ORIGINAL ARTICLE

Synthesis, Spectral Properties and DFT Calculationsof new Ruthenium (II) Polypyridyl Complexes; DNA BindingAffinity and in Vitro Cytotoxicity Activity

Rajender Reddy Mallepally1 & Nagamani Chintakuntla1 & Venkat Reddy Putta1 &

Nagasuryaprasad K2& Ravi Kumar Vuradi1 & Madhuri P2

& Satyanarayana Singh S2&

Ramesh Kumar Chitumalla3 & Joonkyung Jang3 & Nagababu Penumaka4,5 &

Satyanarayana Sirasani1

Received: 10 January 2017 /Accepted: 4 April 2017# Springer Science+Business Media New York 2017

Abstract In this paper a novel ligand debip (2–(4–N,N–d i e t h y l b e n z e n am i n e ) 1H – i m i d a z o [ 4 , 5 – f ] [ 1 ,10]phenanthroline) and its Ru(II) polypyridyl complexes[Ru(L)2(debip)]

2+, (L = phen (1), bpy (2) and dmb (3)) havebeen synthesized and characterized by spectroscopic tech-niques. The DNA binding studies for all these complexes wereexamined by absorption, emission, quenching studies, viscositymeasurements and cyclic voltammetry. The light switchingproperties of complexes 1–3 have been evaluated. Moleculardocking, Density Functional Theory (DFT) and time dependentDFT calculations were performed. The Ru(II) complexes ex-hibited efficient photocleavage activity against pBR322 DNAupon irradiation and exhibited good antimicrobial activity. Alsoinvestigated 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) reduction assay, lactate dehydrogenase(LDH) release assay and reactive oxygen species (ROS) againstselected cancer cell lines (HeLa, PC3, Lancap, MCF-7 andMD-MBA 231).

Keywords Ru(II) polypyridyl complexes . Computationalchemistry . DNA binding . Photocleavage . Antimicrobialactivity

Introduction

Cancer is a highly heterogeneous disease characterized by con-tinuous uncontrolled growth and expansion of abnormal cells[1]. The drugs used to combat cancer belong to one of twobroad categories. The first is cytotoxic (cell killing)drugs and the second is cytostatic (Inhibits cell growth). Anumber of researchers introduced and explored many metalcomplexes as cytotoxic and cytostatic agents such asPlatinum, Ruthenium, and Rhodium etc. One of the rapidlygrowing scenarios of cancer therapy is the use of metal-complexes as anti cancer potential candidates. After the ser-endipitous discovery of antitumor and anti-metastatic proper-ties of Cisplatin, intensive studies have been performed oncytotoxic compounds with more acceptable toxicity profiles[2]. However, Cisplatin possesses inherent drawbacks such asserious side effects, general toxicity, and acquired drug resis-tance [3]. In order to overcome the challenges put forth byplatinum drugs, researchers in the area of medicinal inorganicchemistry are developing non-platinum based chemothera-peutic agents that exhibit enhanced selectivity and non-covalent DNA binding interaction modes (intercalation,groove binding, and external electrostatic binding) [4, 5].

Ruthenium complexes are regarded as the most promisingalternatives to Cisplatin as anticancer drugs, due to their in-triguing structural features and potential applications in vari-ous fields [6]. These complexes offer the potential for reducedtoxicity and can be tolerated in vivo. The various oxidation

* Satyanarayana Sirasanissnsirasani@gmail.com

1 Department of Chemistry, Osmania University,Hyderabad, Telangana State 500007, India

2 Department of Biochemistry, Osmania University,Hyderabad, Telangana State 500007, India

3 Department of Nanoenergy Engineering, Pusan National University,Busan 609-735, Republic of Korea

4 Inorganic & Physical Chemistry Division, CSIR−IICT, Tarnaka,Hyderabad, Telangana 500007, India

5 CSIR-NEERI Kolkata Zonal Laboratory, 1-8, Sector C, East Kolkata,Area Development Projecct, P.O. East Kolkata, Township,Kolkata 700107, India

J FluorescDOI 10.1007/s10895-017-2091-5

states, different mechanism of action, and the ligand substitu-tion kinetics of ruthenium compounds give them advantagesover platinum-based complexes, thereby making them suit-able for use in biological applications [7]. The biological ac-tivity of Ru(II) complexes has been investigated since theearly 1950’s when Dwyer and coworkers have reported theirantibacterial activity. Since then several Ru(II) complexeshave been developed as potential anti-cancer agents. In par-ticular, their DNA binding affinity and photo-triggered DNAdamage have presented them as potential cellular imaging andtherapeutic agents [8–15].

A recent study has been explained the DNA-binding affin-ity by computational calculations with density functional the-ory (DFT). It is shown that the energy of these complexes’frontier molecular orbital, i.e. the highest occupied molecularorbital (HOMO) and the lowest unoccupied molecular orbital(LUMO) are varied and when they intercalating in the DNAbase pairs, the energy of the transition conformation will dif-ferent. According to the frontier molecular theory, an electronwill transfer more easily from a high HOMO to a lowerLUMO, and resulting those complex have the lowest LUMO(in generally, the HOMO of DNA is higher than that of ruthe-nium complexes) will bind to DNA the strongest [16–18].

Some Ru(II) polypyridyl complexes show a cytotoxic po-tency similar to or even better than Cisplatin [19]. The advan-tages of using such Ru(II) polypyridyl complexes as cellulartargeting agents lies in the fact that the structural nature of thepolypyridyl units, solubility, lipophilicity, charge, and theirphotophysical properties. Recently, Ru(II) complexes devel-oped by us and others [20–25].

Herein, we report the synthesis and characterizationof a new ligand debip (2–(4–N,N–diethylbenzenamine)1H–imidazo[4,5–f] [1, 10]phenanthroline) and its three complexes1–3. Moreover, we describe the interaction of the Ru(II)polypyridyl complexes with CT–DNA investigated by absorp-tion, emission, quenching studies, viscosity measurements andlight switch Bon and off^ effect. Molecular Docking, DFT andtime dependent DFT (TDDFT) calculations were performed forthe complexes 1–3. The results were compared with the exper-imental photophysical and electrochemical data, antimicrobialactivity and photocleavage of pBR322DNAof complexes 1–3,also investigated cytotoxicity, apoptosis and cell cycle arrestagainst selected cancer cell lines by flow cytometry. All theseexperiments showed that the complexes 1–3 exhibit efficientactivity in a dose-dependent manner [26–29].

Experimental

Materials

All reagents and solvents were purchased commercially andused without further purification unless otherwise noted.

RuCl3, 1,10–phenanthroline monohydrate, 2,2′–bipypridine,4 ,4 ′–dimethyl 2 ,2 ′ –bipyr id ine , CT–DNA, N,N–Diethylaminobenzaldehyde, Dimethyl sulfoxide (DMSO)and RPMI 1640 were purchased from Sigma Aldrich.Supercoiled pBR322 DNA was obtained from BangaloreGenie. Cancer cell lines (HeLa, PC3, Lancap, MCF-7 andMD-MBA 231) were purchased from NCCS Pune. Doublydistilled water used for preparing various buffers. The DNAhad a ratio of UVabsorbance at 260 and 280 nm of 1.8–1.9:1,indicating that the DNA was sufficiently free from protein[30]. DNA concentration per nucleotide was determined byusing a molar absorption coefficient [6600 M−1cm−1] at260 nm [31].

Analytical Measurements

IR spectra were recorded by means of KBr discs with aShimadzu (IRAffinity-1) Fourier transform, UV–Visible spec-tra were recorded on Bio–spectrophotometer, model Elico BL198, Fluorescence spectra were recorded with spectrofluo-rometer, model Jasco FP-8500 and NMR spectra weremeasured on a Bruker Z–Gradient 400 MHz spectropho-tomete r us ing DMSO–d6 as the so lven t , andtetramethylsilane as the internal standard at room tem-perature, Cyclic voltmeter was carried out on WonATechmultichannel potentiostat/galvanostat (WMPG1000,Gyeonggido, Korea), Viscosity experiments were carried outon Ostwald Viscometer. Electrospray ionization mass spec-trometry (ESI–MS) was recorded on an LQC system(Finnigan MAT, USA) using CH3CN as the mobile phase.All DFT and TDDFT simulations of the three Ru(II) com-plexes were carried out using Gaussian 09 (Revision B.01)chemical program. A flow cytometer (Guava EasyCyte™(Millipore)) was used to study apoptotic-inducing activitiesand cell cycle analysis.

