Hexanuclear Zn(II) and Mononuclear Cu(II) Complexes containing imino phenol ligands: Exploitation of their Catalytic and Biological Perspectives

Article abstract of DOI:10.1002/aoc.5011

A unique hexanuclear zinc(II) (1) and two mononuclear copper(II) (2 and 3) complexes anchored with imino phenol ligand HL¹ and HL² were synthesized with good yield and purity (where HL¹?=?4-tert-butyl-2,6-bis((mesitylimino)methylphenol and HL²?=?5-tert-butyl-2-hydroxy-3-((mesitylimino)methyl)benzaldehyde). These complexes were characterized by utilizing various spectroscopic protocols like NMR, FTIR, UV as well as ESI-Mass spectrometry, elemental analysis and single crystal X-ray diffraction studies. Their potential to bind calf thymus DNA (CT-DNA) was tested utilizing different techniques such as UV–visible and fluorescence spectroscopy. The experiment implies that they interact with CT-DNA via non-intercalative mode with moderate capabilities (K_b ~ 10⁴?M^?1). On the other hand, these complexes have high capabilities to quench the fluorescence of bovine serum albumin (BSA) following the static pathway. In addition, they are active catalysts for the oxidation reaction of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) under aerobic condition. From the recorded EPR signals of all complexes, it has been concluded that the oxidation reaction proceeds via ligand oriented radical pathway instead of metal based redox participation. Kinetic studies using 1–3 indicate that it follows Michaelis–Menten type of equation with moderate to high turnover number (k_cat). Apart from these aspects, complexes 1–3 were screened for their cytotoxic behavior towards HeLa cells (human cervical carcinoma) and found quite active with comparable IC₅₀ values to cisplatin.

Full text of DOI:10.1002/aoc.5011

Received: 2 January 2019
DOI: 10.1002/aoc.5011
Revised: 5 March 2019
Accepted: 22 April 2019
F U L L P A P E R
Hexanuclear Zn(II) and Mononuclear Cu(II) Complexes
containing imino phenol ligands: Exploitation of their
Catalytic and Biological Perspectives
Shyam Sundar Mondal1 | Manosree Chatterjee2 | Ranjay K. Tiwari3,4 | J.N. Behera3,4
Nripen Chanda2 | Sourav Biswas1 | Tanmoy Kumar Saha1
|
1 Department of Chemistry, National
A unique hexanuclear zinc(II) (1) and two mononuclear copper(II) (2 and 3)
complexes anchored with imino phenol ligand HL1 and HL2 were synthesized
with good yield and purity (where HL1 = 4‐tert‐butyl‐2,6‐bis((mesitylimino)
methylphenol and HL2 = 5‐tert‐butyl‐2‐hydroxy‐3‐((mesitylimino)methyl)
benzaldehyde). These complexes were characterized by utilizing various spec-
troscopic protocols like NMR, FTIR, UV as well as ESI‐Mass spectrometry, ele-
mental analysis and single crystal X‐ray diffraction studies. Their potential to
bind calf thymus DNA (CT‐DNA) was tested utilizing different techniques such
as UV–visible and fluorescence spectroscopy. The experiment implies that they
interact with CT‐DNA via non‐intercalative mode with moderate capabilities
(Kb ~ 104 M−1). On the other hand, these complexes have high capabilities to
quench the fluorescence of bovine serum albumin (BSA) following the static
pathway. In addition, they are active catalysts for the oxidation reaction of
3,5‐di‐tert‐butylcatechol (3,5‐DTBC) to 3,5‐di‐tert‐butylquinone (3,5‐DTBQ)
under aerobic condition. From the recorded EPR signals of all complexes, it
has been concluded that the oxidation reaction proceeds via ligand oriented
radical pathway instead of metal based redox participation. Kinetic studies
using 1–3 indicate that it follows Michaelis–Menten type of equation with
moderate to high turnover number (kcat). Apart from these aspects, complexes
1–3 were screened for their cytotoxic behavior towards HeLa cells (human cer-
vical carcinoma) and found quite active with comparable IC50 values to
cisplatin.
Institute of Technology Durgapur, West
Bengal, India, 713209
2 Department of Materials Processing and
Microsystems Laboratory, CSIR‐Central
Mechanical Engineering Research
Institute, West Bengal, India, 713209
3 Department of Chemistry, National
Institute of Science Education and
Research, Bhubaneswar, Odisha, India,
752050
4 Homi Bhabha National Institute,
Mumbai, India, 400094
Correspondence
Tanmoy Kumar Saha, Department of
Chemistry, National Institute of
Technology Durgapur. West Bengal,
India‐713209.
Email: chem.tanmoy@gmail.com
Funding information
Science and Engineering Research Board,
Grant/Award Number: ECR/2016/000681
KEYWORDS
catecholase activity, cytotoxic activity, DNA and protein binding, Hexanuclear zinc (II) complex,
mononuclear copper (II) complex
1 | INTRODUCTION
era, great blooming has been observed on the develop-
ment of Schiff base ligands containing diversified organic
Schiff base ligand is one of the most promising candidates
in coordination chemistry with different denticities in
terms of complex formation with metal ions. In the recent
scaffolds. The interest has been manifested due to their
ease of preparation and formation of stable coordination
complexes with most of the transition metals. In these
Appl Organometal Chem. 2019;e5011.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.5011

