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In vitro and in vivo biological activity screening of Ru(III) complexes involving 6-benzylaminopurine derivatives with higher pro-apoptotic activity than NAMI-A.
JournalofInorganicBiochemistry105(2011)937–948
ContentslistsavailableatScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
In vitro and in vivo biological activity screening of Ru(III) complexes involving
6-benzylaminopurine derivatives with higher pro-apoptotic activity than NAMI-A
Zdeněk Trávníčeka,⁎ , Miroslava Matiková-Maľarováb, Radka Novotnáa, Ján Vančoa,
Kamila Štěpánkováb, Pavel Suchýc
aRegionalCentreofAdvancedTechnologiesandMaterials,DepartmentofInorganicChemistry,FacultyofScience,PalackýUniversity,17.listopadu12,CZ-77146Olomouc,CzechRepublic
bDepartmentofInorganicChemistry,FacultyofScience,PalackýUniversity,17.listopadu12,CZ-77146Olomouc,CzechRepublic
cDepartmentofHumanPharmacologyandToxicology,FacultyofPharmacy,UniversityofVeterinaryandPharmaceuticalSciences,Palackého1–3,CZ-61242Brno,CzechRepublic
a r t i c l e i n f o a b s t r a c t
Articlehistory: Aseriesofnoveloctahedralruthenium(III)complexesinvolving6-benzylaminopurine(L)derivativesasN-donor
Received30December2010 ligandshasbeenpreparedbythereactionof[(DMSO)H][trans-RuCl(DMSO)]withthecorrespondingLderivative.
2 4 2
Receivedinrevisedform4April2011 The complexes 1–12 have the general compositions trans-[RuCl(DMSO)(n-Cl-LH)]⋅xSol (1–3), trans-[RuCl
4 4
Accepted4April2011 (DMSO)(n-Br-LH)]·xSol (4–6), trans-[RuCl(DMSO)(n-OMe-LH)]·xSol (7–9) and trans-[RuCl(DMSO)(n-OH-
Availableonline12April2011 LH)]·xSol(10–12);n=2,3,and4,x=0–1.5 4 ;andSol=HO,DMSO,EtOHand/or(Me)CO.The 4 complexeshave
2 2
beenthoroughlycharacterizedbyelementalanalysis,UV–visible,FTIR,Raman,andEPRspectroscopy,ES+(positive
Keywords:
ionization electrospray) mass spectrometry, thermal analysis, cyclic voltammetry, magnetic and conductivity
Ruthenium(III)complexes
Purinederivatives measurements. The X-ray molecular structure of trans-[RuCl 4 (DMSO)(3-Br-LH)]⋅(Me) 2 CO (5) revealed the
Crystalstructure distortedoctahedralcoordinationinthevicinityofthecentralatom,andalsoconfirmedthatthe3-Br-Lligandis
Antiradicalactivity presentastheN3-protonatedN7-HtautomerandiscoordinatedtoRu(III)throughtheN9atomofthepurine
Invitrocytotoxicity moiety.Thetestedcomplexeshavebeenfoundtobeinvitronon-cytotoxicagainstK562,G361,HOSandMCF7
Invivoantitumoractivity humancancercelllineswithIC N100μMincontrasttothemoderateresultsregardingtheantiradicalactivitywith
50
IC ≈10−3M.Onthecontrary,invivoantitumoractivityscreeningshowedthatthepreparedRu(III)complexes
50
possesshigherpro-apoptoticactivitythanNAMI-A.ThereductionofRu(III)toRu(II)andRu(II)-speciesformationin
tumortissueswasconfirmedbymeansofasimplemethodofdetectionandvisualizationofintracellularRu(II)by
fluorescencemicroscopy.TheoriginalityofthismethodisbasedonthepreparationofaRu(II)-bipyridinecomplex
insitu.
©2011ElsevierInc.Allrightsreserved.
1.Introduction andapromisingalternativetoplatinum-basedsubstances.Ruthenium
complexescanexhibitmoreadvantageousphysico-chemicalproperties
Agreatnumberofcoordinationcompoundshavebeenusedinthe comparedtoPt-involvingdrugs,e.g.coordinationsatiationinconnection
therapyofvariousdiseasesuptonow.Thesuccessfuldevelopmentof with theoctahedral geometry and, as a consequenceof this, another
metal-containinganticancerdrugsclearlystartedwithcis-[Pt(NH ) Cl ], modeofinteractionswithDNA;andarangeofoxidationstatesaccessible
3 2 2
referredtoascisplatin[1,2].Inthesedays,cisplatinanditsanalogsare under physiological conditions [2,7–9]. Additionally, ruthenium
stillonesofthemosteffectivechemotherapeuticagentsinclinicaluse. compoundsaregenerallylesstoxicthantheirplatinumcounterparts,
However,theirhightoxicityandacquiredorintrinsicdrugresistance whichisbelievedtobeduetotheabilityofrutheniumtomimicironin
remain the main drawbacks in their clinical applications. These bindingtobiomolecules,includingserumproteins(e.g.transferrinand
limitationshavepromptedthesearchforalternativechemotherapeutic albumin)[2,9].Indeed,theselectivityofRuspeciesmightbederived
strategies[3–5].Therefore,nowadays,anticancerresearchisfocusedon fromthecapacityofthesecompoundstobetransportedwithtransferrin
the investigation of either “non-classical” platinum-containing intotumorcells,whichintheirhigherironrequirementsoftenover-
complexes or compounds involving other suitable transition metals, expresstransferrin-receptors[10,11].Moreover,theselectivityagainst
e.g.Ru,Rh,Ti,andV[1,2,6].Amongthe“non-platinum”complexes,the solidtumorsandlowertoxicityofsomerutheniumcompounds(with
rutheniumcompoundshaveattractedsignificantattention,sincethey biologicallyaccessiblereductionpotentialsandsubstitutableligands)is
offermanysuitablefeaturesforbecomingnewmetallopharmaceuticals often connected with the “activation-by-reduction” hypothesis, when
ruthenium(III) complexes remain in their relatively inactive Ru(III)
oxidationstatesuntiltheyreachthetumorsite.Inthisenvironment,with
⁎ Correspondingauthor.Tel.:+420585634352;fax:+420585634954. itsloweroxygencontentandpHthaninnormaltissues,reductiontothe
E-mailaddress:zdenek.travnicek@upol.cz(Z.Trávníček). morereactiveRu(II)oxidationstatetakesplace[2,7,12].
0162-0134/$–seefrontmatter©2011ElsevierInc.Allrightsreserved.
doi:10.1016/j.jinorgbio.2011.04.002
938 Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948
Inthepastfewdecades,alargenumberofRu(II/III)complexeshave being tested (named as Seliciclib or CYC202) in the IIb-phase of
been prepared and characterized, while some of them have already clinical evaluations on patients with non-small cell lung cancer
demonstrated the ability to control tumor proliferation, growth and (NSCLC)[28].Todate,onlyoneRu-complexbearingunsubstitutedL
metastasisinpreclinicalmodels[2,7,12–14].Amongthesecomplexes, withthecompositionof[RuCl (DMSO)(LH)]hasbeenprepared[29].
4
theRu(III)complexesinvolvingN-donorheterocyclesasligandshave Theinteractionstudyof[RuCl (DMSO)(LH)]withplasmidicDNAwas
4
beenintensivelystudiedbothduetotheirantitumoractivity,especially performedandtheresultsshoweddifferentmorphologicalchangesin
againstmetastaticcancers[15,16],andinconnectionwiththeirability theDNAforms.Inourpreviousresults,wehavealreadyshownthat
to bind to imine sites of biomolecules with a relatively high affinity coordinationoftheLderivativestosuitabletransitionmetalions(e.g.
[17,18].Todate,tworutheniumagentsinvolvingN-donorheterocyclic Pt,Pd,Cuetc.)canleadtotheformationofcomplexeswithnotable
ligands, NAMI-A {[ImH][trans-RuCl (DMSO)(Im)] (Im=imidazole)} biologicalactivity(invitrocytotoxicity,SOD-likeactivity)[30,31].
4
andKP1019{[IndH][trans-RuCl (Ind) ](Ind=indazole)},haveentered Onthebasisoftheabove-mentionedfacts,wedecidedtoprepare,
4 2
human clinical trials [14]. Despite their structural and chemical fully characterize a series of novel Ru(III)-complexes involving the
similarities, these two Ru(III) complexes show distinct antitumor variously benzyl-substituted 6-benzylaminopurine derivatives, and
behaviors.Inpreclinicalstudies,NAMI-Ahasdemonstratedinhibitory studytheirbiologicalactivity(invitrocytotoxicity,antiradicalSOD-
effectsagainsttheformationofcancermetastasesinavarietyoftumor mimicactivity,invivoantitumoractivity),andmoreover,basedonthe
animalmodelsbutappearstolackdirectcytotoxiceffects,whileKP1019 obtained findings, try to explain the possible mechanism of their
hasshowndirectantitumoractivityagainstawiderangeofprimary biologicalaction.
human tumors by inducing apoptosis. These differences derive from
diverse modes of action of these two complexes. KP1019 is a new 2.Experimentalsection
tumor-inhibitingdrugactingbyamechanisminvolvingaccumulationin
transferrinreceptor-(over)expressingtumorcellsaswellassubsequent 2.1.Materials
reduction to Ru(II) species in reductive tumor environment (the
“activation-by-reduction”mechanism)[19].Ontheotherhand,inthe RuCl ·xH O, 6-chloropurine, 2-methoxybenzylamine, 3-meth-
3 2
caseoftheselectiveantimetastaticactivityofNAMI-A,the“activation- oxybenzylamine, 4-methoxybenzylamine, 2-hydroxybenzylamine
by-reduction”mechanismhasbeenchallengedbyrecentfindings.In hydrochloride, 3-hydroxybenzylamine, 4-hydroxybenzylamine, 2-
fact,althoughreductionofNAMI-Aislikelytooccurinvivo(reducing chlorobenzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine,
agents mightbe glutathioneorascorbic acid),it seemsthatitisnot 2-bromobenzylamine hydrochloride, 3-bromobenzylamine, 4-bro-
specificallyneededfortheactivationandconsequentbiologicalaction. mobenzylamine,2,2′-bipyridineandsolventsusedwerepurchased
DuetoNAMI-ArelativelabilityatphysiologicalpH,activationmayoccur from Aldrich Co. or Acros Co. and used as received.
