Magneto-structural correlation, antioxidant, DNA interaction and growth inhibition activities of new chloro-bridged phenolate complexes

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Paper

Magnetoꢀstructural correlation, antioxidant, DNA interaction and

growth inhibition activities of new chloroꢀbridged phenolate complexes

Perumal Gurumoorthy,^aDharmasivam Mahendiran,^aDurai Prabhu,^bChinnasamy Arulvasu^band

Aziz Kalilur Rahiman,*^a

5

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI: 10.1039/b000000x

A new class of chloroꢀbridged dinuclear nickel(II) and copper(II) phenolate complexes (1–8) were

synthesized from 4ꢀsubstitutedꢀ2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenols (L^1–4) and characterized. The

XRD analysis of complexes 4 and 8 consist of two mononuclear units connected through a bridged chlorine

10 atom that gives dinuclear complexes. The stability of complexes has been determined using a

spectrophotometric method. Complexes 5–8 possess significant antioxidant activity against DPPH radical.

The binding studies of complexes with CTꢀDNA suggest partial intercalative/electrostatic interaction and the

cleavage ability on pBR322 DNA shows the involvement of the hydroxyl radical as an intermediate in the

cleavage reaction. The IC₅₀value of complexes 2, 6 and 8 against HepG2 cell line is comparable with that of

15 cisplatin. To find the extent of nuclear chromatin cleavage, propidium iodide staining and comet assays were

employed. Among the newly synthesized complexes, copper(II) complexes exhibited superior biological

activity when compared to its nickel(II) analogues.

drawbacks have prompted researchers to develop other transition

Introduction

metalꢀbased drugs. In this connection, attempts are being made to

40 replace these drugs with more efficacious, less toxic, and target

specific nonꢀcovalently DNA binding (Intercalation, groove

binding and external static electronic effects) chemotherapeutic

agents. Among the DNA binding modes, intercalation is the most

important one, in which the molecules can intercalate between the

⁴⁵baseꢀpairs of double helix DNA, forming π–π overlapping

interaction, and it is related to the antitumor activity of the

molecule.⁴Nickel⁵and copper⁶complexes are regarded as most

promising alternatives to platinum complexes as anticancer drugs.

Nickel(II) complexes display interesting binding and cleavage

⁵⁰reactivity with the nucleic acids.⁷Copper is known to play

significant role in biological systems, as pharmacological agents

and copper(II) complexes are found to exhibit prominent

antitumor activity.⁸

Phenolic Schiff base ligands have received increasing attention

55 because of their ease of formation, mixed hardꢀsoft donor

character, versatile coordination behavior and their diversified

applications.⁹Transition metal ions play a pivotal role in a vast

number of diverse biological processes, and their metal

complexes have been widely exploited, because of their unique

60 spectral, electrochemical, magnetic and catalytic signatures but

also due to the fact that by changing the ligand environment one

can tune the DNA interaction of a metal complex.¹⁰A further

reason for using metal containing compounds as structural

scaffolds relates to the kinetic stabilities of their coordination

65 spheres in the biological environment.¹¹In past decades, several

phenolic based transition metal complexes were reported as they

are avid DNA intercalative binders as well as DNA cleavers.¹²

DNA is storage and carrier of genetic information in a cell and it

²⁰is the primary intracellular target of anticancer drugs. The

interaction between the small molecules and DNA can cause

DNA damage, blocking the division of cancer cells, and resulting

in cell death.¹The DNA cleavage is an enzymatic reaction which

comprises various biological processes as well as

25 biotechnological manipulation of genetic material. Nowadays,

exploring and designing novel molecules capable of interacting

with DNA, and triggering apoptosis is one of the strategies for

researchers to discover effective DNAꢀtargeted anticancer drugs

for chemotherapy.²

30

In modern medicinal field, widely and effectively used

antitumor drugs are Ptꢀbased cisplatin [cisꢀdiamminedichloro

platinum(II)], carboplatin [cisꢀdiammine(cyclobutaneꢀ1,1ꢀdi

carboxylatoꢀO,O')platinum(II)], and oxaliplatin [((1R,2R)ꢀcyclo

hexaneꢀ1,2ꢀdiamine)(ethanedioatoꢀO,O')platinum(II)], but they

35 possess inherent limitations such as ototoxicity, nephrotoxicity,

peripheral neuropathy and neurotoxicity, nausea, myeolotoxicity,

and tumor resistance.³Hence, the efforts to mitigate the

__________________________________________________________________

^aPostꢀGraduate and Research Department of Chemistry, The New

College (Autonomous),Chennaiꢀ600 014, Tamil Nadu, India

Fax: +91 44 2835 2883

Eꢀmail: akrahmanjkr@gmail.com

^bDepartment of Zoology, University of Madras, Guindy Maraimalai

Campus, Chennaiꢀ600 025, Tamil Nadu, India

† Electronic supplementary information (ESI) available: Tables S1

Scheme S1 and Fig. S1–S10, See DOI: 10.1039/b000000x/

–S5,

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Moreover, transition metal complexes are intensely coloured due

to localized MLCT (metal to ligand charge transfer transition),

free ligands. The resonance signal of the NH proton is appeared

to be shifted significantly. Further, the broad signal (10.94–10.99

and this transition is particularly important as it is perturbed when ⁶⁰ppm) was observed for the OH proton of the methanol. The NMR

complex interacts with DNA, providing a spectroscopic probe.

The foregoing facts stimulated us to focus on the studies of

biological activities of new haloꢀbridged dinuclear complexes

containing phenolate ligands, which is continuation of our recent

spectra gave additional information about the structure of the

complexes, which are consistent with the crystal structure of

complex 4.

5

In DMF medium, complexes 1–8 exhibit two intense bands in

report¹³. Amongst the metal ions chosen, we have opted for bioꢀ ⁶⁵the UV region (250–400 nm) attributed to the intraligand charge

compatible metal ions, Ni and Cu. These metal complexes are

transfer transitions (π–π*, n–π*). The bathochromic shift of n–π*

transition band in complexes suggests the coordination of

azomethine with metal center.¹⁵In the visible region, the

nickel(II) complexes (1–4) shows two absorption bands around

¹⁰better suited for medical applications because of their favorable

rate of ligand exchange and their ability to mimic iron binding to

certain bioꢀmolecules. Herein, we report the synthesis and

characterization of chloroꢀbridged dinuclear nickel(II) and ⁷⁰624–644 and 930–973 nm whereas the copper(II) complexes

copper(II) complexes containing Schiff base ligand, 4ꢀsubstituted

¹⁵ꢀ2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenol. Antioxidant

DNA interaction and growth inhibitory activities of complexes

were performed in order to investigate their biological efficacy.

(5–8) exhibit one absorption band in the region of 655–668 nm

characteristic of metal(II) ion located in distorted octahedral¹⁶and

squareꢀpyramidal¹⁷environment, respectively. The crystal

structure of complexes 4 and 8 have been determined by using

⁷⁵single crystal XRD analysis which is consistent with the

absorption spectral data and hence the proposed structure of

complexes.

Results and Discussion

Synthesis and spectral properties

²⁰The chloroꢀbridged dinuclear nickel(II) and copper(II) complexes

(1–8) have been isolated by direct reaction of ligands (L^1–4) with

an equimolar ratio of metal(II) chlorides in methanol. The ligands

were used without further purification for the complexation

reactions. All complexes are obtained in good yield and, they are

²⁵soluble in H₂O, DMF, DMSO and 5 mM TrisꢀHCl/50 mM NaCl

buffer (pH 7.2). The synthesized metal complexes were

characterized by means of the spectroscopic methods. All the

resulted complexes are characterized by the molar ratio between

the ligand and metal ion (2:2). The following series of complexes

³⁰have been obtained and crystallographically characterized.

IR spectra of complexes are compared with those of free

ligands to gain evidence for the coordination mode with the metal

ions. All complexes exhibit bands in the region of 1655–1637 and

1309–1290 cm^–1due to v(C=N) and v(Ar–O), respectively, which

35 were observed at lower frequency than that of free ligands,

suggest the coordination via imine nitrogen and phenoxide atom,

and further confirmed by single crystal XRD structure of

complexes 4 and 8. A broad band in the region 3452–3379 cm^–1

indicates v(O–H) stretching of methanol/water and water

⁴⁰molecules for nickel(II) and copper(II) complexes, respectively,

and the broadness of O–H stretching is due to hydrogen bonding.

All complexes exhibit a medium intensity bands in the region of

2964–2929 and 1549–1524 cm^–1due to N–H stretching and

bending of piperazinium ion,¹⁴respectively. The ESIꢀMS results

45 of complexes 1–8 exhibit peaks corresponds to the half unit of the

complexes.

Fig. 1 ORTEP representation of complex 4 showing an atom numbering

scheme and displacement ellipsoid (30% probability level). All the

hydrogen atoms are omitted for clarity.

The ¹H NMR spectra (Fig. S1) of diamagnetic nickel(II)

complexes (1–4) show the considerable changes in the chemical

shift of the proton signals when compared with the respective

⁵⁰ligands, which we have reported recently.¹³The complexes

exhibit the signal for the coordinated azomethine proton at 8.42–

8.54 ppm, and these are shifted downfield with respect to the

corresponding resonance in the free ligands, indicating that the

metalꢀnitrogen bond is retained in solution. The signals of

55 aromatic (6.74–7.64 ppm) and aliphatic protons (methyl and

methylene protons; 2.01–3.72 ppm) appear in their usual

positions with slight downfield shifts when compared with the

Description of crystal structure of [(NiL⁴(CH₃OH)₂)₂(µꢀCl)]

(Cl)₃.(H₂O)₂, 4

80 Suitable single crystals of green coloured nickel(II) complex

[(NiL⁴(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂, 4 was obtained by slow

evaporation of the reaction mixture for several days in dark room

which crystallizes in a monoclinic space group C2/c. Half of the

title molecule forms the asymmetric unit and two halves of the

85 molecule are related through centre of inversion. The ORTEP

diagram of the molecule 4 showing the atom numbering scheme

is given in Fig. 1, while their selected bond lengths and angles are

2

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given in Table S1. The measured unit cell dimensions are a =

10.1699(4), b = 16.8523(8), c = 24.8924(11) Å, β = 98.768(2)

and Z = 4. The solved structure contains one mononuclear Ni(II)

hydrogen bonding exhibit between O(2)–HꢀꢀꢀO(1)' and O(2)'–Hꢀꢀ

ꢀO(1). The adjacent molecules are connected through O(3)–Hꢀꢀꢀ

O(4), N(3)–HꢀꢀꢀCl(2) and N(3)–HꢀꢀꢀCl(3) interactions to form a

unit with an half coordinating chorine atom which bridges the ⁴⁵two dimensional supramolecular sheet extending parallel to [0 1

5

another half unit through a 2ꢀfold axis along [0 1 0] direction at

(1/2, y, 3/4) to form the title compound. Apart from these, the

structure contains three non coordinating chloride anions and two

water molecules. The symmetry operation connecting the

bridging chlorine atom is about 1 – x, y, 3/2 – z.

0] plane. The final Rꢀvalue of complex 4 was 0.0333, and the

final electrodensity map contains maximum and minimum peak

heights of 0.581 and –0.500 e.Å^–3, respectively.

