A planar Schiff base platinum(II) complex: Crystal structure, cytotoxicity and interaction with DNA

Article abstract of DOI:10.1248/cpb.c13-00256

A new platinum(II) complex of salphen derivative, namely Schiff base ligand that derived from o-phenylenediamine and 5-chlorosalicylaldehyde was synthesized. The complex possessed a planar mononuclear structure. The in vitro cytotoxicities of the complex

Full text of DOI:10.1248/cpb.c13-00256

March 2014
Regular Article
Chem. Pharm. Bull. 62(3) 221–228 (2014)
221
A Planar Schiff Base Platinum(II) Complex: Crystal Structure,
Cytotoxicity and Interaction with DNA
Yan Peng, Hui Zhong, Zhen-Feng Chen,* Yan-Cheng Liu, Guo-Hai Zhang, Qi-Pin Qin, and
Hong Liang*
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry & Chemical Engineering of Guangxi Normal University; Guilin 541004, P. R. China.
Received April 3, 2013; accepted December 2, 2013
A new platinum(II) complex of salphen derivative, namely Schiff base ligand that derived from o-phen-
ylenediamine and 5-chlorosalicylaldehyde was synthesized. The complex possessed a planar mononuclear
structure. The in vitro cytotoxicities of the complex were evaluated by microculture tetrozolium (MTT) assay
against seven human tumor cell lines with the IC50 values of ca. 11.61 µM. Cell cycle analysis indicated that
the complex induced apoptosis and G1-phase arrest in A549 cells. The results of colony formation assay
showed that the complex could suppress the proliferation and viability of A549 cells. The binding of the
complex to potential target DNA were investigated by fluorescence spectroscopy, viscosity measurements,
fluorescence polarization and agarose gel electrophoresis. The results suggest that the most probable binding
mode of the complex is intercalation.
Key words Schiff base; platinum(II) complex; crystal structure; antitumor activity; DNA binding
The study of platinum(II)-containing anticancer drugs has chasedꢀ fromꢀ Sigma-Aldrich.ꢀ Theyꢀ wereꢀ allꢀ usedꢀ asꢀ receivedꢀ
been a hot topic in medicinal inorganic chemistry over the withoutꢀfurtherꢀpurificationꢀunlessꢀnotedꢀspecifically.
pastꢀfewꢀdecadesꢀsinceꢀcisplatinꢀwasꢀidentifiedꢀasꢀtheꢀveryꢀef-
Instrumentation and Methods The pH value of buffer
1,2)
ficientꢀanticancerꢀagent. Thousands of platinum(II/IV) com- solution was measured on a Sartorius Professional PP-20 pH
plexes have been synthesized and assayed for their in vitro or Meter.ꢀ Elementalꢀ analysesꢀ (C,ꢀ Hꢀ andꢀ N)ꢀ wereꢀ carriedꢀ outꢀ
in vivoꢀ anticancerꢀ activities.ꢀ However,ꢀ onlyꢀ thirtiesꢀ ofꢀ themꢀ onꢀ aꢀ PerkinElmer,ꢀ Inc.ꢀ Seriesꢀ IIꢀ CHNS/Oꢀ 2400ꢀ elementalꢀ
1
13
enteredꢀ clinicalꢀ trials.ꢀ Discoveringꢀ moreꢀ potentꢀ platinum- analyzer.ꢀ H-NMR and C-NMR spectra were recorded on a
based anticancer drugs while with less side effects represents BrukerꢀAV-500ꢀNMRꢀspectrometerꢀwithꢀDMSO-d ꢀasꢀsolvent.ꢀ
6
aꢀ majorꢀ challenge.ꢀ Aꢀ fewꢀ newꢀ strategiesꢀ suchꢀ asꢀ someꢀ trans, Electrosprayꢀ ionizationꢀ (ESI)-MSꢀ spectraꢀ wereꢀ performedꢀ
monofunction in geometry and classical intercalation to DNA, onꢀ Thermofisherꢀ Scientificꢀ Exactiveꢀ LC-MSꢀ Spectrometer.ꢀ
have been applied for designing new platinum(II) complexes Fluorescence emission spectra were performed on a Shimadzu
showed different anticancer mechanisms with cisplatin to RF-5301/PCꢀspectrofluorometer.ꢀDNAꢀviscosityꢀwasꢀmeasuredꢀ
overcome the intrinsic or acquired resistance, moreover, a onꢀ aꢀ Brookfieldꢀ DV-IIꢀ proꢀ digitalꢀ viscometerꢀ equippedꢀ withꢀ
3
–5)
greatꢀofꢀprogressꢀhaveꢀbeenꢀmade.
Schiff bases are versatile ligands and have been play-
UltraꢀLowꢀAdaptorꢀimmersedꢀinꢀaꢀTC-502Dꢀthermostaticꢀbath.
Synthesis. Synthesis of Ligand A mixture of o-phenyl-
enediamineꢀ (1.08ꢀg,ꢀ 0.01ꢀmol),ꢀ 5-chlorosalicylaldehydeꢀ (3.12ꢀg,ꢀ
6
,7)
ing key roles in the development of new bioactive agents,
andꢀ inꢀ particular,ꢀ anticancerꢀ andꢀ antibacterialꢀ agents.ꢀ Metalꢀ 0.02ꢀmol)ꢀ dissolvedꢀ inꢀ distilledꢀ waterꢀ (20ꢀmL)ꢀ wasꢀ refluxedꢀ
complexes of Schiff bases have been shown to be able to withꢀ stirring.ꢀ Theꢀ solutionꢀ wasꢀ continuouslyꢀ refluxedꢀ inꢀ
interact with several primary biogenic macromolecules such water-bathꢀatꢀ80°Cꢀforꢀ6ꢀh.ꢀAfterꢀcoolingꢀtheꢀreactionꢀmixtureꢀ
8
–10)
asꢀ DNA,ꢀ RNAꢀ andꢀ peptides.
Particularly, the Schiff base to the room temperature, the yellow precipitate formed and
metal complexes derived from salicylaldehyde were effective filtrated.ꢀThenꢀtheꢀyellowꢀprecipitateꢀwasꢀre-crystallizedꢀfromꢀ
11–14)
1
forꢀ cleavingꢀ DNA.
In this paper, we attempt to develop methanol,ꢀandꢀdried,ꢀwithꢀaꢀyieldꢀofꢀ71%.ꢀ H-NMR (500 MHz,
new platinum(II) Schiff base anticancer agents that binding DMSO-d ) δꢀ12.93ꢀ(s,ꢀ2H,ꢀAr-OH),ꢀ8.93ꢀ(s,ꢀ2H,ꢀCH= ꢀN ),ꢀ7.78ꢀ
6
non-covalentlyꢀ toꢀ DNA.ꢀ Aꢀ newꢀ planarꢀ platinum(II)ꢀ complexꢀ (d, J= ꢀ2 .6ꢀ Hz,ꢀ 2H,ꢀ H-Ar),ꢀ 7.45–7.38ꢀ (m,ꢀ 4H,ꢀ H-Ar),ꢀ 7.12–6.96ꢀ
13
of Schiff base ligand (H -LCl ) derived from o-phenylene- (m,ꢀ 4H,ꢀ H-Ar).ꢀ C-NMRꢀ (125ꢀ MHz,ꢀ DMSO-d ) δꢀ 162.77,ꢀ
2
2
6
diamineꢀ andꢀ 5-chlorosalicylaldehydeꢀ wasꢀ synthesized.ꢀ Itsꢀ in 159.52,ꢀ 142.52,ꢀ 136.21,ꢀ 133.40,ꢀ 131.27,ꢀ 128.64,ꢀ 127.81,ꢀ 122.99,ꢀ
vitroꢀ antitumorꢀ activitiesꢀ andꢀ itsꢀ influenceꢀ onꢀ cellꢀ cycle,ꢀ cellꢀ 121.28,ꢀ 120.11,ꢀ 119.97,ꢀ 119.53,ꢀ 119.43,ꢀ 119.22.ꢀElemental Anal.ꢀ
apoptosis and colony formation, as well as its binding proper- Calcd for C H Cl N O :ꢀ C:ꢀ 62.35%,ꢀ H:ꢀ 3.66%,ꢀ N:ꢀ 7.27%,ꢀ
2
0
14
2
2
2
tiesꢀtoꢀpotentialꢀtargetꢀDNAꢀwereꢀevaluated.
