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3-Hydroxyflavones vs. 3-hydroxyquinolinones: structure-activity relationships and stability studies on Ru(II)(arene) anticancer complexes with biologically active ligands.
Dalton
Transactions
PAPER
fl vs.
3-Hydroxy avones 3-hydroxyquinolinones:
–
structure activity relationships and stability studies on
Citethis:DaltonTrans.,2013,42,6193
RuII(arene) anticancer complexes with biologically
†
active ligands
AndreaKurzwernhart,a,bWolfgangKandioller,a,bÉvaA.Enyedy,cMariaNovak,a
MichaelA.Jakupec,a,bBernhardK.Kepplera,bandChristianG.Hartinger*a,b,d
RuII(η6-arene) complexes, especially with bioactive ligands, are considered to be very promising com-
pounds for anticancer drug design. We have shown recently that RuII(η6-p-cymene) complexes with
3-hydroxyflavoneligandsexhibitveryhighinvitrocytotoxicactivitiescorrelatingwithastronginhibition
oftopoisomeraseIIα.Inordertoexpandourknowledgeaboutthestructure–activityrelationshipsandto
determinetheimpactoflipophilicityoftheareneligandandofthehydrolysisrateonanticanceractivity,
aseriesofnovel3-hydroxyflavonederivedRuII(η6-arene)complexesweresynthesised.Furthermore,the
impact of the heteroatom in the bioactive ligand backbone was studied by comparing the cytotoxic
activity of RuII(η6-p-cymene) complexes of 3-hydroxyquinolinone ligands with that of their 3-hydroxy-
Received21stSeptember2012, flavoneanalogues.TobetterunderstandthebehaviouroftheseRuIIcomplexesinaqueoussolution,the
Accepted1stNovember2012
stability constants and pK values for complexes and the corresponding ligands were determined.
a
DOI:10.1039/c2dt32206d Furthermore,theinteractionwiththeDNAmodel5’-GMPandwithaseriesofaminoacidswasstudied
www.rsc.org/dalton inordertoidentifypotentialbiologicaltargetstructures.
Introduction observed.2 Two RuIII compounds, namely [ImH][trans-Ru-
(DMSO)(Im)Cl ] (NAMI-A, Im = imidazole) and [IndH][trans-Ru-
4
Ruthenium complexes represent a promising class of metal- (Ind) Cl ] (KP1019, Ind = indazole) (Chart 1), are currently
2 4
based anticancer compounds. The octahedral geometry of
undergoingclinicaltrialswithverypromisingresults.3–5
ruthenium, its binding ability to plasma proteins and the In the course of ruthenium anticancer drug development
number of possible oxidation states in biological environ- programmes, organometallic and especially half-sandwich
ments make it well suitable for drug design.1 Several ruthe- RuII(η6-arene) complexes have more and more demonstrated
niumcomplexeshaveshowninterestingpropertiesinvivo,and their
potential.6–10
Their hydrophobic arene ligand isthought
a generally lower toxicity than for platinum drugs was to facilitate the diffusion through the lipophilic cell mem-
brane.11 The three remaining Ru coordination sites can be
filled with various mono-, bi- or tridentate ligands, which
offers a number of possibilities to modulate biological and
aInstituteofInorganicChemistry,UniversityofVienna,WaehringerStr.42,1090
pharmacological properties by proper ligand selection.12
Vienna,Austria
bResearchPlatform“TranslationalCancerTherapyResearch”,UniversityofVienna, Important examples for this substance type are RuII(η6-arene)
WaehringerStr.42,1090Vienna,Austria complexes of bidentate ethylenediamine, such as RM175
cDepartmentofInorganicandAnalyticalChemistry,UniversityofSzeged,Dómtér7,
H-6720Szeged,Hungary
dTheUniversityofAuckland,SchoolofChemicalSciences,PrivateBag92019,
Auckland1142,NewZealand.E-mail:c.hartinger@auckland.ac.nz;
http://hartinger.wordpress.fos.auckland.ac.nz/;Tel:+6493737955ext83220
†Electronicsupplementaryinformation(ESI)available:Fluorescencespectraof
ligandbandcomplex2inaqueoussolution,measuredandcalculatedUV-vis
absorbance spectra and concentration distribution curves of the RuII(cym)–
ligandbsystem,NMRstudiesonthestabilityof12inaqueoussolution,NMR
spectrademonstratingthebindingabilityof12′totheDNAmodel5′-GMPand
NMRspectrashowingthereactionsof1′,12′and13′withaminoacids.SeeDOI:
10.1039/c2dt32206d Chart1 StructuresofRuanticanceragents.
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(Chart 1), and the RAPTA-type compounds containing the
monodentate 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane
(pta)ligand.RM175bindstoDNAeithercovalentlyviatheN7
of guanine or non-covalently by intercalation of the arene,
leading to cell death by modulation of the p53-p21-bax
pathway.2,13 As opposed to this, the RAPTA compounds have
very different chemical and biological properties. RAPTA-T
(Chart 1) is selectively activated under the hypoxic conditions
of solid tumours and is capable of inhibiting experimental
metastasis models both in vitro and in
vivo.4,14–16
Tethering
ethacrynic acid to the arene ligand of RAPTA led to a com-
poundcapableofovercomingtheglutathionetransferasedrug Scheme1 Synthesis of RuII(η6-arene) complexes 1–13 and formation of the
resistance mechanism of tumour cells and triggered several hydrolysisproducts1’–13’inaqueoussolution.aFromref.20and23.
biological pathwaysinvolving eitherendonucleaseG, caspases
or c-Jun N-terminal kinase.17 This is an example of linking a
the substituent on the in vitro anticancer activity was
biologicallyactive molecule to a metal centre and modulating
studied.20,23Inordertoextendourknowledgeaboutthestruc-
thereby its biological properties. Other related approaches
ture–activity relationships (SARs), a series of RuII(η6-arene)
involve RuII(arene) compounds with ligand systems that
complexes with 3-hydroxyflavones a–c and 3-hydroxyquinol-
resemblethekinaseinhibitorstaurosporine18orcomplexesof
inonesdandewassynthesisedbydeprotonationoftheligands
paullones,whicharecyclin-dependentkinase(CDK)andglyco-
with sodium methoxide and subsequent reaction with the
gen synthase kinase-3 inhibitors.19 More recently, we have
demonstratedthatRuII(cym)(cym=η6-p-cymene)complexesof
respective bis[dihalido(η6-arene)ruthenium(II)] ([RuX
2
(arene)]
2
;
η6-arene=cym,toluene,biphenyl;X=Cl,Br,I),yieldingcom-
3-hydroxyflavonesarepotenttumourcellgrowthinhibitors.20
plexes 1–13 in good to very good yields (Scheme 1). The com-
3-Hydroxyflavonesbelongtothenaturallyoccurringclassof
pounds were characterised by standard analytical methods
flavonoids which are polyphenols of plants, fruits and vege-
(see the experimental part) and were stable for over one year
tables. They are well known for their beneficial effects on
thoughexposedtosunlightandair.
