Solvent induced distortion in a square planner copper(II) complex containing an azo-functionalized Schiff base: Synthesis, crystal structure, in-vitro fungicidal and anti-proliferative, and catecholase activity

Article abstract of DOI:10.1016/j.molstruc.2021.131057

This research work reports the synthesis, single crystal X-ray structure, catechol oxidation, fungicidal and antiproliferative activity of a newly synthesized copper(II) complex, [Cu(L)₂]H₂O. CH₃OH (1) containing an azo-functionalized Schiff base, HL = 2-methoxy-6-((Z)-((4-((E)-phenyldiazenyl)phenyl)imino)methyl)phenol. The crystal structure analysis reveals that the Cu(II) centre exists in a highly distorted square planar geometry. The crystallize water and methanol form a strong intermolecular association through H-bonding. More importantly, the H atoms of the lattice water interact with the O atoms of ligand units leading to 5- and 6-membered cycles through the H-bonding network and distort the square planar geometry. The copper(II) complex has emerged as a bioinspired catalyst in the oxidative transformation of 3,5-di-tert-butylcatechol (DTBC) to o-benzoquinone in methanol with a high turnover number, 4.75 × 10² h^–1. Electrochemical analysis of the copper(II) complex in presence of DTBC recommends the generation of catechol/o-benzosemiquinone redox couple in the course of oxidation. The EPR spectral analysis of 1 in presence of DTBC was found silent and suggested the antiferromagnetic interaction between copper centre and benzosemiquinone species. The copper(II) complex turns out to be a potential fungicidal agent against clinical candida albicans and scanning electron microscope studies confirm the destruction of the fungal cell membrane with the deposition of copper. The IC₅₀ value of the copper complex was determined as 15 μg/mL which suggests the excellent antiproliferative potency of the synthetic compound against the breast cancer cell lines, MCF-7.

Full text of DOI:10.1016/j.molstruc.2021.131057

Journal of Molecular Structure 1245 (2021) 131057
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Solvent induced distortion in a square planar copper(II) complex
containing an azo-functionalized Schiff base: Synthesis, crystal
structure, in-vitro fungicidal and anti-proliferative, and catecholase
activity
Subham Mukherjeea, Chanchal Kumar Pala, Muddukrishnaiah Kotakondab, Mayank Joshic,
Madhusudan Shitd, Prasanta Ghoshe, Angshuman Roy Choudhuryc, Bhaskar Biswasa,∗
a Department of Chemistry, University of North Bengal, Darjeeling-734013, India
b Department of Technology, Anna University, Chennai 600025, India
c Department of Chemical Sciences, Indian Institute of Science Education and Research, Mohali, Sector 81, Knowledge City, S. A. S. Nagar, Manauli PO,
Mohali, Punjab 140306, India
d Department of Chemistry, Dinabandhu Andrews College, Kolkata 700084, India
e Department of Chemistry, Ramakrishna Mission Residential College, Kolkata 700103, India
a r t i c l e i n f o
a b s t r a c t
Article history:
This research work reports the synthesis, single crystal X-ray structure, catechol oxidation,
Received 19 April 2021
Revised 12 June 2021
Accepted 5 July 2021
Available online 10 July 2021
fungicidal and antiproliferative activity of
a
newly synthesized copper(II) complex, [Cu(L)2]H2O
.
CH3OH (1) containing an azo-functionalized Schiff base, HL 2-methoxy-6-((Z)-((4-((E)-
=
phenyldiazenyl)phenyl)imino)methyl)phenol. The crystal structure analysis reveals that the Cu(II)
centre exists in a highly distorted square planar geometry. The crystallize water and methanol form
a strong intermolecular association through H-bonding. More importantly, the H atoms of the lattice
water interact with the O atoms of ligand units leading to 5- and 6-membered cycles through the
H-bonding network and distort the square planar geometry. The copper(II) complex has emerged as a
bioinspired catalyst in the oxidative transformation of 3,5-di-tert-butylcatechol (DTBC) to o-benzoquinone
in methanol with a high turnover number, 4.75 × 102 h–1. Electrochemical analysis of the copper(II)
complex in presence of DTBC recommends the generation of catechol/o-benzosemiquinone redox couple
in the course of oxidation. The EPR spectral analysis of 1 in presence of DTBC was found silent and
suggested the antiferromagnetic interaction between copper centre and benzosemiquinone species. The
copper(II) complex turns out to be a potential fungicidal agent against clinical candida albicans and
scanning electron microscope studies confirm the destruction of the fungal cell membrane with the
deposition of copper. The IC50 value of the copper complex was determined as 15 μg/mL which suggests
the excellent antiproliferative potency of the synthetic compound against the breast cancer cell lines,
MCF-7.
Keywords:
Copper(II)
Electrochemical analysis
Catechol oxidation activity
Schiff base
X-ray structure
Antifungal property
Antiproliferative activity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
important electronic reversibility in photo-isomerisation processes
and they are employed to a greater extent in the development
Nowadays, azo-functionalised Schiff bases grab special attention
for their unique and versatile structural and functional properties
[1-3]. Generally, azo-functionalised compounds are widely used in
dye and pigment industries [4,5]. Besides that azo compounds ex-
hibit remarkable importance in the medicinal and pharmaceutical
field including biological sciences [6-8]. These compounds show
of functional materials, optical computing, and coordination com-
pounds [9-12]. Azo-functionalized Schiff bases form stable coordi-
nation compounds with interesting structural and functional prop-
erties [13]. amongst the various coordination compounds, cop-
per(II) complexes have been paid substantial attention for their
stability in an aerobic environment, good reactivity in solutions
and the interesting flexible geometry [14-20]. Being a d9 system,
copper(II) ion undergoes Jahn-Teller distortion and imposes a dis-
∗
Corresponding author.
E-mail address: bhaskarbiswas@nbu.ac.in (B. Biswas).
https://doi.org/10.1016/j.molstruc.2021.131057
0022-2860/© 2021 Elsevier B.V. All rights reserved.

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
tortion in coordination geometries which facilitates the catalytic
routes for different organic transformations [21-23].
Yield of 1: 0.775 g (~86.4%) Anal. calc. for C41H38N6O6Cu (1): C,
63.60; H, 4.95; N, 10.85; Found: C, 63.65; H, 4.91; N, 10.89. IR (KBr
pellet, cm−1; Fig. S5): 3379 (νOH), 1616,1581 (νC=N), 1437 (νN=N),
1252 (νPh-O); UV–Vis (1 × 10–4 M, λmax(abs), nm, MeOH; Fig. S2):
230, 346.
The copper complexes mediated catalytic transformation of or-
ganic substrates in molecular oxygen environment increase expo-
nentially using oxygen a sink of electrons (oxidase activity) as well
incorporating oxygen atoms the product (oxygenase activity) or
both [24-27]. In the biological world, copper ions in the coordina-
tion of various bio-ligands exist in the functional core of different
metalloproteins [28-30]. At present different scientific groups are
actively engrossed in the catalytic oxidations of organic substrates
based on synthetic copper(II) based coordination compounds [31-
34]. In light of a better understanding of metalloenzymes me-
diated biological oxidation and the emergence of novel antibi-
otics with potential resistance, it is of great importance to design
novel antibiotics, which would destroy the lipid layer as well as
the cell membrane of the pathogen with high selectivity [35,36].
In this perspective, copper-based coordination compounds hold a
great promise to provide future alternatives to the existed antibi-
otics [37,38]. In the context of newly designed copper(II) com-
plexes with high catalytic activities and potential therapeutic val-
ues, this research study describes the synthesis, structural charac-
terization and catalytic catecholase activity of a newly synthesized
square planar copper(II)-Schiff base complex. The fungicidal prop-
erty against candida albicans and the antitumor activity towards
the breast cancer MCF-7 cell lines has also been delineated.
2.2. Physical measurements
FTIR-8400S SHIMADZU spectrometer (Shimadzu, Kyoto, Japan)
was employed to record IR spectra (KBr) of Schiff base and 1
.
ranging 400–3600 cm–1 1H and 13C NMR spectra of the ligand
(HL) were obtained on a Bruker Advance 400 MHz spectrome-
ter (Bruker, Massachusetts, USA) in CDCl3 at 298 K. Steady-state
absorption and other spectral data were obtained on a JASCO
V-730 UV–Vis spectrophotometer (Jasco, Tokyo, Japan). A Perkin
Elmer 2400 CHN microanalyser (Perkin Elmer, Waltham, USA) was
used to perform the elemental analysis. X-band EPR spectra were
recorded on a Magnettech GmbH MiniScope MS400 spectrometer
(equipped with temperature controller TC H03, Magnettech, Berlin,
Germany), where the microwave frequency was measured with an
FC400 frequency counter. The electroanalytical instrument, BASi
Epsilon-EC was employed for recoding the cyclic voltammograms
in CH2Cl2 solutions. The BASi platinum working electrode, plat-
inum auxiliary electrode, Ag/AgCl reference electrode were used
for the measurements. Field emission scanning microcopy images
of the fungal species in presence and absence of the copper com-
plex was recorded with JSM-IT800 FESEM, Japan.
2. Experimental
2.1. Preparation of the SCHIFF base and copper(II) complex
2.3. Crystal structure determination and refinement
(a) Chemicals, solvents and starting materials
X-ray diffraction data of 1 were collected using a Rigaku XtaLAB
Mini diffractometer equipped with Mercury 375R (2 × 2 bin mode)
CCD detector. The data were collected with a graphite monochro-
Highly pure 2-hydroxy-3-methoxybenzaldehyde (Alfa Aeser,
UK), aniline (Merck, India), copper acetate monohydrate (Thomas
Baker, India) and other reagents were purchased from respective
suppliers. All the reagents and chemicals were of analytical grade.
The solvents of spectroscopic grade were used to study the bio-
inspired oxidation of catechol.