byanaquation(i.e.chloridoligandsaresubstitutedbyaqualigands)
[10,20,21].Additionally,thestudiesontheNAMI-Amechanismofaction 2.2.Celllineforinvivotesting
seemtoexcludeDNAastheprimarycellulartarget.Itlikelybindsto
molecules (probably proteins) expressed or relevant only in the The cell line L1210—Mouse DBA/2 lymphocytic leukemia was
metastatic tumor cells [11,22]. Overall, the mechanism of metastasis obtainedfromECACC(EuropeanCollectionofCellCultures),through
control seems to be attributable to the combined effects of anti- Sigma-Aldrich Co. The frozen cells were thawed, reconstituted, and
angiogenicandanti-invasivepropertiesofNAMI-Aontumorcellsand cultivated according to the standard procedure recommended by
bloodvessels [23]. Anyway, forbothNAMI-A and KP1019, a phase I ECACC in the culture medium containing the Fischer's medium
studyhasbeenrecentlyreported.AphaseIIstudyevaluatingKP1019in (Invitrogen), supplemented with 2mM glutamine (Sigma-Aldrich
patientswithadvancedcolorectalcancerisnowbeingplanned[14]. Co.),and10%horseserum(fromcontrolledherd,Sigma-AldrichCo.).
Theexperienceacquiredinthelastyearsintheareaofruthenium
compounds indicates the relationship between the structure and 2.3.Physicalmeasurements
activity for compounds that appear to function by the mechanisms
including the transport into the cell via the transferrin cycle and Elementalanalyses(C,H,N)wereperformedonaFisonsEA-1108
activation by reduction. The nature of the N-donor ligand and/or CHNS-OElementalAnalyzer(ThermoScientific).Infraredspectrawere
presence of DMSO in the coordination sphere of the central atom obtainedonaNexus670FTIRspectrometer(ThermoNicolet)usingthe
influence redox propertiesand antitumoractivityofsuchcomplexes KBr(4000–400cm−1)andNujol(600–200cm−1)techniques.Raman
[24].Forinstance,duetoπ-acceptorpropertiesofS-bondedDMSO,the spectroscopymeasurementswereperformedusinganNXRFT-Raman
reductionpotentialvalues,E ,ofsuchcomplexesareshiftedtohigher Module (Thermo Nicolet) in the 3750–150cm−1 region. Electronic
red
values compared to those without the coordinated DMSO molecule absorptionspectrawererecordedonaLambda40spectrometer(Perkin-
{e.g.E forNAMI-Aequals253mV,andfor[ImH][trans-RuCl (Im) ] Elmer)inthe45,000–10,000cm−1range.Conductivitymeasurements
red 4 2
equals−160mV,bothvsnormalhydrogenelectrode(NHE),measured of the complexes in the N,N′-dimethylformamide (DMF) solutions
inwater}[25].ThehighervaluesofE aremoresuitableforbiological (10−3M)wereperformedonaCond340i/SETconductometer(WTW)
red
application due to similarity of the electrode potential between the at 25°C. Variable temperature magnetization measurements were
tumorandnormaltissues[24]. carriedoutinthetemperaturerangeof2–300Katanappliedmagnetic
Theorganiccompoundscontainingthe6-benzylaminopurine(L) field of 1T on a grounded polycrystalline sample with a Quantum
skeleton belong to the group of aromatic cytokinins which affect a Design MPMS-XL-7 SQUID magnetometer. Isothermal magnetization
variety of important physiological processes such as cell division, measurementswereperformedat2Kwithmagneticfieldsupto7T.
differentiation and senescence [26]. A suitable modification of the Themagneticdatawerecorrectedforthesampleholderandforthe
purineskeletonattheC2and/orN9positionscandramaticallychange diamagnetic contributions estimated using Pascal constant [32]. The
andextendthespectrumofbiologicaleffectsofthesecompounds.The EPR spectra were recorded on a MiniScope MS100 spectrometer
most promising representative of such derivatives, (R)-Roscovitine, (MagnettechGmbH,Germany)attheX-bandfrequencyforthepowder
i.e. 2-[(R)-(1-ethyl-2-(hydroxyethyl)amino)]-6-benzylamino-9- samples at room temperature. TG and DTA (thermogravimetric and
isopropylpurine, can be classified among cyclin-dependent kinase differentialthermalanalysis)measurementsofcomplexes2,3,4and
inhibitors(i.e.enzymesbeingabletoregulatethehumancell cycle 8 were made in air in the temperature range of 25–1000°C using a
and showing a significant antineoplastic activity) [27], and has TG/DTA6200Exstarthermalanalyser(SeikoInstruments,USA)witha
successfullypassedinvivoandpreclinicaltestinganditiscurrently sampleweightof5–15mgandthermalgradientof5°Cmin−1.X-ray
Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948 939
powder diffraction patterns of the thermal decomposition products Table1
were taken on an XRD-7 powder diffractometer (Seifert) and Crystaldataandstructurerefinementfor[RuCl4(DMSO)(3-Br-LH)]⋅(Me)2CO(5).
interpreted using the ZDS system [33] and PDF-2 database [34]. Compound 5
ES+mass spectra were recorded on a Micromass ZMD2000 single
quadrupolemassspectrometer.Themassmonitoringintervalequalled
Empiricalformula C17H23Br1Cl4N5O2Ru1S1
Formulaweight 684.24
50–900m/z. The spectra were collected using a 2.0 cyclical scan and Temperature(K) 110(2)
applyingthesampleconevoltageof20,30or40V,atthesourceblock Wavelength(Å) 0.71073
temperatureof100°C,desolvationtemperatureof150°Canddesolvation Crystalsystem,spacegroup monoclinic
gas flow rate of 322lh−1. The samples for mass spectroscopy were Unitcelldimensions P21/c
a(Å) 7.7069(2)
measuredinDMF/methanol/aceticacidsolutions(c=10−4M).Themass
b(Å) 14.4973(3)
spectrometer was directly coupled to a MassLynx data system. Cyclic c(Å) 21.7987(5)
voltammetry measurements were performed using a Potentiostat/ α(°) 90.00
β(°) 99.090(2)
Galvanostat Model 273 apparatus (EG&G Princeton Applied Research,
γ(°) 90.00
USA)underinertatmosphere(Ar)inathreeelectrodecellat25°C.The V(Å3) 2404.96(10)
working electrode was a platinum–carbon electrode, the auxiliary Z,Dcalc(gcm−3) 4,1.890
electrode was Pt-wire, and the reference electrode was the saturated Absorptioncoefficient(mm−1) 2.870
Ag/AgClelectrode.Thepotentialsweremeasuredin0.1M[nBu N][PF ]/ Crystalsize(mm) 0.25×0.25×0.20
4 6 F(000) 1356
DMF vs Ag/AgCl electrode. All measurements were performed at
θrangefordatacollection(°) 2.81to25.00
laboratorytemperatureand100mVs−1scanrate.
Indexranges(h,k,l) −8≤h≤9
−15≤k≤17
2.4.SinglecrystalX-rayanalysisoftrans-[RuCl (DMSO)(3-Br-LH)]· −25≤l≤24
(Me) CO(5)
4 Reflectionscollected/unique(Rint) 16,886/4227[Rint=0.0279]
2 Data/restraints/parameters 4227/0/284
Goodness-of-fitonF2 1.068
The X-ray data of trans-[RuCl 4 (DMSO)(3-Br-LH)]·(Me) 2 CO (5) FinalRindices[IN2σ(I)] R1=0.0407,wR2=0.1076
were collected on a four-circle κ-axis diffractometer Xcalibur2 Rindices(alldata) R1=0.0516,wR2=0.1121
(OxfordDiffractionLtd.)equippedwithaSaphire2CCDdetectorat
Largestpeakandhole(eÅ−3) 1.339and−1.311
120K. The CrysAlis software package [35] was used for data
collection and reduction. The structure was solved by the SIR97
program[36]incorporatedintheWinGXprogrampackage[37].All weredissectedandselectedorgansandprimarytumorlesionswere
non hydrogen atoms were refined anisotropically on F2 using full takenforfurtherhistologicalexamination.
matrixleast-squareprocedure[SHELXL-97][38]withweight:w=1/ Experimentaldatawereexpressedasthepercentageofmeansurvival
[σ2(F )2+(0.06P)2+9.56P], where P=(F2+2F2)/3. The hydrogen time,%T/Cdefinedastheratioofthemeansurvivaltimeofthetreated
o o c
atomswerefoundinthedifferenceFouriermapsandwererefined animals(T)dividedbythemeansurvivaloftheuntreatedcontrolgroup
usingaridingmodel.DIAMONDwasusedforthepreparationofthe (C).Therewerenodeathsattributabletotoxicityofthetestedcompounds.
