Accepted Manuscript

Synthesis, structure, DNA/BSA interaction and in vitro cytotoxic activity ofnickel(II) complexes derived from S-allyldithiocarbazate

Nanjan Nanjundan, Ponnusamy Selvakumar, Ramaswamy Narayanasamy,Rosenani A. Haque, Krishnaswamy Velmurugan, Raju Nandhakumar,Tamilselvan Silambarasan, Ramamurthy Dhandapani

PII: S1011-1344(14)00309-1DOI: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.009Reference: JPB 9861

To appear in: Journal of Photochemistry and Photobiology B: Bi-ology

Received Date: 22 April 2014Revised Date: 6 October 2014Accepted Date: 11 October 2014

Please cite this article as: N. Nanjundan, P. Selvakumar, R. Narayanasamy, R.A. Haque, K. Velmurugan, R.Nandhakumar, T. Silambarasan, R. Dhandapani, Synthesis, structure, DNA/BSA interaction and in vitro cytotoxicactivity of nickel(II) complexes derived from S-allyldithiocarbazate, Journal of Photochemistry and PhotobiologyB: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.009

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Synthesis, structure, DNA/BSA interaction and in vitro cytotoxic activity of nickel(II) complexes derived from S-allyldithiocarbazate Nanjan Nanjundan,a Ponnusamy Selvakumar,a Ramaswamy Narayanasamy,a* Rosenani A. Haque,b Krishnaswamy Velmurugan,c Raju Nandhakumar,c Tamilselvan Silambarasan,d

and Ramamurthy Dhandapani d aDepartment of Chemistry, Coimbatore Institute of Technology, Coimbatore - 641 014, India.

bThe School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

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

dDepartment of Microbiology, Periyar University, Salem - 636 011, India

* Corresponding author. Fax: + 91-422-2575020

E-mail address: narayanasamycit@gmail.com (R. Narayanasamy)

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Abstract

Two nickel(II) complexes with formula NiL1 and NiL2 (HL1 = S-allyl-4-methoxybenzylidene

hydrazinecarbodithioate, HL2 = S-allyl-1-napthylidenehydrazinecarbodithioate) have been

synthesized and characterized by elemental analysis, FT-IR, NMR, UV-vis spectroscopy and ESI

mass spectrometry. The crystal structure of complex 1 has been determined by single crystal X-

ray diffractometry. Both HL1 and HL2 ligands are coordinated to the metal in thiolate form. In

complexes, squareplanar geometry of the nickel is coordinated with two bidentate ligand units

acting through azomethine nitrogen and thiolato sulfur atoms. To explore the potential medicinal

value of the complexes with calf thymus DNA and bovine serum albumin (BSA) were studied at

normal physiological conditions using fluorescence spectral techniques. The DNA binding

constant values of the complexes were found in the range from 5.02 × 104, 3.54 × 104, and the

binding affinities are in the following order 1 > 2. In addition, nickel complexes 1 and 2 shows

better binding propensity to the bovine serum albumin (BSA) protein, giving a Ksv value 5.8 ×

104 , 4.47 × 104 respectively. From the oxidative cleavage of the complexes with pBR322 DNA,

it is inferred that the effects of cleavage are dose-dependent. In addition, in vitro cytotoxicity of

the complexes assayed against Vero and HeLa cell lines have shown higher cytotoxic activity

with the lower IC50 values indicating their efficiency in killing cancer cells even at various

concentrations.

Keywords:

Nickel(II) complex

DNA / BSA interaction

In vitro anticancer activity

3

Introduction

Numerous biological experiments have demonstrated that DNA is the primary intracellular target

of many anticancer drugs, cancerogens and viruses [1–3]. Knowledge of interactions between

external small molecules and DNA is important to understand how nucleic acids function in

biological systems. Among these small molecules, transition metal complexes containing

bidentate aromatic ligands gained much attention due to their significant physicochemical

properties and possible application as new therapeutic agents [4, 5]. Nickel, an essential element

involved in life process can promote absorption of iron, increase the red blood corpuscle and

synthesis of certain amino-enzymes in the body [6]. Nickel complexes have also drawn much

attention due to their environmental toxicity, carcinogenic nature and chemotherapeutic value in

the past [7]. It has been found that these complexes inhibit DNA repair by interfering with

enzymes or proteins involved in DNA replication and/or DNA repair [8]. The nickel

coordination sphere in both of these metalloenzyme systems contains N and S donor atoms in

unusual 5 or 6 coordinate arrangements with significant distortions from regular geometry. These

distorted configurations often give rise to nickel centres with reversible Ni(II)/Ni(I) and

Ni(III)/Ni(II) couples and low Ni(III)/Ni(II) redox potentials, characteristics which are crucial to

the activity of the enzymes. These unusual structural and electronic features have led to

increased interest in the synthesis of Ni(II) complexes with mixed N, S donating chelates as

structural and spectroscopic models of the active sites [9-11]. Dithiocarbazates constitute an

important class of mixed hard-soft nitrogen-sulfur donor ligands [12, 13]. There is continuing

interest in the coordination chemistry of heterocyclic bidentate ligands containing NS donor set

[14]. S-substituted dithiocarbazate Schiff base compounds, RCH=NNHC(S)SR’ are known to

coordinate via extended π-conjugation through ligand deprotonation [15] and give corresponding

nickel(II) ion complexes that have attracted considerable interest because of the occurrence of

square planar via N2S2 coordination mode [16]. Allyl group in the molecular structure is inclined

for many interesting chemical reactions and can be used as a ligand [17-19]. Although most

previous studies on nickel (II) complexes and their biological studies based on Schiff base

ligands were derived from S-allyldithiocarbazates, they had not received much attention.

