mean IC50 valuesranging from 23.6 to 94.7 mM, compounds 2, 4, 8a, 13, 16, 18,and 20 exhibited high cytotoxic effects against HepG2 cells.Compound 16 had IC50 and SI of 23.6 mM and 3.2, respectively,against HepG2 as compared to sorafenib (25.6 mM. and SI 3.9)indicating a strong potency and selectivity for activated HepG2cell lines. Compound 16, however, exhibited low cytotoxicityagainst liver mesenchymal stem cells (IC50 76.5 mM). In vitroRSC Adv., 2024, 14, 37960–37974 | 37971http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854bRSC Advances PaperOpen Access Article. Published on 28 November 2024. Downloaded on 12/2/2024 3:37:52 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinewound scratch assay indicated that several synthesized hydra-zone derivatives, including compound 16, attenuated HepG2cell migration and wound closure by 40–70%. HepG2 cellapoptosis as determined by assay of active caspase 3 enzyme,showed that the most potent hydrazone derivative, compound16, caused a higher increase in the percentage of cell apoptosis(80%) as compared to sorafenib (75%). Cell cycle analysisshowed that compound 16 reduced the number of cells in S-phase to 4.5% while 1.2% of the cell population was in theG2/M phase which suggest a signicant inhibitory effect ofcompound 16 on the HepG2 cells. Furthermore, compound 16signicantly increased cytochrome C release and signicantlydecreased IL-6 gene expression compared to sorafenib.These ndings suggest that compound 16 might be a potentanticancer candidate for HCC treatment by downregulating theexpression of the IL-6 gene that causes suppression of the JAK/STAT pathway, decreases inammation, and tumorigenesiswith favorable binding interaction and bioavailability. Compu-tational analysis including molecular docking and structure–activity correlations also supported the anticancer potential ofthe synthesized hydrazone derivative 16. Compound 16 dis-played better binding interactions to EGFR4 as compared to 5-FU which further supports the potential of compound 16 as aneffective chemotherapeutic agent. Furthermore, ADME predic-tions supported preferable bioavailability of the synthesizedhydrazones.EGFR and IL-6 signaling pathways crosstalk in multipledownstream signaling pathways and both are linked to tumor-igenesis. Therefore, co-targeting them could represent a prom-ising strategy for effective cancer treatment.54 Findings of thisstudy indicate that compound 16 targets both signalling path-ways by binding to the kinase domain of EGFR4 and decreasingIL-6 expression. Further computational, in vitro, and in vivoevaluations should be considered to support the anticancerpotential of the synthesized novel hydrazone derivatives.Data availabilityThe data supporting this study are available in the mainmanuscript and its ESI.†Author contributionsAll authors contributed equally to the experimental and writingparts depending on their specialty. All authors read andapproved the nal version of this manuscript.Conflicts of interestThe authors declare no conict of interest.AcknowledgementsThe authors of this manuscript express their great gratitude toCollege of Science, Qassim University for providing laboratoryfacilities. This work was funded in part by a grant from theAmerican University in Cairo to Prof. Hassan Azzazy.37972 | RSC Adv., 2024, 14, 37960–37974References1 A. Villanueva, Hepatocellular carcinoma, NEJM, 2019,380(15), 1450–1462.2 A. M. Attallah, M. El-Far, C. A. A. Malak, M. M. Omran,G. E. Shiha, K. Farid, L. A. Barakat, M. S. Albannan,A. A. 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Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4ra05854b Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)... Anti-hepatocellular carcinoma activities of novel hydrazone derivatives via downregulation of interleukin-6Electronic supplementary information (ESI)...