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MONDAL ET AL.
complexes, variable coordination number, oxidation
states of metal ion, type of the attached ligand, kinetic
and thermodynamic prospects do offer the active impetus
to exploit a wide range of pharmaceutical and biological
domains.[1–11] They also have enormous potential to
create versatile platforms for rational strategies of drug
design.
After iron, zinc is a vital trace element for the proper
functioning of various biological processes[28] and is
engrossed in the prodigious number of enzymes in mam-
malians. In the current era, Zn (II) complexes have
grabbed tremendous attention due to their multidimen-
sional aspects in many research areas.[29–36] Current
investigations have depicted that these compounds are
proven to be active in medicinal therapeutic uses,[37,38]
in particular for the treatment of cancer with low
in vivo toxicity and reduced side effects.[29,39–46]
Now, the phenolic Schiff base ligands with nitrogen
and oxygen atoms as donor centers have flexible coordi-
nating characters along with hard and relatively softer
sites. Nonetheless, copper and zinc ions have remarkable
affinity to bind with these types of Schiff base ligands
through chelation.[47–49] In past decades, transition metal
complexes comprising of phenolic moieties in the ligand
skeleton were reported owing to their appreciable bind-
ing and cleaving abilities towards DNA.[3,29,50–52]
All these above facts encouraged us to investigate the
catalytic and biological activities of a unique hexanuclear
Zn(II) and two mononuclear Cu(II) complexes having
imino phenolate ligand. Copper and zinc were selected
for coordination due to their biocompatibility, effective-
ness in the bio‐medical field and low toxicity compared
to the conventional Pt based drugs. Therefore, we report
the synthesis, structural characterization of hexanuclear
zinc(II) and two mononuclear copper(II) complexes
consisting of tert‐butyl((mesitylimino)methyl)phenol
ligand units (HL1–2) (Scheme 1). The potential of these
complexes was investigated towards the DNA binding,
protein binding, catecholase and cytotoxic activities.
Moreover, this is the first example in the literature of
such hexanuclear Zn(II) complex as per our knowledge
whose catecholase and cytotoxic activities were studied.
Cisplatin which is a most popular choice to treat can-
cer brings the metallodrugs in the frontline area against
this threat since its discovery.[12–14] Nevertheless, the
scope of Pt based drugs is still restricted owing to their
toxic side effects[15] and drug resistance phenomena.[16–
19] So, the researchers are keener to develop safe and effi-
cacious anticancer drugs based on endogenous metals
with superior pharmacological aspects. Now, the metal
complexes can target DNA either by the covalent mode
or non‐covalent interactions. In the covalent mode, a
nitrogen base of DNA residue (for example guanine N7)
binds to the complex by replacing the labile ligand pres-
ent in it.[9,20] In contrary, the non‐covalent interaction
involves intercalation, electrostatic effects and groove
binding. Intercalation associates with the π–π stacking
due to the partial insertion of aromatic rings present in
ligand moiety between twin helical strands of DNA.
Besides, the groove and electrostatic bindings associate
the involvement of a metal complex by accessing the
bases near to the edges of the DNA helix within the major
or minor groove. In this context, copper and zinc based
complexes have portrayed promising perspectives
amongst many bio‐compatible metals.
Copper is an extremely important microelement for
living organisms and plays a pivotal role in the proper
function of many metalloenzymes like cytochrome oxi-
dase, tyrosinase, dopamine β‐hydroxylase and superoxide
dismutase.[21] It is also noteworthy that copper (II) based
complexes have been widely exploited in DNA cleavage
due to the production of reactive oxygen species (ROS)
via the redox activity of copper ions. This enables it to
attack at the different sites of DNA leading to rupture of
double‐strand.[22] Due to the apparent permeability of
copper compounds through the membrane of a cancer
cell, it accumulates in the targeted tumor. In recent times,
a series of copper complexes are found active as potent
drugs towards the anticancer activity both in vitro and
in vivo.[9,10,22–27]
2 | EXPERIMENTAL
2.1 | General information and
instrumentation
All the reagents and chemicals were purchased from
commercial sources and used without further purifica-
tion. Solvents were dried and purified according to
SCHEME 1 tert‐Butyl((mesitylimino)
methyl)phenol ligand used for complex
formation

MONDAL ET AL.
3 of 17
standard procedure prior to use. 3,5‐di‐tert‐butylcatechol
(3,5‐DTBC), Ethidium bromide (EB), 3‐(4,5‐dimethyl-
thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT),
bovine serum albumin (BSA) and calf thymus DNA
(CT‐DNA) were obtained from Sigma. The Tris–HCl
buffer solution was prepared with double‐distilled water.
Elemental analysis (% CHN) was performed using a
Thermo Fisher Flash 2000 CHNS funded by DST‐FIST
grant, Govt. of India. Infrared (IR) spectra were recorded
using KBr pellet method in the range 4000–400 cm−1
with a Bruker Alpha FTIR spectrometer. Electronic spec-
tra were recorded using a Shimadzu UV‐1800 spectropho-
tometer in the range 200–800 nm. Fluorescence emission
spectra were measured using a Hitachi F‐2500 spectroflu-
orometer using a 1.0 cm quartz cell. The 1H and 13C NMR
spectra of the zinc (II) complex were collected on a
Bruker Avance 400 spectrometer in CDCl3 at room tem-
perature. EPR Spectra were recorded on a Magnettech
MS‐5000 by Freiberg Instruments, Germany.
(Ar–C), 129.16 (Ar–C), 129.36 (Ar–C), 129.84 (Ar–C),
134.95 (Ar–C), 135.09 (Ar–C), 139.18 (Ar–C), 141.74
(Ar–O), 166.30 (C=N), 173.20 (O–C (CH3)–O), 174.53
(O–C (CH3)–O), 181.16 (O–C (CH3)–O), 206.89 (C=O).
ESI m/z calculated for [M]+; C76H94N4O20Zn6: 1774.08
found 1774.45.
2.3.2 | Complex 2
To a stirred solution of HL1 (0.088 g, 0.2 mmol) in 10 ml
toluene was combined with a solution of Cu(OAc)2·H2O
(0.018 g, 0.1 mmol) in 5 ml toluene. Then the resultant
mixture was refluxed for 12 hr at 112 °C under N2 atm.
The resulting deep green color solution was kept for slow
evaporation. After few days, block shape crystals were
formed suitable for single crystal X‐ray diffraction. Yield:
0.078 g (83%). Anal. Calc. for C60H70CuN4O2: C, 76.44; H,
7.48; N, 5.94. Found: C, 76.64; H, 7.53; N, 6.02. IR (KBr
pellets, cm−1): 2963(m, νC‐H), 2911(w, νC‐H), 2856(w, νC‐
H), 1624(s, νC=N), 1615(s, νC=N), 1591(s, νC=C), 1193(s,
ν
phenolic‐C‐O). ESI m/z calculated for [M +
H]+;
2.2 | Synthesis of Schiff base ligand
C60H70N4O2Cu: 942.76 found 943.49.
The ligand HL1, 4‐tert‐butyl‐2,6‐bis((mesitylimino)
methyl)phenol, was prepared from 4‐tert‐butyl‐2,6‐
diformyl phenol and 2,4,6‐trimethyl aniline according to
reported literature method.[53]
2.3.3 | Complex 3
An ethanolic solution (7 ml) of 2, 4, 6‐trimethyl aniline
(0.135 g, 1 mmol) was added very slowly to an ethanolic
solution (11 ml) of 4‐tert‐butyl‐2,6‐diformyl phenol
(0.206 g, 1 mmol) and the mixture was refluxed for 1 hr.
Then, a methanolic solution (10 ml) of Cu(OAc)2·H2O
(0.090 g, 0.5 mmol) was added and stirred for additional
3–4 hr. The whole experiment was performed under N2
atm. The resulting solution was kept for slow evapora-
tion. Yield: 0.488 g (69%). Anal. Calc. for C42H48N2O4Cu:
C, 71.24; H, 6.79; N, 3.91; Found: C, 71.15; H, 6.89;
N, 3.67. IR (KBr pellets, cm−1): 2962(m, νC‐H), 2912(w,
2.3 | Synthesis of metal complexes
2.3.1 | Complex 1
To a stirred acetonitrile solution of HL1 (0.044 g,
0.1 mmol), ethanolic solution of Zn(OAc)2·2H2O
(0.066 g, 0.3 mmol) was added drop wise for a period of
15 mins. Then, the resulting solution was refluxed for
12 hr under N2 atmosphere. The resulting light yellow
color solution was kept at room temperature for crystalli-
zation. After one week, square shape light yellow crystals
were obtained. Yield: 0.150 g (85%). Anal. Calc. for
C76H94N4O20Zn6: C, 51.39; H, 5.44; N, 3.12. Found: C;
51.79; H; 5.60; N; 3.15. IR (KBr): 2960(m, νC‐H), 2916(w,
ν
C‐H), 2859(w, νC‐H), 1670(s, νC=O), 1618(s, νC=N),
1592(m, νC=C), 1196(m, νphenolic‐C‐O). ESI m/z calculated
for [M]+; C42H48N2O4Cu: 708.38 found 708.31.
ν
ν
ν
C‐H), 2862(w, νC‐H), 1613(s, νC=N), 1592(s, νC=C), 1536(s,
asym–COO), 1478(m, νC=C), 1437(s, νsym–COO), 1196(s,
phenolic‐C‐O). H NMR (CDCl3, 400 MHz): 1.27 (s, CO–
2.4 | X‐ray crystallography
1
Suitable single crystals of zinc (II) and copper (II) com-
plexes (1, 2 and 3) were carefully chosen under a polariz-
ing microscope and mounted at the tip of the thin glass
fiber using cyanoacrylate adhesive. Single crystal struc-
ture determination was carried out on a Bruker AXS
KAPPA‐APEX II diffractometer equipped with a normal
focus, 2.4 kW sealed‐tube X‐ray source (Mo‐Kα radiation,
λ = 0.71073 Å) operating at 50 kV and 30 mA and data
was collected at 100 K. SAINT program used for the
CH3, 6H), 1.34 (s, C (CH3)3, 18H), 2.11 (s, Ar–CH3,
12H), 2.14–2.16 (s, Ar–CH3, 24H), 2.17–2.30 (s, H3C–C(–
O–)2, 18H), 6.76 (s, Ar–H, 2H), 6.89–6.93 (s, Ar–H, 8H),
7.41 (s, Ar–H, 2H), 8.15 (s, CH=N, 2H), 8.38 (s, CH=N,
2H).13C NMR (CDCl3, 100 MHz): 18.30 (C (CH3)3),
20.73 (H3C–C(–O–)2), 20.79 (H3C–C(–O–)2), 23.20 (Ar–
CH3), 30.90 (Ar–CH3), 31.26 (C (CH3)3), 121.08 (Ar–C),
121.88 (Ar–C), 123.88 (Ar–C), 128.24 (Ar–C), 128.83