health due to their antioxidant, antiinflammatory, antiviral
and anticarcinogenic properties. These effects are caused pri-
Behaviourandstabilityinaqueoussolution
marilybythescavengingoffreeradicalsbytheflavonoidstruc-
ture and by interaction with a number of enzymes.21 In order to study the properties of the 3-hydroxyflavone-
Flavonoids are capable of forming stable chelate complexes derived RuII(cym) complexes in aqueous solution, the proton
with a broad range of metal ions, which have already shown dissociation process of the p-fluoro-substituted ligand b, the
biological activity in the treatment of diseases like AIDS, dia- hydrolysis of [RuII(cym)X ]n (n = −1 to 2; X = Cl − , H O or
3 2
betes mellitus, some genetic diseases and also cancer.22 The DMSO,ESI†)andthecomplexformationprocessofthecorres-
RuII(cym) complexes of 3-hydroxyflavones were found to ponding complex 2 were investigated and stability and dis-
exhibit not only high in vitro anticancer activity in human sociationconstantswerestudied.
cancer cell lines but also to inhibit human topoisomerase IIα Proton dissociation process of ligand b. The proton dis-
activity,whichcorrelateswiththeircytotoxicpotency.20 sociationconstant (pK )ofligandbwasdeterminedbyUV-vis
a
Inordertostudytheimpactofthenatureoftheareneand spectrophotometry in 20% (w/w) dimethyl sulfoxide (DMSO)–
halogenido ligands on the stability and cytotoxic activity, a H O because of the poor solubility of the ligand and its
2
seriesofRuII(η6-arene)Xcomplexeswith3-hydroxyflavoneshas complex in pure water. Since flavonoids may suffer from
beensynthesised.Thesepropertiesarecomparedwiththoseof photodegradation,24 spectra were measured at various pH
structurally related 3-hydroxyquinolinone complexes featuring values employing the batch technique instead of continuous
a nitrogen atom in the heterocyclic ligand. These studies are titrations. This guarantees minimal UV exposure and helps
complemented with UV-vis and fluorescence spectroscopy avoiding photolysis, especially at high pH values (Fig. 1). The
experiments to gain information on the stability and pK pH-dependent spectra of the ligand show characteristic
a
valuesofthehydrolysisproductsandligandsystems. changes at increasing pH values. The deprotonation (HL ⇌
L − + H+) attributed to the hydroxyl functional group is
accompanied bya bathochromic shift of the λ and a small
max
Results and discussion increase in intensity. The isosbestic point is constant at
366 nm up to pH 10.4 but shifts at higher pH most probably
Synthesis
due to the photodegradation of the ligand. Therefore, the pK
a
Within the course of a project to prepare 3-hydroxy-4-pyrone value of 8.70 ± 0.01 and the individual spectra of the ligand
complexes, we have reported the synthesis of ruthenium(II)– species(HL,L − ;Fig.1b)werecalculatedonthebasisofdecon-
cymenecomplexeswithvarioussubstituted3-hydroxyflavones, volutedspectrarecordedatpH<10.4.Theλ valuesofboth
max
andtheinfluenceofthesubstitutionpatternandthenatureof the protonated and the deprotonated forms of ligand b are
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identical speciation was found in pure aqueous solution.27
The presence of DMSO can suppress the hydrolysis of
[RuII(cym)X ]n which is then shifted to higher pH values
3
(Fig.S2b†).
Thecomplex formationprocessesof the ruthenium(II)–cym
complex 2 were studied under the same conditions as for
[RuII(cym)X ]n+(Fig.2a)andarecomparedtothemaltol–ruthe-
3
nium(II)–cym system (Fig. 2b).11 The pH-dependent spectral
changes of the ruthenium(II)–cym-containing systems (Fig. 2c)
comparedtothefreeligandsrevealthatthecomplexformation
startsatpH>∼4inbothcases.Thecomplexformationresults
in a significant shift of the λ values, and this new band is
max
different from the bands belonging to the protonated and
deprotonated forms of the metal-free ligands. This band is
especially well-separated in the case of 2 (Fig. 2a) (i.e. λ of
max
−
complex: 436 nm, HL: 342 nm, L : 402 nm). Analysis of
changes in the overlapping ligand and charge transfer (CT)
bands shows the exclusive formation of mononuclear species
[RuII(cym)(L)X]n with a 1:1 metal-to-ligand ratio. By deconvo-
Fig.1 UV-visspectraofligandbatvariouspHvalues(a)andcalculatedindi- lution of the UV-vis spectra (Fig. S3†), a stability constant
vidualabsorbancespectraoftheHLandL−species(b){c =5×10−5M;T=
25°C;I=0.20M(KCl);20%(w/w)DMSO–H O}.HL:λ liga = nd 342nm(λ = log β ([RuII(cym)(L)X]n) = 7.13 ± 0.08 for 2 was determined,
2 max 342nm
10210mol−1dm3cm−1);L−:λ max=402nm(λ 402nm=10755mol−1dm3cm−1). which is in about the same range as that of the maltolato
complex(logβ=7.04±0.05).
At neutral and alkaline pH various parallel processes take
identical to those of the unsubstituted 3-hydroxyflavone a.25 place,namelythecomplex[RuII(cym)(L)X]n+startstohydrolyse
However,itspK valueissignificantlylowerduetotheelectron forming the mixed hydroxido species [RuII(cym)(L)(OH)] and a
withdrawing effect of the fluoro substituent. The pK of the to dissociate giving the tris-hydroxido-bridged dinuclear
a
structurally related pyrone ligand maltol (8.76 ± 0.01), which species [Ru (cym) (OH) ]+ and the metal-free ligand (Fig. 2d).
2 2 3
was also determined under the same conditions, was in the The dissociation of (O,O)-pyrone ligands such as maltol of
samerangeasthatofb.26 mono-ligand complexes is relatively slow.28 However, in the
In addition, the proton dissociation process of b in case of flavonoid complexes, the photodegradation of the
aqueous phase was monitored by fluorimetry (Fig. S1a†) at a ligand is a possible side reaction at pH > ∼10. For these
much lower concentration. The ligand excitation maximum reasons the deconvolution of the spectra becomes more
wasfoundat342nm,andtheemissionspectrumcontainstwo difficult and stability data of the [RuII(cym)(L)(OH)] species
maxima at 504 and 411 nm. The appearance of the two emis- couldonlybeobtainedwithloweraccuracyaslogβ=0.3±0.1
sion bands indicates two pathways for deactivation of the for2and0.1±0.1formaltol.
