Accepted Manuscript

Dissymmetric thiosemicarbazone ligands containing substituted aldehyde armand their ruthenium(II) carbonyl complexes with PPh3/AsPh3 as ancillary ligands:Synthesis, Structural characterization, DNA/BSA interaction and in vitro anticanceractivity

Paranthaman Vijayan, Periasamy Viswanathamurthi, Vaidhyanathan Silambarasan,Devadasan Velmurugan, Krishnaswamy Velmurugan, Raju Nandhakumar, Ray JayButcher, Tamilselvan Silambarasan, Ramamurthy Dhandapani

PII: S0022-328X(14)00322-2

DOI: 10.1016/j.jorganchem.2014.06.026

Reference: JOM 18639

To appear in: Journal of Organometallic Chemistry

Received Date: 2 April 2014

Revised Date: 6 June 2014

Accepted Date: 24 June 2014

Please cite this article as: P. Vijayan, P. Viswanathamurthi, V. Silambarasan, D. Velmurugan, K.Velmurugan, R. Nandhakumar, R.J. Butcher, T. Silambarasan, R. Dhandapani, Dissymmetricthiosemicarbazone ligands containing substituted aldehyde arm and their ruthenium(II) carbonylcomplexes with PPh3/AsPh3 as ancillary ligands: Synthesis, Structural characterization, DNA/BSAinteraction and in vitro anticancer activity, Journal of Organometallic Chemistry (2014), doi: 10.1016/j.jorganchem.2014.06.026.

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Graphical abstract synopsis

Dissymmetric thiosemicarbazone ligands containing substituted aldehyde arm

and their ruthenium(II) carbonyl complexes with PPh3/AsPh3 as ancillary

ligands: Synthesis, Structural characterization, DNA/BSA interaction and in

vitro anticancer activity

Paranthaman Vijayan,a Periasamy Viswanathamurthi,*a Vaidhyanathan Silambarasan,b

Devadasan Velmurugan,b Krishnaswamy Velmurugan,c Raju Nandhakumar,c Ray Jay

Butcher,d Tamilselvan Silambarasane and Ramamurthy Dhandapanie

New dissymmetric Schiff base ligands and their ruthenium(II) carbonyl complexes were

synthesized and characterized by FT-IR, NMR, UV–Vis and single crystal X-ray diffraction. The

synthesized ruthenium(II) complexes were used for biological applications such as DNA/ BSA

interaction and in vitro anticancer activities.

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Graphical abstract pictogram

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Dissymmetric thiosemicarbazone ligands containing substituted aldehyde arm

and their ruthenium(II) carbonyl complexes with PPh3/AsPh3 as ancillary

ligands: Synthesis, Structural characterization, DNA/BSA interaction and in

vitro anticancer activity

Paranthaman Vijayan,a Periasamy Viswanathamurthi,*a Vaidhyanathan Silambarasan,b

Devadasan Velmurugan,b Krishnaswamy Velmurugan,c Raju Nandhakumar,c Ray Jay

Butcher,d Tamilselvan Silambarasane and Ramamurthy Dhandapanie

aDepartment of Chemistry, Periyar University, Salem-636 011, India.

bCentre for Advanced Studies in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai- 600 025, India.

cDepartment of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114, India.

dDepartment of Chemistry, Howard University, 525 College Street NW, Washington, DC 20059, USA.

eDepartment of Microbiology, Periyar University, Salem - 636 011, India.

*Corresponding author E-mail: viswanathamurthi72@gmail.com; Fax: +91 427 2345124

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Abstract

A serious of new dissymmetric ruthenium(II) carbonyl complexes of the type

[Ru(CO)(EPh3)(L1-2)] (1-4), [E = P or As; L1 = N4- (2-Hydroxy-5-chlorobenzylidene)-2-amino-

5-chlorobenzophenone thiosemicarbazone; L2 = N4-(2-Hydroxynaphthalene-1-carbaldehyde)-2-

amino-5-chlorobenzophenone thiosemicarbazone] have been synthesized and characterized by

several spectroscopic studies. The molecular structure of the ligand L1 and the ruthenium(II)

carbonyl complexes (2, 4) have been analyzed by single crystal X-ray studies, and found that the

ruthenium(II) complexes possess a distorted octahedral geometry. The DNA binding studies such

as emissive titration, Ethidium bromide/Methylene blue (EB/MB) displacement assay and

viscometry measurements revealed that the ruthenium(II) complexes bound with calf thymus

DNA through intercalative mode with relatively high binding constant values. Further, the

interactions of the complexes with bovine serum albumin (BSA) were also investigated using

fluorescence spectroscopic methods, which showed that the new complexes could bind strongly

with BSA. The complexes (1-4) were tested for DNA and BSA cleavage activities, and the

results showed that the complexes exhibited good cleavage properties. In addition, the newly

synthesized ruthenium(II) complexes possess better in vitro cytotoxic activities against various

cell lines (MCF-7, Hop62, MDA-MB-435) and AO/EB staining method showed that these

complexes induced apoptosis of MCF-7 cell lines.

Keywords: Ruthenium(II) carbonyl complexes; DNA/BSA interaction; In vitro Anticancer

activity

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1. Introduction

For a half century, the field of metal based anticancer drugs has been dominated by the

precious metal such as platinum [1-3]. Cisplatin has proven to be a highly effective

chemotherapeutical agent for treating various types of cancers like ovarian, testicular, head and

neck carcinomas. Whilst the chemotherapeutical success of platinum is undeniable, it is by no

means the perfect drug. A widely used cancer drug damage DNA and kill normal cells. It is not

effective from many common types of cancer; drug resistance is common, and it has a deplorable

range of side effects, which can include nerve damage, hair loss and nausea [4,5]. To overcome

these limitations, other transition metals based drugs have been developed and tested against

cancer cell lines. Among the transition metal based drugs, ruthenium metal complexes acted as

promising anticancer agents. Researchers have now developed ruthenium complexes well suited

towards pharmacological applications. It can access a range of oxidation states under

physiologically appropriate conditions. Furthermore, the energy barrier to inter conversion

between these oxidation states is relatively low, allowing for ready oxidation state changes when

inside the cell. The mode of action of the ruthenium complexes differs from that of approved

platinum based anticancer drugs [6, 7]. Broadly speaking ruthenium complexes have similar

kinetics for ligand exchange with those of platinum(II) species. It is also generally accepted that

ruthenium(II) complexes are less inert compared to the corresponding ruthenium(III) species,

which can be partially attributed to effective higher nuclear charge(Zeff) [8]. Recently,

ruthenium based compounds namely NAMI-A; KP1019 and KP1339 successfully completed

phase I clinical trials and are scheduled to enter Phase II clinical trials in near future [9]. In

particular, KP1019 and KP1339 are transported by serum protein transferrin into the cells, where

it is reduced to ruthenium(II) species induces oxidative DNA damage resulting in apoptosis [10,

11]. Further recent research in bio inorganic chemistry concern's DNA binding and cleavage by

metal complexes. Because DNA is an important target of antitumor drugs, and it plays a central

role in replication, transcription and regulation of genes [12-14]. The presence of metal-binding

sites in its structure makes it a good target for metal-containing drugs. Metal complexes can be

bound to DNA in a non-covalent interaction such as electrostatic binding, groove binding,

intercalate binding and partial intercalating binding. Many useful applications of these

complexes require that the complexes bind to DNA through an intercalative mode [15-17].

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Literature reveals that the ruthenium(II) carbonyl complexes have a wide range of

applications as antitumour agents who find their origin in intercalative interactions with DNA.

Serum albumins are abundantly found in blood plasma and are often termed transport proteins.

They are circulated in the body several times and act as carriers for numerous exogenous and

endogenous compounds. The most popularly studied albumins are bovine serum albumin (BSA)

and human serum albumin (HSA). Both BSA and HSA have very high conformational

adaptability to a great variety of ligands [18]. Two main approaches have been adopted in the

complex-protein binding studies. One way the binding mechanism have been examined using

absorption, fluorescence and circular dichroism, [19, 20] whereas some groups have studied the

in vivo consequences of binding drugs in addition to other metabolites in the direction of serum

albumins [21-23] In the current work, the authors have chosen BSA as a model protein to

investigate its interaction with the newly synthesized ruthenium(II) carbonyl complexes.

Thiosemicarbazones are versatile ligands and adopt various binding modes with transition

metal and main group metal ions. A number of reasons have been offered as responsible for their

versatility in coordination, such as intramolecular hydrogen bonding, bulkier coligand, steric

crowding on the azomethine carbon atom, and π-π stacking interactions [24-26]. In addition,

thiosemicarbazones and their metal complexes exhibit a wide range of applications that extend

from their use in analytical chemistry through pharmacology to nuclear medicine. Hence our

recent research depends upon the studies of thiosemicarbazones based chelations [27-31]. We

decided to evaluate the effect of ruthenium(II) unsymmetrical thio semicarbazone Schiff base

complexes towards DNA binding/cleavage activity against calf thymus DNA, BSA

binding/cleavage [32, 33] and cytotoxicity under in vitro conditions. In this context, here we

have reported synthesis and crystal structures of ruthenium(II) complexes ( Scheme 1) and their

biological applications.

2. Experimental methods

2.1. Materials and methods

2-amino-5-chlorobenzophenone thiosemicarbazone [34] and the unsymmetrical Schiff base

ligands L1 and L2 were prepared by a modified procedure of the literature methods were shown

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in scheme 1 [35]. The ruthenium(II) precursor complexes [RuHCl(CO)(EPh3)3] (E = P or As)

were prepared according to the literature methods [36-38]. All reagents were purchased from

commercial sources and used as received. Solvents were purified and dried according to

procedures [39]. Calf thymus DNA (CT-DNA), agarose, protein markers and Bovine serum

albumin (BSA) were purchased from Genei, Bangalore and Himedia, India respectively.

RuCl3.3H2O, Ethidium bromide, Methylene blue, Tris(hydroxymethyl) amino methane were

purchased from Sigma-Aldrich. Cell lines of Hop62 (Lung cancer cell line), MDA-MB-435

(Breast cancer cell line), MCF-7 (Breast cancer cell line) were purchased from NCCS, Pune,

India. Elemental analyses (C, H, N, and S) were carried out on a Vario EL III CHNS analyzer at

SAIF-Cochin, India. Infrared spectra were recorded as KBr pellets using a Perkin-Elmer FT-IR

spectrophotometer in the range 4000-400 cm-1. 1H NMR spectra were recorded on a Bruker Ultra

Shield at 300 MHz using DMSO-d6 as solvent and TMS as an internal reference. Electro spray

ionization (ESI) were done using an advanced Q-TOF micro™ mass spectrometer. Electronic

spectra were measured on a JASCO V-570 spectrophotometer. Fluorescence spectral data were

obtained on a JASCO FP-8200 fluorescence spectrophotometer at room temperature. Gel

electrophoresis was carried out by Bio-rad UV Transilluminator and their image has taken by

Sony cyber shot WX60. Single crystal X-ray diffraction data collections were carried out at 173

K on a Bruker Apex-II CCD diffractometer equipped with a liquid nitrogen cryostat. The melting

points were recorded on a technico micro heating apparatus and are uncorrected. Fluorescence

microscopy of apoptosis assays was performed with an OX31 fluorescence microscope

(Olympus, Japan). Stock solutions of ruthenium(II) complexes (1.0 × 10-3 M in DMSO) were

stored at 4°C and prepared required concentrations for all experiments. Data were expressed as

the mean ± the standard deviation from three independent experiments. Double distilled water

was used to prepare buffers. All the stock solutions were stored at 4°C and used after no more

than four days. Solutions of compounds were freshly prepared 1 hour prior to biochemical

evaluation.

