Synthesis of organometallic ruthenium(II) complexes with strong activity against several human cancer cell lines.

JournalofInorganicBiochemistry114(2012)65–74 ContentslistsavailableatSciVerseScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Synthesis of organometallic ruthenium(II) complexes with strong activity against several human cancer cell lines Tânia S. Morais a, Tiago J.L. Silva a,b, Fernanda Marques c, M. Paula Robalo d,e, Fernando Avecilla f, Paulo J. Amorim Madeira g, Paulo J.G. Mendes b, Isabel Santos c, M. Helena Garcia a,⁎ aCentrodeCiênciasMoleculareseMateriais,FaculdadedeCiênciasdaUniversidadedeLisboa,CampoGrande,1749‐016Lisboa,Portugal bCentrodeQuímicadeÉvora,UniversidadedeÉvora,RuaRomãoRamalho59,7002‐554Évora,Portugal cUnidadedeCiênciasQuímicaseRadiofarmacêuticas,InstitutoTecnológicoeNuclear,EstradaNacional10,2686‐953Sacavém,Portugal dÁreaDepartamentaldeEngenhariaQuímica,InstitutoSuperiordeEngenhariadeLisboa,Av.ConselheiroEmídioNavarro1,1959‐007Lisboa,Portugal eCentrodeQuímicaEstrutural,InstitutoSuperiorTécnico,Av.RoviscoPais,1049‐001Lisboa,Portugal fDepartamentodeQuímicaFundamental,UniversidadedaCoruña,CampusdeAZapateria15071,ACoruña,Spain gCentrodeQuímicaeBioquímica,FaculdadedeCiênciasdaUniversidadedeLisboa,CampoGrande,1749‐016Lisboa,Portugal a r t i c l e i n f o a b s t r a c t Articlehistory: Anewfamilyof“RuCp”(Cp=η5-CH)derivativeswithbidentateN,OandN,N′-heteroaromaticligandsrevealed 5 5 Received23February2012 outstanding cytotoxic properties against several human cell lines namely, A2780, A2780CisR, HT29, MCF7, Receivedinrevisedform24April2012 MDAMB231,andPC3.IC valuesweremuchlowerthanthosefoundforcisplatin.Crystalstructureofcompound 50 Accepted24April2012 4 was determined by X-ray diffraction studies. Density functional theory (DFT) calculations performed for Availableonline2May2012 compound1showedelectronicflowfromtherutheniumcentertothecoordinatedbidentateligand,inagreement withtheelectrochemicalstudiesandtheexistenceofametal-to-ligandcharge-transfer(MLCT)bandevidencedby Keywords: spectroscopicdata. Ruthenium(II) Cyclopentadienyl ©2012ElsevierInc.Allrightsreserved. Heteroaromaticligand Organometallic Cytotoxicity DFTcalculations 1.Introduction tumorcelllines[9–11]togetherwiththeextensivelystudiedfamiliesof compoundsbasedon‘Ru(η6-arene)’fragment[12–21].Inaddition,the Study of ruthenium organometallic complexes became, in the compoundsderivedof‘Ru(η5-C H )(CO)’withpyridocarbazoleligands 5 5 recent years, a particular attractive area for the search of new revealed strong and selective inhibitors of protein kinases GSK-3 and antitumordrugs.Theadvantageofrutheniumbasedpharmaceuticals, Pim-1 [22,23] showing also their potentiality as anticancer drugs. relativelytoantitumorplatinum(II)complexescurrentlyusedinclinic Therefore,animportantprospectiveiscertainlyforeseenforruthenium [1–4],canderiveonthereducedtoxicityofrutheniumcompounds. half sandwich structured complexes. In fact, the stabilization of the Thiscrucialfeaturecanbeoriginatedontheabilityofrutheniumto metal center by an aromatic ligand such as ‘η5-C H ’ or ‘η6-C H ’ 5 5 6 6 mimictheironinbindingtobiologicalmolecules.Furthermore,the occupying three coordination positions, constitutes the base for the non-cross-resistance and a novel mechanism of action found for designofthesemolecules.Theremainingcoordinationsitescanbeused rutheniumcomplexes[5,6]incisplatin-resistantcancercellstogether bytheligandthatcanimparttherequiredantitumoractivitytogether withtheprospectofadifferentspectrumofactivity[7,8],putforward withligandsthatcancontroltheelectronicpropertiesattheruthenium thispromisingareaofresearch. center. Ourstudiesinthisfield,pioneeredthefamilyof‘Ru(η5-C H )’based We have been interested in the coordination of N-heteroaromatic 5 5 complexesaspresentingpotentcytotoxicityagainstarangeofhuman molecules to ‘Ru(η5-C H )’ systems and particularly for the complex 5 5 withtheN,N′-bipyridylbidentateligandwefoundinterestingproperties of stability aside the excellent cytotoxicity against several cancer cell lines[11],thussuggestingtheeffectivenessofachelateligandforour ⁎ Correspondingauthorat:FaculdadedeCiênciasdaUniversidadedeLisboa,Edificio purpose. Having this in mind, our strategy was then the selection of C8, Campo Grande, 1749‐016 Lisboa, Portugal. Tel.: +351 217500972; fax: +351 217500088. someN,O‐heteroaromaticmolecules,whichpresentothercoordination E-mailaddress:lena.garcia@fc.ul.pt(M.H.Garcia). sites than nitrogen, as bidentate ligands. We found that this new 0162-0134/$–seefrontmatter©2012ElsevierInc.Allrightsreserved. doi:10.1016/j.jinorgbio.2012.04.014 66 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 family of cationic compounds, besides the good stability to air and 2.2.2.[Ru(η5-C H )(PPh )(2-ap)][CF SO ]2 5 5 3 3 3 moisture presents outstanding cytotoxic properties against several SolidAgCF SO (0.13g,0.5mmol)wasaddedtoasolutionof[Ru(η5- 3 3 humancelllinesnamelyA2780(ovariancarcinoma),A2780CisR(ovarian C H )(PPh ) Cl](0.32g,0.5mmol)indichloromethane(25mL)while 5 5 3 2 carcinoma, cisplatin resistant), HT29 (colon adenocarcinoma), MCF7 stirring.After1h,2-acetylpyridine(0.05mL,0.5mmol)wasadded (breast adenocarcinoma‐hormone dependent, ERα+), MDAMB231 andthemixturewasheatedtorefluxfor5h.Theresultingbrownish (breast adenocarcinoma-hormone independent), and PC3 (prostate redsolutionwascannula-filteredandthesolventevaporatedunder cancer) and revealed excellent antitumor activities, with IC values vacuum. The product was washed with n-hexane (2×10mL) and 50 muchlowerthanthosefoundforcisplatin. recrystallizedfromdichloromethane/n-hexane.Yield:89%.1HNMR [(CD ) CO,Me Si,δ/ppm]:9.75[d,1,H ,3J =5.49Hz],8.26[d,1, 3 2 4 1 HH H ,3J =7.56Hz],8.03[t,1,H ,3J =7.87Hz],7.63[t,1,H ,3J = 2.Materialsandmethods 4 HH 3 HH 2 HH 7.23Hz],7.51[m,3,H (PPh )],7.43[m,6,H (PPh )],7.28[m, para 3 meta 3 6, H (PPh )], 4.80 [s, 5, η5-C H ], and 2.54 [d, 3, H7 (CH )]. 