10

The nickel(II) ion in complex is hexacoordinated and best

described as a distorted octahedral coordination geometry with

two nitrogen atom, an imine (N1) and piperazine (N2), one

phenolate (O1) and two methanol solvent (O2, O3) oxygen atoms

in addition to one bridged chlorine (Cl1) atom between the metal

¹⁵centers Ni(1) and Ni(2). The bond distances of Ni(1)–N(1),

Ni(1)–N(2), Ni(1)–O(1) and Ni(1)–Cl(1) are 2.0085(18),

2.222(2), 1.9974(16) and 2.388(5), respectively. The fifth and

sixth coordination positions of the nickel(II) ion is occupied by

two methanol solvent molecules with Ni(1)–O(2) and Ni(1)–O(3)

²⁰distance of 2.0748(17) and 2.1181(16) Å, respectively and O(2)–

Ni(1)–O(3) angle of 176.51(7)°. The deprotonation of phenolic

hydrogen atom followed by the coordination of nickel with

oxygen atom leads to shortening of carbonꢀoxygen bond and

lengthening of carbonꢀcarbon bond. For example the observed

²⁵C(1)–O(1) bond length is 1.302(3) Å as against 1.351(3) Å for

free bromophenol related Schiff base ligand and C(1)–C(2) and

C(1)–C(6) bond lengths are 1.401(4) and 1.422(3) Å,

respectively, which are higher than the aromatic C–C distances of

the same bromophenol related Schiff base ligand.¹⁸The dimeric

30 complex [(NiL⁴(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂associates with a

Fig. 3 ORTEP representation of complex 8 showing an atom numbering

scheme and displacement ellipsoid (30% probability level). All the

hydrogen atoms are omitted for clarity.

Description of crystal structure of [(CuL⁴Cl)₂(µꢀCl)]Cl.

50 (H₂O)₇, 8

Dark green crystals of complex [(CuL⁴Cl)₂(µꢀCl)]Cl.(H₂O)₇, 8

suitable for Xꢀray diffraction studies was obtained by the slow

evaporation of reaction mixture for several weeks in dark room,

which crystallizes in the monoclinic space group P2₁/n. Similar to

55 complex 4, a chlorine atom forms bridge between the two copper

atoms. The ORTEP representation of the molecule showing the

numbering scheme is given in Fig. 3, while their selected bond

lengths and angles are given in Table S3. The measured unit cell

dimensions are a = 13.8391(4), b = 14.3968(4), c = 20.2234(7) Å,

60 β = 98.6740(10) and Z = 4. The solved structure shows one

chlorine atom bridges two mononuclear Cu(II) complexes

through a 2ꢀfold screw axis along [0, 1, 0] direction at (1/4, y,

1/4) with screw component [0, 1/2, 0] to form the dinuclear title

complex. Apart from these, the structure contains one

65 uncoordinated chlorine atom and seven water molecules occupy

the crystal lattice as free molecules.

Fig. 2 Intraꢀ and intermolecular hydrogen bonding network in complex 4.

Ni(1)–Ni(1)#1 distance of 4.096(4) Å, which is shorter than that

of reported dinuclear chloroꢀbridged Ni(II) complex.¹⁹

Nonꢀcovalent (like πꢀstacking) interactions with aryl hydrogen

and hydrogen bonding network are important class in

35 supramolecular chemistry and crystal engineering.²⁰The

molecular packing in the crystalline solid is constituted by

intramolecular and intermolecular hydrogen and halogen bonds

(Fig. 2) and the hydrogen bonding parameters are listed in Table

S2. The lattice chloride anions are able to act as a proton

40 acceptor, interacting with the partially charged N–H group and

O–H group of lattice water molecules. The intramolecular

The copper(II) ions in complex 8 is pentacoordinated and the

geometry around the copper nuclei is best described as squareꢀ

pyramidal with a large contribution from the distortion constant

⁷⁰(or) structural index parameter (τ) values of 0.002 and 0.335 for

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Cu(1) and Cu(2), respectively (τ = (β–α)/60, where β and α (in º)

are the two largest L–M–L angles O(1)–Cu(1)–N(2)

167.64(13)º; N(1)–Cu(1)–Cl(1) = 167.48(11)º; O(2)–Cu(2)–N(5)

= 173.97(13)º; N(4)–Cu(2)–Cl(3) = 153.82(12)º, a regular

trigonalꢀbipyramid (TBP) and squareꢀbased pyramid (SP) have τ

values of 1 and 0, respectively).²¹The geometry and the bond

and the nature of the substituents at para position of the

60 phenoxide ion in benzene ring influences the redox behavior in

the negative potential range. Reduction process voltammograms

for complexes 1–8 are shown in Fig. S3 & S4 and the results are

given in Table S5.

=

5

In cathodic region, all complexes show two reduction waves at

distances/angles around the metal atoms Cu(1) and Cu(2) differ ⁶⁵different potentials. From the voltammograms, ∆E_pvalue for the

from each other. Among the two metal centers Cu(2) has

comparatively larger distortion than the Cu(1) ion. The adopted

¹⁰five coordination geometry of the two metal ions consists of an

imine [Cu(1)–N(1) = 1.955(3) and Cu(2)–N(4) = 1.964(3) Å] and

Ni(II) complexes for the first and second redox couple are more

than 500 and 400 mV, respectively. This higher ∆E_pvalue is due

to the difference between the original complex and the reduced

species. The cyclic voltammograms of Ni(II) and Cu(II)

piperazine nitrogen atoms [Cu(1)–N(2) = 2.104(3) and Cu(2)– ⁷⁰complexes show the first reduction potential values in the

N(5) = 2.089(3) Å], one phenolate oxygen atom [Cu(1)–O(1) =

1.945(3) and Cu(2)–O(2) = 1.935(3) Å] and a bridged chlorine

¹⁵atom [Cu(1)–Cl(2) = 2.626(12) and Cu(2)–Cl(2) = 2.524(12) Å]

between the two metal ions, Cu(1) and Cu(2). The fifth

following order [ML³]₂> [ML⁴]₂> [ML¹]₂> [ML²]₂. The

observed less negative potential of the first reduction process for

complexes containing ligands L^3&4, when compared to other

complexes is due to the presence of electron withdrawing

coordination site of the metal ions is occupied by the chlorine 75 substituent at the para position of phenoxide ion in the phenyl

atom at a distance of 2.270(12) and 2.325(12) Å for Cu(1)–Cl(1)

and Cu(2)–Cl(3), respectively. As observed for complex 8, the

²⁰deprotonation of phenolic oxygen atom leads to shortening of

C(4)–O(1) = 1.315(5), C(17)–O(2) = 1.310(5) Å bonds and the

ring and reduced at less negative potential, which decreases the

electron density around the metal ions and favors easy reduction.

Based on the observations, the reduction processes for Ni(II)

and Cu(II) complexes are irreversible. The following steps may

coordination of copper(II) atoms with O(1), O(2) leads to the 80 involve in the reduction process:

lengthening of the C(3)–C(4) = 1.411(6), C(16)–C(17) = 1.411(6)

M^II₂→ M^IM^II→ M^I₂

Å and C(4)–C(5) = 1.419(6), C(17)–C(18) = 1.414(6) Å bonds

²⁵compared to the bromophenol related Schiff base ligand.¹⁸The

Cu(1)–Cu(2) distance of 4.612(8) Å and Cu(1)–Cl(2)–Cu(2) bond

angle of 127.11(5)º is higher than that reported for the mono

chloroꢀbridged Cu(II) complex [Cu(2)–Cu(3) = 4.016(2) Å and

Cu(2)–Cl(2)–Cu(3) = 106.56(5)º],²²due to lesser bending angle.

30 Table S4 shows the hydrogen bonding parameters and Fig. S2

shows the representation of intraꢀ and inter molecular hydrogen

bonding of complex 8.

In complex 8, the bond lengths around both metal atoms Cu(1)

and Cu(2) are differ from each one. Cu(2) metal atom displays

³⁵lesser bond lengths with phenolic oxygen (O1), piperazine

nitrogen (N5) and bridgedꢀchlorine (Cl2) atom whereas higher

distance with imine nitrogen (N4) atom when compared to same

with respect to Cu(1) metal atom. But both the metal atoms Cu(1)

and Cu(2) exhibit lower coordination bond lengths with tridentate

⁴⁰ligand and bridgedꢀchlorine atoms than that of nickel atoms Ni(1)

and Ni(2) of complex 4, which implies the bond strength between

metal and donor atoms.

The cyclic voltammograms of oxidation process for Ni(II)

complexes 1–4 are shown in Fig. S5 and the oxidation peak

values are given in Table S5. The nature of the oxidation process

⁸⁵is quasiꢀreversible and the following steps may involve in the

oxidation process:

Ni^II₂⇌ Ni^IIINi^II⇌ Ni^III

2

When compared to the chloro complex, the bromo complex

gives higher values of E¹and E²_1/2. This is due to the addition

1/2

90 of an electron to the bromo complex is more difficult than the

chloro complex as the basicity of the bromide ion is higher when

compared to the chloride ion.

Determination of stability constants and thermodynamic

parameters

95 The stability constants (ln K) of the selected complexes in 90%

aqueous DMF were determined at different ionic strengths (I =

0.05, 0.10 & 0.20) and temperatures (25, 35 & 45±0.2 ºC) using a

spectrophotometric method. It was found that the stability

constant value of the complexes is proportional to the ionic

100 strength and inversely proportional to the temperature (Table 1).

Thermodynamic stability constants (ln K^ө) and thermodynamic

parameters (∆H^ө, ∆S^ө& ∆G^ө) of the complexes were derived from

the stability constants.²⁶The plots of ln K^өversus 1/T at zero

ionic strength gave linear curves (Fig. 4), showing that ∆H^өand

105 ∆S^өare essentially independent of temperature over the

temperature range considered. The ∆H^өand ∆S^өvalues were

calculated from the plots (–∆H^ө/R = slope and ∆S^ө/R = intercept,

at 1/T = 0). The value of ∆G^өwas calculated for each metal–

ligand system by using the following equation, ∆G^ө= –RT ln K^ө.

110 The results of thermodynamic parameters implies the exothermic

nature (–∆H^ө) of the metal–ligand interaction, and spontaneous

(+∆S^ө& –∆G^ө) formation of the complexes.

Electrochemical properties

The molar conductivity values in DMF solution (10^–3M) at

45 25 °C, for complexes 1–4 and 5–8 are in the range 282–294 and

125–137 ꢁ^–1cm²mol^–1, due to trisꢀunivalent and uniꢀunivalent

electrolytic behaviours, respectively.²³The electrochemical

properties of complexes have gained our attention because, the

redox properties provides information for its further application

50 in electrochemistry and catalytic reactions. Generally, the

electrochemical properties of complexes depend on a number of

factors such as chelation, axial ligation, degree and distribution of

unsaturation and substitution pattern.²⁴The redox behavior of all

complexes has been investigated by cyclic voltammetry under

55 similar experimental conditions.

The phenolic metal complexes usually undergo reduction at

cathodic potentials, because of the negative influence and hard

nature of phenoxide atoms in the ligand.²⁵The complex structure

4

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range 20–300 K at a constant applied magnetic field of 5000 Oe.

The results of the magnetic measurements for complex 8 is

²⁵displayed in terms of molar susceptibility (χ_M), effective magnetic

moment (ꢁ_eff), and χ_MT product versus thermal variation in Fig. 6.

The molar susceptibility χ_Mvalue increases smoothly from 0.9 ×

10^–3cm³mol^–1with decrease in temperature from 300 K, and

between 80 to 20 K a rapid increase from 2.5 × 10^–3cm³mol^–1to

30 9 × 10^–3cm³mol^–1is observed. The change of magnetic moment

with temperature in the dinuclear copper(II) complexes usually

supported by the following factors: contribution of orbital

momentum, intramolecular magnetic interaction between two

metal centers and antiferromagnetic interaction.²⁷The effective

35 magnetic moment of complex 8 decreases slowly from 1.50 ꢁ_Bat

300 K to 1.35 ꢁ_Bat 60 K and then decreases rapidly to 1.18 ꢁ_Bat

20 K indicating strong intramolecular antiferromagnetic exchange

interaction between two Cu(II) nuclei. As T lowers from 300 to

55 K, the χ_mT product of 8 decreases monotonously from 0.28 to

⁴⁰0.22 cm³K mol^–1and then rapid decrease to 0.18 cm³K mol^–1at

20 K, due to temperatureꢀindependent paramagnetism. The

observed behavior suggests the existence of an intramolecular

antiferromagnetic interaction between copper(II) centers. The

susceptibility data were leastꢀsquares fitted to the modified

⁴⁵BleaneyꢀBowers equation for Cu(II) dimers.²⁸

7.62

7.56

7.50

7.44

2

4

6

8

0.020

0.025

0.030

0.035

0.040

1/T

Fig. 4 The plot of thermodynamic stability constant of complexes 2, 4, 6

and 8 versus 1/T.

