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Synthesis, Cellular Uptake and Biological Activity Against Pathogenic Microorganisms and Cancer Cells of Rhodium and Iridium N‐Heterocyclic Carbene Complexes Bearing Charged Substituents
FULL PAPER
DOI:10.1002/ejic.201300820
Synthesis, Cellular Uptake and Biological Activity Against
Pathogenic Microorganisms and Cancer Cells of Rhodium
and Iridium N-Heterocyclic Carbene Complexes Bearing
Charged Substituents
Peter V. Simpson,*[a] Claudia Schmidt,[b] Ingo Ott,[b] Heike Bruhn,[c]
and Ulrich Schatzschneider*[a]
Keywords: Bioinorganic chemistry / Biological activity / Medicinal chemistry / Antitumor agents / Antibiotics / Carbene
complexes / Rhodium / Iridium
Four new N-heterocyclic carbene (NHC) rhodium and irid- plexes showing moderate to high activity, whereas the ex-
ium complexes decorated with anionic or cationic pendant change of metal from rhodium to iridium had a negligible
groups to increase their water solubility and biological ac- effect.MostpromisingwastheactivityonTrypanosomabru-
tivityhavebeensynthesisedandcharacterised.Thelipophil- cei,withIC
50
valuesintherangeof150–400nMandaselec-
icity of the complexes was determined and the complexes tivityindex (SI)of upto 50.General toxicityon mammalian
that containedcationic phosphonium groups canbe consid- cell lines was a general problem, though, and needs to be
ered delocalised lipophilic cations (DLCs). All complexes mitigatedinfuturework.Cellularuptakestudiesclearlycon-
weretestedfortheirantibacterial,antiparasiticandantican- firmed that the cationicphosphonium groups facilitated up-
ceractivity,withonlythephosphonium-functionalisedcom- take,whichwaslinkedwithhigherbiologicalactivity.
Introduction lectivelytakenupbymitochondriaandcouldtriggerCa2+-
sensitivemitochondrialswelling,withactivitydependenton
Since the 1990s, metal complexes that bear N-hetero-
the lipophilicity of the complexes,[3] while also interacting
cyclic carbene (NHC) ligands have seen a rapid growth in
with protein selenols.[4] The antimitochondrial activity of
theiruseasextremelyactivecatalystsinaplethoraofbond-
thegoldcomplexeswasattributedtotheircationicandlipo-
forming reactions.[1] In more recent years, however, com-
philic nature, which confers properties known from the
plexes of NHC ligands have also emerged as potential bio-
class of delocalised lipophilic cations (DLCs). DLCs can
activeagents,oftendemonstratinghighanticancerandanti-
be selectively taken up by mitochondria of cancer cells, as
infective activity.[2] The intrinsic inertness of many metal–
increased transmembrane potentials are commonly found
NHCcomplexestowardairandmoisture,atleastforsome
under these conditions.[5] Thus the authors demonstrated
time, coupled with the ability to easily functionalise NHCs
the accumulation of lipophilic NHC gold complexes in
withdifferentsubstituents,hasallowed foradetailedstudy
mitochondria of cancer cells, which led to cell death by in-
of structure–activity relationships (SARs) to be made. In
duction of mitochondrial apoptotic pathways.[3,4]
particular, metal–NHC complexes of silver and gold have
In this paper, we report the synthesis of four rhodium
attractedthemostattentionsofarduetotheirhighactivity
and iridium NHC complexes in which the NHC ligands
as anti-infective and anticancer agents, respectively.[2] For
havebeenfurnishedwitheitheranionicorcationicsubstitu-
example,thegroupsofBaker,Berners-PriceandFilipovska
ents, in the form of sulfonate or phosphonium groups.
showed that complexes of the type [Au(NHC) ]X were se-
2 NHC complexes bearing charged groups have attracted
considerableattentioninrecentyearsascatalystsforbond-
[a] Institut für Anorganische Chemie, Julius-Maximilians-
UniversitätWürzburg, forming reactions in aqueous systems,[6] but their use in a
AmHubland,97074Würzburg,Germany biological context has been less explored so far. Although
E-mail:peter.v.simpson@gmail.com
ulrich.schatzschneider@uni-wuerzburg.de thereareexamplesofNHCcomplexesthatcontaincationic
http://www-anorganik.chemie.uni-wuerzburg.de/en/research/ tertiary amines, to the best of our knowledge the inclusion
prof_dr_u_schatzschneider
of phosphonium groups in NHC complexes has hitherto
[b] InstitutfürMedizinischeundPharmazeutischeChemie,
TechnischeUniversitätBraunschweig, notbeenreported.Itisourhypothesisthatthecationicand
Beethovenstrasse55,38106Braunschweig,Germany lipophilic nature of the triphenylphosphonium group will
[c] InstitutfürMolekulareInfektionsbiologie,Julius-Maximilians-
confer properties consistent with DLCs to the NHC com-
UniversitätWürzburg,
Josef-Schneider-Strasse2/D15,97080Würzburg,Germany plexes, which might increase their biological activity owing
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to increased uptake. To this effect, the anti-infective, anti- temperature overnight, using tetra-n-butylammonium
parasitic and anticancer activity of the complexes was chloride as a solubilising agent, and were isolated in yields
evaluated in relation to the overall charge and lipophilicity of approximately 38 and 56%, respectively. Following
of the complexes. We have also included in the complexes workup, rhodium complex 4a was obtained as the neutral
an iodoarene substituent that is amenable to further func- sulfonicacid(pathway1),assupportedbyelementalanaly-
tionalisation, withthe future aim offorming bioconjugates sis, and is readily soluble in dimethyl sulfoxide, methanol,
with suitable carrier molecules such as peptides or sugars dichloromethane and chloroform but poorly soluble in
using bioorthogonal coupling reactions.[7] waterandethanol.SimilarisolationofanAuIII–NHCcom-
plexthatalsobearsaneutralsulfonicacidmoietybymeans
oftransmetalationfromanAg–NHCcomplexwasrecently
Results and Discussion reported.[9]
In fact, treatment of a suspension of 4a in water with
Synthesis and NMR Spectroscopic Studies
sodiumhydroxide ledtodissolutionof thecomplexconsis-
Imidazoliumsalts1and2werepreparedbythereaction tentwiththedeprotonationofthesulfonicacidgroup.Con-
of 1-[(4(cid:2)-iodophenyl)methyl]imidazole with 1,3-propane versely, theiridium complex4b wasisolated asthe charged
sultone or 1-bromopropyl-3-triphenylphosphonium brom- sulfonatespecies(Scheme2)andpossessesexcellentsolubil-
ide and were isolated as white solids in yields of 90 and ity in water. Treatment of an aqueous solution of 4b with
75%, respectively. The silver complex 3 was synthesised by hydrochloric acid, however, led to the slow formation of
the treatment of 1 with silver(I) oxide in water at 50°C for a precipitate, presumably the neutral sulfonic acid species.
