Antibacterial, DNA interaction and cytotoxic activities of pendant-armed polyamine macrocyclic dinuclear nickel(II) and copper(II) complexes

Article abstract of DOI:10.1016/j.saa.2014.03.058

A series of dinuclear nickel(II) and copper(II) complexes (1-6) of hexaaza macrocycles of 2,6-diformyl-4-methylphenol with three different benzoyl pendant-arms, 2,2′-benzoyliminodi(ethylamine) trihydrochloride (L), 2,2′-4-nitrobenzoyliminodi(ethylamine) trihydrochloride (L') and 2,2′-3,5-dinitrobenzoyliminodi(ethylamine) trihydrochloride (L') have been synthesized and characterized by spectral methods. The electrochemical studies of these complexes depict two irreversible one electron reduction processes around E¹_pc = -0.62 to -0.76 V and E² _pc = -1.21 to -1.31, and nickel(II) complexes (1-3) exhibit two irreversible one electron oxidation processes around E¹_pa = 1.08 to 1.14 V and E²_pa = 1.71 to 1.74 V. The room temperature magnetic moment values (μ_eff, 1.52-1.54 BM) indicate the presence of an antiferromagnetic interaction in the binuclear copper(II) complexes (4-6) which is also observed from the broad ESR spectra with a g value of 2.14-2.15. The synthesized complexes (1-6) were screened for their antibacterial activity. The results of DNA interaction studies indicate that the dinuclear complexes can bind to calf thymus DNA by intercalative mode and display efficient cleavage of plasmid DNA. Further, the cytotoxic activity of complexes 2, 5 and 6 on human liver adenocarcinoma (HepG2) cell line has been examined. Nuclear-chromatin cleavage has also been observed with PI staining and comet assays.

Full text of DOI:10.1016/j.saa.2014.03.058

Accepted Manuscript
Antibacterial, DNA Interaction and Cytotoxic Activities of Pendant-Armed
Polyamine Macrocyclic Dinuclear Nickel(II) and Copper(II) Complexes
P. Arthi, A. Haleel, P. Srinivasan, D. Prabhu, C. Arulvasu, A. Kalilur Rahiman
PII:
S1386-1425(14)00469-7
http://dx.doi.org/10.1016/j.saa.2014.03.058
SAA 11891
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
20 January 2014
1 March 2014
20 March 2014
Please cite this article as: P. Arthi, A. Haleel, P. Srinivasan, D. Prabhu, C. Arulvasu, A. Kalilur Rahiman,
Antibacterial, DNA Interaction and Cytotoxic Activities of Pendant-Armed Polyamine Macrocyclic Dinuclear
Nickel(II) and Copper(II) Complexes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
(
2014), doi: http://dx.doi.org/10.1016/j.saa.2014.03.058
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ANTIBACTERIAL, DNA INTERACTION AND CYTOTOXIC ACTIVITIES OF
PENDANT-ARMED POLYAMINE MACROCYCLIC DINUCLEAR NICKEL(II)
AND COPPER(II) COMPLEXES
a
a
b
c
c
a,
P. Arthi , A. Haleel , P. Srinivasan , D. Prabhu , C. Arulvasu A. Kalilur Rahiman *
,
a
Post-Graduate and Research Department of Chemistry, The New College (Autonomous),
Chennai-600 014, India
b
Department of Bioinformatics, Alagappa University, Karaikudi-630 003, India
c
Department of Zoology, University of Madras, Guindy Maraimalai Campus,
Chennai-600 025, India
_
_________________________________________________________________________
Abstract
A
series of dinuclear nickel(II) and copper(II) complexes (1–6) of
hexaaza macrocycles of 2,6-diformyl-4-methylphenol with three different benzoyl
pendant-arms, 2,2‟-benzoyliminodi(ethylamine) trihydrochloride (L), 2,2‟-4-nitrobenzoyl
iminodi(ethylamine) trihydrochloride (L‟) and 2,2‟-3,5-dinitrobenzoyliminodi(ethylamine)
trihydrochloride (L”) have been synthesized and characterized by spectral methods. The
electrochemical studies of these complexes depict two irreversible one electron reduction
1
2
processes around E = 0.62 to –0.76 V and E = –1.21 to –1.31, and nickel(II) complexes
pc
pc
1
(
1-3) exhibit two irreversible one electron oxidation processes around E = 1.08 to 1.14 V
pa
2
and E = 1.71 to 1.74 V. The room temperature magnetic moment values (µ , 1.52–1.54
pa
eff
BM) indicate the presence of an antiferromagnetic interaction in the binuclear copper(II)
complexes (4–6) which is also observed from the broad ESR spectra with a g value of 2.14–
2
.15. The synthesized complexes (1–6) were screened for their antibacterial activity. The
results of DNA interaction studies indicate that the dinuclear complexes can bind to calf
thymus DNA by intercalative mode and display efficient cleavage of plasmid DNA. Further,
the cytotoxic activity of complexes 2, 5 and 6 on human liver adenocarcinoma (HepG2) cell
line has been examined. Nuclear-chromatin cleavage has also been observed with PI staining
and comet assays.
1

_________________________________________________________________________________
Keywords: Pendant-armed macrocycles; Dinuclear complexes; Antibacterial activity;
DNA interaction; Cytotoxicity; Apoptosis.
*
Corresponding author: Tel: +91 44 2835 0297; fax: +91 44 2835 2883
E-mail address: akrahmanjkr@gmail.com
_________________________________________________________________________________
. Introduction
The chemistry of macrocyclic compounds has become a fruitful research area due to
_
1
the ability of these systems to interact with different substrates, such as metal ions or anionic
species [1]. The term “macrocycle” is defined as a cyclic macromolecule or a cyclic
compound with nine or more members [2]. Coordination chemists generally define a
macrocycle more narrowly as a cyclic molecule with three or more potential donor atoms that
can coordinate to a metal center. Cabbiness and Margerum introduced the term “macrocyclic
effect” to explain the stability of macrocyclic polyamine when compared to their acyclic
analogs [3]. Much effort has now been focused on pendant-arm macrocyclic complexes due
to the fact that the ligating groups attached to the macrocyclic backbone can offer additional
donor groups to produce important changes in the control of the stability, selectivity,
stereochemistry and certain thermodynamic parameters [4, 5] or promote the formation of
dinuclear or polynuclear metal complexes with interaction between the metal centers as well
as act as host for organic cations with different properties and applications [6]. The following
two ways are involved in the process of making pendant-arm macrocyclic complexes: (i) The
electrophilic substitution or addition reactions have been used in connecting the functional
group with the ring framework (ii) “opened cryptands” can be generated by using analogous
functional precursors with variable aromatic dialdehydes in the presence of certain metal ions
[
7].
2