In this present work, Schiff base metal complexes from S-allyldithiocarbazate and nickel(II)

complexes have been synthesized and characterized. The interaction of nickel(II) complexes with

4

CT-DNA / BSA protein is investigated by fluorescence spectroscopy. DNA cleavage ability has

been monitored by gel electrophoresis in the presence of the complexes with pBR322 DNA. In

vitro cytotoxic activities of the complexes are tested by MTT assays against human cervical

HeLa cancer cell lines and normal Vero cell lines. The synthetic route of Schiff base ligands and

their nickel(II) complexes are shown in Scheme 1.

Experimental

Materials and methods

All chemicals and reagents were obtained from commercial sources and used as received unless

otherwise stated. Solvents were distilled using appropriate drying agent. Ni(OAc)2.4H2O,

Methylene blue (MB) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich

and used as received. The calf-thymus DNA (CT-DNA) and pBR322 DNA was purchased from

Bangalore GeNei, Bangalore, India. The Vero and human cervical HeLa cancer cell lines was

obtained from the National Centre for Cell Science (NCCS), Pune, India and grown in Dulbeccos

Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were

maintained at 370C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were

passaged weekly, and the culture medium was changed twice a week. Stock solutions were

stored at 4oC and used within 4 days. Concentrated stock solutions of metal complexes were

prepared by dissolution of calculated amounts of metal complexes in a corresponding amount of

solvent and were diluted suitably with the corresponding buffer to the required concentrations for

all experiments. All measurements about interactions of the complexes with CT-DNA were

conducted using solutions of the corresponding complex containing 50 mM NaCl / 5 mM Tris–

HCl at room temperature (pH = 7.2).

Physical measurements

Microanalyses of carbon, hydrogen, nitrogen and sulfur were carried out using Vario EL III

Elemental analyzer at SAIF–Cochin, India. The IR spectra of the ligand and its complexes were

obtained as KBr pellets on a Nicolet Avatar model spectrophotometer at 4000 - 400 cm-1 range.

Electronic spectra of the ligand and its complexes have been recorded in NaCl/Tris-HCl buffer

using a Shimadzu UV-1650 PC spectrophotometer at 800 - 200 nm range. Emission spectra were

5

measured with a Jasco FP 6600 spectrofluorometer. 1H-NMR spectra were recorded in Jeol

GSX-400 instrument at room temperature using tetramethylsilane as the internal standard in

dimethylsulfoxide (DMSO-d6) as solvent. ESI-MS spectra were recorded using LC-MS Q-ToF

Micro analyzer (Shimadzu) in the SAIF, Punjab University, Chandigarh. Melting points were

checked on a Technico micro heating table.

Ni(CH3COO)2.4H2O

H3CO

NNH

H

S

S

NNH

H

S

S

CH3OH

H3CO

N

H N

S

S

OCH3

N

HN

S

S

Ni

N

H N

S

S

N

HNS

S

Ni

Scheme 1 Synthetic route of nickel(II) complexes.

Crystallography

Suitable crystals for X-ray diffraction studies were grown from CH2Cl2/ethanol mixture. X-ray

diffraction data were collected at 293K using a SHELXL-97 detector diffractometer using

monochromated MoKα (k = 0.7107300 Å) radiation. Calculations, structure refinement,

molecular graphics and the material for publication were performed using SHELXTL and PLATON

(Spek, 2009) software packages.

6

Synthesis of Schiff base ligands (HL1)

Schiff base was prepared as per the procedure adopted in a previous study [20]. A mixture of 5

mL (5.06 g, 0.1 mol) hydrazine hydrate and 5.6 g (0.1 mol) KOH in 30 mL of ethanol at 5oC, a

solution of carbon disulphide 6.1 mL (7.61 g, 0.1 mol) and allyl bromide 8.6 mL (12.10 g, 0.1

mol) were added successively with continuous stirring in an ice bath. To this mixture, a solution

of 4-methoxybenzaldehyde 12.14mL (13.6 g, 0.1 mol) in ethanol (50 mL) was added heating and

stirring was continued for 10 min. Then, the pale yellow solid formed was separated, washed

with water and dried under vacuum. The compound was crystallized from dichloromethane/

methanol (15:15 v/v) as yellow color single crystals formed after 3 days at 27oC. Yield: 89%;

color: pale yellow, M.P. 162-165oC. Anal. Calc. for C12H14N2OS2 requires: C, 54.11; H, 5.30; N,

10.52; S, 24.07. Found: C, 54.02; H, 5.28; N, 10.41; S, 24.15 %. Selected IR data (KBr pellet,

cm-1): 3,107 (s) ν(NH), 1,599 (s) ν(C=N), 1,097 (s) ν(C=S), 1,028 (s) ν(N–N), 983 (m) ν(C–S–

S),1275 ν(C–O–C). 1H NMR (300 MHz, DMSO-d6,δ, ppm): 3.99 (2H, SCH2), 5.1 (2H, CH2),

5.75-5.86 ( H, CH), 6.98-7.60 (4H, aromatic), 7.9 (1H, CH=N), 11.6 (1H, NH), 4.1 (3H, OCH3);

UV-Vis (Tris HCl buffer, nm); 237, 364.

