← Back
Studies of ruthenium(ii)-2,2′-bisimidazole complexes on binding to G-quadruplex DNA and inducing apoptosis in HeLa cells
NJC
PAPER
Studies of ruthenium( )-2,20-bisimidazole complexes
II
on binding to G-quadruplex DNA and inducing
Citethis:DOI:10.1039/c3nj00542a
apoptosis in HeLa cells†
Yu Xia, Qingchang Chen, Xiuying Qin, Dongdong Sun, Jingnan Zhang and Jie Liu*
Threeruthenium(II)complexes[Ru(bpy)
2
(biim)]2+(1),[Ru(phen)
2
(biim)]2+(2)and[Ru(p-mopip)
2
-(biim)]2+
(3) (where bpy is 2,20-bipyridine, phen is 1,10-phenanthroline, biim is 2,20-bisimidazole and p-mopip is
2-(4-methoxyphenyl)-imidazo-[4,5f]phenanthroline), have been synthesized and characterized. The
interactionsofhumantelomericDNAoligomers50-G
3
(T
2
AG
3
)
3
-30 (HTG21)withruthenium(II)complexes
were investigated via UV-vis, fluorescence resonance energy transfer (FRET) melting assay, polymerase
chain reaction (PCR) stop assay, and circular dichroism (CD) measurements. The results indicated that
thethreeruthenium(II)complexescouldstabilizetheformationofhumantelomericG-quadruplexDNA,
andcomplex2 wasfoundtobe themost efficient.Invitro cytotoxicity assaybyMTTalso showedthat
Received(inMontpellier,France) complex2wassuperiortocomplexes1and3ininhibitingthegrowthofcancercells.Telomericrepeat
22ndMay2013, amplification protocol (TRAP) showed that complexes 2 and 3 led to an inhibition of the telomerase
Accepted21stAugust2013 activity,andcomplex2wasthesignificantlybetterinhibitor.Flowcytometricanalysisandevaluationof
DOI:10.1039/c3nj00542a mitochondrial membrane potential demonstrated that complex 2 inhibited the growth of HeLa cells
through induction of apoptotic cell death, as evidenced by the depletion of mitochondrial membrane
www.rsc.org/njc potentialinHeLacells.
1 Introduction Anumberofsmallmoleculeshavebeenreportedtoefficiently
stabilizeG-quadruplexDNA,andrecentlysomemetalcomplexes
ThehumantelomericDNAconsistsofthetandemrepeatsequence as G-quadruplex DNA stabilizers were reported.10–14 The metal
[TTAGGGCCCTAA] n .Severalkilobasesofthissequencearepaired canplayamajorstructuralroleinorganizingtheligandsintoan
withacomplementarystrandtoformduplexDNA,butabouttwo optimal structure for G-quadruplex DNA interaction. It is likely
hundred bases are unpaired as a single-stranded overhang.1 thatsuchstabilizationoccursduetop–pinteractionsbetweenthe
This single G-rich strand has the inclination to adopt higher- large aromatic ligandsof the ruthenium(II) complexesand DNA
order and functionally useful four-stranded structures called guanineresidues.Ruthenium(II)complexeshaveprominentDNA
G-quadruplexes.2RecentstudiessuggestthattheG-quadruplex binding properties.15 For example, the typical ruthenium
structures exist in vivo3 and such structures form in telomeric complex [Ru(bpy) (dppz)]2+ (dppz = dipyrido[3,2-a:20,30-c]phena-
2
DNAatspecifictimesduringthecellcycle.4Ithasbeenshown zine) is known as DNA ‘‘light switch’’.16,17 Such complexes can
that the stabilization of G-quadruplexes can effectively inhibit tightly intercalate between the duplex DNA base pairs and
telomeraseactivitywhichisexpressedinover85%oftumorcell stabilize the DNA.18 Some of them have been studied as syn-
linesbutinrelativelyfewnormalcelltypes5andultimatelyalter theticrestrictionenzymes,nucleicacidprobes,anticancerdrugs,
telomeremaintenance.6,7Telomeremaintenanceisverysignif- andDNAfootprintingagents,etc.19–21Ricklingetal.studiedthe
icant for the unlimited proliferative potential of cancer cells.8 actionofthedinuclear[(tap) Ru(tpac)Ru(tap) ]4+complex(tapc=
2 2
Consequently,theinhibitionoftelomeraseactivitybyinducing tetrapyridoacridine) with the [TTAGGG] sequence. They found
4
G-quadruplex formation is very important for developing new that the complex damaged the sequences by photobridging
anticancerdrugs.9 intramolecularly two or more G bases with the metallic [(tap)
2-
Ru(tpac)Ru(tap) ]4+.Thephotoinducedbridgingprocess‘‘froze’’
2
DepartmentofChemistry,JinanUniversity,Guangzhou510632,P.R.China. thefoldedG-quadruplexconformation.22Thomasetal.reported
E-mail:tliuliu@jnu.edu.cn;Fax:+86-20-8522-1263 that the dinuclear tppz-based systems could bind with high
†Electronic supplementary information (ESI) available: ESI-MS and 1H NMR
affinity to quadruplex DNA at high ionic strengths through
s a p n e d ct D ra N o A f -b c i o n m d p in le g xe c s on 1 s , t 2 an an ts d K 3 b (F ((cid:2) ig. 1 S 0 1 6 ,S M 2 (cid:3) a 1 n ) d of S3 c ) o . m Ab p s le o x r e b s an 1 c , e 2 sp a e n c d tr 3 a . (l S m ee ax/ D n O m I ) : the 22-mer d(AG 3 [T 2 AG 3 ] 3 )[G 3 ] human telomeric sequence.23
10.1039/c3nj00542a Theinternationalcommunityhaswidelyrecognizedthatsome
Thisjournalis c TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013 NewJ.Chem.
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
View Journal
Paper NJC
DNA oligomers HTG21 were purchased from Shanghai Sangon
BiologicalEngineeringTechnology&Services(Shanghai,China)
andusedwithoutfurtherpurification.TheconcentrationofHTG21
was determined by measuring the absorbance at 260 nm after
melting. Single-strand molar absorptivities were calculated from
mononucleotide data using a nearest-neighbour approximation.
