Benzoato bridged dinuclear Mn(II) and Cu(II) compounds involving guest chlorobenzoates and dimeric paddle wheel supramolecular assemblies: Antiproliferative evaluation and theoretical studies

Article abstract of DOI:10.1016/j.poly.2021.115409

Two new benzoato bridged Mn(II) and Cu(II) dinuclear coordination compounds viz. [Mn₂(bipy)₂(4-Clbz)₂(H₂O)₄]2(4-Clbz) (1) and [Cu₂(bz)₄(2-MePy)₂] (2) (bipy = 2,2?-bipy

Full text of DOI:10.1016/j.poly.2021.115409

Journal Pre-proofs
Benzoato Bridged Dinuclear Mn(II) and Cu(II) Compounds involving Guest
Chlorobenzoates and Dimeric Paddle Wheel Supramolecular Assemblies: An‐
tiproliferative Evaluation and Theoretical Studies
Pranay Sharma, Debasish Dutta, Rosa M. Gomila, Antonio Frontera, Miquel
Barcelo-Oliver, Akalesh K. Verma, Manjit K. Bhattacharyya
PII:
S0277-5387(21)00391-0
DOI:
Reference:
https://doi.org/10.1016/j.poly.2021.115409
POLY 115409
To appear in:
Polyhedron
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Accepted Date:
28 June 2021
30 July 2021
Please cite this article as: P. Sharma, D. Dutta, R.M. Gomila, A. Frontera, M. Barcelo-Oliver, A.K. Verma, M.K.
Bhattacharyya, Benzoato Bridged Dinuclear Mn(II) and Cu(II) Compounds involving Guest Chlorobenzoates and
Dimeric Paddle Wheel Supramolecular Assemblies: Antiproliferative Evaluation and Theoretical Studies,
Polyhedron (2021), doi: https://doi.org/10.1016/j.poly.2021.115409
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Benzoato Bridged Dinuclear Mn(II) and Cu(II) Compounds involving Guest
Chlorobenzoates and Dimeric Paddle Wheel Supramolecular Assemblies:
Antiproliferative Evaluation and Theoretical Studies
Pranay Sharmaa, Debasish Duttaa, Rosa M. Gomilab, Antonio Frontera*b, Miquel Barcelo-
Oliverb, Akalesh K. Verma*c, Manjit K. Bhattacharyya*a
aDepartment of Chemistry, Cotton University, Guwahati-781001, Assam, India
bDepartament de Química, Universitat de les Illes Balears, Crta de Valldemossa km 7.7,
07122 Palma de Mallorca (Baleares), Spain
cDepartment of Zoology, Cell & Biochemical Technology laboratory, Cotton University,
Guwahati-781001, India
*Corresponding authors E-mail:
amanjit.bhattacharyya@cottonuniversity.ac.in
btoni.frontera@uib.es
cakhilesh@cottonuniversity.ac.in
Abstract
Two new benzoato bridged Mn(II) and Cu(II) dinuclear coordination compounds viz.
[Mn2(bipy)2(4-Clbz)2(H2O)4]2(4-Clbz) (1) and [Cu2(bz)4(2-MePy)2] (2) (bipy = 2,2´-
bipyridine, 4-Clbz = 4-chlorobenzoate, bz = benzoate, 2-MePy = 2-methylpyridine) have
been synthesized and characterized using elemental analysis, TGA, spectroscopic (FTIR,
electronic, EPR) and single crystal X-ray diffraction techniques. Crystal structure analysis of
compound 1 reveals the enclathration of guest 4-Clbz moiety in the supramolecular host
tetramer which provides rigidity to the crystal structure. Further analysis reveals the presence
of cooperative (π–π)2/(π–π)1/(π–π)2 ternary assemblies involving alternate coordinated and
uncoordinated 4-Clbz moieties. In compound 2, intermolecular C‒H∙∙∙O, C‒H∙∙∙π and C‒H∙∙∙C
contacts are involved in the formation of dimeric paddle wheel supramolecular assemblies
which stabilizes the layered architecture. The cooperative interplay of H-bonding and π-
stacked ternary assemblies of 1 has been studied theoretically using DFT calculations, MEP
surface and NCI Plot computational tools. The large interaction energies along with the NCI
plot index analysis confirm that the π-stacking assemblies in combination with the H-bonds
are strong binding motifs. In vitro anticancer activity of the compounds considering
cytotoxicity and apoptosis assays reveal significant concentration dependent cytotoxicity in
Dalton's lymphoma (DL) cancer cell lines with nominal effects in normal healthy PBMC
cells. Molecular docking study reveals the efficient interaction of the compounds with the
antiapoptotic BCL family target proteins.

Keywords: Dinuclear; Cooperative; Enclathration; Paddle wheel; Apoptosis.
1. Introduction
Metal-organic frameworks of coordination compounds elucidate significant attraction
from the researchers in recent times because of their countless functional properties such as
catalysis, sensors, semiconductor devices and various biological applications [1,2]. The
design, synthesis and development of coordination compounds is strongly driven by various
synthetic factors, viz. coordination behavior of the central metal ion, characteristics of the
organic ligands, metal to ligand ratio, the reaction environments etc [3,4]. The proper fusion
of these synthetic attempts in controlled manner can provide suitable approaches to explore
self-assembly processes with interesting structural topologies of desired dimensionalities [5].
The suitable N-donor ligands in combination with aromatic bridging carboxylates have been
widely exploited for the construction of metal-organic compounds [6].
Aromatic carboxylate ligands have attracted considerable attention from the crystal
engineering viewpoint owing to their multiple coordination motifs, thermal stability and
ability to form bridging compounds with transition metals [7]. Carboxylate moieties of
organic ligands can exhibit variety of coordination modes viz. terminal, bridging, chelating,
syn-anti, anti-anti etc.; which can be effectively exploited to design and develop poly-nuclear
coordination compounds with fascinating structural topologies and dimensionalities [8].
Many research groups have explored the potential biological applications such as anticancer,
antimicrobial, antibacterial, antifungal activities of dinuclear carboxylates of transition metals
[9-10]. Such compounds also possess myriad applications in wide range of other fields viz.
catalysis, magnetism, non-linear optics etc [11]. 2,2′-bipyridine, a heterocyclic N-donor
bidentate organic moiety, has been extensively used to design coordination compounds
because of its chelating behavior [12]. It is a biologically important organic ligand as most of
the coordination compounds involving bipyridine possess potential biological applications
[13]. Coordination compounds involving 2-methylpyridine have been also reported to exhibit
antiproliferative activities against human cancer cell lines [14].
Non-covalent interactions in supramolecular assemblies of coordination compounds
involving aromatic ligands play very crucial constructive roles not only in molecular
recognition but also in crystal engineering, molecular biology along with various other fields
of chemical sciences [15]. Because of their wide range of potential applications, myriad
efforts have been put in to explore and quantify the non-covalent interactions observed in
supramolecular self-assembly processes [16]. Compared to coordinate covalent bonds, non-
covalent interactions are much weaker organizing force; however, the larger numbers and