Scheme 1

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2.2. Single crystal X-ray diffraction studies

Suitable single crystals of ligand L1 and complexes 2, 4 with approximate dimensions of 0.40

× 0.30 × 0.12, 0.30 x 0.25 x 0.25 and 0.25 x 0.30 x 0.30 mm3 were mounted on a glass fibers

with epoxy cement. The crystals were cut into a fitting size (less than collimator cross section

diameter). The crystal data collections were performed with an automated 'Xcalibur, Ruby,

Gemini' at 123K (L1) and Bruker SMART APEX 2 CCD diffractometer at 293K (2, 4) using

graphite monochromatized Mo (Ka) (λ = 0.71073 Å). Reflection data were recorded using the ω

and φ scans with frame width of 0.5o scan technique. The structures were solved and refined by

full-matrix least-squares techniques on F2 using the SHELXS-13/97 program [40]. The

absorption corrections were done by the multi-scan technique. All data were corrected for

Lorentz and polarization effects, and the non-hydrogen atoms were refined anisotropically.

Hydrogen atoms were generated using SHELXL-13/97, and their positions calculated based on

the riding mode with thermal parameters equal to 1.2 times that of associated C atoms, and

participated in the calculation of the final R-indices.

2.3. Synthesis of unsymmetrical Schiff base ligand

2.3.1. Synthesis of L1

Thiosemicarbazone (0.4557 g, 5 mmol) in 25 ml methanol was taken in a 50 ml round

bottom flask fitted with a magnetic stir-bar and to this 5-chlorosalicylaldehyde (1.22 g, 10 mmol)

was added drop wise with constant stirring. Immediate formation of yellow precipitate of the

corresponding unsymmetrical Schiff base was observed and the stirring was continued for 1 h.

The Schiff base was then filtered off, washed with ethanol and dried under vacuum. Yield:

0.3094g, 70%; Colour: Pale yellow; MP: 161-169oC; Anal. cal. for C21H16N4SOCl2 (%): C,

56.89; H, 3.64; N, 12.64; S, 7.23. found (%): C, 56.66; H, 3.32; N, 12.41; S, 6.85. IR (KBr, cm-1)

3158(ms) ν(-N1H); 3342(ms) ν(-N2H2); 3457(m) ν(O-H); 1614(s) ν(C=N4); 1578(s) ν(C=N3),

828(m) ν(C=S). UV-vis (5% DMSO and 95% Tris-HCl buffer), λ max (nm): 211, 236. 1H NMR

(300 MHz, DMSO-d6, δ, ppm): 12.04 (s, 1H, OH); 8.30 (s, 1H, NH); 8.52, 9.25 (s, 2H, NH2);

8.95 (s, 1H, CH=N); 6.82-7.75 (m, 11H, aromatic). On slow evaporation of the

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methanol/dimethylforma -mide, the Schiff base ligand L1 was obtained as orange crystals after

20 days.

2.3.2. Synthesis of L2

It was prepared in a manner identical to that described for above synthetic pathway with

thiosemicarbazone (0.4557g, 5 mmol) and 2-hydroxy-1-napthaldehyde (0.6105g, 5 mmol) Yield:

0.3069g, 67%; Colour: yellow; MP: 146-153oC; Anal. cal. For C25H19N4SOCl (%): C, 65.42; H,

4.17; N, 12.21; S, 6.99. Found (%): C, 65.07; H, 3.95; N, 12.02; S, 6.67. IR (KBr, cm-1)

3150(ms) ν(-N1H); 3355(ms) ν(-N2H2); 3454(m) ν(O-H); 1612(s) ν(C=N3); 1593(s) ν(C=N4),

825(m) ν(C=S) . UV-vis (5% DMSO and 95% Tris-HCl buffer), λ max (nm): 215, 234. 1H NMR

(300 MHz, DMSO-d6, δ, ppm): 14.23 (s, 1H, OH); 8.32 (s, 1H, NH); 8.51, 9.72 (s, 2H, NH2);

9.33 (s, 1H, CH=N); 6.98-8.08 (m, 14H, aromatic).

2.4. Synthesis of ruthenium(II) complexes

2.4.1. Synthesis of [Ru(CO)(PPh3)L1] (1)

A suspension of [RuHCl(CO)(PPh3)3] (0.100 g, 0.105mmol) in methanol/chloroform mixture

(20 mL) was treated with L1 (0.0481 g, 0.105 mmol) and the mixture was gently refluxed for 6h.

During this time, the color changed to orange. After this time, it was cooled and filtered, washed

with small amounts of cold methanol, water and excess of ether and then dried in vacuum. Yield:

0.4914g, 59%; Colour: Brown; MP: 253-261oC; Anal. cal. for C40H30Cl2N4O2PRuS (%): C,

57.63; H, 3.63; N, 6.72; S, 3.85. Found (%): C, 57.29; H, 3.36; N, 6.30; S, 3.58. ESI-MS

(CH3OH): m/z = 835 ( [M + H]+ ). IR (KBr, cm-1) 3336(ms) ν (-N2H2); 1601(s) ν(C=N4); 1570(s)

ν(C=N3), 779 (m) ν(C-S) , 1443(s) ν{Ph(P-Ph)}. UV-vis (5% DMSO and 95% Tris-HCl buffer),

λ max (nm): 214, 239, 351. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.63, 9.85 (s, 2H, NH2); 9.19

(s, 1H, CH=N); 6.96-8.01 (m, 11H, aromatic). 31P NMR (162 MHz, DMSO-d6): δ (ppm) = 30.24

2.4.2. Synthesis of [Ru(CO)(PPh3)L2] (2)

It was prepared by adopting the procedure used for the synthesis of complex 1 by the

suspension of [RuHCl(CO)(PPh3)3] with L2. The resulting solution was filtered and kept aside

for crystallization by slow evaporation. When the concentrated solution was kept for a week,

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yellow colored crystals suitable for X-ray diffraction were obtained. Yield: 0.5519g, 65%;

Colour: Brown; MP: 250-257oC; Anal. cal. for C44H33ClN4O2PRuS (%): C, 62.22; H, 3.92; N,

6.60; S, 3.78. Found (%): C, 62.01; H, 3.62; N, 6.18; S, 3.38. ESI-MS (CH3OH): m/z = 850 ( [M

+ H]+ ). IR (KBr, cm-1) 3346(ms) ν(-N2H2); 1604(s) ν(C=N4); 1587(s) ν(C=N3), 786 (m) ν(C-S)

,1442(s) ν{Ph(P-Ph)}. UV- vis (5% DMSO and 95% Tris-HCl buffer), λ max (nm): 218, 238, 395.

1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.60, 9.98 (s, 2H, NH2); 9.19 (s, 1H, CH=N); 7.01-8.10

(m, 11H, aromatic). 31P NMR (162 MHz, DMSO-d6): δ (ppm) = 32.46.

2.4.3. Synthesis of [Ru(CO)(AsPh3)L1] (3)

It was prepared by adopting the procedure used for the synthesis of complex 1 by the

suspension of [RuHCl(CO)(AsPh3)3] with L1. Yield: 0.5524g, 63%; Colour: Brown; MP: 257-

265oC; Anal. cal. for C40H30AsCl2N4O2RuS (%): C, 54.74; H, 3.45; N, 6.38; S, 3.65. Found (%):

C, 54.29; H, 3.12; N, 6.14; S, 3.47. ESI-MS (CH3OH): m/z = 879 ( [M + H]+ ). IR (KBr, cm-1)

3344(ms) ν(-N2H2); 1600(s) ν(C=N4); 1571(s) ν(C=N3), 776 (m) ν(C-S) , 1458(s) ν{Ph(As-Ph)}.

UV-vis (5% DMSO and 95% Tris-HCl buffer), λ max (nm): 213, 237, 390. 1H NMR (300 MHz,

DMSO-d6, δ, ppm): 8.58, 9.81 (s, 2H, NH2); 9.15 (s, 1H, CH=N); 6.99-8.10 (m, 11H, aromatic).

2.4.4. Synthesis of [Ru(CO)(AsPh3)L2] (4)

It was prepared by adopting the procedure used for the synthesis of complex 1 by the

suspension of [RuHCl(CO)(AsPh3)3] with L2. The solvent was removed under vacuum, and the

residue was washed with diethylether and recrystallized from CHCl3/CH3OH to yield big red

crystals suitable for X-ray diffraction. Yield: 0.5536g, 62%; Colour: Brown; MP: 244-251oC;

Anal. cal. for C44H33AsClN4O2RuS (%): C, 59.16; H, 3.72; N, 6.27; S, 3.59. Found (%): C,

58.82; H, 3.81; N, 6.01; S, 3.29. ESI-MS (CH3OH): m/z = 894 ( [M + H]+ ). IR (KBr, cm-1)

3344(ms) ν(-N2H2); 1604(s) ν(C=N4); 1588(s) ν(C=N3), 784 (m) ν(C-S) ,1454 (s) ν{Ph(As-Ph)}.

UV-vis (5% DMSO and 95% Tris-HCl buffer), λ max (nm): 213, 237, 397. 1H NMR (300 MHz,

DMSO-d6, δ, ppm): 8.58, 9.95 (s, 2H, NH2); 9.20 (s, 1H, CH=N); 7.09-8.15 (m, 11H, aromatic).

2.5 DNA binding and cleavage experiments

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2.5.1 Luminescence titration

Luminescence measurement was performed to clarify the binding affinity of ruthenium(II)

carbonyl complexes by emissive titration at room temperature [41]. The complexes were

dissolved in mixed solvent of 5% DMSO and 95% Tris-HCl buffer (5mM Tris-HCl / 50mM

NaCl buffer for pH-7.2) for all the experiments and stored in 4o C for further use. Tris-HCl buffer

was subtracted through base line correction. The excitation wavelength was fixed by the

emission range and adjusted before measurements. Emissive titration experiments were

performed with a fixed concentration of the solution of metal complexes (25 µM). While

gradually increasing the concentration of DNA (0-40 µM), the emission intensities were

recorded in the range of 245-350 nm at an excitation wavelength of 240 nm at room temperature

(298K). Titrations were manually done by using a micropipette for the addition of CT-DNA. It is

noteworthy here that the DNA in double distilled water does not show any luminescence.

Further support for the binding of the complexes to DNA via intercalation was obtained

from emission quenching experiments. EB/MB is a planar cationic dye, well-known to

intercalate into the CT-DNA independently. While EB/MB is most fluorescent compounds, EB-

DNA and MB-DNA adduct is a strong emitter on excitation near 520 and 600 nm respectively

[42]. For all the experiments, DNA was pretreated with EB/MB in the ratio [DNA] / [EB or MB]

= 10 for 30 minutes at 37oC. Then the titration solutions were added to this mixture of EB-

DNA/MB -DNA, and the change in the fluorescence intensity was measured.

Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a

constant temperature of 30.0°C (± 0.1) in a thermostatic bath. DNA samples of approximately

200 base pairs in length were prepared by sonication in order to minimize complexities arising

from DNA flexibility. Titrations were performed by addition of small volume of concentrated

stock solutions of metal complex to a solution of calf thymus DNA in the viscometer. The flow

time was measured with a digital stopwatch, and each sample was tested three times to get an

average calculated time. Relative viscosities for CT-DNA in the presence and absence of the

complex were calculated from the relation g = (t - to)/to, where t is the observed flow time of

DNA-containing solution and to is the flow time of Tris–HCl buffer alone. Data were presented

as (η/η0)1/3 versus binding ratio (R = [Ru]/[DNA] = 0.0–2.4), where η is the viscosity of CT–

DNA in the presence of the complex, and η0 is the viscosity of CT–DNA alone.

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2.5.2. DNA cleavage experiment

For the gel electrophoresis, experiments supercoiled pBR322 DNA (100 µM) in 5% DMSO

was treated with the solution of fixed concentration of ruthenium(II) complexes (50 µM) in 5mM

Tris-HCl / 50mM NaCl buffer (pH-7.2) was incubated at 37oC in absence/presence of

complexes. The DNA, compound and sufficient buffer were premixed in a vial, and the reaction

was allowed to proceed for 1 hour at 37 °C before the addition of ethylene glycol and loading

onto an agarose gel. A loading buffer containing 0.25% bromophenol blue, 0.25% xylenecyanol,

and 30% glycerol (3 mL) were added, and the agarose gel electrophoresis for 1.5 - 2 h at 60 V on

1% agarose gel in Tris borate EDTA buffer. The gel was stained with 0.5 µg/ml of EB in Tris-

HCl buffer. The ability of new complexes efficiency for the conversion of supercoiled DNA

(SC) Form I to Form II (NC) was measured and visualized using UV illuminator, photographed.

2.6. Protein binding and cleavage studies

2.6.1. Tryptophan Quenching Experiment

Quenching of the tryptophan residues of BSA was done using selected ruthenium(II)

complexes as quenchers. To the stock solutions of BSA in 5 mM Tris-HCl / 50mM NaCl buffer

at pH-7.2, increments of the quencher were monitored for the protein binding studies and the

emission signals at 344 nm (excitation wavelength at 280 nm) were recorded after each addition

of the quencher. The excitation and emission slit widths and scan rates were maintained constant

for all the experiments. Titrations were manually done by using a micropipette for the addition of

metal complexes. The Io/I versus [Q] plots were constructed using the corrected fluorescence

data taking into account the effect of dilution.

2.6.2. Protein cleavage studies

Protein cleavage experiments were carried out by incubating BSA (4 µM) with complexes in

Tris-HCl buffer for 4 h at 37oC according to the literature [43]. The samples were dissolved in

the loading buffer (24 µL) containing SDS (7% w/v), glycerol (4% v/v), Tris-HCl buffer (50mM,

pH 6.8), mercaptoethanol (2% v/v) and bromophenol blue (0.01% w/v). The samples were then

loaded on a 3% polyacrylamide stacking gel. [Separating Gel / Stacking gel concentration 4.05/

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2.75ml distill water, 2.5 ml/ 1.7 tris buffer (PH- 8.8/6.8), acromade 3.3/ 2.3 ml, SDS 250/ 100 µl,

APS 250/100 µl, Temed 75/50 µl]. The above mixture was subjected to gel electrophoresis (10%

SDS PAGE) as described by Laemmli [44]. The gel electrophoresis that was done initially at 50

V until the dye passed into the separating gel from the stacking (3%) gel, followed by setting the

voltage to 100 V for 1.5 h. Staining was done with CBR-250 (coomassie brilliant blue R-250)

solution (acetic acid-methanol-water = 1:2:7 v/v) and destaining were done with water-methanol-

acetic acid mixture (5:4:1 v/v) for 4 h. The gels, after destaining, were scanned and the images

were photographed.

2.7. Cytotoxicity assay in vitro 2.7.1. MTT assay Cell viability was assessed by the MTT (3,4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium

bromide) method. MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in

living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an

insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional

to the number of viable cells [45]. MCF-7 cells were maintained in a humidified atmosphere

containing 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented

with 100 units of penicillin, 100µgmL-1 of streptomycin and 10% fetal bovine serum. In brief,

MCF-7 cells with a density of 1 × 106 cells per well were precultured in 96-well microtiter plates

for 48 h under 5% CO2. The ruthenium(II) complexes were added to the micro wells containing

the cell culture at concentrations of 5-200µM. Then each well was loaded with 10 µL MTT

solution (5 mg mL-1 in PBS pH = 7.4) for 4 h at 37 °C. The purple formazan crystal was

dissolved in 200 µL DMSO, and the cell viability was determined by measuring the absorbance

of each well at 540 nm using a BIORAD ELISA plate reader. All experiments were performed in

triplicate, and the percentage of cell viability was calculated according to the following equation.

Inhibition rate (IR %) = OD (control) - OD Drug treated cells / OD (control) × 100.

The IC50 values were determined by nonlinear regression analysis using Origin 6.0 software.

2.7.2. SRB assay

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The sulforhodamine B (SRB) assay is used for cell density determination, based on the

measurement of cellular protein content. This assay allows the measurement of drug-induced

cytotoxicity and cell proliferation for large-scale drug screening applications [46]. The cell lines

HOP62 (human lung), MDA-MB-435 (human breast) were used for in vitro antitumor screening

activity. These human malignant cell lines were procured and grown in RPMI-1640 medium

supplemented with 10% fetal bovine serum (FBS) and antibiotics to study the growth pattern of

these cells. The proliferation of the cells upon treatment with chemotherapy was determined

using the semi-automated sulphorhodamine-B (SRB) assay [47]. Cells were seeded in 96-well

plates at an appropriate cell density to give optical densities in the linear range (from 0.5 to 1.8)

and were incubated at 37°C in a CO2 incubator for 24 h. Stock solutions of the complexes were

prepared as 100 mg mL-1 in DMSO and four dilutions (i.e., 10 µL, 20 µL, 40 µL, 80 µL) were

tested in triplicates, each well receiving 90 µL of cell suspension and 10 µL of the drug solution.

Appropriate positive control (Adriamycin) and controls were also run. The plates with cells were

incubated in a CO2 incubator with 5% CO2 for 24 h followed by the addition of the drug. Again,

the plates were incubated further for 48 h. The experiment was terminated by gently layering the

cells with 50 µL of chilled 30% TCA (in the case of adherent cells) and 50% TCA (in the case of

suspension cell lines) for cell fixation and then being kept at 4 °C for 1 h. Plates were stained

with 50 µL of 0.4% SRB for 20 minutes. The bound SRB was eluted by adding 100 µL, 10 mM

Tris (pH 10.5) to each of the wells. The absorbance was read at 540 nm with 690 nm as the

reference wavelength. All experiments were repeated three times with various concentrations.

2.7.3. Apoptosis assay by acridine orange/ethidium bromide (AO/EB) staining method

Apoptotic studies were performed with a staining method utilizing AO and EB. The cells

were seeded at a density of 1×106 cells/well. After attachment, the MCF-7 cells were treated with

ruthenium(II) complex (50µM) and incubated for 24 h at 37oC in CO2 incubator. After the

treatment period, the cells were washed with ice-cold phosphate-buffered saline (PBS). Both

AO/EB was added at a concentration of 1 mg/ml. The MCF-7 cells were mounted on a slide, and

the images were observed under a fluorescent microscope in green filter with excitation at 350

nm and emission at 460 nm.

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3. Results and discussion

3.1. Synthesis of ruthenium(II) complexes

The present work deals with the synthesis of new ruthenium(II) complexes from

unsymmetrical Schiff base ligands and ruthenium precursor complexes, [RuHCl(CO)(EPh3)3] (E

= P or As) at 1:1 mol ratio by the direct reaction as shown in Scheme 1. The unsymmetrical

Schiff base ligands coordinated as dibasic tetradentate [ONNS] mode in all the ruthenium(II)

complexes 1-4. All the complexes have been obtained in good yield, quite stable in air and

soluble in most of the organic solvents such as C2H5OH, CH3OH, CH2Cl2, CHCl3, DMF and

DMSO. The analytical data of the complexes agreed well with the proposed molecular formulae.

3.2. Spectroscopic data

The IR spectra of the unsymmetrical benzophenone thiosemicarbazone ligands and their

corresponding complexes were compared in the region 4000-400 cm-1. The ligands (L1, L2)

showed a sharp peak at 1604-1614 cm-1 corresponding to ν(C=N4) of the azomethine group. In

the IR spectra of ruthenium(II) complexes (1-4), ν(C=N4) was shifted to lowering frequencies

(1588-1604 cm-1) indicating the azomethine group bound to the ruthenium metal atom [48,49].

The broad band due to ν(O-H) and ν(-N1H) appeared in the region 3340-3465 cm-1 and 3150-

3172 cm-1 respectively in ligands have completely disappeared after complexation showed that

the OH group in ligands undergoes deprotonation prior to coordination and N1H group involved

in thione-thiol tautomerization since it contains a thioamide (-N1H-C=S) functional group. In the

spectra of ligands, the peak appeared at 3338-3348 cm-1 was assigned to terminal N2H2 group

[50]. It can be clearly determined that the above stretching vibration bands are shifted to lower

frequencies and weakened in the spectra of complexes. Furthermore, a strong band appeared at

818-834 cm-1 in the spectra of the ligand is indicative of ν(C=S). The lowering of this band in the

form of ν(C-S) can be attributed to negative coordination of the ligands and ruthenium metal

atom through the thio-sulfur atom [51, 52]. All the complexes display a medium to strong band

in the region 1962−1945 cm-1, which is attributed to the terminally coordinated carbonyl group

(C≡O) and is observed at a slightly higher frequency than in the precursor complexes. Moreover,

EPh3 (E= P or As) were also present in an expected region at 1440-1460 cm-1 [26, 50]. Hence all

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the above facts are in good agreement with the complexes coordinated as a binegative

tetradentate mode (ONNS).

Comparison of the 1H NMR spectra of the free thiosemicarbazone ligands with that of new

ruthenium complexes was assigned based on the observed chemical shift. The spectra of the

unsymmetrical Schiff base ligands displayed a weak singlet at 12.04-14.23 ppm and sharp

signals at 8.30-8.32 ppm because of OH and N1H protons repectively. However, the NMR

spectra of ruthenium(II) complexes did not register any signal corresponding to OH and N1H,

revealing that the ligands adopted thione-thiol tautomerism (-N1H-C=S), followed by

deprotonation prior to coordination with the metal ion. In addition, all the ligands showed a sharp

singlet for the azomethine (-H-C=N4) proton at 8.95-9.33 ppm and two singlets in the range of

8.51-8.52 and 9.25-9.72 ppm for terminal N2H2 [53]. However, in the case of spectra of

ruthenium(II) thiosemicarbazone complexes, the signal corresponding to the azomethine proton

gets shifted downfield due to the participation of azomethine nitrogen in coordination with the

metal ion and were observed at 9.15-9.20 ppm for all the complexes and also the terminal N2H2

which is shifted to downfield and appear at 8.58-8.63 and 9.81-9.95 ppm respectively [54]. The

multiple protons of the aromatic ring moiety of the ligands and metal complexes were observed

as multiplets in the range of 6.83-8.15 ppm. In order to confirm the presence of

triphenylphosphine, 31P NMR spectra were recorded. The singlet observed at 30.24 and 32.46

ppm, suggested that presence of triphenylphosphine in complexes 1, 2 respectively. The positive

ion ESI mass spectra of the unsymmetrical Schiff base ruthenium(II) complexes 1-4 showed

major peaks at m/z = 835, 850, 879 and 894 respectively, which have been assigned to the [M +

H]+ .

3.3. X-ray crystallography

The crystal structure of the ligand L1 and ruthenium(II) complexes 2, 4 has been established

by single crystal X-ray analysis. The ORTEP views of the ligand L1 and the complexes 2, 4

along with the atom numbering scheme are given in Fig. 1, 2 respectively. Crystallographic data

and bond parameters are given in Tables 1 and 2.

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Fig. 1.

Fig. 2.

Table 1

Table 2

3.3.1. Crystal structure of ligand L1

The ligand L1 crystallized monoclinic space group P 21/n with 4 independent molecule

within the unit cell. The azomethine bond length C15–N4 (1.287(1) Å) is confirmed to a formal

C=N double bond length (1.28 Å) and the thione form is confirmed by the C8-S1 bond length of

1. 685(1) Å is very close to a formal C=S bond length (1.60 Å). The thione sulfur atom S1 is

trans to azomethine nitrogen atom N1, which is confirmed by a torsion angle of -169.99(7) for

S1–C8–N2–N1 bond.

3.3.2. Crystal structure of complex 2 and 4

Complexes 2 and 4 crystallized in the triclinic belonging to the Pī space group. The

ruthenium(II) ion adopts a distorted octahedral geometry with the binding of the ligand as dibasic

tetradentate (ONNS donor) manner, and the fifth site is occupied by a PPh3/AsPh3 ligand

whereas sixth site is occupied by CO group. In the complexes presence of CO group as a strong

π-acidic ligand has probably forced the bulky PPh3/AsPh3 ligand to take up mutually trans

position for a steric reason [55]. In complex 2, ruthenium atom is in octahedral environment the

trans angles of N1–Ru1–P1, 172(8)° and N4−Ru1−S1, 155.58(7)°. The carbonyl group is trans

to the coordinated O1 atom (C26–Ru1– O1) with an angle of 179.19(12) deviate from linearity

and N1,S1 chelation (four member ring) leads to small S1–Ru–N1 bite angle 67.53(8)° indicated

a slight deviation from the expected linear trans geometry, suggesting distortion in the octahedral

coordination geometry. The selected Ru-ligand distances [Ru1–C26] 1.823(3) Å, [Ru1–N1]

2.113(3) Å, [Ru1–N4] 2.053(2) Å, [Ru1–O1] 2.105(2) Å, [Ru1–S1] 2.4154(8) Å are comparable

with distances found in previously reported ruthenium complexes containing PPh3 in trans

position [56, 57].

In complex 4, ruthenium atom is in octahedral environment the trans angles of N1–Ru1–As1,

174.01(6)° and N4−Ru1−S1, 152.20(7)°. The carbonyl group is trans to the coordinated O1 atom

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(C26–Ru1–O1) with an angle of 178.27(14)°deviate from linearity and N1,S1 chelation (four

member ring) leads to small S1–Ru–N1 bite angle 64.72(6)° indicated a slight deviation from the

expected linear trans geometry, suggesting distortion in the octahedral coordination geometry.

The selected Ru-ligand distances [Ru1–C26] 1.895(4) Å, [Ru1–N1] 2.328(3) Å, [Ru1–N4]

1.905(2) Å, [Ru1–O1] 2.153(2) Å, [Ru1–S1] 2.2879(10) Å, [Ru1–AS1] 2.5543(11) Å are

comparable with distances found in previously reported ruthenium complexes containing AsPh3

in trans position [58-60]. In addition, a molecule of chloroform was found in the unit cell.

Further, there exists a hydrogen bonding between the solvated chloroform molecule and the

benzophenone thiosemicarbazone hydrogen (H6) of the ligand moiety. The unit cell packing

diagram of the ligand L1 and complex 2, 4 along with hydrogen bonding is given in Fig. 3. The

unit cell packing of the complexes was assembled through the intermolecular hydrogen bonding

via N—H…O contacts. The ligand L1 and complex 2, 4 inter atomic hydrogen bonding

distances are given in Table 3.

Fig. 3

Table 3

3.4. DNA interaction studies

3.4.1. DNA binding by fluorescence spectra

Fluorescence spectrophotometer is universally employed to determine the binding

characteristics of compounds with DNA, as binding to the macromolecule leads to changes in the

emissive spectra of the same [41]. Compounds that bound to DNA through intercalation are

characterized by the change in emissive intensity (hypochromism) and bathochromic shift in

wavelength, due to a strong stacking interaction between the aromatic chromophore of the test

compounds, and DNA base pairs. The extent of hypochromism is commonly consistent with the

strength of intercalative interaction [61a, 61b]. In the present study, the emissive spectral titration

experiment was used to monitor the interaction of selected ruthenium(II) complexes 1-4 with

CT-DNA. The fluorescence titration results of ruthenium(II) complexes with increasing

concentration of CT-DNA were illustrated in Fig. 4. Upon incresing concentration of CT-DNA

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to the test complexes, the intensity band of the complexes 1 and 2 at 259 nm exhibited

hypochromism of 31.31 and 52.33% with the red shift of 2 and 1nm respectively. However, the

emissive band of complex 3 and 4 exihibted hypochromism of 29.75 and 45.44% with the red

shift of 3 and 2 nm respectively. The intensity of bands decreased from the original intensities

based on the DNA binding enhancement. The intrinsic binding constants were obtained

according to the following Scatchard equation [62].

Fig. 4.

CF = CT [(I/ I0)–P] / [1–P]

Where, CT is the concentration of the probe (complex) added; CF is the concentration of the

free probe, and I0 and I are its emission intensities in the absence and in the presence of DNA,

respectively. P is the ratio of the observed emission quantum yield of the bound probe to the free

probe. The value of P was obtained from a plot of I/I0 versus 1/[DNA] such that the limiting

emission yield is given by the y-Intercept. The amount of bound probe (CB) at any concentration

was equal to CT -CF. The Scatchard plots of r/Cf versus r for complexes 1-4 with increasing

concentration of CT-DNA were shown in Fig. 5. A plot of r/CF vs r (= CB / [DNA]) gave the

binding isotherm, and the best fit of the data resulted in the intrinsic binding constant (Kb) values

[63]. It has been found that binding constant values of selective ruthenium(II) complexes 1-4

were 4.49 × 104, 4.78 × 105, 5.8 × 103, 7.41 × 104 respectively. These observations imply that

the complex 2 could bind completely DNA via intercalation mode, even though other complexes

also bind with DNA via the same mode [64]. Finally, the overall binding ability of the

complexes is in the order 2 > 4 > 1 > 3. Though it has been observed that the compounds can

bind to DNA by intercalation from the fluorescence spectral studies, such a binding mode needs

to be proved through some more experiments.

Fig. 5.

3.4.2. Competitive DNA binding studies

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A comparative competitive DNA binding study with ethidium bromide (EB) and methylene

blue (MB) was undertaken to identify the binding mode and their binding affinity of

ruthenium(II) complexes [65]. EB and MB are a planar cationic dye, which is widely used as a

sensitive fluorescence probe for native DNA. EB and MB emit intense fluorescent light in the

presence of DNA due to its strong intercalation between the adjacent DNA base pairs. The

fluorescence of EB or MB increases after intercalating into DNA. If the metal complexes

intercalate into DNA, it leads to a decrease in the binding sites of DNA available for EB and MB

resulting in decrease in the fluorescence intensity of the EB-DNA and MB-DNA system. The

emission spectra of both DNA-EB and DNA-MB system with increasing concentration of

selected complexes were shown in Fig. 6. This illustrates that, as the concentration of the

complexes increases, the emission band at 601nm for EB exhibited hypochromism up to 33.19

and 45.47 % of the initial fluorescence intensity respectively. The emission band at 684 nm for

MB exhibited hypochromism up to 17.50 and 38.55% respectively. The apparent DNA binding

constant (Kapp) was calculated from a Stern-Volmer plot of the observed intensities against

complex concentration used to titrate with DNA pretreated with EB and MB. According to the

classical Stern-Volmer equation [66].

Fig. 6.

F0 / F = Kq [Q] + 1 Where, F0 is the emission intensity in the absence of compound, F is the emission intensity in

the presence of compound, Kq is the quenching constant, and [Q] is the concentration of the

compound. The Kq value is obtained with a slope from the plot of F0/F versus [Q]. The Stern -

Volmer plots of F0/F versus [Q] was shown in Fig. 7. The quenching constant (Kq) values were

obtained from the slope, which was 8.88 × 103, 1.88 × 104, 1.06 × 103 and 1.33 × 103 for the

complexes 1-4 respectively. Further the apparent DNA binding constant (Kapp) values were also

calculated using the following equation [55].

Fig. 7.

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KEB [EB] = K app [complex] and KMB [MB] = K app [complex]

Where, KEB or KMB = 1.0 × 10-7 M-1 is the DNA binding constant of EB and MB; [EB] or

[MB] is the concentration of EB or MB (7.5µM) and [complex] is the concentration of the

complex used to obtain 50% reduction in fluorescence intensity of DNA pretreated with EB and

MB. The experimental result’s data were found to be 6.66 × 105, 1.41 × 106, 7.95 × 104 and 9.97

× 104 respectively for 1-4. From these observed data for using EB, we could see that the complex

2 has more binding affinities. Similarly, using MB for the complex 4 has shown more binding

affinities. From these experimental data, it is seen that the order of comparative binding affinities

of the complexes is in the order 2 > 1 for EB and 4 > 3 for MB, which is in agreement with the

results observed from the emission spectra. Furthermore, the observed quenching constants and

binding constants of the ruthenium(II) complexes 2 and 4 suggested that the interaction of the

complexes with DNA should be of intercalation, due to the presence of the naphthalene ring

system in one compartment of the ligand L2.

3.4.3. Viscosity measurements

The viscosity of the DNA solution was sensitive to the addition of organic drugs and metal

complexes and relative viscosity measurements have proved to be a reliable method for the

assignment of the mode of binding compounds to DNA. In classical intercalation, the

intercalating agents were expected to elongate the double helix to accommodate the complexes

in between the bases, leading to an increase in the viscosity of DNA. This behavior contrasts

with the classical groove binders which have no effect on DNA length or solution viscosity. On

the other hand, non-classical or partial intercalators bend or kink the DNA helix, thereby

decreasing the viscosity of DNA. As a validation of the above verdict, viscosity measurements of

complexes (1-4) on the relative viscosities of CT-DNA were shown in Fig. 8. When

ruthenium(II) complexes (1-4) were treated with CT-DNA (200 µM) and the concentrations of

ruthenium complexes (0-120 µM) are increased from a ratio of R= 0-2.4 (1/ R = [Ru] / [DNA]),

the relative viscosity of DNA increases steadily (Fig. 8) in the order 2 > 4 > 1 > 3. The viscosity

changes revealed that all the complexes (1-4) intercalate between the base pairs of DNA. This

result is consistent with our foregoing hypothesis.

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Fig.8.

3.4.4. Cleavage of pBR322 DNA by ruthenium(II) complexes

To assess the DNA cleavage ability of ruthenium(II) complexes, pBR322 supercoiled DNA

was incubated with fixed concentrations (50 µM) of the respective complexes 1-4 in 5 mM Tris-

HCl/50 mM NaCl buffer at pH 7.2 for three hours without addition of any reductant. Upon gel

electrophoresis of the reaction mixture, a concentration dependent DNA cleavage was observed

during which Form I was converted into Form II. From the fixed concentration of all the

complexes (50 µM), cleave SC DNA (Form I) to NC (Form II). The complexes did not require

any addition of external agents to affect DNA cleavage activity. Moreover, the control DNA lane

2 did not exhibit any cleavage activity under the same experimental conditions. From this,

clearly the ruthenium(II) complexes 1-4 (lane 3-6) alone are responsible for the cleavage of DNA

were shown in Fig. 9. The involvement of reactive oxygen species (hydroxyl, superoxide, singlet

oxygen, and hydrogen peroxide) in the nuclease mechanism can be inferred by monitoring the

quenching of the DNA cleavage in the presence of radical scavengers in solution [67a]. In our

study, no inhibition of DNA cleavage upon interaction with ruthenium(II) was observed even in

the presence of scavengers of hydroxyl radicals (DMSO and mannitol), singlet oxygen (sodium

azide and L-histidine), and superoxide radical scavengers (SOD). This indicates that the cleavage

of DNA probably follows a hydrolytic cleavage mechanism which is unaffected by external free

radicals. Moreover, inhibition or promotion of DNA cleavage is not observed appreciably under

aerobic and anaerobic conditions suggesting that oxidative cleavage is not a factor [67b]. Hence

the DNA cleavage observed here is expected to occur through a hydrolytic process (Fig. 9.) [68-

69].

Fig. 9.

3.5. Protein binding studies

3.5.1. Fluorescence quenching of BSA with ruthenium(II) complexes

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The three amino acid residue’s tryptophan, tyrosine and phenylalanine in Bovine Serum

Albumin (BSA) have fluorescence properties, and so BSA emits fairly intensely an internal

source of fluorescence when it is excited. The relative ratio of fluorescence intensity for these

three amino acids residues is 100:9:0.5, and thus the intrinsic fluorescence intensity of BSA

when excited at 295 nm mainly comes from the tryptophan residues such as Trp-134 and Trp-

212 more exposed to the environment [70]. The emission is sensitive to the changes in the local

environment of the tryptophan and so can be attenuated by binding of a small molecule or near

this residue. The emission intensity depends upon the degree of exposure of two tryptophan side

chains in BSA. Hence the intrinsic fluorescence spectra of ruthenium(II) complexes folding with

BSA protein at room temperature to provide considerable secondary structures leading to change

with tryptophan environments. A solution of BSA (5 µM) was titrated with various

concentrations of the complexes 1-4 (0-50 µM). The effects of the complexes on the fluorescence

emission spectrum of BSA were given in Fig. 10.

Fig. 10.

Addition of the complexes to the solution of BSA resulted in a significant decrease of the

fluorescence intensity from the initial fluorescence intensity of BSA accompanied by a red shift

of 78 nm for the complex 1 and blue shift of 33 nm for the complexes 2-4 respectively. The

observed red shift and blue shift is mainly due to the fact that the active site in protein is buried

in a hydrophobic environment. The observed shift is mainly due to the interaction of the

complexes with the active site of BSA protein. Furthermore, fluorescence quenching data were

analyzed with the Stern-Volmer equation and Scatchard equation. From the plot of I0/I Vs [Q]

the quenching constant (Kq) can be calculated from Fig. 11B. If it is assumed that the binding of

compounds with BSA occurs at equilibrium, the equilibrium binding constant can be analyzed

according to the Scatchard equation [71].

Fig. 11A.

Fig. 11B.

Table 4

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Log [I0 - I / I] = log K bin + n log [Q]

Where, Kbin is the binding constant of the compound with BSA and n is the number of

binding sites. The number of binding sites (n) and the binding constant (Kbin) have been found

from the plot of log (I0 - I) / I versus log [Q] (Fig. 11A.). The calculated Kq, Kbin, and n values

are given in Table 4. The value of n for complex 2 clearly indicates two binding sites availed to

react with residues, while remaining complexes one binding site could be availed to react with

residues from BSA hydrophobic environment. From these experimental data, it is seen that the

order of binding affinities of the complexes is 2 > 4 > 1 > 3. It is further confirmed that complex

2 interacts with BSA more strongly than the other complexes, due to the presence of the

naphthalene ring system in one compartment of the ligand L2. The larger values of Kq and Kbin

indicate a strong interaction between the BSA and the new complexes.

3.5.2. Protein cleavage studies

The protease activity of ruthenium complexes 1 and 2 (50 µM) against BSA (4 µM) in the

phosphate buffer medium was studied by SDS-PAGE in Tris-HCl pH 7.2 staining with CBR-250

at room temperature. The SDS PAGE gel diagrams showed that the complex 1 displays moderate

protein cleavage activity [72]. The major cleavage sites of complex 2 with BSA showed more

electrophoretic bands corresponding to protein fragments of 60kDa, 90kDa, 100KDa, 140kDa

and 160kDa as reaction products (Fig. 12.). The complex 2 showed significant smearing or

fading of the BSA band indicating that non-specific binding of the complexes to BSA leads to

cleavage by band fragments, which is supported by BSA binding studies.

Fig. 12

3.6. Anticancer activity in vitro

3.6.1. MTT assay

Ruthenium complexes were considered as a potential target for anticancer activity, where it

binds to DNA through intercalation along with electrostatic interaction of the ionic side chain

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with the DNA phosphate groups. The complexes exert their anticancer action through covalent

binding. Moreover the complex have been reported to exercise its anticancer properties due to

combination of a lysosomotropic effect, the inhibition of some enzymes that regulate cell cycle

which potentiate apoptosis and it is known that complex have the ability to inhibit the

DNA/RNA polymerase activities. Hence, we proposed that our complexes enter into the tumour

cells, bind to DNA inside the cells and inhibit the tumor cell growth. Since all the present

ruthenium(II) complexes have the ability to strongly bind and cleave DNA, which is considered

as one of the key requirements for a drug to act as an anticancer agent, the cytotoxicity of the

complexes against MCF-7 human breast cancer cell line has been investigated by MTT assay in

aqueous buffer solution and the results are compared with the widely used drug cisplatin.

Complexes were dissolved in DMSO, and blank samples containing same volume of DMSO

were taken as controls to identify the activity of solvent in the cytotoxicity experiment. Cisplatin

was used as a positive control to assess the cytotoxicity of the test compounds [73]. Interestingly

on comparison of the IC50 value of the complexes with literature value (which was calculated by

the same experimental method) of cisplatin [74a, 74b], the inhibitory activity of the

ruthenium(II) complexes against MCF-7 cell line is about three times higher than that of cisplatin

for 48-hour incubations (Table 5). The results were analyzed by cell viability curves and

expressed with IC50 values in the studied concentration range from 5 to 200 µM. The activity that

corresponds to the inhibition of cancer cell growth at a maximum level is shown in Fig. 13. The

antitumor activities of ruthenium(II) complexes are found in the following order 2 > 3 > 1. From

the above results, we observed that complex 2 showed better activity when compared to the rest

of the other complexes, which may be due to the presence of the naphthalene ring system in one

compartment of the ligand L2 discussed in the previous section.

Fig 13

Table 5

3.6.2. SRB assay

In vitro anticancer activity of ruthenium complexes 1-3 have been evaluated in terms of GI50

(concentration of drug required to decrease the cell growth to 50%, compared with that of the

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untreated cell number), TGI (concentration of drug required to decrease the cell growth to 100%,

compared with that of the untreated cell number during drug incubation) and LC50 (concentration

of drug required to decrease the cell growth by 50% of the initial cell number prior to the drug

incubation) values against human tissue of origin Hop62 (lung cancer), MDA-MB-435 (breast

cancer) by SRB assay. All the assays were performed 3 times at various concentrations. The

results showed that the complexes in vitro cytotoxic antitumor activity towards Hop62 and

MDA -MB-435 cell lines. Interestingly the complex 2 acts as a potent decrease the cell growth

against MDA-MB-435 then Hop62 cell line with pronounced GI50 values <10 µg mL−1 [75]. The

results were presented in Table 6. The antitumor activities were found in the following order 2 >

3 > 1. This result is consistent with our foregoing hypothesis.

Table 6

3.6.3. Apoptosis study by the AO/EB staining method

The results of MTT and SRB assays demonstrated that the complex 2 showed more

cytotoxicity than the complexes 1, 3 against the tumor cell lines tested. Hence the complex 2 is

selected as the test compound to further confirm the cytotoxicity of the complexes by apoptosis

assay. Generally cell death can be divided into two types: necrosis (accidental cell death) and

apoptosis (programmed cell death) [76]. Necrotic cells undergo cell lysis and lose their

membrane integrity, and severe inflammation is induced [77]. Apoptotic cells, however, are

transformed into small membrane-bound vesicles (apoptotic bodies) which are engulfed in vivo

by macrophages, and no inflammatory response is found [78]. Harmless removal of cells (cancer

cells, for example) is one consideration in chemotherapy [79]. The AO/EB staining assay can

detect the difference in membrane integrity between necrotic and apoptotic cells. AO is a vital

dye and can stain both live and dead cells. EB stains only cells that have lost their membrane

integrity. The Fig. 14. Clearly shows control cells are viable and uptake the dye acridine orange

(cells appear bright green with normal cell morphology) whereas the cells treated with complex 2

uptake the dye ethidium bromide and appears red orange with cell shrinkage indication of cell

death. So the complex 2 were found to induce the cell death by necrosis. It can be confirmed

from the results that the complex 2 have potent anticancer properties against MCF-7 cell lines.

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Fig. 14.

4. Conclusion

We have reported the synthesis of new ruthenium(II) complexes of unsymmetrical Schiff base

ligands and their characterization by various several spectral techniques. The molecular

structures of ligand L1 and complexes 1 and 3 have been confirmed by single-crystal X-ray

studies in which the complexes possess distorted octahedral geometry with the Schiff base ligand

coordinated to dibasic tetra dentate ONNS fashion. Using emission and titration methods, it was

proposed that all the complexes bound to CT-DNA via intercalating mode. In addition, all the

complexes had been found to promote the photo cleavage of pBR322 DNA and the cleavage

involved a hydrolytic mechanistic pathway. From the protein binding studies, the results showed

that the mechanism of quenching of BSA was found to be a static one indicating that the

complexes could bind to BSA via hydrophobic residues. In vitro cytotoxicity evaluation showed

that ruthenium(II) complexes displayed high antitumor activity against MCF-7, Hop62, MDA-

MB-435 cell lines and can induce the cell death by necrosis. In all the above experimental

results, we observed that complex 2 has the most significant activity, which may be due to the

presence of the naphthalene ring system in one compartment of the ligand.