Synthesis and Characterization of Ligand debipand Complexes 1–3

Synthesis of Ligand

The novel ligand (debip) was synthesized [32, 33], withm i x t u r e o f p h e n - d i o n e ( 2 . 5 0 mM ) , N , N –Diethylaminobenzaldehyde (3.50 mM), ammonium acetate(50.0 mM) and glacial acetic acid (15 ml) was refluxed for4 h. Above solution was cooled to room temperature anddiluted with water, dropwise addition of conc. NH3 gave ayellow precipitate, which was collected washed with H2Oand dried. The crude product recrystallized with C5H5N.H2O and dried (yield: 75%). Anal. data for C23H21N5: calc.C, 75.20; H, 5.72; N,19.07; found: C, 75.12; H, 5.61; N,19.06. ESI–MS Calc: 367; found: 366.

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Synthesis of [Ru(phen)2(debip)](ClO4)2. 2H2O (1)

A mixture of cis- [Ru(phen)2Cl2]2H2O (0.5 mM), debip(0.5 mM) and ethanol (15 mL) was refluxed for 8 h underN2 atmosphere. When the light purple color solution is obtain-ed, it was cooled to room temperature and an equal volume ofsaturated aqueous NaClO4 solution was added under vigorousstirring. The red solid was collected and washed with smallamounts of water, ethanol and diethyl ether, then dried undervacuum (yield: 72%). Anal. data for RuC47H41N9Cl2O12: cal.C,48.49; H, 3.52; N, 10.83; found: C,48.46; H, 3.51; N, 10.81.ESI-MS calc: 1063; found: 1064. IR (KBr, cm−1): 1605(C =N), 1525 (C = C), 1288 (C–C), 3074 (C–H), 622 (Ru–N).

Synthesis of [Ru(bpy)2(debip)](ClO4)2. 2H2O (2)

This complex was synthesized as described above complex 1by taking a mixture of cis- [Ru(bpy)2Cl2]2H2O (0.5 mM),d eb i p ( 0 . 5 mM) (y i e l d : 71%) . Ana l . d a t a f o rRuC43H41N9Cl2O10: calc. C, 46.27; H, 3.67; N, 11.30; found:C, 46.25; H, 3.65; N, 11.29. ESI–MS calc: 1015; found: 1016.IR (KBr, cm−1): 1606 (C = N), 1473 (C = C), 1277 (C–C),3081 (C–H), 620 (Ru–N).

Synthesis of [Ru(dmb)2(debip)] (ClO4)2. 2H2O (3)

This complex was synthesized as described above complex 1by taking a mixture of cis- [Ru(dmb)2Cl2]2H2O (0.5 mM),d eb i p ( 0 . 5 mM) (y i e l d : 70%) . Ana l . d a t a f o rRuC47H49N9Cl2O10 calc. C, 48.16; H, 4.18; N, 10.76; found:C, 48.15; H, 4.16; N, 10.74. ESI–MS calc: 1071; found: 1072.IR (KBr, cm−1): 1611 (C = N), 1555 (C = C), 1273 (C–C),3082 (C–H), 622 (Ru–N).

Synthetic route of above debip ligand and its Ru(II) com-plexes 1-3 shown in Scheme 1 and NMR data of Ru(II) com-plexes depicted in Table 1.

Computational Studies

DFT Studies

All DFT and TDDFT simulations of the three Ru(II) com-plexes were carried out using Gaussian 09 (Revision B.01)ab initio quantum chemical program [34]. Ground state geom-etry optimization and the vibrational frequency analysis of thecomplexes were performed by employing a hybrid Becke [35,36], three-parameter, Lee–Yang–Parr, exchange-correlationfunctional (B3LYP) [37].We used 6-31G (d,p) basis functionsfor the H, C, and N atoms. In addition, a Bdouble-ξ^ qualitybasis set consisting of Hay and Wadt’s effective core poten-tials (LanL2DZ ECP) [38–40] was used for the Ru atom. Nosymmetry constraints were imposed during the geometry

optimizations. Vibrational frequency analysis was carried outto confirm that each configuration is indeed a local minimumon the multivariate potential energy surface. The contour plotsfor the frontier molecular orbitals are generated with the GaussView visualization program [41]. On the basis of the opti-mized structures of the electronic ground state (S0),the UV-vis absorption spectra of the three Ru[II] complexeswere obtained by using the TDDFT formalism. To mimic theexperimental conditions, we performed the TDDFT calcula-tions in dimethyl sulfoxide solution. The absorption propertiesof the three complexes in dimethyl sulfoxide solvent werecalculated in association with the polarizable continuum mod-el [42, 43]. as implemented in Gaussian 09. The TDDFT cal-culations were performed by using the B3LYP functional andthe mixed basis set as described above (vide supra). This levelof theory has been shown to give reliable results for structural,electronic and optical properties of Ru complexes [44].

Molecular Docking Studies

Accelrys Discovery Studio (version 2.5) was used in the presentstudy to design lead molecules and estimate the docking interac-tions of complex of drug and protein binding, the number ofbonds formed by ligand with the target. For Receptor ligandinteraction (Docking), LibDock [45] module in DiscoveryStudio is used in the present study, LibDock is a high-throughput algorithm for docking ligands into an active bindingsite on the receptor, which is also a site-features docking algo-rithm. The crystal structure of human DNA topoisomerase1(TOP1) receptor was downloaded from RCSB PDB (PDB ID-1T8I), after downloading the pdb format of the protein; all thewatermolecules of the proteinwere removed by usingDiscoveryStudio and stabilizing the charges, filling the missing residuesand generation of the side chains, according to the parametersavailable. After preparation of protein, CHARMm force fieldwas applied with before running LibDock and the active sitewithin the protein was identified. The receptor may have manyactive sites but the one of the interest should be selected.Ruthenium complexes were sketched using tools Chemsketch,then applied CHARMm force field andwas used to dock into thetarget binding site. The resulting poses with higher LibDockscore were investigated and the interacting ligand target complexwas examined. The scoring functions have been used to estimateligand-binding affinity to screen out active and inactive com-pounds during the process of virtual screening [46].

DNA-Binding and Photocleavage Studies of Ru(II)Complexes

The DNA-binding experiments were performed at room tem-perature. Tris buffer (5 mM tris-(hydroxymethyl)aminomethane(tris)hydrochloride, 50 mM NaCl, pH 7.0) wasused for absorption, luminescence experiments and viscosity

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measurements. TAE buffer (Tris-acetate-EDTA buffer, pH 8.2)was used for DNA photocleavage experiments.

The absorption experiment of the Ru(II) complex in tris buff-er was performed using a fixed concentration (20 μM) of com-plex to which increments of the DNA stock solution were added.The mixture of Ru-DNA solutions was allowed to incubate for5–10 min before the absorption spectra were recorded. The in-trinsic binding constant (Kb) measured by monitoring the chang-es in absorption of the MLCT band of complex with increasingconcentration of DNA using the following equation [47].

where, [DNA] is the concentration of DNA, Ɛa, Ɛf and Ɛbcorresponds to the apparent absorption coefficient Aobsd/[Ru], the extinction coefficient for the free complex, and theextinction coefficient for the complex in the fully bound form,respectively. In plots of [DNA]/(Ɛa–Ɛf) vs [DNA], Kb is givenby the ratio of the slope to the intercept.

In the emission experiment fixed metal complex concentra-tion (10 μM) was taken and to this varying concentration (10–100 μM) of DNAwas added. The excitation wavelength was

fixed and the emission range adjusted before measurements.The fraction of the ligand bound calculated from the relationCb = Ct[(F–F0)/Fmax–F0)], where Ct is the total complex con-centration, F is the observed fluorescence emission intensity ata given DNA concentration, F0 is the intensity in the absence ofDNA and Fmax is the fully bound DNA to complex. Bindingconstant (Kb) was obtained from amodified Scatchard equationplot of r/Cf vs r, where r is the Cb/[DNA] and Cf is the concen-tration of free complex [48].

Emission quenching experiments were carried out at roomtemperature by using [Fe(CN)6]

4− as a quencher in Tris–HClbuffer. The Stern–Volmer quenching constant KSV, can bedetermined by using Stern–Volmer equation I0/I = 1 +KSV[Q], where I0 and I are the emission intensities in theabsence and presence of quencher [Fe(CN)6]

4−, KSV is thelinear Stern–Volmer constant and [Q] is the quencher concen-tration. The molecular light-switch studies of Ru(II) com-plexes were performed by using equimolar concentrations(0.01 M) of CoCl2 and EDTA [49, 50].