TheformationofintramolecularG-quadruplexeswascarriedoutas
follows:theoligonucleotidesamples,dissolvedindifferentbuffers,
wereheatedto901Cfor5min,gentlycooledtoroomtemperature,
andthenincubatedat41Covernight.Buffer:10mMTris-HCl,
pH = 7.4. Solutions of DNA in 5 mM Tris-HCl/50 mM NaCl
buffer in water gave a ratio of UV absorbance at 260 and
280nm,A /A ,of1.9.TheconcentrationofDNAinnucleo-
260 280
tide phosphate (NP) was determined by UV absorbance at
260 nm after 1:100 dilution. The molarabsorptivity, e , was
260
determinedtobe6600M(cid:3)1cm(cid:3)1.Stocksolutions werestored
Scheme1 Syntheticroutesforthepreparationofcomplexes1,2and3.
at41Candusedafternomorethan4days.
2.2 Physicalmeasurements
ruthenium complexes exhibit great cytotoxicity toward cancer Microanalysis (C, H and N) was carried out using a Perkin-
cellsbutlowtoxicitytonormalcells,theyareeasilyabsorbedby Elmer 240C elemental analyzer. Electrospray ionization mass
tumortissueandrapidlyexcretedfromthebody.24,25
spectrometry (ESI-MS) was performed on a LQC system (Finngan
Itisveryinspiringthatthepromisingrutheniumanticancer MAT,USA)usingCHCNasthemobilephase.1HNMRspectrawere
3
agents,[ImH][trans-RuCl 4 (Im)(DMSO)](NAMI-A,whereDMSO= recorded on a Varian Mercury-plus 300 NMR spectrometer with
dimethyl sulfoxide and Im = imidazole), [IndH][trans-RuCl 4 (Ind) 2 ] d 6 -DMSO as the solvent and SiMe 4 as an internal standard at
(KP1019, where Ind = indazole) and trans-[RuCl 4 (Ind) 2 ]IndH 300 MHz at room temperature. All chemical shifts were given
(NKP-1339),26–28havesuccessfullyenteredclinicaltrials.These
relative to TMS (tetramethylsilane). UV-Vis spectra were measured
reports make ruthenium complexes containing imidazole and on a Perkin-Elmer Lambda-850 spectrophotometer. Circular
their derivative ligands draw much attention.29,30 In the past dichroism (CD)spectrawererecordedona JascoJ-810spectro-
few years, our group has been committed to the research on polarimeter.
antitumor properties of ruthenium complexes including their
design, synthesis, structural modification, biological activity 2.3 Synthesisandcharacteristics
and mechanisms.31 In our previous studies, we found that Ruthenium(III) chloride hydrate (Alfa Aesar), ethanedial
ruthenium complexes containing methylimidazole ligands, (Sigma),1,10-phenanthroline(Sigma),and4-methoxybenzalde-
such as [Ru(MeIm) 4 (N–N)]2+ [N–N = tip, iip, dppz, dpq], had hyde (Sigma) were used. The compounds 2,20-bisimidazole,
some antitumor activity. We designed three new complexes 1,10-phenanthroline-5,6-dione, p-mopip and cis-[Ru(bpy) Cl ](cid:4)
2 2
[Ru(bpy) 2 (biim)]2+(1),[Ru(phen) 2 (biim)]2+(2)and[Ru(p-mopip) 2 - 2H 2 O and cis-[Ru(phen) 2 Cl 2 ](cid:4)2H 2 O were prepared and charac-
(biim)]2+ (3), all the three complexes have bisimidazole as the terizedaccordingtomethodsdescribedintheliterature.33–35
mainligand(Scheme1).ItwaswellknownthattheHlinkingto 2.3.1 Synthesis of cis-[Ru(bpy) (biim)](PF ) . A mixture of
2 62
the N atom of bisimidazole may form hydrogen-bonding with cis-[Ru(bpy) Cl ](cid:4)2H O (0.26 g, 0.5 mmol), biim (0.067 g,
2 2 2
the functional groups located on the edges of DNA to favor 0.5 mmol), and ethylene glycol (20 mL) was refluxed for 6 h
interactionofthemainligand,andthussuchaligandstrength- under argon. The cooled reaction mixture was diluted with
enstheDNA-bindingaffinityofthecomplex.32Theinteraction
water(50mL).Saturatedaqueoussodiumhexafluorophosphate
of human telomeric G-quadruplex DNA with the three com- solution was added under vigorous stirring, and filtered. The
plexes has been studied. Importantly, complex 2 exhibited darkredsolidwascollectedandwashedwithsmallamountsof
potentantitumoractivitiesandeffectivelyinhibitedtelomerase water,anddiethylether,thendriedundervacuum,andpurified
activity.Thebiologicalpropertiesofcomplex2,themostactive by column chromatography on alumina using acetonitrile–
rutheniumcomplexamongthethree,werestudiedbyshowing toluene (12:1 v/v) as the eluant. The solvent was removed
itsapoptosis-inducingactivitiesandrelatedsignalingpathways under reduced pressure and red microcrystals were obtained
inHeLatumorcells. (0.25g,60%yield).1HNMR(300MHz,[D ]DMSO,251C):d8.75
6
(d,J=7.8Hz,4H,bpy-H
6
,H 60),8.11(t,J
1
=7.5Hz,J
2
=6.9Hz,
2 Experimental 4H, bpy-H 3 , H 30), 7.84 (d, J = 6.5 Hz, 4H, bpy-H 4 , H 40), 7.46
(t,J=6.5Hz,4H,bpy-H
5
,H 50),7.56(d,J=6.2Hz,2H,biim-H
5
,
2.1 Reagentsandmaterials H 50), 6.44 (d, J = 6.3 Hz, 2H, biim-H
4
, H 40). MS (ESI+, CH
3
CN):
Allthereagentsandsolventswerepurchasedfromcommercial m/z=547.1[M]+(seeFig.S1,ESI†).C H N Ru(547.58):calcd
26 22 8
sourcesandusedwithoutfurtherpurification.Humantelomeric C37.29,H2.65,N13.38%;foundC37.27,H2.66,N13.35%.
NewJ.Chem. Thisjournalis cTheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013
.92:83:80
3102/90/81
no
yendyS
fo ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
NJC Paper
2.3.2 Synthesisofcis-[Ru(phen) (biim)](PF ) .Amixtureof concentration due to dilution at the end of each titration were
2 62
cis-[Ru(phen) Cl ](cid:4)2H O (0.284 g, 0.5 mmol), biim (0.067 g, negligible.