figures [39]. Crystal data and structure refinement for 5 are
summarizedinTable1. 2.6.1.Histologicalandhistochemicalevaluations
Thesamplesfrominvivoantitumoractivitytestingwerefixedin
2.5.Invitrocytotoxicitytesting 10% neutral buffered formaldehyde, dehydrated by an increasing
amountofalcohol(30%–50%–70%–80%–95%–100%),clearedinxylene
Thepreparedcompounds1,2,7,8,9,10and11weretestedbythe and embedded in paraffin. Paraffin block preparations were
calcein acetoxymethyl (AM) assay for in vitro cytotoxicity against hematoxilin and eosin stained. Immunohistochemical detection of
malignant melanoma (G361), chronic myelogenous erythroleukemia apoptosisbyTdTenzymewascarriedoutusingtheApoTagPeroxidase
(K562),osteogenicsarcoma(HOS)andbreastadenocarcinoma(MCF7) DetectionKit—TUNEL(MilliporeCo.).
humancancercell,asdescribedpreviouslyintheliterature[40andthe Duetohighcellularity,thesemiquantitativemethodwasusedforthe
referencestherein].Thecellsweremaintainedinplastictissueculture evaluationofmitosis,apoptosis,andnecrosisinwell-circumcisedand
flasksandgrownonDulbecco'smodifiedEagle'scellculturemedium diffusetumorsamples.Thereforeitwasalsoimpossibletocalculatethe
(DMEM)at37°Cin5%CO atmosphereand100%humidity.Thecell relevantapoptoticandmitoticindexes,andonlythenumberofmitotic
2
suspension of approximate density of 1.25×105cellsmL−1 was andapoptoticfiguresinthreerandomlyselectedfieldswerecalculated
redistributed into 96-well microtitre plates (Nunc, Denmark). The at1000×magnification.Themeanvaluesofthesedeterminationswere
testedcomplexeswereaddedafter12hofpreincubation.Incubation used.Thenecrotizedsectionswereexcludedfromtheevaluation.The
lastedfor72h,afterwhichthecellswereincubatedfor1hwithcalcein detection of apoptotic cells was carried out based on the following
AM.Fluorescenceofthelivecellswasmeasuredat485/538nm(ex/em) morphologicalcharacteristics:chromatincondensing,chromatinmar-
with Fluoroskan Ascent (Labsystems, Finland). The presented IC ginationunderthenuclearmembraneandformingofapoptoticbodies.
50
valuesequaltoarithmeticmeansdeterminedfromthreevalues. AlltheevaluationswereconfirmedbytheTUNELapoptosisdetectionkit.
Furthermore, necrosis focuses were assessed in the whole tissue
2.6.Evaluationofinvivoantitumoractivity section, at 40× magnification. The evaluation was semi-quantitative,
basedonthepercentageofnecroticareasintheviewfieldofatleastthree
The implantation procedure—106 viable L1210 cells (leukocytic differentsamples.Thescalefrom0to4wasused,where0=without
leukemia) were administered to DBA/2 mice (female 9–12weeks, necrosis,1=upto25%,2=upto50%,3=upto75%and4=upto100%
20–30g) i.p. on day 0. After the induction period of 10days, the ofthenecroticareasintheviewfield.Sectionsforthisevaluationwere
animalgroupsweretreatedwiththesamedoses(50mg/kg,days10, hematoxylinandeosinstained.
11,and12)ofNAMI-AandtestedRu(III)complexes1,3,5,7,9,and
11.Theanimalswereweigheddailyandobservedseveraltimesaday 2.6.2.InsituvisualizationofintracellularRu(II)byfluorescence
for the signs of tumor progression, sudden death, and sacrificed if microscopy
theirbodyweightdecreasedbelow80%ofthestartingweightorif Inadditiontostandardhistochemicalprocedures,wedevelopeda
otherseveretoxicologicalproblemswereseen.Thesacrificedanimals newsimplemethodforthequalitativedetectionofintracellularRu(II)
940 Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948
byinsituformationofafluorescentRu(II)-complexwith2,2′-bipyridine. 2.8.2.Generalprocedureforthepreparationofcomplexes1–12
Abriefdescriptionofthismethodisasfollows:thesaturatedsolutionof TheS1solution,containing2.5mmolof[(DMSO) H][trans-RuCl
2 4
2,2′-bipyridine in the standard fixing solution, containing equal (DMSO) ], was diluted with 2mL of ethanol. To this solution, the
2
volumesofacetoneandmethanol,wasaddedontoaparaffinembedded equimolar amount of the corresponding 6-benzylaminopurine (L)
slideandlefttoreactatroomtemperaturefor3h.Thestainedsamples derivative in a mixture of DMSO and another solvent (10/90v/v;
wereevaluatedbyafluorescencemicroscopeequippedwithadigital acetonefor3,5,7,9–12,chloroformfor4,6,8orethanolfor1,2)was
camera (Olympus BX52 Research Microscope) using the multi- added.Thereactionmixturewasheatedupto45–60°Candstirred
excitation source and 650nm filter. The visual differences between untilanorangesolutionformed.Orangeororange-redmicrocrystals
thesamplesfromtherutheniumcomplex-treatedgroupsandsamples wereobtainedafterafewhoursordays.Thecrystalswerecollectedby
fromtheuntreatedcontrolgroupweredescribed. filtration,washedwithcoldacetoneandsmallportionofdiethylether
anddriedinvacuumdesiccator.
2.6.3.Statisticalmethods
Experimentaldataweresubjectedtostatisticalanalysisusingthe 2.8.2.1. trans-[RuCl 4 (DMSO)(2-Cl-LH)]DMSO (1). Yield: 60%. Anal.
one-wayANOVAmethod.Differencesofpb0.05wereconsideredtobe Calcd. for C 16 H 23 N 5 Cl 5 O 2 Ru 1 S 2 (659.9): C, 29.1; H, 3.5; and N, 10.6.
significantly different from controls, or other selected groups, Found: C, 29.5; H, 3.3; and N, 10.4. IR (cm−1; m = medium, w =
respectively. weak, s = strong, vs = very strong): 3253m, 3217m ν as (N―H),
3131m, 3056m ν(C―H) , 3020m ν (C―H), 2978m, 2924m ν
ar as s
(C―H), 1659m ν(CN), 1617m, 1559m δ(N―H), 1464w, 1447m
2.7.SOD-mimicactivitytesting
ν(C―C) , 1119s ν(SO), 1062s ρ(N―H), 1021s ρ(CH ), 459w
ar 3
ν(Ru―N), 430m ν(Ru―S), 331s ν(Ru―Cl), and 376w δ(C―S―O).
TheSOD-mimicactivityofthecomplexes2,3,8,11and12,asthe Raman(cm−1):3056mν(C―H) ,3016mν (C―H),2995m,2924s
representativesofthewholegroupofcomplexes1–12,wastested ar as
ν(C―H), 1661m ν(CN), 1512s δ(N―H), 1115m ν(SO), 314vs
byanindirectchemicalmethod[41].Theindirectmethodisbased s
ν(Ru―Cl),and270mν(Ru―N).ES+MS:(m/z)260[(2-Cl-L)+H]+,
on the concurrent competitive reaction between the tested
and579[M–H]+(molecularpeak).
compoundsandtheleuko-formoftetrazoliumdye(XTT)withthe
saturatedDMSOsolutionofpotassiumsuperoxide.Thereactionof
2.8.2.2. trans-[RuCl (DMSO)(3-Cl-LH)](Me) CO0.5DMSO (2). Yield:
the leuko-form of XTT with superoxide leads to the formation of 4 2
70%.Anal.Calcd.forC H N Cl O Ru S (678.8):C,31.8;H,3.9;
orangecoloredwater-solubleXTT-formazane(λ max =480nm).The andN,10.3.Found:C, 1 3 8 1. 2 7 6 ;H 5 ,3 5 .6 2 ; .5 and 1 N 1 , .5 10.2.IR(cm−1):3247m,
dependence of the formazane formation inhibition percentage on the
3202m ν (N―H), 3059m ν(C―H) , 3022m ν (C―H), 2970m,
inhibitor concentration proceeds in accordance with the first-order as ar as
2924m ν(C―H), 1704m ν(CO), 1657m ν(CN), 1612m, 1567m
reactionmodel,whichcouldbelinearizedusingthelogarithmicscalingof s
δ(N―H), 1466w, 1447m ν(C―C) , 1117s ν(SO), 1075m ν(C―Cl),
concentration.TheSOD-mimicactivityofcompoundswasexpressedas ar
1061sρ(N―H),1020sρ(CH ),451wν(Ru―N),430mν(Ru―S),and
theconcentrationthatcausedthe50%inhibitionoftheXTT-formazane 327sν(Ru―Cl).Raman(cm− 3 1):3055mν(C―H) ,3014mν (C―H),
formation (IC ) and it was compared to the standard of bovine Cu, ar as
50 2994m, 2924s ν(C―H), 1661m ν(CN), 1511s δ(N―H), 1120m
Zn-superoxidedismutase. s
ν(SO), 314vs ν(Ru―Cl), and 269m ν(Ru―N). ES+MS: (m/z) 260
[(3-Cl-L)+H]+,579[M–H]+.
2.8.Preparationsofcompounds
2.8.2.3. trans-[RuCl (DMSO)(4-Cl-LH)]·DMSO·0.5H O (3). Yield: 60%.
4 2
Thereferencecompoundusedininvivoantitumortesting,[ImH] Anal.Calcd.forC H N Cl O Ru S (668.9):C,28.7;H,3.6;andN,10.5.
16 24 5 5 2.5 1 2
[trans-RuCl 4 (DMSO)(Im)](NAMI-A),waspreparedaccordingtothe Found:C,29.0;H,4.0;andN,10.5.IR(cm−1):3245m,3210mν as (N―H),
already reported procedure [42], and its purity was checked by 3154m,3068mν(C―H) ,3010mν (C―H),2916mν(C―H),1663m
ar as s
comparison of the results of elemental analyses and spectral data ν(CN), 1621m, 1580m δ(N―H), 1490m, 1468w ν(C―C) , 1118s
(UV–visibleandFTIR)withthepublishedones. ν(SO), 1091m ν(C―Cl), 1059s ρ(N―H), 1025m ρ(CH a ) r , 484w
3
ν(Ru―N), 431m ν(Ru―S), 334vs ν(Ru―Cl), and 380w δ(C―S―O).
2.8.1.Startingcompounds Raman(cm−1):3061mν(C―H) ,3018mν (C―H),2993m,2924sν
ar as s
The6-benzylaminopurine(L)derivatives,6-(2-chlorobenzylamino) (C―H),1659mν(CN),1521sδ(N―H),1124mν(SO),313vsν(Ru―Cl),
purine(2-Cl-L),6-(3-chlorobenzylamino)purine(3-Cl-L),6-(4-chloro- and268mν(Ru―N).ES+MS:(m/z)260[(4-Cl-L)+H]+,579[M–H]+.
benzylamino)purine(4-Cl-L),6-(2-bromobenzylamino)purine(2-Br-L),
6-(3-bromobenzylamino)purine (3-Br-L), 6-(4-bromobenzylamino) 2.8.2.4.trans-[RuCl (DMSO)(2-Br-LH)]·DMSO·H O·0.5EtOH(4).Yield:
4 2
purine (4-Br-L), 6-(2-methoxybenzylamino)purine (2-OMe-L), 6-(3- 65%.Anal.Calcd.forC H N Br Cl O Ru S (745.4):C,27.4;H,3.8;
17 28 5 1 4 3.5 1 2
methoxybenzylamino)purine (3-OMe-L), 6-(4-methoxybenzylamino) andN,9.4.Found:C,27.2;H,3.6;andN,9.3.IR(cm−1):3244m,3205m
purine (4-OMe-L), 6-(2-hydroxybenzylamino)purine (2-OH-L), 6-(3- ν (N―H),3157m,3066mν(C―H) ,3020mν (C―H),2980m,2923m
as ar as
hydroxybenzylamino)purine (3-OMe-L), and 6-(4-hydroxybenzyla- ν(C―H), 1660m ν(CN), 1614m, 1566m δ(N―H), 1470w, 1442s
s
mino)purine (4-OH-L), were prepared as described in the literature ν(C―C) , 1118s ν(SO), 1065m ν(C―Br), 1060s ρ(N―H), 1021s
ar
[43]andtheirpuritywasverifiedbyelementalanalysis,FTIRandRaman ρ(CH ),487wν(Ru―N),431m ν(Ru―S),334s ν(Ru―Cl), and 380w
3
spectroscopy. δ(C―S―O). Raman (cm−1): 3060m ν(C―H) , 3016m ν (C―H),
ar as
The initial solution (S1) containing [(DMSO) H][trans-RuCl 2991m, 2924s ν(C―H), 1658m ν(CN), 1513m δ(N―H), 1123m
2 4 s
(DMSO) ]waspreparedbyaslightlymodifiedpreviouslypublished ν(SO), 314vs ν(Ru―Cl), and 268m ν(Ru―N). ES+MS: (m/z) 304
2
procedure [44]. 0.5g (2.4mmol) of RuCl ⋅xH O was partially [(2-Br-L)+H]+,623[M–H]+.
3 2
dissolved in 2.4mL of DMSO, and consequently, 0.4mL of 37% HCl
solutionwasadded.Thereactionmixturewasintensivelystirredat 2.8.2.5. trans-[RuCl (DMSO)(3-Br-LH)]·(Me)CO (5). Yield: 60%. Anal.
4 2
80°Cfor20min.Thedeepredsolutionwasthenheatedupto100°C Calcd.forC H N Br Cl O Ru S (684.3):C,29.8,H,3.4;andN,10.2.
17 23 5 1 4 2 1 1
whilethecolorturnedtobrightorange.Then,thesolutionwascooled Found: C, 30.2; H, 3.1; and N, 10.3. IR (cm−1): 3284m, 3253m ν
as
downand7mLofacetonewasadded.Theresultingsolution(S1)was (N―H),3152,3057mν(C―H) ,3022mν (C―H),2954m,2924mν
ar as s
lefttostandatroomtemperaturefornext2daysandthenusedforthe (C―H),1704ν(CO),1657vsν(CN),1612m,1564mδ(N―H),1465w,
synthesisofRu(III)complexes1–12. 1446s,1421mν(C―C) ,1118sν(SO),1061sρ(N―H),1021sρ(CH ),
ar 3
Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948 941
486w ν(Ru―N), 431m ν(Ru―S), 334s ν(Ru―Cl), and 380w 3128m,3062mν(C―H) ,3021mν (C―H),2923mν(C―H),1661vs
ar as s
δ(C―S―O). Raman (cm−1): 3052m ν(C–H) , 3016m ν (C―H), ν(CN), 1615m, 1590w δ(N―H), 1443m, 1400m ν(C―C) , 1117s
ar as ar
2993w, 2923s ν(C―H), 1662m ν(CN), 1123s ν(SO), 1061m ν(SO),1022sρ(CH ),1062sρ(N―H),453wν(Ru―N),430mν(Ru―S),
s 3
ρ(N―H),317vsν(Ru―Cl),and270mν(Ru―N).ES+MS:(m/z)304 334s ν(Ru―Cl), and 383w δ(C―S―O). Raman (cm−1): 3051m
(3-Br-L)+H]+,623[M–H]+. ν(C―H) ,3014mν (C―H),2995w,2925sν(C―H),1660mν(CN),
ar as s
1620m, 1517s δ(N―H), 1124s ν(SO), 317vs ν(Ru―Cl), and 270m
2.8.2.6. trans-[RuCl (DMSO)(4-Br-LH)]·DMSO (6). Yield: 55%. Anal. ν(Ru―N).ES+MS:(m/z)242[(3-OH-L)+H]+,561[M–H]+.
4
Calcd.forC H N Br Cl O Ru S (704.3):C,27.3;H,3.3;andN,9.9.
16 23 5 1 4 2 1 2
Found: C, 27.2; H, 3.0; and N, 9.8. IR (cm−1): 3240m, 3201m ν 2.8.2.12. trans-[RuCl (DMSO)(4-OH-LH)]·1.5EtOH (12). Yield: 45%.
as 4
(N―H), 3126m, 3068m ν(C―H) , 3014m ν (C―H), 2922m ν Anal.Calcd.forC H N Cl O Ru S (632.4):C,32.3;H,4.3; andN,
ar as s 17 27 5 4 3.5 1 1
(C―H), 2881m, 1659m ν(CN), 1615m, 1572w δ(N―H), 1441w, 11.1.Found:C,32.4;H,4.1;andN,10.7.IR(cm−1):3240m,3203mν
as
1402s ν(C―C) , 1117s ν(SO), 1078m ν(C―Br), 1058s ρ(N―H), (N―H),3120m,3070mν(C―H) ,3012mν (C―H),2954m,2922m
ar ar as
1022sρ(CH ),493mν(Ru―N),431sν(Ru―S),335sν(Ru―Cl),and ν(C―H), 1660vs ν(CN), 1616m, 1575w δ(N―H), 1457m, 1448m,
3 s
379w δ(C―S―O). Raman (cm−1): 3061m ν(C―H) , 3014m ν 1400mν(C―C) ,1116sν(SO),1021mρ(CH ),1069sρ(N―H),484w
ar as ar 3
(C―H), 2994m, 2924s ν(C―H), 1662m ν(CN), 1511m δ(N―H), ν(Ru―N), 431m ν(Ru―S), 340s ν(Ru―Cl), and 382w δ(C―S―O).
s
1128mν(SO),315vsν(Ru―Cl),and272mν(Ru―N).ES+MS:(m/z) Raman(cm−1):3062mν(C―H) ,3015mν (C―H),2923sν(C―H),
ar as s
304[(4-Br-L)+H]+,623[M–H]+. 1665m ν(CN), 1515s δ(N―H), 1121s ν(SO), 315vs ν(Ru―Cl), and
270wν(Ru―N).ES+MS:(m/z)242[(4-OH-L)+H]+,561[M–H]+.
2.8.2.7.trans-[RuCl (DMSO)(2-OMe-LH)]·0.5EtOH·H O(7).Yield:65%.