In recent years, metal complexes of pendant-arm macrocyclic polyamines are of
growing interest for applications in biological and medicinal chemistry, in addition to
catalysis. Macrocyclic polyamines bearing pendant-arms can effectively bind specific metals
with suitable complexation kinetics and stability while attached to a tumor-targeting vector or
peptide. These compounds are used in the radiopharmaceuticals for positron-emission
tomography (PET), different phases of biological testing against HIV or antitumor activity
and DNA interaction [8–10]. Studies on the interaction of pendant-arm macrocyclic
complexes with DNA not only gives information about the reactive modes for protein-DNA
interactions and probes of DNA structure but also explains the techniques of molecular
biology and drug design [11]. In some cases, the toxicity of macrocyclic polyamines was
found to decrease with the increased anti-HIV activity when they are coordinated with
bivalent metals like nickel and copper [12]. Based upon these criteria researchers are further
involved to exploit their derivatives. Nickel(II) and copper(II) complexes have shown notable
different biological activities. Nickel was long thought not to be a metal of biological
importance. However, Zerner discovered that urease is a nickel enzyme [13]. Since then,
other important enzymes that depend on nickel for activity have been identified. For this
reason, the synthesis of complexes oriented to mimic metal sites of different types of
metalloproteins constitutes an important branch in both inorganic and organic chemistry [14].
The role of copper compounds as pharmaceutical drugs in the treatment of numerous chronic
diseases is well established. The macrocyclic ligands in the complexes are smaller than
enzymes which allow the attack at the macromolecular region which are beyond the reach of
enzymes. Due to its unique properties it is used as adjuvant in PCR diagnostics, cleaving
agents and attacking agents [15, 16].
It is significant to note that pendant-arm macrocyclic nickel(II) and copper(II)
complexes have been rarely used for bimetallic biosite [17, 18]. This has prompted us to
3

design and synthesis a series of dinuclear nickel(II) and copper(II) complexes with pendant-
armed polyamine macrocycles of diethylenetriamine derivatives with N-substituted groups
(at the secondary amine nitrogen atom) and 2,6-diformyl-4-methylphenol by template
method. The biological significance of the synthesized pendant-armed complexes has been
investigated in term of antibacterial, DNA interaction and cytotoxic activities.
2
2
. Experimental section
.1. Materials
2
,6-Diformyl-4-methylphenol has been synthesized according to the literature
method [19]. Benzoyl chloride, nitrobenzoyl chlorides, diethylenetriamine, salicylaldehyde
and metal(II) perchlorate salts were commercial products (from Merck and Aldrich). Solvents
were dried and purified before being used according to standard procedure.
Tetra(n–butyl)ammonium perchlorate (TBAP) used as supporting electrolyte in the
electrochemical measurements was purchased from Fluka and recrystallized from hot
methanol. (Caution! TBAP is potentially explosive; hence care should be taken in handling
the compound). Tris-HCl buffer (5mM Tris-HCl/50 mM NaCl, pH 7.2), Tris =
Tris(hydroxymethyl)aminomethane, solution was prepared using deionized double distilled
water. Calf thymus DNA (CT-DNA) and pBR322 DNA were purchased from Bangalore
Genei (India).
2
.2. Physical measurements
The elemental analysis (C, H and N) was measured with a Carlo Erba elemental
1
analyzer Model 1106. H NMR spectral data were collected on Varian-VNMRS-400 in
CDCl and DMSO solution with tetramethylsilane (TMS) as an internal standard at ambient
3
temperature. IR spectra were recorded on ABB Instruments, MB-3000 spectrophotometer
–
1
using KBr pellets in the range 4000–400 cm . The mass data of ligands were obtained on a
JEOL GC mate GC-MS spectrometer and the mass spectra for complexes were taken on
4

Q-TOF 6000 ESI mass spectrometer. Electronic spectra of complexes were recorded on
Perkin Elmer Lambda-45 spectrophotometer, in the range of 200–1100 nm using 1 cm quartz
micro cuvettes. X-band EPR spectra were recorded on a Varian EPR-E 112
spectrophotometer using diphenylpicrylhydrazine (DPPH) as the reference. Room
temperature magnetic moments were recorded on a PAR Vibrating sample magnetometer
model 155. Cyclic voltammograms were obtained on a CHI-602D electrochemical analyzer
using a three-electrode cell in which a glassy carbon electrode was the working electrode, a
saturated Ag/AgCl electrode was the reference electrode and platinum wire was used as the
auxiliary electrode. A ferrocene/ferrocenium (1+) couple was used as an internal standard and
+
E1/2 of the ferrocene/ferrocenium (Fc/Fc ) couple under the experimental condition was 470
mV. Tetra(n-butyl)ammonium perchlorate (TBAP) was used as the supporting electrolyte
–
3
(
0.1 M) and all complex solutions were around 10 M concentration. All electrochemical
measurements were carried out in solutions purged with pure nitrogen for 30 min in advance.
2
2
.3. Synthesis of ligands
.3.1. 2,2’-Benzoyliminodi(ethylamine) trihydrochloride (L)
A solution of diethylenetriamine (1.08 mL, 10 mmol) in ethanol (20 mL) was added
dropwise to a solution of salicylaldehyde (2.1 mL, 20 mmol) in ethanol (20 mL). The mixture
was stirred for 2 h and then refluxed for 6 h. Then the reaction mixture was cooled to room
temperature, and a solution of benzoyl chloride (1.16 mL, 10 mmol) in ethanol (20 mL)
followed by Na CO (1.06 g, 10 mmol) were added. The resulting solution was refluxed for
2
3
3
6 h, cooled to room temperature, an excess solid Na CO was filtered off and the filtrate was
2 3
concentrated. A yellowish hygroscopic product 2,2‟-(benzoyliminodiethylene)bissalicylidene
was obtained which was used without further purification.
–
1
Yield: 3.25 g (78%). Analytical data for C H N O : Selected IR (KBr) (ν/cm ):
25
25
3
3
1
3
442 ν(OH), 1732 ν(C=O), 1631 ν(C=N). H NMR (δ/ppm in CDCl ): 2.97 (t, 8H, CH ), 3.58
3 2
5

(
t, 4H, CH ), 6.86 (t, 4H, Ar-H), 7.22 (d, 4H, Ar-H), 7.30 (d, 2H, Benzylic-H), 7.44 (t, 3H,
2
benzylic-H), 8.36 (s, 2H, CH=N), 13.46 (br s, 2H, Ar-OH).
,2‟-(benzoyliminodiethylene)bissalicylidene (3.25 g, 0.0078 mol) was suspended in
M HCl (50 mL). The reaction mixture was maintained under reflux for 4 h, cooled and
2
6
filtered to eliminate salicylic acid. The solution was concentrated under reduced pressure up
to 10 mL. The solid (L) obtained on addition of few drops of ethanol was filtered off, washed
with ethanol and dried under vacuum.
L Yield: 1.36 g (55%); Colour: Pale brown; M.P: 230 °C. Analytical data for
C H Cl N O: Calculated (%): C, 41.72; H, 6.36; N, 13.27. Found: C, 41.66; H, 6.48; N,
11
20
3
3
–
1
1
1
3.02. Selected IR (KBr) (ν/cm ): 2979 ν(NH), 1700 ν(C=O). H NMR (δ/ppm in D O): 2.91
2
+
(
m, 8H, CH ), 8.40 (s 5H, Ar-H), 10.09 (br s, NH ). Mass EI m/z: 207.27 [L–3HCl].
2
,
2
.3.2. 2,2’-4-Nitrobenzoyliminodi(ethylamine) trihydrochloride (L’)
This compound was obtained by following the procedure similar to that for L, using
-nitrobenzoyl chloride in place of benzoyl chloride.
4
2
,2‟-(4-nitrobenzoyliminodiethylene)bissalicylidene: Yield: 3.52 g (76%). Analytical
–
1
data for C H N O : Selected IR (KBr) (ν/cm ): 3444 ν(OH), 1728 ν(C=O), 1632 ν(C=N),
2
5
24
4
5
1
1
543, 1350 ν(NO ). H NMR (δ/ppm in CDCl ): 3.06 (t, 4H, CH ), 3.74 (t, 4H, CH ), 6.85
2 3 2 2
(s, 2H, Ar-H), 6.93 (s, 2H, Ar-H), 7.20 (s, 2H, Ar-H), 7.29 (s, 2H, Ar-H), 8.21 (d, 4H,
Benzylic-H ), 8.29 (s, 2H, CH=N), 13.37 (br s, 1H, Ar-OH).
L’ Yield: 1.45 g (52%); Colour: Brown; M.P: 220 °C. Analytical data for
C H Cl N O : Calculated (%): C, 36.53; H, 63.10; N, 15.49. Found: C, 36.47; H, 36.65;
11
19
3
4
3
-1
N, 15.24. Selected IR (KBr) (ν/cm ): 1730 ν(C=O), 1522 ν (NO ), 1376 ν (NO ),
asym
2
sym
2
1
2
1
900 ν(N-H stretching). H NMR (δ/ppm in D O): 2.88 (m, 8H, CH ), 8.39 (s, 4H, Ar-H),
2 2
+
0.02 (s, NH ). Mass EI m/z: 252.27 [L‟–3HCl].
6

