A new rhodium(I) NHC complex inhibits TrxR: In vitro cytotoxicity and in vivo hepatocellular carcinoma suppression.

EuropeanJournalofMedicinalChemistry183(2019)111721 ContentslistsavailableatScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech Research paper A new rhodium(I) NHC complex inhibits TrxR: In vitro cytotoxicity and in vivo hepatocellular carcinoma suppression Rong Fan a,1, Mianli Bian a,1, Lihong Hu a, Liu Wukuna,b,c,* aSchoolofPharmacy,NanjingUniversityofChineseMedicine,Nanjing,210023,China bStateKeyLaboratoryofNaturalMedicines,ChinaPharmaceuticalUniversity,Nanjing,210009,China cStateKeyLaboratoryofCoordinationChemistry,NanjingUniversity,Nanjing,210023,China a r t i c l e i n f o a b s t r a c t Articlehistory: Thioredoxinreductase(TrxR)isoftenoverexpressedindifferenttypesofcancercellsincludinghepa- Received24June2019 tocellularcarcinoma(HCC)cellsandregardedasatargetwithgreatpromiseforanticancerdrugresearch Receivedinrevisedform anddevelopment.Here,wehavesynthesizedandcharacterizedninenewdesignedrhodium(I)N-het- 27August2019 erocycliccarbene(NHC)complexes.Allofthemwereeffectivetowardscancercells,especiallycomplex Accepted18September2019 1e was more active than cisplatin and manifested strong antiproliferative activity against HCC cells. Availableonline21September2019 Invivoanticancerstudiesshowedthat1esignificantlyrepressedtumorgrowthinanHCCnudemouse Keywords: modelandamelioratedliverlesionsinachronicHCCmodelcausedbyCCl4.Notably,amechanisticstudy revealedthat1ecanstronglyinhibitTrxRsystembothinvitroandinvivo.Furthermore,1epromoted Rhodium(I)NHCcomplexs Thioredoxinreductase intracellular ROS accumulation, damaged mitochondrial membrane potential, promoted cancer cell Apoptosis apoptosisandblockedthecellsintheG1phase. Reactiveoxygenspecies ©2019ElsevierMassonSAS.Allrightsreserved. Hepatocellularcarcinoma 1. Introduction mechanism. The thioredoxin (Trx) system is one of the most importantmembersintheredoxcontrolsystem,whichcomprises Hepatocellularcarcinoma(HCC)isacommonlymalignantcan- NAPDH, Trx and thioredoxin reductase (TrxR) [8e11]. As a redox cer with high mortality, and its death rate ranks third among all regulatoryprotein,TrxcanbeoxidizedbyabundantROS. cancerdeathratesinChina[1,2].Morespecifically,thenumberof TrxR activates Trx by reduction and provides electrons in HCCcasesinChinaoccupiedalargeproportionoftheHCCcasesin numerous oxidation/reduction cycles. Many studies have shown theworld[3].Andthefive-yearsurvivalrateofHCCisbelow5%, that TrxR involved in multifarious physiological and pathological whichisoneoftheworstprognosiscancers[4].Oralsorafenibis processes in bodies, including apoptosis, cancer and chronic currentlytheonlydrugthatsignificantlyprolongsthesurvivalof inflammation[12]. patients with advanced HCC. However, none of the new molecu- In previous years, several studies have reported that Trx and larly targeted drugs, including sorafenib, that has been approved TrxRareoverexpressedinHCC[13,14];TrxRcontributestotumor for marketing, have achieved very satisfactory results. Therefore, growththroughthehypoxia-induciblefactor1pathwayandshows improvedHCCtreatmentmethodsstillneedtobedeveloped[5,6]. acrucialroleinmaintainingcancerphenotypeandmetastasis[14]. Redoxdysregulationstemmingfrommetabolicalterationsand Targeted inhibition of TrxR leads to the production and accumu- relyingonmitogenicandsurvivalsignalsthroughreactiveoxygen lation of ROS in HCC cells, inhibition of cell proliferation and species (ROS) embodies an exceptional sensitivity to tumor cells mitochondrial membrane damage [13]. Accumulating evidences thatcanbeselectivelytargetedbyredoxchemotherapeuticagents demonstrate that TrxR/Trx is an important modulator in tumor [7]. Redox control has become an essential biological regulatory development [10,15].Inthe nudemousexenograft modelof HCC cells,theprogressionsoftumorwereslowerandthesizesoftumor weresmallerintheTrxRknockdowntumorgroupthanintheTrxR normal tumor group [16e18]. Therefore, targeting and inhibiting * Corresponding author. School of Pharmacy, Nanjing University of Chinese TrxR/TrxisahopefulstrategyforHCCtreatment. Medicine,Nanjing,210023,China, E-mail addresses: liuwukun0000@njucm.edu.cn, liuwukun0000@hotmail.com As Trx/TrxR is involved in the pathological process of cancer, (W.Liu). inhibition of TrxR is a significant clinical goal. Recently, some 1 RongFanandMianliBiancontributedequallytothiswork. https://doi.org/10.1016/j.ejmech.2019.111721 0223-5234/©2019ElsevierMassonSAS.Allrightsreserved. 2 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 important studies have reported TrxR inhibitors (e.g. metal- NMR spectra of ligands (1d-9d) and complexes (1e-9e) were containinginhibitors)[10,19e23],someofwhichexhibitedstrong characteristic of complex formation, as shown in Table S1. The TrxRinhibitioneffectsandgoodantiproliferativeeffectsonmany carbenecarbonchemicalshiftincreasedbyapproximately45ppm tumorcells,suchasgold(I)NHCcomplexes[24e31].Interestingly, after coordination to rhodium. From the positive ESI mass spec- rhodium(I) NHC compounds, which possess a square-planar ge- trum,wecanobservethepeakof[M-X] þ (whereX¼BrorCl)for þ ometry as cisplatin, also inhibited TrxR activity in Ott and co- compounds1d-9dand[M-Cl] peaksforcomplexes1e-9e. workers’ studies [32,33]. The ion rhodium(I) is isoelectronic with Crystals of 1e were grown byslowly diffused n-hexane into a platinum(II). thick 1e dichloromethane solution. As shown in Fig. 1, the Rh- Generally, TrxR inhibitors display anticancer effects by pro- Ccarbenedistanceof1eis1.945Å.Crystallographicof1ehasgivenin motingtheapoptosisoftumorcellswhileHCCiscloselyrelatedto TableS2. the overexpression of TrxR. To develop an inhibitor that can effectivelyinhibittheoverexpressionofTrxRinHCCcells,here,we present the synthesis, characterization, and cytotoxicity of nine 2.2. InteractionwithGSHandNAC rhodium(I) NHC compounds possessed pharmacologically active 4,5-diarylimidazoles (Schemes 1 and 2, Table 1) [28,34,35]. The Forfurtherstudy,thestabilityoftherhodium(I)NHCcomplexes most promising rhodium(I) complex can inhibit TrxR, increase inthepresenceofbiologicallyimportantthiolswereexamined.We intracellularROSaccumulationandpromoteHCCcellsapoptosis.In testedthereactivityof1einthepresenceofglutathione(GSH),N- addition, this complex has also been shown to significantly sup- acetylcysteine (NAC) and deuteroxide in acetonitrile-d3 byexam- presstumorgrowthinanHCCnudemousemodelandameliorate ining the 1H NMR spectra (Fig. S1). When 1e was treated with liverlesionsinachronicHCCmodelcausedbyCCl4. equivalentGSH,severalchangesinthespectra(Fig.S1A)couldbe observed over time: at 5.53ppm, 2.92ppm, 3.21ppm new peaks appeared,inwhichthe5.53ppmpeakwasenhancedovertime,and 2. Resultsanddiscussion accordingtotheliteraturethatthepeaksat3.26ppmwereattrib- utedtotheb-CHcysgroupsofGSSG[37],indicatingtheexistenceof 2.1. Synthesisandcharacterization reactions between 1e and GSH. When 1e was treated with NAC (Fig.S1B),thepeakat5.53ppmalsoappeared,andaseriesofpeaks The4,5-diarylimidazoleprecursorsweresynthesizedintermsof (8.68ppm,7.40ppm,7.22ppm,4.66,and4.07ppm)appearedand literaturemethods[28,34,35],andtherhodium(I)NHCcomplexes were enhanced over time. The 5.53ppm peak may be the RheS werepreparedbytheclassicalsilverconversionmethod(Scheme complex signal, and the 8.68ppm, 7.40ppm, 7.22ppm, 4.66ppm, 2)[32,33,36].NHCs(1d-9d)weretreatedwithahalfequivalentof and4.07ppmpeaksmaybeattributedtotheNHCligandsignals, Ag2Oindichloromethanetogenerateasilverintermediate;then,a which indicated that 1e can react with NAC and lose its NHC li- halfequivalentofbis[chloride(1,5-cyclooctadiene)rhodium(I)]was gands.Asacontrol,thestabilityofcomplex1eindeuteroxidewas added,andapproximately6hlater,therhodium(I)NHCcomplexes tested(Fig.S1C).The1HNMRspectraof1eindeuteroxidewasnot wereobtainedandpurifiedbyfiltrationoverCelite,columnchro- observed significant changes after 96h, indicating that 1e was matographyandrecrystallization.Thestructureswereanalyzedby relativelystableindeuteroxide.The1HNMRspectrachangesof1e 1H,13C NMR,andESI-MS, andthepurityof thecomplexes deter- andGSH/NAC werecausedbythereaction between1eandGSH/ mined by elemental analysis. Compared to 1d-9d, the 1H NMR NAC, rather than by the reaction with deuteroxide. From these spectraof1e-9eappearedtheprotonsignalsof1,5-cyclooctadiene. experimental results, we speculated that 1e could interact with ThechemicalshiftchangesofNHC-Ccarbenesignalsbetweenthe13C mercaptobiomacromolecules. Scheme1. Thestructuresof1e-9e. R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 3 Scheme2. Synthesisroutesforrhodium(I)NHCcomplexes(1e-9e). Table1 