Acknowledgements

The author of the manuscript (Dr. P. V.) acknowledge the University Grants Commision

(UGC), Government of India, New Delhi, for financial support in the form of a major research

project No. F.No.40-66/2011 (SR) to one of the authors (P. V.). The author (R. N) thankful to

DST for financial assistance (Project No.SR/FT/CS-95/2010). The authors would like to thank

Dr. G. Sekar, Dept of chemistry, IIT, Madras for helping ESI mass spectral analysis and

ACTREC, Tata Memorial Centre, Mumbai for carrying out the anticancer activities.

Appendix A. Supplementary material

CCDC 985978 (for L1), 943914 (for 2) and 943913 (for 4), contain the supplementary

crystallographic data for this paper. These data can be obtained free of charge via

http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data

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Centre, 12 Union Road, Cambridge CB2 1EZ, UK; [Fax: +44–1223/150 336033; e-mail:

deposit@ccdc.cam.ac.uk].

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References

1. A. Dorcier, W. H. Ang, S. Bolano, L. Gonsalvi, L. Juillerat-Jeannerat, G. Laurenczy, M.

Peruzzini, A. D. Phillips, F. Zanobini and P. J. Dyson, Organometallics 25 (2006) 4090-

4096.

2. P. M. Takahara, C. A. Fredrick and S. J. Lippard, J. Am. Chem. Soc. 118 (1996) 12309-

12321.

3. P. Schluga, C. G. Hartinger, A. Egger, E. Reisner, M. Galanski, M. A. Jakupec and B. K.

Keppler , Dalton Trans. 14 (2006) 1796-1802.

4. D. Wang and S. J. Lippard, Nature Rev. Drug Discov. 4 (2005) 307-320.

5. P. C A. Bruijnincx and P. J. Sadler, Current Opinion in Chemical Biology 12 (2008) 197-

206.

6. J. X. Ong, C. W. Yap and W. H. Ang, Inorg. Chem. 51 (2012) 12483-12492.

7. E. Reisner, V. B. Arion, M. F. C. Guedes da Silva, R. Lichtenecker, A. Eichinger, B. K.

Keppler, V. Y. Kukushkin and A. J. Pombeiro, Inorg. Chem. 43 (2004) 7083-7093.

8. C. F. Chin, Q. Tian, M. I. Setyawati, W. Fang, E. S. Q. Tan, D. T. Leong and W. H. Ang ,

J. Med. Chem. 55 (2012) 7571-7582.

9. I. Kostova., Cur. Med. Chem. 13 (2006) 1085-1107.

10. P. Heffeter, K. Bock, B. Atil , M. A. Reza Hoda, W. Körner, C. Bartel , U. Jungwirth, B.

K. Keppler, M. Micksche, W. Berger and G. Koellensperger, J. Biol. Inorg. Chem. 15

(2010) 15737-15748.

11. I. Bratsosa, S. Jednera, T. Gianferrarab, and E. Alessio, Chimia. 61 (2007) 692-697.

12. E. Gallori, C. Vettori, E. Alessio, F. G. Vilchez, R. Vilaplana, P. Orioli, A. Casini and L.

Messori, Arch. Biochem. Biophys. 376 (2000) 156-162.

13. S. Anbu , M. Kandaswamy , Pon. Sathya Moorthy , M.Balasubramanian and M.N.

Ponnuswamy, Polyhedron 28 (2009) 49-56.

14. V. Rajendiran, M. Murali, E. Suresh, M. Palaniandavar,V. S. Periasamy and

M. Abdulkader Akbarsha, Dalton Trans. 26 (2008) 2157-2170.

15. M. Ravera, S. Baracco, C. Cassino, P. Zanello and D. Osella, Dalton Trans.15 (2004)

2347-2351.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

28

16. B. Selvakumar, V. Rajendiran, P. U. Maheswari, H. Stoeckli- Evansb and M.

Palaniandavar, J. Inorg. Biochem. 100 (2006) 316-330.

17. Q. Q. Zhang, F. Zhang, W. G. Wang and X. L. Wang, J. Inorg.Biochem. 100 (2006)

1344-1352.

18. F. A. French and E. J. J. Blanz, Cancer Res. 25 (1965) 1454-1458.

19. P. Filipe, P. Morlière, L. K. Patterson, G. L. Hug , C. Maziere, J. P. Freitas, A. Fernandes

and R. Santus, Biochim. Biophys. Acta. 1572 (2002) 150-162.

20. J. Ruiz, C. Vicente, C. D. Haro and D. Bautista, Inorg. Chem. 52 (2013) 974-982.

21. U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17-53.

22. C. V. Kumar and A. Buranaprapuk, J. Am. Chem.Soc. 121 (1999) 4262-4270.

23. R. Jaiswal, M. A. Khan and J. Mussarat, J. Photochem. Photobiol. B. 67 (2002) 163-170.

24. H. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall, R. O. Gould and P. J. Sadler, J.

Am.Chem. Soc. 124 (2002) 3064-3082.

25. W. Y. Lee, Y. K. Yan, P. P. F. Lee, S. J. Tanb and K. H. Limb, Metallomics 4 (2012)

188-196.

26. E. Ramachandran, D. Senthil Raja, N. P. Rath and K. Natarajan, Inorg. Chem.52 (2013)

1504-1514.

27. R. Ramachandran, G. Prakash, S. Selvamurugan, P. Viswanathamurthi, J. G. Malecki and

V. Ramkumar Dalton Trans. 43 (2014) 7889-7902.

28. P. Anitha, P. Viswanathamurthi, B. Misini, W. Linert, Monatsh Chem. 144 (2013) 1787-

1795.

29. R. Manikandan, P. Viswanathamurthi, K. Velmurugan, R. Nandhakumar, T. Hashimoto

and A.Endo, J. Photochem.Photobiol.B. 130 (2014) 205-216.

30. R. Manikandan, N. Chitrapriya, Y. J. Jang and P. Viswanathamurthi, RSC Adv. 3 (2013)

11647-11657.

31. P. Anitha, R. Manikandan, A. Endo, T. Hashimoto and P. Viswanathamurthi,

Spectrochim. Acta Part A. 99 (2012) 174-180.

32. N. H. Khan, N. Pandya, N. C. Maity, M. Kumar, M. Rajesh, P. R. I. Kureshy, S. H. R.

Abdi, S. Mishr, S. Das and H. C. Bajaj, Eur. J. Med. Chem. 46 (2011) 5074-5085

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

29

33. V. Rajendiran, R. Karthik, M. Palaniandavar, H. S. Evans, V. S. Periasamay, M.A

Akbarsha, B. S. Srinag and H. Krishnamurthy, Inorg. Chem. 46 (2007) 8208-8221.

34. H. H. Nguyen, J. D. Castillo Gomez and U. Abram, Inorg. Chem. Comm.26 (2012) 72-

76.

35. Y. D. Kurt and B. Ulkuseven, Reviews in Inorg. Chem. 25 (2011) 1-12

36. K. Natarajan and U. Agarwala, Inorg. Nucl. Chem. Lett., 14 (1978) 7-10.

37. R. A. Sanchez-Delgado, W. Y. Lee, S. R. Choi, Y. Cho and M. J. Jun, Trans. Met. Chem.,

16 (1991) 241-244.

38. N. Ahmed, J. J. Levison, S. D. Robinson, M. F. Uttley, E. R. Wonchoba and G. W.

Parshall, Inorg. Synth.45 (1974) 15-19.

39. A. I. Vogel, Text Book of Practical Organic Chemistry, 5th ed, Longman, London, 1989.

40. G.M. Sheldrick, SHELXL97, Program for Crystal Structure Refinement, University of

Göttingen, Germany, 1997.

41. Q. Yu, Y. Liu, J. Zhang, F. Yang, D. Sun, D. Liu, Y. Zhou and J. Liu, Metallomics 5

(2013) 222-231.

42. N. Shahabadi, M. Falsafi and N. Hosseinpour Moghadam, J. Photochem.Photobiol.B.,

122 (2013) 45-51.

43. R. Loganathan, S. Ramakrishnan, E. Suresh, A. Riyasdeen, M. A. Akbarsha and M.

Palaniandavar, Inorg. Chem. 51 (2012) 5512-5532.

44. U. K. Laemmli, Nature 227 (1970) 680-685.

45. M. Blagosklong and W. S. EI-diery, Int. J. Cancer.67 (1996) 386-392.

46. V. Vichai and K. Kirtikara, Nat. Protoc.1 (2006) 1112-1116.

47. F. Arjmand, F. Sayeed, S. Parveen, S. Tabassum, A. S. Juvekar and S. M. Zingdeb,

Dalton Trans. 42 (2013) 3390-3401.

48. D. X. West, I. S. Billeh, J. P. Jasinski, J. M. Jasinski and R. J. Butcher, Transit. Met.

Chem. 23 (1998) 209-224.

49. R. Prabhakaran, V. Krishnan, K. Pasumpon, D. Sukanya, E. Wendel, C.

Jayabalakrishnan, H. Bertagnolli and K. Natarajan, Appl. Organometal. Chem. 20 (2006)

203-213.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

30

50. J. G. Malecki A. Maron, M. Serda and J. Polanski, Polyhedron, 56 (2013) 44-54.

51. R. C. Chikate and S. B. Padhye , Polyhedron, 24 (2005) 1689-1700.

52. K. Alomar, M. A. Khan, M. Allain and G. M. Bouet, Polyhedron, 28 (2009) 1273-1280.

53. A. D. Naik, S. M. Annigeri, U. B. Gangadharmathy, V. K. Revankar and V. B. Mahale, J.

Mol. Stuct. 616 (2002) 119-127.

54. P. Kalaivani, R. Prabhakaran, F. Dallemer, P. Poornima, E. Vaishnavi, E. Ramachandran,

V. Vijaya Padma, R. Renganathan and K. Natarajan, Metallomics, 4 (2012) 101-113.

55. P. Kalaivani, R. Prabhakaran, P. Poornima, F. Dallemer, K. Vijayalakshmi, V.

Vijayapadma and K. Natarajan, Organometallics, 31 (2012) 8323-8332.

56. N. Chitrapriya, V. Mahalingam, M. Zeller and K. Natarajan, Polyhedron, 27 (2008) 1573-

1580.

57. P. Sengupta, R. Dinda, S. Ghosh and W. S. Sheldrick, Polyhedron, 20 (2001) 3349-3354.

58. F. Basuli, S. M. Peng and S. Bhattacharya, Inorg. Chem. 39 (2000) 1120-1127.

59. F. Basuli, S. M. Peng and S. Bhattacharya, Inorg. Chem. 40 (2001) 1126-1133.

60. R. Prabhakaran, R. Huang, R. Karvembu, C. Jayabalakrishnan, and K. Natarajan, Inorg.

Chim. Acta. 360 (2007) 691-694.

61. a) S. Satyanarayana, J. C. Dabrowiak and J. B. Chaires, Biochemistry, 31 (1992) 9319-

9324. b) Y. J. Liu, Z. H. Liang, Z. Z. Li, C. H. Zeng, J. H. Yao, H. L. Huang, F. H. Wu,

Bio metals 23 (2010) 739–752.