FoundꢀC:ꢀ62.31%,ꢀH:ꢀ3.69%,ꢀN:ꢀ7.30%.
Synthesis of Complexꢀ ꢀT heꢀ mixtureꢀ ofꢀ Pt(DMSO) Cl
2
2
Experimental
(0.041ꢀg,ꢀ 0.1ꢀmmol),ꢀ H -ClL ꢀ (0.046ꢀg,ꢀ 0.12ꢀmmol),ꢀ ethanolꢀ
2 2
Materials All the chemical reagents and solvents were of (1ꢀmL),ꢀ dimethylꢀ sulfoxideꢀ (DMSO)ꢀ (1ꢀmL)ꢀ wereꢀ heatedꢀ toꢀ
analyticalꢀ grade.ꢀ PtCl , 1,2-diaminobenzene, 5-chlorosalicyl- 80°Cꢀ andꢀ wasꢀ stirredꢀ forꢀ 12ꢀh.ꢀ Afterꢀ coolingꢀ toꢀ theꢀ roomꢀ
2
aldehydeꢀ wereꢀ purchasedꢀ fromꢀ Alfa-aesar.ꢀ GelRedꢀ wasꢀ pur- temperature,ꢀtheꢀreddish-brownꢀcrystalsꢀwereꢀobtained.ꢀYield:ꢀ
chasedꢀ fromꢀ Biotium.ꢀ Agarose,ꢀ ethidiumꢀ bromideꢀ (EB),ꢀ calfꢀ 0.049ꢀg,ꢀ 56%.ꢀ Singleꢀ crystalsꢀ suitableꢀ forꢀ X-rayꢀ diffractionꢀ
thymus DNA (ct-DNA) and pUC19 plasmid DNA were pur- analysis were harvested by slow evaporation of the re-crystal-
1
lizedꢀ ethanolꢀ solution.ꢀ H-NMRꢀ (500ꢀMHz,ꢀ DMSO-d ) δꢀ 9.57ꢀ
6
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
(s, 1H, CH= ꢀN ),ꢀ 8.32ꢀ (s,ꢀ 2H,ꢀ CH= ꢀN ,ꢀ Ar),ꢀ 8.23ꢀ (d,ꢀ J=7.7ꢀHz,ꢀ
*
ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀchenzfubc@yahoo.com;ꢀhliang@gxnu.edu.cn
© 2014 The Pharmaceutical Society of Japan

2
22
Vol.ꢀ62, No.ꢀ3
1
H,ꢀH-Ar),ꢀ7.90ꢀ(s,ꢀ1H,ꢀH-Ar),ꢀ7.54ꢀ(d,ꢀJ=11.0ꢀHz,ꢀ2H,ꢀH-Ar),ꢀ heavilyꢀmoisturizedꢀenvironmentꢀsuppliedꢀwithꢀ5%ꢀCO .ꢀTheꢀ
2
7.37ꢀ (dd,ꢀ J= ꢀ2 5.1,ꢀ 17.2ꢀHz,ꢀ 4H,ꢀ H-Ar),ꢀ 6.95ꢀ (d,ꢀ J=9.0ꢀHz,ꢀ 1H,ꢀ next day, the cells were treated with various concentrations
13
H-Ar).ꢀ C-NMRꢀ (125ꢀMHz,ꢀ DMSO-d ): δꢀ 161.78,ꢀ 148.68,ꢀ ofꢀ theꢀ complexꢀ orꢀ 0.1%ꢀ DMSOꢀ forꢀ 24ꢀh.ꢀ Floatingꢀ andꢀ adher-
6
1
47.35,ꢀ140.82,ꢀ134.36,ꢀ133.04,ꢀ128.79,ꢀ128.27,ꢀ126.82,ꢀ123.90,ꢀ entꢀcellsꢀwereꢀcollectedꢀandꢀfixedꢀinꢀcoldꢀ70%ꢀethanolꢀatꢀ4°Cꢀ
+
116.96.ꢀ ESI-MS:ꢀ m/z 578 [M+H] .ꢀ Elemental Anal.ꢀ Calcdꢀ forꢀ overnight.ꢀAfterꢀwashing,ꢀtheꢀcellsꢀwereꢀsubsequentlyꢀstainedꢀ
C H Cl N O Pt:ꢀ C:ꢀ 41.54%,ꢀ H:ꢀ 2.09%,ꢀ N:ꢀ 4.84%,ꢀ Foundꢀ C:ꢀ with 50µg/mL propidium iodide (PI) and 100µg/mL RNase A
2
0
12
2
2
2
41.58%,ꢀH:ꢀ2.11%,ꢀN:ꢀ4.89%.
forꢀ1ꢀhꢀinꢀtheꢀdarkꢀandꢀsubjectedꢀtoꢀflowꢀcytometricꢀanalysisꢀinꢀ
X-Ray Crystallography The data collection of single orderꢀ toꢀ determineꢀ theꢀ percentageꢀ ofꢀ cellsꢀ atꢀ specificꢀ phasesꢀ
crystal of the complex was carried out on an Agilent Super- ofꢀ theꢀ cellꢀ cycle.ꢀ Flowꢀ cytometricꢀ analysisꢀ wasꢀ performedꢀ
Nova CCD diffractometer equipped with graphite monochro- usingꢀ aꢀ FACSCaliburꢀ flowꢀ cytometerꢀ (Becton-Dickinson,ꢀ Sanꢀ
mated MoKα radiation (λ= ꢀ0 .71073ꢀÅ)ꢀ atꢀ roomꢀ temperature.ꢀ Jose,ꢀCA,ꢀU.S.A.)ꢀequippedꢀwithꢀaꢀ488ꢀnmꢀargonꢀlaser.ꢀAꢀhighꢀ
Theꢀ structuresꢀ wereꢀ solvedꢀ withꢀ directꢀ methodsꢀ andꢀ refinedꢀ number of events were evaluated for each sample and the cell
15,16)
usingꢀ SHELX-97ꢀ programs.