Thesubstituentgroupsofcompounds1a-1e,2a-2e,3a-3e,5d-5e,6d-6e,7d-7e,8d- 8e. Compounds R1 R2 X 1a-1e F Et Br 2a-2e MeO Et Br 3a-3e Br Et Br 5d-5e F Cl 6d-6e F Br 7d-7e F Cl 8d-8e F Cl 2.3. Invitroantiproliferativeactivity Complexes 1e-9e were tested for antiproliferative activity in HCCcells(HepG2),breastcancercells(MCF-7),andhumancolon cancer cells (HT-29) by the MTTassay. Cisplatin was used as the positivecontrol.AsoutlinedinTable2,1eshowedlowerIC50values Fig.1. Thecrystalstructureof1e. in all threecarcinoma cell lines comparedtocisplatin. When the aromatic ring para location is substituted by fluorine, methoxy, bromineorabenzenering(1e-4e),asmallersubstituentsizeleads totheproperlipophilicityofthecomplexesbeingthekeyforthe tostrongerantiproliferativeactivityagainstMCF-7cellsandHepG2 antiproliferativeactivities.Complex9ewaslessactivethan1e-8e cells(sizeofsubstituents:F<Br<OMe<Ph;antiproliferativeactiv- againstallthreecelllines,possiblybecausethe4,5-diarylmoiety ity:F(1e)>Br(3e)>OMe(2e)>Ph(4e)).ThesubstituentsattheN- expanded the conjugated region of the NHC and increased its atomsseemedtoplayalargeroleinanticanceractivities,because stability. compoundswithanN1-aliphaticchain(1e,6e)exhibitedequivalent Inaddition,theantiproliferativeactivityof1e(themostactive orhigherantiproliferativeactivitythanthosewithanN1-aromatic complexagainstHepG2cellswithanIC50valueof1.33mM)intwo chain(5e,7e-8e)againstallthreecelllines.Thisresultmaybedue other HCC cell lines and one normal liver cell line (LO2) were 4 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 Table2 imidazoliumsalt1dagainstpurifiedTrxRenzymeweremeasured Theantiproliferativeactivityof1e-9eagainstMCF-7,HT-29andHepG2celllines by the DTNB assay [38]. Compared to non-rhodium-containing (IC50values,Mean±SD[mM]). compound 1d (EC50 >10mM), 1e displayed a stronger inhibitory Compound MCF-7 HT-29 HepG2 effectonTrxRenzyme(EC50:1.89±0.44mM),suggestingTrxRmay Cisplatin 4.95±1.16 9.51±2.02 2.18±0.23 indeedbeabiologicaltargetfor1e.Inaddition,wealsomeasured 1e 4.47±1.96 6.51±1.24 1.33±0.21 theTrxRinhibitoryactivityof1eincells. 2e 6.21±0.89 11.33±2.14 5.84±0.61 Briefly, different concentrations of 1e were used to dispose of 3e 4.12±0.98 6.78±2.12 4.96±1.86 HepG2cellsfor48hand72h.ATrxRAssayKitwasusedtomeasure 4e 7.72±0.79 5.17±1.24 8.87±2.79 5e 17.58±3.12 13.44±1.68 6.01±2.48 theactivityofTrxRand1eshowedTrxRinhibitoryactivity(withan 6e 3.83±0.21 6.04±1.61 4.04±1.92 IC50 z2.5mM)bothdose-andtime-dependently,indicatingthat1e 7e 5.54±0.92 6.48±1.61 4.46±1.45 may represent a fresh kind of TrxR inhibitor with potential for 8e 3.72±0.84 7.36±2.12 3.86±0.02 further research. Besides, we investigated the effect of 1e on 9e 16.19±2.13 15.63±2.45 16.55±2.89 intracellularmRNAlevelsofTrxRinHepG2cellsbyreal-timePCR analysis.AsshowninFig.2C,1eeffectivelyreducedtheexpression ofTrxR.Thesealterationswereconfirmedbyimmunofluorescence evaluated. As showed in Fig. S2A, 1e exhibited strong anti- analysis(Fig.2D).Consideringthattheunderlyingmechanismof1e proliferative activity (IC50 values of 1.78mM in Hep3B, 4.01mM in mayinhibittheoverexpressedTrxRinHCCcells,wesubsequently SMMC-7721) in HCC cell lines. However, no selectivity was examinedtherelationshipbetweenTrxRexpressionlevelsand1e observedforHCCcellsversusLO2normalcells(IC50 ¼2.18mM).To treatmentconcentrationsindrug-sensitiveHepG2cellsbywestern obtainasafedoseof1eforfurtherstudy,weevaluatedtheALTand blotting(Fig.2B).TrxRexpressionwasupregulatedinHCCcells,and LDHlevelsinanormallivercellline(LO2)(Figs.S2BandS2C).The theexpressionwasdecreasedby1einadose-dependentmanner. doses of 1.5mM, 3mM, and 6mM of 1e caused minimal harm to Insummary,alltheseresultsconfirmthatTrxRcouldbeapossible normalcells;inotherwords,thesedosesaresafe.Thus,followup biologicaltargetforcomplex1e. studiesweremainlyfocusedoncomplex1e. 2.5. Fluorescencemicroscopy 2.4. TrxRinhibition Thecellularuptakeanddistributionofdrugsintumorcellsisthe Asmentionedabove,TrxRwasoverexpressedinvariouscancer key to the anticancer activity of drugs [39]. To investigate the cellsincludingHCCcellsandstudieshaveshownthatrhodium(I) subcellular distribution of rhodium(I) NHC complexes, we chose NHC complexes can bind with TrxR [32,33,36]. Thus, we further complex5e,whichpossessesalargeconjugatedstructureandcan testedthereactivityof1ewithTrxRinpurifiedTrxRenzymeand emit fluorescence at a specific wavelength. The emission spectra HepG2 cells (Fig. 2A). The inhibitory activity of TrxR by 1e and andexcitationspectraof5eareshowninFig.S3.Duetotheintrinsic Fig.2. QuantificationofTrxRactivityinHepG2cellstreatedwith1eat37(cid:2)Cfor48hand72h(A),WesternblotofHepG2cellstreatedwith1efor24h(B),mRNAlevelsofTrxRin HepG2cellstreatedwith1efor24h(C),immunofluorescenceanalysisofHepG2cellstreatedwith1efor24h(D,originalmagnification,40(cid:3)).Errorbars:S.D.,n¼3.statistical significanceofdifferencesinmeanvalues:*p<0.05and***p<0.001. R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 5 emissionof5e,thelocalizationofthecomplexinHepG2cellswas analysetheproductionofROSin1e-treatedHepG2cells.Thelevel further investigated byfluorescence microscopy. Afterincubating ofROSgenerationwashigherincellstreatedwith1e(6mM)thanin HepG2 cells with 5e (10mM) for 24h and Red-Mito-Tracker dye untreatedcells (Fig. 3C and D).Then, wetestedthe protection of (200nM, a mitochondria-specific reactive dye) for 0.5h, we HepG2 cells by NAC when HepG2 cells were incubated with the observed an obvious blue fluorescence in the cytoplasm. By the indicated concentrations of NAC and 1e for 48h. NAC as an anti- merge of all the luminescent spots (caused by both 5e and Red- oxidant can remove excess ROS in cells [41]. Interestingly, treat- Mito-Trackerdye),wecanseethatthebluefluorescencewasspe- ment with 1e prominently reduced the viability of HepG2 cells cifically localized in mitochondria (Fig. 3A). Accordingly, we can (Fig.S4),whichwaspartiallyorcompletelyabrogatedbytreatment speculatethatrhodium(I)NHCcomplexesweremainlydistributed with1mMe4mMNAC,respectively.Thesefindingssuggestedthat inmitochondriaandmayinteractwithmitochondrialTrxR. 1ecanactivateROS-inducedapoptosisinHepG2cells. 2.7. MMPandATP 2.6. ROSanalysis Studies have shown that high levels of ROS can trigger mito- IntracellularaccumulationofROS,whichdamagesDNAthrough chondrial membrane permeabilization and mitochondrial mem- oxidative stress, disrupts mitochondrial membrane potential, brane potential depolarization, leading to mitochondrial causing mitochondrial dysfunction leading to apoptosis and cell dysfunctionandactivationofintrinsicapoptoticpathway[42].We death[40].TrxactsasanintracellularROSscavengerwhoseactivity usedtheJC-1kittoinvestigateMMPupontreatmentwith1e.After isaffectedbyTrxRactivity.WhentheactivityofTrxRwasinhibited, treatment with 1e, we observed a dose-dependent decrease in theactivityofTrxwasalsoinhibited,resultinginaccumulationof mitochondrialmembranepolarization(orangefluorescenceturned ROS in cancer cells. Thus, HepG2 cells were inoculated with 1e greenfluorescence).Inparticular,6mMof1eledtohigherdamage (1.5mM,3mM,and6mM)for24h,fluorescenceanalysiswasusedto to the membrane compared with the control group, as expected (Fig.3E). MMPisvitaltoATPgenerationandATPisessentialtotheefflux ofthedrug,whileATPisderivedfromATPhydrolysisbyATPases [43].Toverifywhetherourcompound1ewouldaffectATPase,we treated HepG2 cells with 1e at different concentrations (1.5mM, 3mM,and6mM),andtheresultsshowedthatsamplestreatedwith 1e had significantly lower ATPase activity than that in the DMF control (Fig. 3B). These data indicated that 1e inhibited ATPase activity. 