cumulative effects of such interactions play increasingly crucial roles in the development of
coordination network topologies [17]. The cooperativity between intra- and intermolecular H-
bonding, as well as aromatic π-stacking interactions have developed as an intriguing research
topic in metal-organic chemistry [18]. The cooperative π-stacking interactions are remarkably
important from the biological point of view, as they have been reported to provide stability in
protein structures or nucleic acids and also in the recognition of drugs by enzymes [19].
However, the cooperative interplay of hydrogen bonding and aromatic π-stacking interactions
involving 4-Clbz moieties are scarcely explored in the literature. The enclathration of anionic
molecules in the supramolecular host cavities of crystal structures unfolds new dimensions
for molecular association topologies in coordination compounds [20].
To explore the role of non-covalent interactions in dinuclear coordination compounds;
herein; we have reported the synthesis and crystal structures of two new Mn(II) and Cu(II)
compounds involving benzoato ligands and characterized them using FT-IR, electronic, EPR
spectroscopy, TGA and elemental analysis. Crystal structure analysis of the compound 1
reveals the presence of cooperative (π–π)2/(π–π)1/(π–π)2 ternary assemblies involving
alternate coordinated and uncoordinated 4-Clbz moieties. The enclathration of lattice 4-Clbz
moieties in the supramolecular tetrameric host cavities of compound 1 provides additional
reinforcement to the crystal structure. Single crystal X-ray analysis reveals that the compound
2 has a dinuclear paddle wheel structure where the two Cu(II) centers are bridged by four
benzoato ligands. Various non-covalent interactions such as C‒H∙∙∙O, C‒H∙∙∙π and C‒H∙∙∙C
contacts are involved in the stabilization of the supramolecular dimeric paddle wheel
structural assemblies in compound 2. The biologically relevant cooperative ternary π-stacking
interactions observed in the crystal structure of compound 1 have been further studied
theoretically using DFT calculations, MEP surface and NCI Plot computational tools. In vitro
anticancer activities of the compounds have been carried out using cell cytotoxicity and
apoptosis assays. We also aim to investigate the light microscopy based cell morphology
study of the compounds to corroborate the apoptotic cell death. We also aim to perform the in
silico molecular docking of the structures of the compounds with the antiapoptotic target
proteins to elucidate the structure activity relationship.
2. Experimental
2.1 Materials and methods
All the chemicals used in the present study, viz. manganese chloride tetrahydrate,
copper chloride dihydrate, 4-chlorobenzoic acid, 2,2´-bipyridine, benzoic acid and 2-methyl
pyridine were obtained from commercial sources and used without further purification.

Perkin Elmer 2400 Series II CHNS/O analyser was used to carry out the elemental analysis of
the compounds. KBr phase FT-IR spectra of the compounds were recorded using Bruker
Alpha II Infrared spectrophotometer in the frequency range 4000-500 cm-1. The electronic
spectra were recorded using Shimadzu UV-2600 spectrophotometer. For solid state spectra,
BaSO4 powder was used as reference (100% reflectance). Room temperature magnetic
moments of the compounds were calculated at 300 K using Sherwood Mark 1 Magnetic
Susceptibility balance by Evans method. X-band EPR spectra of the crystalline compounds
were recorded using JEOL JES-FA 200 instrument. Thermogravimetric curves of the
compounds were obtained under the flow of N2 gas using Mettler Toledo TGA/DSC1 STARe
system at the heating rate of 10°C min−1.
2.2 Syntheses
2.2.1 Synthesis of [Mn2(bipy)2(4-Clbz)2(H2O)4] 2(4-Clbz) (1)
MnCl2·4H2O (0.197 g, 1 mmol) and bipy (0.156 g, 1mmol) were dissolved in 10 mL
of de-ionised water and mechanically stirred at room temperature for two hours. Then, Na-4-
Clbz (0.356 g, 2 mmol) was added and mechanically stirred for another one hour (Scheme 1).
The resulting solution was kept unperturbed in cooling condition for crystallization; from
which pale yellow block shaped single crystals suitable for single crystal X-ray diffraction
was obtained after a few days. Yield: 0.474 (85%). Anal. calcd. for C24H20N2O6Cl2Mn: C,
51.63%; H, 3.61%; N, 5.02%; Found: C, 51.52%; H, 3.55%; N, 4.92%. IR spectral data (KBr
disc, cm−1): 3381(s), 1545(s), 1418(s), 1366(s), 1195(s), 1099(w), 978(m), 908(m), 850(s),
805(m), 729(s), 678(s) (s, strong; m, medium; w, weak; br, broad; sh, shoulder).
2.2.2 Synthesis of [Cu2(bz)4(2-MePy)2] (2)
CuCl2·2H2O (0.170 g, 1 mmol) and sodium benzoate (0.288 g, 2 mmol) were mixed
in a round bottomed flask in 10 mL of de-ionised water and mechanically stirred at room
temperature for two hours (Scheme 1). After two hours, 2-MePy (0.1 mL, 1 mmol) was added
to the resulting solution and kept for continuous stirring for another one hour. The resulting
solution was kept unperturbed in cooling condition for crystallization. Blue block shaped
crystals suitable for single crystal X-ray diffraction was obtained by the slow evaporation of
the mother liquor after several days. Yield: 0.661 g (83%). Anal. calcd. for C40H34Cu2N2O8:
C, 60.22%; H, 4.30%; N, 3.51%; Found: C, 60.15%; H, 4.25%; N, 3.47%. IR spectral data
(KBr disc, cm−1): 3117(br), 2667(br), 1692(s), 1609(s), 1538(s), 1430(s), 1347(s), 1290(s),