20handsubsequentlyisolatedasaslightlyhygroscopicso- Complex 4a can also be prepared in a comparable yield
lidin94%yield(Scheme1).Complex3possessestwoNHC by treatment of zwitterion 1 with [{Rh(μ-OCH 3 )(cod)} 2 ],
ligands coordinated to a AgI centre, similar to other Ag– sodium hydride and tetra-n-butylammonium chloride in
NHC complexes with pendant sulfonate groups,[8] and dis- dichloromethane at room temperature for 3d (pathway 2),
plays excellent solubility in water, dimethyl sulfoxide, N,N- although an analogous reaction in the absence of sodium
dimethylformamide,methanolandacetonitrile,andmoder- hydride did not lead to the formation of 4a. The 1H and
ateorpoorsolubilityindichloromethaneandacetone.The 13C NMR spectra of 4a,b display two doublets associated
1Hand13CNMRspectraof3in[D ]DMSOareconsistent with the inequivalent benzylic hydrogen atoms (geminal 2J
6
withtheproposedcomposition.Thesignalduetothecarb- couplings of 15.6 and 15.1Hz) and carbene carbon signals
ene carbon was observed at δ = 179.1ppm, which is in a at δ = 174.6 and 178.6ppm, respectively. The carbene car-
similar range to other complexes of the type [Ag(NHC) ] bon signal in 4a appears upfield relative to a similar sulf-
2
that bear pendant sulfonate groups.[8] onate-functionalised Rh–NHC complex prepared by Syska
andco-workers,[10]possiblyduetodifferentinteractionsbe-
tweenthesulfonateandsulfonicacidgroupswiththemetal
centre.Comparisonofthecarbenecarbonchemicalshiftin
4b to known compounds cannot be made as the only re-
ported Ir–NHC complexes with pendant sulfonate groups
containanNHCderivedfrombenzimidazole[10]orabiden-
tate bis(NHC) ligand bound to an IrIII centre.[11]
Scheme2. Synthesis of sulfonate-functionalised Ir–NHC complex
(4b).
Scheme1. Synthesis of Ag–NHC (3) and sulfonic acid function-
In situ generation of an Ag–NHC complex by the reac-
alisedRh–NHC(4a)complexes.
tion of phosphonium bromide 2 with silver(I) oxide in
Rhodium and iridium complexes 4a,b were prepared by dichloromethane followed by treatment with [{M(μ-
treating two equivalents of 3 with [{M(μ-Cl)(cod)} ] (M = Cl)(cod)} ] (M = Rh, Ir), afforded Br/Cl “halide-
2 2
Rh, Ir; cod = cyclooctadiene) in dichloromethane at room scrambled” complexes, as observed by characteristic frag-
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Scheme3.Synthesisofphosphonium-functionalisedRh–Ir–NHCcomplexes(5a,b).
ments in the mass spectrum consistent with [M – Br]+ and firming the increased lipophilic properties relative to 4a,b
[M–Cl]+ionsinthepositive-modeESImassspectra.These conferred by the triphenylphosphonium groups.
complexes were converted to bromo compounds 5a,b by
stirring with an excess amount of potassium bromide in
acetone,andwereobtainedinoverallyieldsof32and42%,
Biological Properties
respectively, from 2 (Scheme3). Complexes 5a,b are air-
stablesolidsthatarereadilysolubleinwater,dimethylsulf- Complexes4a,b and5a,b weretested fortheir antibacte-
oxide,dichloromethaneandchloroformbutmoderatelysol- rial, antiparasitic and anticancer activity on a broad panel
uble in methanol and acetone. The carbene carbon signals oforganismsandcelllines.Furthermore,threemammalian
in 5a,b were observed at δ = 181.0 and 176.7ppm in the cell lines were included in the studies to investigate their
13CNMRspectra,whereasthe31PNMRspectrashowssin- differentialactivity.The minimuminhibitoryconcentration
glets at δ = 24.1 and 24.0ppm, respectively. Unfortunately, (MIC)andminimumbactericidalconcentration(MBC)de-
repeated attempts to grow crystals of any of the complexes termined for the four title complexes on a panel of eight
suitable for X-ray diffraction were unsuccessful. different bacterial strains is collected in Table1, together
It is understood that the lipophilicity of a substance with data on gentamicin and tetracycline used as reference
greatly influences its bioavailability, and as such, the n-oct- compounds.
anol/water partition coefficient of complexes 4a,b and 5a,b Complexes 4a,b with the anionic sulfonate group on the
atpH7.4wasdeterminedbytheshake-flaskmethod.Lipin- NHCligandwereessentiallyinactiveonallbacterialstrains
ski’s “rule of five” describes the drug-likeness of a com- tested. Compounds 5a,b with the cationic alkyl phosphon-
poundbyvariouspropertiesandstipulatesthatthelogD iumsubstituentonthecarbene,ontheotherhand,showed
7.4
valueshouldbebelowfiveforadruglikecompound.[12]Val- better activity, with low micromolar (5–20μm) MIC/MBC
ues of logD of 1.45(cid:3)0.09 and 0.79(cid:3)0.09 were deter- valuesdeterminedinparticularonGram-positiveS.aureus,
7.4
minedforcomplexes4aand4b,respectively,consistentwith S. epidermis, E. faecalis and E. faecium, whereas they were
theneutral4abeingmorelipophilicthantheioniccomplex much less active on theother four Gram-negative bacterial
4b.Complexes5aand5bpossesscomparablelipophilicities strains. This is readily explained by the fact that the pepti-
of 2.86(cid:3)0.24 and 2.61(cid:3)0.22, respectively, thereby con- doglycanlayeronthemembraneofGram-positivebacteria
Table1.Minimuminhibitoryconcentration(MIC)andminimumbactericidalconcentration(MBC)[μm]forcomplexes4a,band5a,bon
apanelofeightdifferentbacteriastrains.[a,b]
S.aureus S.epidermidis E.faecalis E.faecium
MIC MBC MIC MBC MIC MBC MIC MBC
4a (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d.
4b (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d.
5a 5 10 5 5 5 20 5 20
5b 5 10 2.5 5 5 10 5 5
gentamicin 0.17 n.d. n.d. n.d. 3 n.d. n.d. n.d.
tetracycline n.d. n.d. 2 n.d. n.d. n.d. 3 n.d.
E.coli P.aeruginosa Y.pseudotuberculosa Y.pestis
MIC MBC MIC MBC MIC MBC MIC MBC
4a (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d.
4b (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d. (cid:4)40 n.d.