4 2
Anal.Calcd.forC H N Cl O Ru S (618.3):C,31.1;H,4.1;andN,11.3. 3.Resultsanddiscussion
16 25 5 4 3.5 1 1
Found:C,30.7;H,3.8;andN,11.4.IR(cm−1):3234m,3207mν (N―H),
as
3086m,3068mν(C―H) ,3018mν (C―H),2942w,2922wν(C―H), 3.1.Synthesesandgeneralproperties
ar as s
1661mν(CN),1614w,1601w,1577wδ(N―H),1441w,1402sν(C―C) ,
ar
1112s ν(SO), 1059s ρ(N―H), 1055sh ρ(C―O), 1025s ρ(CH ), 457w TheDMSO/acetone/HClsolution(S1)containingthe[(DMSO) H]
3 2
ν(Ru―N),434sν(Ru―S),335sν(Ru―Cl),and382wδ(C―S―O).Raman [trans-RuCl (DMSO) ]complex(2.5mmol)wasusedforthesynthesis
4 2
(cm−1):3062mν(C―H) ,3015mν (C―H),2994m,2924sν(C―H), ofallRu(III)complexes1–12.Itisnecessarytonotethatthesolution
ar as s
1664m ν(CN), 1511m δ(N―H), 1122m ν(SO), 316vs ν(Ru―Cl), and waspreparedasdescribedinSection2.8.1butusedafterstandingfor
267mν(Ru―N).ES+MS:(m/z)256[(2-OMe-L)+H]+,575[M–H]+. 2daystoachievehighyieldsandpurityofcomplexes1–12.Originally,
weprepared[(DMSO) H][trans-Ru(DMSO) Cl ]inthesolidstatefor
2 2 4
2.8.2.8. trans-[RuCl (DMSO)(3-OMe-LH)]·DMSO·0.5(Me) CO (8). subsequentsyntheticpurposes.Thisapproach,however,resultedin
4 2
Yield: 40%. Anal. Calcd. for C H N Cl O Ru S (684.5): C, 32.4; thefinalproductswhichwereisolatedeitherwithlowyieldsorpoor
18.5 29 5 4 3.5 1 2
H, 4.3; and N, 10.2. Found:C, 32.2; H, 4.2; and N, 10.0. IR (cm−1): purity.Thus,thereactionoftheS1solutioncontaining[(DMSO) H]
2
3288w,3249m,3205mν (N―H),3082m,3057mν(C―H) ,3012m [trans-Ru(DMSO) Cl ]dilutedbyethanolorCHCl withasolutionof
as ar 2 4 3
ν (C―H), 2954m, 2927m ν(C―H), 1705 ν(CO), 1658vs ν(CN), thecorrespondingorganiccompoundaffordedtheRu(III)complexes
as s
1616m, 1602w δ(N―H), 1441m, 1403m ν(C―C) , 1118s ν(SO), 1–12ofthecompositionstrans-[RuCl (DMSO)(n-Cl-LH)]⋅xSol(1–3),
ar 4
1066sρ(N―H),1024sρ(CH ),465wν(Ru―N),430mν(Ru―S),334s trans-[RuCl (DMSO)(n-Br-LH)]⋅xSol (4–6), trans-[RuCl (DMSO)
3 4 4
ν(Ru―Cl),and376wδ(C―S―O).Raman(cm−1):3065mν(C―H) , (n-OMe-LH)]⋅xSol (7–9) and trans-[RuCl (DMSO)(n-OH-LH)]⋅xSol
ar 4
3015m ν (C―H), 2995w, 2925s ν(C―H), 1658m ν(CN), 1616m, (10–12); n=2, 3, and 4; x=0–1.5; SolH O, DMSO, EtOH and/or
as s 2
1514sδ(N―H),1124sν(SO),315vsν(Ru―Cl),and267mν(Ru―N). (Me) CO.Thecompositionsandstructuresofthepreparedcomplexes
2
ES+MS:(m/z)256[(3-OMe-L)+H]+,575[M–H]+. havebeendeterminedusingavarietyofphysicaltechniques,mainly
by single crystal X-ray analysis in the case of trans-[RuCl (DMSO)
4
2.8.2.9. trans-[RuCl (DMSO)(4-OMe-LH)]·DMSO (9). Yield:50%.Anal. (3-Br-LH)]·(Me) CO(5).
4 2
Calcd. for C H N Cl O Ru S (655.4): C, 31.2; H, 4.0; and N, 10.7. ES+MSexperimentswereperformedforallthecomplexesinthe
17 26 5 4 3 1 2
Found:C,31.6;H,3.8;andN,10.5.IR(cm−1):3240m,3203mν (N―H), DMF/methanol/aceticacidsolutions(c=10−4M)(seeExperimental
as
3120m,3070mν(C―H) ,3012mν (C―H),2954m,2922mν(C―H), Section).Experimentalandcalcd.ES+MSvaluesforeachcompound
ar as s
1660vsν(CN),1611m,1568wδ(N―H),1442s,1400sν(C―C) ,1115s were identical to a significant last figure above the decimal point.
ar
ν(SO),1023sρ(CH ),1057sρ(N―H),451wν(Ru―N),431mν(Ru―S), Togetherwiththemolecularpeaksofthecomplexes,theadductswith
3
340s ν(Ru―Cl), and 382w δ(C―S―O). Raman (cm−1): 3061m sodium and potassium, usually with higher intensity than the
ν(C―H) ,3010vsν (C―H),2949w,2927sν(C―H),1661mν(CN), molecular peaks, were also observed in the spectra. All the mass
ar as s
1508mδ(N―H),1121mν(SO),317sν(Ru―Cl),and267mν(Ru―N). spectra of 1–12 contained the fragment corresponding to the
ES+MS:(m/z)256[(4-OMe-L)+H]+,575[M–H]+. appropriate organic L derivative. The molar conductivity values
measured in N,N′-dimethylformamide (DMF) varied from 7.5 to
2.8.2.10.trans-[RuCl (DMSO)(2-OH-LH)]·DMSO·H O(10).Yield:60%. 19.8Scm2mol−1andthusshowedthatallthecomplexesbehavedas
4 2
Anal.Calcd.forC H N Cl O Ru S (659.4):C,29.1;H,4.0;andN,10.6. non-electrolytesinthesolventused(Table3)[45].Thermalbehavior
16 26 5 4 4 1 2
Found:C,29.5;H,4.0;andN,10.3.IR(cm−1):3248m,3212mν (N―H), of complexes 2, 3, 4 and 8, as the representative examples, was
as
3155m,3060mν(C―H) ,3019mν (C―H),2924mν(C―H),1659vs studied by simultaneous TG and DTA analyses. The results of the
ar as s
ν(CN), 1616m, 1575w δ(N―H), 1457m, 1448m, 1400m ν(C―C) , analysesshowedthatthecoursesofthermaldegradationsofallthe
ar
1116sν(SO),1021mρ(CH ),1069sρ(N―H),484wν(Ru―N),433m complexeswereverysimilar,andmoreover,thepresenceofsolvent
3
ν(Ru―S), 336vs ν(Ru―Cl), and 389w δ(C―S―O). Raman (cm−1): molecules of crystallization was also clearly proved. The detailed
3065mν(C―H) ,3015mν (C―H),2993m,2923sν(C―H),1656m interpretation of the obtained TG and DTA curves for complex 2 is
ar as s
ν(CN), 1511s δ(N―H), 1123s ν(SO), 316vs ν(Ru―Cl), and 266m giveninSupplementarydata(seeFig.S1).
ν(Ru―N).ES+MS:(m/z)242[(2-OH-L)+H]+,561[M–H]+.
3.2.X-raystructureoftrans-[RuCl (DMSO)(3-Br-LH)]·(Me) CO(5)
4 2
2.8.2.11.trans-[RuCl (DMSO)(3-OH-LH)]·DMSO·H O(11).Yield:50%.
4 2
Anal.Calcd.forC H N Cl O Ru S (659.4):C,29.1;H,4.0;andN,10.6. The molecular structure of trans-[RuCl (DMSO)(3-Br-LH)]⋅
16 26 5 4 4 1 2 4
Found:C,28.7;H,3.8;andN,10.7.IR(cm−1):3244m,3207mν (N―H), (Me) CO (5) is depicted in Fig. 1. Selected bond lengths and angles
as 2
942 Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948
arepresentedinthelegendtoFig.1,whilehydrogenbondparameters Table2
are given in Table 2. The asymmetric unit of 5 contains the slightly Hydrogenbondparameters(Å,°)for[RuCl4(DMSO)(3-Br-LH)]·(Me)2CO(5).
distortedoctahedralcomplextrans-[RuCl 4 (DMSO)(3-Br-LH)]andone D–H···A D(D–H) d(H···A) d(D···A) b(DHA)
acetonemoleculeofcrystallization.TheRu(III)atomishexacoordinated
N3–H3A⋅⋅⋅Cl2 0.88 2.85 3.317(4) 115.2
byfourchloridoligandsinabasalplane,onemoleculeofS-coordinated N3–H3A⋅⋅⋅Cl3 0.88 2.67 3.252(4) 124.6
DMSO, and one molecule of protonated 6-(3-bromobenzylamino) N3–H3A⋅⋅⋅O2i 0.88 2.17 2.827(6) 131.5
purine (3-Br-LH). The organic N-donor ligand is represented by the N6–H6A⋅⋅⋅O1ii 0.88 2.11 2.967(5) 165.1
N3-protonated N7-tautomer which is coordinated to ruthenium(III) N7–H7A⋅⋅⋅O1ii 0.88 1.96 2.770(5) 153.1
N7–H7A⋅⋅⋅Cl2ii 0.88 2.76 3.263(4) 117.8
throughtheN9atomofthepurinemoiety.ThemoleculeofDMSOis
situatedinthetranspositiontotheN-donorligand. Symmetrytransformationsusedtogenerateequivalentatoms:(i)−x+1,−y+1,−z+1;
(ii)−x+1,y+1/2,−z+3/2.
Thefourchloridoligandsarebondedtothemetalinanearlyideal
planewiththemaximaldeviationfromthemeanplanebeing0.0238(6)
ÅfortheCl2atom.TheRu―Clbonddistancesrangefrom2.336(2)Åto
2.346(2)Å. Similar bond lengths were found in trans-[RuCl (DMSO) of the coordination site N9. The C8–N9–C4 angle is due to the
4
(GuaH)],whereGuaHN(3)-protonatedguanine[2.341(2)–2.366(2)Å] presenceoftheRu―N9bondsignificantlyenhanced;itequals103.1
and trans-[RuCl (DMSO)(6-BuapH)], where 6-BuapHN3-protonated (3)°in3-Br-LHchlorideand104.6(4)°in5.AlsotheN9―C8bondwas
4
6-butylaminopurine [2.312(3)–2.360(3)Å] [46,47]. Todate,29 struc- foundtobelongerin5[1.344(6)Å]thanintheuncoordinatedcation
tures involving the trans-[RuCl (DMSO)(NL)] fragment (where NL [1.329(4)Å].Moreover,alsotheC8–N7–C5angleisbiggerin5thanin
4
standsforanyN-donorligand)havebeendepositedintheCambridge 3-Br-LHchloride;107.5(4)°ascomparedwith106.3(2)°,respectively.