13C 2.1.Generalprocedure orto 3 5 5 3 NMR[(CD ) CO,δ/ppm]:157.15(C ,2-ap),152.04(C (C_O),2-ap), 3 2 1 6 137.29(C ,2-ap),134.24(CH,PPh ),131.82(Cq,PPh ),131.30(CH, Syntheses werecarriedoutunderdinitrogen atmosphereusing 3 3 3 PPh ),130.88(C ,2-ap),129.78(C ,2-ap),129.68(C ,2-ap),129.59 current Schlenk techniques and the solvents used were dried by 3 4 5 2 (CH, PPh ), 72.11 (C H ), 24.91 (C , 2-ap). 31P NMR [(CD ) CO, δ/ standard methods [24]. Starting material [Ru(η5-C 5 H 5 )(PPh 3 ) 2 Cl] ppm]: 49 3 .68 [s, PPh ] 5 . F 5 TIR [KBr, cm 7 −1]: 3059 (m), 2963 (w 3 ) 2 , 2928 was prepared following the method described in the literature 3 (m),2856(vw),1638(w),1585(vw),1503(vw),1435(s),1370(w), [25]. 1H, 13C and 31P NMR spectra were recorded on a Bruker 1315(w),1263(vs),1224(s),1151(s),1094(s),1031(s),852(w), Avance 400 spectrometer at probe temperature. The 1H and 13C 745(m),695(s),637(s),572(w),514(s),433(w).ESI-HRMS:calc. chemical shifts(s=singlet;d=duplet;t=triplet;m=multiplet for[M+]544.090075,found544.09124.Elementalanalysis(%)found: for1H)arereportedinpartspermillion(ppm)downfieldfrominternal C,52.40H,3.71;N,1.90;S,5.0.Calc.forC H NSPF O Ru·0.2CH Cl Me Siandthe 31PNMRspectraarereportedinppmdownfieldfrom 31 27 3 4 2 2 4 (715.65):C,52.36;H,3.80;N,1.96;S,4.5. externalstandard,85%H PO .FTIRspectrawererecordedinaMattson 3 4 SatelliteFTIRspectrophotometerwithKBrpellets;w=weak,vw= 2.2.3.[Ru(η5-C H )(PPh )(isoquinpk)][CF SO ]3 very weak, m = medium, s = strong, and vs = very strong. ESI- 5 5 3 3 3 AgCF SO (0.13g,0.5mmol)wasaddedtoasolutionof[Ru(η5C HRMSspectrawereacquiredinanApexUltraFTICRMassSpectrometer 3 3 5- H )(PPh ) Cl] (0.32g, 0.5mmol) in dichloromethane (25mL). The equipped with an Apollo II Dual ESI/MALDI ion source, from Bruker 5 3 2 resultant orange solution was stirred and after 1h, 1-isoquinolinyl Daltonics,anda7TactivelyshieldedmagnetfromMagnexScientific. phenyl ketone (0.12g, 0.5mmol) was added. After refluxing for 4h Elemental analyses were obtained at our laboratories (Laboratório thesolutionturnedfromorangetopurple.Thereactionmixturewas de Análises, Instituto Superior Técnico), using a Fisons Instruments cooledtoroomtemperature,filteredandthesolventremovedunder EA1108 system. Data acquisition, integration and handling were reducedpressure;theproductwaswashedwithn-hexane(2×10mL) performed using a PC with the software package EAGER-200 and recrystallized from dichloromethane/n-hexane. Yield: 87%. 1H (CarloErbaInstruments).Electronicspectrawererecordedatroom NMR[(CD ) CO,Me Si,δ/ppm]:9.81[d,1,H ,3J =6.31Hz],8.15[d, temperature on a Jasco V-560 spectrometer in the range of 3 2 4 1 HH 1,H ,3J =8.23Hz],8.05[d,1,H ,3J =6.17Hz],7.77[m,2,H + 200–900nm. 14 HH 2 HH 13 H ,3J =7.36Hz],7.59[m,2,H +H ,3J =7.47Hz],7.50[m,2, 15 HH 12 16 HH H +H ,3J =7.36Hz],7.42[m,2,H +H ,3J =6.60Hz],7.33[m, 4 7 HH 5 6 HH 2.2.SynthesisofRu(II)complexes 15,H(PPh )],5.06[s,5,η5-C H ]. 13C[(CD ) CO,δ/ppm]:149.76(C , 3 5 5 3 2 1 isoquinpk),149.29(C (C_O),isoquinpk),134.16(CH,PPh ),133.70 10 3 2.2.1.[Ru(η5-C H )(PPh )(bopy)][CF SO ]1 (CH,PPh ),131.82(C ,isoquinpk),131.70(CH,PPh ),131.43(C + 5 5 3 3 3 3 11 3 13 Toastirredsolutionof[Ru(η5-C H )(PPh ) Cl](0.32g,0.5mmol)in C ,isoquinpk),131.03(C ,isoquinpk),130.90(C ,isoquinpk),129.78 5 5 3 2 15 3 9 dichloromethane(25mL)wasaddedAgCF SO (0.13g,0.5mmol).The (C ,isoquinpk),129.51(Cq,PPh ),129.54(C +C ,isoquinpk),129.44 3 3 8 3 5 6 resulting orange solution was stirred for 1h at room temperature (C +C , isoquinpk), 128.77 (C +C , isoquinpk), 127.26 (C , 4 7 12 16 14 followed by addition of 2-benzoylpyridine (0.09g, 0.5mmol). The isoquinpk), 125.71 (C , isoquinpk), 80.43 (C H ). 31P [(CD ) CO, δ/ 2 5 5 3 2 reaction mixture was stirred at room temperature for further 20h, ppm]:48.54[s,PPh ].FTIR[KBr,cm−1]:3053(m),2927(vw),2855 3 withchangeofcolorfromorangetoviolet.Thesolutionwasseparated (vw), 1586 (w), 1544 (vw), 1478 (w), 1435 (m), 1393 (w), 1309 fromtheprecipitateofAgClbycannula-filtrationandthesolventwas (vw),1271(vs),1224(w),1149(m),1092(m),1030(s),998(vw), evaporated under vacuum. The residue was washed with n-hexane 925(vw),869(w),734(w),749(m),700(s),637(s),524(m),510 (2×10mL)andrecrystallizedfromdichloromethane/n-hexane.Yield: (s),459(m).ESI-HRMS:calc.for[M+]656.121376,found656.12304. 93%. 1H NMR [CDCl , Me Si, δ/ppm]: 9.89 [d, 1, H , 3J =5.65Hz], Elementalanalysis(%)found:C,58.3;H,3.70;N,1.65;S,4.0.Calc. 3 4 1 HH 7.99[d,1,H ,3J =7.61Hz],7.83[t,1,H ,3J =7.19Hz],7.71[t, forC H NSPF O Ru·0.2CH Cl (827.77):C,58.33;H,3.82;N,1.69;S, 4 HH 3 HH 40 31 3 4 2 2 1,H ,3J =7.05Hz],7.61[t,1,H ,3J =7.61Hz],7.49[t,2,H + 3.9. 2 HH 10 HH 9 H ,3J =7.19Hz],7.38[m,5,H (PPh )+H +H ],7.30[m,6, 11 HH para 3 8 12 H (PPh )],7.20[m,6,H (PPh )],4.75[s,5,η5-C H ].13CNMR 2.2.4.[Ru(η5-C H )(PPh )(dpk)][CF SO ]4 meta 3 orto 3 5 5 5 5 3 3 3 [CDCl ,δ/ppm]:157.90(C ,bopy),149.74(C (C_O),bopy),135.74 AgCF SO (0.13g, 0.5mmol) wasaddedto astirredsolution of 3 1 6 3 3 (C , bopy), 134.20 (C , bopy), 133.71 (C , bopy), 133.45 (CH, [Ru(η5-C H )(PPh ) Cl] (0.32g, 0.5mmol) in dichloromethane 3 7 10 5 5 3 2 PPh ), 133.34(CH, PPh ), 131.60 (C , bopy), 130.99 (Cq, PPh ), (25mL).Theresulting orange solution wasstirredfor1hatroom 3 3 4 3 130.64+130.56 (C +C , bopy), 129.45 (C , bopy), 129.07 (C + temperature followed by addition of di(2-pyridyl)ketone (0.09g, 8 12 2 9 C ,bopy),128.99(CH,PPh ),128.89(C ,bopy),77.65(C H ). 31P 0.5mmol).Afterrefluxing for4hthesolutionturnedfrom orangeto 11 3 5 5 5 NMR [CDCl ,δ/ppm]:48.48[s,PPh ].FTIR[KBr, cm−1]:3057(m), magenta. The solution was separated from the AgCl precipitate by 3 3 1597(w),1558(w),1516(w),1481(w),1435(m),1338(s),1262 cannula-filtration and the solvent evaporated under vacuum. The (vs),1223(s),1155(s),1093(m),1028(s),997(w),820(w),752 residuewaswashedwithdiethylether(2×10mL)affordingmagenta (m),700 (s),636(s),525(m),511(m).ESI-HRMS:calc.for[M+] crystals after recrystallization from dichloromethane/n-hexane. Yield: 606.105725, found 606.107070. Elemental analysis (%) found: C, 85%. 1H NMR [(CD ) CO, Me Si, δ/ppm]: 8.54 [d, 2, H +H , 3J = 3 2 4 1 11 HH 55.95;H,3.65;N,1.80;S,4,00.Calc.forC H NSPF O Ru·0.2CH Cl 5.29Hz],8.22[d,2,H +H ,3J =7.38Hz],8.03[tt,2,H +H ,3J = 36 29 3 4 2 2 4 8 HH 3 9 HH (777.72):C,55.91;H,3.81;N,1.80;S,4.12. 