2 2 2
0.5 mmol), and ethylene glycol (20 mL) was refluxed for 6 h
under argon. The cooled reaction mixture was diluted with
2.5 Fluorescenceresonanceenergytransfer(FRET)
water(50mL).Saturatedaqueoussodiumhexafluorophosphate
solution was added under vigorous stirring, and filtered. The Thefluorescentlabeledoligonucleotide,F21T(50-FAM-G [T AG ] -
3 2 33
darkredsolidwascollectedandwashedwithsmallamountsof TAMRA-30, FAM:6-carboxyfluorescein, TAMRA:6-carboxytetra-
water,anddiethylether,thendriedundervacuum,andpurified methylrhodamine), used as the FRET probe was diluted in
by column chromatography on alumina using acetonitrile– Tris-HCl buffer (10 mM, pH 7.4) containing KCl (60 mM) and
toluene (13:1 v/v) as the eluant. The solvent was removed thenannealedbyheatingto921Cfor5min,followedbyslow
under reduced pressure and red microcrystals were obtained coolingtoroom temperature,overnight. Fluorescence melting
(0.22g,49%yield).1HNMR(300MHz,[D ]DMSO,251C):d8.79 curves were determined using a Bio-Rad iQ5 real time PCR
6
(d,J=7.7Hz,2H,phen-H ),8.66(d,J =8.3Hz, 2H,phen-H ), detection system, by using a total reaction volume of 20 mL,
2 9
8.35 (m, J = 7.2 Hz, J = 5.5 Hz, 6H, phen-H , H , H ), 8.11 with labeled oligonucleotide (1 mM) and different concentra-
1 2 4 7 5
(d,J=6.7Hz,2H,phen-H ),8.01(d,J =7.9Hz, 2H,phen-H ), tionsofcomplexesinTris-HClbuffer(10mM,pH7.4)contain-
6 8
7.43 (d, J = 7.1 Hz, 2H, phen-H ), 7.68 (d, J = 6.2 Hz, 2H, ingKCl(60mM).Fluorescencereadingswithanexcitationwave
3
biim-H
5
, H 50), 6.39 (d, J = 5.6 Hz, 2H, biim-H
4
, H 40). MS (ESI+, at 470 nm and detection at 530 nm were taken at intervals of
CH CN): m/z = 595.2 [M]+ (see Fig. S2, ESI†). C H N Ru 11Covertherange30–951C,withaconstanttemperaturebeing
3 30 22 8
(595.62): calcd C 40.69, H 2.50, N 12.65%; found C 40.65, maintained for 30 s prior to each reading to ensure a stable
H2.52,N12.61%. value.ThemeltingoftheG-quadruplexwasmonitoredaloneor
2.3.3 Synthesis of cis-[Ru(p-mopip) (biim)](PF ) . A mix- in the presence of various concentrations of complexes. Final
2 62
ture of cis-[Ru(p-mopip) Cl ](cid:4)2H O (0.43 g, 0.5 mmol), biim analysisofthedatawascarriedoutbyusingOrigin6.0.
2 2 2
(0.067 g, 0.5 mmol), and ethylene glycol (20 mL) was refluxed
for 6 h under argon. The cooled reaction mixture was diluted
2.6 PCRstopassay
withwater(50mL).Saturatedaqueoussodiumhexafluorophos-
phatesolutionwasaddedundervigorousstirring,andfiltered. The oligonucleotide HTG21 (50-G [T AG ] -30) and the corre-
3 2 33
The dark red solid was collected and washed with small spondingcomplementarysequence(HTG21rev,ATCGCTTCTCG-
amountsofwater,anddiethylether,thendriedundervacuum, TCCCTAACC) were used. The reactions were performed in 1 (cid:2)
and purified by column chromatography on alumina using PCR buffer, containing each oligonucleotide (10 pmol each),
acetonitrile–ethanol (8:1 v/v) as the eluant. The solvent was dNTPs (0.16 mM), Taq polymerase (2.5 U), and different con-
removed under reduced pressure and red microcrystals were centrationsofcomplexes.Reactionmixtureswereincubatedina
obtained (0.32 g, 54% yield). 1H NMR (300 MHz, [D ]DMSO, thermocycler under the following cycling conditions: 94 1C for
6
251C):d9.05(d,J=7.1Hz,4H,p-mopip),8.91(d,J=7.8Hz,4H, 3 min, followed by 30 cycles of 94 1C for 30 s, 58 1C for 30 s,
p-mopip), 8.33 (m, J = 8.8 Hz, J = 7.2 Hz, 4H, p-mopip), 8.00 and 72 1C for 30 s. PCR products were then analyzed on a
1 2
(d,J =8.5Hz, 4H,p-mopip), 7.81 (d,J=8.5Hz, 4H, p-mopip), nondenaturingpolyacrylamidegel (15%)in1 (cid:2) TBE andsilver
6.4–7.6 (d, J = 6.2 Hz, 4H, biim-H
4
, H 40, H
5
, H 50). MS (ESI+, stained.
CH CN): m/z = 887.3 [M]+ (see Fig. S3, ESI†). C H N O Ru
3 46 34 12 2
(887.91): calcd C 46.91, H 2.91, N 14.27%; found C 46.95,
2.7 Circulardichroism(CD)
H2.94,N14.22%.
CD experiments were performed on a JASCO-J810 circular
dichroismspectrophotometer.Aquartzcuvettewith4mmpath
2.4 Absorbancespectra
length was used for the spectra recorded over a wavelength
Absorbance spectra titrations were carried out at room tem- range of230–400nm at1nm bandwidth, 1nmstep size, and
perature to determine the binding affinity between DNA and 0.5 s time per point. The oligomer HTG21 was diluted from
thecomplex.Initially,3mLwatersolutionsoftheblankbuffer stock to the correct concentration (2 mM) in Tris-HCl buffer
and the ruthenium complex sample (10 mM) dissolved in (10 mM, pH 7.4) and then annealed by heating to 90 1C for
distilledwaterwereplacedinthereferenceandsamplecuvettes 5min,graduallycooledtoroomtemperature,andincubatedat
(1.0 cmpath length),respectively,and thenthe firstspectrum 4 1C overnight. Then, CD titration was performed at a fixed
wasrecordedintherangeof200–600nm.Duringthetitration, HTG21 concentration (2 mM) with various concentrations
1–10 mL aliquot of buffered DNA solution was added to each (2.0molequiv.)ofthecomplexesinbufferat251C.Aftereach
cuvettetoeliminatetheabsorbanceofDNA,andthesolutions additionofthecomplexes,thereactionwasstirredandallowed
were mixed by repeated inversion. Complex–DNA solutions to equilibrate for at least 3 min until no elliptic changes were
wereincubatedfor5minbeforeabsorptionspectrawererecorded. observed and a CD spectrum was collected. Buffer baseline
Thetitrationprocesseswererepeateduntiltherewasnochangein was collected in the same cuvette and subtracted from the
the spectra for four titrations at least, indicating that binding samplespectra.Finalanalysisofthedatawascarriedoutusing
saturationhadbeenachieved.Thechangesinthemetalcomplex Origin6.0.