2.8. Releaseofcytochromecandapoptosis Studyhasdemonstratedthatmostmetal-basedantitumordrugs normallykilltumorcellsbyactivatingtheapoptoticpathway[44]. In addition, ROS can promote a series of mitochondria-related eventssuchasapoptosisbyaffectingMMP.Thus,theapoptosisof HepG2cellsinducedby1ewasresearchedbypropidiumiodide(PI) stainingandFITC-AnnexinVstaining.ForHepG2cellstreatedwith 1e,wemeasuredthestateofapoptosisbyflowcytometry.HepG2 cells were treated with 1e for 72h, separately. The results demonstratedthat1eenhancedapoptosisinHepG2cellsinadose- dependent manner(Fig. 4A). The initialsynthetic caspases in the cellsareinactivezymogenformandthenactivatedbyhydrolysisof the N-terminal sequence. Activated caspase can hydrolyze the correspondingsubstrateandinduceapoptosisbycascadeamplifi- cation [45]. Thus, we also investigated the protein levels of the cleavedactiveformsofcaspases.Theresultsdemonstratedthat1e dose-dependentlyupregulatedtheexpressionofapoptosis-related factors Cleaved-Caspase-3 and Cleaved-Caspase-9 (Fig. 4B). Hoechst33258stainingwasusedtoverifytheabilityof1e-induced apoptosis by analysing the morphology of the bare cell nucleus (Fig. 4C). HepG2 cells were treated with 1e for 24h and then stainedbyHoechst33258morphologicallydetectingapoptosisvia fluorescencemicroscopy.Thecontrolcellsdisplayedaweakregular bluefluorescence.Whilein1e-treatedgroup,brightbluefluores- cencewasobservedinmostHepG2cells,andthenucleiappeared hypercondensed(brighterbluestained).WhenHepG2cellswere treated with complex 1e (6mM) for 24h, the obviously increased Fig. 3. The uptake of 5e (10mM) in HepG2 cells (A, original magnification, 40(cid:3)), apoptotic nuclei containing condensed chromatin demonstrated ATPaseactivityinHepG2cellsaftertreatedwith1efor24h(B),ROSpositivecellsafter that apoptosis of HepG2 cells was occasioned by 1e in a dose- treatedwith1efor24h(C),ROSgenerationaftertreatedwith1efor24h(D,original dependent way. These results suggested that 1e induced magnification,20(cid:3)),mitochondrialmembranedamageimagedbyJC-1stainingafter treatedwith1efor24h(E,originalmagnification,40(cid:3)).Errorbars:S.D.,statistical apoptosisofHepG2cellsalongwithcaspaseactivation. significanceofdifferencesinmeanvalues:*p<0.05,**p<0.01and***p<0.001. Apoptosis leads to changes in mitochondrial membrane 6 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 Fig.4. ApoptosisofHepG2cellstreatedwith1eatdifferentconcentrationsfor72h(A),WesternblotanalysisofHepG2cellsaftertreatmentwith1eatdifferentconcentrationsfor 24h(B),andHoechst33258stainingaftertreatmentwith1efor24h(C,originalmagnification,40(cid:3)).Errorbars:S.D.,n¼3.Statisticalsignificanceofdifferencesinmeanvalues: *p<0.05,**p<0.01and***p<0.001. permeability, AIF/Bak transfers from mitochondria to nucleus. A dose-dependent expression level decrease was caused by the IncreasedBax/Bcl-2ratiopromotescytochromecsetfreeintothe treatment with 1e. Additionally, the upregulation of cell cycle cytosol and further triggers the cysteine protease cascades. PARP control inhibitors of Cyclin-CDK complexes (p15, p21 and p27) cleavage involves the response of cells to apoptotic signal stimu- causes to cell cycle arrest in the G0/G1 phase. Particularly, over- lation and the repair of DNA damage, and serves as a marker of expressionofp21andp27activatesBaxthroughdifferentinhibi- apoptosis. Therefore, the effects of 1e on PARP cleavage was tion signals, attenuating cancer cell proliferation and inducing examined by Western blot assay. As shown in Fig. 5A and B, the apoptosis(Fig.6C). results demonstrated that 1e enhanced the apoptotic family- related proteins localized to the mitochondrial inner membrane 2.10. InteractionwithDNA inHepG2cellsinadose-dependentmanner.Thesewereconsistent withthefindingsfromFITCAnnexinVstaining. It is widelyaccepted that DNA symbolizes a primary targetof anticancer metal compounds [46e52] and rhodium(I) NHC com- 2.9. Cellcycleanalysis plexes have also been proven to target DNA [53e55]. To more clearly find out the anticancer activity of our rhodium(I) NHC Next,theresultof1eoncellcycleprogressionwasstudiedbyPI complexes, the binding ability of 1e to DNA was investigated by staining. Cells were treated with 1e for 72h, showing that the UVeVis absorption, fluorescence emission and CD spectroscopy proportionofcellsthataccumulatedintheG0/G1phaseincreased (Fig. S5). Sodium dodecyl sulfate (SDS) was added as a probe to compared tothe S and G2/M phases (Fig. 6A). Cycle-related pro- confirm whether 1e interacts with DNA via electrostatic in- teins’ levels were studied by Western blot analysis (Fig. 6B). The teractions. When 1e was combined with SDS, the UV spectra expression levels of Cyclin D1 and Cyclin E1 were significantly changed.Nosignificantdistinctionwasobservedbetweentheab- reduced,whilethelevelsofCDK4andCDK6wasalmostunchanged. sorptionspectraof1eintheabsenceandpresenceofSDS(Fig.S5A). R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 7 Fig.5. Theexpressionofapoptosis-inducingfactor(AIF)byimmunofluorescenceanalysis(A),theexpressionofapoptosis-inducingfactor(AIF)andBax/Bcl-2byWesternblot(B). Statisticalsignificanceofdifferencesinmeanvalues:*p<0.05,**p<0.01and***p<0.001. From the UVeVis spectrum, the ct-DNA titration experiment be usedtoidentifywhether thecompoundand ct-DNA actin an showed a clear isosbestic point at 296nm. (Fig. S5B). Forthe ab- insertionornon-insertionmode.AsshowninFig.S5C,theEB-ct- sorptionbandatapproximately258nm,aslighthyperchromicity DNA system produced the strongest fluorescence emission at and an ~2nm blueshift were occurred upon addition of ct-DNA. 613nmwithout1e;upontitrationof1e,thefluorescenceintensity These results indicated that the changes in absorption spectra was weakened, progressively, which revealed the presence of a observed after the addition of ct-DNA were not due to the elec- pattern of insertion of 1e with DNA. The fluorescence quenching trostaticbindingofthe1etothepolyanionDNAphosphateback- constant (Kq) was analyzed through the Stern-Volmer equation bone, but rather to the binding of 1e to the DNA grooves or its accordingtopreviousliterature[56].Acirculardichroismspectral involvementintheinsertionofp-pstackinginteractions. assayof1e(Fig.S5D)showedthat1eboundtoct-DNA,andwhen Inaddition,thebindingmodesof1eandct-DNAwereevaluated the 1e/ct-DNA ratio reached 6/1, the intensity of positive and bymonitoringthefluorescencespectraincompetitivebindingct- negativeopticalrotationhaschangedalot,furtherconfirmingthat DNA experiments between 1e and ethidium bromide (EB). After the DNA bindingmode of 1e occurs viathe intercalation binding the small molecule compound supersedes EB, the fluorescence mode. Considering that 1e0s analogue complex 5e was mainly emission intensity of the EB-ct-DNA system ([EB]¼2.0(cid:3)10 (cid:4)6M, localized in mitochondria, we speculate that this complex may [ct-DNA]¼2.0(cid:3)10 (cid:4)5M) will be changed significantly, which can react with mitochondrial DNA [57e59], which requires further 8 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 chronic liver injury, which led to pathological morphological changesinliver,includinginflammation,steatosis,andnecrosis.To assess the protective effect of 1e (10mg/kg), we used mice to explore its therapeutic potential in preventing or treating liver injury.Wethereforeanalyzedbiochemicalmarkersofliverinjury afterCCl4treatmentfor14weeks.Allmiceweresacrificedafterthe experiment.AllanimalliversdealtwithCCl4hadcirrhoticfeatures withmicronodularpalesurfaces,whichindicatedthatthemouse livercirrhosis model was successfullymodeled (Fig. 8A). In addi- tion, H&E staining showed that treatment with 10mg/kg of 1e significantlyamelioratedthemorphologicalchangesinlivertissue (Fig.8B).Theaccumulationofcollagenaccompaniesfibrogenesis; therefore, liver tissue sections were stained with Sirius red and Masson’s reagent to examine collagen expression in the mouse liver.Thisanalysisshowedthatcollagenwasmarkedlydepositedin theCCl4-injuredliverbutthattheamountwasreducedintheliver ofmiceinthe1etreatmentgroups(Fig.8CandFig.S6A).During chronicliverdisease,inflammationiscommonlyaccompaniedby hepatocellularinjury.Severeinflammatorycellinfiltrationinlivers exposed to CCl4 was suppressed by 1e, as characterized by a (cid:4) decrease in the number of intrahepatic CD45 and F4/80-positive cells(Fig.8DandFig.S6B).AsdemonstratedbytheELISAresults, 1e treatment also decreased the serum levels of IL-6 and IL-8 (Fig. 9A). Thus, we proposed that 1e protected the liver against CCl4-inducedinjurybysuppressinginflammation.ThelevelsofAST, ALT, ALP, and LDH were effectively increased in two groups (the modelgroup)anddecreasedby1einCCl4-treatedmice(Fig.9B). Moreimportantly,theexpressionofTrxRinserumandlivertissue washighinCCl4-injuredliver,andtreatmentwith1esignificantly reversedthesechanges(Fig.10AandB).Overall,theseresultscan providemoreinvivoevidencethattreatmentof1edidreducethe accumulation of collagen and inhibit the inflammation in HCC modelmice. Fig.6. Effectof1eoncellcycleprogression(A),WesternblotanalysisofHepG2cells aftertreatmentwith1eatdifferentconcentrationsfor24h(B,C).Errorbars:S.D., 3. Conclusions n¼3.Statisticalsignificanceofdifferencesinmeanvalues:*p<0.05,**p<0.01and ***p<0.001. In summary, our study demonstrates that a novel rhodium(I) complex 1e triggers antiproliferative effects in vitro and exhibits researchtoprove. promisinginvivoanticancereffectsinHCC.Toourknowledge,this studyshowedforthefirsttimeoftheinvivobiologicalactivityof rhodium(I)NHCcomplex.Thiscomplexinhibitedtheexpressionof 2.11. Invivoantitumorefficacy the TrxR system and induced the accumulation of ROS, the apoptosis of cancer cells (Fig. 11). Moreover, 1e inhibited the To assess the potency of complex 1e in inhibiting HCC tumor mitochondrial apoptotic pathway, causing high expression of the growthinvivo,weconstructedHepG2tumorxenograftmodelsin apoptogenic factor Bax and low expression of the antiapoptotic nude mice by subcutaneously injecting HepG2 cells in the expo- cytokine Bcl-2. Besides,1e induced DNA damage and led to cell nential phase intothe right armpit of the mice. When successful cyclearrest.Inlightofthisreportweproposethat1erepresentsa modeling,thetumorvolumeofnudemicewere180e200mm3,and newTrxRinhibitorandapotentialcandidateforthetreatmentof these tumor model nude mice were randomly divided into two HCC. groups(n¼4):(1)thevehicle-treatedgroupand(2)thecomplex 1e-treated(10mg/kg)group.Duringthe14-daytreatmentperiod, 4. Experimentalsection there was significant weight loss in the 1e treatment group (Fig. 7A). Treatment with 10mg/kg 1e remarkably inhibited the 4.1. Materialsandmethods growth of HepG2 tumor volume (Fig. 7B). Compared with the control, the tumor weight of mice treated with 1e 10mg/kg was All the reagents for synthesis were purchased from Energy significantlyreduced,andthetumorgrowthinhibitionratereached Chemical(China)andusedasreceivedwithoutfurtherpurification. 