The non-hydrogen atoms cycle distribution was analyzed using Cell Quest software
wereꢀ locatedꢀ inꢀ successiveꢀ differenceꢀ Fourierꢀ synthesis.ꢀ Theꢀ (Becton-Dickinson).ꢀTheꢀresultsꢀwereꢀpresentedꢀasꢀtheꢀnumberꢀ
finalꢀ refinementꢀ wasꢀ performedꢀ byꢀ full-matrixꢀ least-squaresꢀ of cells versus the amount of DNA as indicated by the intensi-
methods with anisotropic thermal parameters for no-hydrogen tyꢀofꢀtheꢀfluorescenceꢀsignal.ꢀAllꢀexperimentsꢀwereꢀconductedꢀ
2
atoms on F .ꢀ Theꢀ hydrogenꢀ atomsꢀ wereꢀ addedꢀ theoreticallyꢀ threeꢀtimes.
andꢀridingꢀonꢀtheꢀconcernedꢀatoms.ꢀTheꢀparamertersꢀusedꢀin-
tensityꢀcollectionꢀandꢀrefinementsꢀareꢀsummarizedꢀinꢀTableꢀ1ꢀ usingꢀAnnexinꢀV-fluoresceinꢀisothiocyanateꢀ(FITC)/PIꢀapopto-
togetherꢀwithꢀtheꢀcrystalꢀdata.
sisꢀ detectionꢀ kitꢀ (Becton-Dickinson).ꢀ Cellsꢀ atꢀ theꢀ logarithmicꢀ
Apoptosis Assay Cell-apoptosis analysis was conducted
Cytotoxicity Assay in Vitro The tumor cell lines A549, phase of growth were harvested using cold phosphate buff-
MDA-MB-231,ꢀHepG2,ꢀT24,ꢀMG-63,ꢀMGC-803,ꢀHUVECꢀandꢀ ered saline (PBS) and collected by centrifugation for 6min at
6
theꢀ normalꢀ lungꢀ fibroblastꢀ cellꢀ linesꢀ wereꢀ obtainedꢀ fromꢀ theꢀ 1.000×g.ꢀCellsꢀwereꢀresuspendedꢀatꢀaꢀdensityꢀofꢀ1×10 cells/
Shanghaiꢀ Cellꢀ Bank,ꢀ Chineseꢀ Academyꢀ ofꢀ Science.ꢀ Allꢀ cellsꢀ mL in 1×binding buffer, stained with FITC-labeled annexin
were grown in RPMI-1640 medium supplemented with 10% V and PI for 20min and immediately analyzed in a FACScan
3
(
v/v) fetal bovine serum, 2mM glutamine, 100U/cm penicil- FlowꢀCytometerꢀ(Becton-Dickinson).
3
lin, and 100U/cm ꢀ streptomycinin,ꢀ inꢀ aꢀ highlyꢀ humidifiedꢀ
Colony Formation Assay The colony formation assay is
atmosphereꢀofꢀ95%ꢀairꢀwithꢀ5%ꢀCO ꢀatꢀ310ꢀK.ꢀTheꢀcytotoxici- aꢀ methodꢀ forꢀ analyzingꢀ cellꢀ proliferationꢀ andꢀ survival.ꢀ A549ꢀ
2
ties of the complex and the ligand against all cell lines were cellsꢀ wereꢀ seededꢀ intoꢀ aꢀ 6-wellꢀ plate.ꢀ Afterꢀ theꢀ cellsꢀ wereꢀ
17)
examinedꢀ byꢀ theꢀ MTTꢀ assay. Three replicate tests were grownꢀ toꢀ 70–80%ꢀ confluence,ꢀ theꢀ cellsꢀ wereꢀ treadedꢀ withꢀ
conducted.ꢀTheꢀgrowthꢀinhibitoryꢀrateꢀofꢀtreatedꢀcellsꢀwasꢀcal- the complex at the concentrations of 10, 20, 30µmol/L, re-
culatedꢀ usingꢀ theꢀ formula:ꢀ (ODcontrol−ODtest)/ODcontrol×100%.ꢀ spectively.ꢀ Afterꢀ 24ꢀh,ꢀ theꢀ cellsꢀ wereꢀ trypsinized,ꢀ aboutꢀ 500ꢀ
The experiments were carried out following the procedure cells treaded with the complex were seeded into a new 6-well
reportedꢀ inꢀ theꢀ literature.ꢀ Theseꢀ compoundsꢀ wereꢀ incubatedꢀ plate,ꢀandꢀallowedꢀtoꢀgrowꢀforꢀ8–14ꢀd.ꢀCellsꢀwereꢀthenꢀwashedꢀ
with the cell lines for 24h, 48h and 72h, respectively, at the severalꢀtimesꢀwithꢀPBSꢀandꢀfixedꢀforꢀ10ꢀminꢀinꢀ4%ꢀparaform-
18)
concentrationsꢀofꢀ 1.25ꢀµg/mL,ꢀ 2.5ꢀµg/mL, 5µg/mL, 10µg/mL, aldehydeꢀandꢀstainedꢀwithꢀ0.1%ꢀcrystalꢀvioletꢀforꢀ5ꢀmin. The
0µg/mL. wells were washed with PBS several times, and the colonies
Cell Cycle Analysis A549 cells were seeded into a 6-well formedꢀ wereꢀ countedꢀ subsequently.ꢀ Theꢀ inhibitoryꢀ effectꢀ ofꢀ
2
5
plate (3×10 cells/well) and grown in RPMI 1640 medium the complex on the colony formation of A549 cells was calcu-
supplemented with 10% fetal bovine serum (FBS) at 37°C in a lated as the percentage of visible colony numbers in the drug-
treated groups compared with that in the untreated control
19)
groups.