Thisjournalis c TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013 NewJ.Chem.
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
Paper NJC
2.8 Cellculture 2.11 Flowcytometricanalysis
CellswereculturedinaRPMI1640mediumsupplementedwith Thecellcycledistributionwasanalyzedusingflowcytometryas
heat inactivated fetal bovine serum (FBS, 10%), penicillin previously described. Cells exposed to the complexes were
(100 mg mL(cid:3)1), and streptomycin (100 mg mL(cid:3)1). Cells were trypsinized and washed with PBS. After adding 70% ethanol
maintained at 37 1C in a 5% CO incubator, and the medium and overnight fixation at (cid:3)20 1C, the trypsinized cells were
2
waschangedtwiceweekly. stainedwithpropidiumiodide(PI)for4hinthedark.TheDNA
content was measured using a Epics XL-MCL flow cytometer
2.9 MTTassay (Beckman Coulter, Miami, FL) and the cell cycle distribution
was analyzed by MultiCycle software (Phoenix FlowSystems,
HeLa (human cervical cancer), A549 (human lung carcinoma)
San Diego, CA). Apoptotic cells with hypodiploid DNA content
and HepG2 (human hepatocellular liver carcinoma) cells were
weremeasuredbyquantifyingthesub-G1peakinthecellcycle
growninaRPMI1640mediumsupplementedwithFBS(10%),
penicillin(100mgmL(cid:3)1)andstreptomycin(100mgmL(cid:3)1).They pattern.Eachexperimentpersamplewasdeterminedbyrecording
wereincubatedat371Cinahumidifiedincubatorwith5%CO 10000events.
2
and95%air.Cellsattheexponentialgrowthphasewerediluted
2.12 Evaluationofmitochondrialmembranepotential(DW )
to 2.5 (cid:2) 103 cells mL(cid:3)1 with RPMI 1640, and then seeded in m
96-wellcultureclusters(Costar)atavolumeof100mLperwell, The mitochondrial membrane potential was measured by the
andincubatedfor24hat371Cin5%CO .Thenthecellswere JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolylcarbo-
2
treated with various concentrations of the complex (5, 10, 25, cyanine iodide) probe purchased from Beyotime Institute of
50,75,100,150and200mmolL(cid:3)1);themediumanddrug-free Biotechnology.Firstly,cellsin6-wellplatesweretrypsinizedand
control samples were prepared simultaneously. After incuba- resuspended in 0.5 mL of PBS buffer containing 10 mg mL(cid:3)1 of
tion of the cells for up to 48 h, MTT (100 mL, 5 mg mL(cid:3)1) JC-1.After10minincubationat371Cinthedark,thesupernatant
solution was added to each well. After a further period of wasimmediatelyremovedbycentrifugation.Afterbeingwashed
incubation (4 h at 37 1C in 5% CO ) the cell lysate (100 mL) and resuspended in PBS, the stained cells were immediately
2
was added to each well. After 12 h at 37 1C, the plates were analyzed using flow cytometry. The percentage of the green
analyzed on a microplate reader at a wavelength of 570 nm fluorescence from JC-1 monomers was used to demonstrate
(the absorbance of the complexes at this wavelength can be thecellsthatlostDC m .
neglected). The percent growth inhibitory rate of treated cells
was calculated as (A (cid:3) A /A (cid:3) A ) (cid:2) 100%,
control drug control cell-free 3 Results and discussion
where A is the mean value calculated by using the data from
three replicate tests. The IC values were determined by 3.1 DNA-bindingstudiesbyabsorbancespectroscopy
50
plotting the percentage viability versus concentration on a Electronic absorbance spectroscopy is one of the most useful
logarithmicgraphandreadingtheconcentrationatwhich50% ways to investigate the interactions of complexes with DNA.36
ofcellswereviablerelativetothecontrol. ComplexbindingtoDNAusuallyresultsinhypochromismand
red shift. The extent of the hypochromism and red shift
2.10 TRAPassay parallels the binding affinity.37 The absorbance spectra of
The telomerase extract was prepared from HepG-2 cells. A complexes 1, 2 and 3 in the absence and presence of HTG21
modified version of the TRAP assay was used in this experi- (ataconstantconcentrationofcomplexes,[Ru]=10mM)were
ment. PCR was performed in a final 50 mL reaction volume obtained.WiththeincreaseinconcentrationofHTG21,allthe
composedofa45mLreactionmixcontaining20mMTris-HCl absorbance bands of the complexes displayed clear hypochro-
(pH8.0),50mMdeoxynucleotidetriphosphates,1.5mMMgCl , mism. The hypochromism (H%), as defined by H% = 100%
2
63 mM KCl, 1 mM EDTA, 0.005% Tween 20, 20 mg mL(cid:3)1 BSA, (A free (cid:3)A bound )/A free ,ofMLCTbandsatabout480nmof1,2and
3.5pmolofprimerHTG21(50-G (T AG ) -30),18pmolofprimer 3 were 13%, 29% and 23% with slight red shifts, respectively
3 2 33
TS(50-AATCCGTCGAGCAGAGTT-30),22.5pmolofprimerCXext (Fig. 1 and eqn (1), ESI†). Hypochromism and red shifts
(50-GTGCCCTTACCCTTACCCTTACCCTAA-30),2.5UofTaqDNA indicated strong interactions between the three complexes
polymerase, and 100 ng of telomerase. Complexes or distilled and HTG21. The intrinsic binding constants K b of 1, 2 and 3
water was added at a volume of 5 mL. PCR was performed in
weremeasuredtobe1.15(cid:2)106M(cid:3)1,2.91(cid:2)107M(cid:3)1and1.5(cid:2)
an Eppendorf Master cycler equipped with a hot lid and 107 M(cid:3)1, respectively, from the decay of the absorbance (see
incubated for 30 min at 30 1C, followed by 92 1C 30 s, 52 1C TableS1andFig.S4,ESI†).ThebindingconstantK b ofcomplex
30 s, and 72 1C 30 s for 30 cycles. After amplification, 8 mL of 2islargerthanthatofcomplexes1and3,whichindicatesthat
loading buffer (containing 5 (cid:2) Tris-Borate-EDTA buffer (TBE complex 2 is bound to HTG21 more tightly than complexes
buffer), 0.2% bromophenol blue, and 0.2% xylene cyanol)was 1and3.
addedtothereaction.A15mLaliquotwasloaded ontoa10%
3.2 Thestudiesofthermodynamicstabilization
non-denaturing acrylamide gel (19:1) in 1 (cid:2) TBE buffer and
electrophoresed at 200 V for 1 h. Gels were fixed and then To evaluate the stabilization of complexes 1, 2 and 3 for
stainedwithAgNO . G-quadruplex F21T (sequence: 50-FAM-G [T AG ] -TAMRA-30,
3 3 2 33
NewJ.Chem. Thisjournalis cTheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
NJC Paper
Fig.2 (a) Melting curves of G-quadruplex F21T (1 mM) in the absence of
complexes(black)andinthepresenceofcomplex1(red),2(blue)and3(green)
ataconcentrationof1mMinTris-HClbuffer(10mM,pH7.4)with60mMKCl.(b)
DNAstabilizationtemperatureversustheconcentrationofcomplexes1,2and3
bindingtoF21T.
stabilization effect of these three complexes on G-quadruplex
F21T.TheDT valueofcomplex2wasthehighest,indicating m
that complex 2 possessed a stronger stabilizing ability to the
F21TG-quadruplexthancomplexes1and3.