1150(m), 1009(m), 933(s), 876(s), 799(s), 716(s), 557(s) (s, strong; m, medium; w, weak; br,
broad; sh, shoulder).
Scheme 1 Synthesis of the compounds 1 and 2.
2.3 Crystallographic data collection and refinement
X-ray crystallographic structures of the compounds were determined using Bruker
D8 Venture diffractometer with graphite monochromatized Cu/Kα radiation (λ = 1.54178 Å).
Semi-empirical absorption correction, as well as scaling and merging the wavelength were
performed using SADABS [21]. Crystal structures were solved by direct method and refined
by full matrix least squares technique with SHELXL-2018/3 [22] using the WinGX [23]
software. All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms,
except those attached to oxygen atoms of water molecules were placed at their calculated
positions and refined in the isotropic approximation. The structural diagrams were drawn
using Diamond 3.2 [24]. Crystallographic data of the compounds 1 and 2 are tabulated in
Table 1.
2.4 Computational Methods
The energies of the supramolecular assemblies studied in the present study were
computed at the RI-PB86-D3/def2-TZVP [25,26] level of theory using the crystallographic
coordinates and the Turbomole 7.0 program [27]. Grimme’s D3 dispersion [25] correction
has been used since it is convenient for the correct evaluation of non-covalent interactions
where dispersion effects are important like π-stacking interactions. The basis set
superposition error for the calculation of interaction energies has been corrected using the
counterpoise method [28]. The high spin configuration for the Mn(II) ions has been used in
the calculations. The NCI plot [29] isosurfaces have been used to characterize non-covalent

interactions. They correspond to both favorable and unfavorable interactions, as differentiated
by the sign of the second density Hessian eigenvalue and defined by the isosurface color. The
color scheme is a red-yellow-green-blue scale with red for ρ+ (repulsive) and blue for ρ−
cut
cut
(attractive). The MEP surface calculations have been performed at the same level of theory
and using the 0.001 a.u. isosurface as best estimate of the van der Waals surface.
2.5 Cell line and drug preparation
Short term (24 hours) in vitro antiproliferative activities of the compounds 1 and 2
have been explored in Dalton’s lymphoma (DL) malignant cancer cell lines. To investigate
the cytotoxic potential of the compounds in normal healthy cells, peripheral blood
mononuclear cells (PBMC) were used. The cancerous cells (DL) were cultured using RPMI-
1640 medium supplemented with 10% FBS, gentamycin (20 mg/mL), streptomycin (100
mg/mL) and penicillin (100 IU) in a CO2 incubator at 37°C with 5% CO2. The 80% confluent
of exponentially growing cells were sub-cultured and used to test cytotoxicity and apoptosis
inducing abilities of the compounds. All the chemicals and buffer solutions were autoclaved
followed by sterilization by filtration through 0.22 micron membranes. All the experimental
results obtained in the present study were compared with cisplatin, a positive reference drug.
2.6 MTT based cell viability assay
Colorimetric MTT assay was used to investigate the cytotoxic potential of the
compounds 1 and 2 in DL cancer cell lines [30]. This technique basically depends on the
ability of NADPH dependent cellular oxidoreductase enzymes to reduce MTT dye viz. [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to purple colour insoluble
formazan crystals [31]. The investigations were carried using different concentrations (0,
0.01, 0.1, 0.5, 1, 5 and 10 μM) of the compounds 1 and 2 prepared in PBS (pH = 7.4) in 96
cell culture plates (Thermo Scientific, Cat. No: 265301). Peripheral blood mononuclear cells
(PBMC) were used to evaluate the cytotoxicity in normal healthy cells. The IC50 profiles of
the compounds against DL and PBMC cell lines were determined from the dose response
curves using non-linear curve fit. For the IC50 curves, different concentrations of the
compounds in log scale were plotted on X-axis, whereas the corresponding cell cytotoxicities
were plotted on Y-axis. For the IC50 calculation in DL cell lines, concentrations ranging from
0.01 to 100 μM were used, whereas, in normal cells (PBMC) the tested concentration ranges
from 0.01 to 400 μM. The non-linear curve fit function used for IC50 calculations for the
different compounds was as follows:

y = A1 + (A2-A1)/(1 + 10^[(LOG × 0-x)*p)]
(Where, A1 = bottom asymptote, A2 = top asymptote, LOG × 0 = center and p = hill slope).
2.7 Apoptosis study
Acridine orange and ethidium bromide (AO/EB) dual staining method was employed
to investigate the apoptosis inducing abilities of the compounds in DL cells [32]. The post
treatment morphological alterations followed by differential staining (green and red/orange)
in the cells clearly distinguishes the viable and apoptotic cells. AO is a vital dye that can
penetrate inside the cells and can stain the viable cells showing green fluorescence; whereas
EB can stain only the apoptotic cells showing red/orange fluorescence [33]. After treating
with different concentrations of the compounds for 24 hours, the treated cells were stained
with equimolar mixture of AO/EB and covered with a cover slip in dark for three minutes.
The morphological features of control and treated cells were examined using fluorescence
microscope (Leica Microsystems, Germany) and photographed. About 1000 cells were
counted and the percentage of apoptotic cells was determined. To further corroborate the
apoptotic cell deaths, light microscopy based cell morphology was also studied using Giemsa
stain (Sigma Aldrich).
2.8 Molecular docking simulation
To elucidate the possible mode of action of the compounds, molecular docking study
has been carried out with antiapoptotic target proteins viz. BCL-2 (PDB: 2O22), BCL-XL
(PDB: 2YXJ) and MCL-1 (PDB: 6QGD) using fully functional Molegro Virtual Docker
(Trial MVD 2010.4.0) software for Windows [34]. The docking run was conducted using a
GRID of 15 Å radius and 0.30 resolution with number of runs: 10 runs; maximum
interactions: 1500; maximum population size: 50; maximum steps: 300; neighbour distance
factor: 1.00; maximum number of poses returned: 5 [35]. Finally, the best docked pose was
further analyzed and visualized using Chimera software (https://www.cgl.ucsf.edu/chimera/)
and
Discovery
Studio
Visualization
BIOVIA
(https://www.3dsbiovia.com/products/collaborative-science/biovia-discovery-studio/) [36].
2.9 Statistical analysis
The experimental results were expressed as mean ± S.D. All the experments were
repeated for three times (n = 3). The data was analyzed by ANOVA considering *P ≤ 0.05
followed by post hoc, Tukey’s rank test.