Tableꢀ 1.ꢀ CrystalꢀDataꢀandꢀStructureꢀRefinementꢀforꢀ[PtL2]
Spectroscopic Studies on DNA Interaction The DNA
bindingꢀ studiesꢀ wereꢀ performedꢀ byꢀ firstꢀ preparingꢀ aꢀ stockꢀ
Empiricalꢀformula
C H Cl N O Pt
20 12 2 2 2
solutionꢀ ofꢀ concentrationꢀ ofꢀ 2.0×10− mol·L by dissolving
3
−1
Formula weight (M)
Crystal system
Space group
aꢀ(Å)
578.31
−3
−1
theꢀcomplexꢀintoꢀDMSO.ꢀTris-bufferꢀsolutionꢀ(5×10 mol·L
Monoclinic
Tris, 50×10− mol·L NaCl, adjusted to pH= ꢀ7 .3ꢀ byꢀ hydro-
chloricꢀ acid)ꢀ wasꢀ preparedꢀ usingꢀ doubleꢀ distilledꢀ water.ꢀ Aꢀ
Tris-buffer solution of ct-DNA gave a ratio of UV absorbance
atꢀ260ꢀnmꢀandꢀ280ꢀnmꢀofꢀ1.85ꢀ:ꢀ1,ꢀindicatingꢀthatꢀtheꢀDNAꢀwasꢀ
3
−1
P2 /n
1
12.0481(8)ꢀÅ
7.9838(4)ꢀÅ
18.6451(9)ꢀÅ
90.00°
bꢀ(Å)
cꢀ(Å)
2
0)
α (°)
sufficientlyꢀfreeꢀfromꢀprotein. The concentration of ct-DNA
stock solution was determined spectrophotometrically as
2×10− M per nucleotide by employing a molar absorption co-
β (°)
106.248(6)°
90.00°
3
γ (°)
3
3
−1
−1
V/(Å )
1721.82(16)Å
4
efficientꢀ(6600ꢀM ·cm )ꢀatꢀ260ꢀnm.ꢀTheꢀct-DNAꢀsolutionꢀwasꢀ
Z
thenꢀ storedꢀ atꢀ 4°Cꢀ withinꢀ oneꢀ weekꢀ beforeꢀ use.ꢀ Inꢀ aꢀ typicalꢀ
F(000)
1096
−4
DNAꢀcompetitiveꢀbindingꢀexperiment,ꢀaꢀsolutionꢀofꢀ1.0×10
M
θ range for data collection (°)
Reflectionsꢀcollectedꢀ/ꢀunique
5.58–52.74°
−5
DNAꢀandꢀ1.0×10 MꢀEBꢀwithꢀaꢀ[DNA]/[EB]ꢀratioꢀofꢀ10ꢀ:ꢀ1ꢀwasꢀ
17112 / 3495 [R(int)=0.0391]
1.067
−3
−1
prepared.ꢀ Smallꢀ aliquotsꢀ ofꢀ 2.0×10 mol·L of the complex
wereꢀ graduallyꢀ addedꢀ intoꢀ theꢀ EB–DNAꢀ solutionꢀ toꢀ increaseꢀ
theꢀ [complex]/[DNA]/[EB]ꢀ ratiosꢀ fromꢀ 1ꢀ:ꢀ10ꢀ:ꢀ1ꢀ toꢀ 10ꢀ:ꢀ10ꢀ:ꢀ1.ꢀ
Fluorescenceꢀ emissionꢀ spectraꢀ wereꢀ recorded.ꢀ Theꢀ excitationꢀ
2
Goodness-of-fitꢀonꢀF
Final R indices [I>2σ(I)]
R indices (all data)
R =0.0240ꢀωR =0.0535
1
2
R =0.0297ꢀωR =0.0562
1
2

March 2014
223
andꢀ emissionꢀ slitꢀ widthsꢀ wereꢀ bothꢀ 10ꢀnm.ꢀ Theꢀ quenchingꢀ Results and Discussion
constant K of the complex was obtained from restricted linear
Synthesis and Characterization The tetradentate Schiff
q
fittingꢀofꢀI /I versus [quencher] plot based on the classic Stern– base ligand (H -L) was synthesized via the condensation
0
2
21)
Volmer equation: I /I=1+K ×[quencher], where I and I are reaction of o-phenylenediamine and 5-chlorosalicylaldehyde
theꢀ peakꢀ emissionꢀ intensitiesꢀ ofꢀ EB–DNAꢀ systemꢀ inꢀ theꢀ ab- (Chart 1), and characterized by elemental analysis, H-NMR,
sence and presence of the complex (the quencher), respective-
0
q
0
1
13
C-NMRꢀspectroscopy.
ly.ꢀ Inꢀ theꢀ fluorescenceꢀ polarizationꢀ experiment,ꢀ theꢀ workingꢀ
The corresponding platinum(II) complex was prepared by
solutionꢀwasꢀpre-incubatedꢀforꢀ40ꢀminꢀbeforeꢀuse.ꢀTheꢀdegreeꢀ the reaction of cis-Pt(DMSO) Cl with H -L in the presence
2
2
2
ofꢀ polarizationꢀ wasꢀ determinedꢀ byꢀ measuringꢀ fluorescenceꢀ ofꢀ ethanolꢀ andꢀ DMSOꢀ underꢀ solvothermalꢀ conditionsꢀ (Chartꢀ
1
intensities parallel and perpendicular with respect to the plane 1), and was characterized by elemental analysis, H-NMR,
13
of linearly polarized excitation light at 595nm under 350nm
C-NMR,ꢀandꢀESI-MSꢀspectroscopyꢀasꢀwellꢀasꢀsingleꢀcrystalꢀ
UVꢀ lightꢀ excitation.ꢀ Theꢀ slitꢀ widthsꢀ wereꢀ setꢀ asꢀ 5ꢀnmꢀ andꢀ X-rayꢀdiffraction.
−
5
−1
5
nm for E and E ,ꢀ respectively.ꢀ 1.0×10 mol·L GelRed
Crystal Structure Description The crystallographic data
x
m
−1
−
5
andꢀ 7.0×10 mol·L ꢀct-DNAꢀwereꢀpreparedꢀinꢀaꢀ2.0ꢀmLꢀTris- andꢀ structureꢀ refinementꢀ detailsꢀ forꢀ theꢀ complexꢀ wereꢀ sum-
−
3
−1
NaClꢀ bufferꢀ solution.ꢀ Smallꢀ aliquotsꢀ ofꢀ 2.0×10 mol·L of marizedꢀ inꢀ Tableꢀ 1.ꢀ Theꢀ selectedꢀ bondꢀ lengthsꢀ (Å)ꢀ andꢀ bondꢀ
the complex were added into the GelRed/ct-DNA solution to anglesꢀ(°)ꢀforꢀtheꢀcomplexꢀwereꢀgivenꢀinꢀtheꢀcaptionꢀofꢀFig.ꢀ1.ꢀ
increaseꢀ [complex]/[DNA]ꢀ ratios.ꢀ Theꢀ fluorescenceꢀ polariza- Asꢀ shownꢀ inꢀ Fig.ꢀ 1,ꢀ theꢀ platinum(II)ꢀ isꢀ four-foldꢀ coordinatedꢀ
tion was measured at about 15min after each addition of the by the Schiff base ligands (H -ClL ) via the imine nitrogen
2
2
complex.ꢀ Allꢀ spectroscopicꢀ analysesꢀ wereꢀ performedꢀ atꢀ roomꢀ atomsꢀ andꢀ theꢀ oxygenꢀ atomꢀ ofꢀ theꢀ hydroxylꢀ groups.ꢀ Theꢀ
temperature.ꢀ Inꢀ aꢀ typicalꢀ DNAꢀ viscosityꢀ measurement,ꢀ ct- platinum atom resides in a square-planar coordination envi-
−
3
−1
DNAꢀwasꢀdissolvedꢀinꢀBPEꢀbufferꢀ(6×10 mol·L Na HPO , ronment.ꢀTheꢀbondꢀanglesꢀatꢀtheꢀplatinumꢀatomꢀ(N1–Pt1–N2,ꢀ
2
4
−3
−1
−3
−1
2
×10 mol·L NaH PO , 1×10 mol·L Na EDTA,ꢀ pH= N1–Pt1–O1,ꢀ N2–Pt1–O2ꢀ andꢀ O1–Pt1–O2)ꢀ wereꢀ inꢀ theꢀ rangeꢀ
2 4 2
3
−
7
.2)ꢀ toꢀ prepareꢀ aꢀ 22ꢀmLꢀ workingꢀ solutionꢀ ofꢀ 1.0×10 M.ꢀ Theꢀ ofꢀ 85.0–95.0°.ꢀ Theꢀ averageꢀ Pt–Nꢀ andꢀ Pt–Oꢀ distancesꢀ wereꢀ
complex was then added with increasing [complex]/[DNA] 1.964ꢀ andꢀ 2.000ꢀÅ,ꢀ respectively,ꢀ resemblingꢀ thoseꢀ reportedꢀ
ratiosꢀrangingꢀfromꢀ0.01ꢀtoꢀ0.09ꢀinꢀeveryꢀ0.01ꢀinterval.ꢀCircu- for platinum(II) complexes bearing the N O ligand donor
2
2
2
6–28)
latingꢀ bathꢀ temperatureꢀ wasꢀ maintainedꢀ atꢀ 35.0± ꢀ0 .5 ° C .ꢀ V i s - set.