62. C. Shobha Devi, D. Anil Kumar, Surya S. Singh, N. Gabra, N. Deepika, Y.

Praveen Kumar and S. Satyanarayana, Eur. J. Med. Chem. 64 (2013) 410-421.

63. S. Anbu, M. Kandaswamy and M. Selvaraj, Polyhedron, 33 (2012) 1-8.

64. C. Rajarajeswari, R. Loganathan, M. Palaniandavar, E. Suresh, A. Riyasdeen, and M. A.

Akbarshae, Dalton Trans. 42 (2013) 8347-8363.

65. M. Matson, F. R. Svensson, B. Norden and P. Lincoln, J. Phys. Chem. B, 115 (2011) 1706-1711.

66. E. S. G. Baron, R. H. DeMeio and F. Klemperer, J. Biol. Chem. 112 (1936) 625-640.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

31

67. a) Y.J. Liu, Z. H. Liang, X. L. Hong, Z. Z. Li, J. H. Yao, H. L. Huang, Inorg. Chim. Acta

387 (2012) 117–124. b) D. Senthil Raja, N. S. P. Bhuvanesh and K. Natarajan, Inorg.

Chem. 50 (2011) 12852-12866.

68. A. M. Thomas, G. C. Mandal, S. K. Tiwary, R. K. Rath and A. R. Chakravarthy, J. Chem.

Soc., Dalton Trans. 9 (2000) 1395-1396.

69. D. S. Sigman, M. D. Kuwabara, C. B. Chen and T. W. Bruice, Methods. Enzymol. 208

(1991) 414-433.

70. C. X. Wang, F. F. Yan, Y. X. Zhang and L. Ye, J. Photochem. Photobiol.A. 192 (2007)

23-28.

71. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic/ Plenum

Press, New York, 1999.

72. V. Rajendiran, R. Karthik, M. Palaniandavar, H. S. Evans, V. S. Periasamay, M. A

Akbarsha, B. S. Srinag and H. Krishnamurthy, Inorg. Chem. 46 (2007) 8208-8221.

73. F. A. Beckford, M. Shaloski, G. Leblanc, J. Thessing, L. C. L. Alleyne, A. A. Holder, L.

Li and N. P. Seeram, Dalton Trans. 48 (2009) 10757-10764.

74. a) D. Senthil Raja, N. S. P. Bhuvanesh and K. Natarajan, Dalton Trans. 41 (2012) 41

4365-4377. b) H. L. Huang, Z. Z. Li, Z. H. Liang, J. H. Yao, Y.J. Liu, Eur. J. Med. Chem.

46 (2011) 3282-3290.

75. T. Lammers, P. Peschke, V. Ehemann, J. Debus, B. Slobodin, S. Lavi and P. Huber,

Molecular Cancer, 6 (2007) 65-78.

76. I. Vermes, C. Haanen, Adv. Clin. Chem. 31 (1994) 177-246.

77. R. Van Furth, T.L. Van Zwet, J. Immunol. Methods. 108 (1988) 45-51.

78. J.S. Savill, P.M. Henson, J.E. Henson, C. Haslett, M.J. Walport, A.H. Wyllie, J.

Clin.Invest. 83 (1989) 865-875.

79. N.A. Thornberry, Y. Lazebnik, Science. 281 (1998) 1312-1316.

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Table and Figure Captions

Table 1 Crystal data and structure refinement for ligand L1 and complex 2, 4.

Table 2 Selected geometrical parameters of the ligand L1 and the complex 2, 4.

Table 3 Hydrogen bonds for L1 and complexes 2, 4 [Å and °].

Table 4 Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) for

the interactions of complexes with BSA.

Table 5 IC50 (µM) value of ruthenium(II) complexes and cisplatin against human breast cancer

cell line (MCF-7).

Table 6 Summary of the screening data (SRB assay) of selected complexes against different

tumor cell lines.

Scheme 1. Synthesis of new ruthenium(II) carbonyl complexes.

Fig. 1. ORTEP view of the molecular structure and atom labeling scheme of ligand L1. Thermal

ellipsoids are drawn at the 30% probability level.

Fig. 2. ORTEP view of the molecular structure and atom labeling scheme of complexes 2 and 4.

Thermal ellipsoids are drawn at the 30% probability level. All hydrogen atoms and chloroform

molecule have been omitted for clarity.

Fig. 3. Packing diagram of ligand L1 and ruthenium complexes 2, 4

Fig. 4. Fluorescence spectra of selected complexes 1(A), 2(B), 3(C), 4(D) in 5mM Tris-HCl

buffer at pH 7.2 and arrows an indicate absence and presence of incresing amounts of CT-DNA

concentration. [Complex=25 µM (▫▫▫▫▫▫▫ lines)], DNA = 0-50 µM.

Fig. 5. Scatchard plots of r/Cf Vs r for complexes 1(A), 2(B), 3(C) and 4(D) with incresing

concentration of CT-DNA.

Fig. 6. Fluorescence quenching curves of EB bound to CT-DNA in presence of complexes 1(A),

2(B) and MB bound to CT-DNA in presence of complexes 3(C), 4(D) in 5mM Tris-HCl buffer at

pH 7.2. Arrow shows an indicate emission intensity changes upon incresing concentration of

complexes. [DNA=7.5 µM], [EB & MB] = 7.5 µM and complexes [0-50 µM].

Fig. 7. Stern–Volmer plots of the EB-DNA fluorescence titration for complexes 1, 2 and MB-

DNA fluorescence titration for complexes 3, 4.

Fig. 8. Effect of increasing amounts of ruthenium(II) complexes 1-4 on the relative viscosity of

calf thymus DNA at 28 (±0.1) °C. The total concentration of DNA is 200 µM.

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Fig. 9. Cleavage activity of unsymmetrical ruthenium(II) complexes (1-4) monitored by 1%

agarose gel containing 5mM Tris-HCl / 50mM NaCl buffer (pH-7.2), the effect of hydrolytic

cleavage of CT-DNA incubated at 37oC with a fixed concentration of the complex for incubation

3 hr. Lane 1: DNA ladder, Lane 2: DNA control, Lane 3-6: DNA+ Complex 1-4 (50µM)

Fig. 10. The emission spectrum of bovine serum albumin (BSA) (1 µM; λex = 280 nm, λem =

344 nm) in the presence of increasing amounts (0-50 µM) of the complexes 1(A), 2(B), 3(C) and

4(D). The arrow shows that the emission intensity decreases upon the increase in concentration

of the compounds.

Fig. 11. Stern-Volmer plots (A) and Scatchard plots (B) of the fluorescence titration of the

complexes [1-4] with BSA.

Fig. 12. SDS page analysis showing cleavage of BSA (4µM) with complexes (50 µM) 1 and 2 in

5mM Tris-HCl / 50mM NaCl buffer (pH-7.2) buffer medium for an exposure hour incubated at 6

hr. Lane 1: Molecular marker, Lane 2: BSA control, Lane 3: Complex 1 + BSA, Lane 4:

Complex 2 + BSA.

Fig. 13. Growth inhibition plot showing cytotoxic effect of the complexes 1-3 in MCF-7 cells

line.

Fig. 14. Analysis of cell death induced by ruthenium complexes identified by AO/EB staining.

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Table 1 Crystal data and structure refinement for ligand L1 and complex 2, 4

L1 2 4 Identification code Shelxl Shelxl shelxl Empirical formula C21 H16 Cl2 N4 O S C45 H32 Cl4 N4 O2 P Ru S C45 H32 Cl4 As N4 O2 Ru S Formula weight 443.34 966.65 1010.59

Temperature (K) 123(2) K 293(2) K 293(2) Wavelength (Å) 0.71073 Å 0.71073 Å 0.71073 Å Crystal system Monoclinic Triclinic Triclinic Space group P 21/n Pī Pī Unit cell dimensions a (Å) 12.32585(18) Å 10.9778(5) Å 12.6775(1) b (Å) 10.33238(15) Å 11.2239(5) Å 10.4943(3) c (Å) 15.6696(3) Å 17.4630(8) Å 16.871(4) α (°) 90° 82.846(3)° 85.678(5) β (°) 96.8006(14)° 84.314(2)° 86.567(3) γ (°) 90° 88.810(2)° 89.767(5) Volume (Å3) 1981.56(5) Å3 2124.35(17) Å3 2234.1(5) Å3 Z 4 2 2 Density (calcd) MgM-3 1.486 Mg/m3 1.511 Mg/m3 1.504 Mg/m3 Absorption coefficient 0.454 mm-1 0.751 mm-1 1.411 mm-1

F(000) 912 978 1016

Crystal size ( mm3) 0.40 x 0.30 x 0.12 0.30 x 0.25 x 0.25 0.25 x 0.30 x 0.30

Theta range for data collection

2.981 to 41.056° 2.05 to 24.54° 2.33 to 27.56°

Index ranges -19<=h<=22, -12<=h<=12 -16<=h<=16 -19<=k<=18 -13<=k<=13 -13<=k<=13 -22<=l<=28 -20<=l<=20 -21<=l<=21 Reflections collected 46136 36739 39823 Independent reflections

12876 [R(int) = 0.0360]

7073 [R(int) = 0.0346] 10320 [R(int) = 0.0376]

Max.and min. transmission

1.00000 and 0.84927

0.8345 and 0.7661 0.9253 and 0.8554

Data/restraints/parameters

12876 / 0 / 263 7073 / 14 / 544 10320 / 14 / 561

Goodness-of-fit on F2 1.051 1.046 1.054

Final R indices [I>2sigma(I)]

R1= 0.0462, wR2= 0.1133

R1= 0.0355, wR2= 0.0925

R1= 0.0363, wR2= 0.0941

R indices (all data) R1= 0.0674, wR2 = 0.1252

R1= 0.0433, wR2 = 0.0988

R1= 0.0544, wR2= 0.1155

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Table 2 Selected geometrical parameters of the ligand L1 and the complex 2, 4