Viscosity measurements were carried out on OstwaldViscometer, immersed in a thermostated water bath maintainedat 30 ± 0.1 °C. DNA samples approximately 200 base pairs in

Synthetic route of debip ligand

N

N

O

O

4 hrs Reflux

Glacial CH 3COOH

CH 3COONH 4

1,10- Phenanthroline - 5,6 -dione

N, N Diethyl benzaene amine imidazophenanthroline [debip]

NCH 2

H 2C

CO

HCH 3

CH 3

N

N N

NN

CH2

H H2C CH3

CH3

Synthetic route of complexes 1, 2 and 3.

Cis-[Ru (phen) 2Cl2]2+

Cis-[Ru (dmb) 2Cl2]2+

Cis-[Ru (bpy) 2Cl2]2+

Ethanol+ water8 hrs reflux

Ethanol+ water8 hrs reflux

Ethanol+ water8 hrs reflux

debip

N

N N

NN

CH2

H H2C CH3

CH3

Ru

N

N

N

N

H2C

CH2

H

N

CH3

CH3

N

N

NN

2+

1.

Ru

N

N

N

N

H2C

CH2

H

NN

N

NN

CH3

CH32+

Ru

N

N

N

N

H2C

CH2

H

NN

N

NN

CH3

H3C

CH3

H3C

CH3

CH32+

2.

3.

Scheme 1 Synthetic route ofdebip ligand and itscomplexes 1–3

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average length were prepared by sonication in order to mini-mize the complexities arising from DNA flexibility [51]. Flowtime was measured with a digital stopwatch, and each samplemeasured three times, then an average flow time calculated.Data were presented as (η/η0)

1/3 vs [Ru(II)]/[DNA], where ηis the viscosity of DNA in the presence of the complex, and η0is the viscosity of DNA alone. Viscosity values calculated fromthe observed flow time of DNA-containing solutions (t > 100 s)corrected for the flow time of the buffer alone (t0) [30].

Cyclic voltammogram were recorded by using standardthree electrode cell containing a platinum foil as working elec-trode, platinum wire as counter electrode and saturated calo-mel electrode (SCE) as reference electrode, 50 mg of Ru(II)complexes 1–3 dissolved in 100 ml of acetonitrile containing1 M Et4NBF4 at 10 mg/s scan rate [52].

The antibacterial tests were performed by the standard discdiffusion method [53]. All the Ru(II) complexes werescreened for antibacterial activity against standard microor-ganisms such as Staphylococcus aureus (S.aureus) andEscherichia coli (E.coli). The Mueller Hinton agar was pre-pared and poured into sterile Petri plates and allowed to dry,and inoculate 0.2 mL of bacterial culture which has 106 CFU/

mL concentrations. Filter paper discs approximately 5 mm indiameter were placed in the previously prepared agar plates.The complexes 1–3 (40 µg/mL) in DMSO were applied onpaper disc with the help of a micropipette and the agar plateswere then incubated at 37 °C. After 24 h of incubation, eachplate was examined. The resulting zones of inhibition wereuniformly circular with a confluent lawn of growth. The di-ameters of the zones of complete inhibition were measured (inmm), including the diameter of the disc where the DMSOwasused as the negative control.

For the gel electrophoresis assay, supercoiled pBR322DNA was treated with Ru(II) complexes (1–3) (20, 40and 80 μM) in TAE buffer and the samples were irra-diated with a UV lamp (365 nm) for 30 min. A loadingbuffer bromophenol blue (6×) was added and the sam-ples were analyzed by 0.8% agarose gel electrophoresisat 50 V for 2 h. The gel was stained with 2 μL (1 μg/mL) ethidium bromide is a fluorescent dye and it inter-calates between bases of nucleic acids and provides anopportunity to detect nucleic acid fragments in gels, andthen photographed by illumination with UV light using Geldoc system [54].

Fig. 1 Optimized singletgeometries of the complexes1–3 obtained at B3LYP/6-31G(d,p)/LanL2DZ level oftheory. Hydrogens are omittedfor the sake of clarity

Table 1 The 1H and 13C[1H] NMR data of 1–3 complexes

Complex 1H NMR data (400 MHz, DMSO-d6, TMS) 13C[1H] NMR data (100 MHz, DMSO-d6,Major peaks)

1 δ 9.2 (d, 6H), 8.6 (d, 6H), 8.2 (s, 4H), 8.0 (t, 6H), 7.7 (d, 2H),6.8(d, 2H), 3.2 (q, 2H, −CH2), 1.2 (t, 3H, N-CH3).

156.3, 151.2, 150.6, 142.4, 136.6, 130.1, 122.3,113.6, 47.5(N-CH2), 20.1(N-CH3).

2 δ 9.1 (d, 2H), 8.8(d, 4H), 8.7 (d, 4H), 8.2 (d, 2H), 7.9 (t, 4H),7.0(t, 4H), 6.6 (d, 2H) 6.3 (d, 2H), 3.2 (q, 2H, N- CH2), 1.1(t, 3H, N-CH3).

157.4, 150.2, 137.4, 136.5, 127.6, 126.3, 124.6,121.4, 114.5.47.2(N-CH2),19.9(N-CH3)

3 δ 9. 1 (s, 4H d), 8.8 (d, 4H), 8.2 (d, 2H), 7.9 (t, 4H), 7.52 (t, 2H),7.3(t, 4H), 6.8 (d, 2H) 6.7 (d, 2H), 3.3 (q, 4H, N-CH2),1.3 (t, 6H, N-CH3), 2.5(s, 12H, −CH3) .

157.9, 150.6, 149.8147.6, 128.5, 120.4,114.7,47.0(N-CH2), 26.4 (CH3), 19.8(N-CH3)

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In Vitro Studies

In Vitro Cytotoxicity Assay

The cytotoxicity in vitro assay for ruthenium complexeswas assessed used by standard 3-(4,5-Dimethylthiazole-2-yl)-2,5-Diphenyltetraazolium bromide (MTT) assay Cellswere placed in 96-well microassay culture plates (8 × 103

cells per well) in 200 μL and were grown overnight at37 °C in a 5% CO2 incubator. Control wells were

prepared by addition of culture medium (200 μL). Wellscontaining culture medium without cells were used as anegative control and Cisplatin was used as a positive con-trol. DMSO was used as the vehicle control. A stocksolution of Cisplatin (10 mM in DMSO) was freshly pre-pared for every experiment. After 48 h, 20 μL of MTTsolution [5 mg/mL in phosphate buffered saline (PBS)]was added to each well and the plates were wrapped inaluminum foil and incubated for 4 h at 37 °C. The purpleformazan product was dissolved by addition of 100 μL of

Fig. 2 The electron densitycontours of frontier molecularorbitals of the complexes 1–3,obtained from DFT calculations

Table 2 Simulated absorptionwavelengths (λcal), oscillatorstrengths (f) and coefficient ofconfiguration interaction (CI)with the dominant contribution toeach transition for complexes 1, 2and 3. H and L denote HOMOand LUMO, respectively

Complex Transitions λcal(nm) f CI Coefficient Dominant Contribution

1 S0➔S1 532 0.1193 0.5224 H ➔ L (55%)

S0➔S2 506 0.0215 0.5245 H ➔ L + 2 (55%)

S0➔S3 498 0.0018 0.6998 H ➔ L + 1 (98%)

S0➔S4 474 0.0049 0.6999 H ➔ L + 3 (98%)

S0➔S5 468 0.0045 0.7026 H ➔ L + 4 (99%)

S0➔S6 451 0.3158 0.6880 H ➔ L + 5 (95%)

2 S0➔S1 536 0.1111 0.5375 H ➔ L (58%)

S0➔S2 513 0.0252 0.5374 H ➔ L + 2 (58%)

S0➔S3 507 0.0027 0.7024 H ➔ L + 1 (99%)

S0➔S4 459 0.0005 0.6736 H-1➔ L + 1 (91%)

S0➔S5 458 0.0019 0.6300 H-1 ➔ L (79%)

S0➔S6 452 0. 3225 0.6878 H ➔ L + 3 (95%)

3 S0➔S1 526 0.1394 0.5662 H ➔ L (64%)

S0➔S2 493 0.0061 0.5446 H ➔ L + 2 (59%)

S0➔S3 486 0.0051 0.6762 H ➔ L + 1 (91%)

S0➔S4 458 0.0063 0.5934 H-1➔ L + 2 (70%)

S0➔S5 458 0.0010 0.4904 H-1 ➔ L (48%)

S0➔S6 457 0.0017 0.4597 H-1➔ L + 1 (42%)

S0➔S7 448 0.3490 0.6895 H ➔ L + 3 (95%)

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100% DMSO to each well. The absorbance was moni-tored at 620 nm using a 96-well plate reader. The stocksolutions of the metal complexes were prepared inDMSO, and in all experiments the percentage of DMSOwas maintained in the range of 0.1–2%. DMSO by itselfwas found to be nontoxic to the cells up to a concentra-tion of 2%. Data were collected for three replicates eachto obtain the mean values. The IC50 values were deter-mined by plotting the percentage viability vs concentra-tion on a logarithmic graph and reading the concentrationat which 50% of cells remained viable relative to thecontrol [55].