45%.(Fig.7CandD). Cisplatin was purchased from Yuanye Bio. (Shanghai, China). Many animal models have provided pertinent information on DeuteratedsolventswerepurchasedfromCambridgeIsotopeLab the pathogenesis and molecular mechanisms of HCC, which is Inc.(CIL).Solutionsforallspectroscopicandbiologicalstudieswere critical for preclinical studies of drugs [60]. Chronic exposure of prepared with solvents of HPLC grade and doubly distilled water mouselivertohepatotoxincarbontetrachloride(CCl4)isamature fromMillipore(>18.2MU).Antibodies,suchasactivatedcaspases- method of inducing liver damage and promoting fibrosis and 3,caspase-9,p15,p21,p27,NAPDH,forWBorIF,werepurchased cirrhosis[61].Hence,tofurtherevaluatetheanticanceractivitiesof fromAbcamCo.(China).JC-1assaykitwaspurchasedfromKeyGen 1eagainstHCCinvivo,wechoseCCl4-inducedchronicHCCmice. Co.(Jiangsu,China);ROSassaykit,ATPassaykit,mitochondriaand InjectionofCCl4intothemiceapproximatelyfor14weeksinduced nucleiIsolationkitwerepurchasedfromBeyotimeBiotechnology R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 9 Fig.7. Effectoftreatmentsonthemeanbodyweightofmice(A),tumorgrowthcurvesinmicereceivingthe1etreatmentschedule.Valuesarethemean±SEM,n¼4/group(B), representativeimagesoftumorsexplantedfromcontrolandtreatedmiceatsacrifice(upperpanel)andrelativetumorweight(lowerpanel)aftertumorchallenge;valuesarethe mean±SEM(C)and(D).Valuesarethemean±SEM.Thesignificancewasdeterminedbyone-wayANOVAfollowedbyTukey’smultiplecomparisonpost-test;eachgroupwas comparedtothecontrol.(***p<0.001). (Jiangsu, China). TrxR activity assay kit, purified TrxR (rat liver), d 10.94 (s, 1H, NCHN), 7.58 (d, J¼8.4Hz, 4H, ArphH), 7.14 (d, ATPaseactivityassaykitandCalf-thymus(ct-DNA)werepurchased J¼8.4Hz, 4H, ArphH), 4.33 (q, J¼7.3Hz, 4H, NCH2CH3), 1.51 (t, fromSigma-Aldrich Co.; MitoTracker ®Red CMXRos (M7512) was J¼7.3Hz,6H,NCH2CH3).13CNMR(126MHz,CDCl3,20(cid:2)C):d137.44 purchasedfromYeasen(Shanghai,China). (NCHN),132.88,131.81,123.53,125.52 (ArphC),130.90 (NC¼CN), 1HNMRand13CNMRspectrawereobtainedonBrukerAvance 43.62(NCH2CH3),15.82(NCH2CH3).ESI-MS(þ)[m/z]:435.0[M-Br] þ . III HD, 500M spectrometers. The tetramethylsilane signal as a 4,5-di([1,10-biphenyl]-4-yl)-1,3-diethyl-1H-imidazol-3-ium bro- reference. The purities of 1e-9e (>95%) were performed by the mide4d.Yield78%;whitesolid.1HNMR(500MHz,CDCl3,20(cid:2)C): centreforinstrumentalanalysis ChinaPharmaceuticalUniversity, d 10.96 (s, 1H, NCHN), 7.66 (d, J¼8.2Hz, 4H, ArphH), 7.58 (d, Nanjing, andobtainedonPE2400Series II.UVeVisspectrawere J¼7.3Hz,4H,ArphH),7.46(t,J¼7.6Hz,4H,ArphH),7.41e7.33(m, recordedonaShimadzuUV2400spectrophotometer.Fluorescence 6H,ArphH),4.41(q,J¼7.3Hz,4H,NCH2CH3),1.56(t,J¼7.3Hz,6H, excitation and emission spectra were recorded on a Cary Eclipse NCH2CH3). 13C NMR (126MHz, CDCl3, 20(cid:2)C): d 143.23, 139.31, Varian fluorescence spectrophotometer (band pathways were 130.76, 129.02, 128.26, 127.95, 127.06, 123.64 (ArphC), 131.54 5nm).CDspectrawereobtainedfromvibrationalCDspectrometer (NC¼CN),137.06(NCHN),45.52(NCH2CH3),15.99(NCH2CH3).ESI- Bruker PMA50. Fluorescent microscopy was performed using a MS(þ)[m/z]:429.2[M-Br] þ . LeicaDMi8fluorescencemicroscope.ESI-MSspectraweregotfrom 3-(anthracen-9-ylmethyl)-1-ethyl-4,5-bis(4-fluorophenyl)-1H- theAgilent6125.X-RaySingleCrystalDiffractometerwasBRUKER imidazol-3-ium chloride 5d. Yield 67%; yellow solid. 1H NMR Smart APEX II CCD, Flow Cytometer was BD C6 Plus. MTT and (500MHz, DMSO‑d6, 20(cid:2)C): d 8.87 (s, 1H, NCHN), 8.70 (s, 1H, protein assays were quantified by PerkinElmer Fusion Reader AranthH), 8.23 (d, J¼8.2Hz, 2H, AranthH), 8.19 (d, J¼8.7Hz, 2H, (PackardBioScienceCompany).Analysesindicatedbythesymbols AranthH),7.72(dd,J¼8.6,5.4Hz,2H,AranthH),7.64(m,4H,ArphH), oftheelementsorfunctionswerewithin±0.4%ofthetheoretical 7.54(dd,J¼8.6,5.4Hz,2H,AranthH),7.34(dd,J¼17.7,2.2Hz,4H, values. ArphH),6.16(s,2H,NCH2Aranth),3.97(q,J¼7.1Hz,2H,NCH2CH3), 0.99(t,J¼7.2Hz,3H,NCH2CH3).13CNMR(126MHz,CDCl3,20(cid:2)C): d 164.63, 164.51, 162.63, 162.50, 133.06, 132.99, 132.72, 132.65, 4.2. Synthesisandcharacterization 120.85,120.82,120.70,120.67,116.58,116.40,116.08,115.91(ArphC), 136.16 (NCHN), 132.13, 131.31 (NC¼CN), 131.19, 130.84, 130.32, The synthesis of 1a-3a,1b-3b,1c-4c,1d-8d were seen in sup- portinginformation. 129.29,127.98,125.41,123.20,122.83(AranthC),45.49(NCH2Aranth), 43.56(NCH2CH3),15.39(NCH2CH3).ESI-MS(þ)[m/z]:475.2[M-Cl] þ . 3-(cyclopropylmethyl)-1-ethyl-4,5-bis(4-fluorophenyl)-1H-imida- 4.2.1. Generalprocedureforthesynthesisof1d-8d zol-3-ium bromide 6d. Yield 72%; white solid.1H NMR (500MHz, Bromoethane or halohydrocarbon (10.0mmol) and imidazole CDCl3,20(cid:2)C):d10.85(s,1H,NCHN),7.32e7.26(m,4H,ArphH),7.15 1c-4c(2.0mmol)weredissolvedin30mLCH3CN,andrefluxedfor (m,4H,ArphH),4.37(q,J¼7.3Hz,2H,NCH2CH3),4.11(d,J¼7.4Hz, 2e4d. Then removed the solvent in vacuo, purified by silica gel 2H,NCH2C3H5),2.19(s,1H,NCH2CHCH2CH2),1.52(t,J¼7.3Hz,3H, columnchromatography. NCH2CH3), 0.71 (q, J¼5.7Hz, 2H, NCH2CHCH2CH2), 0.50 (q, 3d. 4 Y ,5 ie - l b d is 7 (4 0 - % b ; ro p m al o e p y h e e l n lo y w l)-1 so ,3 l - id di . e 1 t H hy N l- M 1H R -i ( m 50 id 0 a M zo H l- z 3 , - C iu D m Cl3, br 2 o 0 m (cid:2) i C d ) e : J¼5.1Hz,2H,NCH2CHCH2CH2).13CNMR(126MHz,CDCl3,20(cid:2)C): 10 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 Fig.8. Thelivermorphologicalchangesaftertreatmentwithvehicle,CCl4,andbothCCl4and1e(A),liverissueconventionalslicewithH&Estainingaftertreatmentwithvehicle, CCl4,andbothCCl4and1e(B,originalmagnification,20(cid:3)),liverissueconventionalslicewithSiriusredstainingaftertreatmentwithvehicle,CCl4,andbothCCl4and1e(C,original magnification,20(cid:3)),liverissueconventionalslicewithCD45staining(D,originalmagnification,20(cid:3)).(Forinterpretationofthereferencestocolourinthisfigurelegend,the readerisreferredtotheWebversionofthisarticle.) d 164.71, 162.70, 132.83, 132.76, 132.68, 132.61, 121.01, 120.98, 4.2.2. Generalprocedureforthesynthesisof1e-9e 120.89,120.86,116.87,116.85,116.69,116.67(ArphC),136.80(NCHN), One equivalent of the imidazolium cholrine/bromine 1d-9d 131.07, 130.98 (NC¼CN), 52.77 (NCH2C3H5), 43.55 (NCH2CH3), (0.20mmol)and0.6equivalentofAg2O(27.8mg,0.12mmol)were 15.83 (NCH2CH3), 11.10 (NCH2CH), 4.89 (NCH2CHCH2CH2). ESI- added,andthen15mLofanhydrousdichloromethanewasadded, MS(þ)[m/z]:339.1[M-Br] þ . andreactedovernightunderN2atmosphereatambienttempera- 1-ethyl-4,5-bis(4-fluorophenyl)-3-(naphthalen-1-ylmethyl)-1H- ture. After that, 0.5 equivalent of bis[chloride(1,5-cyclooctadiene) imidazol-3-ium chloride 8d. Yield 38%; white solid. 1H NMR rhodium(I)] (49.3mg, 0.10mmol) was added in the mixture, and (500MHz,DMSO‑d6,20(cid:2)C):d9.82(s,1H,NCHN),8.02e7.98(m,1H, continuedtostirfor6hindarkness.Thenthesolutionwasfiltered ArnaphH),7.95(d,J¼8.5Hz,2H,ArnaphH),7.62e7.55(m,4H,ArphH), overaCelitebed(281nm)andthefiltrateconcentratedinvacuo. 7.47(t,J¼7.7Hz,1H,ArnaphH),7.42(dd,J¼8.5,5.4Hz,2H,ArphH), Next,theresiduewaspurifiedbysilicagelcolumnchromatography, 7.34(d,J¼8.8Hz,2H,ArphH),7.19(dd,J¼16.9,8.0Hz,3H,ArnaphH), and recrystallized from CH2Cl2/n-hexane to get the yellow crys- 5.93(s,2H,NCH2Arnaph),4.16(q,J¼7.2Hz,2H,NCH2CH3),1.31(t, tallinesolid. J¼7.2Hz, 3H, NCH2CH3). 13C NMR (126MHz, DMSO‑d6, 20(cid:2)C): Chlorido (h2,h2-cycloocta-1,5-diene)(4,5-bis(4-fluorophenyl)-1,3- d 164.41, 164.30, 162.44, 162.33, 133.88, 133.81, 133.74, 133.67, diethylimidazol-2-ylidene) rhodium(I) 1e. Yield 71%; yellow solid. 