2
.3.3. 2,2’-3,5-Dinitrobenzoyliminodi(ethylamine) trihydrochloride (L”)
This compound was obtained by following the procedure similar to that for L, using
,5-dinitrobenzoyl chloride in place of benzoyl chloride..
3
2
,2‟-(3,5-dinitrobenzoyliminodiethylene)bissalicylidene: Yield: 3.48 g (68.8%).
–
1
Analytical data for C H N O : 13.69. Selected IR (KBr) (ν/cm ): 3444 ν(OH), 1730
2
5
23
5
7
1
ν(C=O), 1630 ν(C=N), 1543, 1350 ν(NO ). H NMR (δ/ppm in CDCl ): 2.83 (s, 4H, CH ),
2
3
2
3
.64 (s, 4H, CH ), 6.81 (d, 4H, Ar-H), 7.25 (d, 4H, Ar-H), 8.89 (m, 3H, Benzylic-H), 8.45
2
(s, 2H, CH=N) 13.43 (br s, Ar-OH).
L” Yield: 1.52 g (38%); Colour: Brown; M.P: 225 °C. Analytical data for
C H Cl N O : Calculated (%): C, 32.48; H, 4.46; N, 17.22. Found: C, 32.42; H, 4.58; N,
11
18
3
5
5
–
1
1
1
6.97. Selected IR (KBr) (ν/cm ): 2961 ν(NH), 1729 ν(C=O), 1522 νasym(NO2),
1
376 ν (NO ). H NMR (δ/ppm in D O): 2.91 (m, 8H, CH ), 8.41 (s, 3H, Ar-H), 10.01
sym
2
2
2
+
”
(
s, NH ). Mass EI m/z: 297.91 [L –3HCl].
2
.4. General procedure for synthesis of pendant-armed polyamine macrocyclic
dinuclear nickel(II) and copper(II) complexes
A solution of NaOH (3 mmol) in ethanol (10 mL) was added to a suspension of
hydrochloride salt of the appropriate amine (L, L’, L”) (1 mmol) in ethanol (10 mL). The
mixture was stirred at room temperature for few minutes and filtered. Then the precipitate
was washed with ethanol (10 mL) and the combined filtrate was added dropwise to a solution
of M(ClO ) .6H O (1.5 mmol) and 2,6-diformyl-4-methylphenol (1 mmol) in ethanol (20
4
2
2
mL) over a period of 30 min. The resulting solution was stirred for 4 h and refluxed for 5–7
h. The precipitate thus obtained was filtered, washed with cold ethanol, followed by diethyl
ether and dried under vacuum.
7

1
2
.4.1. [Ni L ](ClO ) , (1)
2 4 2
Yield: 0.46
g
(42%); Colour: Brownish yellow. Analytical data for
[
C H N O Ni ](ClO ) (FW = 1075.04 g/mol): Calculated (%): C, 44.69; H, 3.56; N, 10.42.
4
0
40
6
4
2
4 2
–
1
Found: C, 44.70; H, 3.76; N, 10.97. Selected IR (KBr) (ν/cm ): 1645 ν(C=N), 1091 and
–
2
–1
6
25 (s) v[ClO uncoordinated]. Conductance (Λ /S cm mol ) in DMF: 139.
4
m
2
2
.4.2. [Ni L ](ClO ) , (2)
2 4 2
Yield: 0.40
g
(40%); Colour: Brownish yellow. Analytical data for
[
C H N O Ni ](ClO ) (FW = 985.05 g/mol): Calculated (%): C, 48.77; H, 4.09; N, 8.53.
40 38 8 8 2 4 2
–
1
Found: C, 48.78; H, 4.29; N, 9.08. Selected IR (KBr) (ν/cm ): 1643 ν(C=N),
–
1
531 νasym(NO ), 1332 ν (NO ), 1092 and 624 (s) v[ClO uncoordinated]. Conductance
2
sym
2
4
2
–1
(
Λ /S cm mol ) in DMF: 142.
m
3
2
.4.3. [Ni L ](ClO ) , (3)
2
4 2
Yield: 0.51
g
(43%); Colour: Brownish yellow. Analytical data for
[
C H N O Ni ](ClO ) (FW = 1165.04 g/mol): Calculated (%): C,41.23; H, 3.11; N,
4
0
36 10 12
2
4 2
–
1
1
2.02. Found: C, 41.24; H, 3.31; N, 12.57. Selected IR (KBr) (ν/cm ): 1647 ν(C=N), 1529
–
νasym(NO ), 1340 ν (NO ), 1096 and 625 (s) v[ClO4 uncoordinated]. Conductance
2
sym
2
2
–1
(
Λ /S cm mol ) in DMF: 150.
m
1
2
.4.4. [Cu L ](ClO ) , (4)
2
4 2
Yield: 0.52 g (50%); Colour: Green. Analytical data for [C H N O Cu ](ClO )
4 2
4
0
40
6
4
2
(FW = 1021.21 g/mol): Calculated (%): C,47.04; H,3.75; N,10.97. Found: C, 47.05; H, 3.95;
–
1
–
N, 11.52. Selected IR (KBr) (v/cm ): 1646 v(C=N), 1105 and 624 (s) v[ClO uncoordinated].
4
2
–1
Conductance (Λ /S cm mol ) in DMF: 148. g = 2.14. µ = 1.52 B.M.
m
eff
8