5a 40 40 (cid:4)40 n.d. 40 (cid:4)40 20 (cid:4)40
5b 40 40 (cid:4)40 n.d. 40 40 20 40
gentamicin 2.8 n.d. 2.8 n.d. 2.8 n.d. 2.8 n.d.
tetracycline n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
[a]Fullnames:Staphylococcusaureus,Staphylococcusepidermis,Enterococcusfaecalis,Enterococcusfaecium,Escherichiacoli,Pseudomo-
nasaeruginosa,Yersiniapseudotuberculosa,Yersiniapestis.[b]n.d.:notdetermined.
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ismadeup ofanetworkofN-acetylmuramine acidandN- Table2.IC values[μm]ofcomplexes4a,band5a,btowardLeish-
50
acetylglucosamine with embedded lipo-teichoic acids, thus maniamajoraswellasTrypanosomabruceitogetherwithtwoheal-
leading to an overall negative charge,[13] which is attractive
thymammaliancelllines.[a]
to the positively charged phosphonium groups. Essentially L.major T.brucei 293T J774.1
no difference in activity was found between the rhodium 4a (cid:4)100 (cid:4)40 n.d. (cid:4)100
compound5aanditsiridiumanalogue5b,therebysuggest- 4b (cid:4)100 (cid:4)40 n.d. (cid:4)100
ing that the nature of the metal centre is most likely not a 5a 8.9(cid:3)0.12 0.41(cid:3)0.39 1.8(cid:3)0.25 7.3(cid:3)0.04
5b 10.5(cid:3)0.68 0.15(cid:3)0.01 2.8(cid:3)0.51 7.6(cid:3)1.05
decisive factor in the antibacterial effects. For some bacte-
Pentamidin n.d. 0.005 n.d. n.d.
rial strains, the activity of the rhodium/iridium complexes
5a,b even approached that of the organic reference drugs [a]n.d.:notdetermined.
gentamicin and tetracycline, in particular for S. epidermis,
E.faecalisandE.faecium.However,thegeneraltoxicityon Finally,sincerhodium–andiridium–NHCcomplexes,al-
mammaliancelllinesturnedouttobeasignificantproblem beitwithdifferentsubstituents,havealreadybeentestedon
(seebelow).Althoughtheantibacterialactivityofrhodium anumberofcancercelllines,thefourtitlecompoundswere
NHC complexes is almost entirely unexplored, the earliest also evaluated for their cytotoxic potential on HT-29 hu-
report of their use in this context came from Cetinkaya et man colon carcinoma and MDA-MB 231 human breast
al.in1996.[14]Theauthorscomparedtheactivityofaseries
carcinoma as well as the (not tumour-derived) L-929 mu-
of rhodium and ruthenium NHC complexes with various rine connective tissue fibroblast cell line, the results of
Gram-positive and Gram-negative bacterial strains. The whicharesummarisedinTable3.Inlinewiththeotherbio-
rhodium NHC complexes were more active than the ruth- activity data, the lipophilic cationic complexes 5a and 5b
enium derivatives; they exhibited antibacterial activity showedmoderatetolowmicromolarIC valuesagainstthe
50
against the Gram-positive bacterial strains E. coli and S. HT-29 and MDA-MB 231 cell lines but were also active
aureus, although no activity was observed against Gram- against the L-929 cell line. The latter activity compares
negativestrains.Thisledtotheconclusionthattherhodium quite well with IC values determined on the J774.1 and
50
NHC complexes showed greater potential than analogous 293T cell lines (see above). However, complexes 4a and 4b
ruthenium derivatives. with the anionic sulfonate groups were essentially inactive
Furthermore, the activity of the four title compounds toward all cell lines tested.
was tested on two common tropical parasites, Leishmania
major and Trypanosoma brucei, with the latter one being Table3.IC values[μm]forcomplexes4a,band5a,boncancerous
50
the causative agent of African sleeping sickness. As far as andhealthycelllines.
we are aware, transition-metal–NHC complexes have never HT-29 MDA-MB231 L-929
before been evaluated in this context. IC values for 4a,b
50 4a (cid:4)100 (cid:4)100 67.3(cid:3)9.1
and 5a,b against L. major and T. brucei as well as mouse 4b (cid:4)100 (cid:4)100 (cid:4)100
macrophages (J774.1) and human embryonal kidney cells 5a 9.0(cid:3)2.2 14.7(cid:3)5.6 13.4(cid:3)1.5
(293T) that served as a control are collected in Table2. As 5b 30.5(cid:3)2.4 24.1(cid:3)4.3 24.1(cid:3)5.3
already observed for the bacterial strains, the anionic com-
pounds 4a,b were essentially inactive against all cell lines To check whether the differences in cationic versus an-
tested. The phosphonium compounds 5a,b, however, ioniccompoundsareduetodifferencesincellularinternal-
showed low micromolar activity toward L. major and sub- izationorsomeothermechanism,theuptakeofthesecom-
micromolar activity toward T. brucei, but unfortunately plexesbytheHT-29celllinewasstudiedinatime-depend-
were also quite toxic toward mammalian cells. Although entmanner.Forthispurpose,thecellswereexposedto4a,b
anti-leishmanial activity could only be observed at concen- and 5a,5b over a period of 8h and the metal levels were
trations comparable to or even higher than the IC values measured after different incubation periods by using high-
50
on the normal cells, the selectivity index (SI) for 5a,b to- resolution continuum source atomic absorption spec-
wardT.bruceiwas calculatedtobeintherange of4to50, troscopy (HRCS-AAS; see the Experimental Section for
dependingonthecompoundandreferencecellline.Again, more details). Values are obtained as picomol of metal per
only negligibledifferences in anti-leishmanial/anti-trypano- milligramprotein orasthemolar cellularmetalconcentra-
somal activity were observed for the rhodium compound tionderivedfromthat(Figure1).Infact,for4aand4b,the
relativetotheiridiumone.Althoughthesearequitepromis- cellular metal levels were below the detection limit of the
ingresults,[15]themetal–NHCcomplexesarestillabouttwo usedAASmethod,whichclearlyconfirmsthattheabsence
orders of magnitude less active than the reference drug of biological activity of these species is the consequence of
pentamidin, which has low nanomolar activity under sim- theirlowbioavailability.Incontrast,with5aand5b,detect-
ilar conditions and a selectivity index of approximately able metal levels were determined, thus indicating that the
3500. Thus, these metal–NHC compounds miss the defini- cationic and lipophilic phosphonium group of these com-
tion and activity criteria for hit and lead development as plexesfacilitatesthecellularuptake(Figure1).Theiridium
defined by the WHO/TDR (World Health Organization, NHCcomplex5breachedstablevaluesfasterthantherho-
Special Programme for Research and Training in Tropical dium analogue5a. The maximum cellularconcentration of
Diseases).[16] bothcomplexes(intherangeof5–7μm)waslessthan50%
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of the exposure concentration (15μm) and thus generally gold(I)–NHC complexes.[17a] Still, the incorporation of an
lower than those observed recently with other metal–NHC iodoarenegroup inthe complexesmight providean appro-
complexes under similar experimental conditions.[17] priate site for the conjugation of the NHC complexes to
suitable targeting groups such as peptides or other bio-
logicalcarriermolecules,whichmightleadtomoreselective
activityonmicrobialoraberranthumancellsonly.Thisre-
search is ongoing in our laboratory and results will be re-
ported in due course.