3.3 InhibitionofamplificationofHTG21
Inordertofurtherevaluatetheabilityofcomplexes1,2and3to
stabilize G-quadruplex DNA, polymerase chain reaction (PCR)
stopassaywascarriedouttoascertainwhethercomplexeswere
Fig.1 Absorbancespectraofcomplexes1(a),2(b)and3(c)inbufferat251Cin boundtothetestoligomer(50-G (T AG ) -30)andstabilizedthe
3 2 33
thepresenceofincreasingamountsofHTG21.[Ru]=10mM,[DNA]=0–10mM G-quadruplex structure.39 In the presence of the complexes
fromtoptobottom.Arrowsindicatethechangeinabsorbanceuponincreasing
tested, the template sequence 50-G (T AG ) -30 was induced
theDNAconcentration. 3 2 33
into a G-quadruplex structure that blocked the hybridization
withacomplementaryprimersequence.Inthatcase,the50 to
mimicking the human telomeric repeat, where FAM is the 30 primer extension by DNA Taq polymerase was arrested and
donor fluorophore 6-carboxyfluorescein and TAMRA is the the final double-stranded DNA PCR product could not be
acceptor 6-carboxytetramethylrhodamine), FRET melting detected. The inhibitory effect of 1 and 3 was gradually
experiments were carried out.38 Fig. 2a shows the melting enhanced as the concentrations were increased from 2.5 to
curves of G-quadruplex F21T (0.2 mM) in the presence of 1, 2 15 mM with no PCR product detected at 17.5 mM (Fig. 3).
and 3. It was clear that 1, 2 and 3 at a concentration of 1 mM Meanwhile, 2 at a concentration of 10.0 mM could completely
could raise the melting temperature of the G-quadruplex by inhibittheformationofthePCRproduct.TheIC valuesof1,2
50
about81C,131Cand111C,respectively.Fig.2bshowsthatthe and 3 were estimated to be 16.28 ((cid:5)1.07), 7.45 ((cid:5)0.63) and
stabilization of G-quadruplex DNA depended on the concen- 13.14((cid:5)0.83)mM,respectively.ThePCRassayfurtherindicated
tration of complexes. The results demonstrated an obvious thatcomplex2isabetterG-quadruplexbinder.
Thisjournalis c TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013 NewJ.Chem.
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
Paper NJC
Fig.3 Dose-dependent inhibition of HTG21 PCR amplification by complexes
1,2and3.
3.4 Circulardichroism(CD)spectra
Circular dichroism (CD) spectroscopy can be used to monitor
the formation of the G-quadruplex structure.40 Fig. 4 displays
theCDspectraforthetitrationofHTG21oligonucleotidewith
increasingamountsofcomplexes.Intheabsenceofsalt,theCD
spectrum of the HTG21 was found to have a major positive
bandat253nmandapositivebandnear295nm(Fig.4a,black
line),whichindicatesthecoexistenceofasinglestrand,parallel
andantiparallelG-quadruplex.41Upontitrationwithcomplexes
1,2and3intoHTG21oligonucleotide,dramaticchangesinthe
CDspectrawereobserved.
AsshowninFig.4a,themaximumat253nmwasgradually
suppressed and shifted toward 245 nm, while the band cen-
teredatabout293nmincreaseddramaticallywithanincrease
intheconcentrationof1.Meanwhile,amajornegativebandat
about261nmstartedtoappear.TheCDspectrumofthisnew
DNAconformationwasvirtuallyconsistentwiththeCDspectra
of antiparallel G-quadruplexes described in previous studies,
where the major positive band was usually observed around
290nmwithanegativebandat265nmandasmallerpositive
bandat246nm.42As theconcentrationof1furtherincreased
to7mM,astrongshoulderlocatednear275nmasthespectral
characteristic of a parallel G-quadruplex appeared. These
results indicated that 1could induce the guanine-rich DNA to
formthemixedparallel–antiparallelG-quadruplexstructure.At
the same time, a strong and positive induced CD signal was
observedbetween320and380nm.ThisinducedCDsignalwas
additionalevidencefortheinteractionbetweentheG-quadruplex
andcomplex1.43
The CD spectra of complex 2 titrated into the HTG21
oligonucleotide in the absence of salt was similar to, but not
identical, that of complex 1 in the wavelength region below
300 nm. In Fig. 4b, it can be seen that titration of 2 into this
DNA solution resulted in significant changes in the CD spec-
trum, including the observation of enhancement of the maxi-
mum at 290 nm and suppression of the band at 252 nm,
shifting to 260 nm, and the emergence of a strong shoulder
at 275 nm and a negative peak near 240 nm. The presence of
these signals suggested that 2 can also induce the HTG21 to
form the mixed parallel–antiparallel G-quadruplex structure.44 Fig.4 CD spectra of the HTG21 in the presence of increasing amounts of
complexes1(a),2(b)and3(c).[HTG21]=2mM,in10mMTris-HCl,pH=7.4,
Itisworthnotingthattheshouldernear275nminducedby2is
thecomplexconcentrationwas0–10mM.(d)Illustrationofhowthreeruthenium
much more dramatical than that induced by 1. The reason
complexes induce single-stranded human telomeric DNA to form a mixed
seemstobethattheplanararrangementofthephenringsand G-quadruplex.
NewJ.Chem. Thisjournalis cTheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
NJC Paper
Table1 IC50values(mM)ofthecomplexes1,2and3after48htreatmentin
threeselectedcelllines
IC (mM)
50
Complexes HeLa A549 HepG2
1 89.4(cid:5)2.3 127(cid:5)1.5 103(cid:5)1.8
2 13.5(cid:5)0.5 19.2(cid:5)0.7 29.7(cid:5)0.6
3 67.2(cid:5)1.5 32.4(cid:5)0.9 59.1(cid:5)0.8
CDDP 7.6(cid:5)0.4 13.6(cid:5)0.3 26.8(cid:5)0.5
theirappropriatespacingmake2idealtobestackedontopof
theguaninetetrads.