3 Results and Discussion
3.1 Synthesis and general aspects
Compound 1 has been prepared by reacting one equivalent of MnCl2·4H2O, one
equivalent of bipy and two equivalents of Na-4-Clbz at ambient conditions in water medium.
Similarly, compound 2 has been isolated by the reaction between one equivalent of
CuCl2·2H2O, two equivalents of sodium benzoate and one equivalent of 2-MePy at room
temperature in water medium. Compounds 1 and 2 are fairly soluble in water and in common
organic solvents like methanol, DMSO etc. Compounds 1 and 2 exhibit room temperature μeff
values of 5.72 and 1.59 BM respectively, which indicate the presence of five and one
unpaired electron(s) per Mn(II) and Cu(II) centers in the compounds 1 and 2 respectively
[37]. The room temperature μeff values are found to be lower than that expected on the basis
of spin-only value; which may be due to spin exchange between the metal ions in the solid
state of the compounds owing to short metal···metal distances [37,38]. Similar magnetic
studies have also been reported for dinuclear Mn(II) and Cu(II) coordination compounds
[39].
3.2 Crystal structure
Fig. 1 represents the molecular structure of compound 1. Selected bond lengths and
bond angles around the Mn(II) centre have been summarized in Table 2. Compound 1
1
crystallizes in triclinic crystal system having P space group. In the crystal structure of
compound 1, there are two 4-Clbz bridged Mn(II) centers with similar coordination
environments. Each Mn(II) centers are hexa-coordinated with one bidentate bipy, two
coordinated water and two bridging 4-Clbz moieties. The di-positive charge of the cationic
complex moiety of compound 1 is compensated by two uncoordinated 4-Clbz molecules. X-
ray crystallographic analysis reveals that the cationic dinuclear Mn(II) complex moiety
possesses a crystallographic inversion center of symmetry, with the inversion centre located
at the midpoint of the compound. The coordination geometry around the Mn(II) centers are
distorted octahedron with the axial sites are occupied by the coordinated water molecules
(O1W and O2W); whereas the equatorial positions are occupied by N1, N13 from bipy and
O1´, O3´ from bridging 4-Clbz. The equatorial atoms viz. N1, N13, O1´ and O3´ are distorted
from the mean equatorial plane with the mean r.m.s. deviation of 0.0047 Å. The average Mn–
O and Mn–N bond distances are almost consistent with the previously reported Mn(II)
complexes [40]. The distance between the two Mn(II) centers is found to be 3.969(9) Å.

Fig. 1 Molecular structure of [Mn2(bipy)2(4-Clbz)2(H2O)4] 2(4-Clbz) (1).
Table 1 Crystallographic data and structure refinement details for the compounds 1 and 2
Parameters
Formula
1
2
C24H20N2O6Cl2
Mn
C40H34Cu2N
2O8
Formula
weight
558.26
797.77
Temp, [K]
Crystal
system
293(2)
Triclini
c
293(2)
Triclinic
Space group
a, [Å]
1
P
1
P
10.0290(
8)
7.5777(5)
b, [Å]
c, [Å]
11.0584(8)
15.2655(11)
10.9867(
8)
16.2890(
13)
α, [°]
β, [°]
γ, [°]
V, [Å3]
Z
101.499(3)
98.506(3)
107.141(3)
1168.31(14)
2
99.595(2)
90.724(2)
91.746(2)
1768.6(2)
2
Absorption
coefficient
(mm−1)
F(0 0 0)
7.091
1.960
570
82
0
D (calcd),
[Mg/m3]
1.578
1.498

Index
−9≤h≤9
−12≤h≤12
ranges
,
–
,
12≤k≤1
−13≤k≤13
3,
,
−18≤l≤18
−19≤l≤1
9
Crystal size,
[mm3]
0.38
0.34
0.21
×
×
0.33 × 0.32 ×
0.28
θ range, [°]
6.058
to
5.504
136.638
to
133.95
2
Independent
Reflections
Reflections
collected
Refinement
method
4075
6293
14237
25342
Full-
matrix
Full-
matrix
least-
least-
squar
es on
F2
squares on
F2
Data/restrai
nts/
4075/1/3
20
6293/14/5
27
parameters
Goodness-
of-fit on F2
1.092
1.098
Final
indices
>2σ(I)]
R
[I
R1
0.0788,
wR2
0.2021
R1
0.0880,
wR2
0.2140
=
R1
=
0.0421,
wR2 =
0.1143
=
=
=
R
indices
R1
=
(all data)
0.0424,
wR2 =
0.1146
Largest hole
1.97
0.38 and -0.45
and
peak
and -
0.54
[e·Å−3]
Table 2 Selected bond lengths (Å) and bond angles (°) of Mn(II) and Cu(II) centers in 1 and
2 respectively
Bond
lengths
of 1 (in
Å)
Bond
angles
of 1 (in
Deg)
Mn1–
O1'
2.151(3)
2.180(3)
2.145(3)
2.141(3)
O1'–
Mn1–
O1W
O1'–
Mn1–
N1
O1'–
Mn1–
N13
83.11(12)
143.91(13)
82.44(13)
84.96(13)
Mn1–
O1W
Mn1–
O2W
Mn1–
O3'#2
O1W–
Mn1–
N1

Mn1–
N1
2.323(4)
2.302(4)
O1W–
Mn1–
N13
O2W–
Mn1–
O1'
111.73(13)
90.09(13)
160.12(14)
Mn1–
N13
O2W–
Mn1–
O1W
O2W–
Mn1–
N1
O2W–
Mn1–
N13
O3'#2–
Mn1–
O1'
110.76(13)
85.69(13)
130.02(13)
O3'#2–
Mn1–
O1W
O3'#2–
Mn1–
O2W
O3'#2–
Mn1–
N1
O3'#2–
Mn1–
N13
N13–
Mn1–
N1
88.23(12)
81.87(12)
83.32(13)
144.89(14)
70.67(14)
Bond
lengths
of 2 (in
Å)
Cu1B–
O12B#2
Bond
angles
of 2 (in
Degree)
O1A–
Cu1A–
O3A#1
O1A–
Cu1A–
O10A
O1A–
Cu1A–
O12A#1
O(4)#2–
Cu(1)–
N(1)#2
O1A–
Cu1A–
N19A
O3A#1–
Cu1A–
Cu1A#1
1.9717(2)
1.9621(2)
1.9739(2)
1.9736(2)
1.9846(2)
2.2197(2)
2.202(2)
167.09(7)
88.03(7)
91.77(7)
96.52(12)
101.50(7)
84.28(5)
Cu1A–
O1A
Cu1A–
O3A#1
Cu1A–
O10A
Cu1A–
O12A#1
Cu1A–
N19A
Cu1B–
N19B
O3A#1–
Cu1A–
88.83(7)
O12A#1
Cu1B–
1.9646(16)
O3A#1–
91.40(7)