Experimental Section
General:AllreactionswerecarriedoutinSchlenktubesunderan
atmosphereofdinitrogenbyusinganhydroussolventspurifiedac-
cording to standard procedures. The compounds 1-[(4(cid:2)-iodophen-
yl)methyl]imidazole,[18]1-bromopropyl-3-triphenylphosphonium
bromide[19]and[{Rh(μ-Cl)(cod)} ],[20]and[{Ir(μ-Cl)(cod)} ][21]
2 2
werepreparedaccordingpublishedprocedures.Allchemicalswere
purchasedfromcommercialsourcesandusedasreceived.1H,13C
and 31P NMR spectra were recorded with Bruker DRX 300 (1H
Figure1. Cellular metal accumulation in HT-29 cells exposed to
15μmof5aand5b,respectively,overaperiodof8hasdetermined 300.13MHz, 13C 75.47MHz) and Avance 500 (1H 500.13MHz,
with HRCS-AAS. Metal concentrations obtained with 4a and 4b 13C125.77MHz)spectrometers.The1Hand13Cspectrawerecal-
underessentiallythesameexperimentalconditionswerebelowthe ibratedagainsttheresidualprotonsignalofthesolventthatserved
detectionlimit(datanotshown). as an internal reference, whereas the 31P{1H} NMR spectra were
referenced to external 85% H PO . When necessary, assignments
3 4
weredeterminedbyHSQC (one-bond1H–13Ccorrelation)and
HMBC (2/3-bond 1H–13C correlation) 2D experiments. The ESI
Conclusion mass spectra were recorded with a Bruker micrOTOF mass spec-
trometer.Theelementalcompositionsofthecompoundswerede-
Aseriesofrhodium–andiridium–NHCcomplexesbear- terminedwithaVarioMicrocubeanalyser.
ing anionic and cationic substituents was synthesised and
3-{1-[(4-Iodophenyl)methyl]-1H-imidazol-3-ium-3-yl}propane-1-sulf-
theirbiologicalpropertiesinantibacterial,antiparasiticand
onate(1):1,3-Propanesultone(0.62mL,7.04mmol)wasaddedto
anticancer assays were evaluated. This represents the first a solution of 1-[(4(cid:2)-iodophenyl)methyl]imidazole (400mg,
timeinwhichNHCcomplexesthatbearsulfonateorphos- 1.41mmol) in acetonitrile (15mL) and heated at reflux for 20h.
phonium groups have been tested in a biological context, The resulting precipitate was collected, washed with acetonitrile
which seems unusual given the rapid emergence of metal– (3(cid:5)5mL) anddiethylether (3(cid:5)5mL) andair-driedto afford1
NHCcomplexesasbioactiveagents.Complexes5aand5b, asawhitesolid,yield514mg,90%.1HNMR(300.13MHz,[D 6 ]-
which bear terminal phosphonium groups, have properties DMSO):δ=9.31(s,1H,imidazolylH2),7.86(d,3J H,H =1.8Hz,
1H,imidazolylH5),7.83(d,3J =8.2Hz,2H,ArCH),7.79(d,
of delocalised lipophilic cations. This has led to their in- H,H
3J =1.8Hz,1H,imidazolylH4),7.26(d,3J =8.2Hz,2H,
creased activity relative to the neutral and anionic com- H,H H,H
Ar CH), 5.41 (s, 2 H, benzyl CH ), 4.34 (t, 3J = 7.0Hz, 2 H,
plexes 4a and 4b, which are essentially inactive in all the 2 H,H
NCH ), 2.44 (t, 3J = 7.2Hz, 2 H, SCH ), 2.13 (m, 2 H,
assayscarriedout.Complexes5aand5bexhibitlowmicro- 2 H,H 2
NCH CH ) ppm. 13C NMR (75.47MHz, [D ]DMSO): δ = 137.8
2 2 6
molar MIC/MBC in antibacterial assays on Gram-positive
(ArCH),136.5(C2),134.5(ArC),130.6(ArCH),122.9(C5),122.5
S. aureus, S. epidermis, E. faecalis and E. faecium, low (C4), 95.3 (ArCI), 51.4 (benzyl CH ), 48.0 (NCH ), 47.3 (SCH ),
2 2 2
micromolar to sub-micromolar activity on L. major and T. 26.1(NCH CH )ppm.MSESI:m/zcalcd.428.97461[M+Na]+,
2 2
brucei and moderate, low- to mid-micromolar IC values found 428.97190;calcd. 834.95944 [2M +Na]+, found 834.95738;
50
onhumancolonandbreastcancercelllines.Essentiallyno calcd. 1240.94385 [3M + Na]+, found 1240.94021; calcd.
difference in biological activity was observed for the rho- 1646.92835 [4M + Na]+, found 1646.92559. C 13 H 15 IN 2 O 3 S
(406.24):calcd.C38.43,H3.72,N6.90,S7.89;foundC38.41,H
dium compound compared to the iridium one, which sug-
3.46,N6.91,S7.78.