StructuralDatabase[48].Itwasfoundthatthecalculatedaveragevalues Itcanbecausedbythedifferentnetworksofhydrogenbondspresent
ofthebondlengthsRu–N[2.112Å]andRu–S[2.283Å]aresomewhat in the crystalstructures of thetwo compounds. The intramolecular
higherthanthosefoundin5[Ru–N9=2.104(4)andRu–S1=2.2603 N3―H⋅⋅⋅Cl2 and N3―H⋅⋅⋅Cl3, and intermolecular N3―H⋅⋅⋅O2,
(13)Å].The3-Br-LHligandcontainsnearlyplanarbenzene,pyrimidine N7―H⋅⋅⋅Cl2 and N7―H⋅⋅⋅O1 hydrogen bonds (Fig. 2) were found
and imidazole ring systems. The imidazole and pyrimidine rings are tocontributetothestabilizationofthecrystalstructureof5.
nearlycoplanarwiththedihedralangleof0.622(2)°.Themeanplanesof
thebenzeneringandpurineskeletonmakeadihedralangleof49.49 3.3.FTIR,RamanandUV–visiblespectroscopy
(8)°.
The X-ray structure of 6-(3-bromobenzylamino)purin-3-ium TheFTIR(4000–200cm−1)andRaman(3750–150cm–1)spectros-
chloride has been already determined [49]. Both 3-Br-LH chloride copy confirmed the presence of the organic N-donor, DMSO and
andthecoordinatedligandarerepresentedastheN3-protonatedN7 chloridoligandsinallthecomplexes.Themid-FTIRspectraofcomplexes
tautomers. If we compare the geometric parameters of the N9- 1–12showedthecharacteristicabsorptionbandscorrespondingto:ν
as
coordinated 3-Br-LH with the previously published data of 3-Br-LH (N―H)at3288–3201cm−1;ν(C―H) at3070–3056cm−1;ν(CN)at
ar
chloride,themostsignificantdifferencescanbefoundinthevicinity 1663–1657cm−1; ν(C―C) at 1470–1398cm−1 and ν(S O) at
ar coord.
1119–1112cm−1.Inthefar-FTIRspectra,theintensivepeaksdetected
at430–434cm−1and340–327cm−1maybeassignedtotheν(Ru―S),
and ν(Ru―Cl) vibration, respectively. The presence of the peak
correspondingto ν(Ru―S)clearlyconfirmedthecoordinationofthe
DMSOligandtotheRuatomviatheSatom.ν(Ru―N)isrepresentedby
weakpeaksinthe451–493cm−1region.TheRamanspectraexhibited
resolvedabsorptionpeakscorrespondingtoν (C―H)inCH (2923–
as 2
2927cm−1); ν (C―H) in S–CH (3014–3018cm−1); ν(C―H) in
as 3 s
S–CH (2991–2996cm−1) and ν(C―H)in O–CH (2948–2950cm−1
3 3
and2836–2938cm−1).Theintenseabsorptionbands,varyingfrom310
to 315cm−1, are assignable to ν(Ru―Cl). The peaks, with medium
intensity, observed at 266–270cm−1 correspond to ν(Ru―N). The
assignment was performed in accordance with the literature data
[44,50,51].
Octahedral low spin d5 complexes have the 2T ground state
2g
correspondingtothet5 electronicstate.Basedonthisstatement,many
2g
d–dtransitionsmaybeexpectedinconnectionwiththe2T groundstate.
2g
Theelectronicabsorptionspectraofcomplexes1–12weremeasuredin
DMFandmethanol.Threeabsorptionbandswereobservedat20,746–
20,920cm−1, 24,390–25,707cm−1, and 32,787–36,496cm−1 both in
thesolutionandsolidstate.Thespectraofcomplex2bothinmethanol
andsolidstateareshowninFig.3.Theintensivebandsatca.34,000cm−1
are assignable to the ligand-to-metal (Cl→Ru) charge-transfer (CT)
transitions,probablyconnectedwiththetransitionsoriginatingfromthe
π-level of molecular orbitals of chlorido ligands to the incompletely
occupiedmetalt level(CT1)[52].Thenextintensivebands(CT2)atca.
2g
25,000cm−1 can be assigned to the metal-to-ligand (Ru→3-Cl-LH)
Fig.1.Themolecularstructureof5togetherwiththeatomnumberingscheme.The
acetonesolventmoleculeofcrystallizationwasomittedforclarity.Thenon-Hatomsare electron transitions. The bands observed at ca. 20,800cm−1 may be
drawnasdisplacementellipsoidsatthe50%probabilitylevel.Selectedbondlengths(Å) assignedtothespin-allowed2T →2A transitions,correspondingtoν
2g 2g 1
and angles (°): Ru–N9, 2.104(4); Ru–S1, 2.2603(13); Ru–Cl1, 2.3356(13); Ru–Cl2, [53].Thecalculatedvaluesofmolarabsorptioncoefficientconnectedwith
2.3472(12);Ru–Cl3,2.3453(12);Ru–Cl4,2.3411(12);Br1–C12,1.908(5);S1–O1,1.489
the last-mentioned transition clearly correspond to d–d transitions
(4);S1–C17,1.762(6);O2–C20,1.217(8);N9–Ru–S1,176.58(11);Cl1–Ru–Cl3,177.63
(5); Cl4–Ru–Cl2, 176.36(5); C2–N1–C6, 119.4(4); C2–N3–C4, 117.0(4); C8–N7–C5,
(Table3).Theenergydifferencebetweenthegroundstateandthefirst
107.5(4);C8–N9–C4,104.6(4);andC6–N6–C9,122.1(4). excited state, assuming pure t
2g
→e
g
quantization, can be calculated
Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948 943
Fig.2.Thesystemofhydrogenbondsinthecrystalstructureof5.TheH-atomsnotinvolvedintohydrogenbondsareomittedforclarity.Symmetrycodes:(i)−x+1,−y+1,−z+1;
(ii)−x+1,y+1/2,−z+3/2.
using the relation: ν =10Dq−3F −20F , with F =10F =1000 for4andg=2.30,Θ=+0.22Kfor6.Thehighervaluesofg-factorsare
1 2 4 2 4
[54,55].ThevaluesofligandfieldparametersBandβwerecalculated inagreementwiththeroomtemperaturevaluesofeffectivemagnetic
tobe345–357cm−1,and0.55–0.57,respectively[53].Thedecreasein moments,2.01μ for4and1.99μ for6,andareexpectedforpseudo-
B B
valuesoftheRacah'sparameterBascomparedtothefree-ionvalue octahedral low-spin Ru(III) complexes with t5 e0 configuration with
2g g
(627cm−1) indicates that a dominantly covalent character of bonds S=1/2.ThepositiveWeissconstantsreflectintermolecularinteractions
occursinthevicinityofthecentralRu(III)atom.Theobtainedβvalues oftheferromagneticoriginandalsoexplaintheincreaseoftheeffective
mayindicatethedecreaseineffectivepositivechargevaluesoftheRu magneticmomentsbelow25Kforbothcompounds(Figs.4andS2).In
(III) ion. This conclusion was confirmed by the calculation of the ordertoanalyzebothtemperatureandfielddependentexperimental
effectiveioniccharges,Z*[56],whichwerefoundtorangefrom1.03e- magnetic data of the complexes, the following spin Hamiltonian for
to 1.13e- for the complexes 1–12. Similar values of the ligand field S=1/2wasused[57]
parametersB(395–424cm−1)andβ(0.63–0.68)werealsodetermined
D E
for other Ru(III) complexes, e.g. [RuCl x (H 2 O) y L 2 ], where L stands for H ˆ =μ Bg S ˆℏ−1+zj S ˆ S ˆℏ−1; ð1Þ
1-phenyl-1,2-propanedione-2-oxime; 2,3-butanedione monoxime or B iso z z T z
α-benzilmonoxime,x=1or2,andy=0or1[55].
wheretheZeemantermandmolecular-fieldcorrectionareincluded.
3.4.MagneticdataandEPRspectra Only isotropic g-value was considered, zj is the common molecular-
fieldparameterand〈Ŝ〉 isthethermalaverageofthespinprojection.
z T
Themagneticdatawereinvestigatedindetailfortwocompounds4 Molar magnetization was calculated through the partition function
and6.First,thetemperaturedependencesofthemagneticsusceptibility M =N kT(∂lnZ/∂B).ThemeanvalueofŜ,asgivenintheEq.(1),was
mol A z
werefittedusingCurie–Weisslawandresulteding=2.31,Θ=+0.27K
Table3
Electronicspectraldata,ligandfieldparameters,andmolarconductivitydataforRu(III)
complexes1–12.
Complex 2T2g →2A[ 2 a g ] CT1[a][cm−1] CT2[b][cm−1] 10Dq λ M [a]
[cm−1](ε,M−1 (ε,M−1cm−1) (ε,M−1cm−1) [cm−1] [Scm2
cm−1) mol−1]
1 20,790(473) 24,691(2392) 35,971(2188) 25,790 13.7
2 20,876(516) 24,509(1863) 33,113(1794) 25,876 8.2
3 20,920(454) 24,570(2318) 32,894(1920) 25,790 17.5
4 20,790(448) 24,570(1804) 36,101(1732) 25,790 18.3
5 20,790(498) 24,630(1715) 35,714(1756) 25,790 13.2
6 20,790(456) 24,449(3046) 35,971(2992) 25,790 16.3
7 20,790(416) 24,390(2110) 36,496(2075) 25,790 7.5
8 20,876(560) 24,691(1455) 33,113(1304) 25,876 13.7
9 20,790(385) 24,509(2012) 36,232(1987) 25,790 19.0
10 20,833(395) 24,509(2392) 36,101(2038) 25,833 12.0
11 20,746(378) 24,691(1953) 33,113(1722) 25,790 14.6
12 20,920(402) 25,707(2005) 32,787(1890) 25,920 19.8
[a]MeasuredinDMFsolution(c=10−3M).