cosity values, η (unit: cP) were obtained directly by running
In Vitro Antitumor Activity Assay The in vitro cytotox-
0
#ꢀspindleꢀinꢀworkingꢀsamplesꢀatꢀaꢀrateꢀofꢀ30ꢀrpm.ꢀDataꢀwereꢀ icity of H -L and its complex were evaluated by MTT assay
2
presented as η/η versus [complex]/[DNA] ratio, in which η0 against seven typical human tumor cell lines covering lung,
0
and η are the viscosities of the DNA solutions in the absence breast, liver, bladder, saos and gastric cancers and normal
andꢀ presenceꢀ ofꢀ theꢀ complex,ꢀ respectively.ꢀ Theꢀ influenceꢀ ofꢀ lungꢀfibroblastꢀcells.ꢀTheꢀinhibitoryꢀeffectsꢀofꢀH -L and com-
2
solventꢀ DMSOꢀ uponꢀ theꢀ measuredꢀ viscosityꢀ valuesꢀ wasꢀ cor- plex on the growth of selected tumor cells were determined
rectedꢀbyꢀusingꢀaꢀDMSOꢀviscosityꢀvalueꢀofꢀ2.03ꢀcPꢀmeasuredꢀ after tumor cells being incubated with 20µM of complex and
atꢀ 35.0°C.ꢀ Allꢀ DNAꢀ bindingꢀ experimentsꢀ wereꢀ conductedꢀ atꢀ
2
2)
leastꢀinꢀduplicate.
Agarose Gel Electrophoretic Assay In plasmid DNA
unwindingꢀ experiments,ꢀ DMSOꢀ stockꢀ solutionꢀ ofꢀ theꢀ com-
plex was diluted to the concentrations of 20, 40, 60, 80 and
100 µMꢀ withꢀ TBEꢀ bufferꢀ (TBE:ꢀ Tris-Boricꢀ acid–EDTAꢀ bufferꢀ
solution).ꢀ Theꢀ complexꢀ ofꢀ variousꢀ concentrationsꢀ andꢀ 0.5ꢀµg
DNA were mixed and brought the volume up to 25µL using
TBEꢀbufferꢀsoꢀthatꢀelectrophoresisꢀofꢀeachꢀsampleꢀcanꢀbeꢀre-
peatedꢀtwice.ꢀAllꢀsamplesꢀwereꢀincubatedꢀatꢀ25°Cꢀinꢀdarkꢀforꢀ
4
h, and every 12µL of each sample mixed with 2µL DNA
loading buffer was electrophoresed through a 1% agarose gel
pre-mixedꢀwithꢀGelRed)ꢀimmersedꢀinꢀTBEꢀbufferꢀforꢀ60ꢀminꢀ
(
underꢀ aꢀ 5ꢀV/cmꢀ electronicꢀ field.ꢀ Thenꢀ theꢀ gelꢀ wasꢀ visualizedꢀ
andꢀphotographedꢀusingꢀaꢀBIO-RADꢀimagingꢀsystemꢀunderꢀaꢀ Fig.ꢀ 1.ꢀORTEPꢀViewꢀofꢀComplexꢀ[PtL ] Showing Atom Labeling, Ther-
2
2
3–25)
malꢀEllipsoidsꢀAreꢀDrawnꢀatꢀtheꢀ30%ꢀProbability
UV-Visꢀtransilluminator.
Statistics The data processing included the Student’s t-
test with p≤0.05ꢀtakenꢀasꢀsignificanceꢀlevel,ꢀusingꢀSPSSꢀ13.0.
Selectedꢀbondꢀlengthsꢀ(Å)ꢀandꢀbondꢀanglesꢀ[°]:ꢀPt1–N1ꢀ1.963(3),ꢀPt2–N2ꢀ1.965(3),ꢀ
Pt1–O1ꢀ 2.002(3),ꢀ Pt1–O2ꢀ 1.997(3);ꢀ N1–Pt1–N2ꢀ 83.75(15),ꢀ N1–Pt1–O1ꢀ 96.03(13),ꢀ
N2–Pt1–O2ꢀ 95.61(13),ꢀ O1–Pt1–O2ꢀ 84.63(12),ꢀ N1–Pt1–O2ꢀ 179.06ꢀ (13),ꢀ N2–Pt1–O1ꢀ
179.31ꢀ(12).
Chart 1

2
24
Vol.ꢀ62, No.ꢀ3
Tableꢀ 2.ꢀ InhibitionꢀRatesꢀ(%)ꢀofꢀtheꢀLigandꢀandꢀComplexꢀagainstꢀSixꢀHumanꢀTumourꢀCellsꢀatꢀ20ꢀµM
A549
MDA-MB-231
HepG2
T24
MG63
MGC-803
Ligand
26.7± 3.6
71.6± 2.8
30.5± 2.3
89.8± 4.0
29.4± 3
82.5± 5.12
22.6± 4.5
68.8± 1.88
28.8± 3.92
79.6± 3.64
23.9± 1.22
54.5± 2.21
Complex
Fig.ꢀ 2.ꢀThe IC50 of the Complex against Seven Human Tumor Cell Lines Comparing with Cisplatin
H -Lꢀ forꢀ 72ꢀhꢀ (Tableꢀ 2).ꢀ Asꢀ shownꢀ inꢀ Tableꢀ 2,ꢀ theꢀ complexꢀ the populations of the S and G2 phase decreased only to a
2
exhibited enhanced cytotoxicity to all selected tumor cells certainꢀextentꢀcomparedꢀwithꢀtheꢀcontrolꢀcells.ꢀWeꢀspeculatedꢀ
comparedꢀtoꢀfreeꢀligand.ꢀTheꢀinhibitionꢀofꢀselectedꢀtumorꢀcellsꢀ that the decrease of populations in S and G2 phases was the
by complex were all more than 50%, particularly, the inhibi- directꢀresultꢀofꢀtheꢀpopulationꢀincreaseꢀinꢀG1ꢀphase.ꢀSimulta-
tionꢀofꢀMDA-MB-231ꢀandꢀHepG2ꢀwereꢀasꢀhighꢀasꢀ89.8%ꢀandꢀ neously, 3-dose–response studies indicated that the complex
82.5%,ꢀrespectively.ꢀTheꢀin vitro antitumor activities of com- exhibited a more profound dose–effect relationship, as shown
plexꢀwereꢀfurtherꢀquantifiedꢀbyꢀdeterminingꢀtheꢀcorrespondingꢀ by the G1-phase arrest at doses of 5, 10, 20µMꢀ(Fig.ꢀ3).ꢀHow-
2
9)
IC ꢀ values.ꢀ Asꢀ shownꢀ inꢀ Fig.ꢀ 2,ꢀ theꢀ complexꢀ exhibitedꢀ con- ever,ꢀcisplatinꢀresultedꢀinꢀpopulationꢀincreaseꢀinꢀtheꢀSꢀphase.