Table 1 The stability constants (ln K) at different temperatures

and ionic strengths (I = mol/dm³), and the thermodynamic

parameters

χ_M= Ng²β²/3kT[(1–P)(3+e^–2J/kT)^–1] + 0.45(P/T) + N_α

ln K

Temp

K

–∆G^ө/ –∆H^ө/ ∆S^ө/

kJ/mol kJ/mol J/mol K

Complex

ln K^ө

I = 0.20 I = 0.10 I = 0.05

Where, χ_M– molar magnetic susceptibility; N – Avogadro’s

number; g – average gyromagnetic ratio fixed at 2.18 obtained

from EPR measurements; β – Bohr magneton; k – Boltzmann

⁵⁰constant; J – singletꢀtriplet energy separation; P – percentage of

monomeric impurities (0.0015); N_α– temperature independent

paramagnetism (TIP) usually assumed to be 120 × 10^–6cm³mol^–1

for Cu(II) dimers. From the best fit of BleaneyꢀBowers equation,

the singletꢀtriplet energy separation, –2J, is calculated to be 228

⁵⁵cm^–1at room temperature. The obtained –2J value and increase of

χ_M, the decrease of ꢁ_effand χ_MT product upon lowering the

temperature suggest the presence of an intramolecular

antiferromagnetic interaction between the two Cu(II) centers via

chlorideꢀbridge. The observed lower –2J value for complexes 7

⁶⁰(220 cm^–1) and 8 (228 cm^–1), when compared to complexes 5

298 7.660 7.636 7.625 7.613 18.86

308 7.617 7.605 7.599 7.593 19.44

318 7.612 7.595 7.586 7.578 20.04

298 7.496 7.486 7.478 7.472 18.51

2

17.86 62.64

4

6

8

308 7.485 7.474 7.469 7.463 19.11 9.11 61.76

318 7.474 7.463 7.456 7.451 19.70

298 7.628 7.622 7.619 7.616 18.87

308 7.633 7.616 7.604 7.596 19.45 18.71 62.68

318 7.629 7.601 7.595 7.582 20.05

298 7.606 7.555 7.534 7.508 18.60

7.566 7.536 7.522 7.506 19.22

7.575 7.530 7.513 7.491 19.81

9.59 62.12

308

318

5

Magnetic properties

EPR and VSM studies: The 3d⁹configuration of Cu(II) with

squareꢀpyramidal geometry have magnetic moment

approximately given by the spinꢀonly value of √3ꢁ_B. The room

10 temperature magnetic moment (ꢁ_eff) values for the chloroꢀbridged

copper(II) complexes 5–8 were calculated by the equation, ꢁ_eff

10.0

0.30

0.25

7.5

0.20

χ

=

2.828[χ_MT]^1/2and ranges from 1.49 to 1.52 BM, which is lower

than the spinꢀonly value of 1.73 BM for high spin Cu(II) center

with S = 1/2, suggests the presence of intramolecular antiferro

15 magnetic interaction between Cu(II) nuclei. Xꢀband EPR spectra

of all the dry and powdered copper(II) complexes exhibit a broad

signal (Fig. S6) with ‘g_||’ value 2.15–2.19, and the similar results

obtained in DMF solution implies that the squareꢀpyramidal

geometries of the complexes, as observed in the Xꢀray crystal

²⁰structure of complex 8, is retained in solution.

1.50

5.0

2.5

χ

1.35

ꢀ

1.20

0

75

150

225

300

T (K)

Temperatureꢀdependent magnetic susceptibilities of dry and

powdered complexes 5–8 were measured in the temperature

Fig. 5 Temperature dependence of χ_M, ꢁ_effand χ_MT product for complex

8 in the range 20–300 K.

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(236 cm^–1) and 6 (239 cm^–1) is due to the presence of electron

withdrawing substituents on the phenyl ring which decreases the

electron density on the copper(II) nuclei i.e. reduction in electron

The DPPH radical is a stable free radical which shows a strong

absorption band at 517 nm in visible spectrum due to the

presence of an odd electron. As this electron becomes paired off

density on the copper nuclei are less favorable for effective ⁶⁰in the presence of a free radical scavenger, this absorption

5

coupling resulting in a lower –2J value.²⁹

vanishes resulting in decolorization stoichiometrically with

respect to the number of electrons taken up. The reactivity of free

radicals can be neutralized by the donation of an electron or

hydrogen. Lower the IC₅₀values, greater the hydrogen donating

⁶⁵ability and thus the antioxidant activity of the free radical

scavengers. We have compared the abilities of the present

complexes to scavenge DPPH radicals with those of the wellꢀ

known standards including natural antioxidant vitamin C and

synthetic antioxidant BHT (Butylated toluene) under the same

⁷⁰experimental conditions. The IC₅₀values (Table 2) obtained from

in vitro radical scavenging assay strongly support that complexes

presented in this work possess excellent scavenging activity

against DPPH radical, which is more or less equal to the standard

Magnetoꢀstructural correlation: The strong antiferromagnetic

interaction can be explained as follows: The magnetoꢀstructural

parameters of complex 8 are compiled and the results are

consistent with those of related mono chloroꢀbridged

¹⁰complexes.³⁰In a squareꢀpyramidal geometry around the Cu(II)

ions, the unpaired electron mainly resides in the d_{x –y}orbital i.e.

2

magnetic orbital, and a halfꢀfilled band formed by combination of

such orbitals correspond to the highest occupied band (HOB).

The two copper(II) nuclei have a squareꢀpyramidal geometry with

¹⁵a apices occupied by the bridgedꢀchlorine for both Cu(1) and

Cu(2), coupling can occur through this chloroꢀbridge. In a series

of mono chloroꢀbridged copper(II) dimeric complexes, Hatfield

et. al.³¹has suggested that a smooth correlation exists between

the exchange parameter J and the ratio θ/R (where θ is the

²⁰magnitude of the angle at the Cu(1)–Cl–Cu(2) bridge and R is the

Cu–Cl distance). The overall magnetic behaviour can be expected

for values of the quotient θ/R lower than approximately 40 and

higher than 57 while antiferromagnetic character appears when

this quotient θ/R is between these two values. Cortés et. al.³²have

25 reported that the increase in distortion of squareꢀpyramidal

toward the trigonal bipyramidal geometry causes an effective

antioxidants (vitamin

C and BHT). Among these tested

⁷⁵complexes, copper(II) complexes show better scavenging activity

than nickel(II) analogous, because of the availability of an odd

electron in copper(II) ion, which decreases the capacity to

stabilize unpaired electrons and there by arrest the free radicals.

The antioxidant efficacy (IC₅₀) of the present complexes

80 decreases in the order of 6 (85.3±1.6) > 5 (88.1±0.9) > 8

(92.9±4.8) > 7 (95.8±3.3) > 2 (124.1±1.2) > 1 (131.8±3.6) > 4

(166.3±1.4) > 3 (186.5±0.9). The scavenging effects of the

copper(II) complexes is significantly higher than that of nickel(II)

complexes containing the same ligands. These results clearly

⁸⁵indicate that the synergic combination of ligand and metal ions

plays vital role in the design of a potential antioxidant. The

antioxidant property of different ligands follows the same order

by changing metal ions. The presence of an electron releasing

group at the para position to the phenoxoꢀligand shows higher

⁹⁰radical scavenging activity than that with an electron withdrawing

group. Although the mechanism of radical scavenging activity of

complexes under study remains unclear, these experimental

results are helpful in designing more effective antioxidant agents

against DPPH radical and also other free radicals.

mixing of magnetic orbitals with the d_zorbital and leads to

2

stronger magnetic interactions and also observed an increase in

antiferromagnetic coupling as the Cu–Cl–Cu angle increases. The

³⁰increase in distance between metal centers influencing the

exchange parameter J which becomes more negative. Thus, the

observed –2J value (–228 cm^–1) found for chloroꢀbridged

copper(II) complex 8 can be explained by the presence of a

relatively higher Cu(1)–Cl(2)–Cu(2) angle of 217.11(5)° and less

³⁵Cu(1)–Cu(2) distance of 3.954 Å.³³

Antioxidant studies

Many bioorganic redox processes generate free radicals such as

superoxide anion radical (O₂^·–) and hydroxyl radical (OH^·) that

may induce oxidative damage in various components of the body

40 (lipids, proteins and DNA) and have been implicated in aging and

a number of lifeꢀlimiting chronic diseases (cancer, hypertension,

cardiac infarction, atherosclerosis, rheumatism, cataracts, etc.,).³⁴

Efforts to counteract the damage caused by these species are

gaining acceptance as a basis for novel therapeutic approaches

45 and the field of preventive medicine is experiencing an upsurge

of interest in useful antioxidants. The hydroxyl radical is by far

the most potent and therefore the most dangerous oxygen

metabolite, elimination of this radical is one of the major aims of

antioxidant administration. DPPH assay is widely used for

50 assessing the ability of radical scavenging activity and it is

measured in terms of IC₅₀values. The hydroxyl radical in

aqueous media was generated by the Fenton system. To explore

the DPPH radical scavenging ability of the newly synthesized

dinuclear complexes, we have carried out the experiments with

55 the hope of developing potential antioxidants and therapeutic

agents for certain chronic diseases.

95

Table 2 In vitro antioxidant and cytotoxicity assays for complexes

IC₅₀± SD (ꢂM),

DPPH

Comet assay

score (%)

HepG2

Complexes

24 h

ꢀ

48 h

1

131.8 ± 3.6

ꢀ

2

124.1 ± 1.2 18.7 ± 0.3 5.4 ± 0.7

64.99

3

186.5 ± 0.9

166.3 ± 1.4

88.1 ± 0.9

85.3 ± 1.6

95.8 ± 3.3

92.9 ± 4.8

144.6 ± 1.8

91.4 ± 3.1

ꢀ

4

ꢀ

5

ꢀ

6

7

9.3 ± 0.4 2.3 ± 0.6

82.12

ꢀ

8

15.6 ± 1.1 3.9 ± 0.5

73.38

Vitamin C

BHT

Cisplatin

ꢀ

10.2 ± 0.5 2.5 ± 0.3

6

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DNA binding studies

nucleotide of DNA such as guanine N7) and/or nonꢀcovalent

(intercalative, electrostatic and surface (major/minor groove)

binding) interaction. In general, hypochromism and

²⁰hyperchromism are the known spectral changes typical of metal

complexes associated with DNA helices.

The hyperchromic effect is due to surface binding while the

hypochromism accompanied by a significant red/blue shift is

characteristic of strong π–π stacking interaction between the

²⁵aromatic chromophore ligand of a metal complex and the

aromatic rings of DNA bases with the extent of hypochromism

commonly consistent with the strength of intercalation.³⁵The π*

orbital of intercalated ligand of a complex is coupled with the π

orbital of DNA bases. This coupling results in decreasing π–π*

³⁰transition energy and bathochromism is observed. The coupling π

orbitals, which are also partially filled by electrons, lowers the

Biological investigations have been performed in TrisꢀHCl/NaCl

buffer, so it was necessary to check the stability of complexes (1–

8) in this buffer. A negligible absorbance and current change was

observed after 48 h in electronic spectra and cyclic voltammetry,

respectively. Little absorbance/current change without any

considerable shift in wavelength/potential predicted the stability

of complexes in this buffer.

