A new C,N-cyclometalated osmium(ii) arene anticancer scaffold with a handle for functionalization and antioxidative properties.

ChemComm COMMUNICATION A new C,N-cyclometalated osmium( ) II arene anticancer scaffold with a handle for Citethis:DOI:10.1039/c8cc06427j functionalization and antioxidative properties† Received7thAugust2018, Enrique Ortega, a Jyoti G. Yellol,a Matthias Rothemund,b Accepted4thSeptember2018 Francisco J. Ballester, a Venancio Rodr´ıguez, a Gorakh Yellol,a DOI:10.1039/c8cc06427j Christoph Janiak, c Rainer Schobert b and Jose´ Ruiz *a rsc.li/chemcomm Aseriesofsixosmium(II)complexesofthetype[(g6-p-cymene)Os(C^N)X] compoundscanexhibitadistinctiveabilitytomodulatethelevel (X=chloridooracetato)containingbenzimidazoleC^Nligandswith of intracellular ROS, which are key signalling molecules within an ester group as a handle for further functionalization have been cancer cells associated with tight redox regulation and tumor synthesized.TheyexhibitIC valuesinthelowmicromolarrangeina progression.6 While several metallodrugs have been described 50 panel of cisplatin (CDDP)-resistant cancer cells (approximately as ROS-generating agents that cause oxidative stress,5,7 some 10(cid:2) more cytotoxic than CDDP in MCF-7), decrease the levels of examples of reduction of ROS have also been reported.8–10 In intracellularROSandreducetheNAD+coenzyme,andinhibittubulin fact, abrogating ROS signalling has been established as an polymerization. This discovery could open the door to a new large effectivestrategytoinhibitcancercellproliferation.11 familyofosmium(II)-basedbioconjugateswithdiversemodesofaction. Werecentlyreportedaseriesofhalf-sandwich‘‘piano-stool’’ C,N-cyclometalatedruthenium(II)anticancercomplexesbearing Mechanistically, CDDP and carboplatin exert their anticancer the benzimidazole pharmacophore with promising biological activitythroughtheformationofplatinum–DNAadducts,inter- activity,12andanoctahedralbenzimidazoleiridium(III)conjugate feringwithtranscription,DNAreplicationandmitosisandthus to tumor-targeting vectors based on octreotide peptide.13 leadingtocelldeath.1However,duetotheissuesofresistance Accordingly, in continued efforts towards developing novel, and toxicity the development of new cancer treatments is bettermetallodrugs,herewediscloseaseriesoforganometallic crucial.Inthisway,animpressivenumberofmetalcomplexes osmium(II) complexes of the type [(Z6-p-cymene)Os(C^N)X] have been explored as chemotherapeutic agents.2 Thus the (Fig. 1A) containing a 2-arylbenzimidazole C^N ligand which ruthenium(III) complex NKP-1339 is undergoing clinical trials incorporatesanestergroupforfurtherfunctionalization.They forcancertreatment,3aandRuII(Z6-arene)complexeshavebeen were synthesized using the generalized procedure shown in investigatedfortheirtunabilityandnovelmodesofaction.3b–e Fig.1A.Thecorrespondingbenzimidazoleligandsweretreated However,the5dmetalionOs(II),theheaviercongenerofRu(II), with the p-cymene osmium(II) dimer [(p-cymene)OsCl 2 ] 2 and hasattractedcomparativelylessattentionasa chemotherapeutic sodiumacetatetoobtainthecorrespondingosmiumcomplexes agent,4anditsclinicalapplicabilityforcancertreatmentremains (1–6)inmoderatetogoodyield(47–68%),isolatedaschlorido tobedetermined.Themechanismofactionofosmium(II)-based oracetatoderivativesdepending,probably,onthesolubilityof anticancer agents in vitro often involves cell-cycle progression themonomer.Thestructuresof1–6wereconfirmedby1Hand blockage and the induction of apoptosis through the generation 13CNMR,IR(Fig.S1–S15intheESI†)andESI-MSspectrometry, of reactive oxygen species (ROS).5 Interestingly, organometallic elementalanalysisandX-raycrystallography(for1and6). In the 1H NMR spectra of 1–6 the disappearance