gests that the metal only plays a minor role in the activity
mechanism. In particular for T. brucei, an IC value of (3-{1-[(4-Iodophenyl)methyl]-1H-imidazol-3-ium-3-yl}propane-1-
50
150nm and selectivity index of 20–50 for iridium complex triphenylphosphonium)dibromide(2):1-Bromopropyl-3-triphenyl-
5b are quite encouraging, but still the relatively low micro- phosphonium bromide (524mg, 1.13mmol) was added to a solu-
tionof1-[(4(cid:2)-iodophenyl)methyl]imidazole(300mg,1.06mmol)in
molar activity of both 5a and 5b toward different normal
acetonitrile(15mL) andheated atreflux for2d. Thesolvent was
mammaliancelllines provedtobea limitingfactor.Evalu-
removedundervacuumandtheresultingsolidsubjectedtocolumn
ationofthecellularmetalaccumulationclearlysuggesteda
chromatography on silica using a gradient elution with dichloro-
positive contribution of the phosphonium group. The cat-
methane/methanoltoaffordawhitegum.Thegumwasdissolved
ionic and lipophilic character of 5a and 5b leads to en- in dichloromethane, filtered through Celite and dried under vac-
hancedbioavailability,aneffectthathasalsobeenobserved uum. The resulting reside was triturated with diethyl ether (3(cid:5)
recently in a study that compares neutral and cationic 5mL)andn-pentane(3(cid:5)5mL)anddriedundervacuumtoafford
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2 as a hygroscopic white solid, yield 597mg, 75%. 1H NMR benzyl CHH), 5.60 (d, 2J = 15.6Hz, 1 H, benzyl CHH), 4.86
H,H
(500.13MHz,[D ]DMSO):δ=9.37(s,1H,imidazolylH2),7.94– (m, 1 H, olefinic cod CH), 4.80 (m, 1 H, olefinic cod CH), 4.72
6
7.98(m,3H,ArCH),7.86(m,1H,imidazolylH4),7.85(m,1H, (m, 1 H, NCHH), 4.62 (m, 1 H, NCHH), 4.23 (m, 1 H, olefinic
imidazolylH5),7.81–7.85(m,6H,ArCH),7.84(d,3J =8.3Hz, cod CH), 3.94 (m, 1 H, olefinic cod CH), 2.55–2.69 (2 m, 2 H,
H,H
2 H,ArCH), 7.79–7.83 (m, 6H, ArCH), 7.29 (d,3J = 8.3Hz, SCH ),2.39–2.45(2m,2cod,4HCH ),2.26(m,2H,NCH CH ),
H,H 2 2 2 2
2H,ArCH),5.44(s,2H,benzylCH ),4.41(t,3J =6.9Hz,2 2.04–2.20 (2 m, 2 cod, 4 H, CH ) ppm. 13C NMR (125.77MHz,
2 H,H 2
H,NCH ), 3.72(m,2 H,SCH ),2.16 (m,2H, NCH CH )ppm. [D ]DMSO): δ = 174.7 (C2), 137.6 (ArCH), 136.8 (ArC), 129.5
2 2 2 2 6
13CNMR(125.77MHz,[D ]DMSO):δ=137.7 (ArCH),136.6 (ArCH), 123.4 (C4), 122.5 (C5), 97.15 (cod CH), 96.6 (cod CH),
6
(C2),135.1(d,4J =3.0Hz,ArCH),134.4(ArC),133.6(d,3J 94.0(ArCI),85.5(codCH),84.7(codCH),53.1(benzylCH ),49.6
C,P C,P 2
= 10.3Hz, ArCH), 130.7 (ArCH), 130.3 (d, 2J = 12.6Hz, (NCH ), 48.3 (SCH ), 31.1 (cod CH ), 30.1 (cod CH ), 28.6 (cod
C,P 2 2 2 2
ArCH), 122.8 (C4), 122.6 (C5), 118.0 (d, 1J = 86.3Hz, Ar C), CH ),28.5(codCH ),26.6(NCH CH )ppm.MSESI:m/zcalcd.
C,P 2 2 2 2
95.4 (ArCI), 51.4 (benzyl CH ), 48.7 (NCH ), 22.6 (NCH CH ), 638.96618[M–Cl–H+Na]+,found638.96764;calcd.1254.94359
2 2 2 2
17.7(PCH ) ppm.31P{1H}NMR(202.46MHz, [D ]DMSO):δ= [2M–2Cl–2H+Na]+,found1254.94562;calcd.1870.91809[3M–
2 6
24.1 (RPPh ) ppm. MS ESI: m/z calcd. 587.11090 [M – 3Cl–3H+Na]+,found1870.92617;calcd.2486.89389[4M–4Cl–
3
2Br–H]+, found 587.11101; calcd. 669.03071 [M – Br]+, found 4H+Na]+,found2486.90108.C H ClIN O RhS(652.78):calcd.
21 27 2 3
669.03598.C H Br IN P·0.5H O(748.27): calcd. C49.17,H C38.64,H4.17,N4.29,S4.91;foundC38.87,H4.55,N4.30,S
31 30 2 2 2
4.13,N3.70;foundC49.11,H4.40,N3.60. 4.65.
Silver Complex 3: Silver(I) oxide (143mg, 0.62mmol) and 1 IridiumComplex4b:[{Ir(μ-Cl)(cod)} ](61mg,0.090mmol)and3
2
(250mg, 0.61mmol) were added to degassed water (20mL) and (85mg, 0.090mmol) were added to degasseddichloromethane
heatedat50°Cfor20h.Sodiumchloride(40mg,0.68mmol)was (10mL)andstirredatroomtemperaturefor3h.Tetra-n-butyl-
added and the mixture was stirred for an additional 5min. The ammonium chloride (100mg) and sodium chloride (20mg) were
mixture was filtered through a 0.2μm nylon membrane filter and thenaddedandthemixturestirredovernight.Thesolventwasre-
concentratedundervacuum.Theresiduewasdissolvedinmethanol moved and the residue subjected to column chromatography on
(2mL),precipitatedwithdiethyletherandthencooledto4°Cfor silica by using a gradient elution with dichloromethane/methanol
2h. The resulting precipitate was collected, washed with diethyl toafford,uponconcentrationundervacuum,awaxyyellowsolid.
ether(3(cid:5)5mL)andbrieflyair-driedtoafford3asaslightlyhygro- Thewaxysolidwastrituratedwithcoldbrine(3(cid:5)0.5mL),dried
scopicwhitesolid,yield 273mg, 94%. 1H NMR(500.13MHz, undervacuum,thentheresiduewasdissolvedinaminimalamount
[D ]DMSO): δ = 7.69 (d, 3J = 8.3Hz, 2 H, ArCH), 7.58 (d, ofN,N-dimethylformamideandfilteredthroughCelite.Theresidue
6 H,H
3J =1.7Hz,1H,imidazolylH5),7.55(d,3J =1.7Hz,1H, was dried under vacuum for 2d at 40–50°C, then dissolved in a
H,H H,H
imidazolylH4),7.06(d,3J =8.3Hz,2H,1H,d,ArCH),5.32 minimalamountofdichloromethaneanddroppedintorapidlystir-
H,H
(s,2H,benzylCH ),4.28(t,3J =6.8Hz,2H,NCH ),2.44(t, ringn-pentane.Theresultingprecipitatewascollected,washedwith
2 H,H 2
3J = 7.2Hz, 2 H, SCH ), 2.10 (m, 2 H, NCH CH ) ppm. 13C n-pentane(3(cid:5)5mL)anddiethylether(3(cid:5)5mL)andair-driedto
H,H 2 2 2
NMR (125.77MHz, [D ]DMSO): δ = 179.1 (C2), 137.5 (ArCH), afford 4b as a yellow solid, yield 77 mg, 56%. 1H NMR
6
137.2 (ArC), 129.7 (ArCH), 122.3 (C5), 122.3 (C4), 94.1 (ArCI), (500.13MHz, [D ]DMSO): δ = 7.75 (d, 3J = 8.3Hz, 2 H,
6 H,H
53.5 (benzyl CH ), 49.9 (NCH ), 47.7 (SCH ), 27.7 (NCH CH ) ArCH),7.40(d,3J =2.0Hz,1H,imidazolylH4),7.21(d,3J
2 2 2 2 2 H,H H,H
ppm.MSESI:(m/z)calcd.964.83831[M+Na]+found964.83722. = 8.3Hz, 2 H, ArCH), 7.17 (d, 3J = 2.0Hz, 1 H, imidazolyl
H,H
C H I N NaO S ·1.5H O: m/z calcd. 860.47, found 941.32. H5),5.70(d,2J =15.1Hz,1H,benzylCHH),5.47(d,2J =
26 28 2 4 6 2 2 H,H H,H
C H I N NaO S ·1.5H Ocalcd.C32.25,H3.23,N5.79,S6.62; 15.1Hz, 1 H, benzyl CHH), 4.52 (m, 1 H, NCHH), 4.36–4.42 (2
26 28 2 4 6 2 2
foundC32.01,H3.27,N5.62,S6.24. m,2H,2olefiniccodCH),4.32(m,1H,NCHH),2.93(m,1H,
olefiniccodCH),2.78(m,1H,olefiniccodCH),2.48–2.54(m,2
Rhodium Complex 4a: Method A: [{Rh(μ-Cl)(cod)} ] (39mg,
2 H, SCH ), 1.98–2.25 (4 H, 2 m, 2 cod CH ), 2.16 (m, 2 H,
0.080mmol) and 3 (75mg, 0.080mmol) were added to degassed 2 2
NCH CH ), 1.43–1.69 (4 H, 2 m, 2 cod CH ) ppm. 13C NMR
dichloromethane(10mL)andstirredatroomtemperaturefor20h. 2 2 2
(125.77MHz, [D ]DMSO): δ = 178.6 (C2), 137.2 (ArCH), 137.1
Tetra-n-butylammonium chloride(100mg) was added tothe mix- 6
(ArC), 130.1 (ArCH), 121.7 (C4), 120.9 (C5), 93.8 (ArCI), 82.3
ture,thuscausingasolutiontoform,whichwasstirredovernight.