122.01,121.98,121.85,121.82,116.74,116.57,116.40(ArphC),136.76 1H NMR (500MHz, CDCl3, 20(cid:2)C): d 7.12 (dd, J¼8.3, 5.4Hz, 4H, (NCHN), 133.60, 130.36, 130.15, 129.59, 129.22, 127.42, 126.79, ArphH), 7.01 (dd,J¼8.3Hz, 5.4Hz, 4H,ArphH),5.05 (s,2H, CH¼), 126.58,125.89,123.15(ArnaphC),48.76(NCH2Arnaph),131.55,131.48 4.77(m,2H,NCH2CH3),4.54(m,2H,NCH2CH3),3.43(s,2H,CH¼), (NC¼CN), 43.37 (NCH2CH3), 15.22 (NCH2CH3). ESI-MS(þ)[m/z]: 2.50e2.36(m,4H,CH2),2.02e1.93(m,4H,CH2),1.25(t,J¼7.2Hz, 425.1[M-Cl] þ . 6H, NCH2CH3). 13C NMR (126MHz, CDCl3, 20(cid:2)C): d 182.46 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 11 Fig.9. ELISAmeasurementofIL-6,IL-8levelsinserum(A),determinationofserumALT,AST,ALPandLDHlevels(B)aftertreatmentwithvehicle,CCl4,andbothCCl4and1e.Forthe statisticsofeachpanelinthisfigure,dataareexpressedasmean±SD(n¼5);###p<0.001comparedwithVehicle,*p<0.05,**p<0.01and***p<0.001comparedwithgroupCCl4. Fig.10. ELISAandWesternblotmeasurementofTrxRlevelsinserum(A)andinliver tissue(B)aftertreatmentwithVehicle,CCl4,andbothCCl4and1e.Forthestatisticsof each panel in this figure, data are expressed as mean±SD (n¼5); ###p < 0.001 comparedwithVehicle,***p<0.001comparedwithgroupCCl4. (NCNeRh), 163.71, 161.73, 132.03, 124.77, 115.97, 115.80 (ArphC), 130.78 (NC¼CN), 98.35,98.41 (CH¼), 68.33, 68.44 (CH¼), 44.21 (NCH2CH3),32.94(CH2),28.21(CH2),16.12(NCH2CH3).ESI-MS(þ) Fig.11. Proposedanticancermechanismsof1e. þ [m/z]:523.1[M-Cl] .Anal.CalcdforC27H30ClF2N2Rh:C,58.02;H, 5.41 C ; h N lo , ri 5 d .0 o 1 (h ; 2 F ,h o 2 u - n cy d c : lo C o , c 5 t 7 a . - 9 1 0 ,5 ; - H di , e 5 n . e 4 ) 3 ( ; 4, N 5 , -b 5 i . s 0 ( 3 4 . -methoxyphenyl)- N 15 C 9 H .4 2 9 C , H 1 3 3 ) 1 . . 1 5 3 1 C ,1 N 2 M 1.2 R 7, (1 11 2 3 6 .9 M 5 H ( z A , r C ph D C C ) l , 3 1 , 3 2 1 0 .1 (cid:2) 5 C ( ) N : C d ¼ 18 C 0 N .9 ) 2 ,9 ( 7 N .9 C 9 N , e 97 R . h 9 ) 4 , s 1 o ,3 l - id d . ie 1 t H hyl N im M i R da ( z 5 o 0 l- 0 2- M yl H id z e , n C e D ) C r l h 3 o , d 2 i 0 um (cid:2)C ( ) I) : d 2e 7 . .0 Y 5 ie ( l d d , J 9 ¼ 1.8 8 % .7 ; H y z e , ll 4 o H w , ( ( C C H H 2 ¼ ), ), 2 6 8 8 .9 .3 1 3, (C 6 H 8. 2 2 ) 1 , 1 ( 6 C . H 24 ¼) ( , N 5 C 5 H .1 2 9 CH (O 3 C ). H E 3 S ), I- 4 M 4. S 0 ( 8 þ) ( [ N m C / H z] 2 : CH 54 3 7 ), .2 32 [ . M 95 - ArphH),6.80(d,J¼8.7Hz,4H,ArphH),5.02(s,2H,CH¼),4.73(m, Cl] þ . Anal. Calcd for C29H36ClN2O2Rh: C, 59.75; H, 6.22; N, 4.81; 2H,NCH2CH3),4.50(m,2H,NCH2CH3),3.76(s,6H,OCH3),3.42(s, Found:C,59.70;H,6.15;N,5.03. 2H,CH¼),2.41(m,4H,CH2),1.95(m,4H,CH2),1.22(t,J¼7.1Hz,6H, Chlorido (h2,h2-cycloocta-1,5-diene)(4,5-bis(4-bromophenyl)-1,3- 12 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 diethylimidazol-2-ylidene)rhodium(I)3e.Yield33.5%;yellowsolid. C29H32ClF2N2Rh:C,59.55;H,5.51;N,4.79;Found:C,59.49;H,5.56; 1HNMR(500MHz,CDCl3,20(cid:2)C):d7.46(d,J¼8.2Hz,4H,ArphH), N,4.98. 7.01(d,J¼8.2Hz,4H,ArphH),5.06(s,2H,CH¼),4.78(dq,J¼14.2, Chlorido(h2,h2-cycloocta-1,5-diene)(3-benzyl-4,5-bis(4- 7.1Hz,2H,NCH2CH3),4.56(dq,J¼14.0,7.0Hz,2H,NCH2CH3),3.43 fluorophenyl)-1-ethylimidazol-2-ylidene) rhodium(I)7e. Yield: 62%; (s,2H,CH¼),2.44(m,4H,CH2),1.98(m,4H,CH2),1.25(t,J¼7.1Hz, yellowsolid.1HNMR(500MHz,CDCl3,20(cid:2)C):d7.19e7.12(m,5H, 6H, NCH2CH3). 13C NMR (126MHz, CDCl3, 20(cid:2)C): d 182.96 ArbenzylH),7.07e6.99(m,4H,ArphH),6.86e6.78(m,4H,ArphH),6.11 (NCNeRh),132.05,131.60,127.57,123.22(ArphC),130.70(NC¼CN), (d,J¼15.3Hz,1H,NCH2Ph),5.73(d,J¼15.3Hz,1H,NCH2Ph),5.09 98.55, 98.50 (CH¼), 68.48, 68.37(CH¼), 44.29 (NCH2CH3), 32.93 (p, J¼8.1Hz, 2H, CH¼), 4.81 (dq, J¼14.3, 7.2Hz,1H, NCH2CH3), (CH2), 28.87 (CH2), 16.22 (NCH2CH3). ESI-MS(þ)[m/z]: 701.7 [M- 4.70(dq,J¼14.3,7.2Hz,1H,NCH2CH3),3.54(m,1H,CH¼),3.33(m, HþNa] þ , 645.1 [M-Cl] þ . MALDI-TOF/TOF MS: 644.9907 [M-Cl] þ . 1H,CH¼),2.56e2.46(m,1H,CH2),2.43e2.33(m,2H,CH2),2.17(m, Anal.CalcdforC27H30Br2ClN2Rh:C,47.64;H,4.44;N,4.12;Found:C, 1H, CH2), 2.06e1.90 (m, 3H, CH2), 1.83 (m, 1H, CH2), 1.31 (t, 47.69;H,4.42;N,4.17. J¼7.2Hz, 3H, NCH2CH3). 13C NMR (126MHz, CDCl3, 20(cid:2)C): Chlorido(h2,h2-cycloocta-1,5-diene)(4,5-di([1,10-biphenyl]-4-yl)- d 183.62,183.21 (NCNeRh),163.74,163.58,161.75,161.60,132.32, 1,3-diethylimidazol-2-ylidene) rhodium(I) 4e. Yield 31%; yellow 132.26,132.04,131.97,124.63,124.60,124.55,124.53,115.96,115.78, solid.1HNMR(500MHz,CDCl3,20(cid:2)C):d7.56(dd,J¼11.7,8.1Hz, 115.45,115.28(ArC),136.94,128.27,127.47,127.28(ArbenzylC),131.47, 8H, ArphH), 7.42 (t, J¼7.6Hz, 4H, ArphH), 7.35 (d, J¼7.4Hz, 2H, 131.21 (NC¼CN), 98.94, 98.88, 98.39, 98.33 (CH¼), 68.92, 68.81, ArphH),7.26(d,J¼7.7Hz,4H,ArphH),5.07(s,2H,CH¼),4.86(m, 68.61,68.49(CH¼),53.25(NCH2Ph),44.31(NCH2CH3),33.34(CH2), 2H, NCH2CH3), 4.64 (m, 2H, NCH2CH3), 3.49 (s, 2H, CH¼), 32.40(CH2),29.15(CH2),28.46(CH2),16.19(NCH2CH3).ESI-MS(þ) 2.54e2.36 (m, 4H, CH2),1.99 (m, 4H, CH2),1.31 (t, J¼7.2Hz, 6H, [m/z]: 585.1 [M-Cl] þ . Anal. Calcd for C32H32ClF2N2Rh: C,61.89; H, NCH2CH3).13CNMR (126MHz,CDCl3,20(cid:2)C): d181.85 (NCNeRh), 5.19;N,4.51;Found:C,61.78;H,5.28;N,4.67. 141.10,140.00,130.57,128.84,127.85,127.69,127.19,126.97(ArphC), Chlorido(h2,h2-cycloocta-1,5-diene)(4,5-bis(4-fluorophenyl)-3- 131.39 (NC¼CN), 98.39, 98.24 (CH¼), 68.47, 68.36 (CH¼), 44.30 (naphthalen-1-ylmethyl)-1-ethylimidazol-2-ylidene) rhodium(I) 8e. (NCH2CH3),32.97(CH2),28.92(CH2),16.33(NCH2CH3).ESI-MS(þ) Yield:23%;yellowsolid.1HNMR(500MHz,CDCl3,20(cid:2)C):d8.12(d, [m/z]:639.42[M-Cl] þ .MALDI-TOF/TOFMS:639.2273[M-Cl] þ .Anal. J¼8.3Hz, 1H, ArnaphH), 7.85 (d, J¼7.9Hz, 1H, ArnaphH), 7.73 (d, CalcdforC39H40ClN2Rh:C,69.38;H,5.97;N,4.15;Found:C,69.15; J¼8.2Hz, 1H, ArnaphH), 7.58 (t, J¼7.2Hz, 1H, ArnaphH), 7.53 (t, H,5.97;N,4.22. J¼7.2Hz,1H, ArnaphH), 7.32 (t, J¼7.7Hz,1H, ArnaphH), 7.22 (dd, Chlorido(h2,h2-cycloocta-1,5-diene)([3-(anthracen-9-ylmethyl)]- J¼8.6,5.3Hz,2H,ArphH),7.05(t,J¼8.6Hz,2H,ArphH),6.97(dd, 4,5-bis(4-fluorophenyl)-1-ethylimidazol-2-ylidene) rhodium(I) 5e. J¼5.3Hz,2H,ArphH),6.93(d,1H,ArnaphH),6.86(d,J¼16.5Hz,1H, Yield63%;yellowsolid.1HNMR(500MHz,CDCl3,20(cid:2)C):d8.36(d, NCH2Arnaph),6.71(t,J¼8.6Hz,2H,ArphH),6.00(d,J¼16.5Hz,1H, J¼8.9Hz,2H,AranthH),8.24(s,1H,AranthH),7.86(d,J¼8.4Hz,2H, NCH2Arnaph),4.99(p,J¼8.1Hz,2H,CH¼),4.90(dq,J¼14.3,7.2Hz, AranthH), 7.52e7.46 (m, 2H, AranthH), 7.43e7.37 (m, 2H, AranthH), 1H, NCH2CH3), 4.65 (dq, J¼14.1, 7.1Hz, 1H, NCH2CH3), 3.54 (t, 7.32 (d, J¼15.2Hz,1H, NCH2Aranth), 6.94 (dd, J¼8.6, 5.4Hz, 2H, J¼6.9Hz,1H,CH¼),3.12(s,1H,CH¼),2.54e2.44(m,1H,CH2),2.29 ArphH), 6.85 (t, J¼8.6Hz, 2H, ArphH), 6.53 (d, J¼15.1Hz, 1H, (m,1H,CH2),2.00e1.87(m,2H,CH2),1.78(m,2H,CH2),1.51e1.39 NCH2Aranth), 6.03e5.95 (m, 2H, ArphH), 5.93 (t, J¼8.7Hz, 2H, (m,2H,CH2),1.35(t,J¼7.2Hz,3H,NCH2CH3).13CNMR(126MHz, ArphH),5.21e5.10(m,2H,CH¼),4.86e4.74(m,2H,NCH2CH3),3.69 CDCl3, 20(cid:2)C): d 184.44,184.03 (NCNeRh),163.76,163.45,161.77, (s, 2H, CH¼), 2.63e2.54 (m, 1H, CH2), 2.44e2.31 (m, 3H, CH2), 161.47,132.10,132.04,124.74,124.71,124.19,124.16,116.04,115.87, 2.14e2.07(m,1H,CH2),2.04e1.90(m,3H,CH2),1.31(t,J¼7.2Hz, 115.57,115.39(ArphC),133.45,133.25,128.67,127.82,126.49,125.95, 3H, NCH2CH3).13C NMR (126MHz, CDCl3, 20(cid:2)C): d 183.11,182.70 125.01, 124.05, 122.60 (ArnaphC), 131.11, 130.28 (NC¼CN), 99.08, (NCNeRh), 163.54, 162.45, 161.56, 160.48, 131.97, 131.90, 131.24, 99.03,97.78,97.72(CH¼),69.13,69.01,68.59,68.47(CH¼),49.93 131.17,124.50,124.47,123.68,123.66,115.61,115.44,113.67,113.50 (NCH2Arnaph), 44.40 (NCH2CH3), 33.55 (CH2), 31.84 (CH2), 28.85 (ArC), 131.84, 131.21 (NC¼CN), 131.07, 130.82, 128.77, 128.65, (CH2), 28.16 (CH2), 16.32 (NCH2CH3). ESI-MS(þ)[m/z]: 635.2 [M- þ 126.53,126.03,124.89,124.60(AranthC),99.09,99.03,98.51,98.46 Cl] . Anal. Calcd for C36H34ClF2N2Rh: C, 64.44; H, 5.11; N, 4.17; (CH¼),69.55,69.44,67.95,67.88(CH¼),48.46(NCH2Arnath),44.53 Found:C,63.94;H,5.07;N,4.37. (NCH2CH3), 33.59 (CH2), 32.44 (CH2), 29.32 (CH2), 28.31 (CH2), Chlorido(h2,h2-cycloocta-1,5-diene)(1,3-diethylimidazol-2- 16.25 (NCH2CH3). ESI-MS(þ)[m/z]:743.14 [MþNa] þ . Anal.Calcd for ylidene)rhodium(I)9e.Yield54%;yellowsolid.1HNMR(500MHz, C40H36ClF2N2Rh:C,66.63;H,5.03;N,3.88;Found:C,66.56;H,4.99; CDCl3,20(cid:2)C):d6.87(s,2H,CH¼),5.02(s,2H,CH¼),4.69(m,2H, N,4.15. NCH2CH3),4.52(m,2H,NCH2CH3),3.31(s,2H,CH¼),2.46e2.37(m, Chlorido(h2,h2-cycloocta-1,5-diene)([3-(cyclopropylmethyl)]-4,5- 4H,CH2),1.94(m,4H,CH2),1.54(t,J¼7.3Hz,6H,CH2CH3).13CNMR bis(4-fluorophenyl)-1-ethylimidazol-2-ylidene) rhodium(I) 6e. Yield (126MHz,CDCl3,20(cid:2)C):d181.60(NCNeRh),119.75(NCH]CHN), 40%;yellowsolid.1HNMR(500MHz,CDCl3,20(cid:2)C):d7.19e7.11(m, 98.23,99.17(CH]CH),67.91,67.80(CH¼),45.59(NCH2CH3),32.95 4H,ArphH),7.08e6.97(m,4H,ArphH),5.10(d,J¼6.9Hz,1H,CH¼), (CH2), 28.87 (CH2),16.31 (NCH2CH3). ESI-MS(þ)[m/z]: 335.09 [M- 5.05(dd,J¼12.6,7.8Hz,1H,CH¼),4.79e4.70(m,2H,NCH2CH3), Cl] þ ,Anal.CalcdforC15H24ClN2Rh:C,48.60;H,6.53;N,7.56;Found: 4.66(td,J¼14.2,7.1Hz,1H,NCH2CH(cyclopropane)),4.32(dd,J¼14.0, C,48.65;H,6.61;N,7.53. 