Lactate Dehydrogenase (LDH) Release Assay

The LDH is a cytoplasmic enzyme retained by viable cellswith intact plasma membranes, but released when there isdamage. The LDH catalyzes the conversion of lactate to py-ruvate upon reduction of NAD+to NADH/H+; the added tet-razolium salt is then reduced to formazan. All the three com-plexes were processed under identical conditions and LDHassay was performed according to the manufacturer’s instruc-tions (Biovision, K 311–400, California, USA). Absorbancewas determined in the multimode reader using 96 well platereaders at 492 nm [56, 57].

Detection of Reactive Oxygen Species

The assay was performed in a similar way to previously pub-lished protocol briefly; the cells were treated with 10 μMDCFH-DA at 37 °C for 30 min, and the formation of ROS(fluorescence) was quantitated with excitation at 495 nm andemission at 538 nm. All values were corrected for the autofluo-rescence and fluorescent intensities were normalized by theamount of total protein in each well. The data has been comput-ed from three independent experiments carried out in duplicates.

Apoptosis Assay

To assess the apoptotic activity of the Ru(II) complexes, HeLaCells 1 × 106 cells per well were cultured and treated withRu(II) complexes at their IC50 dose for 24 h and centrifugedat 4 °C. The cells were then washed in 1 mL of Ice cold PBS(phosphate buffered saline) twice for 2 min. The pellets wereresuspended in 500 μL of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) binding buffer and washed at4 °C for 5 min. Resuspended the cells in 70 μL of HEPESbinding buffer and add 5 μL of Annexin V, incubate for15 min at room temperature in the dark. After incubation indark at room temp analysis was done by flow cytometer.Apoptosis cells appear green, and morphological changes

Fig. 3 Simulated UV-visible absorption spectra of the three Ruthenium complexes (B1,B2 and B3) obtained at B3LYP/6-31G(d,p)/LanL2DZ level oftheory. The spectra are broadened by using the Gaussian convolution with FWHM = 1000 cm-1

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such as cell blebbing and formation of apoptotic bodies will beobserved [58].

Cell Cycle Analysis

Acridine Orange (AO) is a nucleic acid selective metachro-matic stain useful for cell cycle determination. Adjust cellsuspension at a concentration of 1 × 106 cells/mL in PBS.Add Buffer I (20 mM citrate-Phosphate, 1 mM EDTA, 0.2sucrose, 0.1% Triton X-100) 0.5 mL at 40 °C, agitate to sus-pend and incubate for 10 min. Add Buffer II (10 mM Citrate-Phosphate, 0.1 M NaCl) with AO 0.5 mL at 40 °C, agitate tosuspend. The ruthenium complexes were read and analyzedby a flow cytometer [59–61].

Results and Discussion

Computational Chemistry

DFT Studies

The DFT/TDDFT calculations were performed for complexes1, 2 and 3 to gain insight into their structural, electronic, elec-trochemical and photophysical properties. All the complexes

have pseudo-octahedral coordination geometry around the Rumetal center. The optimized ground state geometries of thestudied Ru(II)complexes are depicted in Fig. 1.The importantgeometrical parameters around the metal center are given inTable 2. The bond length between the nitrogen of ancillaryligands (phen, bpy and dmb) and Ru metal ranges from 2.113to 2.127 Å. For all the three complexes the correspondingbond distances are almost similar and the bond angles areca. 78°. The reported geometrical parameters (bond lengthsand bond angles) are not significantly varied from complex 1–3. The Kohn–Sham orbitals of the complexes 1, 2, and 3 aredisplayed in Fig. 2. From the electron density distributionplots, the HOMO of 1, 2, and 3 are mainly localized on liganddebip, which also extended to imidazol and benzene rings.The LUMO are localized over the ancillary ligands (phen,bpy and dmb) and metal center. Interestingly, the HOMO ei-genvalue for all the three complexes is found be similar andwhich is −5.17 eV. The similar eigenvalue of HOMO may bedue to the presence of identical ligand. On the other hand, theLUMO energies of Ru(II) complexes 1, 2, and 3 are −2.47,−2.51, and −2.42 eV, respectively. The increased LUMO en-ergy for the complex 3 is due to the substitution of electrondonating methyl groups over two bipyridine rings. The

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Fig. 5 The electronic absorption spectrum of complex 1 in Tris bufferupon addition of CT-DNA. The arrow shows the hypochromism uponincreasing CT–DNA concentration. Plots of [DNA]/(Ɛa − Ɛf) vs [DNA]for the titration of DNAwith Ru(II) complexes

Fig. 4 Receptor-ligand interactions [Ru(phen)2debip]2+ compound with

human DNA topoisomerase I

Table 3 The LibDock score and docking interactions of the ruthenium complexes (1–3) with human DNATOP 1

Complex Lib Dock score k.cal/mol Interacting residues Interacting atoms

1 150.721 DC112, DA113, DT10, ASN722, DG12,ASP533, THR718, TGP11

complex:N9–D:DA113:H61complex:H65–B:DT10:H73complex:C25–D:DC112:N4

2 138.692 DA113, ARG364, DG12, DT110, LEU721,THR718, ASN722, ASP533

C:DG12:O6 – complex:H60complex:H65–D:DT110:H73A:LEU721:HD21–complex:C2

3 126.360 DT10, DA113, DG12, THR718, ASN722,ARG364, ASP533, TGP11

complex:N9 –D:DA113:H61complex:H65 –B:DT10:H73

J Fluoresc

calculated HOMO-LUMO gaps of the complexes 1, 2, and 3are 2.70, 2.66, and 2.75 eV, respectively. Due to the negligibledifference in HLG, considerable shifts in the absorption spec-tra cannot be expected in the transition from complexes.

The UV-visible absorption spectra of the complexes weresimulated in dimethyl sulfoxide solution and are depicted inFig. 3. It can be seen from the Fig. 3 that the TDDFTsimulationsreproduced themain bands that were observed in the experimen-tal UV-visible spectrum. The most intense singlet transition inthe low-energy region of the three Ru(II) complexes locatedaround 450 nm. The calculated excitation wavelengths withthe largest oscillator strengths and coefficient of configurationinteraction together with the dominant contribution to each tran-sition were tabulated in Table 2. From the TDDFT data, it can beobserved that the substitution of methyl groups on bipyridineligand have hardly any influence on the absorption behavior.

Molecular Docking Studies

Molecular docking is used to predict the structure of theintermolecular complex formed between two molecules.

Docking small molecules into larger macromolecules isa complex and difficult task. In this article, we per-formed detailed molecular docking to further explorethe molecular factors that determine the binding affinityand binding energy of Ru(II) complexes 1, 2, and 3 withhuman DNA TOP1. DNA TOP1 is over-expressed intumor cells and is an important target in cancer chemo-therapy. The ruthenium complexes 1, 2, and 3 weredocked into the active site pocket of TOP1, usingLibDock module in Discovery Studio. Docking scoreof the complexes was tabulated in Table 3. Amongthem, complex 1 shown the highest dock score of150.721 kcal/mol (Fig. 4). The active site pocket resi-dues interacted with ruthenium complexes, included ami-no acids of protein and DNA nucleic acids. Therefore,these residues play an important role in substrate bind-ing and consequently metabolic activity towards the ru-thenium complexes. A higher score indicates a strongerreceptor-ligand binding affinity. The docking result re-vealed that the receptor-ligand complex was stabilizedby hydrophobic interactions.