L1 2 4 Interatomic distances (Å) C(8)-S(1) 1.685(1) C(26)-Ru(1) 1.823(3) C(26)-Ru(1) 1.895(4) C(7)-N(1) 1.292(1) N(1)-Ru(1) 2.113(3) N(1)-Ru(1) 2.328(3) C(8)-N(3) 1.324(1) N(4)-Ru(1) 2.053(2) N(4)-Ru(1) 1.905(2) N(2)-N(1) 1.373(1) O(1)-Ru(1) 2.105(2) O(1)-Ru(1) 2.153(2) C(15)-N(4) 1.287(1) P(1)-Ru(1) 2.3010(8) As(1)-Ru(1) 2.5543(11) C(17)-O(1) 1.352(1) S(1)-Ru(1) 2.4154(8) S(1)-Ru(1) 2.2879(10) Bond angles (°) C(17)-O(1)-H(1) 109.5 O(2)-C(26)-Ru(1) 178.6(3) O(2)-C(26)-Ru(1) 175.3(3) C(7)-N(1)-N(2) 116.52(8) C(1)-N(1)-Ru(1) 100.6(2) C(1)-N(1)-Ru(1) 100.2(2) C(8)-N(2)-N(1) 119.95(8) N(3)-N(1)-Ru(1) 132.3(2) N(3)-N(1)-Ru(1) 141.33(17) C(15)-N(4)-C(14) 121.15(8) C(15)-N(4)-Ru(1) 127.8(2) C(15)-N(4)-Ru(1) 127.0(2) O(1)-C(17)-C(18) 118.11(9) C(14)-N(4)-Ru(1) 113.01(19) C(14)-N(4)-Ru(1) 109.7(2) O(1)-C(17)-C(16) 122.47(8) C(25)-O(1)-Ru(1) 125.67(18) C(25)-O(1)-Ru(1) 131.01(17) N(3)-C(8)-N(2) 116.98(9) C(33)-P(1)-Ru(1) 107.54(15) C(33)-As(1)-Ru(1) 125.53(11) N(3)-C(8)-S(1) 124.11(8) P(1)-Ru(1)-S(1) 105.14(3) As(1)-Ru(1)-S(1) 109.57(3) N(2)-C(8)-S(1) 118.88(8) N(1)-Ru(1)-S(1) 67.53(8) N(1)-Ru(1)-S(1) 64.72(6) N(1)-C(7)-C(1) 117.58(8) O(1)-Ru(1)-S(1) 86.75(6) O(1)-Ru(1)-S(1) 90.43(6) N(1)-C(7)-C(9) 122.86(8) N(4)-Ru(1)-S(1) 155.58(7) N(4)-Ru(1)-S(1) 152.20(7) N(3)-C(8)-N(2) 116.98(9) C(26)-Ru(1)-S(1) 93.22(10) C(26)-Ru(1)-S(1) 88.62(10) N(3)-C(8)-S(1) 124.11(8) N(1)-Ru(1)-P(1) 172.46(8) N(1)-Ru(1)-As(1) 174.01(6) O(1)-Ru(1)-P(1) 92.29(6) O(1)-Ru(1)-As(1) 84.20(6) N(4)-Ru(1)-P(1) 97.91(7) N(4)-Ru(1)-As(1) 96.59(8) C(26)-Ru(1)-P(1) 88.50(10) C(26)-Ru(1)-As(1) 97.48(13) O(1)-Ru(1)-N(1) 85.63(9) O(1)-Ru(1)-N(1) 93.81(8) N(4)-Ru(1)-N(1) 89.11(10) N(4)-Ru(1)-N(1) 88.66(9) C(26)-Ru(1)-N(1) 93.61(12) C(26)-Ru(1)-N(1) 84.88(12) N(4)-Ru(1)-O(1) 84.36(9) N(4)-Ru(1)-O(1) 83.06(9) C(26)-Ru(1)-O(1) 179.19(12) C(26)-Ru(1)-O(1) 178.27(14) C(26)-Ru(1)-N(4) 95.35(12) C(26)-Ru(1)-N(4) 97.21(14) C(1)-S(1)-Ru(1) 79.28(11) C(1)-S(1)-Ru(1) 86.41(11) C(27)-P(1)-Ru(1) 117.56(12) C(27)-As(1)-Ru(1) 115.89(16)

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Table 3 Hydrogen bonds for L1 and complexes 2 and 4 [Å and °] Compound D–H….A d(D–H) d(H…A) d(D….A) <(DHA) L1 O(1)-H(1)-N(4) 0.84 1.89 2.6242(11) 145.7 N(3)-H(3)-S(1) 0.88 2.73 3.4822(11) 144.9 N(3)-H(3)-O(1) 0.88 2.62 3.1966(14) 123.8 C(15)-H(15A)-S(1) 0.95 2.85 3.7378(10) 156.7 C(19)-H(19A)-Cl(1) 0.95 2.78 3.6996(10) 156.7 C(21)-H(21A)-S(1) 0.95 2.92 3.7934(10) 153.5 2 N(2)-H(2A)-O(1) 0.839 2.421 2.944 114.24(2) 4 N(2)-H(2A)-O(1) 0.839 2.421 2.944 114.24(2) aSymmetry operation: Ligand L1: 'x, y, z'; '-x+1/2, y+1/2, -z+1/2'; '-x, -y, -z'; 'x-1/2, -y-1/2, z-1/2' Complex 2: 'x, y, z' ; '-x, -y, -z', Complex 4: 'x, y, z' ; '-x, -y, -z'; b[D= donator, A=acceptor]

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Table 4 Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) for the interactions of complexes with BSA. Complex Kq (M

-1) Kbin (M-1) n-value

1 3.36×104 ±(0.09) 5.44×104± (0.11) 1.49 2 2.33×105 ±(0.14) 7.71×104 ±(0.13) 2.22 3 4.03×104 ±(0.07) 4.12×104 ± (0.16) 1.55 4 5.30×104 ±(0.11) 7.29×104 ± (0.10) 1.41

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Table 5 IC50 (µM) value of ruthenium(II) complexes and cisplatin against Human breast cancer cell line (MCF-7)

Complex IC50 (µM)a MCF-7

1 4.67 ± 0.2 2 3.78 ± 0.6 3 4.45 ± 0.7 Cisplatinb 12.75±0.8

a Fifty percent inhibitory concentration after exposure For 48 h in the MTT assay.

bData from [74].

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Table 6 Summary of the screening data (SRB assay) of selected complexes against different tumor cell lines

Hop62 MDA-MB-435 Complex aLC50

aTGI aGI50 aLC50

aTGI aGI50 1 >100 >100 55.5±2.49 >100 >100 36.5±4.52 2 >100 72.4 32.6±4.12 >100 89.4 >0.1±0.03 3 >100 89.1 41.8±3.97 >100 >100 >3.01±2.35 ADR >100 >100 >0.1±0.05 >100 76.5 >0.1±0.02 a = µg mL−1 aGI50 = growth inhibition of 50% (aGI50) calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, drug concentration result in a 50% reduction in the net protein increase. Tz = absorbance measurements at time zero. Ti = test growth in the presence of a drug at the different concentration levels. aTGI = tumor growth inhibition, aLC50 = lethal concentration of 50% (aLC50). aGI50 value <10 µg mL−1 is considered to demonstrate activity. ADR = Adriamycin (taken as positive control compound).

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Ru

O

4N 1N

S

3N

Ph

2NH2

H

Cl

CO

EPh3

Cl

OH

4N 1NH

S

3N

Ph

2NH2

H

Cl

Cl

E = P or As (1, 3)

[RuHClCO(EPh3)3]

CH3OH / CHCl3

Ru

O

4N 1N

S

3N

Ph

2NH2

H

Cl

CO

EPh3OH

4N 1NH

S

3N

Ph

2NH2

H

ClE = P or As (2, 4)

[RuHClCO(EPh3)3]

CH3OH / CHCl3

Scheme 1. Synthesis of new ruthenium(II) carbonyl complexes.

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Fig. 1. ORTEP view of the molecular structure and atom labeling scheme of ligand L1. Thermal ellipsoids are drawn at the 30% probability level.

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Fig. 2. ORTEP view of the molecular structure and atom labeling scheme of complexes 2 and 4. Thermal ellipsoids are drawn at the 30% probability level. All hydrogen atoms and chloroform molecule have been omitted for clarity.

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Fig. 3. Packing diagram of ligand L1 and ruthenium complexes 2, 4

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Fig. 4. Fluorescence spectra of selected complexes 1(A), 2(B), 3(C), 4(D) in 5mM Tris-HCl buffer at pH 7.2 and arrows an indicate absence and presence of incresing amounts of CT-DNA concentration. [Complex=25 µM (▫▫▫▫▫▫▫ lines)], DNA = 0-50 µM.

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Fig. 5. Scatchard plots of r/Cf Vs r for complexes 1(A), 2(B), 3(C) and 4(D) with incresing concentration of CT-DNA.

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Fig. 6. Fluorescence quenching curves of EB bound to CT-DNA in presence of complexes 1(A), 2(B) and MB bound to CT-DNA in presence of complexes 3(C), 4(D) in 5mM Tris-HCl buffer at pH 7.2. Arrow shows an indicate emission intensity changes upon incresing concentration of complexes. [DNA=7.5 µM], [EB & MB] = 7.5 µM and complexes [0-50 µM].

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Fig. 7. Stern–Volmer plots of the EB-DNA fluorescence titration for complexes 1, 2 and MB-

DNA fluorescence titration for complexes 3, 4.

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Fig. 8. Effect of increasing amount of 1 (▪), 2 (▼), 3 (•), and 4 (▲) on the relative viscosities of

CT-DNA in 5 mM Tris–HCl buffer.

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Fig. 9. Cleavage activity of unsymmetrical ruthenium(II) complexes (1-4) monitored by 1% agarose gel containing 5mM Tris-HCl / 50mM NaCl buffer (pH-7.2), the effect of hydrolytic cleavage of CT-DNA incubated at 37oC with a fixed concentration of the complex for incubation 3 hr. Lane 1: DNA ladder, Lane 2: DNA control, Lane 3-6: DNA+ Complex 1-4 (50µM)

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Fig. 10. The emission spectrum of bovine serum albumin (BSA) (1 µM; λex = 280 nm, λem = 344 nm) in the presence of increasing amounts (0-50 µM) of the complexes 1(A), 2(B), 3(C) and 4(D). The arrow shows that the emission intensity decreases upon the increase in concentration of the compounds.

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Fig. 11. Stern-Volmer plots (A) and Scatchard plots (B) of the fluorescence titration of the complexes [1-4] with BSA.

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Figure. 12. SDS page analysis showing cleavage of BSA (4µM) with complexes (50 µM) 1 and 2 in 5mM Tris-HCl / 50mM NaCl buffer (pH-7.2) buffer medium for an exposure hour incubated at 6 hr. Lane 1: Molecular marker, Lane 2: BSA control, Lane 3: Complex 1 + BSA, Lane 4: Complex 2 + BSA.

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Fig. 13. Growth inhibition plot showing cytotoxic effect of the complexes 1-3 in MCF-7 cells

line.

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Fig. 14. Analysis of cell death induced by ruthenium complexes identified by AO/EB staining.

MCF-7 cells without treatment (A) and in the presence of complex 2 (B). MCF-7 cells were treated with ruthenium(II) complex (50µM) and incubated for 24 h at 37oC. Cells in a, b and c are living, apoptic and necrotic cells, respectively.

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Research Highlights

Dissymmetric thiosemicarbazone ligands containing substituted aldehyde arm

and their ruthenium(II) carbonyl complexes with PPh3/AsPh3 as ancillary

ligands: Synthesis, Structural characterization, DNA/BSA interaction and in

vitro anticancer activity

Paranthaman Vijayana, Periasamy Viswanathamurthi*a, Vaidhyanathan Silambarasanb,

Devadasan Velmuruganb, Krishnaswamy Velmuruganc, Raju Nandhakumarc, Ray Jay Butcherd,

Tamilselvan Silambarasane and Ramamurthy Dhandapanie

� New ruthenium(II) carbonyl complexes were synthesized and structurally

characterized.

� The complexes exhibited intercalative mode of interaction with CT-DNA and bind with BSA via static interaction.

� The MTT and SRB assays demonstrated that the complexes possess cytotoxic

activity against various cancer cell lines.

� The mode of cell death was observed by AO/EB staining method.