(codCH),82.2(codCH),52.5(benzylCH ),51.2(codCH),51.0
The solvent was removed and the residuesubjected to column 2
(cod CH), 48.9 (NCH ), 48.5 (SCH ), 33.2 (cod CH ), 33.0 (cod
chromatography on silica using a gradient elution with dichloro- 2 2 2
CH ),29.2(codCH ),28.9(codCH ),26.8(NCH CH )ppm.MS
methane/methanoltoafford,uponconcentrationundervacuum,a 2 2 2 2 2
ESI (positive mode): m/z calcd. 729.02362 [M – Cl]+, found
waxyyellowsolid.Waterwasaddedtothewaxysolid,whichwas
729.02347;calcd.1433.05514[2M–2Cl–Na]+,found1433.05647;
stirred until a yellow precipitate began to form. The mixture was
(negativemode) calcd. 741.00270[M– Na]–,found 741.00177.
cooled to 4°C for 2h, then the resulting precipitate collected,
washedwithwater(6(cid:5)5mL),air-dried,washedwithdiethylether C 21 H 26 ClIIrN 2 NaO 3 S (764.07): calcd. C 33.01, H 3.43, N 3.67, S
(3(cid:5)5mL)andair-driedtoafford4aasayellowsolid,yield40mg, 4.20;foundC33.50,H3.87,N3.72,S3.85.
36%.MethodB:[{Rh(μ-OCH )(cod)} ](45mg,0.092mmol),1 RhodiumComplex5a:Silver(I)oxide(34mg,0.15mmol) and2
3 2
(75mg,0.18mmol)andtetra-n-butylammoniumchloride(100mg) (200mg, 0.27mmol) were added to degasseddichloromethane
wereaddedto degassed dichloromethane(5mL) andstirred at (7mL) and the mixture stirred in darkness for 20h. [{Rh(μ-
roomtemperaturefor20h.AnalysisbyTLCindicatednoreaction Cl)(cod)} ] (72mg, 0.15mmol) was added and stirring continued
2
hadoccurred,probablyduetothepoorsolubilityof1inthatsol- overnight.Themixturewasfilteredthrougha0.2μmnylonmem-
vent.Sodiumhydride(8mg,0.2mmol)wasaddedandthemixture branefilterandconcentratedundervacuum.Theresiduewassub-
stirredovernight.SubsequentworkupwasthesameasmethodA, jectedtocolumnchromatographyonsilicausingagradientelution
yield 45mg, 39%. 1H NMR (500.13MHz, [D ]DMSO): δ = 7.82 withdichloromethane/methanoltoafford,uponconcentrationun-
6
(d, 3J = 8.3Hz, 2 H, Ar CH), 7.59 (d, 3J = 1.8Hz, 1 H, der vacuum, a yellow solid. The solid was dissolved in a minimal
H,H H,H
imidazolylH5),7.43(d,3J =1.8Hz,1H,imidazolylH4),7.18 amount of dichloromethane, filtered through Celite and concen-
H,H
(d, 3J = 8.3Hz, 2 H, ArCH), 5.77 (d, 2J = 15.6Hz, 1 H, tratedundervacuumtoaffordthe“halide-scrambled”complex
H,H H,H
Eur.J.Inorg.Chem.2013,5547–5554 5552 ©2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
www.eurjic.org FULL PAPER
(120mg, 48%). This solid (53mg, 0.058mmol) andpotassium (C4), 121.6 (C5), 118.3 (d, 1J = 86.2Hz, ArCH), 94.0 (ArCI),
C,P
bromide (70mg, 0.58mol) were then added to degassed acetone 82.6(codCH),82.1(codCH),52.6(benzylCH ),52.5(codCH),
2
(8mL)andstirredovernight.ThemixturewasfilteredthroughCe- 52.4(codCH),49.3(NCH ),33.1(codCH ),32.5(codCH ),29.6
2 2 2
lite and concentrated under vacuum. The resulting solid was dis- (cod CH ), 29.0 (cod CH ), 23.3 (NCH CH ), 18.3 (PCH ) ppm.
2 2 2 2 2
solvedinaminimalamountofdichloromethaneanddroppedinto 31P{1H}NMR(202.46MHz,[D ]DMSO):δ=24.0(RPPh )ppm.
6 3
rapidly stirring n-pentane. The resulting precipitate was collected, MS ESI: m/z calcd. 967.08650, found 967.08233 [M – Br]+.
washed with n-pentane (3(cid:5) 5mL), diethyl ether (3(cid:5) 5mL), and C H Br IIrN P(1047.66):calcd.C44.71,H3.94,N2.67;found
39 41 2 2
air-driedtoafford5aasayellowsolid,yield37mg,67%.1HNMR C44.65,H3.82,N2.39.
