Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II), Cu(II) and Zn(II): Synthesis, characterization and biological studies

Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II),

Cu(II) and Zn(II): Synthesis, characterization and biological studies

a

b

c

Nanjan Nanjundan , Ramaswamy Narayanasamy* , Steven Geib , Krishnaswamy Velmurugan ,

c

d

Raju Nandhakumar , Manickam Dakshinamoorthi Balakumaran , Pudupalayam Thangavelu

d

Kalaichelvan

a

Department of Chemistry, Coimbatore Institute of Technology ,Coimbatore-641 014, India.

b

Department of Chemistry, University of Pittsburgh, PA 15260 USA.

c

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

d

Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy

Campus, Chennai - 600 025, Tamil Nadu, India.

*

To whom correspondence should be addressed, e-mail: narayanasamycit@gmail.com

Fax: + 91-422-2575020.

Abstract

Transition metal complexes of acetophenone-S-allyldithiocarbazate [H-(Ap-sadtc)] of the

2

+

2+

general formula [M-(Ap-sadtc) ] (Ni , Cu , Zn ) (1-3) were synthesized and characterized by

2

1

13

elemental analysis, UV-Vis, FT-IR, H NMR, C NMR and mass spectroscopy. The molecular

structures of the ligand and its complexes 1-3 were confirmed by both theoretical as well as

experimental methods. The interactions of the complexes with biomolecules, such as calf thymus

DNA (CT-DNA) and bovine serum albumin (BSA), were explored using absorption and

emission spectral techniques. All the complexes synthesized were found to be most promising

due to their large binding affinity towards different bio-macromolecules and higher Kcat values in

catechol oxidation (3,5-DTBC) / phosphate hydrolysis (4-NPP) reactions. The in vitro anticancer

activities using MTT and a morphological staining assay indicate that the complexes are active

against the (Hela) cervical cancer cell line, which is consistent with our above mentioned

hypothesis.

Keywords: Schiff base complexes, X-ray/DFT study, DNA/BSA binding activities, catalytic

efficacy, in vitro anticancer activities.

1. Introduction

Transition metal complexes have become of increasing importance in synthetic chemistry,

coordination chemistry, homogenous catalysis and biological chemistry [1]. Platinum based

drugs have been found to be potentially active for their diverse clinical cancer treatment [2-6].

The dose of cisplatin that can be applied to patients is limited by its high toxicity, and the side

effects of cisplatin therapy include damage to the kidney (nephrotoxicity), hearing (ototoxicity),

peripheral neuropathy and neurotoxicity, nausea and vomiting. Therefore, considerable attempts

are being made to replace this drug with suitable alternatives, and numerous transition metal

complexes have been synthesized and tested for their cytotoxicity. In general, Schiff base metal

complexes have widespread applications in electrochemistry, nanotechnology, catalysis, material

science and as potential therapeutic agents. Amongst these, N,S bidentate Schiff base ligands

derived from thiosemicarbazone or dithiocarbazate have attracted considerable interest. The

versatile applications of dithiocarbazate ligands containing metal atoms coordinated with sulfur

and nitrogen donors due to their soft-hard nature are attractive and allow them to show a

structure-activity relationship (SAR) [7]. The first row transition elements play a vital role in the

synthesis of several coordination complexes due their different oxidation states which facilitate

their structural, stereochemical, spectroscopic and electrochemical properties. Among the various

transition metals, the present study is focused on nickel, copper and zinc metal ions based on

their widespread industrial and biological applications [8-11].

The transition metal ions (Ni, Cu and Zn) attached to the donor atoms of the bis(dithiocarbazate)

Schiff base ligand form stable metal ion complexes. Metal-based anticancer drugs exhibit

enhanced selectivity and novel modes of DNA interaction, like non-covalent interactions that

mimic the mode of interaction of biomolecules. The propensity for the interaction of metal

complexes with calf thymus DNA (CT-DNA) is of paramount importance to understand the

mechanism using emission spectral titrations. On the other hand, the design of protein targeting

metal-based anticancer agents with potential in vitro toxicity has gained importance recently

[

12,13]. Changing the ligand environment towards a specific target is one way of tuning the

selectivity of a drug molecule. In another way, a change in the metal environment plays a

significant role in the binding of the metal complex to a biomolecule, such as DNA or protein.

Generally, nickel and copper metal ions have redox nature, which may promote the

transformation of biomolecules through an oxidation pathway [14]. In addition, the redox

inactive zinc metal ion behaves in a different way [15]. Thus, these three mononuclear metal

complexes have unique structural features which justify their selection for the present

investigation.

Many studies have been carried out to establish a relationship between copper-mediated substrate

oxidation and structural parameters, which will influence catecholase activities. Crucial factors,

such as metal–metal distance, electrochemical properties, exogenous bridging ligand, ligand

structure and pH of the medium, do play a major role in the catecholase activity [16-21].

Recently, it was documented that the presence of a positive charge center close to the metal

center might enhance activity. Therefore, much attention has been focused on biomimetic and

bioinspired catalysts of transition metal-based complexes containing hard and soft donor atoms

[

22,23]. Among the metal complexes, M(II) complexes with N,O and N,S-donor ligands have

profound catalytic efficacy, particularly in catalyzing 3,5-DTBC to 3,5-DTBQ. Furthermore,

active research on hydrolytic catalysis of phosphoric and carboxylic acid esters involving

biochemical processes have gained utmost importance. The backbones of DNA, RNA and ATP

macromolecules show significant biochemical behavior involving the formation or cleavage of

P-O bonds [24-34]. The hydrolyses of phosphate monoester bonds in model complexes, such as

4

-nitrophenyl phosphate (4-NPP), exhibit specific reactions which indicate the reactivity of

metalloenzymes with DNA. When compared to other transition metals, Ni, Cu and Zn ions

possess very favorable catalytic activity from the reactant and product release viewpoint [35-45].

Influenced by the above facts, it was considered worthwhile to undertake the synthesis and

characterization of the ligand [H-(Ap-sadtc)] and the mononuclear complexes [Ni-(Ap-sadtc)

2

]

(

1), [Cu-(Ap-sadtc) ] (2) and [Zn-(Ap-sadtc) ] (3), which confirm the presence of quite

2

appreciable tetrahedral distortions about the metal centers. The ligand and its complexes 1-3 are

envisaged by applying density functional theory (DFT) calculations that determine the quantum

mechanical (QM) reactivity descriptors, viz. the highest occupied molecular orbital (HOMO) and

lowest unoccupied molecular orbital (LUMO). A comparative study of the biomolecular

interactions (DNA/BSA) of the metal complexes were conducted along with their related anti-

cancer activities. The results of the present investigation confirm that the metal complexes could

bind to DNA in the intercalation mode and BSA exhibiting the static quenching mechanism.

These three structurally similar complexes exhibit good catecholase and hydrolytic activities.

Moreover, the cytotoxicity of the metal complexes against the HeLa cell line was assessed by an

MTT assay and cell death analysis by AO/EB and DAPI staining methods.