Synthesis of Schiff base ligands (HL2)

It was prepared by a similar procedure as described for HL1 from S-allyldithiocarbazate and

1-napthaldehyde. Yield: 81%; Color: Yellow plates, M.P. 171-175oC; Anal. Calc. for

C15H14N2S2 requires: C, 62.90; H, 4.93; N, 9.78; S, 22.39. Found: C, 62.78; H, 4.90; N, 9.70; S,

22.45 %. Selected IR data (KBr pellet, cm-1): 3,120 (s) ν (N–H), 1,610 (s) ν(C=N), 1,095 (s)

ν(C=S), 1,040 (s) ν(N–N), 975 (m) ν(C–S–S). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.80

(2H, SCH2), 5.2 (2H, CH2), 5.70-5.81 (1H, CH), 6.92-7.88 (7H, aromatic), 8.1 (1H, CH=N),

12.98 (1H, NH); UV-Vis (Tris HCl buffer, nm); 250, 372.

Synthesis of nickel(II) Schiff base complexes

All the new metal complexes were prepared according to the following general procedure. A

warm methanolic solution (15 mL) containing HL1/HL2 (2 mmol) was added to an methanolic

solution of (15 mL) [Ni(OAc)2.4H2O ] (1 mmol). The resulting brown color solution was heated

7

for 5 minutes. Dark brown colored crystalline needle plate was obtained on slow evaporation,

were filtered, washed with methanol, dried under vacuo.

Synthesis of [Ni (HL1)2] (1)

Yield: 75%; Color: Brown; M.P.: 212-223ºC; Anal. Calc. for C24H26N4NiO2S4 requires: C,

48.90; H, 4.45; N, 9.51; S, 21.76%. Found: C, 49.45; H, 4.84; N, 9.45; S, 21.65 %. Selected IR

data (KBr pellet, cm-1): 1,585 (s) ν(C=N), 1,024 (s) ν(N–N), 955 (m) ν(C–S–S),1265 ν(C–O–C). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.65 (2H, SCH2), 5.08 (2H, CH2), 5.70-5.82 ( 1H, CH),

6.92-7.85 (8H, aromatic), 8.4 (1H, CH=N), 3.06 (3H,OCH3); UV-Vis (Tris-HCl buffer); 243,

275, 381, 414, 437. ESI-MS, m/z (%): 589.1 [M + H]. This complex 1 was recrystallized from

CH2Cl2 / Ethanol suitable for X-ray work.

Synthesis of [Ni (HL2)2] (2)

Yield: 65%; Color: Brown; M.P.: 233-245ºC. Anal. Calc. for C30H26N4NiS4 requires: C, 57.24;

H, 4.16; N, 8.90; S, 20.37%. Found: C, 57.01; H, 4.88; N, 8.12; S, 20.31%. Selected IR data

(KBr pellet, cm-1): 1,595 (s) ν(C=N), 1,020 (s) ν(N–N), 960 (m) ν(C–S–S). 1H NMR (400 MHz,

DMSO-d6, δ, ppm): 3.72 (2H, SCH2), 5.14 (2H, CH2), 5.65-5.78 ( H, CH), 6.8-7.75 (14H,

aromatic), 8.5 (1H,CH=N); UV-Vis (Tris HCl buffer, nm); 236, 271, 385, 413, 442 ESI-MS, m/z

(%): 629.1 [M + H].

DNA binding studies For the evaluation of antitumor property of any new compound, DNA binding is the predominant

property looked for in pharmacology and hence, the interaction between DNA and nickel(II)

complexes is of paramount importance [21]. Hence, the binding mode and tendency of nickel(II)

complexes to CT-DNA were studied with different physicochemical methods at room

temperature.Luminescence measurement was performed to clarify the binding affinity of

nickel(II) complexes by emissive titration at room temperature. The complexes were dissolved in

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

pH = 7.2) for all the experiments and stored at 4oC for further use. Tris-HCl buffer was

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

8

range and adjusted before measurements. Emissive titration experiments were performed with a

fixed concentration of metal complexes (25 µM). While gradually increasing the concentration

of DNA (0-25 µM), the emission intensities were recorded in the range of 390-450 nm at an

excitation wavelength of 414 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 experiment. MB is a planar cationic dye, well-known to intercalate into the

CT-DNA independently. While MB is most fluorescent compounds, MB-DNA adduct is a strong

emitter on excitation near 600 nm. For all the experiments, DNA was pretreated with MB in the

ratio [DNA] / [MB] = 10 for 30 minutes at 37oC. Then, the titration solutions were added to MB-

DNA mixture, fluorescence intensity changes were measured.

DNA cleavage studies

The interaction of complexes with supercoiled pBR322 DNA was monitored using agarose gel

electrophoresis. In reactions using supercoiled pBR322 plasmid DNA in 5% CH3OH–5 mM

Tris-HCl 50 mM NaCl buffer (0.5:9.5 v/v) at pH 7.2 was treated with metal complexes at various

concentrations in the same buffer. For photocleavage studies, reactions were carried out under

illuminated conditions at 365 nm (12 W) monochromatic light source. The samples were then

incubated for 1 h in the dark for 370C and analysed for the photocleaved products using gel

electrophoresis as discussed below. A loading buffer containing 25% bromophenol blue, 0.25%

xylene cyanol and 30% glycerol (3µL) was added and electrophoresis performed at 60 V for 5 h

in Tris–acetate–EDTA (TAE) buffer (40mM Tris-base, 20mM acetic acid, 1mM EDTA) using

1% agarose gel containing 1.0 µg mL−1 ethidium bromide [22] . The gels were viewed in a Gel

doc system and photographed.