InFig.4c,itcanbeseenthatthebandsat252and292nm
greatly weaken with the addition of complex 3 into the same
DNA solution, without the appearance of any new bands. The
extraordinary result showed that complex 3 had no ability to
induce the formation of mixed G-quadruplex structures. The Fig.5 Complex 2 induced apoptosis in HeLa cells. (A) The change of cell
spectral changes indicate that 1 and 2 can induce human morphologyobservedusinganinvertedmicroscope((cid:2)200).Cellsweretreated
telomeric DNA to form a mixed/hybrid type G-quadruplex with different concentrations of complex 2 for 48 h. (B) Complex 2 induced
apoptotic morphological changes of HeLa cells. HeLa cells were treated with
structure(Fig.4d).
complex 2 for 48 h, stained with Hoechst 33342 and photographed using
fluorescence microscopy ((cid:2)400). The red arrows indicate the condensed or
3.5 Invitrocytotoxicityassay
fragmentednucleusandmulti-blebbingcells,respectively.
Invitrocytotoxicityassayofcomplexes1,2and3wasevaluated
by means of MTT assay against three human cancer cell lines
(usingcisplatinasthepositivecontrol)includingHeLa(human fluorescence microscopy. Apoptotic and necrotic cells can be
cervical cancer), A549 (human lung carcinoma) and HepG2 distinguished from one another influorescence microscopy.46
(humanhepatocellularlivercarcinoma).45Afterthecancercells After treatment of HeLa cells with 2 for a period of 48 h,
were incubated at various concentrations of tested complexes apoptoticbodiesdisplayingdifferentsizeandirregularmorphology
for48h,eachcomplexexhibiteddifferentantitumoractivities. were observed as shown in Fig. 5B. However, staining bright,
TheIC valuesofthreecomplexesandcisplatinareshown condensed chromatin and fragmented nuclei did not appear in
50
inTable1.Theresultsindicatedthatthecancercellstestedare the normal control group. The cells treated with 2 did exhibit
susceptibletothecomplexes.AsshowninTable1,IC values morphologicfeaturesofapoptosis,andfurtherquantitativeanaly-
50
of 1 on three cell lines range from 89.4 mM to 127 mM, which sisofchangesinapoptosisisnecessary.
exhibited moderate cytotoxic activity. Compared to 1, the IC
50
valueof3islower,whichshowsthat3ismoreactiveinthecell
3.6 TRAPassay
linestestedthan1.Itisinterestingthatcomplex2exhibitedthe
Inordertoexaminetheabilityofthecomplexestoinhibitthe
highest antiproliferative activities in three cancer cell lines
telomeraseactivity,thetelomericrepeatamplificationprotocol
among the three complexes, as evidenced by the lowest IC
50 (TRAP)assaywasperformed.47Complexes2and3weretested
values. Notably, 2 exhibited a broad spectrum of inhibition
inthisexperiment.Fig.6showstheinvitroinhibitoryeffectof
on human cancer cells, with IC values ranging from 13.5 to
50
2 and 3. The process of inhibition of telomerase activity was
29.7 mM, indicating the high cytotoxic effects of 2 on cancer
investigated in a dose-dependent manner, and the number of
cells.Itisalsoworthnotingthat2showsadistinctpreference
bands obviously decreased with respect to the control, in the
forHeLacells,thusHeLacellswerechosenasacellmodelfor
drugconcentrationrangeof1–20mM.Thetwocomplexestested
further investigation of the mechanisms underlying the anti-
proliferativeactionof2.
To clarify how complex 2 affected HeLa cell growth, the
changes in cell morphology were examined using an inverted
microscope (Fig. 5A). We found that HeLa cells treated with
complex 2 for 48 h exhibited marked morphologic signs of
apoptosis in a dose-dependent manner, where lots of cells
became round, detached cells increased and adherent cells
gradually decreased, then a large number of suspended
cellsappeared,accompaniedwithcelldebris,apoptoticbodies
and other characteristics. To observe the morphologic charac-
teristics of apoptotic nuclei, HeLa cells were stained with
Hoechst33342afterexposureto2(10mM)anddetectedusing Fig.6 Theinfluenceofcomplexes2and3onthetelomereactivityofHeLacells.
Thisjournalis c TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013 NewJ.Chem.
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
Paper NJC
Fig.8 Complex2inducedthedepletionofmitochondrialmembranepotential
(DC m)inHeLacells.Cellstreatedwithdifferentconcentrationsofcomplex2were
analyzedusingJC-1flowcytometry.Thenumberineachdotplotrepresentsthe
Fig.7 Quantitativeanalysisofcomplex2inducedapoptoticcelldeath(48h)in
HeLacellsusingflowcytometry.Thecellstreatedwithdifferentconcentrationsof
percentageofcellsthatlostDC m.
complex2for48hwerecollectedandstainedwithPIafterfixation.
pathways associated with the initiation of apoptotic cascades.
led to an inhibition of the telomerase activity, but there were Therefore, mitochondrial dysfunction was investigated by mea-
differences in the extent of inhibition. Fig. 6 clearly revealed suringchangesinthemitochondrialmembranepotential(MMP,
that 2 exhibited greater telomerase inhibitory activity than DC m ) using flow cytometry after staining live cells with the
complex 3. A number of methods have been previously used cationic dye JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-
to confirm that complex 2 is a more effective stabilizer of dazolylcarbocyanine iodide). JC-1 exhibits potential-dependent
HTG21, and shows stronger inhibition activity against cancer accumulationinmitochondria,indicatedbyafluorescenceemis-
cells. According to all the above results, we can infer that sion shift from red to green. As depicted in Fig. 8, complex 2
telomeraseisoneofthetargetsforinhibitingtumorsinduced significantly induceda dose-dependent decline ofDC m in HeLa
bytherutheniumcomplexes. cells. The cell ratio of mitochondrial depolarization increased
roughlybynearly20times,changingfrom1.1%(control)to2.9%
3.7 Cellcyclearrestandinductionofapoptosis (2.5 mM), then 6.9% (5 mM), finally up to 18.7% (10 mM). Its
accumulation in HeLa cells then induced the loss of DC , led
Theinhibitionofcancercellproliferation,thecessationofcell- m
to the release of mitochondrial contents and apoptosis-related
cycleprogressionandtheinductionofapoptosishaveallbeen
factors such as cytochrome c and other apoptosis-inducing fac-
targeted in chemotherapeutic strategies for the treatment of
tors, then DNA damage occurred and morphological changes
cancer.48Wethereforeevaluatedwhethercomplex2alteredthe
appeared.ThedeclineofDC furtherconfirmedthat2induced
cell cycle of HeLa cells and induced apoptosis of HeLa cells m
apoptosis in HeLa cells through the endogenous mitochondria-
using flow cytometric analyses. The cell-cycle phase distribu-
mediatedpathway.
tion in HeLa cells after 48 h exposure to different concentra-
tionsof2wasanalysed.