˚
mated Mo-Kα radiation (λ=0.71073 A) at 150 K using ω scans. The
data were reduced using CrysAlisPro 1.171.39.7f [40] and the space
group determination was done using Olex2. The structure was re-
solved by the dual space method using SHELXT-2015 [41] and re-
fined by full-matrix least-squares procedures using the SHELXL-
2015 [42] software package through the OLEX2 suite [43]. The
observed Rint is high as the crystals of the copper(II) complex
weren’t diffracted to a great extent. Further, as a d-block metal,
copper absorbs x-ray sometimes leading to additional residual den-
sity around copper. This fact accounts for the presence of residual
density around the copper ion in this structure.
(b) Synthesis of the Schiff base and copper(II) complex
4-aminoazobenzene was prepared by using a previously re-
ported well-established synthetic procedure [39]. 2-hydroxy-3-
methoxy benzaldehyde (1.52 g, 10 mmol) was condensed with 4-
aminoazobenzene (1.97 g, 10 mmol) under reflux for 8 hr in EtOH.
The Schiff base was extracted as a reddish-orange crystalline prod-
uct and dried in a vacuum desiccator.
Then, the yellowish brown coloured gummy product was
extracted and stored in vaccuo over CaCl2 for use. Yield: 0.317 g
(~90.8%). Anal. Calc. for C20H17 N3O2 (HL): C, 72.49; H, 5.17; N,
12.68; Found: C, 72.53; H, 5.20; N, 12.75. IR (KBr, cm–1; Fig. S1):
3446 (νOH2), 1618, 1593 (νC=N), 1468 (νN=N), 1260 (νPh-O); UV–
Vis (λmax, nm; Fig. S2): 224, 356; 1H NMR (δ ppm, 400 Mz, CDCl3;
Fig. S3) δ = 12.90 (s, 1H), 9.06 (s, 1H), 6.91–8.04 (Ar-H, 12H), 3.88
(s, 3H) ppm. 13C NMR (400 MHz,CDCl3; Fig. S4): 206.9,192.4
2.4. Catecholase activity of the copper(II) complex (1)
The catecholase activity was examined by the treatment of
1 × 10−4 M solution of Cu(II) complex with 1 × 10−3 M of 3,5-
di-tert-butylcatechol (DTBC) in methanol under an aerobic atmo-
sphere. absorbence vs. wavelength (wavelength scans) of the solu-
tion was monitored through a spectrophotometer at a regular time
interval of 8 minutes for ~1.6 h in the wavelength range from 300–
600 nm [44-45]. All the experiments were performed under aero-
bic conditions at room temperature.
(C-N=N-C);
164.90
(HC=N);
153.31,152.91,151.31,150.84(Ar-
124.42,
OH);148.85,143.27(Ar-N=C);131.98,129.94,125.60,
123.0,122.94,120.57,119.78,118.0,116.40,113.84(Ar-C); 56.50, 40.55,
39.93,31.10 (–OCH3).
Kinetic experiments were also performed with a spectropho-
tometer to determine the efficiency of catalytic oxidation of DTBC
by the Cu(II) complex in MeOH [44-45]. The kinetics of cat-
alytic transformation of DTBC was performed following the ini-
tial rate method. The catalytic oxidation was monitored as a func-
tion of time on the growth of the o-quinone species at 398 nm
[46]. 0.04 mL of the solution of copper complex with a con-
stant concentration of 1 × 10–3 M was added to 2 mL solution
of DTBC of a particular concentration (varying its concentration
from 1 × 10–3 M to 1 × 10–2 M) to achieve the ultimate concen-
tration as 1 × 10–3 M. The conversion of DTBC to 3,5-di-tert-o-
The copper(II) complex containing azo-functionalized Schiff
base was synthesized through the reaction of Cu(OAc)2 (0.199 g,
1 mmol) with HL (0.69 g, 2 mmol) in MeOH-DCM solvent mixture.
The reaction solution turned instantly to deep brown upon drop-
wise addition of Cu(OAc)2 to HL and the total solution was stirring
for 30 mins. The resultant solution was filtered and kept for slow
evaporation. After a week, a green crystalline product of the cop-
per complex was separated. The compound was dried over silica
gel.
2

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
butylquinone was monitored with time at a wavelength 398 nm
(time scan) in MeOH [44-47]. To determine the dependence of
rate on substrate concentration, kinetic analyses were performed
in triplicate.
6.25 mg/mL) and the standard drug, fluconazole (50, 25, 12.5, 6.25,
3.125, 1.5625 mg/mL) were added to the respective controls in
2.0 mL MIC tubes. 108 CFU/ml 100 μL of the tested were added
into each MIC test tube. MIC tubes were incubated overnight at
37 °C for 24 h.
The involvement of aerobic oxygen in the oxidation of DTBC
was examined by the presence of hydrogen peroxide following a
reported procedure [47]. In the oxidation of DTBC in MeOH, H2SO4
was added to reach pH 2 of the solution. After a certain time,
the water of equal volume was further supplied to stop the oxi-
dation. The o-quinone species were isolated using DCM as an ex-
tractant. 10 % KI solution (1 mL) and 3% ammonium molybdate so-
2.5.4. Antifungal activity of copper(II) complex using scanning
electronic microscope
To examine the mode of action of copper(II) complex on the
fungal species, fungi cultures were obtained from MIC samples and
centrifuged. Thereafter, the fungi cells were collected and sputter-
coated with a thin layer of gold-palladium. The coated fungal cells
were fixed on a glass coverslip and observed under a scanning
electron microscope.