7.1Hz,1H,NCH2CH(cyclopropane)),3.48(t,J¼7.2Hz,1H,CH¼),3.41 (s,1H,CH¼),2.53e2.36(m,4H,CH2),2.06e1.92(m,4H,CH2),1.27 (t,J¼7.2Hz,3H,NCH2CH3),1.14(m,1H,NCH2CH(cyclopropane)),0.38 4.3. X-raycrystallographicanalysis (m,2H,CH2(cyclopropane)),0.25e0.18(m,1H,CH2(cyclopropane)),(cid:4)0.16 (m,1H,CH2(cyclopropane)).13CNMR(126MHz,CDCl3,20(cid:2)C):d182.68, Monocrystal of 1e was obtained by slowly diffusing n-hexane 182.67 (NCNeRh), 163.72, 161.73, 132.31, 132.25, 132.14, 132.07, intoconcentratedsolutionof1eofdichloromethane.Thedatawere 125.34,125.31,124.86,124.83,115.95,115.88,115.77,115.70(ArphC), collectedat173(2)KonaBRUKERSmartAPEXIICCDarea-detector 131.05,130.79(NC¼CN),98.49,98.44,98.28,98.22(CH¼),68.78, diffractometer with graphite-monochromated Mo Ka radiation 68.66, 68.25, 68.14 (CH¼), 53.86 (NCH2CH(cyclopropane)), 44.29 (l¼0.71073Å). Single crystal structure analysis and correction (NCH2CH3),33.23,32.61(CH2),29.24,28.58(CH2),16.15(NCH2CH3), wereperformedusingtheSHELX-2014kitprogram.TheCambridge 11.61 (NCH2CH (cyclopropane)), 5.01 (CH2 (cyclopropane)), 4.33 (CH2 Crystallographic Data Centre: CCDC 1916369. DOI: 10.5517/ (cyclopropane)). ESI-MS(þ)[m/z]: 549.2 [M-Cl] þ . Anal. Calcd for ccdc.csd.cc22b4cy. R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 13 4.4. Growthinhibitoryassay 4.10. Immunofluorescencestaining The antiproliferation effect of rhodium(I) compounds and Theimmunofluorescenceanalysiswascarriedoutaccordingto cisplatin were measured using the MTT cytotoxicity assay. The previouslyreportedmethod[64].ThenucleiofHepG2cellswere processesweredescribedas:adding0.5%MTT(0.5mg/mL,DMSO) dyed by 40,6-Diamidino-2-phenylindole (DAPI). HepG2 cells were reagenttothecellstreatedwiththerhodium(I)complexes,incu- seededin24-wellplatesandculturedfor24h,thentreatedwith1e batingfor4hat37(cid:2)C,5%CO2inahumidifiedenvironment,then (1.5mM, 3mM, and 6mM) for 24h. Incubating HepG2 cells with carefullydiscardingtheculturemedium,adding200mLDMSOper antibodiesovernightat4(cid:2)C,followedbytreatingwithanti-mouse well.The96-wellplatewasshakenonashakeratalowspeeduntil or anti-rabbit IgG for 2hat ambient temperature and then the fully dissolved the formazan (about 10min). Detected the absor- HepG2cellswerewashedwithPBSforthreetimes.Immunofluo- banceofeachwellat490nm. rescencewasdetectedwithafluorescencemicroscope(LeicaDMi8, originalmagnification40(cid:3)). 4.5. Stabilityanalysis 4.11. Westernblottinganalysis per 1 io e d w o a f s 96 in h cu a b n a d te 1 d H w NM ith R 1 s 0 p % ec D tr 2 a O w þ ere 90 ta % ke a n ce i t n on d i i t f r fe il r e e - n d t 3 t o im ve e r s. a HepG2 cells (1(cid:3)106) were inoculated into a 10cm diameter tissueculturedish(10mL/well),andculturedat37(cid:2)C,5%CO2ina humidified environment for 24h, and then treated 1e for 24h, 4.6. InteractionwithGSHandNAC carefully discarded the medium, and washed with cold PBS for threetimes.LysedwithRIPAbufferinanicebath,thencentrifuged, Thesolutionwith50%acetonitrile-d3 þ50%D2Ocontainingthe andthesupernatantswerecollectedandquantifiedbyDCProtein complex1e(2mM)andGSH(2mM)orNAC(2mM)wasanalyzed Assay(Bio-Rad).Samples(15e45mg/lane)wereseparatedby12.5% by1HNMRatdifferenttimes. SDS-PAGEgel,Tris-Glycineasthesurgingsolution.Afterthesepa- rationprocess,theproteinswereimprintedontothePVDFmem- branes and blocked overnight inTBST (20mM Tris-HCl, pH¼7.6, 4.7. PurifiedTrxRenzymeassay[38,62] 0.1%V/VTween-20)containing5%BSAatroomtemperature.The DMF or with 1e (1.5mM, 3mM, 6mM) and rat liver TrxR were blots were incubated with TBST for 1hat ambient temperature, dissolvedin50mLofreactionbufferandthenincubatedin96-well whichcontainedprimaryantibodyand5%BSA.Thenwashedwith plates for 75min. Added 200mL of reaction mixture to each well TBSTforthreetimes,next,incubatedwiththesuitablesecondary and25mL20mMNADPHsolution.Andthereactionwasstartedby antibody for 2h, The bands of protein detected by chem- adding25mLofasolutionof20mMDTNB.Afterappropriatemix- iluminescenceprocedure(ECL,Amersham). ing,recordtheabsorptiondataat405nmwithamicroplatereader. 4.12. Fluorescencespectroscopy TheincreaseinDTNBconcentrationovertimefollowsalineartrend (r2(cid:5)0.99), and the enzyme activity is calculated as its slope Complex 5e was dissolved in DMF to get a clear solution (increasedabsorbancepersecond).Foreachtestcompound,itwas confirmedbynon-interferencetestcomponentsbyusinganega- (100mM), which was then diluted to a suitable concentration in 0.1mM Tris-HCl buffer (pH 7.8). The spectra of 5ewere followed tivecontrolexperimentwithoutanenzymesolution. from 400nm to 480nm (excitation spectra) and from 470nm to 560nm(emissionspectra). 4.8. Quantitativereal-timepolymerasechainreaction 4.13. Cellularuptakeexperiment TrizolreagentwasusedtoextractHepG2RNAaccordingtothe manufacturer’sinstructions.Real-timePCRwasexecutedby7500 HepG2cellswereseededonglassslidesandculturedinDMEM RT-PCRSystemasdescribedpreviously[63].ThemRNAlevelsofthe untilthecelldensityreached60%.Then,theywereincubatedwith targetgeneswerecalculatedandresultsarefromtriplicateexper- 10mMof5eat37(cid:2)C,5%CO2atmospherefor24h.Themediumwas iments. The following primers (GenScript, Nanjing, China) were carefullyaspirated,washedthreetimeswithPBS(1(cid:3))andincu- used: batedwith200nMMitoTracker®RedCMXRos(M7512,Yeasen)for b-actin:(forward)50-TGTGGATCAGCAAGCAGGAGTA-30, 30minat37(cid:2)C.PBS(1(cid:3))waswashedthreemoretimesat37(cid:2)C (reverse)50-TGCGCAAGTTAGGTTTTGTCA-30. andthen fixed with 4%PFA for 15min atroomtemperature.The TrxR:(forward)50-GCCCTGCAAGACTCTCGAAATTA-30, cellswerewashedwithPBS(1(cid:3))threetimesagain,thecellswere (reverse)50-GCCCATAAGCATTCTCATAGACGA-30. storedinPBS(1(cid:3))andfluorescencewascapturedbyaLeicaDMi8 fluorescencemicroscopetovisualize5eandMitoTracker®RedCMX 4.9. CellularactivitiesofTrxRassay Rosrespectively(originalmagnification40(cid:3)). WhenHepG2cellsgrewtoabundanceof70e80%,treatedwith 4.14. IntracellularROSmeasurement different concentrations of 1e and incubated for 48h and 72h. Then,theculturewascarefullydiscardedandwashedtwicewith ROSgenerationinHepG2cellswasassessedbythefluorescent phosphate buffer (PBS). Total cell proteinwas extracted by treat- probe CM-DCFH2-DA (Molecular Probes, Invitrogen). HepG2 cells mentwithRIPAbuffer(150mMNaCl,50mMTris-HCl,pH7.5,0.5% (2(cid:3)104) were inoculated in 24-well plates, incubated for 24h, deoxycholate,2mMEDTA,0.1%SDS,1%TritonX-100,1mMPMSF washedwithPBSbufferandtreatedwith10mMCM-DCFH2-DAfor and1mMNa3VO4)inanicebathandquantifiedusingtheBradford 20minat37(cid:2)Cindarkness.Then,thecellswerewashedwithPBS program.TheactivityofTrxRincellswasdeterminedaccordingto buffer and incubated with different concentrations of 1e (1.5mM, the TrxR activity detection kit (Beijing Solarbio Science & 3mM, 6mM). The fluorescence increase was visualized by a Leica Technology). DMi8fluorescentmicroscopy(originalmagnification20(cid:3)). 14 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 4.15. MeasurementofMMP size of tumor every 3 days and calculate the tumor volume (TV) using the following formula: TV (mm3)¼width2 (length/2). The HepG2cells(2(cid:3)104cells/mL)wereseededoncoverslipsina inhibitionratesoftumorgrowth(IRT)werecalculatedasfollows: 24-wellplateandincubatedfor24hinsupplementalmediumand IRT¼100%(cid:3)(M1 eM2)/M1;M1representsthemeanweightofthe thentreatedwith1eorDMFin5%CO2cultureconditionsat37(cid:2)C tumorintheblankgroupandM2representsthemeanweightofthe for 2h. After that, the cells were treated with JC-1 (5mM) for tumorinthedrugtreatmentgroup. 30min, washed with PBS buffer for twice, the cell staining was observed with a fluorescence microscope (original magnification 4.19.1. Chroniclivercancermodelcausedbytetrachloromethane 40(cid:3)). All experiments involving mice wereapproved by the institu- tional and local ethics committee (Beijing Weitong Lihua Labora- 4.16. Cellcyclearrest tory Animal Technology Co., Ltd., China). According to National Institutes of Health guidelines, all mice were gained humane HepG2 cells (2(cid:3)104cells/well) were seeded in 6-well plates sympathy. Four-week-old male BALB/c mice (18e22g) were and cultured for 24h followed by treatment with DMF or 1e randomly divided into 3 groups of 5 mice each, and all animals (1.5mM, 3mM or 6mM) for another 72h. Cellular DNA flow cyto- werefedunderstandardizedconditionswithalight/darkcycleof metrickits(NanjingKeyGenBiotech)wereusedaccordingtothe appropriate humidity at room temperature, and provided water manufacturer’s protocol to determine the stage of the cell cycle, andstandardpathogen-freefeed.Grouponewasthevehiclecon- specificallyG0/G1,S,orG2/M,byflowcytometry(FACSCalibur;BD, trol,inwhichthemiceweretreatedwithoutCCl4or1etreatment. FranklinLakes,NJ,USA). Group two was the CCl4 model group. Group three was drug treatment groups, inwhich the mice received CCl4 and 1e at the 4.17. Apoptosisanalysis sametime.GroupstwoandthreewereinjectedwithCCl4(5mg/kg) three times a week and treated for 14 weeks to induce hepatic AnannexinV-FITCapoptosisassaykit(NanjingKeyGenBiotech carcinoma. Groups three was intraperitoneally injected with 1e Co., Ltd.) was used according tothe manufacturer’s protocol. The (10mg/kg)dailyfortwoweeks.After14weekstreatment,collected percentages of annexin-positive HepG2 cells without PI staining the liver and blood and the livers were fixed in 4% para- weredetermined byflowcytometry(FACSCalibur;BD).Thedata formaldehyde buffer for subsequent Western blot analysis and wereanalyzedusingCellQuestsoftware. histologicalanalysis. 