5
0
siderable antitumor activities with IC50 values of ca. 11.61ꢀµM.ꢀ Therefore, the mechanism of the suppression of tumor cells’
Moreover, the complex had lower IC50 values against A549, growth by the title platinum(II) complexes might be different
MDA-MB-231,ꢀ HepG2ꢀ andꢀ MG-63ꢀ comparedꢀ toꢀ cisplatin.ꢀ fromꢀthatꢀofꢀcisplatin.
The inhibitory activity of complex against HepG2 cells was
The Complex Induced Apoptosis in A549 Cells The
the strongest, with an IC ꢀ valueꢀ ofꢀ 1.06ꢀµM.ꢀ Meanwhile,ꢀ theꢀ inhibitory effect of the complex on cell proliferation has been
5
0
IC ꢀofꢀcomplexꢀagainstꢀA549ꢀalsoꢀreachedꢀ2.269ꢀµM, approxi- further investigated by complex induced cell apoptosis in
5
0
matelyꢀ12ꢀtimesꢀlowerꢀthanꢀthatꢀofꢀcisplatin.ꢀAlthoughꢀtheꢀIC50 A549.ꢀA549ꢀcellsꢀexposedꢀtoꢀdifferentꢀdosesꢀofꢀcomplexꢀwereꢀ
valuesꢀofꢀcomplexꢀagainstꢀT-24,ꢀMGC-803ꢀandꢀHUVEC,ꢀ i.e., examinedꢀbyꢀFACSꢀwithꢀPIꢀandꢀFITC-AnnexinꢀVꢀstaining.ꢀAsꢀ
5.88,ꢀ11.05ꢀandꢀ11.61 ꢀµ M, respectively, were higher than that of shownꢀinꢀFig.ꢀ4,ꢀtheꢀcomplexꢀtriggeredꢀapoptosisꢀinꢀA549ꢀcellsꢀ
cisplatin, the complex exhibited overall broad-spectrum anti- inꢀaꢀdose-dependentꢀmannerꢀatꢀ24ꢀh.ꢀComparedꢀtoꢀtheꢀcontrolꢀ
tumorꢀactivities.ꢀItꢀshouldꢀbeꢀnotedꢀthatꢀtheꢀplatinum(II)ꢀcom- groups,ꢀ theꢀ omplexꢀ inducedꢀ 3.5%,ꢀ 6.5%ꢀ andꢀ 11.7%ꢀ ofꢀ apo-
plex displayed considerable cytotoxicity against the normal ptotic cells in A549 cells at concentrations of 5, 10 and 20µM
lungꢀ fibroblastꢀ cellꢀ WI-38,ꢀ thereby,ꢀ theꢀ platinum(II)ꢀ complexꢀ atꢀ24ꢀh,ꢀrespectivelyꢀ(Fig.ꢀ4).
exhibited little selectivity between cancer cells and normal
Colony Formation Assayꢀ ꢀT heꢀ influenceꢀ ofꢀ presenceꢀ ofꢀ
cells.
the complex on the proliferation and survival of A549 cells
Cell Cycle Arrest in A549 Cells To elucidate the mecha- wasꢀshownꢀinꢀFig.ꢀ5.ꢀTheꢀcellsꢀwithoutꢀtreatedꢀbyꢀcomplexꢀex-
nisms of inhibition of cell proliferation by complex, the cell hibitedꢀtheꢀgreaterꢀproliferationꢀabilityꢀandꢀsurvivalꢀefficiencyꢀ
cycleꢀ arrestꢀ inꢀ A549ꢀ wasꢀ investigatedꢀ usingꢀ fluorescence- inꢀaꢀ6-wellꢀplate.ꢀTheꢀvisualꢀexaminationꢀindicatedꢀthatꢀthereꢀ
activatedꢀ cellꢀ sortingꢀ (FACS).ꢀ Theꢀ resultsꢀ areꢀ shownꢀ inꢀ Fig.ꢀ were more and larger colonies formed in the control groups
3.ꢀ Treatmentꢀ ofꢀ cellsꢀ withꢀ theꢀ platinumꢀ complexꢀ forꢀ 48ꢀhꢀ en- comparedꢀ toꢀ thoseꢀ formedꢀ inꢀ theꢀ treatedꢀ groups.ꢀ Inꢀ addition,ꢀ
hanced cell-cycle arrest at the G1 phase, resulting in popula- as the complex concentration increased from 10 to 30µmol/L,
tionꢀincreaseꢀinꢀtheꢀG1ꢀphaseꢀ(62.07,ꢀ70.97,ꢀ72.24%)ꢀcomparedꢀ theꢀnumberꢀofꢀtheꢀcoloniesꢀformedꢀdecreasedꢀobviously.ꢀTheseꢀ
withꢀtheꢀcontrolꢀcellsꢀ(43.27%)ꢀatꢀdifferentꢀconcentrations,ꢀandꢀ results further supported that the complex could effectively

March 2014
225
Fig.ꢀ 3.ꢀTheꢀEffectꢀofꢀComplexꢀonꢀCellꢀCycleꢀArrestꢀinꢀA549ꢀCellsꢀinꢀThreeꢀDoses
Fig.ꢀ 4.ꢀTheꢀEffectsꢀofꢀComplexꢀonꢀApoptosisꢀinꢀA549ꢀCellsꢀinꢀThreeꢀDoses

2
26
Vol.ꢀ62, No.ꢀ3
Fig.ꢀ 5.ꢀColonyꢀFormationꢀAssayꢀWasꢀPerformedꢀUsingꢀHighlyꢀInvasiveꢀ
A549 Cells Treated Continuously with Complex at 10µmol/L, 20µmol/L,
30µmol/L for 7d
Fig.ꢀ 6.ꢀFluorescenceꢀ Emissionꢀ Spectraꢀ ofꢀ EBꢀ Boundꢀ withꢀ ct-DNAꢀ
−4
−5
(
[DNA]=2.0×10 M,ꢀ [EB]=2.0×10 M) in the Absence (Dashed Line
)
and Presence (Solid Lines—) of Complex with [Complex]/[GelRed] Ra-
tios Ranging from 1:1 to 10:1
inhibitꢀcellꢀproliferationꢀandꢀreduceꢀtheꢀviabilityꢀofꢀA549ꢀcells.
DNA Binding Properties Although there is some evi-
dence to suggest that other biological targets, including RNA
or proteins, may be important in cisplatin binding, it is gener-
Inset:ꢀlinearꢀfittingꢀforꢀquenchingꢀconstantꢀK ꢀbasedꢀonꢀStern–Volmerꢀequation.
q
3
0)
allyꢀacceptedꢀthatꢀDNAꢀisꢀtheꢀprimaryꢀtarget. Similarly, the
interactions between small molecules and DNA are primar-
ilyꢀ responsibleꢀ forꢀ antitumorꢀ activity.ꢀ Theꢀ DNAꢀ replicationꢀ
in tumor cells can be blocked via the intercalations of small
31)
moleculesꢀ betweenꢀ twoꢀ consecutiveꢀ baseꢀ pairsꢀ ofꢀ DNA.
Generally, the active compounds possess an approximately
planar structure as well as some hydrophobic characteristics to
maximizeꢀ intercalations.ꢀ Theꢀ DNAꢀ bindingꢀ propertiesꢀ ofꢀ theꢀ
complex have been studied using several analytical methods,
includingꢀ theꢀ fluorescenceꢀ spectroscopy,ꢀ fluorescenceꢀ polar-
ization, DNA viscosity measurements as well as agarose gel
electrophoresis.