5

Absorption spectral titration: In order to investigate the possible

¹⁰binding mode and propensity of the molecules to CTꢀDNA,

various techniques are studied. Electronic absorption

spectroscopy is one of the most widely used techniques to follow

the interaction of metal complexes with DNA. Literature data

demonstrate that the binding of metal complexes with DNA

¹⁵usually takes place through both covalent (via replacement of a

labile group of complex by a nitrogen donor atom from the

transition

probability

and

concomitantly

results

in

hypochromism.³⁶

Fig. 6 shows the absorption spectra of complexes 4 & 7 in the

35 absence and presence of CTꢀDNA (for other complexes, the

spectra are shown in Fig. S7–S9), and the resulting data is

summarized in Table 3. Upon increasing the CTꢀDNA

concentration, UVꢀVis spectra of complexꢀDNA solutions clearly

show the tendency of hypochromism with blue shift. The

⁴⁰observed changes in the absorption bands of complexes 1–8 at a

ratio (R) of [DNA]/[complex] = 10, unambiguously revealed the

intercalative binding mode of complexes with CTꢀDNA.³⁷The

presence of cationic charge on the coordination sphere would

lead to electrostatic interaction with the negatively charged

⁴⁵phosphate group in the backbone of the DNA. Additionally,

hydrogen bonding is also possible in the interaction of DNA with

complexes. DNA possesses several hydrogen bonding sites in the

major as well as minor grooves. Since, complexes 1–8 contains

–NH groups, there could be hydrogen bonding between

⁵⁰complexes and base pairs in DNA.³⁸However, the hydrogen

bonding is a weak interaction. The observed decrease in the order

of hypochromism reflects the decrease in DNA binding affinities

of complexes in the same order. To compare the binding affinities

of complexes to DNA quantitatively, the intrinsic binding

⁵⁵constant K_bof complexes 1–8 are calculated and found in the

range 1.56 (±0.6)–1.96 (±0.4) × 10⁴M^–1. The binding strength of

complexes are more or less equal to the binding strength of the

previously reported dinuclear complexes,³⁹and lower than that of

the reported classical intercalators (EtBr, in TrisꢀHCl/ NaCl

60 buffer (25:40), pH 7.9 and [Ru(phen)₂(dppz)]²⁺), in which the

binding constants have been found to be in the order of 10⁶–10⁷

M^–1.⁴⁰

(a)

0.35

0.30

0.25

0.20

32

28

24

20

16

12

8

ε

0

2

4

6

10^ꢀ4

8

10

[DNA]

×

M

345

360

375

390

Wavelength (

λ

), nm

0.8

0.7

0.6

0.5

0.4

0.3

0.2

(b)

15

12

9

Hence from the obtained results, it has been found that the

copper(II) complexes exhibit higher binding affinity with CTꢀ

65 DNA than the nickel(II) complexes and, the binding strength of

complexes decreases in the order 6 > 5 > 7 > 8 > 2 > 1 > 3 > 4.

The obtained higher hypochromism and K_bvalue for complex 6

(Complex 2 in the case of nickel(II) analogues) suggest its higher

binding affinity to DNA than that for other complexes. This may

70 be due to the presence of methyl group on the phenyl ring, which

is involved in hydrophobic interaction with the hydrophobic

DNA surface that leads to the enhancement of DNA binding

affinity. Overall, the binding constant values imply the medium

ε

6

3

0

2

4

6

8

10

[DNA]

×

10^ꢀ4

M

325

350

375

400

425

Wavelength (λ), nm

Fig. 6 Absorption spectra of complexes 4 (a) & 7 (b) (50 ꢂM) in Trisꢀ

HCl/NaCl (pH 7.2) buffer upon addition of CTꢀDNA (0–500 ꢂM).

Arrow shows the decrease in absorbance upon increasing concentration

of DNA. Inset: Plot of [DNA]/(ε_a–ε_f) vs [DNA] for absorption titration

of CTꢀDNA with complexes.

binding strength of the dinuclear complexes with CTꢀDNA.

75

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Table 3 Absorption spectral properties of complexes 1–8 bound to CTꢀ

DNA

DNA, cyclic voltammetry was used which is considered to be a

very sensitive analytical technique to determine changes in redox

⁵⁰behaviour of metallic species in the presence of bioꢀmolecules.

The electrochemical investigations of metalꢀDNA interaction can

provide a useful complement to spectroscopic methods and

Change in

Absorption

Complexes λ/nm

R

∆λ/nm ^aH% K_b± SD (10⁴M^–1)

1

2

3

4

5

6

7

8

372 10 Hypochromism

381 10 Hypochromism

382 10 Hypochromism

369 10 Hypochromism

6

3

4

3

18

20

17

15

24

31

24

21

1.68 ± ~0.4

1.76 ± ~0.3

1.62 ± ~0.7

1.56 ± ~0.6

1.85 ± ~0.9

1.96 ± ~0.4

1.83 ± ~0.5

1.77 ± ~0.3

20

359 10 Hypochromism 12

358 10 Hypochromism

372 10 Hypochromism

4

6

8

10

(a)

^aH% = [(A_free–A_bound)/A_free] × 100%.

0

Measurements were made at R = 10, where R = [DNA]/[Complex];

[DNA] = 0–500 ꢂM; [Complex] = 50 ꢂM. ~ shows the decimal (±0.05)

difference in the results.

5

20

Hydrodynamic (Viscosity) measurements: To further investigate

the binding mode and binding intensity of complexes with DNA,

the DNA viscosity variance at 25 °C was studied by changing the

10

(b)

0

¹⁰concentration of complexes. Viscosity of DNA is sensitive to its

length. Intercalating agents are expected to elongate the double

helix to accommodate the ligands in between the base leading to

an increase in the viscosity of DNA. In contrast, a partial/nonꢀ

classical intercalation of ligand may provoke a bend or kink the

¹⁵DNA helix, reduce its effective length and concomitantly

viscosity, whereas groove or electrostatic mode of binding cause

little or no effect on the relative viscosity of DNA solution.

Therefore viscosity measurements are regarded as the least

ambiguous and most critical means of studying the binding mode

²⁰of metal complexes with DNA in solution and provide stronger

arguments for an intercalation mode of binding.^41,42The values of

relative specific viscosity (η/η₀), where η and η₀are the specific

viscosities of DNA in the presence and absence of complex, are

plotted against 1/R ([Complex]/[DNA] = 0.05–0.5).

-0.4

-0.8

-1.2

-1.6

-2.0

Potential (V)

Fig. 7 Cyclic voltammograms of complexes 2 (a) & 6 (b) in the absence

(solid line) and presence of CTꢀDNA (dotted line). [Complex] = [DNA]

= 100 ꢂM and, scan rate was 100 mVs^–1.

viscometric studies (e.g. for nonꢀabsorbing species, and yield

information about interaction with both the reduced and oxidized

55 form of the metal ion).⁴⁵In general, when the redox active metal

complex binds to DNA via intercalation, the potential presents a

positive shift, while in the case of electrostatic interaction, the

potential will shift to a negative direction. If more than one

potential exist simultaneously, a positive and negative shift of E_p¹

⁶⁰and E_p², respectively, the molecule can bind to DNA by both

intercalation and electrostatic interaction.⁴⁰

Since complexes 1−8 possess redox active moieties, their

electrochemical properties are expected to be altered in the

presence of DNA which can be followed by CV studies. On the

⁶⁵incremental addition of CTꢀDNA (R = [DNA]/[Complex]) to the

metal complexes, no new reduction waves (Fig. 7) appeared and

the observed decrease in current intensity is due to the diffusion

of the equilibrium mixture of free and DNAꢀbound complex to

the electrode surface which suggest the existence of same

70 electrochemical behaviour upon addition of CTꢀDNA. The

significant drop of cathodic peak current on the addition of DNA

is observed i.e. the value of i_pcdecreases regularly with increase

in DNA concentration. The slower mass transfer of complexes

bound to DNA fragments leads to a decrease in concentration of

75 the unbound redoxꢀactive species in solution. The decrease in

25

Ethidium bromide (EtBr) usually increases the relative

viscosity due the lengthening of DNA double helix, which results

from intercalation. Upon increasing amount of complexes 1–8,

the obtained plot shows only a minor change in relative viscosity

of DNA (Fig. S10), which indicates groove or electrostatic

30 interaction along with partial intercalative mode of binding

between DNA base pairs and each complex. Furthermore, the

cationic complexes would interact with the polyanionic backbone

of DNA, which supports the possible of electrostatic interaction

between DNA and complexes. The binding ability of complexes

³⁵varies in the order 6 > 5 > 7 > 8 > 2 > 1 > 3 > 4. Thus, complexes

show an increase in viscosity, which is lower than that for the

classical intercalator EtBr.⁴³The highest increase in DNA

viscosity was effected by complex 6, suggesting that complex

dramatically increase the hydrodynamic length of DNA as a

40 consequence of the untwisting of the base pairs and helical

backbone of DNA needed to accommodate the intercalators.⁴⁴

The results were in accordance with UVꢀVis absorption spectral

results.

1

2

voltammetric current with positive (E_p) and negative (E_p)

potential shifts suggests the possible existence of intercalation

along with electrostatic mode of interaction of complexes with

DNA.^40,46The decrease in peak current is much higher for

80 copper(II) complexes than for nickel(II) complexes upon addition

of DNA which suggest the stronger binding affinity of the former

to DNA than that of later and follows the order 6 > 5 > 7 > 8 > 2

Electrochemical titration: The metal complexes of high

45 oxidation state play an important role in bioinorganic chemistry,

because of their biological significance as redox enzyme models.

To further explore the interaction mode between complexes and

8

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> 1 > 3 > 4. The electrochemical studies corroborated well with

the electronic absorption studies and thereby authenticate the

strong interaction of CTꢀDNA with complexes 1−8.

concentration of complexes. The copper(II) complexes shows

(Fig. 8) prominent DNA cleavage to give nicked circular (NC)

form (> 75%), while nickel(II) complexes (Fig. S11) fail to show

⁴⁵the expected efficiency (< 35%).

Nuclease activity

From the obtained results and literature reports,^48,49the

following interaction was concluded between metal ions and

phosphate moiety of DNA. The metal(II) ions may bind with the

phosphoꢀdiester bond of DNA through the coordinate linkage

⁵⁰and/or electrostatic interaction, and the metal ions activate the

central phosphorus atom of DNA, resulting in the formation of

five coordinated phosphorus intermediate. Then, the activated

phosphorus atom is attacked nucleophilically by the metal ions

due to their Lewis acidity via charge neutralization, and finally

⁵⁵one of the P–O ester bond of nucleic acid is cleaved.

5

The degradation of plasmid DNA explored by transition metal

complexes has been interest of researchers. This can be achieved

by targeting the basic components of DNA such as phosphoꢀ

diester linkages, deoxyribose sugar or nucleobases. The nuclease

activity of complexes 1–8 have been studied using supercoiled

¹⁰(SC) pBR322 DNA (33.3 ꢂM) as a substrate in a medium of Trisꢀ

HCl/NaCl (pH 7.2) buffer at 37 °C by gel electrophoresis, in

which the transition from the naturally occurring, covalently

closed circular form to the open circular form and linear form

was monitored. The migration of DNA in gel electrophoresis

¹⁵works under the influence of electric potential. DNA is negatively

charged species, when placed under electric field, it will migrates

toward anode and this migration depends on the size of DNA,

electric field strength, gel density and the buffer nature. When

circular pBR322 DNA is subjected to electrophoresis, relatively

²⁰fast migration will be observed for the intact supercoiled form

(Form I). If scission occurs on one strand (nicking), the

supercoiled form will relax to generate a slowerꢀmoving nicked

form (Form II). If both strands are cleaved, a linear form (Form

III) that migrates between Form I and Form II will be generated.

²⁵The observed results show appreciable nuclease activity by the

copper(II) complexes (5–8) when compared to nickel(II)

analogues (1–4) and no cleavage was observed for controls both

in the absence and presence of H₂O₂. The appearance of

electrophoresis band characteristic of nicked circular form (NC,

³⁰Form II) and the absence of a band corresponding to linear

circular form (LC, Form III) suggest that only singleꢀstrand DNA

cleavage occurs for complexes.