4 of 17
MONDAL ET AL.
integration and scaling of data. The corresponding struc-
tures were solved by the direct method using SHELXT‐
2014 and refined on F2 by a full‐matrix least‐squares tech-
nique using the SHELXL‐2014 program's package. An
empirical absorption correction based on symmetry
equivalent reflections was applied using SADABS.[54]
The graphic programs DIAMOND[55] and ORTEP[56]
were utilized to illustrate the structures. Non‐hydrogen
atoms were refined anisotropically. In the refinement,
hydrogens were treated as riding atoms using the
SHELXL default parameters. The hydrogen atoms were
situated in the Fourier map and were refined with bond
length restraints. In case of 2, the solvent molecule hav-
ing highest Q peak of electron density 1.62 situated near
to C17 (distance between C17 and Q peak is 3.308 Å)
was masked in olex2[57] software. The CCDC reference
numbers of complexes 1–3 are 1852303, 1852304 and
1852305 respectively.
2.5.2 | Ethidium bromide experiment
The ethidium bromide (EB) fluorescence experiment was
performed in Tris–HCl/NaCl buffer (5 mM Tris–HCl,
50 mM NaCl, pH = 7.2). The solutions of all complexes
were titrated into the prepared EB‐DNA solution
(9.75 × 10−5 M EB and 9.75 × 10−5 M CT‐DNA). Fluores-
cence quenching was monitored via recording the change
of fluorescence emission spectra at various complex con-
centrations (0–50 μM). The said samples were excited at
510 nm and emission was documented from 520 to
700 nm.
2.6 | BSA binding studies
A stock solution of BSA was made in phosphate buffer
solution (pH = 7.4) and it was preserved in the dark at
4 °C for 24 hr. The exact concentration of the stock solu-
tion was ascertained spectroscopically following
Bouguer–Lambert–Beer law. The preparation of stock
solutions of complexes was done in DMF. Fluorescence
quenching experiments were performed by keeping con-
stant concentration of BSA (10 μM) and increasing con-
centrations of the aforementioned complexes (0–50 μM)
at room temperature. After addition of the complex, the
system was equilibrated for 10 min prior to the recording
of spectra. These were measured at an excitation wave-
length of 296 nm and the emission spectra from 300 to
500 nm.
2.5 | DNA binding studies
2.5.1 | Absorption spectral titration
The binding interaction between all the complexes and
CT‐DNA was investigated by means of electronic absorp-
tion spectroscopy. At ambient temperature, the absorp-
tion band of CT‐DNA at 260 and 280 nm in 5 mM Tris–
HCl/50 mM NaCl buffer (pH = 7.2) solution produces a
ratio of 1.8–1.9, signifying that the DNA was sufficiently
free from proteins.[58] The stock solutions of CT‐DNA
were prepared in Tris‐ HCl/NaCl buffer and stored at
4 °C for less than 4 days. The concentration of DNA per
nucleotide phosphate was ascertained by using its absorp-
tion intensity at 260 nm taking the molar absorption coef-
2.7 | Cell culture
Human cervical carcinoma cell lines (HeLa) were
acquired from the National Centre for Cell Science
(NCCS), Pune, India. The cells were cultured in DMEM
medium supplemented with 100 units ml−1 penicillin,
10% fetal bovine serum (FBS), and 100 μg ml−1 strepto-
mycin. The cells were incubated maintaining a humidi-
fied atmosphere having 5% CO2 at 37 °C. For the
cytotoxic activities 3.5 mg of all the complexes were dis-
solved in 1 ml DMSO and then further diluted it in com-
plete cultural media. In all the experiments, the
percentage of DMSO was kept within 0.1–1%. DMSO
was non‐toxic to the cells upto 1% concentration.
ficient of 6600 M−1 cm−1 [59a]
The experiments were
.
executed by keeping the complex concentration (50 μM)
constant and varying CT‐DNA concentration (0–50 μM).
The complex‐DNA solutions were incubated for 10 min
at room temperature before the actual measurement. An
equal quantity of CT‐DNA solution was mixed to both
cuvettes for eliminating the absorbance of CT‐DNA itself.
The intrinsic binding constant Kb can be found out using
the following equation:
½DNAꢀ=ðεa − εf Þ ¼ ½DNAꢀ=ðεb − εf Þ þ 1=Kb ðεb − εf Þ (1)
Where εa, εf, and εb correspond to Aobs/[Complex], the
extinction coefficient for the free copper complex, and
the extinction coefficient for the copper complex in the
fully bound form respectively. A plot of [DNA]/ (εa − εf)
vs. [DNA] provides Kb as the ratio of the slope to the
intercept.
2.7.1 | MTT assay
Complex actuated cytotoxicity experiments were done by
using MTT assay method in 96‐well microplates. HeLa
cells were seeded into 96‐well plates at a density of
1.5
×
104 cells/well. Different concentrations of