(500.13MHz,[D ]DMSO):δ=7.92–7.97(m,3H,ArCH),7.82–
6
7.88(m,6H,ArCH),7.77–7.81(m,6H,ArCH),7.76(d,3J PartitionCoefficients(logD 7.4 ):Then-octanol/waterpartitioncoef-
H,H
= 8.3Hz, 2 H, Ar CH), 7.35 (d, 3J = 2.0Hz, 1 H, imidazolyl ficient of compounds 4a,b and 5a,b was determined using the
H,H
H4),7.25(d,3J =8.3Hz,2H,ArCH),7.19(d,3J =2.0Hz, shake-flaskmethod. Phosphatebuffer solution(PBS), doublydis-
1H,imidazolyl H H ,H 5),5.78(d,2J =15.0Hz,1H,b H e , n H zylCHH), tilledwater[100mL,phosphatebuffer,(PO 4 3–)=10mm,(NaCl)=
H,H
5.61(d,2J =15.0Hz,1H,benzylCHH),4.91(m,1H,NCHH), 15m, pH adjusted to 7.4 with hydrochloric acid] and n-octanol
H,H
(100mL)wereshakentogetherusingalaboratoryshaker(IKAKS
4.83 (m, 1 H, olefinic cod CH), 4.78 (m, 1 H, olefinic cod CH),
130 basic) for 72h to allow saturation of both phases. Then, a
4.43(m,1H,NCHH),3.79(m,2H,PCH ),3.26(m,1H,olefinic
2
stock solution ofeach compound (50μL) inn-octanol was mixed
cod CH), 3.15 (m, 1 H, olefinic cod CH), 2.44 (m, 1 H,
withn-octanol(700μL)andbuffer(750μL)for10minwithalabo-
NCH CHH), 2.08–2.34 (2 m, 4 H, 2 cod CH ), 2.23 (m, 1 H,
2 2
NCH CHH), 1.68–1.93 (2 m, 4 H, 2 cod CH ) ppm. 13C NMR ratoryvortexer(VWRAnalogueVortexer).Theresultantemulsion
2 2
was centrifuged (3000g, 5min) to separate the phases. A 500μL
(125.77MHz, [D ]DMSO): δ = 181.0 (C2), 139.3 (ArCH), 137.1
6
(ArC),135.0(d,4J =3.0Hz,ArCH),133.6(d,3J =10.3,Hz, aliquot of each phase was taken and diluted to 1000μL with the
C,P C,P
ArCH), 130.2 (d, 2J = 12.3Hz, ArCH), 128.4 (ArCH), 122.1 appropriatephase.Theconcentrationofeachcomplexintheaque-
C,P
(C4),121.9(C5),118.3(d,1J =86.2Hz,ArCH),97.2(codCH), ousandorganic phasewasdeterminedwithan Agilent8453UV/
C,P
Visdiodearrayspectrophotometerat280nm.ThevalueoflogD
96.9 (cod CH), 94.0 (ArCI), 68.4 (cod CH), 68.2 (cod CH), 53.0 7.4
isdefinedasthelogarithmoftheratiooftheconcentrationofthe
(benzylCH ),49.7(NCH ),32.5(codCH ),32.2(codCH ),28.5
2 2 2 2
complex in the organic and aqueous phase (logD =
(cod CH ), 28.3 (cod CH ), 23.4 (NCH CH ), 18.2 (PCH ) ppm. 7.4
2 2 2 2 2
31P{1H}NMR(202.46MHz,[D ]DMSO):δ=24.1(RPPh )ppm. log{[complex(org)]/[complex(aq.)]}). The values reported are the
6 3
MS ESI: m/z calcd. 879.02701 [M – Br]+, found 879.02607. meanofthreeseparatedeterminations.
C H Br IN PRh(958.35):calcd.C48.88,H4.31,N2.92;found
39 41 2 2 Antibacterial, Antileishmanial and Antitrypanosomal Activity Plus
C48.48,H4.28,N2.95.
Cytotoxicity toward Mammalian Cells: These measurements were
carried out as recently described by Bruhn, Schatzschneider and
Iridium Complex 5b: Silver(I) oxide (24mg, 0.11mmol) and 2
Nordlanderetal.[15]
(150mg, 0.20mmol) were added to degassed dichloromethane
(7mL) and the mixture was stirred in darkness for 2d. [{Ir(μ-
CytotoxicityinCancerand NontumourCells:Theantiproliferative
Cl)(cod)} ](74mg,0.11mmol)wasaddedandstirringwascontin-
2 effectsweredeterminedaccordingtoarecentlyusedmethodwith
ued overnight. The mixture was filtered through a 0.2μm nylon minor modifications.[22] In short: A volume of 100μL of HT-29
membranefilterandconcentratedundervacuum.Theresiduewas coloncarcinomacells(4225cellsmL–1),MDA-MB-231breastcan-
subjectedtocolumnchromatographyonsilicausingagradientelu- cercells(4250cellsmL–1)andL929murineconnectivetissuefibro-
tionwithdichloromethane/methanoltoafford,uponconcentration blastcells(1000cellsmL–1)wastransferredinto96-wellplatesand
undervacuum,ayellowsolid.Thesolidwasdissolvedinaminimal
incubated at 37°C/5% CO for 48h. Stock solutions of the com-
2
amount of dichloromethane, filtered through Celite and concen-
poundsinDMSOwerefreshlypreparedanddilutedwithcell-cul-
trated under vacuum to afford the “halide-scrambled” complex
turemediumtothedesiredconcentrations(finalDMSOconcentra-
(117mg, 59%). This solid (67mg, 0.058mmol) and potassium
tion:0.1%v/v).After72hofexposurethecellbiomasswasdeter-
bromide (80mg, 0.58mol) were then added to degassed acetone
minedbycrystalvioletstainingandtheIC valuewasdetermined
50
(7mL)andstirredovernight.ThemixturewasfilteredthroughCe-
as the concentration that caused 50% inhibition of cell prolifera-
lite and concentrated under vacuum. The resulting solid was dis-
tion. Results were calculated as the mean of two independent ex-
solvedinaminimalamountofdichloromethaneanddroppedinto
periments.
rapidly stirring n-pentane. The resulting precipitate was collected,
washed with n-pentane (3(cid:5) 5mL) and diethyl ether (3(cid:5) 5mL) CellularUptakeStudies:Thecellularmetaluptakewasdetermined
andair-driedtoafford5basayellowsolid,yield51mg,72%.1H accordingtopreviouslydescribedmethods.[23]Inshort:HT-29co-
NMR(500.13MHz,[D ]DMSO):δ=7.92–7.96(m,3H,ArCH), lon carcinoma cells were grown until at least 70% confluency in
6
7.82–7.87 (m, 6 H, ArCH), 7.77–7.83 (m, 6 H, ArCH), 7.75 (d, 75cm2 cell-culture flasks. Stock solutions of the compounds in
3J = 8.3Hz, 2 H, ArCH), 7.39 (d, 3J = 2.0Hz, 1 H, imid- DMSO were prepared and diluted with cell-culture medium to a
H,H H,H
azolylH4), 7.23(d, 3J =2.0Hz, 1H, imidazolylH5), 7.21(d, finalconcentrationof15μmimmediatelybeforeuse(finalDMSO
H,H
3J =8.3Hz,2H,ArCH),5.64(d,2J =14.8Hz,1H,benzyl concentration: 0.1% v/v). The cell-culture medium of the flasks
H,H H,H
CHH),5.49(d,2J =14.8Hz,1H,benzylCHH),4.62(m,1H, wasreplacedwiththemediumthatcontainedthemetalcompound
H,H
NCHH),4.48(m,1H,olefiniccodCH),4.39(m,1H,olefiniccod (10mL) and the flasks were incubated at 37°C/5% CO for 8h.