excitedstate.ThepHdependenceofthefluorescenceemission Based on the increased proton dissociation constants of
spectra shows that the emission intensity is strongly sensitive ligand b and maltol (see above), higher stability constants of
to the pH, and deprotonation results in a significant decrease [RuII(cym)(L)X]nareexpectedin20%(w/w)DMSO–H Othanin
2
oftheintensity.FromthespectralchangesinwaterapK value pure aqueous solution. However, a log β = 9.05 was reported
a
of8.30±0.09wasobtained,whichverifiesthepK determined for the maltolato complex in water,29 which is actually two
a
in 20% (w/w) DMSO–H O and which is again in the same orders of magnitude higher than the constant obtained in a
2
rangeasthepK ofmaltolinaqueoussolution(8.44).25 20% (w/w) DMSO–H O mixture. DMSO complexes of RuII are
a 2
Solutionequilibriaof[RuII(cym)X ] n andcomplex2. Inorder known, and DMSO coordination can suppress the formation
3
tounderstandthebehaviouroftheflavonoidcomplexinaqueous of[RuII(cym)(L)X]ncomplexes.Thespeciationandthestability
solution, theequilibria of thehydrolysis of [RuII(cym)X ]n (n = of 2 and the maltolato complex show very strong similarities
3
−1 to 2; X = Cl − , H O or DMSO) needed to be determined due to similar metal binding sites of the ligands. The fluor-
2
under the same conditions. This was studied in 20% (w/w) escence spectra of ligand b (Fig. S1a†) and complex 2
DMSO–H O by UV-vis spectrophotometric titrations (Fig. S2†). in aqueous solution (Fig. S1b†) show similar features up to
2
Basedonthespectralchanges,stabilityconstantsoftheminor pH ∼ 4. Upon further increasing the pH, a band with high
[Ru (cym) (OH) X ]n (m = 1, 2) and major [Ru (cym) (OH) ]+ intensity at 448 nm develops reaching a maximum at pH ∼ 5
2 2 2 m 2 2 3
dinuclear hydrolysis products were determined as log β [(Ru- and decreasing upon increasing pH. The appearance of this
(cym))
2
H−2 ]2+ = −9.85 ± 0.06 and log β [Ru
2
(cym)
2
H−3 ]+ = strong new band is most probably related to the formation of
−15.11 ± 0.03, respectively (ESI†). As the titrations were per- [RuII(cym)(L)X]n, while the formation of the mixed hydroxido
formed in the presence of 0.2 M KCl, these constants are species [RuII(cym)(L)(OH)] is accompanied by a considerable
regarded as conditional stability constants. Similar but not loss of intensity. Therefore, this latter species seems to be
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themaltolatocomplex,partialhydrolysisanddissociationof2
areprobableatphysiologicalpH.
Reactivitytowardsbiomolecules
In aqueous solution, compounds 1–3,20 5, 7, 9 and 11 are
aquated immediately to the charged aqua species 1′–3′, 5′, 7′,
9′ and 11′, which can further react with biomolecules. The
solubilityof4,6,8and10inaqueoussolutionlimitedinvesti-
gations, however, due to the structural similarity comparable
behaviour is expectable. Several RuII(arene) complexes are
known to bind to the DNA model compound 5′-GMP and
therefore are also able to form adducts with DNA, which is a
possible target for metal-based anticancer
agents.1,2,11,30–33
Similarly, 1–3,20 5, 7,9 and11showinteractions with5′-GMP,
asobserved in1H NMRspectroscopystudies.However, dueto
their low solubility and even lower solubility of their 5′-GMP
adducts, the binding mode and stability of the adducts are
elusive.
The3-hydroxyquinolinone-derivedRuII(arene)complexes12
(Fig. S4†) and 13 show the same aquation behaviour, but
already 5 min after addition of D O the first signs of the
2
hydrolysis side product [Ru (η6-arene) (OH) ]+ were observed 2 2 3
in the 1H NMR spectrum, which increased within 24 h. This
side product is thermodynamically stable and unreactive
towards nucleophiles.7 Compounds 12 and 13 bind immedi-
atelytotheN7atomof5′-GMPasindicatedbyanupfieldshift
of the H8 signal of 5′-GMP from approximately δ = 8.1 to
7.6ppm(Fig.S5†).
To gain more insight into possible interactions with pro-
teins and pharmacokinetic pathways, the reactions of the
representative hydrolysis products 1′, 12′ and 13′ with the
amino acids L-methionine, L-histidine, L-cysteine and glycine
wereinvestigated(Fig.S6–S12†).Thereactivitywasfoundtobe
similartopyrone-derivedRuII(cym)complexes.Allcompounds
reacted immediately with Met and His by replacement of the
aqua ligand with the respective amino acid, which is coordi-
nated to the RuII centre viathe sulphuratom or viathe N1 or
N3atomsoftheimidazolemoiety,respectively.11Inthecaseof
1′, the ligand was cleaved off and precipitated completely
within24h.Thesamebehaviourwasobservedforthe3-hydroxy-
quinolinone-derivedcomplexes.However, after 24 h especially
for 12′ still signals of coordinated quinolinone ligands were
visible. This may be due to a slightly higher stability of the
3-hydroxyquinolinone complexes towards the reaction with
aminoacids.AdditionofCysledtoimmediatedecomposition
of 1′ and to a lower extent of 13′. For 12′ a reaction with Cys
was observed (Fig. 3), but the compound also decomposed
Fig.2 (a)UV-visspectraof2and(b)forcomparisonofamaltolatoRuII(cym)
complex at various pH values. (c) Absorbance values at 402 nm (●) and at
partlywithin24h.Inthecaseofglycine,alsodifferingbehav-
436nm(○)forcomplex2andat322nm(■)andat328nm(□)forthemalto- iour between 3-hydroxyflavone and quinolinone complexes
lato RuII(cym) complex plotted againstthe pH value. (d) Concentration distri- was observed. Glycine reacted immediately with 1′, whereas
butioncurvesofthecomplex2{c complex=5×10−5M(8×10−5Minthecaseof the reaction with 12′ and especially 13′ was significantly
maltol);T=25°C;I=0.20M(KCl);20%(w/w)DMSO–H O;pH=2.5–11.5}.
2 slower. Two minutes after addition only traces of coordinated
glycine (two doublets at approximately δ = 3.1 ppm)11 were
observed in 12′ and only after 18 h in 13′, indicating again
much less fluorescent than [RuII(cym)(L)X]n, but somewhat higher stability of the 3-hydroxyquinolinone complexes con-
morefluorescentthanthemetal-freeligand.Asalsofoundfor cerning reactions with amino acids. However, the cytotoxicity
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carcinoma), SW480 (colon carcinoma) and A549 (non-small
cell lung carcinoma) by means of the colorimetric MTT assay
(Table1).Recently,wehaveshownthatthetypeandespecially
thepositionofthesubstituentonthephenylringoftheligand
have a crucial impact on their biological activity.20 Meta- and
para-substitution led to more cytotoxic compounds, whereas
ortho-substituted or unsubstituted ligand structures showed
lower in vitro potency (Table 1, compare compounds 2 and 3
with 1). These data correlate well with the inhibition of topo-
isomerase IIα activity.20 All synthesised complexes exhibit
promisingtumour-inhibitingpropertieswithIC valuesinthe
50
lowµMrange,whichisveryremarkableforRuII(η6-arene)com-
plexes. In order to determine the effect of the lipophilicityon
the anticancer activity, complexes bearing different arene
ligands were synthesised. The toluene derivatives 8 and 9
exhibit a similaractivity to their RuII(cym) analogues 1 and 3,
whereas the biphenyl complexes 10 and 11 are slightly less
cytotoxic. Therefore, the influence of the arene ligands seems
to be of minor importance for this type of compound. The
same activity pattern was observed for pyrone and especially
thiopyrone-derived RuII(arene) complexes,19 which is in con-
trast to for example ethylenediamine complexes. The latter
Fig.3 Reaction mixtures of 1’ (a) and 12’ (b) with equimolar amounts of compound class showed a strong dependence of cytotoxicity
L-cysteine analysed by 1H NMR spectroscopy after 5 min show immediate on the coordinated arene. The change from benzene to p-
decompositionof1’afteradditionofCys,whereasminoreffectsonthequinol- cymenetobiphenylresultedinalargeincreaseoftheirgrowth
inonesignalsof12’wereobserved.