7.48Hz],7.50[m,3,H (PPh )],7.41[m,6,H (PPh )],7.19[m,6, para 3 meta 3 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 67 H (PPh )],7.14[tt,2,H +H ,3J =6.54Hz],4.78[s,5,η5-C H ].13C Table1 orto 3 2 10 HH 5 5 NMR [(CD 3 ) 2 CO, δ/ppm]: 159.57 (C 1 +C 11 , dpk), 157.05 (C 6 (C=O), Crystaldataandstructurerefinementfor[Ru(η5-C5H5)(PPh3)(dpk)][CF3SO3]·0.5CH2Cl2 dpk),139.07 (C +C ,dpk), 137.87 (C +C , dpk), 134.47 (Cq, PPh ), (4·0.5CH2Cl2). 3 9 5 7 3 134.31(CH, PPh 3 ), 131.49 (CH, PPh 3 ), 129.90 (CH, PPh 3 ), 128.40 Compound 4·0.5CH2Cl2 (C +C ,dpk),127.81(C +C ,dpk),79.19(C H ).31P[(CD ) CO,δ/ pp 4 m]: 5 8 2.64 [s, PPh 3 ]. FT 2 IR [K 1 B 0 r, cm−1]: 3074 5 (m 5 ), 1663 (m 3 ) 2 , 1586 F F o o r r m m u u l l a a weight 8 C 0 35 4 .5 .1 H 6 29ClF3N2O4PRuS (w),1480 (w),1434 (m),1314(m),1263 (vs), 1225(m),1151 (s), T,K 100(2) 1092(m),1031(s),998(w),940(w),820(w),751(s),697(s),637 Wavelength,Å 0.71073 Crystalsystem Monoclinic (s),572(w),527(s),502(m),453(w),421(w).ESI-HRMS:calc.for [M+] 613.09864, found 613.09794. Elemental analysis (%) found: C, S a p /Å acegroup 3 P2 3 1 .8 /c 812(14) 55.35;H,3.85;N,3.55;S,4.0.Calc.forC 35 H 28 N 2 SPF 3 O 4 Ru(761.70):C, b/Å 9.5815(4) 55.20;H,3.71;N,3.60;S,4.20. c/Å 20.2374(8) β/° 91.715(3) V/Å3 1958.59(5) 2.3.X‐raycrystalstructuredetermination Z 4 F000 3256 Three-dimensional X-ray data for [Ru(η5-C 5 H 5 )(PPh 3 )(dpk)] D μ/ c m alc m /g − c 1 m−3 1 0 . . 6 7 2 3 7 2 [CF 3 SO 3 ]·1/2CH 2 Cl 2 (4·0.5CH 2 Cl 2 ) was collected on a Bruker θ/(°) 1.20to25.09 SMART Apex CCD diffractometer at 100(2) K, using a graphite Rint 0.1170 m s c c o a o ll n n ec o m t c e h e d r th o o o m f d f a . r t a R o m e r fl e a e s n c e t d i a o c M n h s o c ‐ w o K v e α e re r r i a n m d g i e a 0 a t . s i 3 u o ° n re in d (λ ω fr = . om O 0 f .7 a t 1 h 0 h e 7 e 6 m 3 8 Å i 5 s ) p 7 h 6 b e y r r e e t fl h o e e f ct d ϕ io a - n t ω a s C G R w r 1 o R y a o 2 s d t ( a n a l e ll s s i s d z - a e o t / f a m - ) fi b m to 3 nF2 0 1 0 0 . . . . 1 0 0 1 8 3 4 4 7 7 0 × 7 0 0,17×0.15 measuredin4·0.5CH Cl ,wereallcorrectedforLorentzandpolarization Largestdifferencespeakandhole(eÅ−3) 1.051and−1.122 2 2 effects, and for absorption by semi-empirical methods based on a R1=Σ∣|Fo|−|Fc|∣/Σ∣Fo ∣. symmetry-equivalent and repeated reflections, 7818 independent b wR2={Σ[w(∣|Fo|2−|Fc|2∣)2]∣/Σ[w(Fo 4)]}1/2. reflections exceeded the significance level |F|/σ(|F|)>4.0. Complex scatteringfactorsweretakenfromtheprogrampackageSHELXT[26]. dried,purifiedbystandardprocedures[24]anddistilledunderdinitrogen The structures were solved by direct methods and refined by full- atmospherebeforeuse. matrix least-squares methods on F2. The non-hydrogen atoms were refinedwithanisotropicthermalparametersinallcases.Thehydrogen 2.5.Cellviabilityassaysinhumantumorcelllines atomswereincludedincalculatedpositionsandrefinedbyusingariding mode.AfinaldifferenceFouriermapshowednoresidualdensityoutside: 1.051and−1.122for4·0.5CH Cl e.Å−3.Aweightingschemew=1/ Thehumantumorcelllines:ovarianA2780andA2780cisR,sen- 2 2 sitiveandresistanttocisplatin,breastMCF7andMDAMB231,colon [σ2(F2)+(0.0684P)2+0.00P] for 4·0.5CH Cl , where P=(|F |2+2| o 2 2 o HT29 and prostate PC3 carcinoma cells (ATCC) were cultured in F|2)/3, were used in the latter stages of refinement. The crystal of c RPMI(A2780/A2780cisR and PC3) or DMEM containingGlutaMax I 4·0.5CH Cl presentsaslightdisorderonthedichloromethanemolecule. 2 2 (MCF7 and MDAMB231) or McCoy's (HT29) culture media (Gibco) This disorder has been refined and two atomic sites for one chlorine supplementedwith10%heat-inactivatedfetalbovineserumand1% atom have been observed and refined with the anisotropic atomic penicillin/streptomycinat37°Cinahumidifiedatmospherewith5% displacement parameters in eachcase. The site occupancy factor was CO (Heraeus, Germany). The cells were adherent in monolayers 0.53435 for Cl(1A). Data CCDC 865796 contain the supplementary 2 and, when confluent, were harvested by digestion with trypsin- crystallographic data for 4·0.5CH Cl . These data can be obtained, 2 2 EDTA (Gibco). Cell viability was evaluated by using a colorimetric viahttp://www.ccdc.cam.ac.uk/conts/retrieving.html,freeofcharge, assaybasedonthetetrazoliumsaltMTT([3-(4,5-dimethylthiazol-2-yl)- orfromtheCambridgeCrystallographicDataCentre,12UnionRoad, 2,5-diphenyltetrazoliumbromide]),whichisreducedbymitochondrial CambridgeCB21EZ,UK;fax:(+44)1223-336-033;ore-mail:deposit@ succinate dehydrogenase in metabolic active cells to insoluble purple ccdc.cam.ac.uk. Crystal data and details of the data collection and formazan crystals [28]. For this purpose, cells were plated in 96-well refinementforthenewcompoundsarecollectedinTable1. sterile plates at a density to ensure exponential growth of untreated controlsamplesthroughouttheexperiment,10–20×103cellsperwell 2.4.Electrochemicalstudies with200μLofmedium.For24hcellswereallowedtosettlefollowed bytheadditionofdilutionseriesofthetestcompoundsinfreshmedium Cyclicvoltammograms(CV's)wereobtainedusinganEG&GPrinceton in aliquots of 200μL per well. Ligands and complexes were first Applied Research Potentiostat/Galvanostat Model 273A equipped with solubilized in DMSO and then in the medium, and added to final ElectrochemicalPowerSuitev2.51softwareforelectrochemicalanalysis, concentrations from 2nM to 200μM (ligands) and 0.2nM to 20μM inanhydrousdichloromethaneoracetonitrilewithtetrabutylammonium (complexes).ThefinalconcentrationofDMSOincellculturemedium hexafluorophosphate (0.1–0.2M) as supporting electrolyte. The didnotexceed1%.Cisplatin(thereferencepositivecontrolforthepair electrochemicalcellwasahomemadethreeelectrodeconfiguration A2780/A2780cisR)wasfirstsolubilizedinsalineandthenaddedatthe cell with a platinum-disk working electrode (1.0mm) probed by a same concentrations used for the other compounds. After continuous Luggincapillaryconnectedtoasilver-wirepseudo-referenceelectrode exposuretothecompoundsfor72h,37°C/5%CO ,themediumwere 