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Fig. 6 The fluorescence spectra of complexes [Ru(phen)2debip]2+ (1),

[Ru(bpy)2debip]2+ (2) and [Ru(dmb)2debip]

2+ (3) with addition of CT–DNA, in Tris buffer with increasing concentration of CT-DNA. The arrow

shows the fluorescence intensity change upon increase of DNA concentra-tion. Inset: Scatchard plot of r/Cf vs r

J Fluoresc

DNA-Binding Studies

Electronic Absorption Spectra

The Electronic absorption spectra of complexes 1, 2 and 3mainly consist of three resolved bands in the range 200–600 nm. The lowest energy bands at 448 nm for complex 1,456 nm for complex 2 and 450 nm for complex 3 are assignedto the metal-to-ligand charge transfer (MLCT) transition,

whereas the bands at the UV region attributed intra ligand(IL) π → π* transitions. For metallo-intercalators, DNA-binding is associated with hypochromism and a red shift inthe MLCT band. In Fig. 5 shows the absorption spectra ofcomplexes 1, 2 and 3 in the presence of increasing concentra-tion of DNA. As increasing the concentration of CT–DNA, atthe MLCT band of complexes 1, 2, and 3 exhibitedhypochromism of 25.2%, 22.6%, and 20.8%, respectively.To further elucidate the binding strength of the Ru(II)

Fig. 8 DNA light switch on andoff experiment of complex 1showing the luminescencechanges where A is luminescenceof complex alone, B isluminescence of complexwith DNA, C is upon addition ofCo2+ to B (Switch off) and D isupon addition of EDTA to C(Switch on)

I 0/I

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Fig. 7 Emission quenchingof complexes 1–3 with[Fe(CN)6]

4 − in the absence ofDNA a, presence of DNA1:30 b and 1:200 c. [Ru] =10 μM,[Fe(CN)6]

4− = 0.1 M

J Fluoresc

complexes, the intrinsic binding constants Kb were deter-mined by monitoring the changes of absorbance in theMLCT band. The values of Kb for complexes 1, 2, and 3 weredetermined 1.81 × 105, 1.64 × 105, and 1.52 × 105 M−1, re-spectively. These spectral characteristics obviously suggestthat the complexes 1, 2, and 3 have interacted with DNAstrongly through a mode that involves a stacking interactionbetween the aromatic chromophore and the base pairs ofDNA.

Luminescence Spectroscopic Studies

The luminescence titration of Ru(II) complexes (1–3) was car-ried out in Tris buffer at room temperature. As shown in Fig. 6,an increase in emission intensity was observed for these com-plexes as increasing the concentration of CT–DNA. The emis-sion intensities of complexes 1, 2, and 3 increased by about1.90, 1.85, and 1.76 times larger than those in the absence ofDNA. The emission intensity enhancement of Ru(II) com-plexes is indicative that the binding of complexes to the hydro-phobic pocket of DNA, since the hydrophobic environmentinside the DNA helix reduces the accessibility of water mole-cules to the complex. Therefore, the mobility of complex isrestricted at the binding site, leading to decrease of the vibra-tional modes of relaxation. The binding constants were calcu-lated 3.1 × 105, 2.0 × 105, and 1.9 × 105 M−1 forcomplexes 1, 2, and 3, respectively. By comparing with bind-ing constant obtained from absorption spectra, although thebinding constant obtained from luminescence titration may

vary. This difference between the two sets of binding con-stants should be caused by the different spectroscopy anddifferent calculation methods.

Emission Quenching Studies

Steady-state emission quenching experiments of complexes1–3 were carried out by using [Fe(CN)6]

4− as a quencher toprovide further information about complexes binding toDNA. In the absence of DNA, the emission of complexeswas efficiently quenched, while in presence of DNA thequenching was small, because the highly negatively charged[Fe(CN)6]

4− would be repelled by the negative charge of theDNA phosphate backbone due to hinder the emission of theDNA-bound complexes. In the quenching plot of I0/I vs [Q],the slope is the KSV (Fig. 7). From Quenching studies, it isclear that the DNA binding ability of complexes follows theorder 1 > 2 > 3. Such a trend is consistent with the observationin electronic absorption titrations, as well as emissiontitrations.

Light Switching Bon and off^ Behavior of Ru(II) Complexes

Light switch on and off behavior of Ru(II) complexes carriedout in the presence of CT–DNA. The luminescence intensityof complexes could be tuned by the introduction of Co2+ ionsfollowed by EDTA. When the complex binds to DNA (switchon), the emission intensity is high, while the emission of theDNA-bound complex is quenched by Co2+ (0.02 mM), thusturning the light switch off. When EDTA is added to the buffersystem containing Co2+-DNA the emission intensity of the

Table 4 Cyclic voltammetry ofRu(II) complexes alone andcomplexes DNAwith CT-DNA

Complexes Complexes Alone Complexes with CT-DNA

Anodic (Epa) Cathodic (Epc) Anodic (Epa) Cathodic (Epc)

1 0.22 V − 0.01 V 0.27 V −0.08 V2 0.20 V −0.03 V 0.26 V −0.07 V3 0.19 V −0.02 0.21 V −0.04 V

Table 5 The antimicrobial activity of complexes 1–3 against S. aureusand E. coli

Compound Bacterial zone of inhibition (mm)

S. aureus E. coli

DMSO – –

Ampicillin 13.2 12.8

1 8.5 7.8

2 7.6 7.2

3 7.1 6.80.02 0.04 0.06 0.08 0.10 0.12

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Fig. 9 Effect of increasing amount of Ethidium bromide a,[Ru(phen)2debip]

2+b, [Ru(bpy)2debip]2+c and [Ru(dmb)2debip]

2+d onrelative viscosity of CT-DNA at 30 ± 0.1 °C

J Fluoresc

complex is recovered again (light switch on) as shown inFig. 8. This indicates that the heterometalic complex (Co2+–complex 1) becomes free again due to the formation ofEDTA–Co2+ strong complex. Similar results obtained for re-maining complexes 2, and 3. The change in luminescence ofthe DNA–bound complex in the presence of Co2+ and EDTAreveals its use in the modulation of drug therapy.

Viscosity Measurements

Viscosity measurements give further clarification about theinteraction between the Ru(II) complex and DNA. Opticaland photophysical probes provide necessary, but not suffi-cient, clues to support the change in the length of DNA uponbinding (i.e. viscosity and sedimentation) are regarded as theleast ambiguous and the most critical tests of a binding modelin solution in the absence of crystallographic study. It is pop-ularly accepted that a partial and/or non-classical intercalationof ligand could bend (or kink) the DNA helix, reduces itseffective length and, concomitantly, its viscosity. A classicalintercalation of a ligand into DNA is known to cause a signif-icant increase in the viscosity of a DNA solution due to anincrease in the separation of the base pairs at the intercalation

site and, hence, an increase in the overall DNA molecularlength. Fig. 9 shows the changes in the relative viscosity ofCT–DNA on the addition of complexes 1, 2, and 3. Uponincreasing the amounts of complexes 1, 2, and 3 the relativeviscosity of CT–DNA solution increases steadily. These re-sults indicate that the complexes 1, 2, and 3 intercalates be-tween the base pair of DNA.

Cyclic Voltammetry Studies

Cyclic voltammetry (CV) is a very useful electroanalytical tech-nique. Many inorganic compounds contain elements that maytake on several different oxidation states. The CV experimentcan provide important information about interaction and theoxidation state of an element in a compound. The cyclic volt-ammetry of Ru(II) complexes (1, 2, and 3) carried out withoutCT–DNA (complex alone) and with CT–DNA, the anodic peakcurrent difference (ΔIpa) was observed in three complexes, itimplies three Ru(II) complexes strongly interacted with CT–DNA, results were shown in Table 4. These results indicate thatcomplexes 1–3 bind to DNA strongly and effectively.

Antibacterial Studies

The antibacterial activity of Ru(II) complexes (1, 2, and 3)was studied against E coli and S. aureus. In these experimentsDMSO used as a negative control and Ampicillin as a positive

Table 6 IC50 values ofcomplexes 1, 2, 3 and Cisplatintoward the selected cell lines

Complex IC50(μM)

HeLa PC3 LanCap Mcf-7 MD-MBA 231

1 32.50 ± 1.2 60.39 ± 1.1 67.17 ± 1.8 60.09 ± 2.4 64.04 ± 2.1

2 41.30 ± 1.5 75.57 ± 1.6 75.7 ± 1.5 76.7 ± 1.8 74.29 ± 1.4

3 49.17 ± 1.4 76.17 ± 1.7 76.1 ± 1.3 76.5 ± 2.1 77.93 ± 2.4

Cisplatin 13.5 ± 1.7 14.2 ± 1.2 14.5 ± 2.5 13.8 ± 2.4 14.7 ± 2.8

Fig. 11 Cell viability of HeLa cell lines in vitro treatment with complexes1–3Fig. 10 a Photocleavage studies of pBR322 DNA, in the absence and

presence of complexes 1–3 after irradiation at 365 nm with UV light for30 min. Control lane plasmid DNA (untreated pBR322), three sets ofconcentrations with 20, 40 and 80 μM, respectively. b. Photoactivatedcleavage of pBR322 DNA in the presence of complex 1 (20 μM) andscavengers (10 μM) after irradiation at 365 nm for 30 min

J Fluoresc

control. Inhibition zone data from Table 5 indicate that threecomplexes showed considerable activity against E.coli andS.aureus. The data further indicated that complexes 1–3 wereeffective against E.coli and S.aureus, but showed less activitywhen compared with Ampicillin as positive control, andDMSO showed a negligible activity. The complex 1 showedthe highest activity 8.5 mm against S.aureus and 7.8 mm to-wards E.coli, whereas the complexes 2 and 3 showed lessactivity against these bacteria. It is evident from our resultsthat all three metal complexes possess antibacterial activity.

Photo-Activated Cleavage of pBR 322DNA

Many ruthenium complexes can cleave DNA under irradia-tion by UV/Vis light. When circular plasmid DNA is subjectto electrophoresis, relatively fast migration will be observedfor the intact supercoiled form (form I). If scission occurs onone strand, the supercoiled will relax to generate a slower-moving open circular form (form II). If both strands arecleaved, a linear form (Form III) will be generated that mi-grates between supercoiled form and nicked circular form.The cleavage of plasmid DNA can be monitored by agarosegel electrophoresis by using different concentrations (20, 40,and 80 μM) of Ru(II) complexes 1, 2, and 3. As shown inFig. 10 (a), three complexes are able to cleave pBR322 DNA.No obvious DNA cleavage was observed for the control inwhich metal complex was absent (DNA alone). With

increasing the concentration of ruthenium complexes, theForm I decrease and Form II increase gradually. Under thesame experimental condition, complex 1 exhibits more effec-tive DNA cleavage activity than complexes 2, and 3. Thedifferent cleaving efficiency may be ascribed to the differentbinding affinity of three Ru(II) complexes to DNA.

To establish the reactive species responsible for thephotoactivated cleavage of the plasmid DNA, the influenceof different potentially inhibiting agents was investigated.The DNA cleavage of the plasmid by complexes 1, 2, and 3was not inhibited in the presence of hydroxyl radical (OH˙)scavengers such as mannitol and DMSO which indicated thathydroxyl radical was not likely to be the cleaving agent.Whereas cleavage plasmid DNAwas inhibited in the presenceof the singlet oxygen (1O2) scavenger Histidine suggestingthat 1O2 is likely to be the reactive species responsible forthe cleavage of plasmid DNA (Fig. 10b).

In Vitro Cytotoxicity Assay

The cytotoxic activity the Ru(II) complexes toward HeLa, PC3,LanCap,Mcf-7, andMD-MBA231 cell lines were investigatedin comparison with the widely used drug Cisplatin was used asa positive control using the MTT assay. The above cells wereexposed to the different concentrations of the ligand Debip andRu(II) complexes for 48 h, the IC50 values of the complexes arelisted in Table 6. Ligand debip is unexpectedly found to display

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J Fluoresc

no cytotoxic activity towards the selected cell lines. Comparingthe IC50 values of complexes 1, 2, 3, and Cisplatin, these com-plexes display lower cytotoxic activities than Cisplatin underidentical conditions as shown in Fig. 11.The Complex 1 sensi-tive to HeLa cells and complex 3 shows the least cell killingability against MD-MBA 231 cells. Complex 2 shows moder-ate cytotoxic activity on the selected cell lines. Comparing theIC50 values of debip and its complexes, the cytotoxic activity isenhanced when the ligand bonded to metal to form Ru(II) com-plexes. Because complexes 1–3 exhibit relative high cytotoxicactivity against HeLa cells, this cell line was used for furtherinvestigation on apoptosis, reactive oxygen species and cellcycle arrest analysis.

Lactate Dehydrogenase (LDH) Activity

The release of the lactate dehydrogenase (LDH) enzymeas a biomarker suggests the loss of membrane integrity,apoptosis, or necrosis. Although treatment of cell linewith Ru(II) complexes at concentrations of 10 μM didnot significantly increase LDH release, indicating thatthe treatment with Ru(II) complexes maintains the integ-rity of plasma membrane in HeLa, PC-3, LaNCap,MCF-7 and MD-MBA231 cells, but when Ru(II) com-plexes concentration increased to >10 μM the LDH ac-tivity in the culture media increased significantly as il-lustrated in Fig. 12.

Fig. 15 Effect of rutheniumcomplexes 1, 2 and 3, andcontrols (−ve control and +vecontrol(cisplatin)) on themechanism of HeLa cell death(apoptosis) evaluated by flow cy-tometry after 24 h of incubation

CisplatinNegativecontrol

Complex-1 Complex-2 Complex-3

Fig. 14 HeLa cells were treatedwith Cisplatin and complexes 1, 2and 3 for 24 h, and then analyzedfor apoptosis by flow cytometry

J Fluoresc

Reactive Oxygen Species Detection

The generation of reactive oxygen species (ROS) plays a crit-ical role in the activation of mitochondrial-initiated eventsleading to apoptosis. The ROS scavenging antioxidant systemcan be disrupted by an increased level of intracellular ROS. Inour experiment, there is strong evidence that ROS are in-volved in the induction of cell apoptosis [62]. To characterizethe effect of the Ru(II) complexes on ROS level of HeLa cells,2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) wasused as the fluorescence probe. DCFH-DA is a a cell-perme-able, non-fluorescent compound that can be cleaved by intra-cellular esterase and then oxidized to a fluorescent derivative,namely dichlorofluorescein (DCF) [63]. As shown in Fig. 13in the control, no obvious point of fluorescence was found.After the treatment of HeLa, PC-3, LaNCap, MCF-7 andMD-

MBA231 cells with Ru(II) complexes 1, 2, and 3 green fluo-rescence points were observed, indicating that these com-plexes can enhance intracellular ROS levels. This result wasfurther confirmed with an automatic micro, plate reader.Ru(II)-treated cells showed a significant concentration-dependent increase in the fluorescence intensity.

Induction of Apoptosis in HeLa Cells by the Complexes

The design of chemotherapeutic drugs in order to understandthe complexities of apoptosis evolved by cancer cells and thedevelopment of strategies to selectively induce apoptosis incancer cells have turned into a unique target in cancer drugdevelopment. Annexin V is a recombinant phosphatidylserine-binding protein that interacts strongly and specifically withphosphatidylserine residues and can be used for the detection

Fig. 17 Bars represent thepercentage of cells present in eachof the cell cycle stages: G0/G1, Sand G2/M. HeLa cells are treatedby cisplatin and rutheniumcomplexes 1, 2 and 3 at theirIC50 value for 24 h

coun

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Fig. 16 The cell cycledistribution of untreated cells(Negative control), cisplatin andruthenium complexes 1–3 onHeLa cells after incubation for24 h at their IC50 value

J Fluoresc

of apoptosis. The percentage of the Annexin V positive(apoptosis) cells were determined and investigated by flow cy-tometry, after treatment with complexes 1–3 at their IC50 valuefor a period of 24 h. The percentage of Annexin V positive cells42.02%, 23.09%, and 17.2% for complexes 1, 2, and 3 respec-tively. DNA distribution histograms of HeLa cells in the ab-sence and presence of complexes 1, 2, and 3 after 24 h incuba-tion shows in Fig. 14. The percentage of Annexin V positivecells was compared with Cisplatin (positive control),and there was a clear correlation of these complexes with theactivity of Cisplatin as shown in Fig. 15. All these morpho-logical changes indicate that the complexes can effectivelyinduce apoptosis in HeLa cells.