2. Experimental

2

.1. Reagents

Potassium hydroxide pellets, hydrazine hydrate, carbon-disulfide, acetophenone, nickel(II)

acetate tetrahydrate, copper(II) acetate monohydrate and zinc(II) acetate dihydrate high purity

reagents were purchased from Merck and used as such. Calf thymus DNA (CT-DNA) and

bovine serum albumin (BSA) were obtained from Genei, Bangalore and Himedia, India

respectively. Ethidium bromide (EB), 3,5-tris(hydroxymethyl) amino methane, di-tert-

butylcatechol (3,5-DTBC), 4-nitrophenyl phosphate disodium salt hexahydrate (4-NPP) and allyl

bromide were purchased from Sigma-Aldrich and used as received. All solvents were dried and

used freshly, distilled unless otherwise stated. Double distilled water was used to prepare buffers.

2

.2. Instrumental methods

Melting points were measured on an Electro-thermal model 9200. C, H, N and S elemental

analyses of the compounds were performed on a CHNS-O model EA 2000 elemental analyzer.

Electronic spectra of the ligand/complexes 1-3 were recorded in DMSO using a Shimadzu UV-

1

650 PC spectrophotometer in the 800-200 nm range. Fourier Transform Infrared spectra of the

synthesized compounds were carried out with a Bruker Scientific 600 Infrared

-1

1

13

spectrophotometer in the spectral range 4000-400 cm using KBr pellets. H and C NMR

spectra were obtained on a Bruker High Resolution console (300.13 MHz, CDCl ). Emission

3

spectra were measured with a Jasco FP 6600 spectrofluorometer. ESI-MS spectra were recorded

using an LC-MS Q-TOF micro analyzer (Shimadzu) in the SAIF, Punjab University,

Chandigarh. The X-ray intensity data were measured on a Bruker Apex II CCD system equipped

with a Cu IMuS micro-focus (λ = 1.54178 Å). The frames were integrated with the Bruker

SAINT software package using a narrow-frame algorithm. Geometry optimization by density

functional theory (DFT) method was performed using the GAUSSIAN09 (B3LYP) package. All

-3

reactions were carried out in an oxygen atmosphere. Stock solutions of complexes 1-3 (1.0×10

o

M in DMSO) were stored at 4 C and the required concentrations were prepared for all

experiments. All the stock solutions were used after no more than four days. Solutions of the

compounds were freshly prepared 1 hour prior to biochemical evaluation. Data are expressed as

the mean ± standard deviation from three independent experiments.

2

.3.1. Synthesis of [H-(Ap-sadtc)]

[

H-(Ap-sadtc)] was synthesized according to the previously published procedure [46,47]. To an

ethanolic solution (30 mL) of 5 mL (0.1 mol) hydrazine hydrate and 5.6 g (0.1 mol) KOH,

carbon disulfide 6.1 mL (0.1 mol) was added dropwise in an ice bath. After 30 min, 8.6 mL (0.1

mol) of allyl bromide was added and the solution was stirred at 5 °C for 1 h. An ethanolic

solution (25 mL) of acetophenone 11.7 mL (0.1 mol) was added to this mixture and heated under

stirring. After 15 mins, a yellow product was separated by filtration, washed with water and

vacuum dried. The product was recrystalised from hot ethanol and single crystals for X-ray

structure analysis were obtained by the slow evaporation method. Yield: 75-80%. Colour:

yellow. MP: 145 °C. Anal. Calc. for C12

H

14

N

2

S : C, 57.64; H, 5.64; N, 11.19; S, 25.61; Found:

2

-1

C, 57.49; H, 5.54; N, 11.06; S, 25.48%. UV-vis (DMSO), λ max (nm): 368. FT-IR (ν cm , KBr):

1

596 ν(C=N); 3057 ν(N-H); 1043 ν(C=S); 982 ν(C-S); 680 ν(C-HAllyl); 1515, 1546, 1561, 1627,

1

693, 1677 ν(C Ar). H NMR (300.13, MHz, CDCl

3

, 25 °C) δ (ppm): 5.20 (m, 2H, -CH

); 7.40-7.93 (d, 5H, CAr). C

); 130.62 (-CH); 37.45 (-CH2);

); 127.09-138.88 (CAr). ESI-MS (Calc., found, m/z):

2

); 5.94

1

3

(

m, H, -CH); 3.88 (d, H, -CH); 9.9 (s, H, -NH); 2.39 (s, 3H, -CH

NMR (300.13, MHz, CDCl , 25 °C) δ (ppm): 119.27 (-CH

00.06 (C=S); 158.19 (C=N); 15.51 (CH

3

2

3

50.38, 250.37.

2

.3.2. Synthesis of complexes 1-3

[

Ni-(Ap-sadtc)

2

] (1)

A boiling methanolic solution (20mL) of Ni(CH

3

COO)

2

.4H O (0.248 g, 1 mmol) was added

2

dropwise to a methanol solution (10 mL) of [H-(Ap-sadtc)] (0.5 g, 2 mmol) with an immediate

colour change from yellow to brownish black. The solution was then refluxed for 1 hour. After

cooling, it was filtered and the filtrate was kept at an ambient temperature for slow evaporation.

After two days, the brownish-black needle-shaped diffraction quality crystals that deposited were

collected by filtration, washed with small amounts of methanol and air dried. Yield: 88%.

o

Colour: brownish black. MP: 175-178 C. Anal. Calc. for C24

H

26

N

4

NiS : C, 51.71; H, 4.70; N,

4

1

0.05; S, 23.01; Found: C, 51.68; H, 4.59; N, 9.98; S, 22.89%. UV-vis (Tris-HCL buffer), λ max

-1

(

nm): 236, 260, 387, 479. FT-IR (ν cm , KBr): 1532 ν(C=N); 923 ν(C-S-S); 688 ν(C-HAllyl);

1

3

1

5

516, 1546, 1561, 1626, 1677, 1693 ν(CAr). C NMR (300.13, MHz, CDCl

19.02 (-CH ); 178.88 (C-S); 22.80 (-CH ); 129.96-139.50 (CAr); ESI-MS (Calc., found, m/z):

57.44, 557.1.

3

, 25 °C) δ (ppm):

2

3

2

.3.3. [Cu-(Ap-sadtc) ] (2)

2

A methanolic solution of Cu(CH

3

COO)

2

.H O (0.199 g, 1 mmol) was added dropwise to a hot

2

acetonitrile solution of [H-(Ap-sadtc)] (0.5 g, 2 mmol). The resulting greenish-black mixture

was refluxed for 1 hour. After cooling, the clear solution was filtered and the filtrate was kept in

a CaCl

filtered and washed with a small amount of petroleum ether/acetonitrile. Yield: 85%. Colour:

greenish black. MP: 190-195 °C. Anal. Calc. for C24 : C, 51.26; H, 4.66; N, 9.96; S,

2.81; Found: C, 51.07; H, 4.50; N, 9.81; S, 22.68%. UV-vis (Tris-HCL buffer), λ max (nm): 238,

2

desiccator in the dark. After one week, square-shaped X-ray quality crystals were

4 4

H26CuN S

2

3

1

-1

14, 393, 493. FT-IR (ν cm , KBr): 1532 ν(C=N); 922 ν(C-S-S); 691 ν(C-HAllyl); 1516, 1546,

561, 1626, 1677, 1693 ν(CAr). ESI-MS (Calcd, found, m/z): 562.3; 562.27.