inhibitory activity related to an increasing size and hydropho-
bicity.34Itmaybethatthechangeinlipophilicitybythemodi-
of 3-hydroxyflavone and quinolinone RuII(cym) complexes was fication of the arene ligand is too marginal in case of
similar (see below), although the MTTassay to determine the lipophilic complexes to outperform the contribution of the
IC 50 values is carried out in an amino acid-containing flavonoid ligand to the lipophilicity. Furthermore, as already
medium. This indicates that the reaction with amino acids shown for analogous pyrone- and thiopyrone RuII(η6-arene)
does not seem to significantly alter their in vitro anticancer derivatives,differenthalidesasleavinggroupsshowonlylittle
potency,mostprobablyduetotheirhigherlipophilicitywhich or no impact on the antiproliferative activity (compare 1, 3,
mayresultinenhancedcellularuptake. 4–7). This can be explained by the quick aquation of the Ru
centre,leadingtothesameaquaproducts.
Invitroanticanceractivity
When changing from 3-hydroxyflavones to 3-hydroxyquino-
The cytotoxic activity of the RuII(arene) complexes was deter- linones as ligands, no improvement of the in vitro anticancer
mined in the human cancer cell lines CH1 (ovarian activity was observed. The quinolinone complexes 12 and 13
Table1 Invitroanticanceractivityof1–13inovarian(CH1),colon(SW480)andnon-smallcelllungcarcinoma(A549)celllinesa
IC [µM]
50
R Y X Arene CH1 SW480 A549
1b H O Cl cym 2.1±0.2 9.6±1.5 20±2
2b p-F O Cl cym 1.7±0.4 7.9±2.1 18±1
3b p-Cl O Cl cym 0.86±0.06 3.8±0.5 9.5±0.5
4 H O Br cym 2.8±0.4 12±1 27±4
5 p-Cl O Br cym 0.86±0.04 3.4±0.4 7.9±0.6
6 H O I cym 1.6±0.2 9.6±1.5 16±1
7 p-Cl O I cym 1.2±0.3 4.7±0.9 8.9±0.8
8 H O Cl tol 3.2±0.1 12±3 19±1
9 p-Cl O Cl tol 0.88±0.17 4.7±0.6 7.8±2.5
10 H O Cl biphen 5.5±1.2 9.2±1.9 28±5
11 p-Cl O Cl biphen 6.3±1.1 21±4 59±1
12 H N–H Cl cym 4.0±0.2 14±1 17±2
13 H N–CH Cl cym 5.3±0.2 12±2 19±1
3
aIC =50%inhibitoryconcentration,96hexposure.bTakenfromref.20and23.tol=toluene,biphen=biphenyl.
50
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exhibit cytotoxic activities in the same range as 1. Also vari- ruthenium(II)], bis[(η6-p-cymene)diiodidoruthenium(II)],36
ation of the unsubstituted 3-hydroxyquinolinone 12 to the 3-hydroxy-2-phenyl-4H-chromen-4-one (a), 2-(4-fluorophenyl)-
1-methylated form in 13 showed no impact on the cytotoxic 3-hydroxy-4H-chromen-4-one (b), 2-(4-chlorophenyl)-3-hydroxy-
activity,indicatingthatthebackboneoftheligandratherthan 4H-chromen-4-one (c), [chlorido{3-(oxo-κO)-2-phenyl-chromen-
the functional group seems to be crucial for the biological 4(1H)-onato-κO}(η6-p-cymene)ruthenium(II)] (1), [chlorido{3-
activityofthistypeofRuII(arene)complex. (oxo-κO)-2-(4-fluorophenyl)-chromen-4(1H)-onato-κO}(η6-p-
cymene)ruthenium(II)] (2), [chlorido{3-(oxo-κO)-2-(4-chloro-
phenyl)-chromen-4(1H)-onato-κO}(η6-p-cymene)ruthenium(II)]
Conclusions (3),23 3-hydroxy-2-phenyl-1H-quinolin-4-one (d) and 3-hydroxy-
1-methyl-2-phenyl-1H-quinolin-4-one (e)37,38 were synthesised
RuII(η6-arene) complexes bearing biologically active ligand accordingtoliteratureprocedures.
systemsexhibitveryinterestingfeaturesandpromisingproper- MeltingpointsweredeterminedwithaBüchiMeltingPoint
ties for anticancer drug design.12 3-Hydroxyflavone-derived B-540 apparatus. Elemental analyses were carried out with a
RuII(η6-arene)complexesarepotentcytotoxicagentswithgood PerkinElmer2400CHNElementalAnalyserattheMicroanaly-
correlation to their topoisomerase IIα inhibitoryactivity.26 We ticalLaboratoryoftheUniversityofVienna.NMRspectrawere
have extended the series of compounds by varying the arene recordedat25°CusingaBrukerFT-NMRspectrometerAvance
and halido ligands to learn about their influence on the bio- IIITM 500 MHz. 1H NMR spectra were measured in CDCl at
3
logicalactivity,aswellascomparedthe3-hydroxyflavonecom- 500.10 MHz and13C{1H}NMR spectraat 125.75 MHz.The 2D
plexes to quinolinone analogues in terms of cytotoxicity and NMRspectrawererecordedinagradient-enhancedmode.
reactivitytowardsbiomolecules.Allcompoundsexhibitinvitro
Syntheticprocedures
anticancer activity mostly in the low µM range and showed
interaction with the DNA model compound 5′-GMP. Substi- General complexation procedure. A solution of [(η6-arene)-
tutionoftheareneandhalidoligandshadonlyaminoreffect RuX(µ-X)] (η6-arene = p-cymene, toluene, biphenyl; X = Cl,
2
on the cytotoxic activity. The 3-hydroxyquinolinone analogues Br, I) in methanol (20 mL) was added to a solution of the
behave similarly to the flavones in aqueous solutions and in ligandandsodiummethoxideinmethanol(20mL).Thereac-
anticanceractivity assays, but are more stable in the presence tion mixture was stirred at room temperature and under an
of amino acids. Extensive solution phase studies by NMR, argon atmosphere for 20 h (except for 8 and 10 which were
UV-visandfluorescencespectroscopyrevealedthatthepara-fluoro stirred for 6 h and 11 and 12 which were stirred for 5 h). The
substituted 3-hydroxyflavone b [2-(4-fluorophenyl)-3-hydroxy- solventwasevaporatedinavacuum;theresiduewasdissolved
4H-chromen-4-one] exhibits a proton dissociation constant in dichloromethane, filtered and concentrated. Pure com-
(pK ) of 8.70 ± 0.01 in 20% (w/w) DMSO–H O and of 8.30 ± plexeswereobtainedbyrecrystallisationfrommethanolorpre-
a 2
0.09inaqueoussolution.Thecomplexformationprocessesof cipitationfrommethanolwithdiethylether.