Cell Cycle Arrest by Flow Cytometry

The effect of ruthenium complexes 1, 2, and 3 on cell cycle ofHeLa cells was investigated by flow cytometry in AO(Acridine Orange) stained cells for 24 h. AO is a nucleic acidselective metachromatic stain useful for cell cycle determina-tion it may distinguish between quiescent, activated, and pro-liferating cells, and may also allow differential detection ofmultiple G1 compartments. Figure 16 showed that the cellcycle distribution after treating HeLa cells with complexes 1,2, and 3 at their IC50 value for 24 h. As shown in Fig. 17, thepercentage of cells at the G0/G1 phase increased from 12.3%in the control to 30.7%, 31.2%, and 30.8% after treatmentwith ruthenium complexes 1, 2, and 3 respectively. Theseresults indicate that complexes 1, 2, and 3 induce the cell cyclearrest in G0/G1 phase.

Conclusions

Ru(II) polypyridyl complexes 1, 2, and 3 have been synthe-sized and characterized by NMR, IR and ESI-MS. From theDFTstudies rendered electro chemical and photophysical prop-erties of Ru(II) polypyridyl complexes. The DNA–bindingproperties with CT–DNA were investigated by spectroscopictitrations, and viscosity measurements. The results indicate thatthe three complexes can intercalate into DNA base pairs. In thecyclic voltammogram of Ru(II) polypyridyl complexes, thecathodic peak current decreased gradually with the addition ofDNA, the decreases in the peak currents are ascribed to thestronger binding between the complex and DNA. Moleculardocking studies were also used to explore the binding affinitiesof ruthenium complexes, these results were in good agreementwith the experimental results. Three complexes can effectivelycleave plasmid DNA, and exhibit good antimicrobial activityagainst E-coli (Gram negative) and S aurous (Gram positive).Cytotoxicity assay in vitro showed that the three rutheniumcomplexes have antitumor activity but less than that of positivecontrol Cisplatin. The complex 1 appeared to have higher

cytotoxicity than complexes 2 and 3 against selected cell lines.The Ru(II)-treated cells showed significant concentration-dependent increase in the fluorescence intensity. The level ofROS generation induced by complex 3 is higher than by com-plex 1. The apoptosis studies showed that the three complexescan effectively induce apoptosis of HeLa cells. The flow cy-tometry analysis of the treatment of HeLa cells with three ru-thenium complexes indicates the induction of G0/G1 phase ar-rest. These results will help to design new cytotoxic rutheniumcomplexes and DNA binding agents.

Acknowledgements We are grateful to DBT New Delhi and UGC NewDelhi for financial support, and CFRD, Osmania University, Hyderabad-07. The authors gratefully acknowledge Korea Research Fellowship pro-gram funded by the Ministry of Science, ICT and Future Planning throughthe National Research Foundation of Korea (2016H1D3A1936765) forperforming the DFT studies.

Compliance with Ethical Standards

Conflict of Interest The authors declared that the article content has noconflicts of interest.

References

1. Blanpain C (2013) Tracing the cellular origin of cancer. Nat CellBiol 15:126–134

2. Galanski M, Jakupec MA, Keppler BK (2005) Update of the pre-clinical situation of anticancer platinum complexes: novel designstrategies and innovative analytical approaches. Curr Med Chem12:2075–2094

3. Li W, Han BJ, Yao JH, Jiang GB, Liu YJ (2015) Cytotoxicityin vitro, cell migration and apoptotic mechanism studies inducedby ruthenium(II) complexes. RSC Adv 5:24534–24543

4. Zhang CX, Lippard SJ (2003) New metal complexes as potentialtherapeutics. Curr Opin Chem Biol 7:481–489

5. Ronconi L, Sadler PJ (2007) Insights into the Acid–Base propertiesof PtIV–Diazidodiam(m)inedihyroxido complexes from multinu-clear NMR spectroscopy. Coord Chem Rev 251:1633–1648

6. Han BJ, Jiang GB, Wang J (2015) Ruthenium(II) polypyridyl com-plexes: synthesis, cytotoxicity in vitro, reactive oxygen species,mitochondrial membrane potential and cell cycle arrest studies.Transit Met Chem 40:153–160

7. El-ajaily MM, Abdullah HA, Al-janga A, Saad EE, and MaihubAA (2015) Zr(IV), la(III), and Ce(IV) chelates with 2-[(4-[(Z)-1-(2-Hydroxyphenyl)ethylidene]aminobutyl)- ethanimidoyl]phenol:synthesis, Spectroscopic Characterization, and AntimicrobialStudies Advances in Chemistry 15

8. Niyazi H, Hall JP, O'Sullivan K, Winter G, Sorensen T, Kelly JM,Cardin CJ (2012) Crystalstructures of lambda-[Ru(phen)(2)dppz](2+) with oligonucleotides containing TA/TA and AT/ATsteps show twointercalation modes. Nat Chem 4:621–628

9. Nonat AM, Quinn SJ, Gunnlaugsson T (2009) Mixed f-d coordi-nation complexes as dual visible- and near-infrared-emitting probesfor targeting DNA. Inorg Chem 48:4646–4648

10. Ryan J, Quinn S, Gunnlaugsson T (2008) Highly effective DNAPhotocleavage by novel "rigid" Ru(bpy)3-4-nitro- and −4-amino-1,8-naphthalimide conjugates. Inorg Chem 47:401–403

11. Zeglis BM, Pierre VC, Barton JK (2007) Metallo-intercalators andmetallo-insertors. Chem Commun (Camb) pp 4565–4579

J Fluoresc

12. Moucheron C, De Mesmaeker AK, Kelly JM (1997) Photoreactionsof ruthenium(II) and osmium(II) complexes with deoxyribonucleicacid (DNA). J Photochem Photobiol B 40:91–106

13. Erkkila KE, Odom DT, Barton JK (1999) Recognition and reactionof metallointercalators with DNA. Chem Rev 99:2777–2795

14. Song H, Kaiser JT, Barton JK (2012) Crystal structure of Delta-[Ru(bpy)(2)dppz](2+) bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation. Nat Chem 4:615–620

15. Gill MR, Thomas JA (2012) Ruthenium(II) polypyridyl complexesand DNA-from structural probes to cellular imaging and therapeu-tics. Chem Soc Rev 41:3179–3192

16. Zheng KC,Wang JP, PengWL, Liu XW, Yun FC (2001) Studies on6,6′- disubstitution effects of the dpq in [Ru(bpy)2(dpq)]2+ withDFT method. J Phys Chem A 105:10899–10905

17. Zheng KC, Liu XW, Wang JP, Yun FC, Ji LN (2003) DFT studieson the molecular orbitals and related properties of [Ru(phen)2(9,9′ -2R- dpq)]2+(R=NH2,OH,H and F). J Mol Struct (THEOCHEM)637:195–203

18. Mei WJ, Liu J, Zheng KC, Lin LJ, Chao H et al (2003)Experimental and theoretical study on DNA-binding andphotocleavage properties of chiral complexes - and -[Ru(bpy)2L](L o-hpip, m-hpip and p- hpip). Dalton Trans 7:1352–1359

19. HotzeACG, BacacM, Velders AH, Jansen BAJ, KooijmanH, SpekAL, Haasnoot JG, Reedij KJ (2003) New cytotoxic and water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes. J MedChem 46:1743–1750

20. Elmes RB, Orange KN, Cloonan SM, Williams DC, GunnlaugssonT (2011) Luminescent ruthenium(II) polypyridyl functionalizedgold nanoparticles; their DNA binding abilities and application ascellular imaging agents. J Am Chem Soc 133:15862–15865

21. Elmes RB, Erby M, Bright SA, Williams DC, Gunnlaugsson T(2012) Photophysical and biological investigation of novel lumi-nescent Ru(II)-polypyridyl-1,8-naphthalimide Troger's bases as cel-lular imaging agents. Chem Commun (Camb) 48:2588–2590

22. Venkat RP, Rjender RM, Srishailam A, Praveen KY, Nagamani C,Deepika N, Nagasuryaprasad K, Satyanarayana singh SS,Satyanarayana S (2015) Design, synthesis, DNA-binding affinity,cytotoxicity, apoptosis, and cell cycle arrest of Ru(II) polypyridylcomplexes. Anal Biochem Methods Biol Sci 485:49–58