2

.3.4. [Zn-(Ap-sadtc) ] (3)

2

An ethanolic solution of Zn(CH

3

COO)

2

.2H O (0.219 g, 1 mmol) was added to a hot

2

dichloromethane solution of [H-(Ap-sadtc)] (0.5 g, 2 mmol), then heated on a water bath for 5

minutes, filtered and kept in a refrigerator. After 15 days, X-ray quality yellow crystals were

obtained, filtered and washed with a small amount of petroleum ether/ethanol. Yield: 82%.

o

Color: Yellow. MP: 160-165 C. Anal. Calc. for C24

H

26

N

4

S Zn: C, 51.10; H, 4.65; N, 9.93; S,

2

1

2.74; Found: C, 51.15; H, 4.51; N, 9.81; S, 22.60%; UV-vis (Tris-HCL buffer), λ max (nm): 238,

-1

60, 398, 497. FT-IR (ν cm , KBr): 1533 ν(C=N); 917 ν(C-S-S); 690 ν(C-HAllyl); 1516, 1546,

1

561, 1626, 1677, 1693 ν(CAr); H NMR (300.13, MHz, CDCl

3

, 25 °C) δ (ppm): 5.15 (m, 2H, -

1

3

CH

2

); 5.87 (m, H, -CH; 2.30 (m, 3H, -CH

, 25 °C) δ (ppm): 119.88 (-CH ); 171.48 (C-S); 161.04 (C-CH

37.32 (CAr). ESI-MS (Calcd, found, m/z): 564.14, 564.10.

3

); 7.41-7.51 (m, 5H, CAr). C NMR (300.13, MHz,

CDCl

3

2

3

); 21.32 (CH ); 127.97-

3

1

2

.4. Crystal structure characterization

Single crystal X-ray data of [H-(Ap-sadtc)] and the three mononuclear complexes 1-3 were

collected by a Bruker Apex II CCD diffractometer using CuK radiation (λ= 1.54178 Å). The

α

linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion

corrections were referred from the International Tables for X-ray Crystallography. The data

integration and reduction were worked out with SAINT software. The structures were solved by

2

direct methods using SHELXTL-2014 and refined on F by full-matrix least-squares using the

SHELXTL-2014 program package. All the non-H atoms were treated anisotropically. The H

atoms attached to carbon atoms were positioned geometrically and treated as riding atoms using

SHELXL-2014 default parameters. The structures of the ligand and the three new mononuclear

metal complexes 1-3 are shown in Fig. 1. Details of the crystallographic data, structure

refinement parameters and selected bond lengths and angles are listed in Tables 1 and 2.

2

.5. Computational methods

The initial geometry for the DFT calculations was based on the measured X-ray diffraction

structure. All theoretical computations were done by using the Gaussian09 program package

[

48]. Geometry optimizations for the thione tautomeric form of the ligand and metal complexes

were performed using the DFT method with Becke’s three parameters hybrid exchange-

correlation functional B3LYP/6-311G. Visualizations of the DFT optimized structures and the

frontier molecular orbitals (HOMO and LUMO) of the ligand and complexes 1-3 were

performed using the chemcraft program package (http://www.chemcraftprog.com). To ascertain

stationary points, a further frequency test was performed. Also, a theoretical investigation of the

natural charge for each atom, QM descriptors, were calculated using the same basic set.

2

.6. DNA binding emission spectral studies

The interaction of the complexes with CT-DNA was investigated by dissolving the complexes 1-

in a solvent mixture of 5% DMSO and 95%Tris-HCl buffer (5 mM Tris-HCl/50 mMNaCl

3

buffer, pH = 7.2) the solutions were stored at 4 °C for further experiments and used within 4

days. Emissive titrations were performed with a fixed concentration of the metal complexes 1-3

(

25µM) but by varying the nucleotide (CT-DNA) concentration from 0 to 10 µM. Before taking

measurements, the mixture was shaken up and incubated at room temperature for 30 min. In

addition, a proper wavelength with CT-DNA was fixed by the emission range and adjusted.

2

.7. Competitive binding fluorescence measurements

Further, to support the competitive binding of complexes 1-3 to DNA via intercalation,

fluorescence emission quenching experiments were performed. The fluorescence quenching

experiments were carried out by adding different concentrations of complexes 1-3 to the EB-

bound CT-DNA solution in 5% DMSO and 95% Tris-HCl buffer. The excitation (550 nm) and

emission (605 nm) fluorescence intensities were recorded at room temperature. EB alone showed

minimal fluorescence and the fluorescence was enhanced greatly with the gradual addition of

CT-DNA until a maximum fluorescence was achieved due to the formation of an intercalative

DNA-EB adduct. Addition of increasing amounts of the complexes 1-3 to the DNA-EB adduct

quenched the fluorescence. For the emission quenching experiments, CT-DNA was pretreated

o

with EB in a ratio [DNA]/[EB] = 10 for 30 minutes at 37 C. The titrants were then added to this

mixture of EB-DNA and the change in fluorescence intensity was measured.

2

.8. BSA binding studies

The fluorescence spectra were recorded at an emission wavelength of 342 nm using a 4mL

quartz cell on a JASCO F6500 spectrofluorometer after each addition of the quencher. Stern–

Volmer and Scatchard equations and graphs were used in order to study the interaction of the

quencher with BSA. Preparation of samples for fluorescence spectra: five 10 mL clean and dried

-6

sample test tubes were taken, Tris–HCl buffer 2.0 mL (pH 7.20), BSA 1.0 x 10 M and the

-6

concentrations of the complexes 1-3 were 10, 20, 30, 40 and 50 x 10 M for each of the five test

tubes. The sixth test tube, containing only BSA solution at pH 7.2, was marked as a “control”.

After mixing, the solutions were allowed to stand for 15 min for maximum binding of the

complexes to BSA.

2

.9. Catechol oxidase activity

In the majority of the investigations dedicated to the study of potential catecholase activity of

biomimicking coordination compounds, 3,5-di-tert-butylcatechol(3,5-DTBC) is chosen as the

model substrate because its low redox potential makes it easily oxidized to the corresponding

quinone (3,5-DTBQ). Since, it prevents over oxidation, such as ring opening, and being stable, it

shows a maximum emission at 435 nm in pure DMSO. Before proceeding into a detailed kinetic

study, the ability of complexes 1-3 were evaluated for the oxidation 3,5-DTBC to 3,5-DTBQ at

-4

room temperature in an oxygen atmospheric condition. For this purpose, 10 M DMSO solutions

-2

of these three metal complexes were treated with 10 M (100 equivalents) of 3,5-DTBC.