The presence of more aromatic moiety and hard Lewis acid

properties could play a vital role in the DNA cleavage process by

hydrolytic pathway.⁴⁹The potency of the dinuclear complexes in

nuclease activity follows the order 6 > 5 > 7 > 8 > 2 > 1 > 3 > 4.

⁶⁰The difference in nuclease activity of complexes may be due to

the influence of the substituted groups in complexes, which

makes it interact with DNA more effectively. The cleavage

ability of complexes might be due to the binding affinity of

complexes with DNA, and the cleavage of DNA by complex is

⁶⁵dependent on the concentration of complexes. To ascertain the

hydrolytic cleavage mechanism, an additional cleavage

experiments were conducted using enzymatic T4 ligase assay.

The cleavage product of SC form by complexes, reacted with T4

ligase enzyme and result implies that 10–20% nicked form moves

⁷⁰to SC form i.e. nicked circular form religated to closed circular

and then supercoiled form. The observed result of T4 ligase

enzymatic assay reveals the hydrolytic DNA breakage

mechanism.

Nuclease activity in the presence of H₂O₂: The damage of

75 plasmid DNA is also dependent on coꢀoxidant used. As shown in

Fig. 9, in the presence of H₂O₂, the degradation of supercoiled

Form I to nicked Form II is induced by complexes, in which the

copper(II) complexes exhibit pronounced cleavage activity than

Nuclease activity in the absence of H₂O₂: In general, the Ni(II)

and Cu(II) complexes cleave DNA by hydrolytic pathway.⁴⁷

³⁵Since the synthesized complexes satisfies one of the primary

criteria for catalyzing hydrolytic cleavage of DNA, i.e.

coordination of the phosphate moiety of DNA to the metal(II)

center of complex, its DNA cleaving ability has been investigated

with different concentration of complexes. The nuclease activity

⁴⁰of complexes was observed in the absence of external agents and

the increasing intensity of NC form was found with increase in

(a)

Form II

Form I

1

2

3

4

5

6

7

8

(b)

(c)

Form II

Form I

(a)

Form II

Form I

1

2

3

4

5

6

7

8

1

2

3

4

5

6

7

8

(b)

Form II

Form I

Form II

Form I

1

2

3

4

5

8

1

2

3

4

5

7

8

Fig. 9 Oxidative cleavage of pBR322 DNA (33.3 µM) by complexes 5

(a), 6 (b) and 8 (c) in the presence of H₂O₂(40 µM) and DMSO (0.2

mM) in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2). Lane 1, DNA

control; lane 2, DNA + 5/6/8 (100 µM); lane 3, DNA + 5/6/8 (100 µM)

+ H₂O₂; lane 4, DNA + 5/6/8 (100 µM) + H₂O₂+ DMSO; lane 5, DNA

+ 5/6/8 (200 µM); lane 6, DNA + 5/6/8 (200 µM) + H₂O₂; lane 7, DNA

+ 5/6/8 (200 µM) + H₂O₂+ DMSO; lane 8, DNA + H₂O₂+ DMSO.

Fig. 8 Hydrolytic cleavage of pBR322 DNA (33.3 µM) by complexes 6

(a) and 8 (b) in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2). Lane 1,

DNA alone; lane 2, DNA + 6/8 (25 µM); lane 3, DNA + 6/8 (50 µM);

lane 4, DNA + 6/8 (75 µM); lane 5, DNA + 6/8 (100 µM); lane 6, DNA

+ 6/8 (150 µM); lane 7, DNA + 6/8 (200 µM); lane 8, DNA + 6/8 (250

µM).

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Span of Incubation 24 h

100

75

50

25

0

the nickel(II) analogues. The oxidative cleavage mechanism was

proposed as follows: When redoxꢀactive metal complexes interact

with DNA in the presence of a coꢀoxidant, it is believed to

produce different oxygen intermediates (reactive oxygen species),

depending on specific complex and conditions. A nonꢀdiffusible

metalꢀperoxo intermediate has been invoked in some cleavage

reactions while in others, Fentonꢀlike chemistry, which invokes

release of freely diffusible hydroxyl (OH^•) or hydroperoxyl

60

2

6

8

5

2

4

6

8

10

•

100

80

60

40

20

0

(HO₂) radical, has been assumed. The metal complexes in the

Span of Incubation 48 h

10 presence of H₂O₂may generate reactive hydroxyl/hydroperoxyl

radical that can damage the deoxyribose ring (C3′), or

alternatively a metalꢀperoxo species may participate directly in

the oxidation of deoxyribose ring.

65

2

6

8

To understand the involvement of reactive oxygen species

¹⁵(ROS), inhibition experiment was carried out with standard

scavengers of reactive oxygen intermediates in the

electrophoresis studies under physiological conditions. DMSO

has little influence on DNA cleavage, suggesting that the

hydroxyl radical is involved in the DNA scission process. A

²⁰possible hydroxyl radical based cleavage reaction is shown in

Scheme S1. In addition, the coordination environment of the

central metal ions and geometry of complexes not only governs

the DNA binding but also determine the nucleolytic action.

Therefore, the difference in nuclease activity of complexes may

²⁵be attributed to their proximity to DNA binding. The nuclease

activity of redox mediated or photo activated metal complexes is

an important element in the characterization of DNA recognition

of transition metal complexes.

2

4

6

8

10

Drug Concentration (ꢂg/mL)

70

Fig. 10

Potency of complexes 2, 6 and 8 on HepG2 cells, after 24 (a)

and 48 h (b) incubation. All determinations are expressed as a percentage

of control (untreated cells).

Cell morphology

PI stained cells

(a)

Growth inhibition activities

30 Cell viability analysis: Hydrolysis of DNA via phosphoꢀdiester

bond is crucial at several stages in the cell cycle, including DNA

repair and excision, integration and signal transduction.⁴⁹

Meanwhile, damage of DNA backbone by hydrolytically has

been reported to be related to antitumor potential,⁵⁰which

³⁵prompts us to evaluate cytotoxicity of complexes. The toxicity of

the complexes was examined against nonꢀcancer cell line (Vero

cell line), and the obtained results show that the negligible

toxicity level (5ꢀ14%) of the complexes against the nonꢀcancer

cell line. In vitro cytotoxicity of the chloroꢀbridged dinuclear

⁴⁰nickel(II) complex 2 and copper(II) complexes 6 and 8 against

human hepatocellular liver carcinoma cell line HepG2 have been

investigated in comparison with the widely used drug, cisplatin

under identical conditions, by MTT reduction assay. The assay is

based on the fact that only live cells reduce yellow MTT but not

45 dead cells to blue farmazone products. Thus, the metabolic

activity of the cells was assessed by their ability to cleave the

tetrazolium rings of pale yellow MTT and form a waterꢀinsoluble

dark blue farmazone crystal. Assay shows that complexes inhibit

the growth of cells. The IC₅₀values (Fig. 10, Table 2) for 48 h

50 incubation are lower than those for 24 h incubation and IC₅₀

values decreases with increasing concentration of complexes,

which clearly indicating that the cytotoxicity of dinuclear

complexes are dose and time dependent.

(b)

(c)

(d)

55

Fig. 11 Morphological and propidium iodide fluorescent staining of

HepG2 cells. Untreated cells (a), treatment of 2 (b), 6 (c) and 8 (d) with

the IC₅₀concentration (20X Magnification).

10 | Journal Name, [year], [vol], 00–00

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60 swelling, cytoplasmic blebbing, and late apoptosis indication of

dotꢀlike chromatin condensation by adopting PI staining. PI

staining of cell nuclei has significantly reduced the microscope

time required for counting and for evaluating gross cell

morphology.

The observed IC₅₀values reveal that the copper(II) complex

exhibited higher in vitro cytotoxicity against selected cancer cell

line than and , and also relatively equal to cisplatin. The metal

complexes exhibit higher mortality at short time with lower

concentration compared to others with higher concentration at

longer duration. The bioꢀactivities of anticancer metal complexes

are dependent on their ability to bind with DNA and damage its

structure resulting in the impairment of its function, which is

followed by inhibition of replication and transcription processes

6

8

2

5

65

The dead cells are viewed as red colour after staining with PI

under the fluorescent light. A very negligible number of PI

positive cells were observed in the control and more number of PI

positive cells was observed in the case of cancer cells treated with

complexes. The observed morphological changes suggest that

¹⁰and, eventually cell death, if the DNA lesions are not properly

repaired.⁵¹Further, it is also observed that the cytotoxicity of

complexes increases with increase in reduction potential or

easilyoxidizable species.⁵²Thus, it is possible to correlate the

cytotoxicity with redox potential. From CV data it is clear that the

¹⁵oxidizing tendency of complexes lies in the order corresponding

with its ligands, L²> L⁴based on the substituent at the para

position and the cytotoxicity follows the same order of an

oxidizing tendency of complexes.

⁷⁰complexes

6 and 8 possesses higher efficacy than 2 in killing the

cells by apoptosis. The mechanism of cell death induced by

complexes on liver carcinoma cell line (HepG2) appears to be

apoptosis, as evidenced by fluorescent PI staining.

Alkaline singleꢀcell gel electrophoresis analysis: The singleꢀcell

⁷⁵gel electrophoresis assay (Comet assay) is considered as a rapid,

simple, nonꢀinvasive, sensitive, visual and inexpensive technique

to asses DNA fragmentation typical of toxic DNA damage and of

an early stage of apoptosis,⁵⁴as compared to other techniques

used for measuring and analyzing DNA strand breaks in

80 mammalian cells. Comet assay gives an image of the changes that

have occurred in the chromatin organization in a singleꢀcell

which is considered more accurate way to detect early nuclear

changes in a cell population. DNA fragmentation is one of the

characteristic features observed in apoptotic cells and is generally

⁸⁵considered as the biochemical hallmark of the apoptosis, mitotic

catastrophe or both and is detected at a singleꢀcell level by the use

of singleꢀcell gel electrophoresis in agarose gel matrix. When a

cell with damaged DNA is subjected to electrophoresis and then

stained with EtBr, it appears as a comet, and the length of the

⁹⁰comet tail represents the extent of DNA damage.⁵⁵Comets form

as the broken ends of the negatively charged DNA molecules

becomes free to migrate in the electric field toward the anode.

Major understand about comet formation based on: (i) DNA

migration is a function of both size and the number of broken

⁹⁵ends of the DNA, and (ii) tail length increases with damage

initially and then reaches a maximum. The comet tail length was

analyzed by CASP software (Fig. S12).

Cell morphology observation: Radical light inverted microscope

20 was used to monitor the microscopic observations (Fig. 11),

wherein treated cells showed obvious cellular morphological

changes indicating unhealthy cells, whereas the control appeared

normal. Control cells were irregular confluent aggregates with

rounded and polygonal cells. The cells treated by complexes

²⁵and appeared to shrink, became spherical shape and cell

spreading patterns were restricted.

2, 6

8

Propidium iodide staining: Two types of cell death can be

observed: An accidental cell death (necrosis) and programmed

cell death (apoptosis). In necrosis, the cells undergo cell lysis and

³⁰lose their membrane integrity, and severe inflammation is

induced. However, apoptotic cells are transformed into small

membraneꢀbound vesicles (apoptotic bodies) which are engulfed

in vivo by macrophages, and no inflammatory response is found.

Harmless removal of cells (cancer cells, for example) is one

35 consideration in chemotherapy. Therefore, induction of apoptosis

is one of the considerations in the development of anticancer

drugs.

Apoptosis is a naturally occurring geneꢀcontrolled process that

plays a major role in tissue homeostasis and elimination of

⁴⁰unwanted cells in animals without affecting normal/unaffected

cells. Most of the cancer cells still remain sensitive to some

apoptotic stimuli from chemo therapeutic agents, and in this

context, the apoptosisꢀinducing ability of drugs seems to be a

primary factor in determining their efficacy.⁵³Therefore, we have

(a)

(b)

45 studied the mode of cell death induced by complexes 2, 6 and 8

by adopting fluorescent staining for morphological assessment of

cell death, singleꢀcell gel electrophoresis (Comet assay) for

detecting DNA fragmentation of the treated cells against control

cells. Fluorescent dye PI is used to differentiate necrotic,

50 apoptotic and normal cells of HepG2 cell line after treatment with

the complexes.