MONDAL ET AL.
5 of 17
complexes (4.26–40.38 μg ml−1) were added to the cells
after overnight preincubation at 37 °C. Untreated cells
were taken as control. After 48 hr of incubation, a freshly
prepared MTT solution (5 mg mL−1) in PBS buffer was
mixed to each well and incubated again at 37 °C for
4 hr. Then, the concerned media were removed and the
formed formazan crystals were then dissolved in 50 μL
of DMSO. The absorbance was measured at 570 nm by
a multi‐well plate reader (Thermo Scientific Multiskan
FC). The experiment was performed in triplicate and
the percentage of cell viability was calculated using the
following equation:
EVOS FL (Life Technologies) inverted fluorescence
microscope. The blebbing of plasma membrane was
examined after 10 hr. Next, Hoechst 33342 (Sigma
Aldrich) staining was carried out once the prominent
deformation in cell membrane was observed. After the
treatment, the cells were washed with PBS buffer for
5 min at room temperature followed by fixation with 4%
paraformaldehyde for 15 min. Cells were washed thrice
with the same buffer followed by incubation with
Hoechst 33342 (1 μg/ml) for 15 min in dark. After the
staining, one time washing with PBS buffer was done
and the plates were allowed to dry for 2 h. Images were
obtained in fluorescent mode of EVOS FL (Life Technol-
ogies) inverted fluorescence microscope and the image
analysis was carried out in PhotoScape software.
Cell viability ð%Þ ¼ ½A570 ðsampleÞ=A570 ðcontrolÞꢀ × 100%:
Where A570 (sample) indicates the reading from the wells
after the treatment with metal complexes and A570 (con-
trol) corresponding to that from the wells with medium
containing 10% FBS only.[59b]
The sensitivity of the cancer cells upon addition of the
complexes was expressed in terms of IC50 value.
3 | RESULTS AND DISCUSSION
3.1 | Synthesis and characterization
Complex 1 and 2 have been synthesized by reacting the
symmetrical ligand, HL1 with Zn(OAc)2·2H2O or
Cu(OAc)2·H2O maintaining the corresponding molar
ratio in a suitable solvent (Scheme 2). However, 3 was
synthesized by the reaction of in situ obtained unsymmet-
rical ligand, HL2 with Cu(OAc)2·H2O in a 2:1 molar ratio
(Scheme 3). Interestingly, it is quite tricky to have com-
plex 3 due to the high chance of double condensed HL1
formation during the reaction instead of HL2. Careful
trapping of the formed HL2 as Cu(II) complex by the
2.7.2 | Analysis of cell death
HeLa cells were cultured on two 35 mm cell culture petri
dishes (1x105 cells/dish) until it reaches 50% confluency.
The culture media was then replaced with serum‐free
media and incubated for 2 hr to attain synchronised cell
cycle. Cells in one tissue culture dish were treated with
40.38 μg/ml of complex 1 and the other was maintained
as control. Images were acquired at 1 hr interval with
SCHEME 2 Synthesis of hexanuclear
Zn(II) (1) and mononuclear Cu(II) (2)
complexes comprising of L1 moieties

6 of 17
MONDAL ET AL.
SCHEME 3 Synthesis of mononuclear Cu(II) complex (3) containing L2 unit
addition (after 1 hr) of Cu(OAc)2·H2O to the resultant
solution of HL2 is the most suitable way to avoid the sec-
ond condensation. However, similar efforts have been
made to prepare a mono‐condensed Zn(II) compound
under the same reaction conditions. Surprisingly, we
were unable to isolate the desired compound as the yield
was very poor along with unidentified side products.
Complexes 1–3 have been characterized by using modern
spectroscopic techniques. Additionally, these compounds
are structurally ascertained by single crystal X‐ray diffrac-
tion studies.
was further confirmed by single crystal X‐ray diffraction
studies. On the other hand, a strong band near about
1670 cm−1 is present in case of 3 which is actually due
to the free –C=O group attached to ligand part.
The molecular ion peak in electrospray ionization
mass spectrometry (ESI–MS) of complexes 1–3 proves
the existence of hexanuclear (for 1) and mononuclear
(for 2 and 3) states for the metal ion in the corresponding
compounds (See Supporting Information†). Elemental
analysis results of 1–3 do exhibit considerable extent of
purity for these compounds.
To check the coordination behavior of the ligand in
the synthesized complexes, IR spectra are recorded and
compared with the free ligand. It has been observed that
complexes 1–3 depict bands in the range of 1618–
1613 cm−1 owing to the stretching of C=N of ligand moi-
ety. The C=N stretching comes at a lower frequency in
the complex in comparison with the free ligand
(νC=N = 1631 cm−1) due to the weakening of double bond
character of C=N. This attributes the formation of the
coordination bond to the metal ion through the nitrogen
atom of imine moiety. Additionally, all the complexes
show bands in the range of 2963–2856 cm−1 due to the
aliphatic C–H stretching of the tert‐butyl group. For com-
plex 1, the νasymmetric and νsymmetric –COO of the acetate
group appear at 1536 and 1437 cm−1 suggesting the bridg-
ing mode of acetate unit. Whereas, two sharp peaks
appear at 1624 and 1615 cm−1 in complex 2 due to the
non‐coordinated and coordinated –C=N moieties. This
3.2 | Single crystal X‐ray diffraction
studies
The crystal data and refinement parameters for 1, 2 and 3
have been summarized in Table 1. From single crystal X‐
ray diffraction studies, it is evident that complex 1
(Figure 1) is having a hexanuclear structure in the solid
state and crystallizes in triclinic system with P‐1 space
group. It consists of six Zn(II) ions and two ligand moie-
ties with several bridging and terminal acetate units.
More interestingly, four Zn(II) ions (namely Zn1 and
Zn2) in the complex are pentacoordinated with distorted
square pyramidal geometry and the other two Zn3 ions
are tetracoordinated with slightly distorted tetrahedron
ascertained from the measured bond angles and bond dis-
tances. The ligand HL1 acts as a tridentate ligand towards