Maintaining the complex concentration as a constant, by adding the different substrate

-2

concentrations (10-50 x 10 M), the immediate reaction progress was monitored by fluorescence

spectrophotometer every 15 minutes over a reaction period of 2 hours, which was indicative for

the formation of the corresponding quinone 3,5-DTBQ.

2

.10. NPP hydrolysis

The rate of hydrolysis of 4-NPP was measured by monitoring the emission spectra at 485nm due

to the release of the P-nitrophenolate anion. In a typical experiment, 2.0 mL of freshly prepared

-2

-3

4

-NPP (10-50 x 10 mol dm ) in dimethyl sulfoxide (DMSO) was transferred into a cuvette in

the sample compartment of the spectrometer. A similar solution was placed in a reference

compartment to correct the hydrolysis in the absence of catalyst. 1.0 mL of Tris-HCl buffer(pH =

7

.2) was added to the reference cuvette to bring the total volume to 3.0 mL and the cuvette was

-3

equilibrated at room temperature for 10 min. The complexes 1-3 (10 mol dm ) were mixed

with 4-NPP separately and the emission spectra were recorded immediately and at regular time

intervals of 15 mins until completion of the reaction.

2

.11. Cell lines and cultural conditions

HeLa and normal Vero cell lines were cultured in RPMI-1640 medium with 10% FBS, 2mML-

glutamine and 1% penicillin/streptomycin under a fully humidified atmosphere 5% CO at 37 °C.

2

.11.1. In vitro cytotoxicity by MTT assay

The effect of the synthesized compounds on the viability of the HeLa cell line was determined by

an MTT (3-[4,5-dimethyl thiozole-2-yl]-2-5-diphenyl tetrazolium bromide) assay. 100µL of the

cell suspensions in growth medium were plated in a 96-well microtitre plate at concentrations of

4

1

x 10 cells/well and incubated for 48 h at 37 °C in a humidified incubator. After 48 hours of

incubation, the cells attained confluence; they were incubated again in the presence of various

concentrations in 0.1% DMSO for 72 h at 37 °C. After removal of the sample solution, they were

washed with phosphate buffered saline (pH 7.2), then 20µL of MTT (5mg/mL) was added to

each well of the plate. The plate was then incubated for 4 h at 37 °C. The solution in each well

including MTT was aspirated and 100 µL of buffered DMSO was added to dissolve the

formazone crystals. The plates were shaken for 5 mins and the optical density was measured on a

microplate ELISA reader at 540 nm with cis-platin as a control. The cytotoxicity was obtained

by comparing the absorbance between the samples and the control. The percentage inhibition

was calculated as follows:

%

inhibition = [Abs (Control) – Abs (Test)/Abs (Control)] x100

IC50 was calculated from the dose-response curve using GraphPad Prism 6 software.

2

.11.2. Apoptotic assays by fluorescence microscopy

6

HeLa cells were seeded onto chamber slides in six-well plates at a density of 1 x 10 cells per

well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% fetal

bovine serum (FBS) and incubated at 37 °C in 5% CO . The medium was removed and replaced

2

with a medium (final DMSO concentration, 0.05% v/v) containing the complexes (50 µM) for 24

h. The medium was removed again and the cells were washed with ice-cold phosphate buffer

saline (PBS) and fixed with formalin (4%, w/v). Cell nuclei were counterstained with acridine

-1

orange (AO) and ethidium bromide (EB) (AO: 100 mg mL , EB: 100 mg mL ) for 10 min. For

DAPI staining, the treated cells were fixed with 80% ethanol at room temperature for 30 min.

The fixative was removed and the cells were washed with PBS 3 times, and then incubated with

−

1

DAPI (1 µg mL ) for 45 min at room temperature in the dark. Both techniques were used to

distinguish viable cells, early apoptotic cells with blebbing and necrotic cells. Acridine orange

intercalates into DNA and gives a green fluorescence and thus the viable cells appear with a

green nucleus, while early apoptotic cells have condensed or fragmented nuclei. EB is taken up

only by the non-viable cells, giving a bright orange nucleus of the dead cells overwhelming the

acridine orange stain. DAPI dye is effective for fixed-cell staining and quantification of DNA

content. HeLa cells were mounted on a slide and the images were observed under a fluorescent

microscope in a green/blue filter on excitation at 350 nm and emission at 460 nm. The cells were

observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan).

3. Results and discussion

3

.1. Synthesis and characterization

The three novel mononuclear complexes 1-3, formed in a high yield of up to 80% from a mixture

of [H-(Ap-sadtc)] and the metal(II) acetate in a 2:1 molar ratio, are shown in Scheme 1. The

complexes are pure, crystalline in nature and completely soluble in ethanol, methanol,

acetonitrile, chloroform, dichloromethane, DMF and DMSO, but are insoluble in hexane,

petroleum ether and diethyl ether. The complexes were characterized by elemental analysis, UV-

1

13

Vis, infrared, H NMR, C NMR and mass spectroscopy, and by single crystal X-ray diffraction

studies.

3

.1.1. Infrared spectra

-1

The FTIR spectrum of the ligand did not show any band at 2570 cm (-SH), which indicates a

solid state thione form. Two strong bands appeared for the ligand at 3057 (-NH) and 1043 (C=S)

-1

cm , which were absent after complexation indicating that these two bands disappeared due to

deprotonation of the ligand and chelation of the thiolate sulfur atom. It suggests that the thione

form has been converted into the thiolate form, so that complexation can occur (Scheme 2)

-1

[

49,50]. A strong vibration observed at 1596 cm for the ligand, corresponding to the C=N band,

-1

was shifted to 1532-1533 cm in the metal complexes due to the interaction of the azomethine

nitrogen atom during bonding [51,52]. The investigated and calculated frequencies of the

dithiocarbazate ligand and its complexes 1-3 are given in Table S1.

3

.1.2. Electronic spectra

The electronic spectral absorption of [H-(Ap-sadtc)] in DMSO solution at 368 nm confirms an

intra-ligand transition, which is shown in Fig. S1. The ligand exhibits an n→π* transition, which

is due to coordination between the nitrogen atom and dithiocarbazate moieties. The spectra of

complexes 1-3 were recorded in Tris-HCl buffer and bands appeared at 236, 260, 238 and 314

nm due to a strong absorption of azomethine nitrogen atom, as shown in Figs. S2-S4. Also,

LMCT broad bands appeared at 389, 393 and 398 nm for the respective complexes 1-3, which

were mainly due to an S→M(II) charge transfer. All the complexes were expected to show the

1

transitions A

1

g→ A

2

g, A

1

g→ B

1

g and A g→ Eg in the visible region, but the prepared

1

complexes showed only a single (d-d) band at 479, 493 and 497 nm, respectively for the

1

A g→ Eg transition as the presence of sulfur covered the other bands.