−
lution were added to the aqueous layer. The development of I3
was monitored through a spectrophotometer to examine the for-
−
mation of I3 band (λmax= 353 nm) which reveals the production
of hydrogen peroxide in the course of catalytic oxidation.
Fungicidal susceptibility studies
2.6. Antiproliferative activity of the copper complex
The fungicidal activity of the copper complex was performed
against clinical candida albicans.
2.6.1. Chemical and reagents for antitumor activity
Dulbecco’s Modified Eagle’s Medium (DMEM), streptomycin,
penicillin-G, L-glutamine, phosphate-buffered saline, (3-[4,5-
2.5.1. Clinical cultures and culture media
The fungicidal property of the copper complex was tested
against clinical candida albicans. The microbial cultures were pro-
cured from the government medical college from Tiruchirappalli,
Tamil Nadu. Muller-Hinton agar media of Himedia Pvt., India was
used for the media. The fungicidal activity was evaluated employ-
ing the Himedia zone reader.
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide;
MTT),
ethylene diamine tetraacetic acid (EDTA), ethanol, and dimethyl
sulfoxide (DMSO) were purchased from Sigma Aldrich Chemicals
Pvt. Ltd (India). All other chemicals were of analytical grade and
purchased from Himedia Laboratories Pvt. Ltd. India.
2.5.2. Inoculums preparation
2.6.2. Cell culture maintenance
A 100μL of clinical C. albicans organisms was inoculated in
5.0 mL of sterile nutrient broth (NB) media, PDB (Potato dextrose
broth) and incubated at 37 °C for 24 h. 200μL of the fresh cul-
ture of the C. albicans was dispensed into 30 mL sterile nutrient
broth and incubated 24 h to standardize the culture to 108 CFU/ml
(colony forming units).
Human breast cancer MCF-7 cell lines were procured from the
cell repository of the National Centre for Cell Sciences (NCCS),
Pune, India. DMEM was used for maintaining the cell line which
was supplemented with 10% fetal Bovine Serum (FBS). The peni-
cillin (100 μg/mL), and streptomycin (100 μg/mL) was added to
the medium to prevent the microbial contamination. The medium
constituting the cell lines was maintained in a humidified environ-
ment with 5% CO2 at 37 °C.
2.5.3. Agar well diffusion method (Kirby-Bauer method)
The fungicidal activity of the copper complex and a standard
marketed drug (Fluconazole-100 mg/2 mL) was studied initially
by using the agar well plate method. C. albicans inoculum was
prepared by using sterile nutrient broth media. Mueller Hinton
agar double strength media were made by autoclaving 0.760 g in
100 ml. Standardized inoculums inoculate the test microorganisms
on the Mueller Hinton agar plates by using sterile cotton swabs.
Four 8 mm diameter agar wells were prepared using sterile cork-
borer, and copper complex 0.1 ml (50 mg/ml) and 0.1 ml Fluoca-
zole (10 mg/ml) were placed on agar well using micropipette un-
der aseptic conditions. Sterile water was used as a negative control.
Agar plates were incubated for 30 min at the refrigerator to diffuse
the formulation into the agar, and finally, plates were incubated at
37˚C for 24 h. Antifungal activity was evaluated by using the Hi-
media zone reader.
2.6.3. MTT assay
The cytotoxicity of the copper complex on the breast cancer
cell lines, MCF-7 was determined through 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay employing the
method reported by Mosmann et al (1983).
50 mg of MTT dye was dissolved in 10 mL of PBS. After vor-
texing for 1 min, it was filtered through 0.45 microfilters. The bot-
tle was wrapped with aluminium foil to prevent light, as MTT was
light-sensitive. The preparation was stored at 4 °C.
Cell viability assay for MCF-7 viable cells was harvested and
counted using a hemocytometer and diluted in DMEM medium to
reach a density of 1 × 104 cells/mL which were seeded in 96 well
plates for each well and incubated for 24 h to allow attachment.
After MCF-7 cells treated with control and the containing differ-
ent concentrations of copper complex 5 to 30 μg/ml were applied
to each well. MCF-7cells were incubated at 37 °C in a humidified
95% air and 5% CO2 incubator for 24 h. After incubation, the drug-
containing cells wash with a fresh culture medium and the MTT
(5 mg/ml in PBS) dye was added to each well, followed by incu-
bated for another 4 h at 37 °C. The purple precipitated formazan
formed was dissolved in 100 μl of concentrated DMSO and the cell
viability was absorbence and measured 540 nm using a multi-well
plate reader. The results were expressed at the percentage of sta-
ble cells to the control. The half-maximal inhibitory concentration
(IC50) values were calculated and the optimum doses were anal-
ysed at the different periods.
Preparation of stock solutions for MIC was done following the
equation 1.
Weight of the powder (mg) =
Volumeofsolution mL × Concentration mg/L
(
)
(
)
(1)
The potency of powder mg/g
(
)
2.5.3. Determination of minimum inhibitory concentration (MIC) for
copper complex against clinical C. albicans
The method of micro-dilution was used to establish the an-
tibacterial potential of the copper complex and respective controls.