2
CH), 4.38 (m, 1 H, NCHH), 3.74 (m, 2 H, PCH ), 2.86 (m, 1 H, Afterthedesiredincubationperiodtheuptakewasstoppedbyre-
2
olefinic cod CH), 2.73 (m, 1 H, olefinic cod CH), 2.36 (m, 1 H, moving the cell-culture medium. The cells were washed with PBS
NCH CHH), 2.14 (m, 1 H, NCH CHH), 2.12, 2.09, 2.08, 1.92, (10mL),thewashingsolutionwasremoved,andthecellswereiso-
2 2
1.70, 1.60, 1.45, 1.33 (6 m, 6 H, 6 cod CHH) ppm. 13C NMR latedaftertrysinizationbycentrifugation.Theobtainedcellpellets
(125.77MHz, [D ]DMSO): δ = 176.7 (C2), 137.2 (ArCH), 136.8 werestoredat–20°Cuntilfurtheruse.Formetalandproteinquan-
6
(ArC),135.0(d,4J =3.0Hz,ArCH),133.6(d,3J =10.2,Hz, tification the pellets were resuspended in demineralized water
C,P C,P
ArCH), 130.3 (d, 2J = 12.5Hz, ArCH), 130.2 (ArCH), 121.8 (1.0mL) and lysed by ultrasonication. The protein content of the
C,P
Eur.J.Inorg.Chem.2013,5547–5554 5553 ©2013Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
www.eurjic.org FULL PAPER
lysateswasdeterminedbytheBradfordmethodandthemetalcon- [4] J.L. Hickey, R.A. Ruhayel, P.J. Barnard, M.V. Baker, S.J.
tentwasdeterminedbyAASasdescribedbelow. Berners-Price, A. Filipovska, J. Am. Chem. Soc. 2008, 130,
12570–12571.
AASMeasurements:Puresamplesoftherespectivecomplexeswere
[5] E.A. Liberman, V.P. Topaly, L.M. Tsofina, A.A. Jasaitis,
used as standards and calibration was done in a matrix-matched V.P.Skulachev,Nat.Biotechnol.1969,222,1076–1078.
manner (meaning all samples and standards were adjusted to the [6] L.-A. Schaper, S.J. Hock, W.A. Herrmann, F.E. Kühn, An-
sameproteinconcentrationbydilutionwithdistilledwater).Triton- gew.Chem.Int.Ed.2013,52,270–289.
X100(1%,20μL)aswellasnitricacid(13%,20μL),inthecase [7] H. Pfeiffer, A. Rojas, J. Niesel, U. Schatzschneider, Dalton
ofrhodium-containingprobes,orhydrochloricacid(18%,20μL), Trans.2009,4292–4298.
inthecaseofiridium-containingprobes,wereaddedtoeachstan- [8] a)A.Almássy,C.E.Nagy,A.C.Bényei,F.Joó,Organometal-
lics 2010, 29, 2484–2490; b) L.R. Moore, S.M. Cooks, M.S.
dardsample(200μL).Fortheiridiumandrhodiummeasurements
Anderson, H.-J.r. Schanz, S.T. Griffin, R.D. Rogers, M.C.
a contrAA 700 high-resolution continuum-source atomic absorp-
Kirk, K.H. Shaughnessy, Organometallics 2006, 25, 5151–
tionspectrometer(AnalytikJenaAG)wasused.Sampleswerein-
5158.
jected (25μL) into coated standard graphite tubes (Analytik Jena [9] E.Tomás-Mendivil,P.Y.Toullec,J.Díez,S.Conejero,V.Mich-
AG) and thermally processed as previously described in more de- elet,V.Cadierno,Org.Lett.2012,14,2520–2523.
tail.[23] Iridium was measured at a wavelength of 263.97nm and [10]H.Syska,W.A.Herrmann,F.E.Kühn,J.Organomet.Chem.
rhodium was quantified at a wavelength of 343.49nm. The mean 2012,703,56–62.
integratedabsorbancesofdouble injectionswereusedthroughout [11]A.Azua,S.Sanz,E.Peris,Chem.Eur.J.2011,17,3963–3967.
the study. The final results were calculated from data obtained in [12]C.A.Lipinski,F.Lombardo,B.W.Dominy,P.J.Feeney,Adv.
two independent experiments and are expressed as picomol of DrugDeliveryRev.1997,23,3–25.
metal permilligram ofcellular protein andas cellularmolar con- [13]W.Vollmer,D.Blanot,M.A.DePedro,FEMSMicrobiol.Rev.
centration [μm]. The procedure for calculating the cellular molar 2008,32,149–167.
[14]B. Cetinkaya, E. Cetinkaya, H. Kucubay, R. Durmaz, Arz-
concentrationinHT-29celllinesisdescribedintheliterature.[24]
neim.-Forsch.1996,46,821–823.
[15]L.Glans,W.Hu,C.Jost,C.deKock,P.J.Smith,M.Haukka,
Acknowledgments H. Bruhn, U. Schatzschneider, E. Nordlander, Dalton Trans.
2012,41,6443–6450.
[16]a) S. Nwaka, A. Hudson, Nat. Rev. Drug Discovery 2006, 5,
P.V.S. thanks the Alexander-von-Humboldt Foundation for a
941–955; b) S. Nwaka, B. Ramirez, R. Brun, L. Maes, F.
postdoctoral fellowship. The authors thank Elena Katzowitsch,
Douglas,R.Ridley,PLoSNeglectedTrop.Dis.2009,3,e440.
SvetlanaSologub,MartinaSchultheisandAntjeFußforthetesting [17]a)R.Rubbiani,S.Can,I.Kitanovic,H.Alborzinia,M.Stefan-
oftheantibacterialandantiparasiticactivityandProf.Dr.Gerhard opoulou,M.Kokoschka,S.Mönchgesang,W.S.Sheldrick,S.
BringmannasthespeakeroftheSFB630“Recognition,Prepara- Wölfl,I.Ott,J.Med.Chem.2011,54,8646–8657;b)L.Oehn-
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