2 and a platinum wire auxiliary electrode. All the experiments were removedandcellswereincubatedwith200μLofMTTsolutioninPBS performed in dinitrogen atmosphere at room temperature. All the (0.5mg/mL). After 3–4h at 37°C/5% CO , the solution was removed 2 potentialsreportedweremeasuredagainsttheferrocene/ferrocenium andthepurpleformazancrystalsformedinsidethecellsweredissolved redox couple as internal standard and normally quoted relative to in200μLDMSObythroughshaking.Thecellularviabilitywasevaluated saturated calomel electrode — SCE (using the ferrocenium/ferrocene by measurement of the absorbance at 570nm by using a plate redoxcoupleEp =0.46or0.40VversusSCEfordichloromethaneor spectrophotometer(PowerWaveXs,Bio-TekInstruments,Winooski,VT, 1/2 acetonitrilerespectively[27]). USA). The cytotoxic effects of the compounds were quantified by Boththesampleandtheelectrolyte(Fluka)weredriedundervacuum calculatingthedrugconcentrationinhibitingtumorcellgrowthby50% forseveralhourspriortotheexperiment.Reagentgradesolventswere (IC ), based on non‐linear regression analysis of dose response data 50 68 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 (GraphPadPrismsoftware).Allcompoundsweretestedin atleast ourcasesthiswasnotstraightforwardduetotheweakintensityof twoindependentexperiments,eachcomprisingeightreplicatesper thebandsobservedinthatregion.AnalysisoftheFTIRspectrumfor concentration. compound4showedthepresenceofν ~1660cm−1provingthe CO preference of ruthenium for the N,N′ chelation, instead the N,O 3.Resultsanddiscussion alternative. 3.1.SynthesisofRu(II)complexes 3.2.Spectroscopicstudies Newcationiccomplexesofgeneralformula[Ru(η5-C H )(PPh )(L)] 1H, 13Cand 31PNMR ofthenew compoundswerecompletedby 5 5 3 [CF SO ]werepreparedbyσcoordinationofligandsL,presentingtwo COSY,HMQCandHMBCstudiesind-acetoneord-chloroformsolutions, 3 3 heteroatoms, namely 2-benzoylpyridine (bopy), 2-acetylpyridine (2- foracompletecharacterizationofthecompounds.Scheme1showsthe ap),1-isoquinolinylphenylketone(isoquinpk)anddi(2-pyridyl)ketone numbering of the coordinated ligands for simplicity of the present (dpk)(Scheme1).Thereactionswerecarriedoutatrefluxorstirringat discussion,whichengagealsotherelevant1Hand13CNMRdatagath- room temperature. The compounds were recrystallized, at room tem- eredonTableS1ofsupplementarymaterial. perature,by slow diffusion ofn-hexanein dichloromethanesolutions. The analysisof the 1H NMR spectra revealedresonances for the ThenewcompoundswerefullycharacterizedbyFTIR, 1H,13Cand η5-cyclopentadienylring,inthecharacteristicrangeofmonocationic 31PNMRspectroscopies;theelementalanalysesandtheaccuratemass ruthenium(II)complexes(4.8–5.1ppm).ThecoordinationoftheN,O measurements were in accordance with the proposed formulations. bidentate ligands reveals the same pattern for compounds 1 to 3 The solid state FTIR spectra of the complexes typically showed the observed through a significant deshielding (~1.2ppm) of the H1 characteristic bands of (η5-C H ) and phenyl aromatic rings in the protonbelongingtotheN-heteroaromaticringwhencomparedtothe 5 5 region3040–3080cm−1,ν appearedat~1435cm−1andCF SO− freeligand.Thisbehaviorisinagreementwithatypicalsigmacoor- C=C 3 3 anion at ~1260cm−1. The coordination of the ketonic functional dinationofthenitrogenatom.Onthecontrary,asignificantshielding grouptorutheniumcenterwasassociatedwiththedisplacementofν was observed for the positions closely located to the oxygen atom CO at~1700cm−1tolowerenergy(compounds1–3).Althoughtheν for suggestinganelectronicflowfromthemetaltotheligandthroughthe CO 1-isoquinolinyl phenyl ketone ligand coordinated to a ruthenium(II) coordinated O atom. In fact, this electronic flow will justify the sig- centerhasbeenfoundat1314cm−1inanoctahedralcomplex[29]in nificant shielding of ~0.7ppm observed for protons H8 and H12 on Scheme1.Reactionschemeforthesynthesesofnew[Ru(η5-C5H5)(PPh3)(L)][CF3SO3]compounds;ligandsarenumberedforNMRspectralassignments. T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 69 compound1and~0.6ppmforprotonsH12andH16oncompound3. For a full characterization of the compounds, having in mind the Moreover,thisfindingisreinforcedby13CNMRdatasincethecarbon electronicchargetransfercharacteristicofthevisibleband,opticalspectra of the CO group of compounds 1–3 revealed shieldings of ~46ppm ofallthecompoundswerealsoobtainedinothersolventsofdifferent and the α carbons present also significant shieldings of ~25ppm. dielectric constant, namely acetone, acetonitrile and dimethylsulfoxide Therefore,adipolarstructureisformedbeingthe δ+chargelocalized (see Table 2). In accordance with the expected solvatochroism, sig- onthemetalcenter,andtheδ−chargeplacedonthedelocalisedthree nificant shifts (up to 20nm) on the maximum of that band, were centeredbondO\C\Catthemetallacyclering.Inthecaseofcompounds foundforallthecompounds,togetherwiththevariationofthemolar 1and3,theextensionofthisπsystemgoesuptotheorthoprotonsofthe absorptioncoefficient,asexemplifiedonFig.2forcompound[Ru(η5- phenylgroup.However,theabsenceofthephenylgroupboundtoCOin C H )(PPh )(2-ap)][CF SO ](2). 5 5 3 3 3 compound2,doesnotallowsuchelectronicextension.Asaresult,the ComparisonoftheresultsshowedthattheλmaximumofthisMLCT dipole(Ru)δ+\(O\C\C)δ−willbeenhancedincompound2,relatively band occurs for compound 2 at higher energy (499nm) than for totheequivalentdipolesgeneratedincompounds1and3,ascanbe compounds1(530nm)and3(554nm).Thisobservationisingood evident from the experimental NMR data and confirmed also by the agreement with our results obtained by 1H, 13C NMR spectroscopy higher oxidation potential of ruthenium center of compound 2 (see thatrevealedhigherbackdonationforcompound2. electrochemicalresults). Forcompound4,whichcoordinationisthroughtwoNatomsofthe bidentateliganddi(2-pyridyl)ketone,1HNMRdatarevealashielding 3.4. Single crystal structure of [RuCp(PPh )(dpk)][CF SO ]·1/2CH Cl 3 3 3 2 2 effectonthearomaticprotonsofthecoordinatedligand.Thiseffect (4·0.5CH Cl ) 2 2 was already found with the coordination of N,N′-bipyridyl to an analogousrutheniummoiety[11]andwasattributedtotheelectronic [Ru(η5-C H )(PPh )(dpk)][CF SO ]·1/2CH Cl (4·0.5CH Cl ) 5 5 3 3 3 2 2 2 2 flowtowardsthearomaticligand,asconsequenceofπ-backdonation crystallizes from dichloromethane/n-hexane solution as dark red involving a d orbital of the ruthenium center and the π*orbital prism crystals (dimensions 0.184×0.154×0.16). Fig. 3 shows an localizedonthenitrogenatoms.Unexpectedly,whileforcompound ORTEP representation of [Ru(η5-C H )(PPh )(dpk)]+ of 4·0.5CH Cl . 