1

3

.1.3. H NMR spectra

The thione and thiol forms of the Schiff base may also exist as either the E or Z conformational

isomer, or as a mixture of both isomers in solution. However, the X-ray crystallographic

structural determination of the ligand shows that in the solid state, it exists solely as the thione

1

form. The H NMR spectrum of the ligand in CDCl

3

exhibits the thione form, as indicated by the

–

NH resonance at 9.9 ppm, showing that in solution, it exists only as the Z isomer [53-55].

1

However, the thiol form may also exist in a CDCl solution of [H-(Ap-sadtc)], the H NMR

3

spectrum displaying a sharp signal for -SH at 4 ppm. The methylene proton of the ligand and

complex 3, appearing at 3.88 and 3.72 ppm respectively, indicates that the allyl sulfur atom is not

involved in coordination. The -CH

2

and -CH protons in the allyl group show multiple peaks at

5

.94-5.96 and 5.17-5.20 ppm, respectively. The aromatic protons appear as doublets within the

range 7.40–7.93 ppm for [H-(Ap-sadtc)] and similar signal peaks are observed for the metal

1

complexes. The -NH and –SH peaks in the H NMR spectrum of the ligand disappeared upon

complexation, which confirms the fact that the N and S atoms are involved in the formation of

the metal complexes.

13

3

.1.4. C NMR spectra

1

3

The C NMR spectra of [H-(Ap-sadtc)] showed signals at 200.06 and 158.19 ppm, which are

attributed to the C=S and C=N groups, respectively. However, in the formation of the metal

complexes 1 and 3, the C=S group experienced a downfield shift at 171.48 and 178.88 ppm, and

the C=N group an upfield shift at 161.04 and 173.94 ppm. These considerable shifts with respect

to [H-(Ap-sadtc)] indicate the involvement of thione sulfur and azomethine nitrogen atoms in

coordination. In addition, C-S carbon atom in [H-(Ap-sadtc)] and complexes 1 and 3 showed

signals at 37.45, 38.58 and 35.59 ppm, respectively. The peaks due to the presence of aromatic

carbons and the methyl group in the ligand and complexes 1 and appeared in the ranges 125.09–

1

39.50 and 15.5-22.80 ppm respectively.

3

.1.5. Crystal structure description of [H-(Ap-sadtc)]

A suitable single crystal was grown from the required amount of hot ethanol by the slow

evaporation method over a time period of one week. A specimen of C12 of approximate

dimensions 0.020 x 0.120 x 0.140 mm was used for the X-ray crystallographic analysis. The

H

14

N S

2 2

final cell constants were based upon the refinement of XYZ-centroids of reflections above 20

σ(I). The calculated minimum and maximum transmission coefficients (based on crystal size)

2

were 0.6370 and 0.9330. The final anisotropic full-matrix least-square refinement on F showed

that [H-(Ap-sadtc)] crystallized in the space group P-1 in the triclinic crystal system. The crystal

structure of the ligand also confirms that it crystallizes as the thione tautomeric form, as

previously predicted by spectroscopic techniques; with the thione sulfur and azomethine nitrogen

atoms in this conformation, the ligand acts as an N,S binding agent. The C=S bond length (1.667

Å) indicates that is a double bond and the C-N bond (1.294 Å) is intermediate between a single

and a double bond, indicating electron delocalization in the N-C-S thioamide group. The N−N

bond distance varied from 1.376(4) to 1.4186(19), 1.396(3) and 1.403(2) Å in the Schiff base to

the complexes, showing that the bond was shorter than a single bond and also indicating

significant π-delocalization of the dithiocarbazate occurred in the C-N-N-C moiety. However,

the rotation of the S-C-S fragment by 180° about the C-N bond orients the donor atom in the

correct position for bidentate coordination.

3

.1.6. Crystal structure description of the metal complexes 1-3

X-ray quality single crystals were isolated from the mother liquors by the slow evaporation

method. Specimens of C24 , of approximate dimensions 0.080 x

.090 x 0.170 and 0.120 x 0.140 x 0.160 mm were used for the X-ray crystallographic analysis.

H26NiN

4

S

4

, and C24

4 4

H26CuN S

0

The ORTEP view of complexes 1-3 at the 25% probability level with the atom numbering

scheme is shown in Fig 1. The calculated minimum and maximum transmission coefficients of

the nickel and copper complexes (based on crystal size) were 0.5360, 0.5530 and 0.7300, 0.6330.

The non-hydrogen atoms were refined anisotropically by applying the full-matrix least-square

2

method on F . The centrosymmetric Ni(II) and Zn(II) complexes crystallized in the monoclinic

space group P121/n1, whereas the Cu(II) complex crystallized in the orthorhombic space group

P

21212, in which the two ligands were symmetrically related to each other and had the same bond

angles and distances. The central metal ion was bis-chelated by the bidentate ligand through the

azomethine nitrogen (N2) and thiolate sulfur (S2) atoms.

The conjugate system of the ligand was influenced by coordination with the metals, as shown by

the N1−N2 bond distance of ligand (1.376(4) Å), with a slight lengthening of the analogous bond

in the complexes (1.4186(19), 1.396(3) and 1.403(2) Å). In addition, the azomethine C9−N2

bond in the nickel complex was found to be 1.282(2) Å, and the C1-N2 bonds in the copper and

zinc complexes were found to be 1.287(3) and 1.285(3) Å, which are slightly shorter than in the

ligand (1.343(5) Å), clearly indicating that the complexation involves deprotonation at the N1

atom. The C4-S2 bond is strongly affected in the nickel (1.7397(18) and 1.7420(17) Å) copper

(

1.739(2) Å) and zinc complexes (1.741(2) and 1.737(2) Å), being longer than the analogous

bond in the Schiff base (1.667(3) Å). So, the C4-S2 bond involved in complexation is a single

bond. The Ni−S (2.1548(5), 2.1583(5) Å), Cu-S (2.2362(7), 2.2363(7) Å), Zn-S (2.2814(5),

2

.2817(5) and Ni−N (1.9320(13), 1.9254(12) Å), Cu-N (2.0011(18) Å), Zn-N (2.0546(15),

.0674(15) Å bond lengths are similar to in the bis-chelated four coordinate mononuclear

complexes 1-3. The Ni(II), Cu(II) and Zn(II) central ions have bite angles of 100.91(5),

1

01.62(11) and 108.48(6)° for N3-M-N1, while the S3-M-S1 bond angles are 90.97(18),

01.98(4) and 115.13(2)° respectively. The geometrical parameters suggest that none of the

complexes show an ideal tetrahedral geometry due to the restricted bite angles of the chelating

dithiocarbazate ligands. The metal ion occupies a position on a twofold axis and adopts a

distorted tetrahedral geometry. From the above facts, the geometry of complex 1 is more

distorted than complexes 2 and 3.