Fluorescence Emission Titration The ability of the
complex binding to ct-DNA was primarily investigated by
fluorescenceꢀ spectroscopyꢀ usingꢀ ethidiumꢀ bromideꢀ (EB)ꢀ asꢀ aꢀ
competitiveꢀDNAꢀbindingꢀprobe.ꢀEBꢀisꢀaꢀclassicalꢀfluorescentꢀ
probe that binding to ct-DNA viaꢀanꢀintercalationꢀmode.
Inꢀ theꢀ competitiveꢀ bindingꢀ experiments,ꢀ theꢀ EB–ct-DNAꢀ
32)
Fig.ꢀ 7.ꢀ Fluorescenceꢀ Polarizationꢀ Analysisꢀ ofꢀ GelRedꢀ asꢀ Intercalativeꢀ
Binding Probe Bound with ct-DNA under the Addition of Complex with
Increasing Concentrations
solutionꢀ ([EB]/[DNA]=1:10) exhibited peak emission at
5
[
DNA]=7.0×10− M, [GelRed]=1.0×10−5 M, the complex was added with increas-
597ꢀnmꢀ uponꢀ 350ꢀnmꢀ UVꢀ lightꢀ excitation,ꢀ indicatingꢀ thatꢀ EBꢀ
−5
ingꢀconcentrationsꢀrangingꢀfromꢀ2.0ꢀtoꢀ10.0×10 M.
molecules intercalated between two adjacent base pairs of ct-
DNAꢀ andꢀ sufficientlyꢀ protectedꢀ fromꢀ fluorescenceꢀ quenchingꢀ molecules in aqueous solution exhibit a weak polarization in
byꢀ polarꢀ solventꢀ molecules.ꢀ Theꢀ emissionꢀ spectraꢀ ofꢀ EB–ct- fluorescenceꢀ emissionꢀ dueꢀ toꢀ theirꢀ rapidꢀ rotationalꢀ diffusion.ꢀ
DNAꢀsystemꢀinꢀtheꢀpresenceꢀofꢀcomplexꢀareꢀshownꢀinꢀFig.ꢀ6.ꢀ However, the rotational diffusion would become restricted
Withꢀ theꢀ [complex]/[DNA]/[EB]ꢀ ratiosꢀ rangingꢀ fromꢀ 1ꢀ:ꢀ10ꢀ:ꢀ1ꢀ when the small molecules intercalate between two consecutive
toꢀ 10ꢀ:ꢀ10ꢀ:ꢀ1,ꢀ theꢀ peakꢀ fluorescenceꢀ intensityꢀ ofꢀ EBꢀ graduallyꢀ baseꢀpairsꢀofꢀDNA,ꢀandꢀconsequently,ꢀtheꢀfluorescenceꢀpolar-
decreasedꢀfromꢀ210.5ꢀtoꢀ141.6,ꢀwithꢀaꢀtotalꢀquenchingꢀpercent- izationꢀ degreeꢀ wouldꢀ increaseꢀ significantly.ꢀ Inꢀ contrast,ꢀ otherꢀ
ageꢀofꢀ32.7%.ꢀTheꢀresultsꢀindicateꢀthatꢀthereꢀexistꢀcompetitiveꢀ external interactions such as groove binding and electrostatic
DNAꢀ bindingꢀ betweenꢀ complexꢀ andꢀ EBꢀ asꢀ aꢀ consequenceꢀ ofꢀ interaction, however, do not exhibit the similar effect, i.e.,
2
6,27)
theirꢀ similarꢀ intercalativeꢀ bindingꢀ modesꢀ toꢀ DNA.
Fur- theyꢀdoꢀnotꢀcauseꢀanꢀobviousꢀchangeꢀinꢀtheꢀfluorescenceꢀpolar-
33)
thermore, the quenching constant, K , for complex, was quan- ization. In this work, GelRed was chosen as the intercalative
q
21)
titativelyꢀ evaluatedꢀ basedꢀ onꢀ theꢀ Stern–Volmerꢀ equation.
bindingꢀprobeꢀforꢀfluorescenceꢀpolarizationꢀanalysis.ꢀAsꢀshownꢀ
3
–1
The K ꢀvalueꢀforꢀcomplexꢀwasꢀfoundꢀtoꢀbeꢀ5.49×10 L·mol , inꢀFig.ꢀ7,ꢀtheꢀpolarizationꢀdegreeꢀofꢀGelRedꢀboundꢀtoꢀct-DNAꢀ
q
indicating a relatively weak intercalative binding ability of the wasꢀ measuredꢀ toꢀ beꢀ 212ꢀ atꢀ aꢀ [DNA]/[GelRed]ꢀ ratioꢀ ofꢀ 7ꢀ:ꢀ1.ꢀ
complexꢀcomparedꢀtoꢀtheꢀclassicꢀintercalativeꢀagentꢀEB.
After the addition of the complex into the GelRed/ct-DNA
Fluorescence Polarization Analysis Fluorescence polar- binaryꢀsystem,ꢀsignificantꢀdecreasesꢀonꢀtheꢀpolarizationꢀdegreeꢀ
ization analysis is a valuable tool for studying the interactions ofꢀGelRedꢀwereꢀobserved.ꢀAsꢀ[complex]/[DNA]/[GelRed]ꢀratioꢀ
between small molecules and DNA, by which the convin- gradually increased from 2:7:1 to 10:7:1, the polarization
15)
cibleꢀ informationꢀ ofꢀ bindingꢀ modeꢀ canꢀ beꢀ inferred. Small degreeꢀofꢀGelRedꢀdecreasedꢀbyꢀ13.0%,ꢀi.e.,ꢀfromꢀ212ꢀtoꢀ184.5.ꢀ

March 2014
227
These results provide additional evidence for the existence of
competitive intercalative binding to ct-DNA between GelRed
and complex, in which the complex may partly displace the
GelRed molecules intercalating between two consecutive
base pairs of DNA and consequently some GelRed molecules
would be forced back into the aqueous solution, leading to a
decreaseꢀinꢀitsꢀfluorescenceꢀpolarization.
DNA Viscosity Measurementsꢀ ꢀW henꢀ smallꢀ planarꢀ mol-
ecules intercalate into two adjacent base pairs of DNA, DNA
double helix is relaxed to accommodate the intercalation,
resultingꢀ inꢀ theꢀ lengtheningꢀ ofꢀ DNAꢀ helix.ꢀ Theꢀ viscosityꢀ ofꢀ
DNA solution is very sensitive to the length of DNA, and the
increased viscosity of DNA solution is generally considered to
be associated with an intercalative binding mode, while other Fig.ꢀ 8.ꢀRelative Viscosity Increments of ct-DNA Solution Bound with
interaction modes, such as groove binding and electrostatic at- Complexꢀ withꢀ Increasingꢀ [Complex]/[DNA]ꢀ Ratiosꢀ inꢀ theꢀ 0.01ꢀ toꢀ 0.09ꢀ
Range
traction, will not induce obvious increase on viscosity of DNA
solutions.
3
4)
EBꢀasꢀclassicꢀintercalativeꢀagentꢀwasꢀsetꢀforꢀcomparison.