5 5 3 2 2 [Ru(η5-C H )(PPh )(N,N′-bipyridyl)][CF SO ] the higher shielding In4·0.5CH Cl ,theasymmetricunitcontainstwocationicruthenium 5 5 3 3 3 2 2 (0.44ppm)wasfoundontheorthoprotons11,inthepresentcompound complexes [Ru(1) and Ru(2)], two CF SO− anions and one 3 3 (4) this enhanced shielding (0.46ppm) was observed on the meta dichloromethanemolecule(seeFig.4).In4·0.5CH Cl ,thephosphine 2 2 protons(H2,H10). ligand turns around P-Ru edge in the two cationic ruthenium complexespresentintheasymmetricunit(seeFig.S1ofsupplementary material).The(η5-C H )ringsturnaroundRu-centroidedge,aswell 5 5 3.3.Electronicabsorptionspectroscopy (see Fig. S1 of supplementary material). In the molecular structure, the ruthenium centers adopt a “piano stool” distribution formed by Theopticalabsorptionspectraofallthesynthesizednewcomplexes theruthenium-(η5-C H )unitboundtothephosphinesandnitrogen 5 5 wererecordedin~10−4to10−6Msolutionsofdichloromethane(see atoms of the dpk. In 4·0.5CH Cl , the nitrogen of the ligand (dpk) 2 2 Table2).Forcomparison,alsotheelectronicspectraoftheuncoordinated bonds through the two nitrogen atoms and one phosphine group ligandsandthe[Ru(η5-C H )(PPh ) Cl]parentcompoundwereobtained occupies the other coordination positions. The distances for Ru\P 5 5 3 2 inthesameexperimentalconditions.Allthestudiedcomplexesshowed bondsareRu(1)–P(1)=2.3207(14)ÅandRu(2)–P(2)=2.3379(14)Å. intense absorption bands in the UV region, in the range 247–326nm, ThedistancesforRu\NbondsareRu(1)–N(1)2.124(4)Å,Ru(1)–N(2) attributed to electronic transitions occurring in the organometallic 2.119(4) Å, Ru(2)–N(3) 2.117(4) Å and Ru(2)–N(4) 2.085(4) Å. The fragment {Ru(η5-C H )(PPh )}+ and coordinated chromophores. In distancebetweenRuandthecentroidoftheπ-bondedcyclopentadienyl 5 5 3 additiontothesetransitions,oneintenseabsorptionband,placedinthe moietyis1.824(5)ÅtoRu(1)(ringslippage0.058Å)and1.816(5)Åto visibleregionbetween450and600nm,wasfoundforallthecomplexes. Ru(2) (ring slippage 0.006Å), in 4·0.5CH Cl . The mean value of the 2 2 Thelocalizationofthisband,togetherwiththecorrespondinghighvalue Ru\Cbonddistancesis2.191(5)ÅinRu(1)and2.181(5)ÅinRu(2). of molar absorption coefficient (ε~105–106M−1cm−1), reveals its Table 3 contains selected bond lengths and angles for compound chargetransfer(CT)character,namelymetaltoligand(MLCT),fromRu 4·0.5CH Cl .Anglesbetweentheplanesformedbythepyridinegroups 2 2 4dorbitalstotheπ*orbitalsoftheligands.Fig.1typifiesthebehavior are25.20(23)°(forC(24),C(25),C(26),C(27),C(28),N(2)andC(30), ofthissetofcompounds. C(31), C(32), C(33), C(34), N(1) planes) and 30.44(21)° (for C(64), Table2 Electronicspectradataforcomplexes(1)–(4),indichloromethane,acetone,acetonitrileanddimethylsulfoxidesolutions. Compound λmax(nm)(εM−1cm−1) CH2Cl2 (CH3)2CO CH3CN DMSO [Ru(η5-C5H5)(PPh3)(bopy)][CF3SO3]1 276(31,300) – 285(16,003) 287(16,818) – 323(6185) – – 529(11,100) 521(5529) 520(6842) 525(7126) [Ru(η5-C5H5)(PPh3)(2-ap)][CF3SO3]2 247(15,800) – 267(22,608) 269(11,385) 326(6130) 335(3768) 325(843) 340(sh) 499(6480) 485(3977) 485(9112) 486(4714) [Ru(η5-C5H5)(PPh3)(isoquinpk)][CF3SO3]3 254(32,353) – –a 271(8397) 316(20,137) 336(5204) –ª 321(7509) 554(21,500) 552(6306) 536(1962) 555(6437) [Ru(η5-C5H5)(PPh3)(dpk)][CF3SO3]4 234(39,979) – 237(26,751) 269(9989) 275(sh) – 276(sh) – 303(sh) – 304(sh) 302(sh) 347(sh) 330(8358) 345(sh) 340(sh) 500(4447) 491(2901) 492(3097) 504(2603) Sh:shoulder. a Bandisnotreliableduetodecompositioninacetonitrile. C(65),C(66),C(67),C(68),N(3)andC(58),C(59),C(60),C(61),C(62), N(4)planes).π–πstackinginteractionsareabsentinthestructures. 3.5.Electrochemicalstudies The activity of metal-based drugs depends largely on their ligand environment and coordination geometry, which also determine the redox properties. The knowledge of metal-centered redox potentials canprovideapowerfultoolforthedesignofnewcomplexesandabetter understanding of the role of metallodrugs in biological applications. Electrochemical studies of a series of [Ru(η5-C H )(PPh )(L)][CF SO ] 5 5 3 3 3 compoundswhereL=bopy,2-ap,isoquinpkanddpk,wereperformed studies [30], is in accordance with an easier reduction of the ligands in dichloromethane and acetonitrile media, to assess their redox be- aftercoordinationtotherutheniummoiety.Sincethereisalittlevariation haviorandreactivityandtherelevantdataareshowninTable4.The onthepotentialsoftheseresponsesacrosstheseries,thepyridylring electrochemicalbehavioroftheligandswasalsostudiedinbothsolvents shouldbetheresponsibleineachcase. revealinginactivitywithinthepotentialwindowusedinthesestudies. The N,N′-coordinated ligand in complex 4 showed a similar Ina0.2M[n-Bu 4 N][PF 6 ]/dichloromethanesolution,thecomplexes electrochemical profile concerning the RuII/RuIII couple and displays were redox-active showing metal (oxidation) and ligand-centered two reduction waves,thefirstone appearing at −0.97V(E ) is irre- pc (reduction)processes.AtypicalexampleisshowninFig.5,withthe versibleandthesecondoneat−1.13V(E )isquasi-reversible.These p/2 cyclic voltammogram of the complex [Ru(η5-C 5 H 5 )(PPh 3 ) (bopy)] tworeductionwavesareassignedtothesuccessivereductionsinthe [CF 3 SO 3 ] (1). The RuII/RuIII coupleprocesswasobserved at positive di(2-pyridyl)ketoneligand,inaccordancewithpreviousstudies[31]. potentialsintherange0.93–1.00Vdependingontheligandidentity. Electrochemicalstudiesinacetonitrilearedistinctlydifferent,since Thepeaktopeakseparationvaluesofthisredoxcouplerangesfrom all the complexes showed an uncoupled oxidation peak in the range 