2
2
.4.5. [Cu L ](ClO ) , (5)
2 4 2
Yield: 0.56 g (60%); Colour: Green. Analytical data for [C H N O Cu ](ClO )
4 2
4
0
38
8
8
2
(FW = 931.22 g/mol): Calculated (%): C, 51.59; H, 4.32; N, 9.02. Found: C, 51.60; H, 4.52;
–
1
N, 9.57, selected IR (KBr) (v/cm ): 1644 v(C=N), 1528 v (NO ), 1330 v (NO ), 1103
asym
2
sym
2
–
4
2
–1
and 625 (s) v[ClO uncoordinated]. Conductance (Λ /S cm mol ) in DMF: 130. g = 2.14.
m
µeff = 1.54 B.M.
3
2
.4.6. [Cu L ](ClO ) , (6)
2
4 2
Yield: 0.50 g (44%); Colour: Green. Analytical data for [C H N O Cu ](ClO )
4 2
40
36 10 12
2
(FW= 1111.20): Calculated (%): C, 43.23; H, 3.26; N, 12.60. Found: C, 43.24; H, 3.46; N,
–
1
1
3.15. selected IR (KBr) (v/cm ): 1647 v(C=N), 1529 v (NO ), 1340 v (NO ), 1106
asym
2
sym
2
–
2
–1
and 624 (s) v[ClO uncoordinated]. Conductance (Λ /S cm mol ) in DMF: 133. g = 2.15.
4
m
µeff = 1.53 B.M.
.5. In vitro antibacterial screening
The antibacterial activity of the pendant-armed macrocyclic nickel(II) and copper(II)
2
complexes 1–6 was evaluated against one Gram –ve Proteus vulgaris and four Gram +ve
Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutants and Streptococcus
pneumoniae strains by agar well diffusion method [20]. These five bacterial strains were
selected on the basis of their clinical importance in causing diseases in humans. All the
microbial cultures were adjusted to 0.5 McFarland standards, which is visually comparable to
8
a microbial suspension of approximately 1.5×10 c.f.u./mL [21]. Five bacterial strains were
pregrown in nutrient broth/Luria Bertaini broth at 37 ºC for 24 h. Nutrient/Luria Bertaini agar
medium was poured into sterilized petri dishes and allowed to solidify. After solidification,
1
00 µL of target bacterial cell cultures were spread over the medium using a spreader and the
wells were punched with 3 mm diameter gel puncher. A different concentration such as 50
µL (250 µg), 100 µL (500 µg) and 200 µL (1000 µg) of complexes was pipetted out into each
9

well against each test bacteria. An empty well inoculated with sterile Nutrient broth/Luria
Bertaini alone served as negative controls. Inoculated plates were incubated for a day at room
temperature. The experiments were performed in triplicates. The antibacterial activity of the
compounds (in term of inhibition of bacterial growth) against bacterial isolates was
determined from the growth in the test plates compared to the respective control plates by
using the formula
%
I (Growth inhibition) = 100(C-T)/C
Where C is the diameter of bacterial growth in control plate and T is the diameter of bacterial
growth in test plate.
2
2
.6. DNA Interaction experiments
.6.1. Electronic absorption titration
The binding analysis of complexes 2, 3, 5 and 6 with CT-DNA was performed at
room temperature. The absorption titration experiments of complexes with CT-DNA was
characterized by measuring their effects on the UV-Vis spectroscopy. The complexes were
dissolved in a mixed solvent of Tris-HCl/NaCl buffer (pH 7.2) containing 5% DMF (solution
was prepared using doubly distilled water). A solution of CT-DNA in the buffer gave a ratio
of UV-Vis absorbance at 260 and 280 nm (A /A ) of ca 1.8 indicating that the CT-DNA
2
60 280
was sufficiently free of protein [22]. Electronic absorption titrations were performed with a
fixed concentration of metal complexes (100 µM) while gradually increasing the nucleotide
concentration from 0 to 40 µM. Due correction was made for the absorbance of DNA itself.
Absorbance values were recorded after each successive addition of DNA solution and
equilibration (ca.10 min). The absorbance band of complexes that get shifted significantly
due to the addition of CT-DNA was chosen to monitor as an indication of the binding
between them. From the obtained data, the intrinsic binding constant K of the metal
b
complexes with CT-DNA was determined using the equation [23],
1
0

[
DNA]/(ε – ε ) = [DNA]/(ε – ε ) + 1/K (ε – ε )
a f b f b b f
Where, [DNA] is the concentration of CT-DNA in base pairs, ε is the extinction coefficient
a
of complex at a given DNA concentration, ε is the extinction coefficient of complex in free
f
solution and ε is the extinction coefficient of complex when fully bound to DNA. A plot of
b
[
DNA]/(ε – ε ) versus [DNA] gave a slope and an intercept equal to 1/(ε – ε ) and 1/K (ε –
b f a f b b
ε ), respectively. The intrinsic binding constant K is the ratio of the slope to the intercept.
f
b
2
.6.2. Fluorescence quenching studies
The binding constant of complexes can also be determined by fluorescence
quenching experiments using a reference ethidium bromide (EB) bound to CT DNA solution
of 5% DMF in Tris–HCl/NaCl buffer (pH 7.2). The changes in fluorescence intensities at 605
nm (545 nm excitation) of EB bound to DNA were measured with different complex
concentrations. EB was non emissive in Tris–HCl/NaCl buffer (pH 7.2) due to fluorescence
quenching of the free EB by the solvent molecules. In the presence of DNA, EB showed
enhanced emission intensity due to its intercalative binding to DNA. A competitive binding
of the metal complexes to CT DNA resulted in the displacement of the bound EB, thereby
decreasing its emission intensity. Stern-Volmer quenching constant [24] was calculated using
the equation I /I = 1 + K [Q] where, I is the emission intensity of EB-DNA in the absence of
o
sv
o
complex, I is the emission intensity of EB-DNA in the presence of complex, [Q] is the
concentration (10 µM) of quencher. K is a linear Stern-Volmer constant given by the ratio
sv
of slope to intercept in the plot of I /I versus Q.
0
2
.6.3. Electrochemical titration
Electrochemical properties of complexes with CT-DNA were studied by cyclic
voltammetry in Tris-HCl/NaCl buffer solution (pH = 7.2) using tetra(n-butyl)ammonium
perchlorate (TBAP) as supporting electrolyte. The concentration of complexes can be taken
1
1

as 100 µM and 200 µM for DNA. Solutions were deoxygenated by purging with N gas for
2
1
0 min prior to measurements.
2
.6.4. DNA Cleavage analysis
DNA cleavage was monitored by agarose gel electrophoresis using the reaction
mixture containing 1 μg of supercoiled DNA pBR322, 250 μg of the pendant-armed
macrocyclic complexes 1–6, 2 μL of 1% DMSO (Reactive oxygen species (ROS) scavenger)
and 5 μL of hydrogen peroxide (40 mmol, oxidizing agent). After incubation at 37 ºC for
2
h, 2 μL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in
H O) was added to each tube . The agarose gel (1 %) containing 1 µg of ethidium bromide
2
(EB) was prepared and the electrophoresis of the DNA cleavage products was performed on
it. The gel was run at 60 V for 2 h in Tris-borate EDTA (TBE) buffer (89 mM Tris-borate,
pH 8.3, 1 mmol EDTA) and the bands were identified by placing the stained gel under an
illuminated UV lamp. After electrophoresis, the proportion of both the cleaved and uncleaved
DNA in each fraction was quantitatively estimated on the basis of the band intensities using
gel documentation system (BIO RAD). The intensity of each band relative to that of the
plasmid supercoiled form was multiplied by 1.43 to take account of the reduced affinity for
ethidium bromide [25].
2
2
.7. Cell Proliferation assay
.7.1. Cell lines and cell culture
Human liver adenocarcinoma (HepG2) cell line was obtained from National Centre
for Cell Science (NCCS), Pune, India. Cells were cultured in Dulbecco‟s modified eagle
medium (DMEM) (St. Louis, Mo, USA) supplemented with 10% (V/V) fetal bovine serum
(FBS) , penicillin 100 µg/mL, streptomycin 20 µg/mL, Kanamycin acid sulphate 20 µg/mL
2
and 7.5% sodium bicarbonate solution. The cells were maintained as monolayers in 25 cm
1
2