MONDAL ET AL.
7 of 17
TABLE 1 Crystallographic data of complexes 1–3
Compound
1
2
3
Empirical formula
C84H106N8O20Zn6
1939.99
293(2)
C60 H70N4O2Cu
942.74
C42H48N2O4Cu
708.36
Formula weight
Temperature/K
100(2)
100(2)
Wavelength
0.71073 Å
Triclinic
P‐1
0.71073 Å
Monoclinic
C2/c
0.71073 Å
Monoclinic
P21/n
Crystal system
Space group
a/Å
11.4722(5)
15.0649(10)
15.1378 (8)
62.435(6)
89.314(4)
84.493(4)
2307.0(2)
1
19.3116(13)
11.3985(13)
25.440(2)
90
13.6798(5)
10.3619(4)
13.8376(5)
90
b/Å
c/Å
α (°)
β (°)
γ (°)
104.418(8)
90
107.312(2)
90
V/Ǻ3
5423.5(9)
4
1872.60(12)
2
Z
ρcalc (g cm−3
)
1.396
1.155
1.256
Absorption coefficient (mm−1
)
1.604
0.447
0.626
F (000)
1006
2012
750
Index ranges
–14 ≤ h ≤ 15
−12 ≤ k ≤ 19
−18 ≤ l ≤ 19
−24 ≤ h ≤ 24
−14 ≤ k ≤ 14
−31 ≤ l ≤ 28
−13 ≤ h ≤ 19
−14 ≤ k ≤ 14
−19 ≤ l ≤ 19
Reflections collected
12908
9911
23463
5562
25393
5712
Independent reflections
Goodness‐of‐fit on F 2
Final R indices [I > 2σ(I)]
1.054
1.177
1.049
R1 = 0.0455
R1 = 0.0668
R1 = 0.0362
wR2 = 0.1047
wR2 = 0.1464
wR2 = 0.1053
R Indices (all data)
R1 = 0.0633
R1 = 0.1095
R1 = 0.0456
wR2 = 0.1178
wR2 = 0.1667
wR2 = 0.0993
Zn(II) ions in which two imine N atoms are anchored
with two different zinc atoms rather than with one zinc
(for example, Zn(1) is appended to N(9) while Zn(2) is
bonded to N(7)). This type of coordination is most stable
from the thermodynamic point of view. In case of square
pyramidal geometry, Zn(1) and Zn(2) are connected
through an oxo bridge and other three coordination posi-
tions are occupied by ligand and acetate unit. The fifth
coordination position of Zn(1) is anchored by an oxygen
atom of a bridging acetate moiety. However, the fifth
coordination position of Zn(2) is fulfilled by a terminal
acetate group. On the other hand, most interestingly,
Zn(3) is attached to O atoms of acetate units to complete
its four coordinations to achieve the tetrahedron geome-
try. In 1, the phenyl rings containing 2,4,6‐trimethyls
are almost perpendicular to the phenyl ring having the
oxygen atom. All the methyl units from acetate moieties
are trans to each other.
The single crystal analysis of complex 2 shows that the
brown colored crystals crystallizes in monoclinic system
with an achiral C2/c space group. Unlike complex 1, com-
plex 2 has a mononuclear structure (Figure 2). The Cu(II)
ion presents at the inversion center and is coordinated
with the surrounding two oxygen and two nitrogen atoms
of L1 units having the distorted square planar geometry.
In this case, the ligand acts as a bidentate chelator rather
than tridentate. Actually, amongst two imine nitrogen
atoms, one is bonded to the metal ion and other remains
free. The coordination with both nitrogen atoms presum-
ably is not thermodynamically stable. The molecules
are arranged in AB‐AB type of stacking and are formed
by two units of HL1 ligands anchoring Cu(II) ions in
the opposite direction to reduce the steric hindrance.
Furthermore, complex 2 gets extra stability from the
intra C–H …. π interaction between C(21)H(21) and
benzene ring of trimethyl aniline unit (as well as C(19)

8 of 17
MONDAL ET AL.
H(19c) and benzene ring of trimethyl aniline) (Figure
S22a†). The C–H …. π interaction ranges from 2.678–
2.790 Å as marked with red color and shown in Figure
S22b†. The stability of these molecules is further
enhanced by the inter C‐H …. π interaction with a dis-
tance of 2.588 Å which are shown with green dotted
lines (Figure S22b†).
On the other hand, the centrosymmetric complex 3
crystallizes in monoclinic system with P21/n space group.
From Figure 3 it is clear that complex 3 has also a mono-
nuclear structure having one Cu (II) ion with two L2
ligand skeletons and has slightly distorted square planar
geometry. In terms of structural coordination, 3 is quite
resemble with 2. The unsymmetrical ligand acts as a true
bidentate unit since it lacks in other imine N atom. The
carbonyl oxygen atoms and two tert‐butyl groups in the
phenyl rings are oriented in the opposite direction. Two
tert‐butyl groups along with 2,4,6‐trimethyl imino units
reside out of the molecular plane. All the bond lengths
are in the expected limits. Like the complex 2, complex
3 is also stabilized by the intramolecular C–H …. π inter-
action between C(7)H(7) and benzene ring of trimethyl
aniline with a distance of 2.656 Å (marked with black
dotted lines in Figure S23†). In addition, the molecule is
further strengthened by the π–π stacking interaction with
a distance of 4.751 Å (displayed in yellow dotted lines in
Figure S23†).
FIGURE 1 The molecular structure of complex 1; thermal
ellipsoids were drawn with 30% probability level; the hydrogen
atoms and solvent molecules have been omitted for clarity; Selected
bond lengths () and bond angles (°): Zn(1)–O(2) 2.113(2), Zn(1)–
N(9) 2.051(3), Zn(1)–O(8) 2.020(2), Zn(2)–O(2) 2.074(2), Zn(2)–
N(7) 2.152(3), Zn(2)–O(11) 2.054(4), Zn(3)–O(1) 1.958(2), Zn(3)–
O(6) 1.997(3), Zn(3)–O(7) 1.982(3); N(9)–Zn(1)–O(2) 86.94(9),
O(4)–Zn(1)–O(2) 93.51(11), N(7)–Zn(2)–O(2) 84.78(9), N(7)–Zn(2)–
O(3) 102.72(11), O(1)–Zn(3)–O(6) 105.71(4), O(1)–Zn(3)–O(7)
107.61(11)
FIGURE 3 The molecular structure of complex 3; thermal
ellipsoids were drawn with 30% probability level and the
asymmetric units were labeled; Selected bond lengths () and bond
angles (°): Cu(1)–O(1)i 1.891(10), Cu(1)–O(1) 1.891(10), Cu(1)–N(1)i
1.998(11), Cu(1)–N(1) 1.998(11); O(1)–Cu(1)–O(1)i 180.00(6), O(1)i–
Cu(1)–N(1)i 92.47(4), N(1)–Cu(1)–N(1)i 180.00(6), O(1)–Cu(1)–
N(1)i 87.53(4), O(1)i–Cu(1)–N(1) 87.53(4), O(1)–Cu(1)–N(1)
92.47(4)
FIGURE 2 The molecular structure of complex 2; thermal
ellipsoids were drawn with 30% probability level and the
asymmetric units were labeled; Selected bond lengths () and bond
angles (°): Cu(1)–O(1) 1.905(2), Cu(1)–N(1) 1.974(3), Cu(1)–O(1)i
1.905(2), Cu(1)–N(1)i 1.974(3); O(1)–Cu(1)–O(1)i 165.19(16), O(1)–
Cu(1)–N(1) 93.05(11), O(1)i–Cu(1)–N(1)i 93.05(11), O(1)i–Cu(1)–
N(1) 88.93(11), O(1)–Cu(1)–N(1)i 88.93(11)