the corresponding ruthenium(II)–cym complex 2 start at pH > [Bromido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}-
∼4,formingmononuclearspecies[RuII(cym)(L)X]nwithastabi- (η6-p-cymene)ruthenium(II)] (4). The reaction was performed
lity constant of log β = 7.13 ± 0.08. At pH ≥ 7, hydrolysis of according to the general complexation procedure using a
[RuII(cym)(L)X]n leads to the mixed hydroxido species (159 mg, 0.67 mmol), NaOMe (40 mg, 0.73 mmol) and [Ru-
[RuII(cym)(L)(OH)] (log β = 0.3 ± 0.1) and partial dissociation (η6-p-cymene)Br ] (200 mg, 0.25 mmol) affording 4 as an
22
giving the tris-hydroxido-bridged dinuclear species orange powder (130 mg, 47%). Mp: 169–171 °C (decomp.); 1H
[Ru (cym) (OH) ]+andthemetal-freeligand.Thestabilitycon- NMR (500.10 MHz, CDCl ): δ = 1.44–1.45 (m, 6H, CH ),
2 2 3 3 3,Cym
stants of the hydroxyflavone-derived ruthenium(II)-cym com- 2.44(s,3H,CH
3,Cym
),3.02–3.08(m,1H,CH
Cym
),5.40–5.41(m,
pounds are therefore in the range of structurally-related 2H, H3/H5 ), 5.68 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H,
Cym
maltolatocomplexes. H2/H6 ), 7.33–7.36 (m, 1H, H7), 7.38–7.41 (m, 1H, H4′),
Cym
Considering stability data and in vitro anticancer activity, 7.46–7.50 (m, 2H, H3′/H5′), 7.56 (d, 3J(H,H) = 8 Hz, 1H, H8),
3-hydroxyflavones seem to be a well-suited ligand system for 7.59–7.63(m,1H,H6),8.22(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,
anticancer RuII(cym)(chlorido) complexes and those represent 1H,H5),8.60(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,2H,H2′/H6′)
apromisingcompoundclassforfurtherdrugdesign. ppm; 13C{1H} NMR (125.75 MHz, CDCl ): δ = 18.9 (CH ),
3 3,Cym
22.7(CH ),31.3(CH ),78.4(C3/C5 ),81.0(C2/C6 ),
3,Cym Cym Cym Cym
95.5 (C4 ), 99.3 (C1 ), 117.9 (C8), 120.1 (C8a), 124.1 (C7),
Cym Cym
Experimental part 124.6 (C5), 127.3 (C2′/C6′), 128.2 (C3′/C5′), 129.3 (C4′), 132.5
(C2),132.6(C6),149.1(C1′),153.8(C4a),154.8(C3),183.5(C4)
Materialsandmethods
ppm; elemental analysis calcd for C H BrO Ru: C 54.35,
25 23 3
Allsolventsweredriedanddistilledpriortouse.Allchemicals H4.20%;found:C54.36,H4.25%.
were purchased from commercial suppliers and used without [Bromido{3-(oxo-κO)-2-(4-chlorophenyl)-chromen-4(1H)-
further purification. Bis[(η6-p-cymene)dichloridoruthenium(II)], onato-κO}(η6-p-cymene)ruthenium(II)] (5). The reaction was
bis[dichlorido(η6-toluene)ruthenium(II)],35 bis[(η6-biphenyl)- performed according to the general complexation procedure
dichloridoruthenium(II)], bis[dibromido(η6-p-cymene)- usingc(191mg,0.70mmol),NaOMe(44mg,0.81mmol)and
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DaltonTransactions Paper
[Ru(η6-p-cymene)Br ] (220mg,0.28mmol)affording5asared [Chlorido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}-
22
powder (210 mg, 64%). Mp: 164–167 °C (decomp.); 1H NMR (η6-toluene)ruthenium(II)] (8). The reaction was performed
(500.10 MHz, CDCl ): δ = 1.43–1.45 (m, 6H, CH ), 2.43 (s, according to the general complexation procedure using a
3 3,Cym
3H, CH ), 3.00–3.07 (m, 1H, CH ), 5.41 (dd, 4J(H,H) = (180mg,0.76mmol),NaOMe(45mg,0.84mmol)and[Ru(η6-
3,Cym Cym
1Hz,3J(H,H)=8Hz,2H,H3/H5 ),5.68 (dd,3J(H,H)=5Hz, toluene)Cl ] (200 mg, 0.38 mmol) affording 8 as an orange
Cym 22
3J(H,H)=5Hz,2H,H2/H6 ),7.33–7.36(m,1H,H7),7.44(d, powder (148 mg, 42%). Mp: 218–220 °C (decomp.); 1H NMR
Cym
3J(H,H) = 9 Hz, 2H, H3′/H5′), 7.54 (d, 3J(H,H) = 8 Hz, 1H, H8), (500.10 MHz, CDCl ): δ = 2.41 (s, 3H, CH ), 5.39 (dd, 3J(H,
3 3,Tol
7.60–7.64(m,1H,H6),8.21(dd,4J(H,H)=1Hz,3J(H,H)=8Hz, H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H2/H6 ), 5.61 (dd, 3J(H,H) =
Tol
1H, H5), 8.55 (d, 3J(H,H) = 9 Hz, 2H, H2′/H6′) ppm; 13C{1H} 5Hz,3J(H,H)=5Hz, 1H,H1 ),5.88–5.90(m,2H,H3/H5 ),
Tol Tol
NMR (125.75 MHz, CDCl ): δ = 19.1 (CH ), 22.5 (CH ), 7.34–7.36 (m, 1H, H7), 7.39–7.42 (m, 1H, H4′), 7.48–7.51 (m,
3 3,Cym 3,Cym
31.3 (CH ), 78.4 (C3/C5 ), 81.0 (C2/C6 ), 95.9 (C4 ), 2H,H3′/H5′),7.57(d,3J(H,H)=8Hz,1H,H8),7.61–7.64(m,1H,
Cym Cym Cym Cym
99.3 (C1 ), 117.8 (C8), 120.0 (C8a), 124.2 (C7), 124.7 (C5), H6),8.24(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,1H,H5),8.61(dd,
Cym
128.4 (C2′/C6′), 128.5 (C3′/C5′), 131.0 (C4′), 132.8 (C6), 134.9 4J(H,H) = 1 Hz, 3J(H,H) = 8 Hz, 2H, H2′/H6′) ppm; 13C{1H}
(C2), 147.9 (C1′), 153.8 (C4a), 154.8 (C3), 183.7 (C4) ppm; NMR (125.75 MHz, CDCl ): δ = 19.1 (CH ), 29.9 (CH ),
3 3,Tol Tol
elementalanalysiscalcdforC H ClBrO Ru·0.25H O:C50.77, 75.1(C1 ),76.7(C2/C6 ),85.2(C3/C5 ),98.9(C4 ),117.8
25 22 3 2 Tol Tol Tol Tol
H3.83%;found:C50.79,H3.77%. (C8), 119.9 (C8a), 124.2 (C7), 124.6 (C5), 127.4 (C2′/C6′), 128.3
[Iodido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}- (C3′/C5′), 129.4 (C4′), 132.3 (C2), 132.7 (C6), 149.4 (C1′), 153.9
(η6-p-cymene)ruthenium(II)] (6). The reaction was performed (C4a),154.6(C3),183.4(C4)ppm;elementalanalysiscalcdfor
accordingtothegeneralcomplexationprocedureusinga(128mg, C H ClO Ru·0.5H O: C 55.64, H 3.82%; found: C 55.87,
22 17 3 2
0.54mmol),NaOMe(33mg,0.61mmol)and[Ru(η6-p-cymene)I ] H3.72%.