3

.1.7. DFT calculations

The density functional theory calculations were carried out in the gas phase to optimize the

structure of [H-(Ap-sadtc)] and its metal complexes 1-3, using coordinates of the crystal

structure from the CIF file. The optimized geometries of [H-(Ap-sadtc)] and the mononuclear

complexes 1-3 are depicted in Fig 2. Selected bond lengths and bond angles of both the ligand

and the complexes are given in Table 2. The calculated values are in good agreement with the

crystallographic data in all cases. The distance for the C=S bond (1.706 Å) in the ligand is

lengthened in the complexes to 1.739, 1.740, 1.805, 1.758, 1.789, and 1.779 Å, suggesting that

the sulfur atom coordinates with the metal complexes. Further, the C-N bond lengths varied from

the ligand to the complexes, indicating that deprotonation occurs on complex formation. The

optimized structures were used in the DFT calculations of vibrational frequencies. The predicted

FT-IR spectra using the B3LYP/6-311G level were correlated with an optimal scaling factor of

0

.9613 [56]. The calculated FT-IR spectra present a strong correlation and are in good agreement

with the experimental data [57]. The frontier molecular orbitals play an important role in the

electric and optical properties, as well as in UV-Vis spectra. The energies of the HOMO and

LUMO orbitals and their orbital energy gap were calculated using the DFT method and pictorial

illustrations of these frontier orbitals for the compounds are shown in Fig 2.

The highest occupied molecular orbital (HOMO) represents the outermost orbital filled by

electrons and behaves as an electron donor, while the lowest unoccupied molecular orbital

(

LUMO) is the first empty innermost orbital unfilled by electrons and behaves as an electron

acceptor. These orbitals are also called the frontier molecule orbitals (FMOs). Positive and

negative regions are shown by red and blue colours, respectively. In the ligand, almost all of the

molecules is localized in the HOMO and LUMO, except the allyl (–CH2-CH=CH2) moiety. The

energy gap between the HOMO and LUMO characterizes the chemical stability of the molecule.

The energy gaps in the case of the title ligand and compounds 1-3 are found to be 4.08, 1.48,

0

.75 and 2.42 eV, respectively. Since, a lower energy gap implies more interaction and less

stability, complex 2 has a higher chemical activity than the other compounds. This observation

suggests that the photo-excitation of the complexes could result in facile electronic transitions

from the HOMO, centered on the dithiocarbazate moiety, to the LUMO, spread over the M(II)

ion and the N, S atoms. It can also explain the remarkable photo-cytotoxicity of the

dithiocarbazate complexes. Additionally, a variety of quantum mechanical (QM) reactivity

descriptors, like the electronic chemical potential (µ) chemical hardness (ƞ) and electrophilicity

(

Ѡ) were calculated with the help of the following equations:

ƞ = (I – A)/ 2

µ = - (I +A)/2

2

Ѡ = µ /2 ƞ

where I = - EHOMO , A = - ELUMO

.

The calculated QM reactivity descriptors in Table 3 also determine the binding strength trends of

the complexes. The chemical hardness is quite useful to rationalize the relative stability and

reactivity of chemical species. Hard species, having a large HOMO–LUMO gap, will be more

stable and less reactive than soft species, having small HOMO–LUMO gap; however, a smaller

HOMO–LUMO gap requires less excitation energy and so the absorption bands of molecules are

shifted towards the visible region. This suggests that the bonding of the metal(II) ion modifies

the charge distribution of the π-electrons of the free ligand. This lower energy gap implies that

the delocalization of π-electrons from the metal complexes will result in good non-linear optical

properties in the order 2 > 1 > 3 > L. The natural charge analysis indicates electrovalent bonding

between the ligand and the corresponding metal complexes as listed in Table S2. The negative

charge of the ligand is mainly localized on the S1, N1 and N2 atoms. Upon coordination of S1 to

the metal center, the electron cloud of the C=S bond is displaced towards the S atom, which is

partially compensated by electron displacement. The calculated charges on the metals were

1

.1434, 1.0069 and 1.5728, comparably lower than the formal charge of +2 for the electronic

8

10

configurations of complexes 1, 2 (d ) and 3 (d ). This confirms electron transmission of the

donor atoms toward the central metal.

3

.2. CT-DNA binding studies

.2.1. Fluorescence emission spectra

The binding studies of small molecules with calf thymus DNA were carried out by using

fluorescence emission spectroscopy. It is well known that any antitumor drug relies on its

binding character, binding mode, selectivity toward specific DNA base pairs and kinetic

procedure followed during its reaction with DNA. An important class of compounds for cancer

chemotherapy involves non-covalent interactions between CT-DNA and the metal complexes,

such as intercalation, groove binding and electrostatic binding [81-84]. Through strong stacking,

non-covalent intercalations usually result in hypochromism/hyperchromism, with or without

blue/red shifts in the fluorescence emission spectra.

In the presence of increasing amounts of CT-DNA (0-10µM) at a constant metal complex

concentration (25µm), the emission spectral technique was used to determine the intrinsic

binding constant (k ). The interactions of complexes 1-3 with DNA have shown that for the

b

emission wavelength region of 412-429 nm, the intensity of the bands decreased from their

original intensities, based on DNA binding enhancement. It suggests that the metal complexes

are electron-acceptors and the DNA molecule is an electron-donor. All three metal complexes

show hypochromism of 27.3, 58.1 and 41%, and a small blue shift, which may be observed when

the DNA duplex is stabilized. A blue shift may also be attributed to improper coupling (due to

conformational changes) of the π* orbital of the intercalated ligand with the π orbital of the base

pairs. The intrinsic binding constant (K

b

) has been determined using the following Scatchard

equation [58]:

C

F

= C

T

[(I/I - P)/1 - P]

0

where C

and I are the emission intensities in the absence and in the presence of DNA, respectively. The

value of P is obtained from a plot of I/I versus 1/[DNA], such that the limiting emission yield is

given by the y-intercept. The amount of bound probe (C ) at any concentration is equal to C

. The slopes of the obtained Scatchard plots of r/ C versus r for the tested compounds with an

increasing concentration of CT-DNA (Fig. 3) gives the intrinsic binding constant (K ) values, as

listed in Table 4.

T

and C

F

are the concentrations of the probe metal complex (25µm) and the free probe, I

0

B

T

−

C

F

b

This observation suggests strong intercalation of the complexes to DNA, with complex 2 having

a comparatively higher intercalation than complexes 1 and 3, also being higher than the observed

values reported earlier [59]. The presence of transition metal ions in the complexes also accounts

for the higher binding extent of the complexes with CT-DNA. It has been reported earlier that a

transition metal complex ultimately leads to an effective binding with the helical structure of

DNA and exposes the embedded base pairs to the helix exterior via normal intercalation [60].

From the results of the binding constants, it is concluded that the complexes 1-3 have a strong

binding affinity with CT-DNA [61].

3

.2.2. EB-DNA displacement study

Ethidium bromide (EB) is a fluorescent dye and also a typical indicator of intercalation, since

upon the formation of soluble complexes with nucleic acids, EB emits intense fluorescence in the

presence of CT DNA due to intercalation of its aromatic ring between adjacent base pairs on the

double helix. The changes observed in the spectra of EB upon its binding to CT-DNA are often

used for the interaction study between DNA and other compounds, such as metal complexes.