Protein binding studies

The excitation wavelength of BSA at 280 nm, emission at 345 nm were monitored for the protein

binding studies. The excitation, emission slit widths and scan rates were maintained constant for

all the experiments. Samples were carefully degassed using pure nitrogen gas for 15 min. Quartz

9

cells (4×1×1 cm) with high vacuum Teflon stopcocks were used for degassing. Stock solution of

BSA prepared in 50 mM phosphate buffer (pH-7.2), stored in the dark at 4°C for further use

Tris-HCl buffer was used for all the titrations. Titrations were manually done by using a

micropipette for the addition of the complexes.

Cytotoxicity Studies

Vero and HeLa cancer cell line was obtained from National Centre for Cell Science (NCCS),

Pune, India, and cell viability was assessed by MTT (3,4,5-dimethylthiazolyl-2-2,5-

diphenyltetrazolium bromide) method. Vero and HeLa cells were maintained in a humidified

atmosphere containing 5% CO2 at 37°C in DMEM medium supplemented with 100 units of

penicillin, 100 µg/mL of streptomycin, and 10% fetal bovine serum. Briefly, Vero and HeLa

cells with a density 1×104 cells per well were precultured in 96-well microtiter plates for 48 h

under 5% CO2. Then, each well was loaded 10 µL MTT solution (5 mg mL−1 in PBS pH-7.4) for

4 h at 37°C. The insoluble formazan was dissolved in 100 µL of 4% DMSO and the cell viability

was determined by measuring the absorbance of each well at 570 nm using Bio-Rad 680

microplate reader. All experiments were performed in triplicate and the percentage of cell

viability was calculated according to the following equation [23].

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

After 48 h, the cells were observed with an inverted phase contrast microscope, photographed

with a Nikon FM 10 camera.

Results and Discussion

Synthesis and characterization

In present work, the synthesis of Schiff base and their complexes in 1:2 ratio by the direct

reaction as shown in scheme 1. 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 are in good agreement with the

proposed molecular formula.

10

The IR spectra of S-allyldithiocarbazate ligands and their corresponding nickel complexes were

compared in the region 4000-400 cm-1. The ligands showed a sharp peak at 1599-1610 cm-1

corresponding to ν(C=N) of the azomethine group. In the IR spectra of nickel(II) complexes 1

and 2, ν(C=N) was shifted to lower frequencies (1585-1595cm-1) indicating that the azomethine

group is bound to nickel atom. The wide band is due to ν(NH) appeared in the region 3107-

3120 cm-1 in ligands that have completely disappeared after complexation showed NH group

involved in thione-thiol tautomerization since, it contains a thioamide (-NH-C=S) functional

group [24]. Furthermore, a well-built band appeared at 1095-1097 cm-1 in the spectra of the

ligand is indicative of ν(C=S). The lower of this band in the form of ν(C-S) can be attributed to

negative coordination of the ligands and nickel atom through the thio-sulfur atom. Hence, all the

above facts are in good agreement with the complexes coordinated as a square planar geometry

via N2S2 fashion. S-allyldithiocarbazate ligands with nickel(II) complexes were assigned, based

on the observed chemical shifts. The spectra of asymmetrical Schiff base ligands displayed a

weak singlet sharp signal at 11.6-12.98 ppm NH protons. However, NMR spectra of nickel(II) S-

allyldithiocarbazate complexes did not register any signal equivalent to NH, revealed that the

ligands adopted thione-thiol tautomerism (-NH-C=S), followed by deprotonation prior to

coordination with the metal ion. In addition, all the ligands showed a sharp singlet for

azomethine (-H-C=N) proton at 7.9-8.1 ppm [25]. The multiple protons of aromatic ring moiety

of the ligands and metal complexes were observed as multiplets in the range of 6.8-7.85 ppm.

The positive ion ESI mass spectra of the unsymmetrical Schiff base nickel(II) complexes 1 and 2

showed major peaks at m/z = 589.1, 629.1 respectively, which have been assigned to the [M +

H]+ . The electronic spectral data for the ligands and their complexes are recorded in the range of

200–900 nm (5 mM Tris-HCl/50 mM NaCl buffer for pH = 7.2). The two bands at 237-250 nm

are attributed π→ π* of ring and dithiocarbazate moiety. The n → π* transition was observed at

364 and 370 nm in the free ligand, but after complexation just a broad band was detected at 375

nm. The intense band at 414 nm is LMCT band. The tail of this band covers all d–transitions.

The crystal structures of nickel(II) complex 1 were determined by single crystal X ray diffraction

method, and ORTEP drawings shown in Fig.1 Crystallographic data and bond parameters are

given in Tables 1 and 2.

11

Fig. 1. ORTEP views of the molecular structure and the atom labeling scheme of complex 1.

Thermal ellipsoids are drawn at 50% probability level. Disorder molecules have been omitted for

clarity.