O1B
Cu1A–
N19A
Cu1B–
O3B#2
1.9754(16)
1.9755(16)
167.42(6)
103.23(7)
89.60(7)
O10A–
Cu1A–
Cu1A#1
O10A–
Cu1A–
O3A#1
86.74(5)
88.60(7)
Cu1B–
O10B
O10A–
Cu1A–
O12A#1
O10A–
Cu1A–
N19A
O3B#2–
Cu1B–
O10B
O3B#2–
Cu1B–
N19B
O10B–
Cu1B–
Cu1B#2
O10B–
Cu1B–
N19B
O12B#2–
Cu1B–
Cu1B#2
O12B#2–
Cu1B–
O3B#2
O12B#2–
Cu1B–
O10B
96.64(7)
81.15(5)
93.80(7)
86.23(5)
88.59(7)
167.37(7)
98.82(7)
167.31(7)
O12B#2–
Cu1B–
N19B
O1B–
Cu1B–
O3B#2
O1B–
Cu1B–
O10B
89.63(7)
89.40(7)
96.05(7)
O1B–
Cu1B–
O12B#2
O1B–
Cu1B–
N19B
#1 1-X, 2-Y, 2-Z; #2 1-X, 1-Y, 1-Z
The neighboring monomeric units of compound 1 are interconnected via C‒H∙∙∙Cl
hydrogen bonding interactions to form the 1D Supramolecular chain along the
crystallographic c axis (Fig. S1). The Cl1´ atom of coordinated 4-Clbz is involved in

C‒H∙∙∙Cl hydrogen bonding interactions with the –CH moiety of bipy having C3‒H3∙∙∙Cl1´
distance of 2.935(1) Å [C3∙∙∙Cl1´ = 3.480(1) Å] (Table 3).
The adjacent 1D chains of 1 are self-assembled by aromatic π-stacking and C‒H∙∙∙O
hydrogen bonding interactions to form the layered assembly along the crystallographic bc
plane (Fig. 2). The coordinated bipy moieties of adjacent 1D chains are involved in π-
stacking interactions having the centroid(N1, C2-C6)-centroid(N13, C7-C11) separation of
3.847 Å. The angle between the ring normal and the vector between the two ring centroids,
slipped angle, is found to be 20.3° [41]. The O1W water molecule is involved in bifurcated
C‒H∙∙∙O hydrogen bonding interactions with the –CH moieties of bipy having C5‒H5∙∙∙O1W
and C8‒H8∙∙∙O1W distances of 2.508(3) and 2.988(3) Å, respectively.
Fig. 2 Layered architecture of compound 1 assisted by π-π and C‒H∙∙∙O hydrogen bonding
interactions. Irrelevant aromatic hydrogen atoms are omitted for clarity.
Detailed investigation on the crystal structure of compound 1 reveals the enclathration
of uncoordinated 4-Clbz moieties in the layered assembly along the bc plane (Fig. 3). The
Cl1A atom of lattice 4-Clbz is involved in C‒H∙∙∙Cl hydrogen bonding interactions with the –
CH moieties of bipy having C3‒H3∙∙∙Cl1A and C10‒H10∙∙∙Cl1A distances of 2.946(1) and
2.983(1) Å, respectively [C3∙∙∙Cl1A = 3.853(4) Å; C10∙∙∙Cl1A = 3.531(4) Å]. The
carboxylate O atom (O1A) of lattice 4-Clbz is involved in C‒H∙∙∙O hydrogen bonding
interactions with –C5H5 moiety with C5‒H5∙∙∙O1A distance of 2.783(3) Å [C5∙∙∙O1A =
3.412(4) Å]. Similarly, the O3A atom is involved in O‒H∙∙∙O hydrogen bonding interactions
with O2W water molecule with O2W‒H2WA∙∙∙O3A distance of 1.963(3) Å [O2W∙∙∙O3A =
2.709(4) Å].

(a)
(b)
Fig. 3(a) Enclathration of lattice 4-Clbz moiety in the supramolecular tetrameric host cavity
of 1; (b) Layered assembly of compound 1. Irrelevant aromatic hydrogen atoms are omitted
for clarity.
The phenyl rings of the alternate coordinated and uncoordinated 4-Clbz moieties of
compound 1 are involved in unusual cooperative (π–π)2/(π–π)1/(π–π)2 ternary assemblies
along the crystallographic ac plane [42] (Fig. 5). (π–π)1 stacking interaction is observed
between the coordinated and uncoordinated 4-Clbz moieties having the centroid(C4A-C9A)–

centroid(C4´-C9´) separation of 3.7924 Å (Fig. 4). Similarly, (π–π)2 interaction is also
observed having the centroid(C4´-C9´)–centroid(C4A-C9A) separation of 3.7939 Å (Fig. 4).
The slipped angles for the (π–π)1 and (π–π)2 interactions are found to be 20.8° and 21.3°,
respectively. There are a few reports of the biological activities of coordination compounds
based on strong cooperative π-stacking interactions with DNA of the target cells [43].
Looking at the biological relevance, this cooperative (π–π)2/(π–π)1/(π–π)2 assembly has been
further studied theoretically (vide infra). Moreover, O‒H∙∙∙O hydrogen bonding interactions
are also observed in the π-stacked ternary assembly involving the coordinated water
molecules (O1W and O2W) and carboxyl O atoms (O1A and O3A) of uncoordinated 4-Clbz
moieties [O1W‒H1WA∙∙∙O1A = 1.893(3) Å; O2W‒H2WA∙∙∙O3A = 1.963(3) Å;
O2W‒H2WB∙∙∙O1A = 2.045(3) Å; O1W‒H1WB∙∙∙O3A = 1.968(3) Å]. Thus, supramolecular
layered architectures of the compound 1 observed along the two almost perpendicular
crystallographic bc and ac planes may result in 3D network architecture of the compound
Fig. 4 Cooperative (π–π)2/(π–π)1/(π–π)2 stacking interactions in compound 1 along the ac
plane. Aromatic H-atoms are omitted for clarity. Aromatic hydrogen atoms are omitted for
clarity.

Fig. 5 Layered architecture of compound 1 along the crystallographic ac plane assisted by the
cooperative (π–π)1/(π–π)2/(π–π)1 stacking interactions. Irrelevant aromatic hydrogen atoms
are omitted for clarity.
Fig. 6 represents the molecular structure of compound 2. Selected bond lengths and
bond angles corresponding to the Cu(II) centers are tabulated in Table 2. Compound 2
1
crystallizes in triclinic crystal system having P space group. Crystal structure analysis
reveals that compound 2 has a di-nuclear paddle wheel structure where the two Cu(II) centers
are bridged by four benzoato ligands. 2-MePy molecules are present at the remaining
coordination site of each Cu(II) ion. The molecular structure of compound 2 contains two
crystallographically similar neutral dimeric units, viz. [Cu2(bz)4(2-MePy)2] with minor
differences in bond angles and bond lengths (Table 2). The values of trigonality index (τ) are
found to be 0.0625 and 0.0172 for Cu1A and Cu1B respectively, which suggests that the
coordination environment for both the Cu(II) centers are square pyramidal [44]. The Cu∙∙∙Cu
distances are found to be 2.670 and 2.673 Å for Cu1A and Cu1B centers respectively. The O-
atoms of bridging bz moieties (O1A, O3A, O10A, O12A for Cu1A; O1B, O3B, O10B, O12B
for Cu1B) are occupying the equatorial positions of the Cu(II) centers, whereas the axial
positions are occupied by N-atoms of coordinated 2-AmPy (N19A for Cu1A; N19B for
Cu1B).