The changes on the viscosity of ct-DNA solution in the
presenceꢀ ofꢀ theꢀ complexꢀ wereꢀ shownꢀ inꢀ Fig.ꢀ 8,ꢀ inꢀ whichꢀ EBꢀ
was employed as the intercalative binding indicator for com-
parison.ꢀ Theꢀ additionꢀ ofꢀ theꢀ complexꢀ resultedꢀ inꢀ anꢀ obviousꢀ
increaseꢀ inꢀ theꢀ viscosityꢀ ofꢀ ct-DNAꢀ solution.ꢀ Asꢀ theꢀ [com-
plex]/[DNA]ꢀratiosꢀgraduallyꢀincreasedꢀfromꢀ0.01ꢀtoꢀ0.09,ꢀtheꢀ
viscosityꢀofꢀDNAꢀsolutionꢀincreasedꢀbyꢀ12.3%,ꢀwhichꢀfurtherꢀ
confirmedꢀ theꢀ intercalativeꢀ bindingꢀ modeꢀ ofꢀ theꢀ complexꢀ toꢀ
ct-DNA.ꢀInꢀcontrast,ꢀEBꢀinducedꢀaꢀtotalꢀ19.7%ꢀincreaseꢀinꢀtheꢀ
viscosityꢀofꢀDNAꢀsolutionꢀunderꢀtheꢀsameꢀcondition.ꢀTheseꢀre-
sultsꢀdemonstrateꢀthatꢀEBꢀexhibitsꢀhigherꢀintercalativeꢀbindingꢀ
35)
abilityꢀtoꢀDNAꢀthanꢀtheꢀcomplex. This is consistent with the
resultsꢀofꢀtheꢀaboveꢀspectroscopicꢀanalyses.
Electrophoretic Gel Shift Assay The interaction mode
of the complex to pUC19 plasmid DNA was examined by
agaroseꢀgelꢀelectrophoresisꢀassay.ꢀpUCꢀ19ꢀDNAꢀaloneꢀconsistsꢀ
Fig.ꢀ 9.ꢀElectrophoreticꢀ Mobilityꢀ Shiftꢀ Assayꢀ ofꢀ Plasmidꢀ pUC19ꢀ DNAꢀ
Treated with the Complex
3
of three forms of DNA with different electrophoretic mobili- (5.49×10 ),ꢀ andꢀ couldꢀ intercalateꢀ intoꢀ DNA.ꢀ Theꢀ intercala-
ties, including supercoiled DNA (Form I), open-circled DNA tiveꢀ bindingꢀ ofꢀ complexꢀ toꢀ DNAꢀ hadꢀ beenꢀ furtherꢀ confirmedꢀ
(
Form II) and linear DNA (Form III) possessing the highest, byꢀ theꢀ resultsꢀ ofꢀ fluorescenceꢀ polarizationꢀ analysis,ꢀ viscosityꢀ
theꢀ lowestꢀ andꢀ moderateꢀ mobility,ꢀ respectively.ꢀ Asꢀ indicatedꢀ measurementsꢀandꢀagaroseꢀgelꢀelectrophoresisꢀassay.
inꢀ laneꢀ 1ꢀ ofꢀ Fig.ꢀ 9,ꢀ pUC19ꢀ DNAꢀ aloneꢀ showedꢀ predominantꢀ
fractionꢀofꢀformꢀIꢀDNA.ꢀHowever,ꢀinꢀtheꢀpresenceꢀofꢀcomplexꢀ Concluding Remarks
at various concentrations of 20, 40, 60, 80 and 100µM, each
A mononuclear planar platinum(II) complex of Schiff base
plasmid DNA sample exhibited obvious variation in electro- ligand that derived from o-phenylenediamine and 5-chloro-
phoreticꢀ mobility,ꢀ asꢀ indicatedꢀ inꢀ lanesꢀ 2–6.ꢀ Particularly,ꢀ theꢀ salicylaldehydeꢀ wasꢀ synthesizedꢀ andꢀ fullyꢀ characterized.ꢀ Theꢀ
supercoiled form DNA (Form I) exhibited an obvious lower complex exhibited potent in vitro cytotoxicities against six
electrophoretic mobility in the presence of the complex at tested human tumor cell lines with IC50 values of ca. 11.61ꢀµM.ꢀ
lower concentrations of 20, 40 and 60µM.ꢀ Itꢀ wasꢀ consistentꢀ The complex induced G1-phase arrest and apoptosis in A549
withꢀtheꢀresultsꢀofꢀpUC19ꢀDNAꢀinꢀtheꢀpresenceꢀofꢀEB,ꢀfurtherꢀ cellsꢀ inꢀ aꢀ dose-dependentꢀ manner.ꢀ Theꢀ bindingꢀ propertiesꢀ ofꢀ
confirmingꢀ thatꢀ theꢀ complexꢀ bindꢀ intercalativelyꢀ toꢀ DNAꢀ asꢀ theꢀ complexꢀ toꢀ DNAꢀ wereꢀ investigatedꢀ byꢀ severalꢀ methods.ꢀ
3
6-37)
EBꢀdoes.
However, because the mobility of Form I DNA The results demonstrate that the most probable binding mode
wasꢀ reducedꢀ moreꢀ significantlyꢀ inꢀ theꢀ presenceꢀ ofꢀ EB,ꢀ theꢀ ofꢀtheꢀcomplexꢀwasꢀintercalation.
intercalative binding strength of the complex to DNA may
beꢀrelativelyꢀweakerꢀcomparedꢀtoꢀthatꢀofꢀEB.ꢀAtꢀaꢀhigherꢀtheꢀ
Supplementary Material Crystallographic data for the
complex concentration of 80µM, the form I DNA was con- structural analysis have been deposited with the Cambridge
verted to its open-circular form (the form II DNA), indicating CrystallographicꢀDataꢀCentre,ꢀCCDCꢀNo.ꢀ897133ꢀforꢀtheꢀcom-
theꢀ cleavageꢀ ofꢀ aꢀ singleꢀ DNAꢀ chain.ꢀ Nevertheless,ꢀ atꢀ aꢀ stillꢀ plex.ꢀTheꢀdataꢀcanꢀbeꢀobtainedꢀfreeꢀofꢀchargeꢀviaꢀhttp://www.
higher complex concentration of 100µM, no obvious DNA seg- ccdc.cam.ac.uk,ꢀorꢀfromꢀtheꢀCambridgeꢀCrystallographicꢀDataꢀ
ments can be detected, which is probably due to the intensive Centre,ꢀ12ꢀUnionꢀRoad,ꢀCambridgeꢀCB21EZ,ꢀUK;ꢀfax:ꢀ(+44)
DNAꢀcleavageꢀbyꢀtheꢀcomplex.ꢀTheseꢀresultsꢀindicatedꢀthatꢀtheꢀ 1223–336–033;ꢀorꢀe-mail:deposit@ccdc.cam.ac.uk.
complex may possess the potent DNA cleavage ability at the
38)
concentrations higher than 80µM.
Acknowledgments This work was supported by Natural
Inꢀ summary,ꢀ theꢀ resultsꢀ ofꢀ fluorescenceꢀ emissionꢀ titrationꢀ Scienceꢀ Foundationꢀ ofꢀ Chinaꢀ (No.ꢀ 21271051),ꢀ Nationalꢀ Basicꢀ
indicated that the complex had a low quenching constant Kq Researchꢀ Programꢀ ofꢀ Chinaꢀ (No.ꢀ 2012CB723501),ꢀ IRT1225ꢀ

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