22
(208mg,0.21mmol)affording6asredcrystals(177mg,70%). [Chlorido{3-(oxo-κO)-2-(4-chlorophenyl)-chromen-4(1H)-
Mp: 131–134 °C(decomp.); 1H NMR(500.10MHz, CDCl 3 ):δ= onato-κO}(η6-toluene)ruthenium(II)] (9). The reaction was per-
1.47–1.48 (m, 6H, CH ), 2.45 (s, 3H, CH ), 3.05–3.12 formed according to the general complexation procedure
3,Cym 3,Cym
(m, 1H, CH ), 5.45 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, usingc(206mg,0.76mmol),NaOMe(45mg,0.84mmol)and
Cym
H3/H5 ), 5.73 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, [Ru(η6-p-cymene)Cl ] (200 mg, 0.38 mmol) affording 9 as red
Cym 22
H2/H6 ), 7.34–7.37 (m, 1H, H7), 7.39–7.42 (m, 1H, H4′), crystals (281 mg, 74%). Mp: 217–219 °C (decomp.); 1H NMR Cym
7.47–7.50 (m, 2H, H3′/H5′), 7.58 (d, 3J(H,H) = 8 Hz, 1H, H8), (500.10MHz,CDCl ):δ=2.40(s,3H,CH ),5.39(dd,3J(H,H)=
3 3,Tol
7.61–7.64(m,1H,H6),8.20(dd,4J(H,H)=1Hz,3J(H,H)=8Hz, 5 Hz, 3J(H,H) = 5 Hz, 2H, H2/H6 ), 5.61 (dd, 3J(H,H) = 5 Hz,
Tol
1H,H5),8.61(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,2H,H2′/H6′) 3J(H,H) = 5 Hz, 1H, H1 ), 5.88–5.91 (m, 2H, H3/H5 ),
Tol Tol
ppm; 13C{1H} NMR (125.75 MHz, CDCl ): δ = 18.6 (CH ), 7.34–7.38 (m, 1H, H7), 7.50 (d, 3J(H,H) = 8 Hz, 2H, H3′/H5′),
3 3,Cym
22.7(CH ),31.9(CH ),77.7(C3/C5 ),80.8(C2/C6 ), 7.52–7.54 (m, 1H, H4′), 7.56 (d, 3J(H,H) = 8 Hz, 1H, H8),
3,Cym Cym Cym Cym
95.0 (C4 ), 99.5 (C1 ), 117.9 (C8), 120.1 (C8a), 124.1 (C7), 7.62–7.65(m,1H,H6),8.24(dd,4J(H,H)=1Hz,3J(H,H)=8Hz, Cym Cym
124.6 (C5), 127.2 (C2′/C6′), 128.2 (C3′/C5′), 129.3 (C4′), 132.5 1H, H5), 8.56 (d, 3J(H,H) = 8 Hz, 2H, H2′/H6′) ppm; 13C{1H}
(C2),132.6(C6),149.1(C1′),153.9(C4a),155.1(C3),183.7(C4) NMR (125.75 MHz, CDCl ): δ = 19.1 (CH ), 29.9 (CH ),
3 3,Tol Tol
ppm; elemental analysis calcd for C H IO Ru·0.25H O: C 75.1(C1 ),76.7(C2/C6 ),85.2(C3/C5 ),98.6(C4 ),117.9
25 23 3 2 Tol Tol Tol Tol
49.72,H3.92%;found:C49.61,H3.68%. (C8), 120.0 (C8a), 124.3 (C7), 124.7 (C5), 128.6 (C2′/C6′/C3′/
[Iodido{3-(oxo-κO)-2-(4-chlorophenyl)-chromen-4(1H)-onato-κO}- C5′), 130.8 (C2), 133.0 (C6), 135.1 (C4′), 148.2 (C1′), 153.9
(η6-p-cymene)ruthenium(II)] (7). The reaction was performed (C4a), 154.6 (C3), 183.6 (C4); elemental analysis calcd for
according to the general complexation procedure using c C H Cl O Ru:C52.81,H3.22%;found:C52.62,H3.14%.
22 16 2 3
(151mg,0.55mmol),NaOMe(36mg,0.67mmol)and[Ru(η6- [Chlorido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}-
p-cymene)I
2
]
2
(217 mg, 0.22 mmol) affording 7 as a deep red (η6-biphenyl)ruthenium(II)] (10). The reaction was performed
powder (190 mg, 68%). Mp: 93–95 °C (decomp.); 1H NMR according to the general complexation procedure using a
(500.10 MHz, CDCl ): δ = 1.45–1.46 (m, 6H, CH ), 2.42 (s, (170mg,0.71mmol),NaOMe(43mg,0.80mmol)and[Ru(η6-
3 3,Cym
3H, CH ), 3.03–3.09 (m, 1H, CH ), 5.44 (dd, 3J(H,H) = biphenyl)Cl ] (200mg,0.31mmol)affording10asadeepred
3,Cym Cym 22
5Hz,3J(H,H)=5Hz,2H,H3/H5 ),5.72(dd,3J(H,H)=5Hz, powder (279 mg, 86%). Mp: 203–206 °C (decomp.); 1H NMR
Cym
3J(H,H)=5Hz,2H,H2/H6 ),7.33–7.36(m,1H,H7),7.43(d, (500.10 MHz, CDCl ): δ = 5.91–5.93 (m, 1H, H1 ),
Cym 3 Biphen
3J(H,H) = 9 Hz, 2H,H3′/H5′),7.54 (d, 3J(H,H) =8 Hz, 1H, H8), 5.96–5.97(m,2H,H2/H6 ),6.01–6.04(m,2H,H3/H5 ),
Biphen Biphen
7.60–7.63(m,1H,H6),8.18(dd,4J(H,H)=1Hz,3J(H,H)=8Hz, 7.32–7.35 (m, 1H, H7), 7.39–7.44 (m, 3H, H3′/H5′, H10 ),
Biphen
1H, H5), 8.54 (d, 3J(H,H) = 9 Hz, 2H, H2′/H6′) ppm; 13C{1H} 7.47–7.51 (m, 3H, H4′, H9/H11 ), 7.55 (d, 3J(H,H) = 8 Hz,
Biphen
NMR (125.75 MHz, CDCl ): δ = 19.1 (CH ), 22.6 (CH ), 1H, H8), 7.60–7.63 (m, 1H, H6), 7.90 (dd, 4J(H,H) = 1 Hz,
3 3,Cym 3,Cym
31.5 (CH ), 78.0 (C3/C5 ), 80.9 (C2/C6 ), 95.6 (C4 ), 3J(H,H) = 8 Hz, 1H, H8/H12 ), 8.16 (dd, 4J(H,H) = 1 Hz,
Cym Cym Cym Cym Biphen
99.3 (C1 ), 117.9 (C8), 120.0 (C8a), 124.3 (C7), 124.6 (C5), 3J(H,H)=8Hz,1H,H5),8.47(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,
Cym
128.3 (C2′/C6′), 128.5 (C3′/C5′), 131.0 (C4′), 132.9 (C6), 134.9 2H, H2′/H6′) ppm; 13C{1H} NMR (125.75 MHz, CDCl ): δ =
3
(C2), 148.0 (C1′), 153.9 (C4a), 155.0 (C3), 183.8 (C4) ppm; 78.4 (C2/C6 ), 78.8 (C1 ), 83.0 (C3/C5 ), 96.9
Biphen Biphen Biphen
elemental analysis calcd for C H ClIO Ru·0.25H O: C 47.03, (C4 ),117.8(C8),120.0(C8a),124.2(C7),124.5(C5),127.4
25 22 3 2 Biphen
H3.55%;found:C46.95,H3.50%. (C2′/C6′), 128.2 (C3′/C5′), 128.8 (C9/C11 ), 129.1 (C8/
Biphen
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Paper DaltonTransactions
C12 ),129.4(C4′),129.6(C10 ),132.1(C2),132.7(C6), 21.3 (CH ), 31.2 (CH ), 37.6 (N–CH ), 77.6 (C3/C5 ),
Biphen Biphen 3,Cym Cym 3 Cym
135.2(C7 ),149.5(C1′),153.9(C4a),154.4(C3),183.3(C4) 79.5 (C2/C6 ), 96.4 (C4 ), 97.9 (C1 ), 116.8 (C8), 120.9
Biphen Cym Cym Cym