MONDAL ET AL.
9 of 17
3.3 | DNA binding ability
3.3.1 | Fluorescence titration
Electron absorption spectroscopy is a widespread and
common protocol in order to explore the binding interac-
tions between metal complex and DNA. The absorption
spectra for 1 in Tris–HCl/NaCl buffer is depicted in
Figure 4 while the absorption spectra for 2 and 3 are
shown in Figure S9–S10†. The absorption bands of these
complexes (1–3) in the range of 406–425 nm in the UV
spectra are assigned to the intraligand π → π* transition.
With a constant concentration of the concerned com-
plexes (50 μM) and increasing amounts of CT‐DNA con-
centration (0–50 μM), considerable hyperchromisms are
observed with a slight red shift. This observation attri-
butes that all the complexes interact significantly with
DNA presumably through non‐intercalative binding
mode via accessing multiple hydrogen bonding sites pres-
ent within both major and minor grooves[60–63] and the
electrostatic interaction between positively charged metal
ion and the negatively charged DNA phosphate skele-
ton.[64,65] The classical intercalation may be ruled out
due to the bulky structure of 1 (non‐planer as well) and
the significant non‐planarity issues (the phenyl rings con-
taining 2,4,6‐trimethyl groups in 2 & 3 are out of the
plane completely) in case of 2 & 3.
To scrutinize further the interacting mode of the afore-
mentioned complexes with DNA, fluorescence quenching
experiments were performed using ethidium bromide
(EB). The fluorescence intensity of EB is very strong in
the presence of DNA owing to the intercalation of EB into
the adjacent base pairs of DNA double helix.[66] However,
EB emits quite weak fluorescence in its free form. After
the addition of a complex, the intercalation between
DNA and the complex causes fluorescence quenching
by knocking out EB.[67] Nevertheless, the electrostatic
interaction may lead to a contraction of the double
stranded DNA through neutralizing the negative charges
of corresponding phosphate moieties present in the DNA
backbone. Consequently, it pushes EB out of the DNA
and governs the quenching of the said fluorescence.[68]
With the increasing concentration of complexes 1–3
(0–50 μM), the fluorescence intensity of EB‐DNA system
was reduced at a considerable rate (Figure 5 for 1 and
Figure S11 − S12† for 2 & 3). It illustrates that 1–3 could
bind to CT‐DNA via the substitution of EB in the EB‐
DNA mixture.
The apparent binding constants (Kapp) of 1–3 were
calculated utilizing the equation 2.[69]
The intrinsic binding constants (Kb) of the complexes
are calculated using the equation 1 (see experimental
part).[9] The binding affinity of the complexes towards
KEB EB ¼ Kapp complex
ꢀ
½
ꢀ
½
(2)
CT‐DNA decreases in the order 1 (Kb = 4.82 × 104 M−1
)
Where the complex concentration was the value at 50%
reduction of the fluorescence intensity of EB and
KEB = 1.0 × 107 M−1, ([EB] = 9.75 × 10−5 M). The appar-
ent binding constants for 1–3 do follow the order 1
(Kapp = 4.4 × 105 M−1) > 2 (Kapp = 3.9 × 105 M−1) > 3
(Kapp = 3.2 × 105 M−1) which are in good agreement with
> 2 (Kb = 1.81 × 104 M−1) > 3 (Kb = 1.32 × 104 M−1).
Nonetheless, these values do imply that the binding capa-
bilities to the CT‐DNA are moderate and comparable with
the literature precedents.[9,63,65]
FIGURE 4 Absorption spectra of complex 1 (50 μM) with
increasing concentration of CT‐DNA (0–50 μM) at room
temperature in Tris–HCl/NaCl buffer (pH = 7.2). Inset: Plot of
[DNA]/(εa − εf) vs. [DNA] for the absorption titration of CT‐DNA
with 1
FIGURE 5 Fluorescence quenching spectra of EB bound to CT‐
DNA by 1 ([complex] = 0–50 μM, λex = 510 nm). The arrow
indicates the change in intensity upon increasing the complex
concentration. Inset: Plot of I0/I vs. [complex]

10 of 17
MONDAL ET AL.
the results found in electronic absorption titration exper-
iments. In addition, the kinetic parameters for DNA bind-
ing are very much comparable with the reported Zn(II)
and Cu(II) complexes (Table 2).
3.4 | Protein binding studies
Serum albumins do play a crucial role in the transporting
of amino acids, steroids, fatty acids, drugs and metal ions.
Due to structural resemblance with human serum albu-
min, bovine serum albumin (BSA) is one of the most
important serum albumin and examined comprehen-
sively. The intrinsic fluorescence of BSA is a predomi-
nantly outcome of tryptophan residues mainly Trp‐134
and Trp‐212. Now, fluorescence spectroscopy is an essen-
tial tool for examining the interacting power of metal
complexes to BSA. The effect of absence and presence of
complex 1 on the fluorescence intensity of BSA is pre-
sented in Figure 6 (similar types of spectra for 2 & 3 are
illustrated in Figure S13 − S14†).
The noteworthy reduction of intensity of the broad
emission spectrum upon the addition of the complex (0–
50 μM) to a constant concentration of BSA (10 μM) actually
points out the interaction of BSA with the complex. Per-
haps this interaction arises owing to the alteration in the
tryptophan residues environment. The quenching of fluo-
rescence may be ascribed using Stern‐Volmer equation.[69]
FIGURE 6 Fluorescence spectra of BSA with gradual increment
of concentration of complex 1 at room temperature.
[BSA] = 10 μM, and [complex 1] = 0–50 μM
prospects. Amongst three complexes, 1 exhibits much
higher binding capabilities towards BSA in comparison
with 2 or 3. However, the Kq values (> 1012 M−1 −1) are
s
greater in comparison to variously diversified quenchers
for biopolymers fluorescence (2 × 1010 M−1 s−1). It signifies
that the static quenching pathway is favoured rather than
the dynamic one.[70] Again, the UV–visible spectra of
BSA were recorded further prior to and after the addition
of the complexes to investigate the structural change
related to the peptide strands in BSA (Figure 7 and Figure
S15 − S16†). The intensity of the absorption maxima is sub-
stantially enhanced on employing of complexes (10 μM)
with slight red shift (2–3 nm). This observation again man-
ifests the formation of a ground state complex associated
with BSA and the aforesaid compounds.[71,72]
F0= F ¼ 1 þ KSV½Qꢀ ¼ 1 þ Kqτ0½Qꢀ
(3)
Where F 0 and F correspond to the fluorescence intensities
in the absence and presence of the concerned quencher, Kq
is the quenching rate constant, τ0 represents the average
lifetime of the biomolecule without the quencher
(~10−8 s),[70] KSV is the Stern‐Volmer quenching constant
and [Q] is the concentration of the quencher. KSV can be
found out from the slope of the plot of F0/F vs. [Q]. The
obtained values of KSV and Kq of complexes 1–3 are sum-
marized in Table 3.
In case of the static mode of quenching, the binding con-
stant (Kb) and the number of binding sites (n) per albumin
can be ascertained utilizing the Scatchard equation.[73]
logðI0–IÞ=I ¼ log Kb þ n log½Qꢀ
(4)
It is very clear from these values that all the complexes
are the potential quenchers of BSA in terms of binding
The Kb and n values are calculated from the intercept and
slope of the double logarithmic curve of log(I0 – I)/I vs.
TABLE 2 Comparison of kinetic parameters for 1–3 with reported Zn(II) and Cu(II) complexes
Complex
Kb (M−1
)
Kapp (M−1
)
Ref.
1
4.82 × 104
1.81 × 104
1.32 × 104
8.92 × 104
1.16 × 104
2.28 × 103
1.35 × 104
4.4 × 105
3.9 × 105
3.2 × 105
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2
3
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[92]
[Zn (Hqasesc)2]2+
Not Determined
1.1 × 105
[93]
[94]
[94]
[Zn2(L)]·(ClO4)2
[Cu(H2L1)Cl]·CH3OH
Not Determined
Not Determined
Cu4(HL2)2(H2L2)2(H2O)(C2H5OH)]·2(ClO4)·2(C2H5OH)