Fig. 6 Molecular structure of [Cu2(bz)4(2-MePy)2] (2). Aromatic hydrogen atoms are omitted
for clarity.
Structural analysis of compound 2 reveals the formation of a dimeric supramolecular
paddle wheel assembly assisted by non-covalent C‒H∙∙∙O, C‒H∙∙∙π and C‒H∙∙∙C contacts (Fig.
7). The coordinated O atoms of bridging bz (O3A and O10A) are involved in C–H⋯O
hydrogen bonding interactions with the –CH moieties of 2-MePy having C4–H4⋯O3A and
C6–H6B⋯O10A distances of 2.931(1) and 2.466(2) Å [C4⋯O3A = 3.852(1) Å; C6⋯O10A
= 3.423(7) Å]. C‒H∙∙∙π interactions are also observed in the dimeric supramolecular assembly
of compound 2 having C14A-Cg and H14A-Cg distances of 3.732 and 3.181 Å, respectively
(Cg is the aromatic ring centroid defined by N19B, C1-C5) [45,46]. Moreover, –CH groups
of the methyl units of 2-MePy moieties are involved in C–H⋯C contacts [47] which also
stabilize the dimeric supramolecular assembly [C6–H6A⋯C21A = 3.177 Å, C(sp3)–
H⋯C(sp3), C6⋯C21A = 3.384 Å; C6–H6C⋯C21A = 3.586 Å, C(sp3)–H⋯C(sp3),
C6⋯C21A = 3.384 Å; C21A–H21E⋯C6 = 3.388 Å, C(sp3)–H⋯C(sp3), C6⋯C21A = 3.384
Å; C21A–H21F⋯C6 = 3.060 Å, C(sp3)–H⋯C(sp3), C6⋯C21A = 3.384 Å]. The neighboring
dimeric supramolecular paddle wheel assemblies are interconnected to form 1D
supramolecular chain along the crystallographic c axis (Fig. 8).

Fig. 7 Dimeric paddle wheel supramolecular assembly formed in compound 2 assisted by
C‒H∙∙∙O, C‒H∙∙∙π and C‒H∙∙∙C contacts. Irrelevant aromatic hydrogen atoms are omitted for
clarity.
The adjacent 1D chains of compound 2 are interconnected via C–H⋯O hydrogen
bonding and C–H⋯C interactions to form the layered architecture along the crystallographic
bc plane (Fig. S2). O1B and O10B atoms of bz are involved in C–H⋯O hydrogen bonding
interactions with the C23A–H23A⋯O1B and C15A–H15A⋯O10B distances of 2.715(1) and
2.679(1) Å respectively. Non-covalent C–H⋯C interactions are observed in the layered
assembly of the compound within expected distances [C25A–H25A⋯C16A = 2.895 Å,
C(sp3)–H⋯C(sp3), C25A⋯C16A = 3.622 Å; C24A–H24A⋯C15A = 2.949 Å, C(sp3)–
H⋯C(sp3), C25A⋯C16A = 3.767 Å] [48].
Fig. 8 1D supramolecular chain of compound 2.

Table 3 Selected hydrogen bond distances (Å) and angles (°) for the compounds 1 and 2.
D–
H⋯
A
d(D–
H)
<(DH
A)
d(D⋯
A)
d(H⋯
A)
1
0.950(
4)
3.853(
4)
2.946(
1)
160.1(
2)
C3‒H3⋯Cl1A
0.950(
4)
3.517(
5)
2.983(
4)
109.5(
2)
C10‒H10⋯Cl1
A
0.950(
4)
3.412(
4)
2.783(
3)
128.5(
3)
C5‒H5⋯O1A
0.874(
3)
2.709(
4)
1.963(
3)
142.3(
3)
O2W‒H2WA⋯
O3A
0.873(
3)
2.736(
4)
2.045(
3)
135.4(
3)
O2W‒H2WB⋯
O1A
0.869(
3)
2.754(
4)
1.893(
3)
170.3(
2)
O1W‒H1WA⋯
O1A
0.870(
2)
2.780(
4)
1.968(
3)
154.8(
2)
O1W‒H1WB⋯
O3A
2
0.950(
1)
3.852(
1)
2.931(
1)
163.4(
8)
C4‒H4⋯O3A
C6–
0.980(
6)
3.423(
7)
2.466(
2)
165.2(
4)
H6B⋯O10A
C15A‒H15A⋯
O10B
0.950(
2)
3.607(
3)
2.679(
1)
165.5(
1)
0.950(
3)
3.498(
3)
2.715(
1)
140.1(
1)
C23A‒H23A⋯
O1B
3.3 Spectral studies
3.3.1 FT-IR spectroscopy
The IR spectral analyses of the compounds have been discussed in detail (See ESI;
Fig. S3). The differences between the asymmetric (as) and symmetric (s) stretching
vibrations for the compounds 1 and 2 ( = 247 and 251 cm-1) indicate that 4-Clbz and bz
moieties behave as bridging O-donor ligands in the compounds [49]. The red shifting of
ν(C=N) stretching frequency of bipy to the lower frequency region in compound 1 indicates
the coordination of N-atoms of bipy to the Mn(II) centers [50]. The absorption peaks for
coordinated 2-MePy in compound 2 are obtained at expected positions (See ESI) [51,52].
3.3.2 Electronic spectroscopy
The electronic spectra of the compounds 1 and 2 were recorded in both the solid and
aqueous phases and discussed in detail (See ESI; Figs. S4 and S5). The spectra of the
compounds indicate the presence of distorted octahedral Mn(II) and square-pyramidal Cu(II)

centers in 1 and 2 respectively [53]. The absorption peaks for the π→π* transition of the
aromatic ligands are obtained at the expected positions [54].
3.3.3 EPR spectral analysis
Fig. 9 depicts the X-band EPR spectrum of the crystalline sample of the compound 1
at room temperature and is characterized by several resonances. The spectrum shows an
intense signal centered on g = 2.27. The spectrum is similar to that of hexa-coordinated
Mn(II) complexes with 6S5/2 ground state [55]. The broad signal of the compound 1 is due to
dipolar interactions and random orientations of the Mn2+ ions [56,57]. Hyperfine splitting of
the Mn(II) ion is not observed in the solid state room temperature spectrum of compound 1
[57]. Similar room temperature solid state X-band EPR has been reported for a dinuclear
Mn(II) compound viz. [{Mn(phen)2}2(µ-4-tBuC6H4COO)2](ClO4)2 (phen
=
1,10-
phenanthroline, tBuC6H4COO = tertiary butyl benzoate) [58].
Fig. 9 X-band EPR spectrum of the crystalline sample of compound 1.