A spectrophotometer (OD595= 0.22) equivalent to 108 CFU/mL
used to fix the bacterial cultures to 0.22 optical density at 595 nm.
Different concentrations of copper complex (100, 50, 25, 12.5,
3

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
Scheme 1. Synthetic route for the copper(II) complex.
Inhibitory of call proliferation %
=
( )
Mean absorbence of the control − Mean absorbense of the sample
Mean absorbence of the control
×10
The IC50 values were determined from the dose-responsive
curve of the copper complex. All experiments were performed at
least three times in triplicate. The values are expressed as mean
SD. The statistical comparisons were performed by one-way analy-
sis of variance (ANOVA) followed by Duncan’s Multiple Range Test
(DMRT), using SPSS version 12.0 for windows (SPSS Inc. Chicago;
http://www.spss.com). The values are considered statistically sig-
nificant if the p-value was less than 0.05.
3. Results and discussion
3.1. Design, synthesis and formulation of the Schiff base (HL) and
copper(II) complex (1)
The azo-functionalized Schiff base (HL) was synthesized by a
reaction between 4-aminoazobenzene and 2-hydroxy-3-methoxy
benzaldehyde under refluxing conditions. The copper(II)-Schiff
base complex was synthesized by reacting copper(II) acetate with
HL in 1:2 mole ratio in a methanol medium. The synthetic pro-
cedure is shown in Scheme 1. Attempts were also made to de-
velop the different structural composition of the copper(II)-Schiff
base complex, perhaps we were unsuccessful to produce the dif-
ferent structure. Both the Schiff base and the copper(II) complex
are soluble in polar solvents like methanol, ethanol, acetonitrile,
chloroform etc.
Fig. 1. Ball and stick drawing of the copper(II) complex with 30% probability. The
hydrogen atoms and the solvent molecules are removed for clarity.
Table 1
Crystallographic data and structure refinement pa-
rameters for 1.
Parameters
1
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
C41H38N6O6Cu
774.31
150
3.2. Description of crystal structure and non-covalent interactions
Monoclinic
P21/c
˚
a (A)
16.104(2)
18.584(2)
13.265(2)
3748.4(9)
4
The crystal structural analysis reveals that the copper complex
crystallizes in a monoclinic system with a P21/c space group. The
ORTEP diagram of the Cu(II) complex is displayed in Fig. 1. The
crystallographic refinement parameter is summarized in Table 1
and bond angles, and bond distances are given in Table 2. The azo-
functionalized Schiff base contains three donor centres in associ-
ation with an azo group, however, during coordination two donor
centres of one ligand unit coordinate with the copper(II) centre ion
as a bidentate chelator. The copper(II) complex adopts two units
of coordinated Schiff base and exists in a highly distorted square
planar coordination geometry. The deviation of copper(II) centric
average bond angles (ࢬL–M–L, 94.23) from the tetrahedral geome-
try is found larger while the average bond angles around copper(II)
centre (ࢬL–M–L, 94.23) are closer to the average value of an ideal
square planar geometry (Table 2). Therefore, bond angle measure-
ments around the copper centre support the existence of a square
planar geometry.
˚
b (A)
˚
c (A)
3
˚
Volume (A )
Z
ρ (gcm–3
μ (mm–1
F (000)
Rint
)
)
1.3721
0.639
1612
0.181
θ ranges (°)
2.6–25
Number of unique reflections
Total number of reflections
Final R indices
Largest peak and hole (eA˚−3
6579
32,338
0.0750, 0.2230
1.00, -0.63
)
crystalline architectures of the copper complex were examined.
It was observed that the crystallized water and methanol form
a very strong intermolecular H-bonding network with the copper
˚
˚
To understand the cause of distortion, the effect of solvate
molecules and the presence of noncovalent interactions in the
complex ranging from 1.91 A to 2.11 A (Fig. S6). Investigation of
the self-assembly of the copper complex exhibits that methanol-
4

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
Table 2
Bond angles and bond distances value of Cu(II) complex.
˚
Bond distances (A)
Cu1 –O2
1.894(3)
1.985(3)
Cu1- O4
1.890(3)
1.978(3)
Cu1 –N1
Cu1 - N4
Bond angles (o)
ࢬO2-Cu1-O4
ࢬO2-Cu1-N1
ࢬO2 -Cu1-N4
88.79(13)
93.17(14)
150.08(14)
ࢬO4-Cu1-N1
ࢬO4-Cu1-N4
ࢬN1-Cu1-N4
145.64(14)
94.78(13)
100.19(14)
Scheme 2. Catalytic oxidation of DTBC to DTBQ.
set of C–H…π interactions in the crystalline phase (Fig. 2, Table
S2).
The Hirshfeld surface analysis of the copper(II) complex over a
definite dnorm was calculated with Crystal Explorer software. The
3
˚
surface volume and area are calculated as 963.85 A and 687.91
2
˚
A . The red highlighted spots indicate the dnorm area and present
close non-covalent interactions in the copper complex with its sur-
rounding molecules (Fig. S7). The contribution of each element in
the non-covalent interactions is given in Table S3. The blue area in
dnorm cites important C–H…π interactions between the azo-linked
phenyl centroid with the H of C-attached to phenyl and methyl
groups. The white denotes no interaction. The interactions in the
copper(II) complex is displayed in Fingerprint plots (Fig. S8).