MONDAL ET AL.
11 of 17
TABLE 3 BSA binding constants of complexes 1–3 and comparison with reported Zn(II) and Cu(II) complexes
Complex
Ksv (M−1
)
Kq (M−1
)
Kb (M−1
)
n
Ref
1
5.95 × 104
2.99 × 104
2.4 × 104
9.59 × 1012
4.84 × 1012
2.9 × 104
3.4 × 103
0.93
0.76
0.52
0.89
0.48
1.03
0.87
This work
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2
3
3.87 × 1012
3.1 × 103
This work
[92]
[Zn (Hqasesc)2]2+
2.01 × 106
9.97 × 104
0. 47 × 105
1.36 × 105
2.82 × 1014
9.97 × 1012
Not Determined
1.19 × 102
9.16 × 103
6.91 × 105
[95]
[94]
[94]
Zn2(L1)2Cl2](ClO4)2·C2H5OH
[Cu(H2L1)Cl]·CH3OH
Not Determined
Not Determined
Cu4(HL2)2(H2L2)2(H2O)(C2H5OH)]·2(ClO4)·2(C2H5OH)
from the damage against pathogen and insects in higher
plants.[78]
Since dicopper(II) units are the centers of activity in
catechol oxidase, the preferred choice to several research
groups is dinuclear Cu(II) systems as catalysts for cate-
chol oxidation reaction.[79–82] In contrast with various
dinuclear Cu(II) systems, a few mononuclear Cu(II) com-
plexes[83–86] and Zn(II) based compounds bearing diversi-
fied ligands with N/O donors were reported in the
literature.[6,51,87–89] Thus, there is an excellent scope of
exploitation of such mononuclear Cu (II) units and
hexanuclear Zn (II) compound as catalysts towards the
conversion of 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) to 3,5‐
di‐tert‐butylquinone (3,5‐DTBQ).
In order to determine the catecholase activity of 1–3,
3,5‐DTBC was utilized as a model substrate. 3,5‐DTBC
is mainly used due to its low reduction potential
(easy oxidation to quinone) and the presence of the
bulky tert‐butyl units preventing further oxidation like
ring opening. However, 3,5‐DTBQ is enormously stable
and has a characteristic absorption peak at around
400 nm in acetonitrile (λmax~402 nm in methanol).
The catalytic activity of complexes 1–3 towards the oxi-
dation of 3,5‐DTBC to 3,5‐DTBQ was investigated in
acetonitrile (in case of 1) and methanol (in case of 2
and 3). In this regard, 1 × 10−4 M solutions of 1–3 were
mixed with 1 × 10−2 M (100 equivalents) of 3,5‐DTBC
under aerobic condition at ambient temperature.
The advancement of the reaction was monitored by
UV–vis spectroscopy as shown in the Figure 8 (for 1)
and Figure S17 − S18† (for 2 and 3) after each 8 minutes
intervals. Upon the addition of the substrate, the
appearance of a new band ~ 400 nm was observed
owing to the formation of 3,5‐DTBQ. The concentration
of the oxidized product was increased with time leading
to the bigger and taller absorption bands. All the said
complexes do behave in a similar fashion and demon-
strate a very smooth conversion of 3,5‐DTBC to 3,5‐
DTBQ. Hence, it proves the efficacy of these complexes
as catalysts for the oxidation of 3,5‐DTBC. Besides this,
the formation of the oxidized products was further
FIGURE 7 UV–Visible spectra of BSA in the absence complex 1
(Black line). The absorption of BSA in the presence of complex 1
(Red line). [BSA] = [complex 1] = 10 μM
log[Q] (Table 3). Table
3 demonstrates that the
hexanuclear Zn (II) complex (1) has a quite higher Kb value
for BSA than the two mononuclear Cu (II) complexes (2
and 3). Based on the Kb values, it points out that the
nuclearity may display a significant role in the protein
binding experiment since the ligand moiety is almost the
same. In addition, the n values are near about one which
reveals the involvement of a single binding site in BSA
for the concerned complexes.
3.5 | Catecholase activity
Catechol oxidase which is a candidate of the type‐III cop-
per proteins uses dioxygen molecule as an oxidant.[74]
This particular enzyme corresponding to the group of
polyphenol oxidases catalyzes the oxidation of several o‐
diphenols to their respective o‐quinones.[75–77] Quinones
are quite reactive molecules which can trigger auto poly-
merization to generate a brown pigment namely melanin.
Perhaps this process is accountable to prevent the tissues

12 of 17
MONDAL ET AL.
Where ν is the rate of the reaction and [S] is the substrate
concentration. The values of aforesaid kinetic parameters
for 1–3 are depicted in Table 4.
From the Table 4, it is clear that complex 1 displays
highest kcat (4.1 × 103 h−1) and Vmax (1.16 × 10−4 M s−1
)
values among all three complexes. So, these complexes
are active catalysts for this oxidation reaction considering
the all the kinetic parameters and the order of the said
activity is 1 > 2 > 3.
Generally, the conversion to quinone from catechol is
a two electron oxidation reaction. Now, it is rather crucial
to understand the probable cause behind the activity of
the complexes for this conversion. Interestingly, either
metal based redox participation or a ligand oriented free
radical route is accountable for that kind of activity.[90a]
To establish the actual cause, we have accomplished
EPR spectral studies of 1 (at 77 K), 2 & 3 (at room temp)
before and after mixing of 3,5‐DTBC with 10−3 M acetoni-
trile solution of concerned complexes in an inert atmo-
sphere. The EPR studies exhibit a sharp signal at g~2 in
case of 1 after mixing the catechol as shown in
Figure 10 which confirm the generation of free radical.
Although, during the experiment, we have found that 1
is an EPR inactive species (Since Zn+2 ion is a d10 sys-
tem). Whereas, the EPR spectra of 2 and 3 represent four
hyperfine peaks (g║ = 2.137 & g┴ = 2.039 for 2 and
g║ = 2.137 & g┴ = 2.035 for 3; A║ = 78.1 G for 2 &
A║ = 78.7 G for 3) in absence of 3,5‐DTBC (see
Figure 11 for 2 and Figure S21† for 3). Now, EPR spec-
troscopy can differentiate the associated ground states
FIGURE 8 UV–vis spectra illustrating the growth of quinone
band at ~ 400 nm after the addition of 100‐fold 3,5‐DTBC to a
solution containing complex 1
confirmed by gas chromatography techniques (Figure
S27–S29).
The kinetic prospects of this oxidation reaction using
1–3 were ascertained through the initial rate method by
analyzing the increment of the quinone band with time.
In this regard, at low concentrations of 3,5‐DTBC, the
rate has first order dependence for all three complexes.
However, saturation kinetics are observed at higher
concentrations of 3,5‐DTBC. Based on the famous
Michaelis–Menten equation of enzyme kinetics, the max-
imum velocity (Vmax), binding constant (KM) and turn-
over number (kcat) were found out from Lineweaver‐
Burk plot of 1/[rate] vs. 1/[S] (Figure 9 for 1 and Figure
S19–20 for 2 & 3†) using the following equation.
2
2
2
(dx –y and dz ) using the g tensor values of the aniso-
2
2
tropic spectra. dx –y will be the ground state when the
complex has elongated octahedral, square pyramidal or
square planar geometries. Whereas, the ground state will
1
ν
KM
1
1
2
¼
×
þ
be dz for a squeezed octahedral or trigonal bipyramidal
Vmax ½Sꢀ Vmax
geometry around the metal ion.[90b] In this case, the axial
EPR spectra along with g║ > g┴ > 2 order emphasize that
both the Cu (II) complexes have square planar geometries
with dx –y orbital as a ground state.[90c–e] Slightly higher
g values accentuate the presence of little distortion in the
Cu (II) complexes which is also clear from single crystal
X‐ray diffraction studies.[90c–e] After the addition of 3,5‐
DTBC to 2 and 3 separately, an additional peak was
appeared at g = 2.004 and g = 1.99 for 2 & 3 respectively
(Figure 11 and Figure S21†). This indicates the involve-
ment of free radical in the oxidation process. Neverthe-
less, the EPR signals of Cu (II) ion in 2 and 3 remain
unchanged after the addition of 3,5‐DTBC which pre-
cludes the possibility of metal based redox participa-
tion.[7] So, from the EPR signals, we can conclude that
the oxidation of 3,5‐DTBC to 3,5‐DTBQ in the presence
of aforementioned complexes goes through a ligand ori-
ented radical pathway.
2
2
FIGURE 9 Plot of the rate (ν) vs. substrate concentration ([S]) for
complex 1. Inset: corresponding Lineweaver−Burk plot of 1/rate
against 1/[substrate]