The spectrum of compound 2 (Fig. 10) with g|| = 2.24 and g⊥ = 2.05 is consistent with
the square-pyramidal geometries found around the Cu(II) centers [59]. The spectrum contains
a broad spectral line without hyperfine splitting which is commonly observed in paddle-
wheel type copper(II) carboxylates [60]. The observed g|| ˃ g⊥ suggests that the dx2-y2 orbital
is the ground state of the Cu(II) ion having distorted square-pyramidal geometry around
penta-coordinated Cu(II) centers; thereby ruling out the possibility of trigonal bipyramidal
geometry [61]. Similar solid state X band room temperature EPR spectrum was reported for a
paddle wheel di-nuclear Cu(II) complex involving 1,3,5-benzenetricarboxylic acid as the
bridging ligand [62].
Fig. 10 X-band EPR spectrum of the crystalline sample of compound 2.
3.4 Thermogravimetric analyses
Thermogravimetric curves of the compounds 1 and 2 were obtained in 25-800°C
under N2 atmosphere at the heating rate of 10°C/min (Fig. S6). For compound 1, the 5.75%
(calcd. = 6.44%) weight loss in the temperature range 70-127C is due to the loss of the four

coordinated water molecules [63]. In the temperature range 128-372°C, the weight loss of
39.72% (calcd. = 42.65%) corresponds to loss of the two lattice 4-Clbz and one coordinated
4-Clbz moieties [64]. The 21.24% weight loss in the temperature range of 375-800°C
corresponds to the loss of remaining coordinated 4-Clbz and 0.5 molecule of bipy [64,65].
For compound 2, in the temperature range of 170-310°C, the weight loss of 42.11% can be
attributed to the loss of two bz and one 2-MePy moieties (calcd. = 43.52%) [66]. In the
temperature range 310-800°C, remaining two molecules of the bz and one molecule of 2-
MePy are decomposed with a weight loss of 41.14% (calcd. = 43.52%) [66].
3.5 Theoretical study
In the present study, theoretical investigation is focused on the analysis of the
cooperative π-stacked assemblies involving alternate coordinated and uncoordinated 4-Clbz
moieties in compound 1, as described in the crystal packing (see Figs. 4 and 5) and hydrogen
bonding (HB) interactions. We have computed the molecular electrostatic potential (MEP)
surface of compound 1 using a centro-symmetric charge neutral assembly, which is shown in
Fig. 11. It consists of the dicationic [Mn2(bipy)2(4-Clbz)2(H2O)4]2+ core interacting with two
uncoordinated 4-Clbz anionic moieties. The MEP surface of this model shows that the H-
atoms of both Mn coordinated water molecules converge to the same region, generating a
large and positive region of MEP (+58 kcal/mol), adequate for interactions with electron rich
atoms or moieties. This large MEP value reveals that the enhanced acidity of these H-atoms
is due to the coordination to Mn(II). The MEP minimum is located at the O-atoms of the
uncoordinated 4-Clbz ligands.

Fig. 11(a) MEP surface of the neutral assembly of compound 1; (b) Assembly used to
generate the MEP surface in the same orientation used to plot the MEP. The energies at some
points of the surface are given in kcal/mol.
Fig. 12 shows two complexation processes that have been used to evaluate the
interesting (π–π)2 and H-bonding interactions between the uncoordinated 4-Clbz moieties and
the neutral model of compound 1 (Fig. 12a), which has been previously used to compute the
MEP surface, i.e. [Mn2(bipy)2(4-Clbz)2(H2O)4]2(4-Clbz). We have used a neutral model to
minimize the effect of the purely coulombic attraction between the counter-ions. The
complexation energy of neutral [Mn2(bipy)2(4-Clbz)2(H2O)4]2(4-Clbz) with two
uncoordinated 4-Clbz is quite large (–36.9 kcal/mol, see Fig. 12b) due to the formation of 4
HBs and two π-stacking interactions. In order to estimate the contribution of the H-bonds we
have computed a theoretical model where formate anions instead of 4-Clbz moieties have
been used (see Fig. 12c). Therefore, the π-stacking interactions are not established and only
the HBs are evaluated. As indicated in Fig. 12, the complexation energy is slightly reduced
by –32.1 kcal/mol, that correspond to the contribution of the four HBs (around –8 kcal/mol
each H-bond). This large interaction energy agrees well with the MEP surface analysis (the
maximum is located at the H-atoms). The contribution of both symmetrically equivalent (π–
π)2 can be estimated by difference (–4.8 kcal/mol), thus revealing the attractive nature of
these π-stacking interactions, which are likely dominated by dispersion effects, since the
anionic nature of the ligands anticipate a repulsive contribution of electrostatics. In fact, the
MEP surface of compound 1 shown in Fig. 11 shows negative value of MEP over the center
of the 4-Clbz ligands (–6 kcal/mol). To further confirm this explanation, we have computed
an extra model where the anionic 4-Clbz molecules have been replaced by neutral
chlorobenzenes (see Fig. 12d), thus diminishing the electrostatic repulsion. The resulting
interaction energy is –18.3 kcal/mol, larger (in absolute value) than that estimated by
difference (–4.8 kcal/mol) due to the neutral nature of the chlorobenzene. This energetic
analysis clearly confirms the unfavorable electrostatic contribution in the (π–π)2 stacking
interaction.

Fig. 12(a) Neutral [Mn2(bipy)2(4-Clbz)2(H2O)4]2(4-Clbz) used as starting point to evaluate
the HBs and (π–π)2 interactions shown as black dashed lines; (b) Assembly of compound 1
analyzed using DFT calculations; (c) Theoretical model of the assembly where two
uncoordinated 4-Clbz ligands have been replaced by formate ligands; (d) Theoretical model
of the assembly where two uncoordinated 4-Clbz ligands have been replaced by
chorobenzene ligands. Distances in Å.
We have also computed the “Non-covalent Interaction plot” (NCI plot) index in order
to characterize both π–π binding modes and HBs in compound 1. The NCI plot index is an
intuitive visualization index that facilitates the visualization of non-covalent interactions and
shows which molecular regions interact. The color scheme is a red-yellow-green-blue scale
with red (repulsive) and blue (attractive). Yellow and green surfaces correspond to weak
repulsive and weak attractive interactions, respectively. Fig. 13 shows the NCI plot index
surfaces of the assembly of compound 1 that includes the cationic [Mn2(bipy)2(4-
Clbz)2(H2O)4]2+ and four uncoordinated 4-Clbz. The π-stacking interactions are characterized
by very extended and green isosurfaces than embrace the whole ligands including the C–Cl
bond. Moreover, the strong nature of the O–H···O hydrogen bonds is confirmed by the
presence of dark blue isosurfaces located between the interacting H and O-atoms. The large
interaction energies along with the NCI plot index analysis confirms that the π-stacking
assemblies in combination with the H-bonds are strong binding motifs, which have a strong
influence on the solid state packing of compound 1.