100to130mVanditisbestdescribedasaquasi-reversibleelectron 0.95–0.99V, assignable to the Ru(II) to Ru(III) oxidation which irre- transferprocess.Moreover,weobservedforallcomplexesanincre- versibilitycouldresultfromdecompositionoftheshort-livedoxidized ment ofthecathodic/anodiccurrentratiowithincreasingofthescan species.Besidesthemetalcenteredprocesses,therutheniumcomplexes rate,whichisconsistentwithachemicalreactionfollowingtheoxidation 1 and 2 display at negative potentials with two additional reduction (ECmechanism). processes.Thefirstuncoupledreductionwave(between−0.69Vand Besides the metal centered processes, the ruthenium complexes −0.84V)showedtobedependentfromtheRuII/RuIIIoxidation(Fig.6), displayoneligand-centeredquasi-reversiblereductionprocessbetween whichsuggestsaprobablefastchemicalreactionfollowingtheoxidation −0.97Vand−1.15V.Thisredoxprocess,inagreementwithprevious 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 400 450 500 550 600 650 700 )1-mc1-M( ε 25000 20000 15000 10000 5000 0 250 350 450 550 650 750 850 Fig. 3. ORTEP plot for the cation complex of [Ru(η5-C5H5)(pdk)PPh3][CF3SO3]·1/ 2CH2Cl2 (4·0.5CH2Cl2). All the non-hydrogen atoms are presented by their 30% probabilityellipsoids.Hydrogenatomsareomittedforclarity. λ (nm) Fig. 2. Electronic spectra of [Ru( η5-C5H5)(PPh3)(2-ap)][CF3SO3] (2) in solvents of differentpolarities,dichloromethane(–•–•–),acetonitrile(—),acetone(…….)and Fig. 4. Capped sticks plot for the asymmetric unit in the compound [Ru(η5-C5H5) dimethylsulfoxide(‐‐‐),in10−4–10−5Msolutions. (pdk)(PPh3)][CF3SO3]·1/2CH2Cl2(4·0.5CH2Cl2). )1-mc1-M( ε 70 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 λ (nm) Fig. 1. Optical spectra of [Ru(η5-C5H5)(PPh3)(2-ap)][CF3SO3] (2) (—) ,in dichloromethane,comparedtotheuncoordinatedligand2-acetylpyridine(2-ap) (–––)and[Ru(η5-C5H5)(PPh3)2Cl]parentcomplex(………..). T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 71 Table3 L/L- Bondlengths[Å]andangles[°]for[RuCp(PPh3)(dpk)][CF3SO3]·1/2CH2Cl2(4·0.5CH2Cl2). 5 µA [RuCp(PPh3)(dpk)][CF3SO3]·1/2CH2Cl2(4·0.5CH2Cl2) RuII/III Bondlengths(Å) Ru(1)–Cpa 1.825(5) Ru(2)–Cpa 1.816(5) Ru(1)–N(1) 2.124(4) Ru(2)–N(3) 2.117(4) Ru(1)–N(2) 2.119(4) Ru(2)–N(4) 2.085(4) Ru(1)–P(1) 2.3207(14) Ru(2)–P(2) 2.3379(14) 1.5 1 0.5 0 -0.5 -1 -1.5 -2 BondAngles(°) Cpa–Ru(1)–N(1) 122.39 Cpa–Ru(2)–N(3) 122.56 E vs SCE (V) Cpa–Ru(1)–N(2) 126.22 Cpa–Ru(2)–N(4) 121.63 Cpa–Ru(1)–P(1) 124.95 Cpa–Ru(2)–P(2) 123.90 Fig. 5. Cyclic voltammogram of complex [Ru(η5-C5H5)(PPh3)(bopy)][CF3SO3] 1 in N(1)–Ru(1)-N(2) 87.93(16) N(3)–Ru(2)–N(4) 87.35(16) dichloromethaneat200mV/s. N(1)–Ru(1)–P(1) 94.24(12) N(3)–Ru(2)–P(2) 97.30(11) N(2)–Ru(1)–P(1) 90.86(11) N(4)–Ru(2)–P(2) 95.38(12) Ru(1)–N(1)–C(30) 124.0(3) Ru(2)–N(3)–C(64) 117.3(3) thecompoundefficiency.Althoughthepresentstudyonlyinvolves4 Ru(1)–N(1)–C(34) 118.3(4) Ru(2)–N(3)–C(68) 123.2(3) compounds, and therefore no generalization can be drawn from the Ru(1)–N(2)–C(28) 124.9(3) Ru(2)–N(4)–C(58) 122.0(3) Ru(1)–N(2)–C(24) 118.7(3) Ru(2)–N(4)–C(62) 118.7(3) relationbetweenourelectrochemicalresultsandthebiologicstudies, itisworthtonotethatcompounds1and2revealedthebestcytotoxic results, presenting compound 2 an oxidation potential 60–80mV oftherutheniummoiety.Thesecondquasi-reversibleprocess(between higherthancompounds1,3and4. −0.95V and −1.09Vvs SCE) could beassignedto a ligand-centered Interestingly, also for compound 2, the reduction process at the process in agreement with the dichloromethane electrochemical data. coordinatedligandwasmoredifficult,beingonepossibleexplanation Complex 4, with the N,N′-coordinated ligand, showed two additional theexpectedstabilizationofthedipoleRuδ+and(O⋯C⋯C)δ−,that processes, one uncoupled oxidation wave at 0.90V and one related wouldreinforcetheintegrityofthebindingRu\O,Nligand. reductionwaveat−0.47V. The N,N′-coordinated ligand in complex 4 showed a similar 3.6.Computationalstudies electrochemicalprofileconcerningtheRuII/RuIIIcoupleanddisplays two reduction waves, the first one at −0.97V(E ) is irreversible pc In order to gain a better view on the electronic properties of andthesecondone,appearingat−1.13V(E ),isquasi-reversible. p/2 these ruthenium compounds, particularly to fully understand the Thesetworeductionwavesareassignedtothesuccessivereductions π-backdonation effect, Natural Bond Analysis[33](NBO)by meansof inthedi(2-pyridyl)ketoneligand,inaccordancewithpreviousresults DFT calculations were performed as implemented in Guassian09 [34], [31]. withcationic[Ru(η5-C H )(PPh )(bopy)]+(1+).Geometryoptimization Noquantitativerelationshipcouldbeestablished,sofar,between 5 5 3 wasdone using M062X [35]functional with a LAN2DZ[36–38] core cytotoxicactivityandredoxpotentialofmetalcomplexes,asobserved potentialintherutheniumandphosphorouscentersanda6–31G(d, by other authors [32]. Nevertheless, it is known that the reductive p) basis set including diffuse and polarizations functions on lighter environment found in the tumors will favor lower oxidation states, atoms.Theobtainedgeometry wascomparedto thecrystallographic andthenRuIIcompoundsmayimplylesssusceptibilityformetabolic structure of cation [Ru(η5-C H )(PPh )(dpk)]+ (4+) and the results degradationandlongersurvivaltime,untiltheyreachthecancercells. 