(c)

(d)

Fig. 11 shows the characteristic morphological changes

brought about in the cells by treatment with complexes 2, 6 and 8.

The results obtained indicate that complexes induce cell death

55 through different modes like mitotic catastrophe, necrosis and

apoptosis. After treating the cells with IC₅₀concentration of

complexes at different time intervals (24 and 48 h), we have

observed cytological changes such as chromatin fragmentation,

biꢀ and multinucleation, cytoplasmic vacuolation, nuclear

Fig. 12 Analysis of apoptotic inducing effect of control (a) and complexes

2 (b), 6 (c) and 8 (d) on HepG2 cells assessed by comet assay.

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Cells treated with complexes shows statistically significant

wellꢀformed comets, whereas the control (untreated) cells fail to

and nuclease activity than the other dinuclear complexes.

Mechanistic studies of the nuclease activity indicated the possible

show a comet or broomꢀlike tail appearance. The typical photos ⁶⁰role of hydroxyl radical as reactive oxygen species. Assay results

of the nucleoids of control cells were uniformly spherical in

shape, reflecting the absence of any DNA damage. However,

upon complete scoring of the nucleoids in experiments, reflecting

a baseline level of DNA singleꢀstrand breaks. Hence, the average

suggest that, complexes

against HepG2 cancer cells. Remarkably, complex

highest anticancer activity with an IC₅₀value of 9.5 ꢂM,

relatively equal to the widely used drug cisplatin (IC₅₀, 10.5 ꢂM)

2

,

6

and

8

exhibit apoptotic cell death

exhibit the

5

6

comet score of tail DNA for control HepG2 cells was 2.7% (Fig. 65 against the same cell line. Apoptotic cell death was further

12). In contrast, comet score for complexes show significant

10 numbers of nucleoids with larger comet tails, indicative of higher

levels of DNA singleꢀstrand breaks. The comet assay score (%)

evidenced from alkaline singleꢀcell gel electrophoresis, and the

assay results show that complexes indeed induce DNA

fragmentation. This implies that a synergic combination of ligand

and metal ion is important in the design of a potential anticancer

of tail DNA for complexes 2, 6 and 8 were 64.99, 82.12 and

73.38, respectively. These results implies that complexes ⁷⁰drug, which correlates well with the ability of complexes to

significantly increased number of tail DNA, tail length, tail

¹⁵moment and olive tail moment in HepG2 cell line when

compared to control cells. The percentage of cells with tail DNA

increased significantly after the cells were treated with complexes

strongly bind and cleave DNA. The results obtained from the

present work would be helpful to design and develop new chloroꢀ

bridged dinuclear complexes as potent therapeutic and anticancer

agents for some chronic diseases.

2

,

6

and

8

(

Table 2). Also, for 24 h treatment the tail length

is longer than others, which is consistent with the

75 Experimental

observed for

6

General methods and materials

20 higher cytotoxicity and the morphological changes observed

inside the cell. This clearly indicates that 6 indeed induce DNA

fragmentation, which is further evidence for its higher ability to

induce apoptotic cell death. On the basis of DNA fragmentation,

the potency of the dinuclear complexes in anticancer activity

Micro analysis (% CHN) was performed using Carlo Erba model

1106 elemental analyzer. IR spectra of ligands and complexes

were recorded using KBr pellets in the range 4000–400 cm^–1on a

1

⁸⁰Bruker Alpha FTꢀIR spectrophotometer. The H NMR spectra of

the nickel(II) complexes were collected on VNMRSꢀ400

²⁵follows the order

6 > 8 > 2.

spectrometer in DMSO (d₆) at room temperature. UVꢀVis spectra

of complexes were recorded in HPLC grade DMF at 25 ºC on

Agilentꢀ8453 spectrophotometer in the range of 200–1100 nm.

Results shows the higher anticancer potency of the copper(II)

complexes, which are comparable with the widely used drug

cisplatin against HepG2 cell line. The present chloroꢀbridged

phenolic complexes exhibit the moderate anticancer activity when

³⁰compared to the reported phenolic complexes against the same

cell line.⁵⁶The outcome of the comet assay shows that a single

cell’s DNA underwent degradation as consequence of direct DNA

damage or rapid apoptosis. The high level of DNA damage

induced by complexes reinforces the above results obtained by

³⁵MTT and fluorescent staining assays. The results of MTT assay,

the morphological assessment of cell death and comet assay

⁸⁵ESI mass spectra were recorded on

a QꢀTof micromass

spectrograph using H₂O as the mobile phase with an approximate

concentration of 1.0 mmol dm^–3. Cyclic voltammograms were

obtained on CHI 602D (CH Instruments Co., USA)

electrochemical analyzer under oxygen free conditions using a

⁹⁰threeꢀelectrode cell with a DMF solution of TBAP (0.1 M) as the

supporting electrolyte. A Pt wire, glassy carbon, and the Ag/AgCl

electrode (saturated KCl solution) were used as counter, working

and reference electrode, respectively. A Ferrocene/ferrocenium

(Fc/Fc+) couple was used as an internal standard. The reported

⁹⁵potentials are relative to the Ag/AgCl electrode and E_1/2of the

Fc/Fc⁺couple. The concentration of complex solutions were

taken around 1.0 × 10^–3M and, the scan rate was 100 mVs^–1.

Molar conductivity was measured with an Elico digital

conductivity bridge model CMꢀ88, using a freshly prepared DMF

100 solution of complex. Xꢀband EPR spectra of the Cu(II)

complexes were recorded at room temperature on a Varian EPRꢀ

E 112 spectrometer. The magnetic measurements were carried out

revealed that complex

6 possesses a very prominent cytotoxicity

than others, which is consistent with its strong DNA binding

involving hydrophobic forces of interaction and efficient DNA

⁴⁰cleavage and potent radical scavenging property.

Conclusions

In summary, a new series of chloroꢀbridged nickel(II) and

copper(II) complexes

Single crystal Xꢀray

45 [(NiL⁴(CH₃OH)₂)₂(

ꢀCl)](Cl)₃.(H₂O)₂, (

Cl.(H₂O)₇, ( ) revealed distorted octahedral and squareꢀpyramidal

1

–

8

were synthesized and characterized.

investigation of complexes

) and [(CuL⁴Cl)₂(

ꢀCl)]

µ

4

ꢁ

on a PAR vibrating sample magnetometer (Modelꢀ155). The χ_MT

8

data were collected over the temperature range 20–300 K with an

105 applied magnetic field of 5000 Oe and instrument was calibrated

using metallic nickel.

geometry, respectively, around the metal ions. Thermodynamic

stability constants and thermodynamic parameters of the

complexes have been determined spectrophotometrically. The

50 temperature dependent magnetic susceptibility measurements of

The ligands 2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenol (L¹),

4ꢀmethylꢀ2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenol

4ꢀchloroꢀ2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenol (L³) and

110 4ꢀbromoꢀ2ꢀ((2ꢀ(piperazinꢀ1ꢀyl)ethylimino)methyl)phenol

(L⁴)

(L²),

the copper(II) complexes (

coupling exchange interaction between the adjacent copper ions.

The antioxidant property implies that complex possesses

5–8) indicate strong antiferromagnetic

6

were prepared by condensation of 1ꢀ(2ꢀaminoethyl) piperazine

with corresponding salicylaldehyde in methanol.¹³All reagents

and chemicals were purchased from commercial sources (SRL

and S.D. Fine chemicals, India; SigmaꢀAldrich, USA) and used

115 without further purification. Solvents were dried and purified

effective radical scavenging activity against DPPH in terms of

55 IC₅₀values. The DNA binding studies implies the partial

intercalation along with electrostatic mode of binding with CTꢀ

DNA. The complex

6 displayed higher DNA binding propensity

12 | Journal Name, [year], [vol], 00–00

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before being used according to standard procedure. Tetra(

ammonium perchlorate (TBAP) used as the supporting electrolyte

in the electrochemical measurements, was purchased from Fluka

n

ꢀbutyl)

36.24; H, 6.08; N, 9.75%. Found: C, 36.22; H, 6.14; N, 9.73%.

Selected IR data (KBr, (O–H str. of H₂O), 2955

, cm^–1): 3423

(N–H str. of >NH₂), 1643 (C=N), 1544 (N–H bend. of

(Ar–O). UVꢀVis (DMF), /nm (

/M^–1cm^–1): 272

(16,285), 359 (6,422), 657 (278) nm. ESIꢀMS (H₂O) display

ν

v

+

v

ε

+

(Switzerland) and recrystallized from hot methanol. The calf 60 >NH₂), 1296

v

λ

5

thymus (CT) and pBR322 DNA were purchased from Bangalore

Genei (India). Ethidium bromide (EtBr) was obtained from

SigmaꢀAldrich (USA).

peaks at: [CuL¹+ 2H]²⁺(298), [CuL¹]⁺(296). Conductance (Λ_M

,

ꢁ^–1cm²mol^–1) in DMF: 131.

cm^–1at 298 K.

g_||= 2.16. ꢁ_eff= 1.51 BM, –2J = 236

Synthesis of dinuclear complexes

65 [(CuL²Cl)₂(ꢁꢀCl)]Cl.(H₂O)₇(6): Yield: 0.64 g (77.6%). Colour:

Green. Anal. Calc. for C₂₈H₅₆Cl₄N₆Cu₂O₉, (FW: 889.68): C,

37.80; H, 6.34; N, 9.45%. Found: C, 37.81; H, 6.38; N, 9.43%.

The chloroꢀbridged dinuclear nickel(II)

10 complexes were prepared by following general synthetic

procedure. To stirred methanolic solution (10 mL) of

NiCl₂.6H₂O (0.232 g, 1 mmol) or CuCl₂.2H₂O (0.170 g, 1 mmol),

a methanolic solution (10 mL) of appropriate ligand L^1–4(1

mmol) was added slowly and stirring was continued for 2 h. The

¹⁵reaction mixture was refluxed for 6 h, filtered hot and kept aside

for slow evaporation. The product was washed with cold

1–4 and copper(II) 5–8

a

Selected IR data (KBr,

ν

, cm^–1): 3452

(N–H str. of >NH₂), 1642 (C=N), 1538

(Ar–O). UVꢀVis (DMF), /nm (

(14,302), 358 (5,428), 668 (221) nm. ESIꢀMS (H₂O) display

peaks at: [CuL²+ 2H]²⁺(311), [CuL²]⁺(309). Conductance (Λ_M

^–1cm²mol^–1) in DMF: 137.

_||= 2.15. _eff= 1.52 BM, –2 = 239

cm^–1at 298 K.

v

(O–H str. of H₂O), 2934

(N–H bend. of

/M^–1cm^–1): 274

+

v

ε

+

⁷⁰>NH₂), 1303

v

λ

,

ꢁ

g

ꢁ

J

methanol and dried in vacuo

.

[(NiL¹(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂(1): Yield: 0.76

g

75 [(CuL³Cl)₂(ꢁꢀCl)]Cl.(H₂O)₇(7): Yield: 0.71 g (76.3%). Colour:

Dark green. Anal. Calc. for C₂₆H₅₀Cl₆N₆Cu₂O₉, (FW: 930.52): C,

33.56; H, 5.42; N, 9.03%. Found: C, 33.54; H, 5.47; N, 9.01%.