MONDAL ET AL.
13 of 17
TABLE 4 Kinetic parameters for the catecholase activity of 1–3 and comparison with reported Zn(II) and Cu(II) complexes
Complex
Vmax (M s−1
)
Km (M)
kcat (h−1
)
Ref.
1
(1.16 0.05) × 10−4
(8.46 0.09) × 10−5
(4.35 0.15) × 10−5
2.58 × 10−5
(3.95 0.04) × 10−3
(5.1 0.07) × 10−3
(3.6 0.10) × 10−3
1.88 × 10−3
4.1 × 103
3.0 × 103
1.5 × 103
9.28 × 102
1.33 × 103
23.58
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2
3
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[6]
[Zn3(L)(NCS)2](NO3)2·CH3OH·H2O
[Zn3(L1)2(μOAc)2 (CH3OH)4]
[CuL (NCO)]
3.0 × 10−4
1.06 × 10−3
[51]
[7]
1.31 × 10−7
2.27 × 10−3
[86]
[Cu (sal‐ppzH)Cl2]
9.85 × 10−3 mmol min−1
5.3 × 10−3
1.182 × 104
3.6 | Anticancer activity
The optimistic outcomes which are acquired from DNA
binding, protein binding and catecholase activity evalua-
tions of complexes 1–3, motivated us further to investi-
gate the cytotoxicity study of the complexes against
HeLa cells (human cervical cancer cell lines).
MTT assay has been utilized to assess in vitro cytotox-
icity of the said complexes. The metabolic action in cells
was scrutinized by their capabilities to reduce the tetrazo-
FIGURE 10 EPR spectrum of complex 1 after the addition of 3,5‐
DTBC. Inset: EPR spectrum of 1 in absence of 3,5‐DTBC
FIGURE 12 The extent of cell viability of HeLa cells after the
treatment with complexes 1–3 for 48 hr
TABLE 5 Summary of IC50 values of 1–3 against HeLa cell lines
Compound
IC50 value (μM)
1
25.3
53
2
3
66.5
15a
Cisplatin
FIGURE 11 EPR spectrum of complex 2 after the addition of 3,5‐
DTBC. Inset: EPR spectrum of 2 in the absence of 3,5‐DTBC
aSee reference 96,97.

14 of 17
MONDAL ET AL.
FIGURE 13 Inverted microscopic
images after staining the nucleus using
Hoechst 33342. (a) control cells show
intact cellular morphology with evenly
stained nucleus and (b) complex 1 treated
cells depict membrane blebbing and
condensed nucleus with intensely staining
lium ring of yellowish MTT to dark blue formazan. This
reduction process is actually based on the fact that the
mitochondrial enzyme succinate dehydrogenase in living
cells (instead of dead cells) is responsible for the cleaving
of the tetrazolium ring. The cytotoxic activity of 1–3
against HeLa cells was examined after 48 hr of incubation
period. All the compounds are potent inhibitor of cell
growth as depicted in Figure 12. Here, cell inhibition is
expressed in terms of IC50 value and summarized in
Table 5. From Table 5, it is quite evident that all the com-
plexes show considerable cytotoxic activity with IC50
values in the range of 25.3–66.5 μM. The potential of
the aforementioned complexes as anticancer drug
decreases in the order 1 > 2 > 3.
Most interestingly, IC50 value of 1 is relatively closer
with famous anticancer drug cisplatin in comparison
with 2 or 3 despite of having a huge structure (in case
of 1). However, all the complexes are less toxic compared
to the cisplatin. The anticancer activity of metal com-
plexes has heavily relied upon DNA binding aspects
which lead to destroy its secondary structure. As a conse-
quence, the replication and transcription processes are
suppressed due to the prevention of normal functioning
of DNA. This triggers the cell death ultimately if the
repair work of DNA is not restored. The observations of
the in vitro cytotoxicity are at par with DNA and protein
binding studies which confirm again the interaction
between DNA and metal complexes.
In order to investigate the mode of cell death, com-
plex 1 was treated with Hela cells (as the IC50 value is
close to that of cisplatin) and monitored the morpholog-
ical changes of the cells with Hoechst 33342 nuclear
staining. After 12 hours of treatment, the treated cells
(Figure 13b) became circular with prominent membrane
protrusion while such cellular features were not
observed in the case of the untreated cells (Figure 13
a). As the formation of the small blister and nuclear
condensation are the hallmark indicators of apoptotic
cells,[91] the study confirmed that 1 has the potential
anticancer activity to cause cell death via an apoptotic
pathway.
4 | CONCLUSIONS
A novel hexanuclear Zn(II) and two mononuclear Cu(II)
complexes containing imino phenolate ligand unit are
prepared and structurally characterized. Subsequently,
the potential of these compounds towards DNA binding,
protein binding, catecholase and cytotoxic activities have
been thoroughly investigated. It is observed that all the
complexes interact to CT‐DNA via non‐intercalative bind-
ing approach which has been monitored by spectroscopic
techniques. Also, they show excellent ability to bind pro-
tein which is an essential requirement for exhibiting anti-
cancer activity. The pathway of quenching of BSA was
originated from the static mode instead of the dynamic
one. On the other hand, they are efficient catalysts for
the oxidation of 3,5‐DTBC to 3,5‐DTBQ under aerobic
condition. Besides, all the complexes have portrayed
noteworthy in vitro cytotoxic activity towards the HeLa
cell lines and the IC50 value was found to be a little bit
higher in case of zinc complex compared to other two
copper compounds. Hoechst 33342 nuclear staining stud-
ies demonstrate that the apoptotic mechanism is the
prime reason of the cell death.
ACKNOWLEDGMENTS
TKS thanks to SERB, Govt. of India for financial support
(File no. ECR/2016/000681) and acknowledges NIT
Durgapur for providing the infrastructure. SSM thanks
to NIT Durgapur for giving the fellowship.
CCDC 1852303 − 1852305 contain the supplementary
crystallographic data for this paper. The data can be
obtained free of charge via www.ccdc.cam.ac.uk/
structures.
CONFLICT OF INTEREST
There are no conflicts to declare.

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How to cite this article: Mondal SS, Chatterjee
M, Tiwari RK, et al. Hexanuclear Zn(II) and
Mononuclear Cu(II) Complexes containing imino
phenol ligands: Exploitation of their Catalytic and
Biological Perspectives. Appl Organometal Chem.
2019;e5011. https://doi.org/10.1002/aoc.5011
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