5 5 3 show anagreementinthe Ru\N bonddistance(2.107Å calculated; Therefore,itisexpectedthatinafamilyofantitumoralruthenium(II) 2.11(8) obtained by crystallography). The Ru\N and Ru\O bonds compounds,ahigheroxidationpotentialshould,inprinciple,increase were not detected in the NBO analysis, showing a predominant Coulomb-typeinteractionbetweenthemetalcenterandtheN,O-ligand. ThisCoulombinteractionwasalsoprovenbytheWibergindices,which T E a le b c l t e ro 4 chemicaldataforcomplexes1–4indichloromethaneandacetonitrile. gave 0.455and 0.400for the Ru\N and Ru\O bondorders, respec- tively. Analysis of the Wiberg indices at the coordinated N,O-ligand Epa Epc Ep1/2 Epa –Epc Ipc/Ipa revealthatC_Odecreasesitsdoublebondcharacteruponcoordination, (V) (V) (V) (mV) thesamehappeningwithC5_Nofthemetallacycle(seenumberingin Dichloromethane Scheme1).Conversely,C5–C6becameshorterrevealingahigherdouble 1 0.99 0.88 0.93 110 0.45 bondcharacter.Thus,thepostulatedeffectofπ-backdonationfromthe [Ru(η5C5H5)(PPh3)(bopy)][CF3SO3] −0.99 −1.07 −1.03 80 1.0 2 1.06 0.94 1.00 120 0.84 [Ru(η5-C5H5)(PPh3)(2-ap][CF3SO3] – −1.15 – – – 3 1.00 0.87 0.94 130 0.8 [Ru(η5C5H5)(PPh3)(isoquinpk)][CF3SO3] −0.92 −1.01 −0.97 90 1.0 4 0.98 0.88 0.93 100 1.0 [Ru(η5-C5H5)(PPh3)(dpk)][CF3SO3] – −0.97 – – – −1.07 −1.19 −1.13 120 1.0 Acetonitrile 1 0.96 – – – – [Ru(η5C5H5)(PPh3)(bopy)][CF3SO3] −0.72 −0.69 – – – −1.03 −0.95 −0.99 80 1.0 2 0.95 – – – – [Ru(η5-C5H5)(PPh3)(2-ap][CF3SO3] – −0.84 – – – −1.05 −1.12 −1.09 70 1.0 1.5 1 0.5 0 -0.5 -1 -1.5 -2 4 0.90 – – – – E vs SCE (V) [Ru(η5-C5H5)(PPh3)(dpk)][CF3SO3] 1.08 – – – – – −0.47 – – – – −0.76 – – – Fig. 6. Cyclic voltammogram of complex [Ru(η5-C5H5)(PPh3)(bopy)][CF3SO3] 1 in −0.91 −0.98 −0.95 70 1.0 acetonitrileat200mV/sshowingthedependencebetweenthefirstreductionwave atnegativepotentialsandtheoxidationwaveofRu(II)toRu(III)oxidation. 72 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 Fig.7.a)HOMOorbitalforcomplex1showingthemaincontributionofthe‘Ru(η5-C5H5)’;b)LUMOorbitalforcomplex1showingtheelectrondensityatthemetallacycle.PPh3 omittedforclarityandorbitalsdrawnwithacontourplotof0.05a.u. rutheniumtothecoordinatedligand,basedonourspectroscopicevi- [Ru(η5-C H )(PPh ) Cl] exhibit IC values (3.5μM) in the range 5 5 3 2 50 dence,canfindsupportbyourDFTcalculations.Moreover,sincethe found for complex3 when studied in theovarian cancer cell lines HOMOorbitaliscenteredat‘Ru(η5-C H )’moietyandtheLUMOorbital A2780. Complexes 1, [Ru(η5-C H )(PPh )(bopy)][CF SO ] and 2 5 5 5 5 3 3 3 shows a significant contribution of the coordinated ligand the lower [Ru(η5-C H )(PPh )(2-ap)][CF SO ] with the respective ligands 2- 5 5 3 3 3 energy transition occurring in the visible region, should be a MLCT benzoylpyridine and 2-acetylpyridine, present exceptional cytotoxic transition.TheHOMOandLUMOorbitalsaredepictedinFig.7.Ascan activitiesagainstallthetumorcelllinesstudiedinparticularthebreast beseenontheLUMO,theelectrondensitiesaremainlylocatedatO, cancercellsMCF7andMDAMB231,andcanalsoovercomeresistance, N,C5andC6atomsofthemetallacyclering. beingequallyactiveinthesensitive(A2780)andresistant(A2780cisR) Chargedecompositionanalysis(CDA)wasalsoperformedtodeter- cell lines. The isoquinolinyl moiety seemed to reduce the cytotoxic minetheelectronsinvolvedinthecoordinationoftheligandaswellas activity of the complex 3 [Ru(η5-C H )(PPh )(isoquinpk)][CF SO ] 5 5 3 3 3 π-backdonation from the metal center. This analysis was made using when compared with 1, 2 and 4. Albeit complex 3 ranked as the QMForgesoftware[39].Wedeterminedaσ-donationof0.801electrons leastactivecomplexofthisseriesforallcelllinestested,presented fromtheligandandaπ-backdonationof0.156electronsfromthemetal highercytotoxicactivitythanthereferencedrugcisplatinevenfor center. themorechemioresistantcancercellsPC3. 3.7.Cytotoxicityinhumantumorcelllines 4.Conclusion Thecytotoxicityofthecomplexeswasassayedinapanelofdif- AnewfamilyofRu(II)three-leggedpianostoolcomplexespossessing ferent human tumor cultured cells within the concentration range O,N and N,N′-heteroaromatic bidentate ligands, was synthesized and from0.2nMto20μM.Theeffectsof1–4andthereferencecompound fully characterized by spectroscopic studies [43]. This family of cisplatinonthegrowthofthesecelllineswereevaluatedafter72h compoundsrevealedthatmetalligandbindingwasstrengthenedbyan continuousexposuretothecompounds.Dose–responsecurveswere electronic flow from the metal center to the σ bonded ligand, clearly performedforeachcompoundandcelllineasshowninTable5and evidenced by the experimental data (1H, 13C NMR and UV–vis.) and Fig. 8. Ru complexes exhibited higher anticancer activities in the proved by our DFT theoretical calculations. Also our electrochemical nanomolarrangewhencomparedtocisplatin.Theorderofpotency studiesbymeansofcyclicvoltammetryshowarelativelyhighvaluefor observed was 1~2>4>3≫cisplatin. IC values found for the Ru theoxidationpotentialofRuII/RuIII(~1V)alongwithaquasi-reversible 50 complexes was in the range 180nM–4.7μM, 210nM–5.2μM, 30– reduction of the coordinated ligand, this suggesting more stability for 490nM, 30–260nM, 80nM–4.8μM and 300nM–5.5μM for the thebondmetal-ligand. A2780, A2780cisR, MCF7, MDAMB231, HT29 and PC3 cell lines, Our cytotoxic studies in A2780, A2780cisR, MCF7, MDAMB231, respectively(Table5,Fig.8).Usingthesameexperimentalconditions, HT29 and PC3 cancer cell lines revealed an exceptional activity of thefreeligandsbopy,2-ap,isoquinpk,dpkandthecounterionCF SO complexes 1–4, with IC values in the nanomolar range following 3 3 50 showed no growth inhibition activities at concentrations as high as order1~2>4>3≫cisplatin.Interestingly,1and2areveryeffective 200μM, which indicate that the complexation with ruthenium was against the highly glycolytic cell lines MCF7 and MDAMB231. This essentialfortheobservedanticanceractivities.Howevertheprecursor latercelllinehas,inaddition,highlymetastaticproperties.Although Table5 IC50values(μM)foundfortherutheniumcomplexesindifferenthumantumorcelllines(72h,37°C). Compound Tumorcelllines IC50(μM) A2780 A2780CisR MCF7 MDAMDB231 HT29 PC3 1 0.19±0.03 0.21±0.04 0.05±0.01 0.03±0.01 0.08±0.01 0.41±0.08 2 0.18±0.02 0.32±0.07 0.03±0.01 0.06±0.02 0.32±0.08 0.30±0.07 3 4.70±1.6 5.20±2.5 0.49±0.10 0.26±0.14 4.8±1.6 5.5±2.1 4 0.46±0.11 0.47±0.16 0.41±0.09 0.23±0.07 0.53±0.14 1.9±0.3 Cisplatin 2.00±0.10a 17±3.0a 28±6.0a 39±5.0a 7.0±2.0a 51±7.0 a Fromreferences[39–42]. -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 log C (M) log C (M) -12 -10 -8 -6 -4 -2 -12 -10 -8 -6 -4 log C (M) log C (M) 150 150 100 100 50 50 0 0 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 log C (M) log C (M) the overall set of cytotoxic studies parallel compounds 1 and 2 we PC3 prostatecancer foresee,onthebaseofourstructuralchemicalevidences,evenbetter SCE saturatedcalomelelectrode strength for 2 in studies in vivo. Further studies are underway