Table 1 Crystal data and structure refinement for complex 1 Complex 1 Identification code SHELXL 97 Empirical formula C13.50H17N2.50Ni0.50O2S2 Formula weight 339.77 Temperature 293(2) Wavelength 0.71073 A˚ Crystal system Triclinic Space group Pī Unit cell a (Å) 8.5274(10)

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b (Å) 11.7318(2) c (Å) 15.8368(3) α (Å) 107.6680(10) β (Å) 98.0150(10) γ(Å) 99.7740(10) Volume (Å3) 1456.61(4) Z 4 Density (calculated) MgM-3 1.549 Absorption coefficient 0.997 mm-1 F(000) 710 Crystal size 0.32×0.23×0.17mm Theta range for data collection(0) 1.38 to 30.13 Reflections collected 32164 Independent reflections

8589

Index ranges -12<=h<=11 -16<=k<=15 0<=l<=22

Max .and min. transmission 0.9485 and 0.6994 Data/restraints/parameters 8589/0/374 Goodness-of-fit on F2 1.059 Final R indices [I>2sigma(I)] R indices (all data)

R1= 0.0487, wR2= 0.1358 R1= 0.0487, wR2= 0.1358

Table 2 Selected geometrical parameters of the complex 1 Bond lengths (Å) Bond angles (0)

Ni(1)-N(1) 1.9252(18) N(1)-Ni(1)-N(3) 178.64(7)

Ni(1)-N(3) 1.9281(17) N(1)-Ni(1)-S(2) 94.36(5)

Ni(1)-S(2) 2.1764(6) N(3)-Ni(1)-S(2) 85.62(5)

Ni(1)-S(1) 2.1820(6) N(1)-Ni(1)-S(1) 85.77(5)

S(1)-C(1) 1.729(2) N(3)-Ni(1)-S(1) 94.30(5)

S(2)-C(10) 1.726(2) S(2)-Ni(1)-S(1) 178.08(2)

S(3)-C(1) 1.741(2) C(1)-S(1)-Ni(1) 95.93(7)

S(3)-C19) 1.823(2) C(10)-S(2)-Ni(1) 96.04(7)

13

S(4)-C(10) 1.745(2) C(1)-S(3)-C(19) 102.62(10)

S(4)-C(22) 1.820(2) C(10)-S(4)-C(22) 102.12(10)

N(1)-C(2) 1.296(3) C(6)-O(1)-C(9) 118.1(2)

N(1)-N(2) 1.414(2) C(15)-O(2)-C(18) 117.56(18)

N(2)-C(1) 1.297(3) C(2)-N(1)-N(2) 114.33(17)

N(3)-C(11) 1.306(3) C(2)-N(1)-Ni(1) 125.40(15)

N(3)-N(4) 1.405(2) N(2)-N(1)-Ni(1) 120.27(13)

N(4)-C(10) 1.300(3) C(1)-N(2)-N(1) 111.71(17)

C(2)-C(3) 1.462(3) C(11)- N(3)- N(4) 114.63(17)

C(11)-C(12) 1.451(3) C(11)-N(3)-Ni(1) 124.52(15)

N(4)-N(3)-Ni(1) 120.85(13)

C(10)-N(4)-N(3) 111.35(17)

N(2)-C(1)-S(1) 124.43(17)

N(2)-C(1)-S(3) 120.82(16)

S(1)-C(1)-S(3) 114.75(11)

N(1)-C(2)-C(3) 132.8(2)

The complex 1 crystallizes in triclinic system with space group Pī with 2 independent molecules

within the unit cell. The Schiff base is coordinated to nickel(II) ion in its deprotonated

iminothiolate form. This form results in delocalization of the negative charge caused by

deprotonation of NH proton as indicated by the intermediate C2-N1, N1-N2, N2-C1 bands. Two

ligands coordinate to nickel(II) atom to form two five-membered chelate rings Ni1-N1-N2-C1-

S1. The coordination of nickel(II) is monomeric and distorted squareplanar with two equivalent

Ni-N1 (1.9252(18) A˚) and Ni-S1 (2.1820(6) A˚) bonds. The coordinated mercapto sulfur and

methine nitrogen atoms in the two ligands are in opposite positions so that the complex has trans-

configuration allyl group moieties on the opposite side and thio S and imine N atoms are cis to

each other [26]. The bond distance C1-N2, 1.297(3) A˚ and C1-S1, 1.729(2) A˚ suggests a C=N

bond and a C-S single bond. One interesting feature of our complex is the presence of two equal

14

C-N bonds (N2-C1, N1-C2). The nickel(II)-donor atom distances, namely Ni-S1 [2.1820(6)A˚],

Ni-N1 [1.9252(18) A˚]. This distortion may be attributed to the restricted bite angles to be

imposed by the planar bidentate Schiff bases and their nickel(II) complexes.

Fluorescence spectroscopic studies

DNA binding is a significant footstep for chemical nuclease activity of the metal complexes.

Therefore, before evaluating the potentials of antitumor activities of the new complexes,

interaction between DNA and the newly synthesized complexes was examined. A complex

bound to DNA through intercalation is characterized by the change in emission (hypochromism)

and red shift in wavelength, due to the intercalative mode involving a stacking interaction with

hydrophobic environment between the aromatic chromophore and the DNA base pairs [27]. The

results of luminescence spectra of the compounds in the absence and presence of CT-DNA are

given in Fig. 2.

Fig. 2. Fluorescence spectra of nickel(II) complexes 1 and 2 in 5 mM Tris-HCl/ 50mM NaCl

buffer at pH=7.2 and arrows indicate absence and presence of increasing amounts of CT‒DNA

concentration at 25oC. [Complex = 25 µM (---- lines)], DNA = 0‒25 µM.

Upon increasing the concentration of DNA to the test complexes, the emission bands of the

complexes 1 and 2 exhibited hypochromism of 72.19% and 65.71% at 414 nm respectively. The

intrinsic binding constants were obtained according to the following Scatchard equation [28].