of one aromaticprotonofthe2-arylbenzimidazoleligandwasobserved, aDepartamentodeQu´ımicaInorga´nicaandRegionalCampusofInternational andtheareneprotonsofp-cymeneexhibitedfournon-equivalent Excellence‘‘CampusMareNostrum’’,UniversidaddeMurcia,andBiomedical doublets.Thepresenceofasingletat1.60ppmfor5and6was ResearchInstituteofMurcia(IMIB-Arrixaca),E-30071Murcia,Spain. assignedtothemethylgroupoftheacetatoligand.Thepositive E-mail:jruiz@um.es;Tel:+34868887455 bOrganicChemistryLaboratory,UniversityBayreuth,Bayreuth, ionESI-MS spectra displayed[M (cid:3) Cl]+ (for 1–4) or[M (cid:3) OAc]+ Universitaetsstrasse30,D-95440,Germany (for 5 and 6) peaks in methanolic solution with the expected cInstitutfu¨rAnorganischeChemieundStrukturchemie,Heinrich-Heine-Universita¨t isotopicdistributionpattern. Du¨sseldorf,Universita¨tsstrasse1,D-40225Du¨sseldorf,Germany The molecular structures of 1 and 6 are shown in Fig. 1B. †Electronicsupplementaryinformation(ESI)available:Synthesis,characterization CrystallographicdataarelistedinTableS4for1andTableS5for dataandbiologicalstudydetails.CCDC1859533(1)and1859534(6).ForESIand crystallographicdatainCIForotherelectronicformatseeDOI:10.1039/c8cc06427j 6(ESI†).Theosmiumcentersin1and6adoptahalf-sandwich Thisjournalis©TheRoyalSocietyofChemistry2018 Chem.Commun. .MA 63:84:9 8102/71/9 no atokaD htuoS fo ytisrevinU yb dedaolnwoD .8102 rebmetpeS 40 no dehsilbuP View Article Online View Journal Communication ChemComm andalsonon-tumorigenichumanendothelialhybridcellsEA.hy926 and Buffalo green monkey cells BGM. For comparison, CDDP cytotoxicity was also evaluated. All Os compounds exhibited high antiproliferative activities against the studied cancer cell lineswithIC valuesinthelowmicromolarrange(seeTable1) 50 andtheywereabletoovercometheacquiredresistancetoCDDP intheA2780cisRcellline(Table1).Theirresistancefactors(RFs) were much lower than that of CDDP (values below 2 vs. 30),9 suggesting that their mode of action is different from that of CDDP. On the other hand, a slight reductionof the anticancer activity towards the multidrug resistant MCF-7, the highly metastatic 518A2 and HCT116wt with respect to A2780 was observed. It is worth noting that 1–6 proved markedly more cytotoxic than CDDP (440 mM) in MCF-7 (10–60-fold), which is inherently resistant to CDDP. Likewise, of interest are the similar IC values obtained in both wildtype HCCT116wt and 50 Fig.1 Synthesisofcomplexes1–6(A).Molecularstructures(B)withatom p53knock-outHCT116(cid:3)/(cid:3)coloncarcinomacells,whichsuggests numberingschemesfor1and6areshownwiththermalellipsoidsatthe that molecular mechanisms underlying cell death induction by 50%probabilitylevel. theOscomplexesmightbep53-independent.Inadditiontothis, the in vitro antiproliferative activity was evaluated against the ‘‘three-legpiano-stool’’geometry.Theselectedbondlengthsand non-tumorigenic EA.hy926 and BGM cell lines to determine anglesof1and6arelistedinTableS6for1andTableS7for6 the differential selectivity for tumor cells. The toxicities of the (ESI†). The Os–chlorido bond length for 1 was found to be complexeswerefoundtobecomparabletothatofCDDPwitha 2.4164 (9) Å, a typical value for organometallic Os complexes.14 slightly highercytotoxicity against cancercells. Overall, 2 and 3 The Os–arene distance for 1 was larger than in 6 (1.714(1) and were the most potent agents with higher selectivity factor (SF) 1.680(1) Å, respectively). C(cid:4)(cid:4)(cid:4)H and H(cid:4)(cid:4)(cid:4)H close intermolecular valuesinalltestedcancercelllines(TablesS1andS2intheESI†). contacts were the most important non-covalent intermolecular ThecellularconcentrationsofmetalsinA2780cellshavingbeen interactionsforthepackingofthesecomplexes(seeFig.S32–S34, exposedto2,3orCDDPfor24hweredeterminedbyICP-MSin