(85.4%). Colour: Green. Anal. Calc. for C₃₀H₅₈Cl₄N₆Ni₂O₈, (FW:

²⁰890.01): C, 40.48; H, 6.57; N, 9.44%. Found: C, 40.45; H, 6.61;

N, 9.42%. Selected IR data (KBr, ν v(O–H str. of

, cm^–1): 3388

+

Selected IR data (KBr,

ν

, cm^–1): 3442

(N–H str. of >NH₂), 1646 (C=N), 1549

(Ar–O). UVꢀVis (DMF), /nm (

(21,815), 358 (9,934), 658 (423) nm. ESIꢀMS (H₂O) display

v

(O–H str. of H₂O), 2949

(N–H bend. of

/M^–1cm^–1): 272

CH₃OH), 2947

bend. of >NH₂), 1292

v

(N–H str. of >NH₂), 1650

v

(C=N), 1549

v

(N–H

+

/M^–1

+

v

ε

v

(Ar–O). UVꢀVis (DMF),

λ/nm (ε

+

⁸⁰>NH₂), 1304

v

λ

cm^–1): 268 (19,184), 372 (11,465), 634 (938), 930 (315) nm. ESIꢀ

25 MS (H₂O) display peaks at: [NiL¹+ H]²⁺(292), [NiL¹]⁺(291).

peaks at: [CuL³+ 2H]²⁺(332), [CuL³]⁺(330). Conductance (Λ_M

,

Conductance (Λ

_M, ꢁ^–1cm²mol^–1) in DMF: 291.

ꢁ^–1cm²mol^–1) in DMF: 125.

cm^–1at 298 K.

g_||= 2.19. ꢁ_eff= 1.49 BM, –2J = 220

[(NiL²(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂(2): Yield: 0.79 g (86%).

Colour: Pale Green. Anal. Calc. for C₃₂H₆₂Cl₄N₆Ni₂O₈, (FW:

918.07): C, 41.86; H, 6.81; N, 9.15%. Found: C, 41.83; H, 6.86;

85 [(CuL⁴Cl)₂(ꢁꢀCl)]Cl.(H₂O)₇(8): Yield: 0.76 g (74.5%). Colour:

Dark green. Anal. Calc. for C₂₆H₅₀Br₂Cl₄N₆Cu₂O₉, (FW:

1019.42): C, 30.63; H, 4.94; N, 8.24%. Found: C, 30.61; H, 4.99;

30 N, 9.12%. Selected IR data (KBr, ν v(O–H str. of

, cm^–1): 3406

+

CH₃OH), 2964

bend. of >NH₂), 1290

v

(N–H str. of >NH₂), 1649

v

(C=N), 1546

v

(N–H

+

N, 8.21%. Selected IR data (KBr,

ν

, cm^–1): 3419

(N–H str. of >NH₂), 1647 (C=N), 1526

v(O–H str. of

v

(Ar–O). UVꢀVis (DMF),

λ/nm (ε

/M^–1

+

H₂O), 2929

v

(N–H

cm^–1): 268 (19,485), 381 (13,905), 644 (1,830), 930 (894) nm.

ESIꢀMS (H₂O) display peaks at: [NiL²+ H]²⁺(307), [NiL²]⁺

+

90 bend. of >NH₂), 1304

v

(Ar–O). UVꢀVis (DMF),

λ/nm (ε

/M^–1

cm^–1): 269 (14,125), 372 (5,734), 655 (262) nm. ESIꢀMS (H₂O)

³⁵(305). Conductance (

[(NiL³(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂(3): Yield: 0.78

Λ

_M, ꢁ^–1cm²mol^–1) in DMF: 294.

display peaks at: [CuL⁴

Conductance (

_M, ꢁ^–1cm²mol^–1) in DMF: 128. g_||= 2.18. ꢁ_eff

1.50 BM, –2

= 228 cm^–1at 298 K.

+

2H]²⁺(362), [CuL⁴]⁺(360).

g

Λ

=

(81.3%). Colour: Green. Anal. Calc. for C₃₀H₅₆Cl₆N₆Ni₂O₈, (FW:

958.90): C, 37.58; H, 5.89; N, 8.76%. Found: C, 37.55; H, 5.93;

J

N, 8.77%. Selected IR data (KBr, ν v(O–H str. of

, cm^–1): 3384

95 Single crystal Xꢀray diffraction

+

40 CH₃OH), 2957

bend. of >NH₂), 1294

v

(N–H str. of >NH₂), 1655

v

(C=N), 1536

v

(N–H

Single crystals of complexes

4

and

8

suitable for XRD

+

v

(Ar–O). UVꢀVis (DMF),

λ/nm (ε

/M^–1

measurements were sorted using polarizing microscope (Leica

DMLSP). Crystals having good morphology were chosen for

threeꢀdimensional intensity data collection. Crystals with

100 dimensions of 0.30 × 0.25 × 0.20 and 0.30 × 0.20 × 0.20 mm for

cm^–1): 268 (15,898), 382 (10,392), 624 (1,637), 954 (765) nm.

ESIꢀMS (H₂O) display peaks at: [NiL³+ H]²⁺(326), [NiL³]⁺

(325). Conductance (

45 [(NiL⁴(CH₃OH)₂)₂(µꢀCl)](Cl)₃.(H₂O)₂(4): Yield: 0.73

(69.7%). Colour: Dark green. Anal. Calc.

Λ

_M, ꢁ^–1cm²mol^–1) in DMF: 282.

complexes

4 and 8, respectively were mounted on a glass fiber

g

for

for diffraction experiment. Xꢀray single crystal data were

collected on a Kappa Apex2 CCD diffractometer equipped with a

fineꢀfocus sealed tube Xꢀray source and graphite monochromated

C₃₀H₅₆Br₂Cl₄N₆Ni₂O₈, (FW: 1047.81): C, 34.39; H, 5.39; N,

8.02%. Found: C, 34.37; H, 5.42; N, 8.05% Selected IR data

(KBr, (O–H str. of CH₃OH), 2952 (N–H str. of

, cm^–1): 3379

(C=N), 1524 (N–H bend. of >NH₂), 1309

(Ar–O). UVꢀVis (DMF), /nm (

/M^–1cm^–1): 268 (17,099), 369

.

¹⁰⁵Mo (

temperature (293 ± 2 K). The intensity data were collected using

and scans with frame width of 0.5°. The frame integration

Kα) radiation in the wavelength (λ) of 0.71073 Å at room

ν

v

+

50 >NH₂), 1637

v

ω

φ

v

λ

ε

and data reduction were performed using Bruker SAINTꢀplus

(Version 7.06a) software. Empirical absorption corrections were

110 applied for complexes, using SADABS program.⁵⁷The structure

was solved by using SIR92⁵⁸and the fullꢀmatrix leastꢀsquares

refinement on F²was performed using SHELXLꢀ97 program.⁵⁹

(10,826), 630 (1,039), 973 (638) nm. ESIꢀMS (H₂O) display

peaks at: [NiL⁴+ H]²⁺(371), [NiL⁴]⁺(370). Conductance (Λ_M

,

ꢁ^–1cm²mol^–1) in DMF: 285.

55 [(CuL¹Cl)₂(ꢁꢀCl)]Cl.(H₂O)₇(5): Yield: 0.63 g (73.1%). Colour:

Green. Anal. Calc. for C₂₆H₅₂Cl₄N₆Cu₂O₉, (FW: 861.63): C,

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Table 4 Crystal data and structure refinement for complexes

4

and

8

Complex

4

8

CCDC

907779

952060

Empirical Formula

Formula Weight

Temperature (K)

Wavelength (Å)

Crystal system

Space group

C₃₀H₅₆Br₂Cl₄N₆Ni₂O₈

1047.85

293 ± 2

0.71073

Monoclinic

C₂₆H₅₀Br₂Cl₄N₆Cu₂O₉

1019.42

293 ± 2

0.71073

Monoclinic

C

2/c

P21/n

a

b

c

α

β

γ

(Å)

(º)

10.1699(4)

16.8523(8)

24.8924(11)

90

98.768(2)

90

4216.4(3)

4

1.651

13.8391(4)

14.3968(4)

20.2234(7)

90

98.6740(10)

90

3983.2(2)

4

1.700

Volume (Å³)

Z

Calculated density (Mg/m³)

Absorption coefficient (mm^–1)

3.094

3.395

F

(000)

Crystal size (mm)

range for data collection (º)

2144

0.30 × 0.25 × 0.20

2.36–28.81

2064

0.30 × 0.20 × 0.20

2.05–26.98

θ

Limiting indices

–13 ≤

23444

5502 [

h

≤ 13, –22 ≤

k

≤ 22, –33 ≤

l

≤ 33

–17 ≤

41141

8638 [

h

≤ 17, –18 ≤

k ≤ 18, –25 ≤ l ≤ 25

Reflections collected

Independent reflections

Max. and min. transmission

Refinement method

Data/restrains/parameters

GOF on F²

R

(int) = 0.0339]

R(int) = 0.0667]

0.5766 and 0.4571

Fullꢀmatrix leastꢀsquares on F²

5502/74/305

1.037

0.592 and 0.501

Fullꢀmatrix leastꢀsquares on F²

8638/6/456

1.055

Final

indices (all data)

Largest diff. peak and hole (e.Å^–3)

R

indices [

I

> 2

σ

(

I

)]

R

1 = 0.0333, w

1 = 0.0574, w

0.581 and –0.5000

R

2 = 0.0740

R

1 = 0.0426, w

1 = 0.0894, w

R

2 = 0.0923

2 = 0.1107

R

R2 = 0.0816

0.716 and –0.617

The scattering factors incorporated in SHELXLꢀ97 were used.

After several cycles of refinement, the positions of hydrogen

atoms were calculated and added to the refinement process.

Molecular graphics, hydrogen bonding and packing figures were

generated by using the softwares ORTEP 3.0⁶⁰and Mercury

3.0.⁶¹Details of the crystallographic data and structure refinement

DNA binding studies

Absorption spectral titration: The application of electronic

absorption spectra in DNA interaction is one of the most effective

³⁰method to examine the binding mode and strength of metal

complexes with DNA. A solution of calf thymus DNA in Trisꢀ

HCl buffer (5 mM TrisꢀHCl/50 mM NaCl, pH = 7.2) gave a ratio

5

parameters for complexes

4 and 8 are summarized in Table 4.

of UV absorbance at 260 and 280 nm (A₂₆₀/A₂₈₀) of about 1.87:1,

CCDC 907779 (for 4) and 952060 (for 8). For crystallographic data in

indicating that the DNA was sufficiently free from proteins.⁶³

³⁵Stock solutions of CTꢀDNA was prepared in TrisꢀHCl/NaCl

buffer and stored at 4 ºC for less than 4 days. The DNA

concentration per nucleotide was determined from the absorption

intensity at 260 nm using the molar absorption coefficient of

6600 M^–1cm^–1.⁶⁴Stock solutions of complexes was prepared by

40 using 5% DMF/TrisꢀHCl (0.5 mL DMF in 10 mL buffer) and

diluting suitably with the corresponding buffer to the required

concentrations for all the experiments. Absorption titration

experiments were carried out by varying nucleic acid

concentration (0–500 ꢂM) and maintaining complex

45 concentration constant (50 ꢂM). ComplexꢀDNA solutions were

allowed to incubate for 30 min at room temperature before

measurements were taken. While measuring the absorption

spectra, equal amounts of DNA was added to both complex

solution and the reference solution to eliminate the absorbance of

50 DNA itself. Absorbances were recorded after each successive

addition of DNA solution at room temperature. Titration curves

were constructed from the fractional change in the absorption

CIF or other electronic format see DOI: 10.1039/b000000x/.

10 DPPH radical scavenging activity

An electron (or) hydrogen atom donation (or) free radical

scavenging abilities of complexes was evaluated from the

bleaching of DPPH in methanolic medium. A 0.1 mM DPPH

solution was used to generate the stable radical⁶²and, the

15 absorbance was measured for this DPPH solution at 517 nm

(A_blank). Complexes of different concentrations (0–250 µM) were

prepared in DMF and added (1 mL) to each DPPH solutions. The

reaction mixtures were incubated for 30 min at room temperature

(in dark) and the absorbance of these solutions (A_sample) was

20 measured at 517 nm against blank. DPPH free radical scavenging

activity (or) inhibition (I%) of DPPH radicals for the various

concentration of complexes were calculated from the following

expression,

I% = (A_blank

– A_sample/A_blank) × 100

25

The IC₅₀values were calculated by plotting I% values as a

function of complex concentrations.

14 | Journal Name, [year], [vol], 00–00

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DOI: 10.1039/C4RA06941B

intensity as a function of DNA concentration.⁴⁰The intrinsic 55 ethidium bromide (1 ꢂg/mL). The gels were run at 50 V for 1 h in

binding constant

K

_bcan be obtained by the following equation:

TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA,

pH 8.3). The bands were viewed and imaged.

[DNA]/(ε_a

–

ε

_f) = [DNA]/(ε_{b f}) + 1/K_bε_bε_f

–

ε

( – )

Ligation enzymatic assay was performed using T4 DNA ligase

to determine whether the cleaved products consistent with

⁶⁰hydrolytic cleavage of DNA. The cleavage product was purified

by DNA gel extraction kit and incubated for 12 h at 16 °C with

1.5 µL of 10X ligation buffer, 1 µL of T4 ligase (4 units) and 2.5

µL of 1 mM ATP. Afterwards, the ligation products were stained

with EtBr, electrophoresed and imaged.

where, [DNA] is the DNA concentration in M (nucleotide),

ε_a– apparent extinction coefficient obtained by calculating

5

A

_obser/[Complex], ε_f– extinction coefficient of complex in its free

form, ε_b– extinction coefficient of complex in the fully bound

form. K_b– is the intrinsic binding constant in M^–1. Each set of

data were fitted to the above equation, and the plot of [DNA]/

10

(

ε_a

–

ε_f

)

versus [DNA] gave a slope and the

_f) and 1/K_bε_{b f}), respectively. The intrinsic

_bwas obtained from the ratio of the slope to

yꢀintercept which are

65 Growth Inhibition studies

equal to 1/(ε_b

binding constant

the intercept.

–

ε

( –ε

Cell line and cell culture conditions: Human hepatocellular liver

carcinoma cell line (HepG2) and Vero cell line were obtained

from National Centre for Cell Science (NCCS), Pune, India. The

cancer cells were cultured in Dulbecco’s Modified Eagles

70 Medium (DMEM) (St. Louis, Mo, USA), supplemented with

10% Fetal Bovine Serum (FBS) (Sigma Aldrich, USA), 100

ꢂg/mL of Streptomycin, 20 µg/mL of Kanamycin acid sulphate

and 7.5% sodium bicarbonate solution (Himedia, Mumbai, India)

in 96ꢀwell culture plates at 37 °C in a humidified atmosphere of

75 5% CO₂in a CO₂incubator (Thermoscientific, Germany). MTT

[3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide, a

yellow tetrazole] was obtained from Himedia. All experiments

were performed using cells from fifteenth passage.

K

Hydrodynamic

(Viscosity)

measurements:

Viscometric

¹⁵experiments were conducted on an Ostwald micro viscometer of

2 mL capacity, immersed in a water bath maintained at 25 ± 0.1

°C. The solutions of DNA (200 µM) and complexes (0–100 µM)

were prepared by using TrisꢀHCl/NaCl buffer (pH = 7.2). Mixing

of the solution was achieved by purging nitrogen gas through

20 viscometer. The flow time was measured with a digital stopwatch

and the experiment was repeated in triplicate to get the concurrent

values. Data were presented as (

[Complex] /[DNA],⁶⁵where

and

DNA in the presence and absence of complex, respectively. The

25 values of and

₀were calculated from the relation,⁴¹

η

/

η₀

)

^1/3versus binding ratio (1/

η₀are the specific viscosity of

R)

η

Cell viability assay (MTT assay): In vitro anticancer potency of

80 complexes 2, 6 and 8 was carried out by using MTT assay as

η

= (t – t_b)/t_b

described previously.⁶⁷HepG2 cells were cultured and seeded

into 96 well plates approximately as 1 × 10⁵cells in each well.

Complexes in the concentration range of 2–10 ꢂg/mL dissolved

in deionized water was added to the wells. After treatment, the

⁸⁵plates were incubated for 24–48 h in order to perform cytotoxic

analysis using MTT assay. MTT was prepared at a concentration

of 5 mg/mL and 10 µL of MTT was added in each well and

incubated for 4 h. Purple color farmazone crystals formed were

then dissolved in 100 µL of dimethyl sulphoxide (DMSO). These

⁹⁰crystals were observed at 570 nm in a multi well ELISA plate

reader. Optical density value was subjected to percentage of

viability (I%) by using the following formula.

where, t_bis the flow time of buffer alone and,

observed flow time for DNA in the absence and presence of

complex. Relative viscosities for DNA were obtained from the

t

is the

³⁰relation, η/η₀.

Electrochemical titration: Electrochemical techniques are best

complementary to other related biophysical techniques that are

applied to study the interaction between redox active molecules

and bioꢀmolecules.⁶⁶The solutions of complexes and DNA were

³⁵prepared by using DMF and TrisꢀHCl/NaCl buffer (pH 7.2),

respectively, in double distilled water. The concentration of

complexes can be taken as 100 ꢂM and also for DNA. Solutions

were deaerated by purging with N₂gas for 5 minutes prior to the

measurements.

Mean OD of untreated cells–Mean OD of treated cells

I% =

× 100

95

Mean OD of untreated cells

40 Nuclease activity

Apoptosis evaluation by cell morphology and propidium iodide

staining: To detect apoptosis, cells were judged according to

their nuclear morphology and disintegration of their cell

membranes, which is indicated by propidium iodide (PI) uptake.⁶⁸

The nuclease activity of complexes 1–8 on supercoiled plasmid

DNA (pBR322) was monitored using agarose gel electrophoresis

technique by determining its ability to convert supercoiled DNA

(Form I) to nicked circular (Form II) and linear forms (Form III).

45 In the cleavage reactions, plasmid DNA (33.3 ꢂM) was treated

with complexes (0–250 ꢂM) and hydrogen peroxide (0.1 mM, coꢀ

reactant) in TrisꢀHCl/NaCl buffer (pH 7.2). To test the

involvement of reactive oxygen species (ROS) during strand

scission, we further investigated the influence of the hydroxyl

50 radical scavenger (DMSO, 10 mM) during the cleavage reaction.

The mixture was incubated for 1 h at 37 ºC. A loading buffer

containing 0.25% bromophenol blue, 0.25% xylene cyanol, 30%

glycerol (3 ꢂL) was added and the electrophoresis of cleavage

products were performed on 0.8% agarose gel containing

100 HepG2 cells were plated at a density of 1 × 10⁶cells/well into a

sixꢀwell plate and incubated overnight. At 90% confluence, the

cells were treated with complexes 2, 6 and 8 at IC₅₀dose, washed

with PBS fixed in methanol and acetic acid (3:1 v/v) for 10 min

and stained with 50 ꢂg/mL of PI for 20 mins. Nuclear

105 morphology of apoptotic cells with condensed/fragmented nuclei

was examined under an Inverted phase contrast/fluorescent

microscope (Radical).

Journal Name, [year], [vol], 00–00 | 15

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DOI: 10.1039/C4RA06941B

6

(a) P. U. Maheswari, S. Roy, H. D. Dulk, S. Barends, G. V. Wezel,

B. Kozlevčar, P. Gamez and J. Reedijk, J. Am. Chem. Soc., 2006,

128, 710−711; (b) C. Marzano, M. Pellai, F. Tisato and C. Santini,

AntiꢀCancer Agent. Med. Chem., 2009, 9, 185–211.

H. Wu, J. Yuan, Y. Bai, G. Pan, H. Wang and X. Shu,

J. Photochem. Photobiol., B, 2012, 107, 65–72.

(a) P. M. May and D. R. Williams, in Metal Ions in Biological

Systems, (Eds: H. Sigel), Marcel Dekker: New York, 1981, 12; (b) T.

Miura, A. Horiꢀi, H. Mototani and H. Takeuchi, Biochemistry, 1999,

38, 11560–11569.

S. Anbu, S. Shanmugaraju and M. Kandaswamy, RSC Adv., 2012, 2,

5349−5357.

Alkaline singleꢀcell gel electrophoresis assay (Comet assay): An

alkaline singleꢀcell gel electrophoresis (SCGE) technique⁶⁹is a

neutral assay, first introduced by Östling and Johanson in 1984

which has been modified and extensively validated over the

years, and is now commonly referred to as the ‘comet assay’.⁷⁰

DNA damage was quantified by adopting comet assay as

previously described.⁷¹Cells used for the comet assay were

sampled from a monolayer during the growing phase, 24 h after

seeding. Cells were treated with complexes at IC₅₀dose and cells

60

65

7

8

5

9

10 were harvested by a trypsinization process. Normal agarose in

PBS (200 ꢂL of 1% solution, pH 7.4) at 65 °C was dropped

gently onto a fully frosted micro slide, covered immediately with

a cover slip, and then placed over a frozen ice pack for ~5 min.

The cover slip was removed after the gel had set. The cell

¹⁵suspension from each samples, was mixed with 1% lowꢀmelting

agarose (LMA) at 37 °C in a 1:3 ratio. This mixture (100 ꢂL) was

quickly applied on top of the gel, coated over the micro slide, and

allowed to set as before. A third coating of 100 ꢂL of 1% LMA

was given on the gel containing the cell suspension and allowed

²⁰to set. After solidification of the agarose, the cover slips were

removed and the slides were immersed in iceꢀcold lytic solution

(2.5 M NaCl, 100 mM NaꢀEDTA, 10 mM Trisꢀbase and 0.1%

Triton X100, 10% DMSO, pH 10) and placed in a refrigerator at

4 °C. All the above operations were performed under low lighting

²⁵conditions to avoid DNA damage due to light. The slides, after

being removed from the lytic solution were placed horizontally in

an electrophoresis tank. The reservoirs were filled with

electrophoresis buffer (300 mM NaOH, 1 mM NaꢀEDTA, pH 13)

until the slides were just immersed in it and allowed to stand in

³⁰the buffer for 20 min (to allow DNA unwinding), after which

electrophoresis was carried out at 0.8 V/cm for 15 min. After

electrophoresis, the slides were removed, washed thrice in

neutralization buffer (0.4 M Tris, pH 7.5), and gently tapped to

dry. Nuclear DNA was stained with 20 ꢂL of propidium iodide

³⁵(50 ꢂg/mL). Photographs were obtained using an Inverted

florescence microscope at 20X magnification. The DNA contents

in the head and tail were quantified by using CASP software.

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Inorg. Chim. Acta, 2003, 342, 158–170; (b) S. Budagumpi, N. V.

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Med. Chem., 2010, 45, 455–462.

11 E. Meggers, Curr. Opin. Chem. Biol., 2007, 11, 287–292.

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Acknowledgements

AKR gratefully thanks UGC, New Delhi, for financial

40 assistance through the Major Research Project grant [F.No.39ꢀ

797/2010 (SR)]. The authors thank to SAIF, IITꢀM and CLRI,

Chennai for Single crystal XRD and EPR studies, respectively.

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DOI: 10.1039/C4RA06941B

Magneto-structural correlation, antioxidant, DNA interaction and

growth inhibition activities of new chloro-bridged phenolate complexes

Perumal Gurumoorthy,^aDharmasivam Mahendiran,^aDurai Prabhu,^bChinnasamy Arulvasu^band

Aziz Kalilur Rahiman,*^a

5

Table of Contents

Control

PI Stained

10

Comet assay

Morphology

15

²⁰Highlights

The consistent stability constants as well as antioxidant, DNA interaction and cytotoxicity efficacy of chloro-bridged complexes have

been established.

Article Doi

Magneto-structural correlation, antioxidant, DNA interaction and growth inhibition activities of new chloro-bridged phenolate complexes

DOI: 10.1039/c4ra06941b

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Authors:

Article abstract of DOI:10.1039/c4ra06941b

Full text of DOI:10.1039/c4ra06941b

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