15

CF = CT [(I/ I0)–P] / [1–P]

where, CT is concentration of the probe (complex) added; CF is 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 and 2 with increase

concentration of CT-DNA were shown in Fig. 3. It has been found that the binding constant

values of selective nickel(II) complexes 1 and 2 were 5.02 × 104, 3.54 × 104 respectively. The

experimental values of Kb clearly indicated that complexes 1 and 2 are bound to DNA via the

intercalative mode. From the results obtained, it has been found that complex 1 binds more

strongly than complex 2.

Fig. 3. Scatchard plots of r/CF Vs r for complexes 1 and 2 with increasing concentration of CT-

DNA.

16

Competitive DNA binding studies

The competitive DNA binding of complexes 1 and 2 have been studied by monitoring the

emission intensity of DNA-bound MB upon adding the complexes. Methylene blue (MB) is a

planar cationic dye which is widely used as a sensitive fluorescence probe for native DNA.

Fig. 4. Fluorescence quenching curves of MB bound to CT‒DNA with presence of complexes 1

and 2 in 5 mM Tris-HCl / 50 mM NaCl buffer at pH-7.2. Arrow showed an indicate emission

intensity changes upon increasing concentration of complexes at 25oC. [DNA =7.5 µM], [MB] =

7.5 µM and complexes [0‒50 µM].

MB emits intense fluorescent light in the presence of DNA due to its strong intercalation

between adjacent DNA base pairs. This illustrate that, as the concentration of the complexes

increases, the emission band at 684 nm exhibits hypochromism upto 79.06 and 68.05% of the

initial fluorescence intensity The fluorescence intensity was observed and clearly indicated that

MB molecules are displaced from their DNA binding sites and are being replaced by the

complexes under investigation. Quenching data were analyzed according to the following Stern-

Volmer equation

F0/F = Ksv [Q] + 1

17

where F0 is the emission intensity in the absence of compound, F is the emission intensity in the

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

compound. Ksvvalue is obtained as a slope from the plot of F0/F versus [Q].

Fig. 5. Stern–Volmer plots of the MB–DNA fluorescence titration with complexes 1 and 2.

In the Stern-Volmer plot (Fig. 5) of F0/F versus [Q], the quenching constant (Ksv) is obtained

from the slope which was 2.9 x 104 M-1, 1.08 x 104 M-1 for the complexes 1 and 2 respectively.

Further, the apparent DNA binding constant (Kapp) was calculated using the following equation,

KMB [MB] = Kapp [complex]

where the complex concentration has the value at a 50% reduction of the fluorescence intensity

of MB, KMB (1.0 × 10-7 M-1) is the DNA binding constant of MB to DNA, [MB] = 7.5µM),

where the binding constants are found to be 2.17 x 106 M-1 and 8.1 x 105 M-1 respectively for 1

and 2. The complex 1 binds to DNA more strongly than the complex 2, which is very well

agreed with the results observed from the emission spectra. The experimental data also suggests

that the complexes bind to DNA via intercalation mode.

DNA cleavage studies

Transition metal-mediated radical production may result in efficient DNA cleavage. Agarose gel

electrophoresis assay is a useful technique to investigate various binding modes of small

18

molecules to supercoiled DNA. Natural-derived plasmid DNA mainly has a closed-circle

supercoiled form (Form I), as well as nicked form (Form II) and linear form (Form III) as small

fractions [29]. Intercalation of small molecules to plasmid DNA can loosen or cleave the

supercoiled form DNA, which decreases its mobility rate and can be separately visualized by

agarose gel electrophoresis method, whereas, simple electrostatic interaction of small molecules

to DNA does not significantly influence the supercoiled form of plasmid DNA. Thus, the

mobility of supercoiled DNA does not change. To assess the DNA cleavage ability of the free

ligand and nickel(II) complex, supercoiled (SC) pBR322 DNA was incubated with five different

concentrations of the complex and further, in two different concentrations of the complexes in 5

mM Tris-HCl / 50 mM NaCl buffer at pH 7.2 for 2 h without the addition of a reductant. Upon

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

observed (Fig. 9). The relatively fast migration form is the intact super coiled form (Form I) and

the slow moving migration is the open circular form (Form II), which was generated from

supercoiled when scission occurred on its one strand. The nickel(II) complex at different

concentrations is able to perform cleavage of pBR322 plasmid DNA ( Fig. 6). The intensity of

supercoiled SC (Form I) diminished gradually and partly converted to nicked form, NC (Form

II). The intensity of the NC (Form II) band increases, whereas, production of the linear form LC

(Form III) of DNA also increases as the concentration of the nickel(II) complex increases from

10 mM to 50 mM.

Fig. 6. Cleavage activity of nickel (II) complexes (1 and 2) monitored by 1% agarose gel

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

19

pBR322 DNA incubated at 37oC with a fixed concentration of the complex for incubation 3

hour. Lane 1: DNA control, Lane 2, 3: Complex 1 (25 µM, 50 µM), Lane 4, 5: Complex 2 (25

µM, 50 µM).

It is obvious that the nickel(II) complex has the ability to cleave the supercoiled plasmid DNA

and this cleavage system does not require addition of any external agents.. In control

experiments, radical scavengers such as, DMSO, KI and MeOH were added to the reaction

mixtures, which did not show any apparent effect on DNA cleavage activity. These observations

suggest that DNA cleavage reaction does not proceed via radical mechanism and indicates the

hydrolytic nature of the cleavage [30].