Before any measurements were taken, the mixture was shaken up and recorded. The fluorescence

spectra of DNA bound EB were obtained at excitation and the emission wavelengths of 541 and

6

05 nm, respectively.

The quenching is due to the reduction of the number of binding sites on DNA that are available

to EB. The fluorescence emission intensity of the DNA–EB system in the absence and presence

of compounds added to DNA pre-treated with EB causes an appreciable reduction in the

emission intensity with increasing complex concentrations. This indicates that complexes 1-3 can

compete with EB, binding stronger to DNA. Furthermore, the quenching data were analyzed

according to the Stern–Volmer equation and Ksv values were obtained from the slope of the plot

of I /I versus [Q]. The quenching plots illustrate that the quenching of EB bound to CT-DNA by

0

complexes 1-3 are in good agreement with the linear Stern–Volmer equation. The Kapp values

were measured from the extent of reduction of the EB emission intensity by the complexes. The

apparent binding constant values were obtained for the compounds using the following equation

[

62-64]:

KEB x [EB] = Kapp [M50%]

-

7

-1

where KEB = 1.0 × 10 M is the DNA binding constant of EB, [EB] is the concentration of EB

(

7.5µM) and [M50%] is the concentration of the compound used to obtain a 50% reduction in

fluorescence intensity of DNA pretreated with EB. The Stern–Volmer constant Kapp values of the

complexes are consistent with the emission titration results. On the basis of spectroscopic

studies, it is concluded that complexes 1-3 bind to CT-DNA in an intercalative mode and

complex 2 binds to CT-DNA more strongly than the other two complexes.

3

.3. BSA interaction studies

.3.1. Fluorescence quenching study

The binding affinity of complexes 1-3 with BSA were examined by the fluorescence spectral

technique. BSA has a strong fluorescence emission band around 342 nm, fixing the excitation

wavelength at 290 nm, and the fluorescence intensity of BSA was quenched drastically when the

concentrations of the complexes 1-3 were increased. Generally, the fluorescence quenching can

be illustrated by the well-known Stern–Volmer equation [65]:

F

0

q o

/F = 1+ KSV [Q] = 1+ K τ [Q]

where F

0

and F are the fluorescence intensities of BSA in the absence and presence of the

complexes, respectively, KSV is the linear Stern–Volmer quenching constant, kq is the quenching

-8

rate constant of the biomolecule, τ

without the quencher and [Q] is the concentration of the quenching complex. The value of Ksv is

obtained from the slope of a linear plot of F /F vs [Q]. Different mechanisms occur during

quenching, which are usually classified as dynamic quenching and static quenching, in which the

o

is the average fluorescence lifetime (10 S) [66] of BSA

0

transient existence of an exited state of the fluorophore and the quencher is referred to as a

dynamic quenching process, while static quenching is due to the formation of a ground-state

complex between the fluorophore and the quencher. In both cases, the fluorescence intensity is

mainly related to the concentration of the quencher. Therefore, the quenched fluorophore can

serve as an indicator for the quenching agent. This linear Stern–Volmer plot indicates that more

than one process contributes to the overall quenching of BSA. It has been reported in many cases

that the fluorophores can be quenched by both collision (dynamic quenching) and by complex

formation with the same quencher (static quenching). This complex formation is facilitated

through hydrogen bonding and hydrophobic interactions. The quenching constant K

q

of the

complexes 1-3 follows the order: 2 > 1 > 3, which is in good agreement with the trend in the

1

0

DNA binding affinities. The maximum possible value for dynamic quenching is (2.0 x 10 L

-1

mol S ), which is comparably lower than the quenching constant of the complexes 1-3. The

1

3

14

3

-1 -1

Stern–Volmer plots show that the high quenching constant values of 10 -10 dm mol s

indicate that strong ground state complex formation contributes significantly to the overall

quenching of fluorescence intensity of BSA [67].

3

.3.2. Determination of binding parameters and binding number

Fluorescence quenching is often a result of energy transfer from a donor (Trp) to an acceptor

metal complex). However, in all the systems and combinations studied, the quenching of Trp

(

fluorescence is static and Forster’s long-range energy transfer is not taking place. When a static

quenching interaction occurs, if it is assumed that the complex binds independently to a set of

equivalent binding sites in BSA, the binding parameters can be determined according to the

Scatchard equation [68,69]:

log (F

0

-F/F) = log K′ + n log[Q]

b

where K′ is the binding constant for the binding of the complex with BSA and n is the number

b

of binding sites per BSA molecule. This gives a straight line in the plots of log(F

0

-F/F) vs

log[Q], as shown in Fig. 6.

The values of n indicate the existence of a single binding site in BSA for complexes 1-3. The

observed values of K , Kbin and ‘n’ indicate a strong interaction between the BSA protein and the

complexes, and the values follow the order: 2 > 1 > 3.

q

3

.4.1. Kinetic studies for catechol oxidase activity

Detailed reactivity and kinetic studies of complexes 1-3 were performed in dimethyl sulfoxide

DMSO) because of their good solubility along with that of the substrate 3,5-DTBC and its

(

product 3,5-DTBQ, as depicted in Scheme 3.

All the complexes show efficient catecholase activity at (pH 7.2) because of the de-protonation

of 3,5-DTBC and subsequent binding to the metal centre before its oxidation. Upon addition of

3

,5-DTBC to the metal complexes, spectral studies were carried out which showed an

immediately increment of the emission bands at 435 nm, proving the oxidation of catechol to

quinone (see Fig. 7). The kinetic studies used the method of initial rate by monitoring the growth

-4

of the quinone band. The oxidation rates and various kinetic parameters were determined by 10

M solutions of the complexes with different concentrations of 3,5-DTBC under aerobic

conditions. In all cases, a first order kinetic dependence was observed at low concentrations of

3

,5-DTBC, whereas at higher concentrations, saturation kinetics were recorded, as shown in

Figs. S15-S17. The observed data were analyzed on the basis of the Michaelis-Menten approach

for the enzymatic kinetic model, which seemed to be appropriate. The values of the Michaelis

binding constant (K ), maximum velocity (Vmax) and the rate constant (Kcat) for the dissociation

M

substrates were calculated for the complexes from graphs of 1/V versus 1/[S] (Fig. 7), known as

the Lineweaver-Burk (double reciprocal) graph, using the equation and the kinetic parameters as

listed in Table 6.

1

/V = { K / Vmax}{1/[S]} + 1/ Vmax

M

Among the three mononuclear complexes reported herein, the highest catecholase activity is

observed for complex 2. If the reduction potential is too negative, the complex shows decreased

catalytic activity due to more difficulty in reduction to copper(I), a more positive reduction

potential of the complex gives higher catalytic activity. The donor atom on the dithiocarbazate

moiety may also help to facilitate the substrate-catalyst interaction by forming a positive channel,

showing better catalytic activity [70].