As shown inFig.7, treatment with2led toa marked dose-
4 Conclusions
dependent increase in the proportion of cells in the G0/G1
phase.Therewere77.1%and86.1%cellsinG0/G1phaseafter In summary, three new ruthenium(II)-2,20-bisimidazole com-
48 h treatment with 2 at concentrations of 5 and 10 mM, plexeshavebeensynthesizedandmadetointeractwithhuman
respectively, compared with 75.6% in untreated cultures; at telomericG-quadruplexDNA.Theexperimentalresultsimplied
the same time, the proportion of cells in the G2/M phase that the three complexes could bind tightly to the human
decreasedsignificantlyfrom4.2%to1.2%.Thisconfirmedthat telomeric DNA. Complex 2 could significantly stabilize the
the 2-treated cells were blocked in the G0/G1-phase. The G-quadruplex structure and exhibited more efficiency in indu-
alteration of cells in the S phase after 48 h treatment with cing the formation of mixed/hybrid type G-quadruplexes com-
different concentrations of 2 was not regular. Furthermore, a paredtocomplexes1and3.Thisresultmightbeexplainedby
populationofsub-G1-phasecells,acharacteristicofapoptosis, thefactthatitsauxiliaryphenligandsareavailableforthep–p
was increased dramatically with an increase in the concen- interactions between the aromatic phen ligands and DNA
trationof2.Theseresultsindicatedthatcelldeathinducedby guanine residues, meanwhile, the great steric hindrance of
complex2ismainlycausedbyinductionofapoptosis. the p-mopip ligands of complex 3 make them difficult to
externally bind to the stacks of guanine quartets within a
3.8 Inductionofmitochondrialdysfunction
quadruplex or intercalating between the stacks. In vitro cyto-
Mitochondriacontrolthelifeanddeathofacell,decidingthe toxicityassaydatashowthatcomplex2exhibitedmorepotent
fate of a cell by controlling the process of apoptosis.49 Mito- antitumoractivitiesagainstcancercelllines,comparedtocom-
chondria act as a point of integration for apoptotic signals plexes1and3.Thisresultshowedthattheantitumoractivityis
originating from both the extrinsic and intrinsic apoptotic closelyrelatedtotheirabilitytointeractwithG-quadruplexDNA,
pathways.50Mitochondrialdysfunctionandthereleaseofapop- andcomplex2couldeffectivelyinhibittumorcellgrowth.TRAP
togenic factors are critical keys in triggering various apoptotic assaydemonstratedthatcomplex2exhibitedstrongerinhibitory
NewJ.Chem. Thisjournalis cTheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
NJC Paper
activity towards telomerase. Thus, the inhibition of telomerase 12 J. E. Reed, A. A. Arnal, S. Neidle and R. Vilar, J. Am. Chem.
activity by inducing G-quadruplex formation is very important Soc.,2006,128,5992–5993.
fordevelopinganticancerdrugs.Complex2inducedHeLacells 13 N. V. Anantha, M. Azam and R. D. Sheardy, Biochemistry,
togenerateasignificantpeakofapoptosisinthesub-G1phase, 1998,37,2709–2714.
indicating that complex 2 inhibits HeLa cells proliferation by 14 H.Yu,X.Wang,M.Fu,J.RenandX.Qu,NucleicAcidsRes.,
induction of apoptosis. Further investigation showed that the 2008,36,5695–5703.
treatmentofthecomplexinHeLacellscouldresultinsignificant 15 B.M.Zeglis,V.C.PierreandJ.K.Barton,Chem.Commun.,
depletion of DC in a dose-dependent manner, and it is a 2007,4565–4579.
m
relatively early event in complex 2-induced apoptosis. Taken 16 M. R. Gill and J. A. Thomas, Chem. Soc. Rev., 2012, 41,
together,theseresultsdemonstratedthatcomplex2couldeffec- 3179–3192.
tively promote the apoptosis of tumor cells by acting on mito- 17 H. Song, J. T. Kaiser and J. K. Barton, Nat. Chem., 2012, 4,
chondrial apoptotic pathways. The present results should be of 615–620.
valueinfurtherunderstandingtheinteractionofG-quadruplexes 18 G.N.Parkinson,M.P.LeeandS.Neidle,Nature,2002,417,
with metal complexes, as well as offer valuable information for 876–880.
designingnewantitumortherapeutics. 19 K.E.Erkkila,D.T.OdomandJ.K.Barton,Chem.Rev.,1999,
99,2777–2796.
20 C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32,
Acknowledgements
215–224.
21 U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia
This work was supported by the National Natural Science
andS.Wo¨lfl,ChemMedChem,2008,3,1104–1109.
Foundation of China (20871056, 21171070, 21371075), the
22 S. Rickling, L. Ghisdavu, F. Pierard, P. Gerbaux, M. Surin,
PlannedItemofScienceandTechnologyofGuangdongProvince
P. Murat, E. Defrancq, C. Moucheron and A. Kirsch-De
(c1011220800060, c1211220800571), the Natural Science Foun-
Mesmaeker,Chem.–Eur.J.,2010,16,3951–3961.
dation of Guangdong Province and the Fundamental Research
23 C.Rajput,R.Rutkaite,L.Swanson,I.HaqandJ.A.Thomas,
FundsfortheCentralUniversities.
Chem.–Eur.J.,2006,12,4611–4619.
24 F. Linares, M. A. Galindo, S. Galli, M. A. Romero, J. A.
Notes and references NavarroandE.Barea,Inorg.Chem.,2009,48,7413–7420.