BSA binding experiment

The protein binding study was performed by tryptophan fluorescence quenching experiments

using bovine serum albumin (BSA, 5 µM) as the substrate in Tris HCl/50 mM NaCl buffer at pH

7.2. Quenching of the emission intensity of tryptophan residues of BSA at 335 nm (excitation

wavelength at 295 nm) was monitored using complexes 1 and 2 as quenchers with increasing

complex concentration [31]. The F0/F versus [complex] plot was constructed using the corrected

fluorescence data taking into account the effect of dilution. Fluorescence spectra of BSA with

various concentrations of complexes were obtained from 290 to 500 nm. Here, the changing

fluorescence intensity is related to both the concentration and nature of the quencher. Therefore,

the quenched fluorophore serves as an indicator to determine the ability of quenching agent.

20

Fig. 7. Emission spectrum of bovine serum albumin (BSA) (1 µM; λex = 280 nm, λem = 344

nm) in the presence of increasing amounts of the complexes 1-2 (0 – 50µM). The arrow shows

that the emission intensity decreases upon the increase in concentration of the compounds.

It is observable that BSA has a strong fluorescence emission peak at 335 nm. 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 2 and 5 nm for the

complexes 1 and 2 respectively. As the data shows, the fluorescence intensity of BSA regularly

decreased and emission maximum undergoes slight red shift of with increasing concentration of

BSA with nickel(II) complexes, suggests more hydrobhobic environment of tryptopen residue. In

addition, fluorescence quenching data were analyzed with the Stern-Volmer and Scatchard

equations. From the plot of Io/I versus [Q], the quenching constant (Ksv) can be calculated (Fig.

8). The binding of complex with BSA occurs at equilibrium, the equilibrium binding constant

can be analyzed according to the Scatchard equation [32].

21

Fig. 8. Stern–Volmer plots (A) and Scatchard plots (B) of the fluorescence titration of the

complexes 1 and 2 with BSA.

Log (I0-I/ I) = log Kbin + n log [Q]

where Kbin is the binding constant of the complex with BSA and n is the number of binding sites.

The binding constant (Kbin) and the number of binding sites (n) have been calculated from the

plot of log [(Io−I)/I] versus log [Q] (Fig. 8). The calculated Ksv, Kbin, and n values are listed in

Table 3.

22

Table 3 Quenching constant (Ksv), binding constant (Kbin), and number of binding sites (n) for

the interactions of complexes with BSA

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

1 5.8× 104 6.75× 104 1.88

2 4.47× 104 6.30× 104 1.68

The calculated value of n is around 2 for all the complexes indicating the existence of two

binding sites in BSA for all the complexes. Moreover, the results showed that complex 1

interacts with BSA more strongly than the other complex 2. From the emission spectra of BSA

and nickel(II) complexes 1 and 2 to a fixed concentration of BSA, a gradual increase in the

intensity of BSA emission was observed while keeping the location of the peak unchanged (a

representative spectrum of complexes is shown in Fig. 7) demonstrating that an interaction

between compounds and BSA occurred through static quenching. Yet again, complex 1 showed

better activity than complex 2.

Anticancer activity in vitro

MTT assay

Schiff base complexes of nickel have recently been investigated for their therapeutic potential

[44] and the positive results obtained from DNA-binding, cleavage and BSA binding studies of

the new nickel(II) complex encouraged us to test its toxicity against Vero cell line, cytotoxicity

against human cancer cell lines namely, human cervical cancer cell line (HeLa) by colorimetric

(MTT) assay in which mitochondrial dehydrogenase activity was measured as an indication of

cell viability [33]. Complexes were dissolved in DMSO and blank samples containing same

volume of DMSO are taken as controls to identify the activity of solvent in this cytotoxicity

experiment. Cisplatin was used as the positive control. The results were analyzed by means of

cell viability cures and expressed with IC50 values [34]. The IC50 values of Vero cell line 58.82,

65.51µg/ml and HeLa cell line 20.28, 25.13 µM showed complexes 1 and 2 respectively. IC50

values in general were found to be highest in Vero cells, compared by HeLa cell line. The nickel

complexes exhibits significant activity against HeLa cell lines, which is almost equal to the

23

activity of the well-known anticancer drug, cisplatin. It clearly demonstrates that upon increasing

the concentration of complexes, an obvious decrease is observed in cell viability (Fig. 9 & 10).

Fig. 9. Cytotoxic effect of nickel(II) complexes against HeLa cell line at different concentrations

(0.1µM, 1.0µM, 10µM, 50µM, 100µM). Cell viability decreased with increasing concentrations

of complexes.

Fig. 10(a). Cell viability (%) against concentrations of nickel(II) complexes 1, 2 on Vero cell

line.

24

Fig. 10(b). Growth inhibition against log10 concentrations of nickel(II) complexes 1 and 2 on

HeLa cell line.

Among the complexes, complex 1 exhibited promising growth inhibitory effect against HeLa

cell lines. Furthermore, the complex 1 displayed higher cytotoxicity than cisplatin to HeLa cells.

Conclusion

We have synthesised as well as characterized new air-stable nickel (II) complexes NiL1 and NiL2,

in which the ligand afforded bis-chelated neutral ML2 complexes with divalent metal ions, acting

in a uninegative bidentate fashion with its NS donor set. The crystal structure of complex 1 has

been established. The binding strength of the compounds with CT-DNA calculated by

fluorescence spectroscopic titrations has shown that complex 1 exhibits the highest Kb value than

the complex 2. Although competitive binding studies with MB showed the potential of the

compounds to displace MB from the MB-DNA complex and confirmed intercalation as the most

possible binding intercalation mode to DNA. Furthermore, protein binding properties of the

complexes examined by the fluorescence spectra suggested that the binding affinity of complex 1

was stronger than complex 2. In addition, in vitro cytotoxicity assay confirmed that both the

complexes performed well against HeLa, cancer cell line. In particular, complex 1 showed higher

cytotoxicity and the IC50 value of the complex 1 is higher than that of cisplatin. The outcome of

above studies would be helpful to understand the mechanism of nickel (II) complexes with DNA

/ BSA protein interaction and new therapeutic agents for certain diseases.

25

Supplementary material

Crystallographic data for complex 1 has been deposited with the Cambridge Crystallographic

Data Centre (CCDC) as supplementary publication no. CCDC-993161. The data can be obtained

free of charge from http://www.ccdc.cam.ac.uk/conts/ retrieving html or from the CCDC (12

Union Road, Cambridge CB2 1EZ, UK; Fax: (+44) 1223-336033; e-mail:

deposit@ccdc.cam.ac.uk).

Acknowledgement We are sincerely thankful to the SAIF Punjab University, Chandigarh for ESI mass facility and

KMCH, Coimbatore, India for carrying out the anticancer activities. This work is supported by

Principal Dr A.S. Ramkumar, Maharaja Prithivi Engineering College, Avinashi. The author

(R.N) thankful to DST for financial assistance (Project No.SR/FT/CS-95/2010). The authors

would like to thank P. Sugumar and M.N. Ponnuswamy, Centre of Advanced Studies in

Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai- 600 025,

India for helping valuable crystal discussion.

26

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30

Scheme and Figure Captions

Scheme 1 Preparation of nickel(II) complexes.

Fig. 1. ORTEP views of the molecular structure and the atom labeling scheme of complex 1.

Thermal ellipsoids are drawn at 30% probability level. Disorder molecules have been omitted for

clarity.

Fig. 2. Fluorescence spectra of nickel(II) complexes 1-2 in 5mM Tris-HCl/ 50mM NaCl buffer at

pH 7.2 and arrows indicate absence and presence of increasing amounts of CT‒ DNA

concentration at 25oC. [Complex = 25µM (---- lines)], DNA = 0 ‒ 25µM.

Fig. 3. Scatchard plots of r/Cf Vs r for complexes 1,2 with increasing concentration of CT-DNA.

Fig. 4. Fluorescence quenching curves of MB bound to CT‒ DNA in the presence of complexes

1, 2 in 5mM Tris-HCl/50 mM NaCl buffer at pH 7.2. Arrow shows emission intensity changes

upon increasing the concentration of complexes at 25oC. [DNA =7.5µM], [MB] = 7.5µM and

complexes [0‒50µM].

Fig. 5. Stern–Volmer plots of the MB–DNA fluorescence titration for complexes 1-2.

Fig. 6. Cleavage activity of nickel(II) complexes (1,2) monitored by 1% agarose gel containing

5mM Tris-HCl / 50mM NaCl buffer (pH-7.2), the effect of hydrolytic cleavage of pBR322 DNA

incubated at 37oC with a fixed concentration of the complex for incubation 3 hr. Lane 1: DNA

control, Lane 2,3: Complex 1(25µM,50µM), Lane4,5: Complex2(25µM ,50µM).

Fig. 7. Emission spectrum of bovine serum albumin (BSA) (1µM; λex = 280 nm, λem = 344 nm)

in the presence of increasing amounts of the complexes 1-2 (0–50µM). The arrow shows that the

emission intensity decreases upon the increase in concentration of the compounds.

Fig. 8. Stern–Volmer plots (A) and Scatchard plots (B) of the fluorescence titration of the

complexes 1,2 with BSA.

Fig. 9. Cytotoxic effect of nickel(II) complexes against HeLa cell line at different concentrations

(0.1µM, 1.0µM, 10µM, 100µM). Cell viability decreased with increasing concentrations of

complexes.

Fig. 10(a). Cell viability (%) against concentrations of nickel(II) complexes 1, 2 on Vero cell

line.

Fig. 10(b). Growth inhibition against log10 concentrations of nickel(II) complexes 1 and 2 on

HeLa cell line.

31

Graphical Abstract for Synthesis, structure, DNA/BSA interaction and in vitro cytotoxic activity of nickel(II) complexes derived from S-allyldithiocarbazate Nanjan Nanjundan,a Ponnusamy Selvakumar,a Ramaswamy Narayanasamy,a* Rosenani A. Haque,b Krishnaswamy Velmurugan,c Raju Nandhakumar,c Tamilselvan Silambarasan,d and Ramamurthy Dhandapanid aDepartment of Chemistry, Coimbatore Institute of Technology, Coimbatore - 641 014, India.

bThe School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

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

dDepartment of Microbiology, Periyar University, Salem - 636 011, India

32

Highlights

� New nickel(II) complexes containing S-allyldithiocarbazates were synthesized and

characterized

� The squareplanar geometry of the complexes was determined by X-ray single crystal

� The complexes interact with DNA via intercalative mode and BSA interact with static

interaction

� A nickel(II) complex showed high cytotoxicity to HeLa tumor cell lines