and Table S8 in the ESI†). There are no significant p(cid:4)(cid:4)(cid:4)p order to investigate the relationship between cellular uptake interactions.9 Hydrolysis of the Os–X bond (X = Cl or OAc) is and cytotoxicity. The results (Fig. S28, ESI†) indicate that relativelyrapidinMeOD-d /D Omixturesasobservedby1HNMR the cellular uptakes of both 2 and 3 are similar and 10-fold 4 2 (Fig.S16andS17,ESI†).Partialreversibilityofthehydrolysisof4 higher than that of Pt. In addition, the amount of osmium wasobservedwhenNaCl(4mM)wasaddedtotheMeOD-d /D O bound to DNA in A2780 cells (as measured by ICP-MS) was 4 2 solution (Fig. S16D, ESI†). The HPLC chromatogram (Fig. S18, below1pgOs/mgDNA,suggestingthatDNAisnotlikelytobe ESI†) of 2 in RPMI culture medium (which contains a high the main target of the present complexes (Table S3 in the concentration of salts) remains unaltered after 24 h, with the ESI†).16 The ability of 2 and 3 to induce apoptosis in A2780 ESI-MSspectrumdisplayingthe[M(cid:3)Cl]+peaks. cellswasalsoevaluated.AsshowninFig.2A,complexes2and3 Theantiproliferativeactivitiesofthesixosmiumcompounds considerably increased the percentage of early apoptotic cells containing a butyl group attached to the benzimidazole C^N (Annexin V+/PI(cid:3)) following 48 h treatment with respect to ligand and a handle for functionalization were evaluated in a controls, whereas the necrotic population (Annexin V(cid:3)/PI+) panel of human cancer cell lines, including cells of the epithelial shows no significant increase. In contrast, the most cytotoxic ovariancarcinomaA2780,CDDP-resistantovariancancerA2780cisR, complex6(Fig.S22intheESI†)contributedtonecroticcelldeath breastcancerMCF7,518A2melanoma,coloncarcinomaHCCT116wt rather than apoptosis induction, which could explain its lack of (wildtype), colon carcinoma HCCT116(cid:3)/(cid:3) (p53 knock-out mutant), selectivityforcancercells. Table1 IC (mM)valuesfor1–6andCDDPafter48h.a 50 Complex A2780 A2780cisR(RF) MCF7 518A2 HCT116wt HCT116[(cid:3)/(cid:3)] EA.hy926 BGM 1 3.6(cid:5)0.7 3.4(cid:5)0.1(0.9) 4.4(cid:5)0.1 6.1(cid:5)0.4 4.5(cid:5)0.1 5.5(cid:5)0.6 5.7(cid:5)0.2 14.2(cid:5)0.5 2 2.0(cid:5)0.2 1.8(cid:5)0.1(0.9) 3.7(cid:5)0.1 4.8(cid:5)0.3 3.8(cid:5)0.4 3.6(cid:5)0.3 4.9(cid:5)0.6 9.8(cid:5)0.7 3 1.9(cid:5)0.1 1.89(cid:5)0.09(1.0) 4.2(cid:5)0.1 4.8(cid:5)0.7 3.8(cid:5)0.2 4.6(cid:5)0.6 4.9(cid:5)0.2 11(cid:5)1 4 2.5(cid:5)0.5 3.0(cid:5)0.5(1.2) 4.9(cid:5)0.1 4.1(cid:5)0.5 4.9(cid:5)0.2 3.8(cid:5)0.1 3.6(cid:5)0.2 7.6(cid:5)0.2 5 2.0(cid:5)0.1 3.7(cid:5)0.2(1.9) 3.1(cid:5)0.2 6.9(cid:5)2.3 8.8(cid:5)0.8 6.9(cid:5)0.8 9.0(cid:5)1.0 7.6(cid:5)0.2 6 0.98(cid:5)0.03 1.0(cid:5)0.1(1.0) 0.76(cid:5)0.03 3.1(cid:5)0.6 2.3(cid:5)0.2 2.9(cid:5)0.1 3.1(cid:5)0.1 1.7(cid:5)0.1 CDDP 1.5(cid:5)0.2 44(cid:5)4(30.6) 47(cid:5)3 2.7(cid:5)0.2 10.3(cid:5)0.2 18.0(cid:5)1.8 5.7(cid:5)0.2 9.8(cid:5)0.4 aThecellviabilitywasdeterminedbytheMTTassayafter48htreatmentandtheIC valueswerecalculatedasdescribedintheExperimental 50 section.Eachvaluerepresentsthemean(cid:5)SDofthreeindependentexperiments.Theresistancefactorsaregiveninparentheses. Chem.Commun. Thisjournalis©TheRoyalSocietyofChemistry2018 .MA 63:84:9 8102/71/9 no atokaD htuoS fo ytisrevinU yb dedaolnwoD .8102 rebmetpeS 40 no dehsilbuP View Article Online ChemComm Communication 2070-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. DCFH-DAwasconvertedtothefluorescentproduct20,70-dichloro- fluorescein (DCF)byROS.Asshown in Fig.2B, the DCF fluores- cenceintensityshowedadose-dependentdecreaseupontreatment with 2 and 3 when compared to CDDP at 24 h. In addition to this, the levels of ROS were monitored by a DCFH-DA assay corroboratingtheantioxidantpropertiesof2,whichinduceda