3.3. Solution property of the Schiff base and copper(II) complex
The Schiff base exhibit characteristic electronic transitions at
224 and 356 nm while the copper(II) complex displayed the elec-
tronic bands at 230 and 346 nm in methanol at room tempera-
ture. The electronic bands at 224 and 365 nm in the Schiff base are
assignable to the presence of π→π∗ and charge transfer (CT) tran-
sitions respectively. The lower energetic electronic band at 346 nm
gets blue-shifted in the complex and is attributed to the CT band
from PhO– → Cu(II) centre exhibited electronic bands at 238, 283
and 400 nm. The electronic spectra are displayed in Fig. S2. The ap-
pearance of the characteristic electronic bands for the Schiff base
and copper complex are in good agreement with the previously
reported values of electronic transitions [48,49]. The high resolu-
tion mass spectrum was recorded in methanol at room tempera-
ture to reveal the purity and homogeneity of copper complex in
solution. The spectrum is displayed in Fig. S9. The mass spectrum
shows a characteristic peak at 723.21 m/z which ensures the ex-
istence of the tetracoordinate copper(II) complex with its struc-
tural integrity and suggests the homogeneity of the compound
in MeOH.
Fig. 2. OH (pink dash lines) and C–H…π (green dashed lines) mediated formation
of a supramolecular architecture for the copper(II) complex. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)
OH(H6A) acts as an acceptor towards the oxygen of water (O5)
while the O5 atom behaves as a donor to develop a strong inter-
molecular association between the crystallized water and methanol
molecules (Fig. S6). Furthermore, the hydrogen atoms of water
(H5A and H5B) play a key role in causing a high distortion in
the molecules. Two H-atoms of water strongly hold two units of
3.4. Catecholase activity of the copper(II) complex (1)
azo-functionalized Schiff base separately through the strong inter-
…
molecular network (H5A…O3, 2.14 A; H5A O4, 2.45 A; H5B…O1,
The catecholase activity of the copper(II) complex has been ex-
amined by considering 3,5-di-tert-butyl catechol (DTBC) as a model
substrate. DTBC contains two bulky t-butyl substituents at the
phenyl ring and helps to lower down the quinone-catechol reduc-
tion potential. The low quinone-catechol reduction potential facil-
itates the oxidation of catechol to the corresponding o-quinone,
DTBQ under ambient reaction conditions (Scheme 2). It is well doc-
umented that DTBQ is quite stable in solution and displays a char-
acteristic absorption peak at 401 nm in methanol [50].
˚
˚
…
˚
˚
2.45 A; H5B O2, 2.11 A, Fig. 2, Table S1). More captivatingly, the
Hs of O5 in the crystallized water form two 5-member cyclic rings
with the phenoxo-O and methoxy-O of the ligand units and as
a result a 6-member cycle is also formed with the copper cen-
tre coordinated by the phenoxo-O (Fig. 2). This intermolecular H-
bonded network amongst the crystallize solvate molecules and lig-
and units account for the cause of high distortion in the chelating
ligands during coordination. It is further documented that for the
involvement of strong intermolecular H-bonding in ligand units,
the methoxy-O didn’t coordinate with the copper centre.
The nature of changes of the spectral bands during the catalytic
oxidation was monitored with a UV–Vis spectrophotometer for a
period of 1.6 h (Fig. 3). The copper complex displays a character-
istic electronic transition at 346 nm. Upon addition of the Cu(II)
complex to the solution of DTBC, a new electronic band at 398 nm
was started to develop with increasing absorbence (Fig. 3). The rise
of the optical band at 398 nm is a definite sign of the oxidation of
DTBC in MeOH [51,52]. The appearance of this new electronic band
at 398 nm in the spectrophotometric scan is assignable to the pro-
duction of the o-benzoquinone species in MeOH.
A close look at the nature of non-covalent interactions suggests
that the mononuclear copper(II) complex interacts with another
complex unit through strong O…H and C–H…π and seems to be
a complex dimer (Fig. 2, Table S2). This complex dimer is further
extended and stabilized through the intermolecular C–H…π inter-
actions between the azo-linked phenyl-C–H and the terminal cen-
troid of the azo-linked phenyl ring. The H of the methyl group adds
more stabilization energy to the complex dimer through another
5

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
Table 3
Comparison of kcat (h−1) values for catalytic oxidation of DTBC by reported cop-
per(II) compounds and 1.