On account of the proposed mechanistic steps of the catecholase oxidase activity of M(II)

complexes, a plausible mechanistic pathway is suggested, as depicted in Scheme 4. Upon

addition of 3,5-DTBC to the metal complexes 1-3, immediately the catechol molecule

coordinates to the complexes and forms an intermediate substrate-complex adduct 1a. This result

indicates a change in coordination environment of the M(II) centre from tetra to penta-

coordinate [71], which may favor electron transfer and equilibration between M(II)-(3,5-DTBC)

and the semiquinone intermediate M(I)-(3,5-DTBQ) (1b, 1c). Its subsequent reaction with

dioxygen may also result in electron reduction, leading to the oxidation of the M(I) ion and

release the quinone molecule along with hydrogen peroxide as a byproduct, 1d [72-75]. After the

quinone molecule is released, the catalyst is regenerated and the catalytic cycle continues [76].

3

.4.2. Kinetics of NPP hydrolysis

The hydrolytic reaction in enzyme-catalysis involves metal ions that are assumed to activate a

water molecule, which forms a hydroxyl group as a nucleophile in the reaction system [77]. The

-

M(II)-bound OH then acts as a nucleophile to attack the phosphate atom of 4-NPP ester, which

leads to hydrolysis (Fig. 8), shown by emission increases at 485 nm due to the formation of 4-

nitrophenolate ions over time (Scheme 5). Since, the catalyst concentration was essentially

constant during measurements, the initial first order rates were measured at different

concentrations of the substrate at pH 7.2 and room temperature. The initial rates obtained for a

range of 4-NPP concentrations were fitted to the Michaelis-Menten equation (Figs. S18-S20) and

linearized by means of the Lineweaver–Burk method, i.e. 1/rate vs. 1/[4-NPP] by changing the

concentration of the substrate (Fig. 8). The kinetic parameters of K , Vmax and the catalytic

M

constant Kcat values are summarized in Table 7. Hydrolysis of 4-nitrophenylphosphate by the

Cu(II), Ni(II) and Zn(II) complexes show that the Cu(II) complex has a higher catalytic activity

than the Ni(II) and Zn(II) complexes, similar to earlier reports [78,79]. All the complexes have

shown better efficacy than catecholase activity.

3

.5.1. In vitro anticancer activity evaluation by an MTT assay

In the present study, the antitumor efficacy of complexes 1–3 was assayed by determining the

viability of HeLa cells using an MTT assay and cis-platin was used as a positive control to assess

the cytotoxicity of the tested mononuclear complexes. The results were analyzed by means of

cell inhibition, expressed as IC50 values which are listed in Table 8. It is notable that complex 2

possesses the most potent inhibitory effect against the HeLa cell line. The IC50 value is well

known for the anticancer drug cisplatin, as shown in Fig. 9. The results of the MTT assay in

Table 8 shows that complexes 1-3 inhibit the growth of cells in a concentration dependent

manner. A low IC50 value implies cytotoxicity at low drug concentrations. The significant

inhibitory activity of the complexes and their binding affinity to CT-DNA/BSA clearly indicates

that the incorporation of the complexes has a marked effect on the cytotoxicity. In addition, an in

vitro cytotoxicity assay conducted on a HeLa cancer cell line versus normal vero cell line has

demonstrated that complexes 1-3 show profound anticancer activity.

3

.5.2. AO-EB/DAPI staining assay

Cell apoptosis is an autonomic ordered programmed cell death in order to maintain homeostasis,

which is controlled by serial genes. The morphological changes of HeLa cells have been

observed under a light microscope. The design of chemotherapeutic drugs, in order to understand

the complexities of apoptosis evolved by cancer cells and the development of strategies to

selectively induce apoptosis in cancer cells, have turned into a unique target in cancer drug

development [80]. The morphological changes induced by complexes 1-3 were evaluated using

AO/EB and DAPI-stained HeLa cancer cells. After treatment of HeLa with 12.5 µM of 1-3,

apoptotic cells with apoptotic features, such as nuclear shrinkage and chromatin condensation,

were found, as shown in Fig. 10. In this figure, yellow arrows show early apoptotic HeLa cells

with membrane blebbing, which is seen at a fixed concentration of complexes 1-3, and blue

arrows exhibit late apoptotic cells with chromatin aggregation, that is highly condensed

chromatin. Necrotic cells having uniform orange to red nuclei with a condensed structure are

observed. The overall results indicate that complexes 1-3 induce cell death by necrosis, which is

in good agreement with the above toxicity results.

4. Conclusions

In conclusion, we have synthesized an N,S donor Schiff base and its mononuclear Ni(II), Cu(II)

and Zn(II) complexes. The characterization of these compounds was carried out using elemental

1

13

analysis, FT-IR, UV-vis, H NMR, C NMR and mass spectrometry. Structural determination

reveals that the complexes are discrete four coordinated, with a slightly distorted tetrahedral

geometry. DFT calculations on all the complexes are in good agreement with the structural

results obtained by X-ray crystallography. The CT-DNA/protein binding properties of the

complexes were examined by fluorescence spectra. All the fluorescence experiments show that

the complexes 1-3 interact strongly with CT-DNA by an intercalative mode and BSA confirmed

that the complexes have a greater binding affinity via the static mode. In this connection, the

catecholase biomimetic catalytic activities of complexes 1-3 have been investigated. The results

obtained show that all the complexes catalyze the aerobic oxidation of catechol to the

corresponding o-quinone. Interestingly, all the three mononuclear complexes provide good

phosphate-hydrolysis catalytic activity that is relevant to the binding of phosphate esters to the

active site of an enzyme that catalyzes the phosphoryl transfer reaction. The cytotoxicity study

shows that the complexes exhibit good cytotoxic activity against HeLa cell lines. Additionally,

apoptotic tests indicate that the complexes show an apoptotic effect on HeLa cells. Based on the

observed results, further studies are planned with these compounds against lung, bladder and

breast cell lines.

Acknowledgement

The author N. N would like to thank P. Sugumar, Centre of Advanced Studies in Crystallography

and Biophysics, University of Madras for his help in the valuable crystallographic discussions.

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

and the Director, CAS in Botany, School of Life Sciences, University of Madras for providing

laboratory facilities to perform the cell lines studies.

Electronic supplementary information (ESI) available

1

UV spectra of the compounds (Figs. S1-S4), FTIR spectra of the compounds (Figs. S5-S8), H

1

3

NMR spectra of the compounds (Figs. S9-S10), C NMR spectra of compounds (Figs. S11-

S13), ESI Mass spectrum of complex 1 (Fig. S14), kinetic Plot (3,5-DTBC) rate vs concentration

of the complexes (Figs. S15-S17), kinetic plot (4-NPP) rate vs concentration of the complexes

(

Figs. S18-S20), DFT and experimental values of IR spectra (Table S1). The natural charge and

electronic configuration (Table S2). CCDC reference numbers 1418965-1418968 for the

compounds. For ESI data in CIF or other electronic format see DOI:

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Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II), Cu(II) and Zn(II): Synthesis, characterization and biological studies

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