25 O.Zava,S.M.Zakeeruddin,C.Danelon,H.Vogel,M.Gratzel
1 N.W.Kim,M.A.Piatyszek,K.R.Prowse,C.B.Harley,M.D. andP.J.Dyson,ChemBioChem,2009,10,1796–1800.
West,P.L.Ho,G.M.Coviello,W.E.Wright,S.L.Weinrich 26 J. M. Rademaker-Lakhai, D. van den Bongard, D. Pluim,
andJ.W.Shay,Science,1994,266,2011–2015. J.H.BeijnenandJ.H.Schellens,Clin.CancerRes.,2004,10,
2 J. Szolom´ajer, G. Paragi, G. Batta, C. F. Guerra, F. M. 3717–3727.
Bickelhaupt, Z. Kele, P. P´ad´ar, Z. Kupih´ar and L. Kov´acs, 27 F. Barragan, D.Carrion-Salip, I. Gomez-Pinto, A. Gonzalez-
NewJ.Chem.,2011,35,476–482. Canto, P. J. Sadler, R. de Llorens, V. Moreno, C. Gonzalez,
3 C. C. Chang, I. C. Kuo, I. F. Ling, C. T. Chen, H. C. Chen, A.MassaguerandV.Marchan,BioconjugateChem.,2012,23,
P. J. Lou, J. J. Lin and T. C. Chang, Anal. Chem., 2004, 76, 1838–1855.
4490–4494. 28 V. Pierroz, T. Joshi, A. Leonidova, C. Mari, J. Schur, I. Ott,
4 K.Paeschke,S.Juranek,D.RhodesandH.J.Lipps,Chromo- L.Spiccia,S.FerrariandG.Gasser,J.Am.Chem.Soc.,2012,
someRes.,2008,16,721–728. 134,20376–20387.
5 A. Paul, B. Maji, S. K. Misra, A. K. Jain, K. Muniyappa and 29 I. Ascone, L. Messori, A. Casini, C. Gabbiani, A. Balerna,
S.Bhattacharya,J.Med.Chem.,2012,55,7460–7471. F. Dell’Unto and A. C. Castellano, Inorg. Chem., 2008, 47,
6 A. M. Burger, F. Dai, C. M. Schultes, A. P. Reszka, M. J. 8629–8634.
Moore, J. A. Double and S. Neidle, Cancer Res., 2005, 65, 30 M. Groessl, E. Reisner, C. G. Hartinger, R. Eichinger,
1489–1496. O. Semenova, A. R. Timerbaev, M. A. Jakupec, V. B. Arion
7 Y.-P. Huang, D.-M. Kong, Q.-M. Chen, H.-X. Shen and andB.K.Keppler,J.Med.Chem.,2007,50,2185–2193.
H.-F.Mi,NewJ.Chem.,2004,28,1488–1493. 31 D. Sun, Y. Liu, D. Liu, R. Zhang, X. Yang and J. Liu,
8 K.Masutomi,E.Y.Yu,S.Khurts,I.Ben-Porath,J.L.Currier, Chem.–Eur.J.,2012,18,4285–4295.
G. B. Metz, M. W. Brooks, S. Kaneko, S. Murakami, J. A. 32 S. Shi, J. Liu, J. Li, K. C. Zheng, X. M. Huang, C. P. Tan,
DeCaprio, R. A. Weinberg, S. A. Stewart and W. C. Hahn, L.M.ChenandL.N.Ji,J.Inorg.Biochem.,2006,100,385–395.
Cell,2003,114,241–253. 33 J. Bolger, A. Gourdon, E. Ishow and J. P. Launay, Inorg.
9 C. Zhao, J. Geng, L. Feng, J. Ren and X. Qu, Chem.–Eur. J., Chem.,1996,35,2937–2944.
2011,17,8209–8215. 34 W.PawandR.Eisenberg,Inorg.Chem.,1997,36,2287–2293.
10 S.N.Georgiades,N.H.AbdKarim,K.Suntharalingamand 35 J. C. Xiao and J. M. Shreeve, J. Org. Chem., 2005, 70,
R.Vilar,Angew.Chem.,Int.Ed.,2010,49,4020–4034. 3072–3078.
11 J. E. Reed, S. Neidle and R. Vilar, Chem. Commun., 2007, 36 Z.-C. Liu, B.-D. Wang, Z.-Y. Yang, Y. Li, D.-D. Qin and
4366–4368. T.-R.Li,Eur.J.Med.Chem.,2009,44,4477–4484.
Thisjournalis c TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013 NewJ.Chem.
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online
Paper NJC
37 A.Tarushi,J.Kljun,I.Turel,A.A.Pantazaki,G.Psomasand 44 Y. Xu, Y. Noguchi and H. Sugiyama, Bioorg. Med. Chem.,
D.P.Kessissoglou,NewJ.Chem.,2013,37,342–355. 2006,14,5584–5591.
38 S. Cogoi, M. Paramasivam, B. Spolaore and L. E. Xodo, 45 X. Zhang, X. Li, S. Ye, Y. Zhang, L. Tao, Y. Gao, D. Gong,
NucleicAcidsRes.,2008,36,3765–8010. M. Xi, H. Meng, M. Zhang, W. Gao, X. Xu, Q. Guo and
39 D.Sun,R.Zhang,F.Yuan,D.Liu,Y.ZhouandJ.Liu,Dalton Q.You,Med.Chem.,2012,8,1012–1025.
Trans.,2012,41,1734–1741. 46 M. Takaya, M. Nomura, T. Takahashi, Y. Kondo,
40 E.M.Rezler,J.Seenisamy,S.Bashyam,M.Y.Kim,E.White, K. T. Lee and S. Kobayashi, Anticancer Res., 2009, 29,
W.D.WilsonandL.H.Hurley,J.Am.Chem.Soc.,2005,127, 995–1000.
9439–9447. 47 X.Chen,J.H.Wu,Y.W.Lai,R.Zhao,H.ChaoandL.N.Ji,
41 R. Rodriguez, G. D. Pantos, D. P. Goncalves, J. K. Sanders DaltonTrans.,2013,42,4386–4397.
and S. Balasubramanian, Angew. Chem., Int. Ed., 2007, 46, 48 Y. Jin, J. Liu, W.-T. Huang, S.-W. Chen and L. Hui, Eur. J.
5405–5407. Med.Chem.,2011,46,4056–4061.
42 J.L.MergnyandJ.C.Maurizot,ChemBioChem,2001,2,124–132. 49 X.Yang,L.Chen,Y.Liu,Y.Yang,T.Chen,W.Zheng,J.Liu
43 E. W. White, F. Tanious, M. A. Ismail, A. P. Reszka, andQ.Y.He,Biochimie,2012,94,345–353.
S. Neidle, D. W. Boykin and W. D.Wilson, Biophys. Chem., 50 C. A. Puckett, R. J. Ernst and J. K. Barton, Dalton Trans.,
2007,126,140–153. 2010,39,1159–1170.
NewJ.Chem. Thisjournalis cTheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2013
.92:83:80
3102/90/81
no
yendyS
fo
ytisrevinU
yb
dedaolnwoD
.3102
tsuguA
22
no
dehsilbuP
View Article Online