Complex
kcat (h−1)(Solvent)
CCDC No
Ref
[Cu(dpa)2NCS]2
4788 (CH3OH)
1456703
[51]
[Cu(dpa)2(NCS)2](ClO4)2
[Cu2(L1∗SSL1∗)](BF4)4
[Cu(phen)(OH2)2(NO3)](NO3)
6.90 × 103(CH3CN)
3.91 × 103(CH3OH)
2.08 × 103(CH3OH)
1.83 × 103 (CH3OH)
4.75 × 102 (CH3OH)
-
[52]
[53]
[45c]
[54]
This
work
1061531
1524681
1513638
2046277
[Cu(2,2-bpy)Cl2]
´
[Cu(2,2-bpy)2(OAc)]
´
+
[Cu(L)2]
the peak at -0.11 V is for the deposition of copper. The cate-
cholase activity was authenticated by the change in cyclic voltam-
mograms of the copper complex in presence of DTBC in CH2Cl2 at
295 K. Cyclic voltammograms of the copper complex in presence
of DTBC produces an anodic peak at -0.30 V perhaps the cathodic
wave of Cu2+/Cu+ couple was disappeared. This peak is a definite
sign of the oxidation of DTBC. Therefore, this peak is assignable to
cat/sq redox couple (cat = catechol, sq = o-benzosemiquinone and
isq = o-iminobenzosemiquinone). The disappearance of Cu2+/Cu+
peak indicates the involvement of Cu2+ in the oxidation processes.
This metal-mediated oxidation of DTBC was also authenticated by
the EPR spectral analysis. The copper complex gives a four-line hy-
perfine structure at g = 2.13 due to 63,65Cu in CH2Cl2 at 295 K
(Fig. S11). However, in the presence of DTBC, the metal com-
plex becomes EPR silent (Fig. S12). This is due to the antiferro-
magnetic coupling between the copper centre and semiquinone
species. These experiments strongly support the metal-mediated
oxidation of DTBC. Further, the fate of molecular oxygen in the
oxidation of DTBC was examined through the production of hy-
drogen peroxide in solution [37,38]. An electronic band at λmax
353 nm confirmed the development of hydrogen peroxide in the
course of catalytic oxidation and thereby, ensured the transfor-
mation of molecular oxygen to H2O2 in the catalytic oxidation of
DTBC. Furthermore, the retention of structural identity of the cop-
per(II) complex was confirmed in the catalytic oxidation of DTBC
by measuring the UV–vis spectrum of the solution after separating
the oxidation product. The solution containing the copper complex
as catalyst produces similar kind of electronic bands at 233 and
350 nm (Fig. S13) as like the electronic bands of the original cop-
per complex displayed in methanol and ensures the retention of
structural identity in catalytic oxidation of DTBC. Therefore, based
on the experimental outcomes, a plausible mechanistic cycle for
the catalytic oxidation of DTBC may be proposed according to the
following scheme 3. A comparison of catalytic oxidation of DTBC
by this Cu(II) complex with some other reported Cu(II) complexes
is furnished in Table 3 [51-54].
Fig. 3. Rise of a new electronic band at 398 nm upon addition of copper complex
1 to DTBC in MeOH with a time interval of 8 mins. Inset: Time vs absorbence plot
at 398 nm.
Fig. 4. Left: Cyclic voltammogram of the copper(II) complex 1 in CH2Cl2; Right:
Cyclic voltammogram of copper(II) complex 1 in presence of DTBC under molecular
oxygen atmosphere in CH2Cl2.
The kinetics for the catalytic oxidation of DTBC was studied to
determine the catalytic performance of the copper(II) complex. The
kinetic parameter of the catalytic oxidation of DTBC was evalu-
ated employing the method of the initial rate. The growth of o-
benzoquinone was monitored as a function of time with respect to
398 nm (Fig. S10) [53]. The nature of oxidation kinetics was exam-
ined by plotting the rate constants vs. concentration of DTBC and
shown in Fig. S10. The first order saturation kinetics of the oxida-
tion reaction seems to be suitable in the Michaelis–Menten model
and can be expressed as the Eq. (2):
V max S
[ ]
V =
(2)
KM + S
[ ]
Where, V indicates the rate of oxidation reaction, Km denotes
the Michaelis-Mentenconstant, Vmax presents the maximum veloc-
ity of the reaction, and [S] is the concentration of the DTBC.
The values of kinetics parameters were determined from
3.6. Fungicidal activity of the Cu(II)-Schiff base complex
The fungicidal activity of the copper complex was assessed with
a well diffusion method against the fungi candida albicans. The re-
sults of the inhibition zone diameters are shown in Table S4. The
antifungal efficiency for the Cu(II) complex was determined by cal-
culating MIC on candida albicans. The minimum inhibitory con-
centration (MIC) value was estimated as 230 μg/mL for the cop-
per(II) complex. Scientific literature suggest that the Cu(II) com-
plex is quite competent to inhibit the growth of candida albicans.
Under a similar experimental condition, the standard fluconazole
shows the MIC value of 6.25 μg/mL (Table S5). The MIC value sug-
gests a good fungicidal potency for the inhibition of the growth
of c. albicans fungal species under experimental conditions. To un-
derstand the origin of fungicidal activity of the copper(II) com-
plex, field emission scanning electron microscopy (FESEM) image
Michaelis−Menten equation as Vmax(MS−1
)
=
1.32
×
10−4
;
KM = 4.52 × 10−3 [Std. Error for Vmax (MS−1) = 7.79 × 10−5; Std.