[b]Measuredinmethanolsolution(c=10−3M).
Fig.3.Methanolic(—)anddiffuse-reflectance(−−−)spectraof2. CT1chargetransfer1,CT2chargetransfer2.
944 Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948
calculatedbytheiterativeprocedureforalldata.Thetemperatureand
fielddependenceofmagnetizationwasfittedtogetherandresultedin
g=2.31andzj=+0.84cm−1for4,andg=2.29andzj=+0.93cm−1
for 6 (Figs. 4 and S2). The positive-values of the molecular-field
parameters express that the intermolecular interactions are of the
ferromagneticnature.Theg-valuesaregreaterthan2.0,whichcanbe
expectedforlow-spinRu(III)complexes.
The room temperature solid state X-band EPR spectra of
complexes 4 and 6 were recorded (see Fig. 5). The low spin d5
configurationisagoodprobeofthemolecularstructureandbonding
duetohighsensitivityofgvaluestosmallchangesinthestructureand
metal-ligand covalence. The EPR spectra of both compounds show
evidenceoftwodifferentgvalueswithg⊥=2.34andg
||
=1.85for4or
g⊥=2.35andg
||
=1.83for6.Themeasuredspectrawerereproduced
using the EasySpin package [58]. The presence of two different g
valuesindicatesanaxialsymmetryoftheRu(III)chromophore,which
isinaccordancewiththecompressedtetragonalstructurefoundin
compound 5 by X-ray analysis (see Section 3.2). Similar EPR
parameter values were also found in other Ru(III) compounds, e.g. Fig. 5. The comparison of measured and calculated EPR spectra of 6 at room
in[RuCl (EPh ) (L)](whereL=abidentateSchiffbaseligand,and temperature.
2 3 2
E=PorAs)wereg⊥=2.03–2.39andg
||
=1.89–1.99[59].
thecomplexes,togetherwiththecyclicvoltammogramsofRuCl ⋅xH O
3 2
andfreeligand2-OMe-L,areshowninFig.S3.
3.5.Cyclicvoltammetry
3.6.Invitrocytotoxicity
Onequasireversiblesingle-electronreductionwave,assignableto
the Ru(III)→Ru(II) process with the peak-to-peak separations Thecomplexes1,2,7,8,9,10and11weretestedfortheirinvitro
rangingfrom78to143mV,wasobservedinvoltammogramsofall cytotoxicityagainsthumancancercelllinesG361,K562,HOSandMCF7.
the complexes (1–12). The values of the reduction potential, E , The IC values for all the tested complexes were determined to be
red 50
rangedfrom −285mV to −170mV (measured in DMF vs Ag/AgCl higher than 100μM. It means that all complexes were found to be
electrode)forthecompounds1–12.Thesevaluesrangefrom−86mV inactiveintheconcentrationrangemeasured.Itshouldbeemphasized
to+29mVaftertheconversionofthevaluestothosebelongingto that NAMI-A itself, as well as its predecessor Na[trans-RuCl (DMSO)
4
normalhydrogenelectrode(NHE).Theyareclosetothosefoundin (Im)]calledNAMIaswellasotherNAMI-A-likecomplexesshowedno
theliteraturefor[IndH][RuCl (DMSO)(Ind)](E =−40mVvsNHE significantinvitrocytotoxicityagainsthumantumorcelllines.Asan
4 red
in a DMF solution) [25]. It has been already found out that the example,aseriesof18ruthenium(III)complexes,structurallyrelatedto
activationbyreductionmightberesponsiblefortheactivityofsome theselectiveantimetastaticdrugNAMI,couldbementioned[62].These
Ru-complexes, e.g. KP1019 (see Introduction). In proliferating cells, complexes were tested on TLXS lymphoma and MCa mammary
the reduction potential is about −240mV vs NHE, while the carcinomahumancelllinestoevaluatethedependenceofthedegree
corresponding value is by about 100mV lower inside the tumor ofcytotoxicityandantimetastaticactivityofsuchcomplexesontheir
cells. This means that biological reducing agents, e.g. glutathione or chemicalproperties.Thediscussedcomplexesshowednosignificantin
ascorbic acid, may be capable of reducing the Ru(III) species to the vitro cytotoxicity with the IC values above 100μM. The study
50
corresponding Ru(II) ones [60]. According to this hypothesis, the concludedthatnoticeableinvivoantitumoractivitymaybeexpected
accessiblereductionpotentialforinvivoactivityofRu(III)complexes incaseofNAMI-likecomplexesforwhichinvitrocytotoxicityispooror
should be in the range of 〈–400mV; +800mV〉 vs NHE [61]. This noneatall.ThisconclusionisapplicabletoNAMI-Aitselfaswell,because
conditionwasaccomplishedforallthepreparedcomplexes1–12.The invitrocytotoxicitywithIC N100μMagainstKBcells(asublineofthe
50
cyclicvoltammogramofcomplex7,asarepresentativeexampleofall ubiquitousKeratin-formingtumorcelllines)wasdeterminedforthis
compound.However,invivoantitumoractivitycomparabletothatfor
cisplatinwasfoundagainstmetastatictumors,i.e.Lewislungcarcinoma
or MCa mammary carcinoma [63]. The absence of in vitro cell
cytotoxicity might be explained by apparent lack of host toxicity of
thesecomplexes[13].
3.7.Invivoantitumoractivity
Theinvivoantitumoractivityofthecomplexes1,3,5,7,9,and11,
andthereferencecompoundNAMI-Awastestedonthemousemodel
ofleukocyticleukemia(L1210).Duetolimitedamountsofthetested
complexes,wedecidedtousethemorerealisticmodel,resembling
the therapeutic use of the tested compounds. The 10day initiation
period in which the primary tumors formed, was subsequently
followed by three days of therapeutic intervention, i.e. the i.p.
application of the tested compounds with the same dosage of
50mg/kg/day. We decided to use this dosage on the basis of very
Fig.4.Magneticpropertiesof4.Left:temperaturedependenceoftheeffectivemagnetic
promisingantitumoractivityresultsofreferencecompoundNAMI-A
moment (calculated from magnetization at B=1T); right: field dependence of
magnetization at T=2.0. Circles—experimental points, lines—calculated using the on model systems involving much shorter time of implantational
best-fitparameters. tumorigenesis and a 7-day therapeutic plan [64]. The direct
Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948 945
confrontationofournewlypreparedRu(III)complexeswithNAMI-A Table5
canthusgiveusthecluewhichofthecomplexesshouldbechosenfor Theresultsofantiproliferative,pro-apoptoticactivityandnecrosis-inductionbyRu(III)
complexes.
thefurtherscreening.
Thepercentagesofmeansurvivaltime,%T/Cdefinedastheratioof Substance Circumscribedtumors Diffusedtumors
themeansurvivaltimeofthetreatedanimalgroups(T)dividedbythe
Mitoses Apoptoses Necroses Mitoses Apoptoses Necroses
meansurvivaloftheuntreatedcontrolgroup(C),werecalculatedand
control 8.5 5 1.5 7.5 5 1
areshowninTable4.Weshouldnotethatdespitetherelativelylarge
NAMI-A 11 10 2 5 3 1
differencesof%T/Cindifferentgroups,noneoftheseresultsprovedto 1 2 3 1 27 8 1
be significantly different from the control, even if the most active 3 4.25 11.75 1.5 7.75 8.25 0.75
compounds 5 and 7 achieved marginal results with p≈0.1. On the 5 4.5 11.5 2.5 6 6.5 1
7 6.6 60.6 2.2 6.3 70.3 1.3
otherhand,whencomparingtheresultsachievedbythegroupofour
9 2.3 10 2.3 6.3 7 0.67
newly synthesized Ru(III) complexes with the results obtained for
11 1.5 17 2.7 8.7 12 1
NAMI-A,significantdifferenceswerefoundforcompounds5,7,1,and
Mitoses—averagenumberofmitosesinthefieldofview,Apoptoses—averagenumber
9(inorderofdecreasing%T/C).Inotherwords,significantlyhigher
ofapoptosesinthefieldofview,Necroses—averageevaluationofnecroses.
lifeextensioneffectwith%T/Crangingfrom100±0.17to106±0.49
wasobservedintheanimalgroupstreatedbycomplexes5,7,1,and9
ascomparedwiththeNAMI-A-treatedgroup. apoptoticactivitywasnotprovedandtheincreasedmitoticactivity
waspreserved.
3.7.1.Histologicalandhistochemicalfindings
3.7.1.3. Evaluation of mitosis, apoptosis, and necrosis in tumor tissue
3.7.1.1.Macroscopicobservations.Allanimals,implantedwiththecell samples. The results of antiproliferative, pro-apoptotic activity and
line L1210, expressed significant tumor infiltration of abdominal necrosis-induction,asdeterminedbythehistologicalandhistochemical
cavity, peritoneal infiltration and well circumscribed tumors in the methods,bythetestedcompoundsaresummarizedinTable5.
place of application. In all animals, the tumor proliferated in the Thesemi-quantitativeevaluationofthetumoractivityrevealedthe
visceral fatty tissue and in the gonadal area, and formed well highest pro-apoptotic activity, when comparing the mitotic rate and
circumscribed focuses. Tumorcell dissemination of the diffuse type necrosis-induction effect, in complex 7-treated group (Fig. 6). The
wasfoundinGALT(gut-associatedlymphoidtissue)inabout75%of treatment with complex 7 would most likely lead to tumor volume
allthetestedanimals.Organswereoftensignificantlyatrophied.The reductionbecauseinthisinvivoscreening,decreasedproliferationwas
presenceofseroustransudatesintheabdominalcavitywasdetected observed, while apoptosis was endorsed and also, necrosis strongly
in 90% of cases. No significant changes between the tested groups induced.Thepositivecontrolcorrespondedtothevaluesofaproliferating
werediscoveredduringmacroscopicexamination.Theintactgroupof tumor.ThereferencesubstanceNAMI-Adidnothaveanyimpactonthe
animalsdidnotshowanychanges. tumor mitotic activity; however, it showed slightly increased pro-
apoptotic activity and induced necrosis in the circumscribed-type
3.7.1.2. Histological examination. The discovered tumors were tumors.Complex11,whencomparingthemitoticrate,expressedhigh
histologicallyidentifiedastwodifferenttypesofmalignantlymphoma pro-apoptoticactivity.Moreover,thenecrosisinductioninthecircum-
originating from the same B-line. B-lymphoma with the presence of scribed-typetumorswasthemostincreasedascomparedtoallotherRu
tangible body macrophages (macrophages with phagocytised debris (III)complexesincludingNAMI-A.Thediffuse-typetumoralsorevealed
fromapoptotictumorcells)andlowermitoticactivitywasdiffuse-type minorchanges.Thecomplexes3,5,and9producedverysimilarresults.