reductionofROSofupto20%in2h(Fig.S25andS26inthe ESI†). ROS are not by-products of cellular metabolism but ratherkeysignallingmoleculesinterveningincancerproliferation pathways.11 Although several organometallic compounds have been described as generators of ROS,5–7 other complexes are knowntoinducecelldeathbyreductivestress.8–10,17Inthisstudy, weshowedthattheadditionof2or3causedadecreaseoftheROS level below the threshold that cancer cells require for survival probably due to the disruption of multiple intracellular redox reactions.Ontheotherhand,mitochondrialmembranepotential (MMP)disruptionisinvolvedinthemodeofactionofnumerous organometallic anticancer compounds.7a,18,19 The treatment of A2780cellswith2or3didnotleadtoasignificantreductionof fluorescenceoftheMMPintegrityindicator,Rhodamine-123dye, comparedtountreatedcontrols(Fig.S23andS24intheESI†).To furthercharacterizethecytotoxiceffectofourOscomplexes,A2780 cellsweretreatedwith2,3orCDDPfor24handanalyzedbyflow cytometry using propidium iodide staining. CDDP induces cell cycle arrests in the S and G phases according to previous 2 reports.7aHowever,themodulationofthecellcycleofA2780cells upontreatmentwith2or3differedfromthatofcellstreatedwith CDDP (Fig. 2C). In fact, 2 and 3 caused a dose-dependent G /G 0 1 arrest with minor effects on the S or G /M phase. These results 2 indicateanactivationofcellcycleblockageinresponsetocellular oxidative statusimbalance asG arrest has been associatedwith 1 lowROSlevels.20 The lack of MMP disturbance and the induced decrease in ROS levels upon cell treatment ruled out ROS-mediated mito- chondrialdysfunctionasatriggerforcelldeath.However,flow cytometry experiments confirmed apoptosis and cell cycle arrest as the mechanism of cell death induction. Rather, the ability of 2 and 3 to effectively participate in the reduction of NAD+toNADHtogetherwiththedepletionofintracellularROS Fig.2 Apoptosisinducingeffectsof2and3after48htreatmentofA2780cells atfinalequitoxicconcentrationsdeterminedbyflowcytometry(A).ROSlevels inducedby2and3after24h(B).CellcycleanalysisofA2780cellstreatedwith2, 3 or CDDP for 24 h (C). Experiments were performed in triplicate, *po0.05,**po0.01,two-tailedStudent’st-test. Then we explored the ability of the osmium complexes to interveneinthereductionofnicotinamideadeninenucleotide (NAD+)toNADHasthisredoxpairisinvolvedinrelevantredox signalling pathways within cells.15 The catalytic formation of NADHwasmonitoredbyUV-VismeasuringtheUVabsorption of NADH at 339 nm (Fig. S19–S21 in the ESI†). For both complexes 2 and 3 an increase in the intensity of the NADH absorption was observed. The turnover frequency reached a maximumof9and10at4hfor2and3,respectively. Fig.3 Effectsontheinvitrotubulinpolymerizationby10mMof2,3and Next, we investigated the intracellular ROS levels after colchicineascontrol,determinedbyODmeasurementsat340nmover treatmentofA2780cellswiththeOscomplexes,detectedusing 90minutesat371C. Thisjournalis©TheRoyalSocietyofChemistry2018 Chem.Commun. .MA 63:84:9 8102/71/9 no atokaD htuoS fo ytisrevinU yb dedaolnwoD .8102 rebmetpeS 40 no dehsilbuP View Article Online Communication ChemComm levelsindicatedashiftintheintracellularredoxbalancetoward P. J. Dyson, Organometallics, 2012, 31, 5677–5685; (d) B. S. Murray, areductivestressenvironmentwhereseveralmetabolicreactions M.V.Babak,C.G.HartingerandP.J.Dyson,Coord.Chem.Rev.,2016, 306,86–114;(e)G.S.Yellol,A.Donaire,J.G.Yellol,V.Vasylyeva,C.Janiak couldbeimpaired,thuscausingaselectivearrestinprogression andJ.Ruiz,Chem.Commun.,2013,49,11533–11535. fromtheG /G toSphase,whichprobablytriggeredtheapoptotic 4 (a) M. Hanif, A. A. Nazarov, C. G. Hartinger, W. Kandioller, 0 1 program. 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