Fig. 13 NCI surface of the assembly of compound 1. The gradient cut-off is sꢀ=ꢀ0.35 a.u. and
the color scale is −0.04ꢀ<ꢀρꢀ<ꢀ0.04 a.u.
3.6 Cell viability assay
Cell-based cytotoxic assays are the most frequently used techniques in preclinical
drug developments to investigate the cytotoxic potentials of compounds/drugs as well as
their impacts on cell proliferation [67]. The MTT based assay has been widely explored to
measure cell proliferation after treatment with the potential drug candidates. Living cells
with active cellular metabolism are capable of converting the MTT dye to a purple colored
formazan product with maximum absorbance near 570 nm [68]. Due to the lack of active
metabolism, nonviable cells cannot convert MTT into formazan crystals, which make this
technique a convenient indicator of only the viable cells [69]. 24 hours treatment with the
compounds 1 and 2 revealed that the compounds induced significant concentration dependent
cytotoxicity in DL cells with negligible effects on healthy normal PBMC cells (Fig. 14). In
comparison, compound 1 induced higher cytotoxicity at higher concentrations (0.5-10 μM)
than that of compound 2. The half-maximal inhibitory concentration (IC50) is one of the most
frequently used parameters to assess the effectiveness of the compounds/drugs in preventing

a specific biological activity. The dose response curves of the compounds 1 and 2 in DL and
PBMC cell lines are shown in Fig. 15.
Fig. 14 Compounds 1 and 2 mediated percentage cell cytotoxicity in DL cells after treatment
with different concentrations. Cisplatin was used as a positive (reference drug) control. Data
are mean ± S.D., n = 3, one way ANOVA, *P ≤ 0.05 as compared to compound 2.

Fig. 15 Dose response curves obtained for calculating IC50 values of the compounds 1 and 2
in DL and PBMC cell lines.
Table S1 depicts the IC50 values of the compounds in DL and PBMC cell lines.
Dimitrijevic et al. have also recently explored the cytotoxic potential of a di-nuclear paddle
wheel Cu(II) complex, viz. [Cu2(S-ipr-thiosal)4(H2O)2]·2H2O (S-ipr-thiosal = S-isopropyl
derivate of thiosalicylic acid) against murine lung cancer cell lines with negligible
cytotoxicity in normal cells [70].

3.7 Apoptosis study
Apoptosis assay helps us to identify and quantify biological events associated with
the programmed cell death, viz. chromatin condensation, nucleus fragmentation, membrane
blebbing, cell membrane permeability, caspase activation, phosphatidylserine (PS)
exposure on the cell surface and DNA fragmentation [71]. Acridine orange (AO) and
ethidium bromide (EB) dual staining assay is a quick and easy technique to investigate the
cell morphology and quantification of apoptosis [72]. AO is capable to enter inside the cells
and causes the nuclei to appear as green; whereas EB is taken up only by the cells with
damaged cytoplasmic membrane integrity and causes the nuclei appear as red [73].
Fig. 16 Morphological features of viable (green) and apoptotic (red) cells observed using
fluorescence microscope. In a light microscopy-based study, a similar observation was made
(arrow: membrane lysis and phagocytosis). Apoptotic nucleus with membrane disruption and
chromatin condensation was observed in treated cells. Cisplatin was used as a reference drug.
Lower panel graph showed percentage of apoptotic cells after treatment with compounds 1
and 2 in DL cell line. Data are mean ± S.D., n = 3, One way ANOVA, *P ≤ 0.05 as compared
to compound 2. Scale bar = 20 μm.

Early apoptosis corresponds to the presence of bright green nucleus with condensed or
fragmented chromatin, while late apoptotic cells contain condensed and fragmented orange
chromatin [74]. The therapeutic efficacy of cisplatin and carboplatin is mediated through
interactions with DNA, RNA and protein, however the cytotoxic effect is predominantly
induced by the production of DNA interstrand and intrastrand crosslinks, which is followed
by apoptosis [75]. 24 hours of treatment with the compounds 1 and caused DL cells to
develop a characteristic apoptotic features (Fig. 16) with a red/orange nucleus. Comparative
analysis reveals that compound 1 induced higher apoptosis than that of compound 2 at all the
concentrations (0.01-10 μM) used and the obtained results can be correlated with the
cytotoxicity assay (MTT). Moreover, it has been well established that cancerous cells possess
higher levels of oxidative stress and hypoxia than healthy normal cells, resulting in more
oxidative DNA damage [76]. This may be the possible reason that the compounds induced
higher cytotoxicity in cancerous cells as compared to normal PBMC cell line. Light
microscopy based cell morphology also revealed cell lysis and apoptotic cell death as
evidenced by degraded plasma membrane (Fig. 16). The cytotoxic activities of two novel di-
nuclear Ni(II) and Co(II) compounds, viz. [Ni2(L)2(H2O)3]·4H2O and [Co2(L)2(H2O)3]·5H2O
(H2L =4,5-bis(pyrazol-1-yl)phthalic acid) have been reported against human cervix and oral
epithelia carcinoma cells [77].
3.8 Molecular docking
Molecular docking simulation has played crucial roles in computer-aided drug design
and considered as an exemplary tool in the current structure based drug designing [78]. It
helps us to examine the intermolecular framework established between a receptor protein and
a small molecule or between two different proteins, as well as to indicate the binding modes
responsible for the inhibition of proteins [79]. The observed cytotoxicity and apoptotic cell
death induced by the compounds 1 and 2 prompted us to investigate the possible mode of
action of the compounds. Antiapoptotic target proteins viz. BCL-2, BCL-XL and MCL-1
have been reported to play significant roles in the initiation and execution of apoptosis
processes by blocking the activities of these target proteins [80]. Docking results with
antiapoptotic target proteins revealed that the compounds 1 and 2 have strong affinity (Table
S2) for antiapoptotic target proteins (Figs. 17-19).