plastic tissue culture flask at 37 ºC in a humidified atmosphere containing 5% CO in air.
2
Exponentially growing cells were used in all the experiments.
2
.7.2. Cell viability assay
The cell viability of complexes 2, 5 and 6 was assessed by MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described by
(
Mosmann [26] . In order to detect the cytotoxicity, the HepG2 cells were grown in DMEM
3
medium containing 10% FBS. For screening experiments, the cells (5×10 cells/well) were
plated in 96-well plates with the medium containing 10% FBS and incubated for 24 h under
5
% CO , 95% O at 37 ºC. Later, the medium was replaced with DMEM containing 1% FBS
2 2
and complexes 2, 5 and 6 (10, 8 and 2 µg/mL, respectively) dissolved in DMSO were added
to the cells incubated at 37 ºC in 5% CO . The test samples were kept in triplicate. Controls
2
were maintained under similar conditions without the influence of compounds under study.
After 24 and 48 h, MTT stock solution (5 mg/mL) in phosphate buffer saline (PBS) was
added to each culture well, equal to one tenth of the original culture volume and the plates
were wrapped with aluminium foil and incubated at 37 ºC for 4 h. At the end of the
incubation period, the medium was removed and the purple formazan product was dissolved
by adding of 100 µL of DMSO to each well and the absorbance was measured at 570 nm
using a multiplate reader. The % of cell viability was evaluated using the following equation:
%
Cell viability = [A570 (sample)/A570 (control)] × 100
Where A570 (sample) refers to the reading from the wells treated with complexes and
A570 (control) refers to that from the wells treated with medium containing 10% FBS. From
this plot, the IC values was calculated.
5
0
2
.7.3. Apoptosis studies
Apoptosis studies were performed with a staining method utilizing propidium iodide
4
(
PI). HepG2 cells plated at a density of 5×10 ꢀcells/well into a six well chamber plate. At
1
3

>
90% confluence, the cells were treated with complexes 2, 5 and 6 at IC dose for 24 h. The
50
cells were washed with PBS fixed methanol and acetic acid (3:1, v/v) for 30 min and stained
with 50 µg/mL propidium iodide for 20 min. Nuclear morphology of apoptotic cells with
condensed/fragmented nuclei was examined under a fluorescent microscope and at least
3
1
× 10 cells were counted to assess apoptotic cell death.
2
.7.4. Alkaline single-cell gel electrophoresis (Comet assay)
A comet assay was performed to determine the degree of oxidative DNA damage
induced by complexes. Adenocarcinoma cells were exposed to complexes 2, 5 and 6 for 24 h
and washed with phosphate buffer saline (PBS). The cells suspension was mixed with 75 µL
of 0.5% low melting agarose (LMA) at 39 ºC and spread on a fully frosted microscopic slide
pre-coated with 200 µL of 1% LMA and then immersed in lytic solution (2.5 M NaCl, 10
mM Na-EDTA, 10 mM Tris–base and 0.1% Trion X-100, 10% DMSO; the last two
compounds were added fresh, pH 10) for 1 h at 4 ºC. The slides were then placed in a gel
electrophoresis apparatus (containing 300 mM NaOH and 10 mM Na-EDTA, pH 13) for 40
min to allow unwinding of DNA and the alkali labile damage. Next, an electrical field (3000
mA, 25 V) was applied for 20 min at 4 ºC to draw the negatively charged DNA towards an
anode. After electrophoresis, slides were washed thrice for 5 min at 4 ºC in a neutralizing
buffer (0.4 M Tris, pH 7.5), followed by staining with 75 µL of propidium iodide (40 µg/mL)
and then the slides were examined using fluorescence microscope at 20X magnification. The
DNA contents in the head and tail were quantified by using CASP software.
1
4

3
. Results and Discussion
For synthesis of ligands (L, L‟ and L”), salicylaldehyde was utilized to protect the two
primary amino groups of diethylenetriamine from nucleophilic additions so that the
benzoylation reaction can take place only at the secondary amine and the resulting ligands
was isolated as its hydrochloride salt (Scheme 1). The Schiff base condensation of ligands
and 2,6-diformyl-4-methylphenol with appropriate hydrated metal(II) perchlorate salt (1:1:1.5
mole ratio) in ethanol gave good yields of analytically pure dinuclear pendant-armed
macrocyclic nickel(II) and copper(II) complexes (Scheme 2). All the complexes were
characterized by elemental analyses consistent with the formulations given in experimental
section. All the complexes are air stable solids, soluble in CH CN, DMF and DMSO. Our
3
attempt to synthesize macrocyclic ligands in the absence of metal ions was unsuccessful and
cyclocondensation reactions did not occur, therefore we tried different metal ions in order to
examine the effect of the template reaction on the occurrence of ring closure. Our results
clearly demonstrate that the nature of the metal template determines the outcome of the
Schiff-base condensation between 2,6-diformyl-4-methylphenol and a difunctional amine
component.
3
.1. Spectral and magnetic studies
The IR spectra provide valuable information regarding the coordination behavior of
–1
the ligand to the metal ion. All the ligands show a sharp band in the region 1597 cm due to
+
NH group of the salt of a primary amine [27]. The IR spectra of ligands show a band in the
3
–
1
region of 1700–1730 cm due to the ν(C=O) vibration. The spectra of complexes revealed
significant differences from those of free ligands as described below. The formation of
complexes of macrocyclic framework is confirmed by the absence of bands characteristic of
+
–1
starting materials: the band due to NH3 group in the region 1597 cm and the
–
1
aldehydic -C=O band at 1680 cm disappeared and band for imine stretching appear as sharp
1
5

–
1
band in the region 1643–1647 cm . The band due to benzoylic ν(C=O) stretching vibrations
are absent in the IR spectra of all complexes suggesting the involvement of this group in
coordination to the metal. A strong absorption in the region of 1528–1531 and
–
1
–1
1
330–1340 cm corresponds to NO stretching vibrations. The bands at 1440–1485 cm
2
were assigned to aromatic ring vibrations and appeared in the same position on complexation
with respect to free ligands. The presence of uncoordinated perchlorate anions in all the
–
1
binuclear complexes are inferred from single broad band around 1100 cm (ν -antisymmetric
3
–
1
stretching) which are not split and a band around 625 cm (ν -antisymmetric bending). The
4
–
1
band around 930 cm (ν -symmetric stretching) due to coordinated perchlorate is not
2
observed and this clearly indicates that no perchlorate ions are coordinated in the complexes
[
28].
1
The H NMR spectra of ligands recorded in DMSO-D show well resolved signals as
6
expected (Fig. 1). The aromatic region shows a sharp singlet at δ 8.3 ppm assigned to the
3
+
+
aromatic protons, and a multiplet at δ 3.3 ppm due to methylene protons. The NH and NH
proton of the ammonium group shows a broad singlet at δ 10.0 ppm. In the mass spectra, the
calculated and observed isotropic patterns are in good agreement. The high resolution mass
spectral data of all the ligands confirm the proposed formulae. The different fragments of the
ligand gave peaks with various intensities at different m/z values. Fig. 2 depicts the mass
spectrum of L”, and its fragmentation pattern is depicted in Fig. S1. The ESI mass spectral
data of all complexes were studied in positive mode in CH CN solution and provided strong
3
evidence for the formation of the macrocyclic dinuclear complexes. Fig. 3 depicts the mass
spectrum of complex 3 and its fragmentation pattern is depicted in Fig. S2. The m/z peak at
9
67 corresponds to molecular weight of complex 3. The intensities of peaks are in accordance
with the abundance of the ions. The observed molecular ion corresponds to the mass of entire
complex excluding two counter ions, perchlorate.
1
6

The electronic spectra of all the binuclear complexes (1–6) were recorded in DMF
medium and the spectral data are given in Table S1. The absorption spectra of complexes
exhibit an intense peak in the range of 232–275 nm assigned to the intra ligand charge
transfer transition (π – π*). A medium intensity broad band in the range of 376–409 nm is due
to ligand to metal charge transfer transition. Complexes 1-3 show three d-d transition bands
2
+
centered at 643–657, 759–771 and 922–935 nm, which are characteristic of Ni in the six
coordination environment of the binuclear complexes whereas complexes 4–6 exhibit one
d-d transition band between 553–587 nm. The electronic spectral features strongly suggest
that the six coordination geometry around the metal ion in the binuclear complexes might be
distorted octahedral [29].
The EPR spectra of dinuclear copper(II) complexes 4–6 were recorded on X-band
frequency 9.4 GH under magnetic field strength 3200 G at room temperature (Fig.S3 & S4).
Z
The g values were evaluated using the relationship hν = gβH. A broad band centered at
g = 2.14–2.15 attributed to the antiferromagnetic interaction between two copper nuclei arises
from the spin-spin coupling of the electrons of both the copper ions [30]. The observed room
temperature magnetic moment values for complexes 4–6 range between 1.52–1.54 BM, lower
than the spin only value. This clearly indicates the occurrence of an antiferromagnetic
exchange interaction between the two copper(II) ions. Magneto structural correlation in
phenoxo-bridged dicopper(II) complexes reveal that the dominant pathway for
superexchange interaction through the oxygen atom involves interaction of the two copper
d
2
2
orbitals and the p orbitals on the oxygen [31].
x –y
3
.2. Electrochemical studies
The molar conductance values for complexes 1–6 in DMF are in the range
2
–1
1
30–170 Λ /S cm mol indicative of 1:2 electrolyte type [32]. The electrochemical
m
–
3
properties of complexes were studied by cyclic voltammetry in DMF containing 10
M
1
7

tetra(n-butyl)ammonium perchlorate as supporting electrolyte. This technique is based on
varying an applied potential at a working electrode in both forward and reverse directions (at
some scan rates) while monitoring the current. The electrochemical properties of the
complexes depend on number of factors such as chelate ring/size, axial ligation, degree and
distribution of unsaturation and substitution pattern in the chelate ring [33].
3
.2.1. Reduction process at cathodic potential
The cyclic voltammograms of complexes 1-6 at cathodic potential was recorded in the
potential range from 0 to –2.0 V (Fig S5). The electrochemical data are summarized in
1
Table S2. The first reduction potential (E ) ranges from 0.62 to 0.76 V and the second
pc
2
reduction potential (E ) lies in the range of –1.21 to –1.31 V. These two reduction waves
pc
II
I
correspond to stepwise one-electron reductions through a M M intermediate to give
I
I
binuclear M M species. The reason for the observed two reduction waves may be due to
electronic exchange between the metal ions; after the first one-electron reduction, some of the
electron density is transferred from the reduced metal ion to the other metal ion, and hence
the second reduction appears at a high negative potential. The two reduction processes are
assigned as follows:
II II
II
I
I
I
M M → M M → M M
3
.2.2. Oxidation process at anodic potential
All the nickel(II) complexes 1–3 show two oxidation processes in the range of 1.08 to
1
.74 V. The cyclic voltammogram of the dinuclear nickel(II) complexes is shown in Fig. S6
1
and the data are summarized in Table S2. The first oxidation potential (E ) ranges from
pa
2
1
.08 to 1.14 V while the second oxidation potential (E ) lies in range of 1.71 to 1.74 V. The
pa
two irreversible oxidation waves correspond to each one-electron transfer process for
complexes. Controlled potential electrolysis experiment indicates that the two oxidation
peaks are associated with stepwise oxidation process at nickel(II) center.
II
II
III
II
III
III
Ni Ni → Ni Ni → Ni Ni
1
8

3
3
.3. Biological studies
.3.1. In vitro antibacterial screening
The in vitro antibacterial screening of complexes 1–6 were performed at fixed
concentration of 150 µg/mL and the results obtained were compared with standard antibiotic,
ciprofloxacin. The mean zone of inhibition values of the investigated complexes are shown in
Fig. S7 & S8 and the data are summarized in Table 1. The antibacterial activities of
complexes follow the order 6 > 5 > 4 and 3 > 2 > 1 for copper (II) and nickel(II) complexes,
respectively. Among these, complex 6 shows good antibacterial activity and 1 shows lowest
activity against tested microorganisms, when compared with the standard drug. Complexes
containing dinitro substituents (3 & 6) in the pendant-arm show maximum antibacterial effect
while minimum activity was exhibited by the complexes without substituents (1 & 4) in the
pendant-arm. The reason for high antibacterial activity of copper(II) complexes can be
explained in terms of the effect of copper(II) metal ion on the normal cell process. The
complexation reaction reduces the polarity of the metal ion by partial sharing of metal ion
positive charge with donor atoms present in the pendant-armed macrocycles and there may be
ꢁ
electron delocalization over the whole chelate ring. This increases the lipophilic character
of the metal chelate and favours its permeation through the lipid layer of the bacterial cell
membranes. These complexes also disturb the respiration process of the cell and thus block
the synthesis of proteins that restricts further growth of the organism and as a result
microorganisms die [34].
It is also assumed that the increase in antibacterial activity is due to the factors such as
dipole moment, conductivity, solubility and cell permeability mechanism influenced by the
presence of metal ion [35]. The antibacterial activity of complexes 1-6 are comparable to
series of N-benzoylated tetraaza macrocyclic complexes reported in the literature [18].
1
9

3
3
.3.2. DNA binding studies
.3.2.1. Absorption spectroscopic studies
DNA being one of the most important targets of many anticancer agents, designing
and developing metal complexes that avidly bind to DNA are of paramount importance [36].
Electronic absorption spectroscopy is an effective method to examine the binding mode and
strength of metal complexes with DNA. In general, hypochromism and/or bathochromism or
hypsochromism are associated with the binding of metal complexes to the DNA helix, due to
the intercalative mode involving a strong stacking interaction between the aromatic
chromophore of complexes and the base pairs of DNA. The extent of hypochromism is
generally consistent with the strength of the intercalative interaction [37]. In the present
investigation, the interaction of the pendant-armed binuclear nickel(II) (2 & 3) and copper(II)
(5 & 6) complexes with CT-DNA in Tris-HCl/NaCl buffer was performed by monitoring the
fixed complex concentration (10 µM, in 5% DMF) to which increments of DNA stock
solutions (from 0 to 100 µM) was added. The absorption spectra of complexes in the absence
and presence of CT-DNA at different concentrations are given in Fig. 4 & 5. With increasing
concentration of duplex DNA to complexes, the absorption gradually decreased along with a
small amount of red-shift, indicating the interaction of aromatic N-containing pendant
macrocycles with CT-DNA. The intrinsic binding constant K for each complex has been
b
obtained from the plot of [DNA]/(ε – ε ) versus [DNA] (Table 2). The binding constants
a
f
follow the order 6 > 5 > 3 > 2 and indicate intercalation between complexes and CT-DNA.
The high binding nature of the metal complexes may be due to the additional π–π* interaction
through the aromatic phenyl rings. Upon intercalation, the π* orbital of the intercalated ligand
may couple with the π orbital of the base pairs of DNA, thus decreasing the π–π* transition
energy leading to bathochromism. On the other hand, the coupling π orbital is partially filled
by electrons, which decrease the transition probabilities and concomitantly leads to
2
0

hypochromism. The complex 6 interacts with DNA to the greatest degree, with binding
2
–1
constant K of 8.5×10 M . The higher DNA binding ability of copper complexes, in
b
particular, can be assigned to its biocompatibility, high nucleobase affinity, and capacity to
possess biologically accessible redox potentials. It can recognize nucleic acids, in a sequence-
specific fashion and bind to them in such a way that their functioning will be altered [38].
These values are very distinctive of metal-based compounds that bind to DNA via
intercalation. The DNA-binding constant of the title complexes are comparable to those of
reported pendant-armed macrocyclic binuclear metal(II) complexes [39, 40].
3
.3.2.2. Ethidium bromide quenching assay
Fluorescence quenching is a useful method to monitor the molecular interactions of
chemical and biological systems because of its high sensitivity. In the present study, we have
investigated the competitive binding of ethidium bromide versus the synthesized
pendant-armed complexes (2, 3, 5 & 6) with calf thymus DNA using fluorescence
spectroscopy to get better insight of DNA binding events. Ethidium bromide (EB) is one of
the most sensitive fluorescence probes that can bind with DNA. The fluorescence of EB
increases in the presence of DNA due to its strong intercalation between the DNA base pairs.
If the complex displaces EB from the DNA-bound EB, the fluorescence intensity decreases
due to the fact that the free EB molecules are much less fluorescent than the DNA bound EB
molecules because the surrounding water molecules quench the fluorescence of free EB [41,
4
2]. The emission spectra of EB bound to CT-DNA in the absence and presence of each
complex have been recorded for [EB] = 10 µM, [complex] = 0–100 µM and the DNA
concentration of 10 µM. The representative emission spectra of complexes 2, 3, 5 & 6 upon
excitation at different wavelength are given in Fig. 6 & 7. In the present work, the
fluorescence intensity of the DNA-bound EB decreases slowly on increasing the
concentration of the pendant-armed nickel(II) and copper(II) complexes. This may be due to
2
1

the displacement of the EB molecule from the intercalating site by the pendant-armed
complexes. The results reveal that all complexes bind with DNA by intercalation mode.
The quenching of EB bound to DNA by the pendant-armed complexes is in good
agreement with the linear Stern-Volmer equation which confirms that complexes are bound
to DNA. The ratio of the slope to the intercept obtained by plotting I /I versus [Complex]
o
yielded the value of K (Table 2). The fluorescence quenching efficiency follows the order
sv
6
> 5 > 3 > 2 indicating a higher quenching by copper(II) complexes as compared to
nickel(II) complexes. These values suggest that complex 6 showed higher quenching
efficiency than the other complexes 2, 3 and 5. The ratio of quenching of the intensities in all
four complexes is different, reflecting more binding of complex 6 with CT-DNA to leach out
more number of EB molecules originally bound to DNA than that of complexes 2, 3 and 5.
The high Ksv values of complexes reveal the intercalative binding of complexes with
CT-DNA. The hydrophobic property of these complexes containing 4-methylphenol group
facilitates the DNA binding. The enhancement of emission intensity is indicative of binding
of these complexes to the hydrophobic pocket of DNA, since the hydrophobic environment
inside the DNA helix reduces the accessibility of water molecules to complex and the
mobility of complexes is restricted at the binding site, leading to decrease of vibrational
modes of relaxation. These results are in agreement with the results obtained from UV-Vis
spectra.
3
.3.2.3. Electrochemical titration
The application of cyclic voltammetry provides useful compliment to the previously
utilized methods of investigation such as UV-Vis and fluorescence quenching experiments to
study interaction between complexes and DNA. It is a very sensitive analytical technique to
determine changes in redox behavior of metallic species in the presence of biologically
important molecules and thus the nature and mode of DNA binding of metal complexes. In
2
2

general, when the 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
1
2
p
shift of E and E , respectively, the molecule can bind to DNA by both intercalation and
p
electrostatic interaction [43].
The typical cyclic voltammograms of complexes 2, 3, 5 and 6 in the absence and
presence of CT-DNA are shown in Fig. 8 and the data are summarized in Table 3. No new
reduction waves appeared on the incremental addition of CT-DNA to complex, but the
cathodic peak current of complexes decreases, suggesting that CT-DNA moiety is bound
strongly to complex. The observed decrease in current intensity is attributed to the diffusion
of an equilibrium mixture of free and DNA-bound complex to the electrode surface and
suggests the existence of the same electrochemical behaviour upon addition of CT-DNA. The
slower mass transfer of complex bound to DNA fragments leads to a decrease in
concentration of the unbound redox active species in solution. The considerable decrease in
1
2
voltammetric current (ipc = 0.70 to 1.50 µA; ipc = 0.80 to 1.45 µA) and positive shift
1
2
(
Epc = –0.69 to –0.75 V; Epc = –1.20 to –1.26 V) suggests the existence of intercalation
mode of binding between complex and nitrogenous bases of DNA. However, according to
Kelly and coworkers [44], the drop of peak currents is due to the reason that the complex
bound to DNA and the product was nonelectroactive, decreasing the concentration of
electroactive species in solution and resulting in the drop of the peak currents. These positive
shifts are considered to be evidenced for intercalation of complexes with DNA due to
hydrophobic interactions.
3
.3.3. DNA cleavage studies
.
There are number of agents which exert their effect by inhibiting enzymes that act
upon DNA. These inhibitions result from the binding of such agents to the enzyme site of
2
3

interaction on the DNA rather than to direct enzyme inactivation. Transition metals have been
reported to inhibit DNA repair enzymes. The DNA cleavage efficiency of complex is
attributed to the different binding affinity of complex to DNA. There has been considerable
interest in DNA cleavage reactions activated by transition metal complexes. The delivery of
metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient
cleavage reaction [45]. Gel electrophoresis is a technique based on the migration of DNA
under the influence of an electric potential. When the original supercoiled form (Form I) of
plasmid DNA is nicked, an open circular relaxed form (Form II) will exist in the system and
the linear form (Form III) can be found upon further cleavage. When circular plasmid DNA is
run on horizontal gel by electrophoresis, the compact Form I migrates relatively faster while
the nicked Form II migrates slowly, and the linearised Form III migrates between Forms I
and II.
The DNA cleavage ability of complexes 1–6 was monitored by agarose gel
electrophoresis on plasmid pBR322 DNA as a substrate in a medium of
5
mM Tris-HCl/50 mM NaCl buffer (pH 7.2) under aerobic conditions with H O as an
2 2
oxidant. Incubation of pBR322 DNA with complexes for 2 h at 37 ºC results in extensive
cleavage of DNA, the supercoiled Form I is first degraded to Form II (relaxed circular) and
then to Form III (linear) in the presence of 100 µM of complex. The loaded pendant-armed
complexes damage DNA more efficiently in the presence of an oxidant and form DNA
adducts that migrate at different rates (Fig. 9 & S9). This may be attributed to the formation
of hydroxyl free radicals which participate in the oxidation of the deoxyribose moiety,
followed by hydrolytic cleavage of a sugar phosphate back bone. All complexes showed
pronounced cleavage activity in the presence of H O which may be due to the increased
2
2
production of hydroxyl radicals. At the same time, the cleavage was diminished significantly
in the presence of free-radical scavenger (DMSO), implying that hydroxyl radicals mediate
2
4

the cleavage. This provides clear evidence for the involvement of hydroxyl radicals in the
DNA strand scission performed by M(II) complexes. These results indicate that the metal
ions play an important role in the cleavage of isolated DNA. As the compounds were
observed to cleave DNA, it can be concluded that these compounds inhibit the growth of
pathogenic organism by cleaving the genome.
3
3
.3.4. Cytotoxicity evaluation
.3.4.1. MTT assay
The cytotoxicity of complexes 2, 5 and 6 against human liver adenocarcinoma
(HepG2) cell line has been investigated by using MTT reduction assay. The assay was based
on the mitochondrial reduction of the tetrazolium salt by actively growing cells to produce
blue water insoluble formazan crystals, i.e., only live cells reduce yellow MTT to blue
formazan products but not dead cells. The observed IC values (Table 2) of the complexes 2,
5
0
5
and 6 at 24 h are lower than those at 48 h indicating that they are dose and time dependent
(
Fig. 10). Further, as revealed by the observed IC values, the potency of the complexes to
5
0
kill the cancer cells follows the order 6 > 5 > 2, revealing that it varies with the mode and
extent of interaction of the complexes with DNA. Interestingly, the complex 6 is more potent
than the complexes 2 and 5. The complexes producing a higher mortality with lower
concentration at short duration of time compared to others with higher concentration and
longer time duration. It is commonly believed that the biological activities of anticancer metal
complexes are dependent on their ability to bind DNA and damage its structure resulting in
the impairment of its function, [46] which is followed by inhibition of replication and
transcription processes, and eventually cell death, if the DNA lesion are not properly
repaired.
2
5

3
.3.4.2. Cytomorphology observation
Microscopic observations were monitored using Nikon light inverted microscope
wherein treated cells showed distinct cellular morphological changes indicating unhealthy
cells, whereas the control appeared normal (Fig. 11). Control cells were irregular confluent
aggregates with rounded and polygonal cells. Synthesized pendant-armed treated cells
appeared to shrink, became spherical in shape and cell spreading patterns were restricted
when compared to control.
3
.3.4.3. Apoptotic activity
Apoptosis (programmed cell death) and necrosis (accidental cell death) are two types
of cell death. Necrotic cells undergo cell lysis and lose their membrane integrity and induce
severe inflammation. Apoptosis plays a major role during the development and homeostasis.
Apoptosis is a process of gene-mediated programmed cell death essential for the elimination
of unwanted cells in various biological systems and no inflammatory response is found.
Harmless removal of cells (says, cancer cells) is one consideration in chemotherapy.
Therefore, apoptosis is one of the considerations in the development of anticancer drugs. The
sequence of changes in cellular morphology of apoptosis includes oligo-nucleosomal DNA
fragmentation, nucleus condensation, DNA leddering and PARP-1 cleavage [47–49].
Apoptosis studies of complexes 2, 5 and 6 at IC50 concentration was performed with a
staining method utilizing propidium iodide (PI) which helps to detect difference in membrane
integrity between necrotic and apoptotic cells (Fig. 12). With the difference in concentration
gradient of complexes, the increase in the apoptotic nuclear morphology such as extensive
chromatin aggregation or nuclear condensation was observed in the treated cells, and even
nuclear fragmentation was found, indicating that the HepG2 cell died through an apoptosis
process. The cytological changes observed are classified into four types according to the
fluorescence emission and morphological features of chromatin condensation in the PI
2
6

stained nuclei: (i) viable cells have uniformly green fluorescing nuclei with highly organized
structure; (ii) early apoptotic cells (which still have intact membranes but have started
undergoing DNA fragmentation) have green fluorescing nuclei; but peri-nuclear chromatin
condensation is visible as bright green patches or fragments; (iii) late apoptotic cells have
orange red fluorescing nuclei with condensed or fragmented chromatin; and (iv) necrotic
cells, swollen to large sizes, have uniformly orange to red fluorescing nuclei with no
indication of chromatin fragmentation [50]. The morphological changes observed for 6
suggest that the cells are committed to apoptotic cell death more efficiently compared to
complexes 2 and 5.
The mechanism of cell death in the HepG2 liver cancer cell line appears to be
apoptosis, as evidenced by PI staining, which identifies early and late apoptotic changes in
cells.
3
.3.4.4. Comet assay
The single-cell gel electrophoresis (comet assay) allows detection of DNA
fragmentation in single cells, and was initially used for DNA damage estimation [51]. Fig. 13
showed the effect of pendant-armed complexes 2, 5 and 6 on HepG2 cells on DNA damage
and tail length. The comet tail length was analyzed by CASP software (Fig. S10). As can be
seen in the representative photos from fluorescence microscopy, 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
single-strand breaks. Hence, the average comet score of tail DNA for control HepG2 cells
was 0.10%. In contrast, other representative photos (Fig. 13 b, c & d) for 24 h at IC values
5
0
show significant numbers of nucleoids with larger comet tails, indicative of higher levels of
DNA single-strand breaks. The comet assay scores of tail DNA were 65.14%, 75.56% and
8
8.48% for complexes 2, 5 and 6, respectively. Therefore, Pendant-armed complexes
2
7

significantly increased number of tail DNA, tail length, tail moment and olive tail moment in
HepG2 liver adenocarcinoma cell line when compared to untreated cells. The results showed
that percentage (Table 2) of cells with tail DNA increased significantly after the cells were
treated with complexes 2, 5 and 6. The high level of DNA damage induced by complex 6
reinforces the results obtained using MTT assay and PI staining assay.
Conclusion
A series of pendant-armed polyamine macrocyclic dinuclear nickel(II) and copper(II)
complexes 1–6 have been synthesized by template cyclocondensation of 2,6-diformyl-4-
methylphenol with three different benzoyl pendant-arms, 2,2‟-benzoyliminodi(ethylamine)
trihydrochloride (L), 2,2‟-4-nitrobenzoyliminodi(ethylamine) trihydrochloride (L‟) and
2
,2‟-3,5-dinitrobenzoyliminodi(ethylamine) trihydrochloride (L”). The in vitro antibacterial
tests revealed that all complexes possess potent antibacterial activities toward Gram-positive
and Gram-negative bacteria. The order of increasing antibacterial activities was 6 > 5 > 4 > 3
>
2 > 1. The results of DNA binding studies suggest that the complexes bound with CT-DNA
through intercalation mode. The binding constants K and K follows the same order of
b
sv
binding affinity towards CT-DNA as 6 > 5 > 3 > 2. The agarose gel electrophoresis studies
show that complexes can promote the oxidative cleavage of plasmid DNA at physiological
pH and temperature in the presence of H O . The higher DNA cleavage efficiency of the
2
2
copper(II) complexes may be considered due to the increased binding ability of complexes to
DNA. In cytotoxicity research, complexes 2, 5 and 6 showed high in vitro cytotoxic
properties against human liver adenocarcinoma cell (HepG2). From the observed IC values,
5
0
the potency of complexes to kill the cancer cells follows the order 6 > 5 > 2. These
complexes bring about apoptosis of the cancerous cell line in a dose and time dependent
manner. Alkaline single-cell electrophoresis (comet assay) shows that these pendant-armed
complexes indeed induce DNA fragmentation, which is a further evidence of apoptosis. The
2
8

results obtained from the present work would be helpful to design and develop new pendant-
armed macrocycles as potent therapeutic and antitumor agents for some major diseases.
Acknowledgements
The authors gratefully acknowledge Dr. N. Sengottuvelan, Department of Chemistry,
Alagappa University, Karaikudi–630 004, India for his fruitful discussions on DNA
interaction studies.
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