(affectingGALTandmesocolon).Wellcircumscribedtumorscouldbe Whentheratesofmitosesandapoptoseswerecompared,theiraction
identifiedassmallcelllymphomas. could be regarded as pro-apoptotic. Complex 1 showed slight pro-
Inpositivecontrol,predominantlywellcircumscribedtumorswith apoptotic activity only in the diffuse-type tumors, however, the
increased mitotic activity, neoangiogenesis (Figs. S4 and S5) and proliferation rate was higher, which means that the tumor growth
diffused necrosis, were observed. After the comparison with the wouldnotbesignificantlyaffected.Alltheevaluationswereconfirmedby
positive control, the most interesting results were observed in the theTUNELapoptosisdetectionkit.
groups treated with complexes 1, 5 and 7 because the findings in Ingeneral,itcanbeconcludedthatthetestedRu(III)complexes
tumorsrevealedintensivenecrosisandapoptoticactivity(Figs.S8,S9, canallbecharacterizedascompoundswithgoodpotentialbecause
S12, S13, and S16); and moreover, this was also detected in the theywereevaluatedtopossesshigherpro-apoptoticactivitythanthe
diffuse-type tumors. On the other hand, in the group treated with reference compound NAMI-A. It should be emphasized that such
complex 3, the diffusion type tumors showed no destruction. The positive results were very encouraging for us because the selected
circumscribed forms, however, revealed sizeable diffusion necroses assaygenerallyrequiresthetestedcompoundstobemoreactiveto
(Figs. S14 and S15), but the increased apoptotic activity was not enabletheinterpretationofresults.Expressingthecompoundactivity
provedandtheincreasedmitoticactivitywaspreserved.Intheanimal bytheratioofthenumberofobservedmitosisandapoptosis,complex
groupsof NAMI-A,9,and 11, both tumortypeswere detected. The 7isthemostpro-apoptoticactivefollowedbycomplexes11,9and5.
diffuse-type tumor exhibited no destruction, but the circumscribed When combining the results of the life extension assay and the
formshowedsignificantdiffusedandzonalnecrosis(Figs.S6,S7,S10, histologicalandhistochemicalobservations,thecompounds5,7and9
S11,S17andS18),especiallyintumorsinfiltratingthevisceralfatty canberegardedaspromisingforfuturestudies,andparticularly,the
tissue. Similarly to the complex 3-treated group, the increased complex 7 which both positively affected life length in the treated
Table4
Theaveragesurvivaltimesofthecompound-treatedgroups(%T/C).
Compound control NAMI-A 1 3 5 7 9 11
⁎ ⁎ ⁎ ⁎
%T/C 100 94 101 104 106 105 100 99
SEM 0.53 0.29 0.35 0.80 0.61 0.49 0.17 0.34
SEM=standarderrorofthemean.
⁎ Statisticallysignificantresult,whencomparingthevaluestoNAMI-Agroup.
946 Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948
Fig.6.MalignantB-lymphoma,isolatedfromtheanimaltreatedwithcomplex7.The Fig.7.FluorescencemicroscopyimageofinsituvisualizationofaRu(II)-bipyridine
pathologicalfindingsarecharacterizedbyzonalnecrosis(markedasN)andmassive complexinthediffusetypeoftumorisolatedfromtheanimaltreatedwithcomplex7.
apoptoticactivity(markedasA).Apoptoticcellsweredetectedbyimmunohistochem- The pathological findings, characterized by zonal necrosis and high intensity of
icaldetectionwiththeApoTagDetectionKit. fluorescence,weremarkedasN.
animalgroupandshowedthemostsignificantpro-apoptoticactivity. reaction speed of the whole superoxide dismutation process. The
Thesepreliminaryresultscouldserveasthebasisforfurtherinvivo Ru―Sbondlengthissomewhatshorterincomplex5ascomparedto
studiesofthemostpromisingcompoundsonthesolidtumormodels. theaveragemeanvaluecalculated for29similar systemsinvolving
the trans-[RuCl (DMSO)(NL)] fragment found in CSD (for detailed
4
3.7.1.4.VisualizationofintracellularRu(II)byfluorescencemicroscopy. informationseeSection3.2).Thiscouldslowdowntheprogressionof
Inanefforttoobtainadeeperinsightintothepossiblemechanismof thedisproportionationprocess.Asmallportionofrepulsiontowards
action of the tested Ru(III)complexes, we successfully developed a thesuperoxideanionwhileapproachingtheRu(III)atomcouldalso
newsimplemethodfordetectionandvisualizationofintracellularRu be caused by the distribution of electron density in the polarized
(II)byfluorescencemicroscopy.Themethodweusedisbasedonthe molecule of the Ru(III) complex involving the protonated ligand of
preparation of an Ru(II)-bipyridine complex in situ. Complexes substituted 6-benzylaminopurine. It is also a known fact, that the
involving the Ru(II)-bipyridine residues, but not Ru(III) complexes, substitution of DMSO ligand in the Ru(III) complexes changes
areknowntoexhibitfluorescenceatca.650nmwhentheexcitation dramatically the redox properties of the complex [7, and the
wavelength of 450nm is applied [65,66]. This feature has already referencestherein]andinfluencestheprogressionofelectrochemical
been successfully used for very sensitive fluorescence staining of reactionofRu(III)complexeswithsuperoxide.Thiscouldbealsothe
proteinsandnucleicacidsbydifferentRu(II)complexeswithvarious reasonwhythetestedRu(III)complexesshowedonlymoderateSOD-
chelating heterocyclic ligands in different biochemical samples, e.g. mimicactivity,representedbytheIC valueswhicharemorethan
50
acrylamidegelsorWesternblotmembranes[67,68].Tothebestofour four orders of magnitude larger than the bovine Cu,Zn-superoxide
knowledge,ourapproachtopreparethefluorescentRu(II)-bipyridine dismutase,usedasastandard.
complexesinsituintheRu(II)-metallatedtissuesisoriginal.
Theanalysisoffluorescentmicroscopyimagesoftumorsisolated
4.Conclusions
from the treated animals led to the findings that the tested Ru(III)
complexes are unequivocally transported to the tumor cells and Herein, we have reported the syntheses and characterization of a
undergo reduction to Ru(II), which was demonstrated by the high seriesoftwelvenewRu(III)complexeswithvariouslybenzyl-substituted
intensityoffluorescenceaftertheapplicationof2,2′-bipyridine(see
6-benzylaminopurine(L)derivatives.Inallcases,thesyntheticprocedure
Fig.7 forfurther details).The highestintensityof fluorescencewas involving the replacement of one DMSO molecule by the organic L
identified in the necrotic tissue. When compared to the tumor derivative in the starting complex anion [trans-RuCl (DMSO) ]– was
4 2
sectionsfromtheuntreatedgroupofanimals,asignificantdifference
used. All complexes were fully characterized by various physical
influorescenceofthetumortissuewasfound.Theuntreatedgroup
techniquesasmononuclearoctahedralRu(III)complexesofthegeneral
tumorsectionsrevealedonlyaninsignificantamountoffluorescence compositions trans-[RuCl (DMSO)(n-Cl-LH)]⋅xSol (1–3), trans-[RuCl
4 4
(Fig.S19). (DMSO)(n-Br-LH)]⋅xSol (4–6), trans-[RuCl (DMSO)(n-OMe-LH)]⋅xSol
4
(7–9)andtrans-[RuCl (DMSO)(n-OH-LH)]⋅xSol(10–12);n=2,3,and4;
4
3.8.SOD-mimicactivity x=0–1.5; Sol=H O, DMSO, EtOH and/or (Me) CO. Moreover, [RuCl
2 2 4
TheSOD-mimicactivityof2,3,8,11and12wastestedandthe
resultsoftheactivitytestsaregiveninTable6.Thereareonlyfew Table6
publicationsregardingtheinteractionofRu(III)complexeswiththe TheresultsofinvitroantiradicalSOD-likeactivitytesting.
superoxideanion,eitherincontextofthemechanismofinteraction Complex IC50(μM) 1/IC50(μM−1)
[69,70] or the scavenging activity of the anion radical by transition
2 2.64×10−3 378
metalcomplexes[55].Onthebasisofpreviouslypublishedstudies, 3 6.09×10−3 164
the redox-based disproportionation of superoxide by the tested Ru 8 5.86×10−3 170
(III)complexescouldbeexpected.Weproposethatthefirstreaction 11 2.83×10−3 353
step,definedbythesubstitutionoftheapicalS-bondedDMSOligand 12 6.05×10−3 165
Cu,Zn-SOD 4.800×10−7 2,083,333
by the superoxide anion radical, could play the crucial role in the
Z.Trávníčeketal./JournalofInorganicBiochemistry105(2011)937–948 947
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