23. Rjender RM, Venkat RP, Praveen KY, Srishailam A, Nambigari N,Satyanarayana S (2014) Synthesis, characterization, DNA binding,light switch "on and off", docking studies and cytotoxicity, ofruthenium(II) and cobalt(III) polypyridyl complexes. J Fluoresc24:803–817

24. Shobha DC, Anil KD, Singh SS, Gabra NM, Deepika N, Yata PK,Satyanarayana S (2013) Synthesis, interaction with DNA, cytotoxic-ity, cell cycle arrest and apoptotic inducing properties of ruthenium(II)molecular "light switch" complexes. Eur J Med Chem 64:410–421

25. Srishailam A, Yata PK, Gabra NM, Reddy PV, Deepika N,Veerababu N, Satyanarayana S (2013) Synthesis, DNA-binding,cytotoxicity, photo cleavage, antimicrobial and docking studies ofRu(II) polypyridyl complexes. J Fluoresc 23:897–908

26. Yamada M, Tanaka Y, Yoshimoto Y, Kuroda S, Shimao I (1992)Synthesis and properties of diamino substistuted di pyrido [3,4-a:2′,3′-c] phenazine. Bull Chem Soc Jpn 65:2007–2009

27. Schatzschneider U, Niesel J, Ott I, Gust R, Alborzinia H, Wolf S(2008) Cellular uptake, cytotoxicity, and metabolic profiling of hu-man cancer cells treated with ruthenium(II) polypyridyl complexes[Ru(bpy)2(N–N)]Cl2 with N–N=bpy, phen, dpq, dppz, and dppn.Chem Med Chem 3:1104–1109

28. Tan CP, Liu J, Chen LM, Shi S, Ji LN (2008) Synthesis, structuralcharacteristics, DNA binding properties and cytotoxicity studies ofa series of Ru(III) complexes. J Inorg Biochem 102:1644–1653

29. Puckett CA, Barton JK (2008) Mechanism of cellular uptake of aruthenium polypyridyl complex. Biochemistry 47:11711–11716

30. Satyanarayana S, Dabrowiak JC, Chaires JB (1993)Tris(phenanthroline)ruthenium(II) enantiomer interactions withDNA: mode and specificity of binding. Biochemistry 32:2573–2584

31. Barton JK, Danishefsky A, Raphael GJ (1984) Site-specific cleav-age of left-handed DNA in pBR322 byA-co(DIP). J AmChem Soc106:2172–2176

32. Chaires JB, Dattagupta N, Crothers DM (1982) Self-association ofDaunomycin. Biochemistry 21:3927–3932

33. Ghosh BK, Chakravorty A (1989) Electrochemical studies of ru-thenium compounds part I. Ligand oxidation levels. Coord ChemRev 95:239–294

34. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,Cheeseman JR, Scalmani G, Barone V, Mennucci B, PeterssonGA, et al., (2009) Gaussian 09, Revision B.01. In Wallingford

35. Becke AD (1993) Semiempirical hybrid functional with improvedperformance in an extensive chemical assessment. J Chem Phys 98:5648

36. Becke AD (1996) Density-functional thermochemistry. IVA newdynamical correlation functional and implications for exact-exchange mixing. J Chem Phys 104:1040–1046

37. Lee C, Yang W, Parr RG (1988) Development of the Colle-Salvetticorrelation-energy formula into a functional of the electron density.Phys Rev B 37:785

38. Hay PJ, Wadt WR (1985) Ab initio effective core potentials formolecular calculations. Potentials for the transition metal atomsSc to hg. J Chem Phys 82:270

39. Hay PJ, Wadt WR (1985) Ab initio effective core potentials formolecular calculations. Potentials for K to au including the outer-most core orbitals. J Chem Phys 82:299

40. Wadt WR, Hay PJ (1985) Ab initio effective core potentials formolecular calculations. Potentials for main group elements Na tobi. J Chem Phys 82:284

41. Dennington R, Keith T, Milliam J (2009) Gauss view, version 5Semichem Inc. Shawnee Mission, KS

42. Miertuš S, Scrocco ET (1981) Electrostatic Interaction of a Solutewith a Continuum. A Direct Utilization of AB Initio MolecularPotentials for the Prevision of Solvent Effects. J Chem Phys 55:117–129

43. Cossi M, Barone V, Cammi RT (1996) Ab initio study of solvatedmolecules: a new implementation of the polarizable continuummodel. Chem Phys Lett 255:327–335

44. Chitumalla RK, Gupta KSV, Malapaka C, Fallahpour Islam RA,Han L, Kotamarthi B, Singh SP (2014) Thiocyanate-freecyclometalated ruthenium(II) sensitizers for DSSC: a combined ex-perimental and theoretical investigation. Phys Chem Chem Phys16:2630

45. Diller DJ, Merz KM Jr (2001) High throughput docking for librarydesign and library prioritization. Proteins 43:113–124

46. Gohlke H, Klebe G (2001) Statistical potentials and scoring func-tions applied to protein-ligand binding. Curr Opin Struct Biol 11:231–235

47. Wolfe A, Shimer GH Jr, Meehan T (1987) Polycyclic aromatichydrocarbons physically intercalate into duplex regions of dena-tured DNA. Biochemistry 26:6392–6396

48. McGhee JD, Hippel VPH (1974) Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding oflarge ligands to a one-dimensional homogeneous lattice. J Mol Biol86:469–489

49. Gale PA (2011) Anion receptor chemistry. ChemCommun 47:82–8650. Jonathan AK, Elaine MB, Thorfinnur G (2012) Synthesis, structur-

al characterisation and luminescent anion sensing studies of aRu(II)polypyridyl complex featuring an aryl urea derivatised 2,2′-bpy auxiliary ligand. Inorg Chim Acta 381:236–242

51. Hui C, Wen JM, Qi WH, Liang NJ (2004) Electronic effectson the interactions of complexes [Ru(phen)2(p-L)]2+

J Fluoresc

(L=MOPIP, HPIP, and NPIP) with DNA. Inorg Chim Acta357:285–293

52. Li G, Liu N, Liu S, Zhang S (2008) Electrochemical biosensorbased on the interaction between copper(II) complex with 4,5-diazafluorene-9-one and bromine ligands and deoxyribonucleic ac-id. Electrochim Acta 53:2870–2876

53. DrewWL, Barry AL, Toole RO, Sherris JC (1972) Reliability of theKirby-Bauer disc diffusion method for detecting methicillin-resistant strains of Staphylococcus aureus. Appl Microbiol 24:240–247

54. Barton JK, Raphael AL (1984) Photoactivated stereospecific cleav-age of double-helical DNA by cobalt(III) complexes. J Am ChemSoc 106:2466–2468

55. Mosmann T (1983) Rapid colorimetric assay for cellular growthand survival: application to proliferation and cytotoxicity assays. JImmunol Methods 65:55–63

56. José CM, Moreno CJ, Puertollano E, Puertollano MA, de PabloMA (2010) The antimicrobial peptide cecropin a induces caspase-independent cell death in human promyelocytic leukemia cells.Peptides 31:1494–1503

57. Aronoff SL, MD, FACP, FACE, Berkowitz K, APRN, BC, FNP,CDE, Shreiner B, RN, MN, CDE, BC-ADM, Want L, RN, MS,CDE, CCRC, BC-ADM, Glucose Metabolism and Regulation:Beyond Insulin and Glucagon

58. Hickman JA (1992) Apoptosis induced by anticancer drugs,Cancer. Metastasis Rev 11:121–139

59. Darzynkiewicz Z (1990) Differential staining of DNA and RNA inintact cells and isolated cell nuclei with acridine orange. MethodsCel Biology 33:285–298

60. Gorczyca W, Hotz MA, Lassota P, Traganos F (1992) Features ofapoptotic cells measured by flow cytometry. Cytometry 13:795–808

61. Hawley ST, HawleyGR (2004) Flow cytometry protocols, 2nd edn.Humana Press, Totowa

62. Shouval RS, Elazar Z (2007) ROS, mitochondria and the regulationof autophagy. Trends Cell Biol 17:422–427

63. Adams DJ, Boskovic ZV, Theriault JR, Wang AJ, Stern AM,Wagner BK, Shamji AF, Schreiber SL (2013) Discovery ofsmall-molecule enhancers of reactive oxygen species that arenontoxic or cause genotype-selective cell death. ACS ChemBiol 8:923–929

J Fluoresc