to highlightthepotentialofthesecomplexesforfutureclinicaluse. Acknowledgment 5.Abbreviations We thank the Fundação para a Ciência e Tecnologia for financial 2-ap 2-acetylpyridine support(PTDC/QUI/66148/2006,PTDC/Qui-qui/101187/2008,PEst-OE/ A2780 ovariancarcinoma QUI/UI0536/2011 and REDE/1501/REM/2005). Tânia S. Morais and A2780CisR ovariancarcinoma,cisplatinresistant Tiago J.L.Silva thanktheFCT fortheir Ph.DGrants (SFRH/BD/45871/ bopy 2-benzoylpyridine 2008andSFRH/BD/64309/2009). Cp η5-cyclopentadienyl Cq quaternarycarbon AppendixA.Supplementarydata DFT Densityfunctionaltheory dpk Di(2-pyridyl)ketone Supplementarydatatothisarticlecanbefoundonlineathttp:// ESI-HRMS electrosprayionization-highresolutionmassspectrometry dx.doi.org/10.1016/j.jinorgbio.2012.04.014. HOMO HighestOccupiedMolecularOrbital HT29 colonadenocarcinoma References isoquinpk 1-isoquinolinylphenylketone LUMO LowestUnoccupiedMolecularOrbital [1] J.M. Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H.M. MALDI matrixassistedlaser Schellens,Clin.CancerRes.10(2004)3717–3727. MCF7 breastadenocarcinoma(hormonedependent,ERα+) [2] C.G.Hartinger,S.Zorbas-Seifried,M.A.Jakupec,B.Kynast,H.Zorbas,B.K.Keppler, J.Inorg.Biochem.100(2006)891–904. MDAMB231 breastadenocarcinoma(hormoneindependent) [3] W.H.Ang,P.J.Dyson,Eur.J.Inorg.Chem.20(2006)4003–4018. MLCT metal-to-ligandcharge-transfer [4] P.J.Dyson,Chimia61(2007)698–703. ytilibaiv ralullec % ytilibaiv ralullec % 150 150 100 100 50 50 0 0 ytilibaiv ralullec % ytilibaiv ralullec % 150 150 100 100 50 50 0 0 ytilibaiv ralullec % ytilibaiv ralullec % T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 73 A2780 A2780 cisR 1 1 2 2 3 3 4 4 MCF7 MDAMB231 1 2 2 3 3 1 4 4 HT29 PC3 1 1 2 2 3 3 4 4 Fig.8.Cellularviability(%)ofthedifferenthumantumorcelllinesaftera72htreatmentwiththerutheniumcomplexes1–4. 74 T.S.Moraisetal./JournalofInorganicBiochemistry114(2012)65–74 [5] W.J.Zeller,S.Fruhauf,G.Chen,B.K.Keppler,E.Frei,M.Kaufmann,Eur.J.Cancer27 [26] G.M.Sheldrick,SHELXL-97:AnIntegratedSystemforSolvingandRefiningCrystal (1991)62–67. StructuresfromDiffractionData(Revision5.1),UniversityofGöttingen,Germany, [6] M.Coluccia,G.Sava,F.Loseto,A.Nassi,A.Boccarelli,D.Giordano,E.Alessio,G. 1997. Mestroni,Eur.J.Cancer29A(1993)1873–1879. [27] N.G.Conelly,W.E.Geiger,Chem.Rev.96(1996)877–910. [7] M.J.Clarke,Coord.Chem.Rev.236(2003)209–233. [28] T.Mosmann,J.Immunol.Methods65(1983)55–63. [8] E.Alessio,G.Mestroni,A.Bergamo,G.Sava,in:A.Sigel,H.Sigel(Eds.),MetalIons [29] J.G.Malecki,J.Coord.Chem.60(2007)1949–1958. inBiologicalSystems.MetalComplexesinTumorDiagnosisandasAnticancer [30] A.S.A.T.dePaula,B.E.Mann,E.Tfouni,Polyhedron18(1999)2017–2026. Agents,vol.42,MarcelDekker,NewYork,2004,pp.323–351. [31] W.L.Huang,J.R.Lee,S.Y.Shi,C.Y.Tsai,Trans.Met.Chem.28(2003)381–387. [9] M.H.Garcia,T.S.Morais,P.Florindo,M.F.M.Piedade,V.Moreno,C.Ciudad,V.Noe, [32] M.Auzias,B.Therrien,G.Süss-Fink,P.Stepnicka,W.H.Ang,P.J.Dyson,Inorg. J.Inorg.Biochem.103(2009)354–361. Chem.47(2008)578–583. [10] V.Moreno,J.Lorenzo,F.X.Aviles,M.H.Garcia,J.Ribeiro,T.S.Morais,P.Florindo, [33] D.Glendening,A.E.Reed,J.E.Carpenter,F.Weinhold,NBO-version3.1,1996. M.P.Robalo,Bioinorg.Chem.Appl.(2010)1–11. [34] M.J.Frisch,G.W.Trucks,H.B.Schlegel,G.E.Scuseria,M.A.Robb,J.R.Cheeseman, [11] V. Moreno, M. Font-Bardia, T. Calvet, J. Lorenzo, F.X. Avilés, M.H. Garcia, T.S. G.Scalmani,V.Barone,B.Mennucci,G.A.Petersson,H.Nakatsuji,M.Caricato,X.Li,H.P. Morais,A.Valente,M.P.Robalo,J.Inorg.Biochem.105(2011)241–249. Hratchian,A.F.Izmaylov,J.Bloino,G.Zheng,J.L.Sonnenberg,M.Hada,M.Ehara,K. [12] Y.K.Yan,M.Melchart,A.Habtermariam,P.J.Sadler,Chem.Commun.38(2005) Toyota,R.Fukuda,J.Hasegawa,M.Ishida,T.Nakajima,Y.Honda,O.Kitao,H.Nakai, 4764–4776. T.Vreven,J.A.Montgomery,J.E.Peralta,F.Ogliaro,M.Bearpark,J.J.Heyd,E.Brothers, [13] S.J.Dougan,P.J.Sadler,Chimia61(2007)704–715. K.N.Kudin,V.N.Staroverov,R.Kobayashi,J.Normand,K.Raghavachari,A.Rendell, [14] C.S.Allardyce,P.J.Dyson,D.J.Ellis,S.L.Heath,Chem.Commun.(2001)1396–1397. J.C.Burant,S.S.Iyengar,J.Tomasi,M.Cossi,N.Rega,J.M.Millam,M.Klene,J.E.Knox, [15] C.S.Allardyce,P.J.Dyson,D.J.Ellis,P.A.Salter,R.J.Scopelliti,J.Organomet.Chem. J.B.Cross,V.Bakken,C.Adamo,J.Jaramillo,R.Gomperts,R.E.Stratmann,O.Yazyev, 668(2003)35–42. A.J.Austin,R.Cammi,C.Pomelli,J.W.Ochterski,R.L.Martin,K.Morokuma,V.G. [16] Y.N.V.Gopal,D.Jayaraju,A.K.Kondapi,Biochemistry38(1999)4382–4388. Zakrzewski, G.A. Voth, P. Salvador, J.J.Dannenberg, S. Dapprich, A.D.Daniels, O. [17] Y.N.V.Gopal,N.Konuru,A.K.Kondapi,Arch.Biochem.Biophys.401(2002)53–62. Farkas,J.B.Foresman,J.V.Ortiz,J.Cioslowski,D.J.Fox,Gaussian,Inc,WallingfordCT, [18] R.E.Morris,R.E.Aird,P.S.Murdoch,H.Chen,J.Cummings,N.D.Hughes,J.Med.Chem. 2009. 44(2001)3616–3621. [35] Y.Zhao,G.D.Truhlar,Theor.Chem.Acc.120(1–3)(2004)215–644. [19] C.Scolaro,A.Bergamo,L.Brescacin,R.Delfino,M.Cocchieto,G.Laurenczy,T.J. [36] P.J.Hay,W.R.Wadt,J.Chem.Phys.82(1)(1985)270–283. Geldbach,G.Sava,P.J.Dyson,Med.Chem.48(2005)4161–4171. [37] W.R.Wadt,P.J.Hay,J.Chem.Phys.82(1)(1985)284–298. [20] G.Süss-Fink,DaltonTrans.39(2010)1673–1688. [38] P.J.Hay,W.R.Wadt,J.Chem.Phys.82(1)(1985)299–310. [21] M.Gras,B.Therrien,G.Süss-Fink,O.Zava,P.J.Dyson,DaltonTrans.39(2010) [39] AdamL.Tenderholt,QMForge,Version2.1,http://qmforge.sourceforge.net. 10305–10313. [40] S.Gama,F.Mendes,F.Marques,I.C.Santos,M.F.Carvalho,I.Correia,J.C.Pessoa,I. [22] H.Bregman,E.Meggers,Org.Lett.8(2006)5465–5468. Santos,A.Paulo,J.Inorg.Biochem.105(2011)637–644. [23] R.Anand,J.Maksimoska,N.Pagano,E.Y.Wong,P.A.Gimotty,S.L. Diamon,E. [41] A.J.Pope,C.Bruce,B.Kysela,M.J.Hannon,DaltonTrans.39(2010)2772–2774. Meggers,R.Marmorstein,J.Med.Chem.52(2009)1602–1611. [42] K.Butsch,R.Gust,A.Klein,I.Ott,M.Romanski,DaltonTrans.39(2010)4331–4340. [24] D.D.Perrin,W.L.F.Amarego,D.R.Perrin,PurificationofLaboratoryChemicals, [43] M.H.Garcia,T.S.Morais,A.I.Tomaz,F.M.Marques,F.Mendes,TransitionMetal seconded.,Pergamon,NewYork,1980,pp.65–371. ComplexesforPharmaceuticalApplications,PatentApplicationPT105890,2011. [25] M.I.Bruce,N.J.Windsor,Aust.J.Chem.30(1977)1601–1604.