Error for Km (M)= 4.53 × 10−4]. The catalytic efficiency for the cat-
alytic oxidation of DTBC was determined as kcat/KM = 1.05 × 105.
The redox activities of the copper complex and its activities to-
ward the biomimetics of the catecholase activities were studied by
electrochemical analysis in CH2Cl2 at 295 K. The redox behaviour
of the copper complex was recorded using ferrocenium/ferrocene
(Fc+/Fc) reference under aerobic atmosphere. The cyclic voltam-
mograms are illustrated in Fig. 4. The copper(II) complex exhibits
a quasi-reversible cathodic wave at -1.13 V due to Cu2+/Cu+ re-
dox species in solution. The anodic peak appears at +0.70 V due
to phenoxide/phenoxide anion radical(O−/O•−) redox couple and
6

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
Scheme 3. Plausible mechanistic cycle for catecholase activity of the copper complex.
cell walls of the fungal species candida albicans became damaged
and shrinked upon the interaction of the Cu(II) complex with the
fungi [55,56]. Most captivatingly, the presence of elemental copper
was detected on the cell wall of candida albicans through the en-
ergy dispersive X-ray spectroscopy (Fig. S14) and is shown as the
orange coloured mark in Fig. 5. Truly, the presence of elemental
copper on cell wall is a remarkable phenomenon to report and in
very limited cases, such type of observation was noted.
3.7. Antiproliferative activity of the Cu(II)-Schiff base complex
MTT assay was employed to determine the cytotoxic effect of
the copper(II) complex on MCF-7 cell lines. The breast cancer cell
lines, MCF-7 were treated with the copper(II) complex in a dose
dependant manner varying the concentration 5 to 30 μg per ml
for 24 h. The results are expressed as a percentage of the control
value in presenting as a cell cytotoxicity ratio for MCF-7 cells and
displayed in Fig. 6. The IC50 value for the copper complex was de-
termined as 15 μg/mL against MCF-7 cells for 24 h. It is well docu-
mented that (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide is a water soluble tetrazolium salt yielding a yellowish
solution when prepared in media. The yellow coloured MTT is re-
duced by mitochondrial dehydrogenase of viable cells yielding a
measurable purple formation product. The viable cells consist of
NADPH-dependant reductase and reduce the MTT reagent to for-
mazon with the development of deep purple formazon species.
Thereafter, the absorbence of formazon solution was measured by
plate reader to examine the cytotoxic effects of the copper com-
plex. The induced morphological changes in MCF-7 cells by Cu(II)
complex (Fig. 7) leads to the shrinkage, detachment and membrane
blebbing which suggests the high anti-proliferative potency of the
copper complex.
Fig. 5. Morphological changes in candida albicans upon treated with the Cu(II) com-
plex.
was recorded for the candida albicans upon treatment with cop-
per(II) complex. The recorded SEM micrographs of candida albicans
cells are shown in Fig. 5. Commonly, the candida albicans fungal
species was found as nearly spherical-shaped cell morphology with
smooth and intact cell walls. It is well observed that the number
of candida albicans was remarkably decreased, and clusterized. The
7

S. Mukherjee, C.K. Pal, M. Kotakonda et al.
Journal of Molecular Structure 1245 (2021) 131057
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Subham Mukherjee: Conceptualization, Formal analysis,
Methodology, Investigation. Chanchal Kumar Pal: Formal analysis,
Methodology, Investigation. Muddukrishnaiah Kotakonda: Formal
analysis, Methodology, Investigation, Validation. Mayank Joshi:
Formal analysis, Validation. Madhusudan Shit: Formal analysis,
Methodology, Investigation. Prasanta Ghosh: Formal analysis,
Visualization. Angshuman Roy Choudhury: Software, Validation.
Bhaskar Biswas: Conceptualization, Writing – review & editing,
Supervision.
Fig. 6. Cellular viability of breast cancer cell lines, MCF-7 cells treated with cop-
per(II) complex in a dose dependant manner for 24 h employing MTT assay.
Acknowledgements
BB thanks Science and Engineering Research Board (SERB), In-
dia for financial support under Empowerment and Equity Oppor-
tunities for Excellence in Science (EEQ/2020/000079). The authors
sincerely acknowledge the Department of Technology of Anna Uni-
versity, Chennai, India for supporting the measurement of biologi-
cal activities.
Fig. 7. Photomicrograph (40 ×) represents morphological changes in MCF-7 cells
such as shrinkage, detachment, membrane blebbing and distorted shape induced by
Cu(II) complex (10 and 15 μg/mL for 24 h) as compared with control. Control cells
showed normal intact cell morphology and their images were captured by light mi-
croscope. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131057.
In true sense, the introduction of the azo-functionalized
copper(II)-Schiff base complex helps to develop reactive oxygen
species (ROS) in the lipid peroxidase in MCF-7 cells. Consequently,
the redox active copper complex generates more reactive free rad-
icals which involve in the destruction of membrane lipid of MCF-7
cells. This phenomenon accounts for the detachment and shrinkage
of the normal cells and increases the apoptosis of MCF-7 cells. This
observation is also supported by previously reported anti-tumour
activities induced by copper complexes [55,56].
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