4.18. DNAbindingtest 4.20. Enzyme-linkedimmunosorbentassay AllspectralanalysesforDNAbindingexperimentsweretested ELISA kits were used to determine the serum levels of tumor in Tris-HCl buffer (5mM, KCl 50mM, pH¼7.8). The ct-DNA necrosis factor, IL-6, IL-8 and TrxR following the manufacturer’s (4.439mM)stocksolutionwasstoredinarefrigeratorat4(cid:2)Cand protocols (Nanjing Sen Bei Jia Biological Technology Co., Ltd., stored for less than 5d before use.1e was dissolved in DMFand Nanjing,China). mixed into a 2mM stock solution, which was diluted tothe cor- responding concentration with Tris-HCl buffer for subsequent 4.21. Biochemicalanalysis spectralexperiments.Similarly,2mMSDSstocksolutionwaspre- paredbydissolvingSDSinTris-HClbuffer. Serumwasseparatedfromwholebloodandassayedforlactate dehydrogenase(LDH)andalanineaminotransferase(ALT),alkaline 4.18.1. UVeVisspectralanalysis phosphatase(ALP)usingacommercialassaykit(NanjingInstitute 1estocksolutionwasdiluted100timeswithTris-HClbufferto ofBioengineering,China),andlevelsofaspartateaminotransferase obtain2.0(cid:3)105Msolution(3mL),thengraduallyaddct-DNAstock (AST)weretestedaccordingtothemanufacturer’sagreement.The solution or SDS stock solution, When the mixture solution had platereaderPerkinElmerFusionReaderwasusedtodeterminethe reactedsufficiently,theUVeVisabsorptionspectrawererecorded. absorbancevalues. 4.18.2. Fluorescencespectralanalysis Notes Fluorescence emission spectroscopy was used to study the competitivebindingexperiments betweenEB orcomplex 1e and Theauthorsdeclarethattheyhavenoconflictofinterest. DNAbyvaryingtheconcentrationof1eandmaintainingthesta- tionaryconcentrationofEB-ct-DNAsystem. Acknowledgments 4.18.3. Circulardichroismspectralanalysis WethankthefinancialsupportsoftheNationalNaturalScience Complex 1e was added to ct-DNA (1.0(cid:3)10 (cid:4)4M) solution and Foundation of China (No. 81703337), the Jiangsu Specially- incubatedat37(cid:2)Cfor5min.Thefinalconcentrationof1ewere0, Appointed Professors programand the Open Project of State Key 2.0,4.0,6.0(cid:3)10 (cid:4)4Mandeachsamplewasrecordedthedatainthe Laboratory of Natural Medicines (No. SKLNMKF201808, rangeof200e320nmbyCDspectrometer. SKLNMKF201712), the State Key Laboratory of Coordination Chemistry, Nanjing University, the Six Talent Peaks Project in 4.19. Tumornudemicemodel JiangsuProvinceofChina(No.SWYY-069). HepG2cells(1.5(cid:3)107/mouse)wereinoculatedsubcutaneously AppendixA. Supplementarydata intomaleBALB/cnudemice(18e22g)tocreateHepG2livercancer mouse models. After the tumor reached 180e200mm3, the mice Supplementarydataassociatedwiththisarticlecanbefoundin wererandomlydividedintotwogroups(n¼4),and1e(10mg/kg) theonlineversion,athttps://doi.org/10.1016/j.ejmech.2019.111721. orthesamevolumeofnormalsalinewasintraperitoneallyinjected ThesedataincludeMOLfileandInChiKeysofthemostimportant everyday,sustained15days,usingaverniercalipertomeasurethe compoundsdescribedinthisarticle. R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 15 References anticanceractivityin2Dand3DspheroidalmodelsofHeLacancercells,Eur.J. Med.Chem.145(2018)291e301. [23] W.Cai,L.Zhang,Y.Song,B.Wang,B.Zhang,X.Cui,G.Hu,Y.Liu,J.Wu,J.Fang, [1] W.Q.Chen,R.S.Zheng,P.D.Baade,S.W.Zhang,H.M.Zeng,F.Bray,A.Jemal, Smallmoleculeinhibitorsofmammalianthioredoxinreductase,FreeRadical X.Q.Yu,J.He,CancerstatisticsinChina,2015,Ca-cancer,J.Clin.66(2016) 115e132. Biol.Med.52(2012)257e265. [24] J.L. Hickey, R.A. Ruhayel, P.J. Barnard, M.V. Baker, S.J. Berners-Price, [2] Y.Jiang,A.Sun,Y.Zhao,W.Ying,H.Sun,X.Yang,B.Xing,W.Sun,L.Ren,B.Hu, A.Filipovska,Mitochondria-targetedchemotherapeutics:therationaldesign C.Li,L.Zhang,G.Qin,M.Zhang,N.Chen,M.Zhang,Y.Huang,J.Zhou,Y.Zhao, of gold(I) N-heterocyclic carbene complexes that are selectively toxic to M.Liu,X.Zhu,Y.Qiu,Y.Sun,C.Huang,M.Yan,M.Wang,W.Liu,F.Tian,H.Xu, cancercellsandtargetproteinselenolsinpreferencetothiols,J.Am.Chem. J.Zhou,Z.Wu,T.Shi,W.Zhu,J.Qin,L.Xie,J.Fan,X.Qian,F.He,F.He,X.Qian, Soc.130(2008)12570e12571. J.Qin,Y.Jiang,W.Ying,W.Sun,Y.Zhu,W.Zhu,Y.Wang,D.Yang,W.Liu, [25] C.Angela,M.Luigi,Molecularmechanismsandproposedtargetsforselected Q.Liu,X.Yang,B.Zhen,Z.Wu,J.Fan,H.Sun,J.Qian,T.Hong,L.Shen,B.Xing, anticancergoldcompounds,Curr.Top.Med.Chem.11(2011)2647e2660. P.Yang,H.Shen,L.Zhang,S.Cheng,J.Cai,X.Zhao,Y.Sun,T.Xiao,Y.Mao, [26] A. Meyer, C.P. Bagowski, M. Kokoschka, M. Stefanopoulou, H. Alborzinia, X.Chen,D.Wu,L.Chen,J.Dong,H.Deng,M.Tan,Z.Wu,Q.Zhao,Z.Shen, S.Can,D.H.Vlecken,W.S.Sheldrick,S.Wolfl,I.Ott,Onthebiologicalprop- X.Chen,Y.Gao,W.Sun,T.Wang,S.Liu,L.Lin,J.Zi,X.Lou,R.Zeng,Y.Wu, ertiesofalkynylphosphinegold(I)complexes,AngewChem.Int.Ed.Engl.51 S.Cai,B.Jiang,A.Chen,Z.Li,F.Yang,X.Chen,Y.Sun,Q.Wang,Y.Zhang, (2012)8895e8899. G.Wang,Z.Chen,W.Qin,Z.Li,C.Chinese,HumanProteomeProject,Prote- omicsidentifiesnewtherapeutictargetsofearly-stagehepatocellularcarci- [27] O. Rackham, A.M.J. Shearwood, R. Thyer, E. McNamara, S.M.K. Davies, noma,Nature567(2019)257e261. B.A. Callus, A. Miranda-Vizuete, S.J. Berners-Price, Q. Cheng, E.S.J. Arner, A. Filipovska, Substrate and inhibitor specificities differ between human [3] F.M.Lu,H.Zhuang,ManagementofhepatitisBinChina,Chin.Med.J.122 (2009)3e4. cytosolicandmitochondrialthioredoxinreductases:implicationsfordevel- opmentofspecificinhibitors,FreeRadicalBiol.Med.50(2011)689e699. [4] A.Jemal,R.Siegel,E.Ward,Y.Hao,J.Xu,M.J.Thun,Cancerstatistics,2009,CA ACancerJ.Clin.59(2009)225e249. [28] W.Liu,K.Bensdorf,M.Proetto,A.Hagenbach,U.Abram,R.Gust,Synthesis, characterization,andinvitrostudiesofbis[1,3-diethyl-4,5-diarylimidazol-2- [5] R.S.Finn,A.X.Zhu,W.Farah,J.Almasri,F.Zaiem,L.J.Prokop,M.H.Murad, ylidene]gold(I/III)complexes,J.Med.Chem.55(2012)3713e3724. K.Mohammed,Therapiesforadvancedstagehepatocellularcarcinomawith [29] T.Zou,C.T.Lum,S.S.Chui,C.M.Che,Gold(III)complexescontainingN-het- macrovascularinvasionormetastaticdisease:asystematicreviewandmeta- analysis,Hepatology67(2018)422e435. erocyclic carbene ligands: thiol “switch-on” fluorescent probes and anti- [6] J.C.Nault,O.Sutter,P.Nahon,N.Ganne-Carrie(cid:1),O.Se(cid:1)ror,Percutaneoustreat- [30] J c . a G n e(cid:1) c r e a r r a d g , e V n . t A s, n A n n e, g T e . w Si C d h en em ,F . e I r n r t o . c E if d e . n E t n y g p l e .5 a 2 nt ( i 2 c 0 a 1 n 3 c ) er 29 d 3 ru 0 g e s 2 , 9 C 3 h 3 e . m.Soc.Rev. mentofhepatocellularcarcinoma:stateoftheartandinnovations,J.Hepatol. 68(2017)783e797. 44(2015)8802e8817. [31] W.Liu,R.Gust,UpdateonmetalN-heterocycliccarbenecomplexesaspo- [7] J.S. Dawane, V.A. Pandit, Understanding redox homeostasis and its role in cancer,J.Clin.Diagn.Res.6(2012)1796e1802. tentialanti-tumormetallodrugs,Coord.Chem.Rev.329(2016)191e213. [8] E.S.J.Arne(cid:1)r,Focusonmammalianthioredoxinreductases–Importantseleno- [32] J.J.Zhang,J.K.Muenzner,M.A.AbuelMaaty,B.Karge,R.Schobert,S.Wolfl, I. Ott, A multi-target caffeine derived rhodium(I) N-heterocyclic carbene proteins with versatile functions, Biochim. Biophys. Acta Bioenerg. 1790 (2009)495e526. complex: evaluation of the mechanism of action, Dalton Trans. 45 (2016) [9] S.E.Eriksson,S.Prast-Nielsen,E.Flaberg,L.Szekely,E.S.J.Arne(cid:1)r,Highlevelsof 13161e13168. thioredoxin reductase 1 modulate drug-specific cytotoxic efficacy, Free [33] L. Oehninger, L.N. Kuster, C. Schmidt, A. Munoz-Castro, A. Prokop, I. Ott, RadicalBiol.Med.47(2009)1661e1671. Achemical-biologicalevaluationofrhodium(I)N-heterocycliccarbenecom- plexesasprospectiveanticancerdrugs,Chem.EurJ.19(2013)17871e17880. [10] J.Zhang,X.Li,X.Han,R.Liu,J.Fang,Targetingthethioredoxinsystemfor cancertherapy,TrendsPharmacol.Sci.38(2017)794e808. [34] W.Liu,K.Bensdorf,M.Proetto,U.Abram,A.Hagenbach,R.Gust,NHCgold halide complexes derived from 4,5-diarylimidazoles: synthesis, structural [11] M.Bian,R.Fan,S.Zhao,W.Liu,Targetingthethioredoxinsystemasastrategy forcancertherapy,J.Med.Chem.62(2019)7309e7321.https://doi.org/10. analysis,andpharmacologicalinvestigationsaspotentialantitumoragents, J.Med.Chem.54(2011)8605e8615. 