ppm; elemental analysis calcd for C H ClO Ru: C 61.42, H (C8a), 123.3 (C5), 123.5 (C7), 128.5 (C3′/C5′), 129.2 (C2′/C6′),
27 19 3
3.63%;found:C61.16,H3.62%. 130.1 (C4′), 130.3 (C6), 132.3 (C2), 136.3 (C1′), 141.8 (C4a),
[Chlorido{3-(oxo-κO)-2-(4-chlorophenyl)-chromen-4(1H)- 152.9 (C3), 174.0 (C4) ppm; elemental analysis calcd for
onato-κO}(η6-biphenyl)ruthenium(II)] (11). The reaction was C
25
H
24
ClNO
2
Ru·CH
2
Cl
2
: C53.52, H 4.66%,N 2.31%;found:C
performed according to the general complexation procedure 53.48,H4.52%,N2.20%.
usingc(193mg,0.71mmol),NaOMe(43mg,0.80mmol)and
[Ru(η6-p-cymene)Cl ] (200 mg, 0.32 mmol) affording 11 as UV-visspectrophotometricandspectrofluorimetric
22
deep red crystals (245 mg, 68%). Mp: 194–197 °C (decomp.); measurements
1HNMR(500.10MHz,CDCl ):δ=5.91–5.93(m,1H,H1 ), Maltol, KCl, KOH, HCl and dimethyl sulfoxide (DMSO) were
3 Biphen
5.95–5.97 (m, 2H, H2/H6 ), 6.02–6.05 (m, 2H, H3/ purchased from Sigma-Aldrich. Stock solutions of maltol, b
Biphen
H5 ), 7.33–7.38 (m, 3H, H3′/H5′/H7), 7.49–7.55 (m, 4H, and2werepreparedina20%(w/w)DMSO–H Omixtureorin
Biphen 2
H6/H8/H9/H11 ), 7.60–7.64 (m, 1H, H10 ), 7.88–7.90 H O.Thestocksolutionof[RuII(cym)X ]nwasobtainedbydis-
Biphen Biphen 2 3
(m,1H,H8/H12 ),8.16(dd,4J(H,H)=1Hz,3J(H,H)=8Hz, solving a known amount of [RuII(cym)Cl ] in water and the
Biphen 22
1H, H5), 8.41 (d, 3J(H,H) = 9 Hz, 2H, H2′/H6′) ppm; 13C{1H} exact concentration (∼5 × 10 −3 M) was determined with pH-
NMR (125.75 MHz, CDCl ): δ = 78.3 (C2/C6 ), 78.5 potentiometric titrations in aqueous solution at 25.0 ± 0.1 °C
3 Biphen
(C1 ), 83.0 (C3/C5 ), 97.1 (C4 ), 117.8 (C8), 120.0 at an ionic strength of 0.20 M (KCl) employing literature data Biphen Biphen Biphen
(C8a), 124.3 (C7), 124.6 (C5), 128.4 (C2′/C6′), 128.6 (C3′/C5′), for[Ru (cym) (OH) X ]n(m=1,2)complexes.29
2 2 2 m
128.9 (C9/C11 ), 129.1 (C8/C12 ), 129.7 (C10 ), A Hewlett Packard 8452A diode array spectrophotometer
Biphen Biphen Biphen
130.6 (C2), 132.9 (C6), 135.1 (C4′, C7 ), 148.4 (C1′), 153.9 was used to record the UV-vis spectra in the interval
Biphen
(C4a), 154.4 (C3), 183.5 (C4); elemental analysis calcd for 200–800 nm. The path length was 1 cm. The measurements
C H Cl O Ru·H O: C 55.87, H 3.47%; found: C 55.86, H for determination of the protonation constants of the ligands
27 18 2 3 2
3.17%. andtheoverallstabilityconstantsofthemetalcomplexeswere
[Chlorido{3-(oxo-κO)-2-phenyl-quinolon-4(1H)-onato-κO}- carriedoutat25.0±0.1°Cina20%(w/w)DMSO–H Omixture
2
(η6-p-cymene)ruthenium(II)] (12). The reaction was performed and at an ionic strength of 0.20 M. The titrations were per-
according to the general complexation procedure using d formed with carbonate-free KOH solutions of known concen-
(172 mg, 0.73 mmol), NaOMe (43 mg, 0.8 mmol) and [Ru(η6- tration (0.20 M). The concentrations of the KOH and HCl
p-cymene)Cl ] (200mg,0.33mmol)toafford12asanorange solutionsweredeterminedbypH-potentiometrictitrations.An
22
powder (195 mg, 59%). Mp: 177–180 °C (decomp.); 1H NMR Orion 710A pH-meter equipped with a Metrohm combined
(500.10 MHz, CD OD): δ = 1.41 (m, 6H, CH ), 2.37 (s, 3H, electrode (type 6.0234.100) and a Metrohm 665 Dosimat
3 3,Cym
CH ), 2.88–2.96 (m, 1H, CH ), 5.57 (d, 3J(H,H) = 5 Hz, burette was used for the pH-potentiometric measurements. 3,Cym Cym
2H, H3/H5 ), 5.81 (d, 3J(H,H) = 6 Hz, 2H, H2/H6 ), TheelectrodesystemwascalibratedtothepH=−log[H+]scale
Cym Cym
7.40–7.44(m,1H,H7),7.55–7.63(m,4H,H3′/H4′/H5′/H6),7.76 in DMSO–water solvent mixtures by means of blank titrations
(d, 3J(H,H) = 8 Hz, 1H, H8), 8.07–8.09 (m, 2H, H2′/H6′), 8.30 (strong acid vs. strong base; HCl vs. KOH), similarly to the
(dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8 Hz, 1H, H5) ppm; 13C{1H} methodsuggestedbyIrvingetal.inpureaqueoussolutions.25
NMR(125.75MHz,CD OD):δ=17.2(CH ),21.3(CH ), Theaveragewaterionisationconstant,pK ,wasdeterminedas
3 3,Cym 3,Cym w
31.2 (CH ), 77.3 (C3/C5 ), 79.6 (C2/C6 ), 95.9 (C4 ), 14.30±0.02at25.0°CandI=0.20M(KCl),whichcorresponds
Cym Cym Cym Cym
98.3 (C1 ), 117.9 (C8), 120.0 (C8a), 122.4 (C5), 123.4 (C7), well to literature data.39 Protonation and stability constants
Cym
128.2 (C3′/C5′), 129.0 (C2′/C6′), 129.4 (C4′), 129.6 (C6), 132.3 and the individual spectra of the species were calculated with
(C2), 135.3 (C4a), 136.3 (C1′), 152.6 (C3), 174.9 (C4) ppm; the computer program PSEQUAD.40 β (M L H) is defined for
p q r
elemental analysis calcd for C H ClNO Ru·0.8CH Cl : C thegeneralequilibriumpM+qL+rH⇌M L H asβ(M L H)=
25 24 2 2 2 p q r p q r
53.90,H4.49%,N2.44%;found:C54.01,H4.78%,N2.27%. [M L H]/[M]p[L]q[H]r where M denotes [RuII(cym)X ]n and
p q r 3
[Chlorido{3-(oxo-κO)-1-methyl-2-phenyl-quinolon-4(1H)- Lthecompletelydeprotonatedligand.
onato-κO}(η6-p-cymene)ruthenium(II)] (13). The reaction was The spectrophotometric titrations were performed on
performed according to the general complexation procedure samples containing either ligand b, maltol or [RuII(cym)X ]n,
3
using e (180 mg, 0.73 mmol), NaOMe (43 mg, 0.8 mmol) and [RuII(cym)X ]n and maltol, or complex 2 in 20% (w/w) DMSO–
3
[Ru(η6-p-cymene)Cl ] (200 mg, 0.33 mmol) to afford 13 as an H O. The concentration of ligands was 5–8 × 10 −5 M and the
22 2
orange powder (157 mg, 46%). Mp: 188–190 °C (decomp.); 1H metal-to-ligand ratios were 1:1 and 1:2 in the case of maltol
NMR (500.10 MHz, CD OD): δ = 1.31–1.33 (m, 6H, CH ), overthepHrange2.0–11.5.Complex2wastitratedataconcen-
3 3,Cym
2.27(s,3H,CH ),2.77–2.85(m,1H,CH ),3.74(N–CH ), trationof5×10 −5Mand[RuII(cym)X ]nat1.8×10 −4M.
3,Cym Cym 3 3
5.45(d,3J(H,H)=5Hz,2H,H3/H5 ),5.67(d,3J(H,H)=6Hz, The pH-dependent fluorescence measurements of b and 2
Cym
2H, H2/H6 ), 7.50–7.53 (m, 3H, H3′/H5′/H7), 7.61–7.66 (m, werecarriedoutonaHitachi-4500spectrofluorimeterwiththe
Cym
3H, H2′/H4′/H6′), 7.72–7.75 (m, 1H, H6), 7.87 (d, 3J(H,H) = 8 excitation at 342 nm in aqueous solution at 25.0 ± 0.1 °C and
Hz,1H,H8),8.44(dd,4J(H,H)=1Hz,3J(H,H)=8Hz,1H,H5) an ionic strength of 0.20 M (KCl). The emission spectra were
ppm; 13C{1H} NMR (125.75 MHz, CD OD): δ = 17.1 (CH ), recorded in a 1 cm quartz cell in the pH range 2.0–11.5 using
3 3,Cym
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10 nm/10 nm slit widths. The samples contained the com- (supplemented with 10% heat-inactivated fetal bovine serum)
poundsat1.5×10 −5Mconcentration. plus20μLperwellMTTsolutioninphosphate-bufferedsaline
Duetothephotosensitivityofband2,thebatchtechnique (5 mg ml
−1).
After incubation for 4 h, the supernatants were
was used for recording the UV-vis and fluorimetric spectra removed, and the formazan crystals formed by viable cells
insteadofcontinuoustitrationsandthesolutionswerekeptin were dissolved in 150 μL DMSO per well. Optical densities at
thedark. 550 nm were measured with a microplate reader (Tecan
Spectra Classic), using a reference wavelength of 690 nm to
Hydrolysis,interactionwith5′-GMPandaminoacids
correct for unspecific absorption. The quantity of viable cells
Hydrolysis and stabilityin water were investigated by1H NMR was expressed in terms of T/C values by comparison to
spectroscopy. Due to the lipophilic character of the organo- untreated controls, and 50% inhibitory concentrations (IC )
50
metallics, all experiments were performed in 10% (v/v) were calculated from concentration–effect curves by inter-
d -DMSO/D O solutions. For the interaction with 5′-GMP, the polation. Evaluation is based on means from at least
6 2
complexes (ca. 0.1 mg mL
−1)
were dissolved in 10% (v/v) threeindependentexperiments,eachcomprisingatleastthree
d -DMSO/D O,yieldingthecorrespondinghighlyreactiveaqua replicatesperconcentrationlevel.
6 2
species.Theaquacomplexeswereconvertedinsitubyaddition
of 50 μL aliquots of 5′-GMP solution (10 mg mL −1) to the
respective 5′-GMP adduct and the reaction was monitored by Acknowledgements
1H NMR spectroscopy. To investigate the reactivity towards
amino acids, the aqua complexes (ca. 0.1 mg mL
−1)
were This work was supported by the Hungarian Research Foun-
treated with equimolar amounts of amino acids and 1H NMR dation OTKA 103905 and É.A. Enyedy gratefully acknowledges
spectrawererecordedafter5minand24h. the financial support of J. Bolyai research fellowship. We
thank the University of Vienna, the Austrian Science Fund
Cytotoxicityincancercelllines
(FWF), the Johanna Mahlke geb. Obermann Foundation, and
Celllinesandcultureconditions. CH1cellsoriginatefroman COST D39 for financial support. We gratefully acknowledge
ascites sample of a patient with papillary cystadenocarcinoma FilipGroznicafordoingpartsofthesyntheticwork.
oftheovaryandwereagiftfromLloydR.Kelland,CRCCentre
for CancerTherapeutics,Instituteof CancerResearch,Sutton,
UK. SW480 (human adenocarcinoma of the colon) and A549 Notes and references
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6202 | DaltonTrans.,2013,42,6193–6202 Thisjournalis©TheRoyalSocietyofChemistry2013
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