1021/acs.jmedchem.8b01595. [35] W.Liu,K.Bensdorf,A.Hagenbach,U.Abram,B.Niu,A.Mariappan,R.Gust, [12] I.S. Harris, A.E. Treloar, S. Inoue, M. Sasaki, C. Gorrini, K.C. Lee, K.Y. Yung, SynthesisandbiologicalstudiesofsilverN-heterocycliccarbenecomplexes D.Brenner,C.B.Knobbe-Thomsen,M.A.Cox,A.Elia,T.Berger,D.W.Cescon, derivedfrom4,5-diarylimidazole,Eur.J.Med.Chem.46(2011)5927e5934. A. Adeoye, A. Brustle, S.D. Molyneux, J.M. Mason, W.Y. Li, K. Yamamoto, [36] L.Oehninger,S.Spreckelmeyer,P.Holenya,S.M.Meier,S.Can,H.Alborzinia, A. Wakeham, H.K. Berman, R. Khokha, S.J. Done, T.J. Kavanagh, C.W. Lam, J. Schur, B.K. Keppler, S. Wolfl, I. Ott, Rhodium(I) N-heterocyclic carbene T.W. Mak, Glutathione and thioredoxin antioxidant pathways synergize to drivecancerinitiationandprogression,CancerCell27(2015)211e222. bioorganometallicsasinvitroantiproliferativeagentswithdistincteffectson cellularsignaling,J.Med.Chem.58(2015)9591e9600. [13] H.Lei,G.Wang,J.Zhang,Q.J.Han,InhibitingTrxRsuppresseslivercancerby [37] H.Petzold,P.J.Sadler,Oxidationinducedbytheantioxidantglutathione(GSH), inducingapoptosisandelicitingpotentantitumorimmunity,Oncol.Rep.40 (2018)3447e3457. Chem.Commun.50(2008)4413e4415. [38] I. Ott, X. Qian, Y. Xu, D.H.W. Vlecken, I.J. Marques, D. Kubutat, J. Will, [14] X.Zheng,W.Ma,R.Sun,H.Yin,F.Lin,Y.Liu,W.Xu,H.Zeng,Butaselenpre- W.S.Sheldrick,P.Jesse,A.Prokop,C.P.Bagowski,Agold(I)phosphinecomplex ventshepatocarcinogenesisandprogressionthroughinhibitingthioredoxin reductaseactivity,RedoxBiol.14(2018)237e249. containing a naphthalimide ligand functions as a TrxR inhibiting anti- proliferative agent and angiogenesis inhibitor, J. Med. Chem. 52 (2009) [15] O.Rackham,A.M.Shearwood,R.Thyer,E.McNamara,S.M.Davies,B.A.Callus, 763e770. A. Miranda-Vizuete, S.J. Berners-Price, Q. Cheng, E.S. Arner, A. Filipovska, Substrate and inhibitor specificities differ between human cytosolic and [39] J.L.Tsai,A.O.Chan,C.M.Che,Aluminescentcyclometalatedgold(iii)-avidin conjugate with a long-lived emissive excited state that binds to proteins mitochondrialthioredoxinreductases:implicationsfordevelopmentofspe- cificinhibitors,FreeRadicalBiol.Med.50(2011)689e699. andDNAandpossessesanti-proliferationcapacity,Chem.Commun.51(2015) [16] L.Bjo€rkhem,H.Teclebrhan,E.Kesen,J.M.Olsson,L.C.Eriksson,M.Bjo€rnstedt, 8547e8550. [40] H.Pelicano,D.Carney,P.Huang,ROSstressincancercellsandtherapeutic IncreasedlevelsofcytosolicthioredoxinreductaseactivityandmRNAinrat livernodules,J.Hepatol.35(2001)259e264. implications,DrugResist.Updates7(2004)97e110. [17] Y.Min-Hyuk,X.Xue-Ming,B.A.Carlson,V.N.Gladyshev,D.L.Hatfield,Thio- [41] S. Keisuke, H. Hitomi, T. Eriko, M. Reiko, I. Masako, I. Yuji, S. Kazuhiko, redoxinreductase1deficiencyreversestumorphenotypeandtumorigenicity S.Makoto,M.Kunitoshi,AntioxidantN-acetyl-L-cysteine(NAC)supplemen- oflungcarcinomacells,J.Biol.Chem.281(2006)13005e13008. tationreducesreactiveoxygenspecies(ROS)-mediatedhepatocellulartumor [18] C.Skogastierna,M.Johansson,P.Parini,M.Eriksson,L.C.Eriksson,L.Ekstro€m, promotion of indole-3-carbinol (I3C) in rats, J. Toxicol. Sci. 36 (2011) L.Bjo€rkhem-Bergman,StatinsinhibitexpressionofThioredoxinreductase1in [42] J 7 .D 75 . e Ly 7 , 8 D 6 . . R.Grubb,A.Lawen,Themitochondrialmembranepotential(Djm)in ratandhumanliverandreducetumordevelopment,Biochem.Biophys.Res. Commun.417(2012)1046e1051. apoptosis;anupdate,Apoptosis8(2003)115e128. [19] C. Anna, F. Alessandra, B. Alberto, P. Pascal, T. Siden, V. Anne, S. Miche(cid:3)le, [43] Z. Chen, T. Shi, L. Zhang, P. Zhu, M. Deng, C. Huang, T. Hu, L. Jiang, J. Li, J. Ge(cid:1)rard, R. Maria Pia, Evidence for targeting thioredoxin reductases with MammaliandrugeffluxtransportersoftheATPbindingcassette(ABC)family inmultidrugresistance:areviewofthepastdecade,CancerLett.370(2016) ferrocenyl quinone methides. A possible molecular basis for the anti- 153e164. proliferative effectof hydroxyferrocifens oncancercells, J.Med. Chem.57 (2015)8849e8859. [44] E.S.J.Arner,A.Holmgren,Thethioredoxinsystemincancer,Semin.Cancer Biol.16(2006)420e426. [20] O. Karaca,V.Scalcon, S.M.Meier-Menches, R.Bonsignore, J.M.J.L.Brouwer, [45] L.U.Xiaoye,X.Y.Zhong,Caspasesandapoptosis,EssaysBiochem.35(2000) F.Tonolo,A.Folda,M.P.Rigobello,F.E.Kuhn,A.Casini,Characterizationof 9e19. hydrophilicgold(I)N-heterocycliccarbene(NHC)complexesaspotentTrxR [46] C.G.Hartinger,P.J.Dyson,Bioorganometallicchemistry–fromteachingpara- inhibitors using biochemical and mass spectrometric approaches, Inorg. Chem.56(2017)14237e14250. digmstomedicinalapplications,Chem.Soc.Rev.38(2009)391e401. [47] A. Wee Han, M. Myatnoezin, S.J. Lippard, Transcription inhibition by [21] J.Zhang,B.Zhang,X.Li,X.Han,R.Liu,J.Fang,Smallmoleculeinhibitorsof platinum-DNA cross-links in live mammalian cells, J. Am. Chem. Soc. 132 mammalianthioredoxinreductaseaspotentialanticanceragents:anupdate, Med.Res.Rev.39(2018)5e39. (2010)7429e7435. [48] G.Gilles,O.Ingo,M.N.Nils,Organometallicanticancercompounds,J.Med. [22] T.S.Reddy,S.H.Priver,N.Mirzadeh,S.K.Bhargava,Synthesisofgold(I)phos- Chem.54(2011)3e25. phine complexes containing the 2-BrC6F4PPh2 ligand: Evaluation of [49] J.J.Wilson,S.J.Lippard,Syntheticmethodsforthepreparationofplatinum 16 R.Fanetal./EuropeanJournalofMedicinalChemistry183(2019)111721 anticancercomplexes,Chem.Rev.45(2014)4470e4495. consequencesforanti-canceractivity,Metallomics4(2012)628e632. [50] S.M. Meier-Menches, C. Gerner, W. Berger, C.G. Hartinger, B.K. Keppler, [58] J.Cao,Y.Zheng,X.W.Wu,C.P.Tan,M.H.Chen,N.Wu,L.N.Ji,Z.Mao,Anti- Structure-activityrelationshipsforrutheniumandosmiumanticanceragents cancer cyclometalated iridium(III) complexes with planar ligands: mito- -towardsclinicaldevelopment,Chem.Soc.Rev.47(2017)909e928. chondrialDNAdamageandmetabolismdisturbance,J.Med.Chem.62(2019) [51] O € .Karaca,S.M.Meier-Menches,A.Casini,F.E.Kühn,Onthebindingmodesof 3311e3322. metal NHC complexes with DNA secondary structures: implications for [59] Z. Zhu, Z. Wang, C. Zhang, Y. Wang, H. Zhang, Z. Gan, Z. Guo, X. Wang, therapyandimaging,Chem.Commun.53(2017)8249e8260. Mitochondrion-targeted platinum complexes suppressing lung cancer [52] L.Ma,W.Na,R.Ma,C.Li,M.K.Tse,G.Zhu,Monochalcoplatin:anactively through multiple pathways involving energy metabolism, Chem. Sci. 10 transported,quicklyreducible,andhighlypotentPt(IV)anticancerprodrug, (2019)3089e3095. AngewChem.Int.Ed.Engl.57(2018)9098e9102. [60] P. Newell, A. Villanueva, S.L. Friedman, K. Koike, J.M. Llovet, Experimental [53] W.Streciwilk,A.Terenzi,F.L.Nardo,P.Prochnow,J.E.Bandow,B.K.Keppler, modelsofhepatocellularcarcinoma,J.Hepatol.48(2008)858e879. I. Ott, Synthesis and biological evaluation of organometallic complexes [61] M.T. Guimara~es, C. Eleazar, F.J.H. Da, L.O.A.C. Rodrigues, S.F. Duarte, bearing bis-1,8-naphthalimide ligands, Eur. J. Inorg. Chem. 26 (2018) R.J.M.A. Fontenelle, G.O.F.H. Ferreira, D.A.L.A. Carneiro, Review of experi- 3104e3112. mentalmodelsforinducinghepaticcirrhosisbybileductligationandcarbon [54] W. Streciwilk, A. Terenzi, X. Cheng, L. Hager, Y. Dabiri, P. Prochnow, tetrachlorideinjection,ActaCir.Bras.27(2012)589e594. J.E. Bandow, S. Wolfl, B.K. Keppler, I. Ott, Fluorescent organometallic rho- [62] K.Wang,C.Zhu,Y.He,Z.Zhang,W.Zhou,N.Muhammad,Y.Guo,X.Wang, dium(I)andruthenium(II)metallodrugswith4-ethylthio-1,8-naphthalimide Z. Guo, Restraining cancer cells by dual metabolic inhibition with a ligands:antiproliferativeeffects,cellularuptakeandDNA-interaction,Eur.J. mitochondrion-targeted platinum(II) complex, Angew. Chem. Int. Ed. 58 Med.Chem.156(2018)148e161. (2019)4638e4643. [55] J.R. McConnell, D.P. Rananaware, D.M. Ramsey, K.N. Buys, M.L. Cole, [63] H.J. Kim, H.S. Yoo, J.C. Kim, C.S. Park, M.S. Choi, M. Kim, H.Choi, J.S. Min, S.R. McAlpine, A potential rhodium cancer therapy: studies of a cytotoxic Y.S.Kim,S.W.Yoon,J.K.Ahn,AntiviraleffectofCurcumalongaLinnextract organorhodium(I)complexthatbindsDNA,Bioorg.Med.Chem.Lett23(2013) againsthepatitisBvirusreplication,J.Ethnopharmacol.124(2009)189e196. 2527e2531. [64] S.Li,Q.Wang,Y.Tao,C.Liu,Swertiamarinattenuatesexperimentalrathepatic [56] D.L.Ma,C.M.Che,Abifunctionalplatinum(II)complexcapableofintercalation fibrosis by suppressing angiotensin II-angiotensin type 1 receptor- andhydrogen-bondinginteractionswithDNA:bindingstudiesandcytotox- extracellularsignal-regulatedkinasesignaling,J.Pharmacol.Exp.Ther.359 icity,Chem.EurJ.9(2003)6133e6144. (2016)247e255. [57] J. Reedijk, Fast and slow versus strong and weak metal-DNA binding: