Evaluation of catacholase mimicking activity and apoptosis in human colorectal carcinoma cell line by activating mitochondrial pathway of copper(II) complex coupled with 2-(quinolin-8-yloxy)(methyl)benzonitrile and 8-hydroxyquinoline

Article abstract of DOI:10.1016/j.bioorg.2020.104479

To evaluate the cytotoxic potential of metal-based chemotherapeutic candidate towards the colorectal cancer, we have synthesized a new copper(II) complex [Cu(qmbn)(q)(Cl)] (1) (where, qmbn = 2-(quinolin-8-yloxy)(methyl)benzonitrile and q = 8-hydroxyquinoline) and structurally characterized by single crystal X-ray, Powder-XRD, FTIR and thermogravimetric analysis (TGA). The structural analysis reveals that copper(II) ions exist in a distorted square pyramidal (τ = ~0.1), with ligation of a chloride ion, oxygen atom and two nitrogen atoms at equatorial position and one oxygen atom at apical position. The cytotoxicity potential of complex 1 was executed against human colorectal cell lines (HCT116), which showed that 1 induces mitochondrion-mediated apoptotic cell death via activation of the Bax (pro-apoptotic protein) caspases-3 and 9 proteins. Interestingly, complex 1 was found to be a good candidate as electron-transfer catalyst which mimics catacholase with high turnover frequency (k_cat = 1.03 × 10² h^?1) for the conversion of the model substrate 3,5-di-tertbutylcatechol (3,5-DTBC) to 3,5-di-tertbutylquinone (3,5-DTBQ). Furthermore, molecular docking studies revealed that complex 1 was successfully localized inside the binding pocket of protein kinase (Akt), which validate the mechanism and mode of interaction of 1 that displayed cytotoxic activity experimentally. The obtained outcomes reveal that the complex 1 could be utilized as an encouraging perspective in the development of new therapeutic candidate for colon cancer.

Full text of DOI:10.1016/j.bioorg.2020.104479

Journal Pre-proofs
Evaluation of catacholase mimicking activity and apoptosis in human colorec-
tal carcinoma cell line by activating mitochondrial pathway of copper(II)
complex coupled with 2-(quinolin-8-yloxy)(methyl)benzonitrile and 8-hy-
droxyquinoline
Arif Ali, Snehasis Mishra, Saima Kamaal, Abdullah Alarifi, Mohd Afzal,
Krishna Das Saha, Musheer Ahmad
PII:
S0045-2068(20)31777-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104479
YBIOO 104479
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 September 2020
7 November 2020
12 November 2020
Please cite this article as: A. Ali, S. Mishra, S. Kamaal, A. Alarifi, M. Afzal, K. Das Saha, M. Ahmad, Evaluation
of catacholase mimicking activity and apoptosis in human colorectal carcinoma cell line by activating
mitochondrial pathway of copper(II) complex coupled with 2-(quinolin-8-yloxy)(methyl)benzonitrile and 8-
hydroxyquinoline, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104479
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Evaluation of catacholase mimicking activity and apoptosis in human
colorectal carcinoma cell line by activating mitochondrial pathway of
copper(II) complex coupled with 2-(quinolin-8-yloxy)(methyl)benzonitrile and
8
-hydroxyquinoline
1
‡
2‡
1
3
3*
Arif Ali, Snehasis Mishra, Saima Kamaal, Abdullah Alarifi, Mohd Afzal, Krishna Das
2
1*
Saha , Musheer Ahmad
1
Department of Applied Chemistry, ZHCET, Aligarh Muslim University, Aligarh, 202002, India
Cancer & Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata
2
7
00032, India
3
Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi
Arabia
‡A.Ali and S. Mishra contributed equally to the article.
1

Abstract
To evaluate the cytotoxic potential of metal-based chemotherapeutic candidate towards the
colorectal cancer, we have synthesized a new copper(II) complex [Cu(qmbn)(q)(Cl)] (1) (where,
qmbn = 2-(quinolin-8-yloxy)(methyl)benzonitrile and q = 8-hydroxyquinoline) and structurally
characterized by single crystal X-ray, Powder-XRD, FTIR and thermogravimetric analysis
(TGA). The structural analysis reveals that copper(II) ions exist in a distorted square pyramidal
(τ = ~0.1), with ligation of a chloride ion, oxygen atom and two nitrogen atoms at equatorial
position and one oxygen atom at apical position. The cytotoxicity potential of complex 1 was
executed against human colorectal cell lines (HCT116), which showed that 1 induces
mitochondrion-mediated apoptotic cell death via activation of the Bax (pro-apoptotic protein)
caspases-3 and 9 proteins. Interestingly, complex 1 was found to be a good candidate as electron-
2
-1
transfer catalyst which mimics catacholase with high turnover frequency (kcat = 1.03 × 10 h )
for the conversion of the model substrate 3,5-di-tertbutylcatechol (3,5-DTBC) to 3,5-di-
tertbutylquinone (3,5-DTBQ). Furthermore, molecular docking studies revealed that complex 1
was successfully localized inside the binding pocket of protein kinase (Akt), which validate the
mechanism and mode of interaction of 1 that displayed cytotoxic activity experimentally. The
obtained outcomes reveal that the complex 1 could be utilized as an encouraging perspective in
the development of new therapeutic candidate for colon cancer.
Keywords: Copper(II) complex; Single crystal; MTT assay; Molecular docking; Mitochondrial
pathway.
2

1
. Introduction
Colorectal cancer (CRC), also recognized as large bowel cancer is the third leading cause of
cancer death worldwide, and its incidence is gradually rising [1,2]. CRC is a solid tumor arises
from the glandular, epithelial cells of the large intestine which encompass aberrant growths in
the appendix and large intestine [3–5]. Deplorably, patients with CRC are first detected by
oncologists in the metastatic CRC (stage IV); so, that is hard for them to make a widespread
recovery [6]. Although innumerable therapeutic regimes such as surgery, chemotherapy has been
extensively applied to treat CRC with different curative care. However, the treatment of CRC is
restricted by the reduced cytotoxicity of existing chemotherapeutic drugs towards cancer cells
due to lack of the tumor suppressor gene p53 [7,8]. The p53 protein is the most crucial tumor
suppressor protein defends the cells from acquiring genetic impairment. Nevertheless, the p53
tumor suppressor gene is normally itself mutated during the development of colorectal cancer
[9–12]. The activation of p53 gene includes a series of phosphorylation events and post
translational modifications which affect the expression of numerous p53-target genes involved in
DNA repair, growth arrest, apoptosis, senescence, angiogenesis, and autophagy [13]. Thus, it is
necessary to maintain the epithelial integrity and to witness the impression of p53 protein
induced by therapeutic candidates is of prime concern. Despite much research and progress made
on the expectation of colon cancer, significant inhibition of tumors is still hardly achieved, and
the treatment with known agents can lead to tumor progression in some cases also. The early-
stage recognition and treatment regimes with proper therapeutic modalities play a pivotal role in
reducing the incidence of colon cancer [14,15].
3

The design and synthesis of metal–organic complexes have attracted remarkable attention
for their tunable and functionalized architectures which proves potential therapeutic agent for
treating cancer with antioxidant activity [16–23]. Structurally, quinoline-bearing heterocyclic
pharmacophores are well-known for their wide range of biological activities such as antifungal
[24], antibacterial [25], antitumor [26], and HIV-1 replication inhibitors [27]. Among the
quinoline core derivatives, 8-hydroxyquinoline (8-HQ) is the most studied lipophilic metal(II)
ion chelator and represents an excellent scaffold with notable features in the area of medicinal
applications [28]. Ortiz et al synthesized quinoline-based berberine derivatives, namely
NAX012, NAX014, and NAX018 and investigated as promising anticancer drugs for their
effects on human colon carcinoma cell lines [29]. Recently, Ai and coworkers reported the newly
designed pyrimido[5,4-c]quinoline-4-(3H)-one compounds as possible candidates for
cytotoxicity against numerous human cancer cell lines (KB, CNE2, MGC-803, GLC-82, MDA-
MB-453, and MCF-7) [30].
In the past few decades, the metal-based drugs have been encouraged after the successful
implementation of cis-platin and its derivatives for their use in the various kinds of tumors [31].
However, dose-dependent toxicity and resistance coupled with a narrow spectrum of activity
associated with these platinum-based drugs restricted their clinical use [32,33]. Therefore, it is
pertinent to synthesize a new therapeutic modalities resulting from endogenous transition metal
ions, whose mode of action is different from existing platinum based drugs. In this line,
copper(II) complexes have been widely studied, which display enhanced cytotoxic profile and
reduced toxicity [34–36]. Copper is a physiologically important metal ion that shows a potential
role in a redox mechanism and triggers the release of reactive oxygen species (ROS) which
induces apoptosis in cancer cells. Copper ions possesses high affinity for nucleobases due to
4

their selective permeability of cancer cell membranes, hence copper complexes are preferred
37–39]. Additionally, copper has been found to be a part of essential cofactor of several
[
enzymes involved in oxidative metabolism, ceruloplasmine, superoxide dismutase, ascorbic acid
oxidase and tyrosinase and so on [40].
In the present article, we have designed and fabricated a new copper(II) complex
[Cu(qmbn)(q)(Cl)] (1). The cytotoxic activity of the complex 1 was investigated against HCT
1
16 (human colon cancer cell line) to examine its potential as promising chemotherapeutic drugs
towards colorectal cancer. Furthermore, catacholase mimics activity of complex 1 has also been
explored which shows good turnover frequency number for the conversion of the model
substrate 3,5-di-tertbutylcatechol (3,5-DTBC) to 3,5-di-tertbutylquinone (3,5-DTBQ).
2
. Experimental section
.1. Materials
2
Reagent grade 2-(bromomethyl) benzonitrile and 8-hydroxyquinoline were purchased from
Sigma Aldrich and used as received. All solvents and K CO , CuCl ·2H O, KI, NaOH were
2
3
2
2
obtained from Thermo Fisher Scientific, India.
.2. Physical measurements
2
-1
Infrared spectra were collected (KBr disk, 4000-400 cm ) utilizing Thermo Scientific iS50
FTIR. Microanalysis was done using a CE–440 elemental analyzer (Exeter Analytical Inc.).
Thermal Gravimetric analysis (TGA) was obtained on a Mettler Toledo Star System (heating rate
°
-1
of 10°C/min). Powder X-ray diffraction patterns (CuK radiation with scan rate 4 min at 298K)
α
°
°
were measured from labX XRD-6100 X-ray diffractometer instrument in the range of 5 -40 at
4
0kV and 30mA. Uv-visible and photoluminescence spectra were carried out with Thermo
5

Scientific Evolution 201 UV-visible spectrophotometer and Perkin-Elmer LS55 fluorescence
spectrophotometer.
2
.3. Crystallography
Single crystal X-ray data of complex 1 was collected with a Bruker SMART APEX CCD
diffractometer using monochromatic Mo-Kα radiation (λ =0.71073 Å) at 100(2) K. The linear
absorption coefficients, and the anomalous dispersion corrections were mentioned from the
International Tables for X–ray crystallography [41]. Using Olex2 [42], the structure was solved
with the olex2.solve [43] structure solution program using Charge Flipping and refined with the
olex2.refine [43] refinement package using Gauss-Newton minimization. All hydrogen atoms
were located in difference Fourier maps in the structures and refined isotropically. All non–H
atoms were refined anisotropically. The crystallographic data for complex 1 was summarized in
Table 1. Selected bond parameters of complex 1 were given in Table S1.
Table 1. Crystal and structure refinement data for complex 1
CCDC
2003694
Empirical formula
C H ClCuN O
2
2
6
18
3
Formula weight
Temperature/K
Crystal system
Space group
503.45
100(2)
orthorhombic
Pbc21
a/Å
b/Å
c/Å
α/°
β/°
7.9452(7)
23.018(2)
23.2430(16)
90
90
γ/°
Volume/Å
Z
90
4250.7(6)
8
1.5733
1.184
3
3
ρ g/cm
calc
μ/mm-1
F(000)
Crystal size/mm
2060.6
0.38 × 0.26 × 0.17
3
6

Radiation
Mo Kα (λ = 0.71073)
2
Θ range for data collection/° 5.12 to 50
-
3
10 ≤ h ≤ 10, -30 ≤ k ≤ 30, -31 ≤ l ≤
1
Index ranges
Reflections collected
63245
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
7484 [R = 0.2253, R
7484/1/595
1.028
= 0.1667]
sigma
int
Final R indexes [I>=2σ (I)] R = 0.0618, wR = 0.1360
1
2
Final R indexes [all data]
Largest diff. peak/hole / e Å 0.83/-0.94
R = 0.1217, wR = 0.1766
1 2
-3
2
.3.
.3.1. Synthesis of 2-(quinolin-8-yloxy)(methyl)benzonitrile(qmbn)
-hydroxyquinoline (2g, 13.7 mmol) and dry K CO (5g, 36.17mmol) were mixed in a round-
Synthesis
2
8
2
3
bottom flask containing acetonitrile (60 mL) under an inert atmosphere. The mixture was
°
allowed to stir for 1 hr at 90 C. Now, the mixture was added with 2-(bromomethyl)benzonitrile
(2.69g, 13.7 mmol), and the resulting solution was refluxed for 24h. After completion of the
reaction, the solution was allowed to cool at room temperature and poured slowly in ice water
(100 mL) with constant stirring to give a white muddy solid precipitate that was collected by
°
filtration and dried under vacuum. Yield: 3.3g (70%). M.P.: 120 C. Elemental analysis (%):
Calcd. For C H N O (260.27): C, 78.44; H, 4.65; N, 10.76%; Found: C, 78.54; H, 4.56, N,
1
7
12
2
-
1
1
1
7
2
0.71%. IR (cm ): 3398(s), 2924(m), 2922(m), 2824(w), 2224(w), 1619(w), 1564(w), 1503(m),
463(m), 1379(m), 1324(m), 1262(m), 1177(w),1100(m), 1027(s), 856(w), 794(m), 753(m),
13(w), 668(w), 547(w),. ESI-MS: m/z (100%) 261.1046 [M+1].
.3.2. Synthesis of complex [Cu(qmbn)(q)(Cl)] (1)
A mixture containing ligand (qmbn)(0.02g) and 8-hydroxyquinoline (q) (0.02g) was dissolved in
mL methanol in a round bottom flask and added dropwise aqueous solution of CuCl ·2H O
3
2
2
7

°
(
0.08g) and then added 5 drops of NaOH (1 M) solution and allowed to stir for 6h at 90 C. The
resulting solution was cooled down and then filtered. The clear green colored filtrate was kept
for evaporation at room temperature. After 2-3 weeks, the green block shape crystals were
°
obtained, which was suitable for single crystal X-ray analysis. Yield: 75%, M.P.: 186 C.
Elemental analysis (%): Calcd. For C H ClCuN O (503.45): C, 62.03; H, 3.57; N, 8.35%;
2
6
18
3
2
-1
Found: C, 62.41; H, 3.36, N, 8.25%. IR (cm ): 3380(w), 3215(w), 3033(m), 2921(w), 2817(w),
229(w), 1658(s), 1614(w), 1572(w), 1502(s), 1462(s), , 1104(s), 818(s), 741(w), 573(m) (Fig.
S1).
.4.
2
2
Cell lines and chemicals
Cell lines (human colorectal carcinoma, HCT116) were purchased from NCCS, Pune, India.
Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin–neomycin (PSN)
antibiotic, fetal bovine serum (FBS), trypsin and ethylenediaminetetraacetic acid (EDTA) were
purchased from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic ware was purchased
from NUNC (Roskilde, Denmark). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was procured from SRL, India. Chromium nitrate was bought from Sigma-
Aldrich.
2
.5.
Cell culture
The human colorectal carcinoma cell line HCT116 was cultured in DMEM with 10% fetal
°
bovine serum (FBS) and 1% antibiotic (PSN) at 37 C in a humidified atmosphere under 5%
CO . After 75–80% confluency, the cells were harvested with 0.025% trypsin and 0.52 mM
2
EDTA in phosphate buffered saline (PBS), and seeded at a required density to allow them to re-
equilibrate a day before the start of experiment [44].
8

2
.6.
The MTT assay was carried out to estimate the cell viability [45]. The HCT 116 and HEK 293
Human embryonic kidney 293) were plated in 96-well plates and treated with or without
MTT assay
(
different concentrations of complex 1 for 12h. After 12hr, MTT solution was added and
incubated for four hours. After the incubation period of MTT, formazan was solubilized with
acidic isopropanol and the absorbance of the solution was measured at 595 nm using an ELISA
reader.
2
.7.
Quantification of apoptosis and necrosis using flow cytometry
Determination Apoptosis and necrosis were analyzed by flow cytometry using an Annexin-V
FITC/PI apoptosis/necrosis detection kit (Calbiochem, CA, USA) [46]. Treated cells (1×106)
were washed and stained with Annexin-V-FITC and PI in accordance with the manufacturer′s
instructions. The percentages of viable, apoptotic (early and late) along with necrotic cells were
estimated by flow cytometry (BD LSR Fortessa, San Jose, CA, USA).
2
.8.
Determination of intracellular ROS (iROS)
Mitochondria are the primary source of the amplification of reactive oxygen species (ROS)
production in mammalian cells and it plays a major part in the stimulation of apoptosis in a
variety of cells [47]. To evaluate the intercellular ROS, we incubated the treated cells with 10
°
μM H2DCFH-DA (2′, 7′-dichlorofluorescein diacetate) at 37 C for 25 min before the analysis by
flow cytometer (BD LSR Fortessa, Becton Dickinson, Franklin Lakes, NJ, USA). The increment
of DCF fluorescence directly reflects the generated ROS inside cells which were represented as
mean fluorescence intensity of DCF [48].
9

2
.9.
Assessment of protein localization using immunofluorescence
HCT 116 cells were plated on cover slips and incubated for 24h. After the incubation period,
cells were treated with 9µM complex 1 and incubated for 12h. Untreated was taken as control.
After treatment of complex 1, control/treated HCT 116 cells were washed twice for 10 min each
in PBS (0.01 M) and incubated for 1hr in blocking solution containing 2% normal bovine serum,
°
and 0.3% Triton X-100 in PBS. After blocking, the cells were incubated overnight at 4 C with
the respective primary antibody (Bax and cytochrome c) followed by washing and incubation
with respective fluorophore-conjugated secondary antibodies for 2 h. The slides were then
counterstained with 6-diamidino-2-phenylindole (DAPI) and MitoTracker Red and mounted with
the Prolong Anti-fade Reagent (Molecular Probe, Eugene, OR, USA). Stained cells were
examined using a confocal laser-scanning microscope (FV 10i, Olympus, Japan) [49].
2
.10. Caspase-3/9 activity assays
Treated cells were exposed to caspase-3 and caspase-9 colorimetric assay using commercially
available kits allowing to the manufacturer instruction (BioVision Research Products, Mountain
View, CA) respectively [22].
2
.11. Scratch assay
6
HCT 116 cells were seeded in 6-well plates at the concentration of 2×10 cells per well and
°
incubated at 37 C overnight. Cell monolayers that converged almost 100% were wounded with a
sterile 100 μL pipette tip. Remove detached cells from the plates carefully with PBS and add
DMEM. HCT 116 cells were left either untreated or treated with the indicated doses of complex
1
. After the incubation for 12h, medium was replaced with PBS and the scratched areas were
photographed using an Olympus microscope [22].
1
0

2
.12. Statistical analysis
Results were represented as Mean ± SEM of the multiple data points. Statistical importance in
the deference was calculated by the analysis of variance (ANOVA) using OriginPro (version 8.0)
software where p < 0.05 was considered as significant.
2
.13. Catalytic oxidation of 3, 5-DTBC
-4
The catecholase mimics activity of the complex 1 (10 M) was tested with 100 fold of equivalent
ratio of 3, 5-di-tertbutylcatechol (3, 5-DTBC) in ethanol as a solvent at room temperature under
aerobic condition. The catalytic activity of 3,5-DTBC to 3,5-DTBQ were recorded
spectrophotometrically at a wavelength 393 nm using absorbance versus max (nm) plot at 5min
time interval. The rate of oxidation of substrate was determined in a concentration dependence
manner using 30, 50, 70 and 100 equivalent of substrate as previously reported method [50,51].
2
.14. Molecular docking
Molecular structure of the complex 1 was generated and optimized using chemical computing
In., MOE 2017.09. This structure was then imported to MOE database. 3D crystal structure of
Akt-1-inhibitor complexes (PDB ID: 3MVH) having resolution of 2.01 Å was downloaded from
protein data bank [52]. Ligand was extracted from 3MVH and prepared for molecular docking.
Before optimization, all water molecules were detached through sequence editor of MOE.
Complex 1 was optimized and protonated by using MM force field and semi-empirical PM3
methods for docking analysis. After preparation of complex 1 as a drug candidate for the targeted
receptor, it was subjected to molecular docking systematic conformational search set at default
-1
parameters with RMS gradient of 0.01 kcal mol employing Site Finder. Several conformational
cycles were run in order to get a docked pose as accurate as possible. The best binding pose was
chosen based on energetics ground [53]. The interaction energy of drug with 3MVH was
1
1

calculated at each step of the docking simulation, whereas, all remaining parameters were fixed
at default settings.
3
. Results and discussion
.1. Synthesis and general characterization
3
A new copper(II) complex namely, [Cu(qmbn)(q)(Cl)] (1) was fabricated with pharmacophore
scaffolds 2-(quinolin-8-yloxy)(methyl)benzonitrile and 8-hydroxyquinoline (Scheme 1). The
complex 1 was stable towards air and moisture and soluble in water on sonication. The
composition of complex 1 was established by microanalytical, TGA, PXRD and FTIR
techniques (Fig. S1-S3).
(a) Synthesis of 2-(quinolin-8-yloxy)(methyl)benzonitrile (qmbn)
Br
N
CN
O
K CO / MeCN
Reflux: 24 hr
2
3
+
N
CN
OH
qmbn
(b) Synthesis of complex 1
Cl
N
Cu
O
N
O
N
i) CH OH + H O
3 2
N
CuCl2 2H O
O
+
+
2
ii) NaOH / Reflux: 6hr
OH
CN
N
Complex 1
Scheme 1. Synthetic scheme for (a) 2-(quinolin-8-yloxy)(methyl)benzonitrile (qmbn) and (b)
complex 1.
-1
The bands observed in the FTIR at 2900 and 2229 cm can be attributed to ʋ(C-H) and
free nitrile ʋ(-C≡N) stretching absorption frequencies, respectively [54]. The band at 1580 cm-1
could be due to the vibration of pyridine ring of uncoordinated 8-hydroxyquinoline which was
1
2

-1
shifted to 1502 cm in the complex 1. The stretching frequency of ʋ(Cu-O) and ʋ(Cu-N) in the
-1
complex 1 was observed at 655 and 570 cm , respectively [55] (Table 2 and Fig. S1).
o
Thermal analysis of CP1 was carried out in N atmosphere at the rate of 10 C per
2
°
minute. Complex 1 exhibited thermal stability upto ~170 C, beyond this temperature framework
decomposed (Fig. S2). Moreover, Powder-XRD patterns of complex 1 (experimental or as-
synthesized) were in good agreement with the simulated patterns (single crystal-XRD), which
confirmed the bulk phase purity of the sample (Fig. S3). Due to different orientations of the
crystalline sample, some diffraction peaks were observed with low intensity.
–
1
Table. 2. Comparison of selected IR stretching vibration wavenumbers, υ(cm ) between free
ligand and complex 1.
Assignment
ʋC-H
Ligand
2918
2219
1619
1460
-
Complex 1
2921
ʋ-C≡N
2229
1614
1468
655
ʋ(C=N)
ʋ(C=C)
ʋ(Cu-O)
ʋ(Cu-N)
-
570
3
.2.
Structural description
The structure of complex 1 (Fig. 1) has been confirmed by single crystal X-ray diffraction
measurement. The complex 1 crystallizes in an orthorhombic crystal system with Pbc2 space
1
group. The asymmetric unit contains two crystallographically independent complex units. Each
-1
complex unit comprises of one q ligand, one qmbn ligand and one coordinated chloride ion
Fig. 1a). Using Addison’s parameter [56], the coordination geometry around Cu1 (τ = 0.106)
(
5
and Cu2 (τ = 0.145) can be described as distorted square pyramidal. The two equatorial position
5
-1
of each Cu(II) ion is occupied by the O and N atom of q ligand, chloride ion and N atom of the
1
3

qmbn ligand. The apical position of the each Cu(II) is coordinated by the O atom of the qmbn
ligand (Fig. 1b-c).
Fig. 1 Representation of (a) asymmetric unit and (b and c) coordination environment of Cu(II)
ions in complex 1. The C-atoms of ligand Hq is shown in purple color.
Fig. 2. A view of (a) crystal packing showing herringbone pattern along with crystallographic a-
axis and (b) supramolecular layers formed via H-bonding, π···π and C-H···π interactions in
complex 1.
1
4

The single crystal packing analysis revealed the interesting intermolecular interactions within the
crystal system. Each crystallographically independent units of complex 1 are linked through C-
H···Cl, C-H···O and C-H···N interactions, and forming 2D herringbone pattern. Further, the
intricate array of several π···π and C-H···π interactions result in the generation of overall 3D
supramolecular framework (Fig. 2) [57–59]. The important H-bonding parameters are given in
Table 3. However, non-covalent interactions are not only playing a pivotal role in the
supramolecular architecture stability but also efficiently interacting with biological systems for
diverse functions.
Table 3 Selected H-bonding parameters in complex 1.
D-H…A
C2-H2…O3
d(D-H)Å
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
d(H...A)Å
2.51
d(D…A)Å
3.4261(3)
3.5451(3)
3.2413(3)
3.3714(3)
3.3993(3)
3.2378(3)
3.4662(3)
3.5866(3)
˂(D-H…A)°
170
C6-H6…Cl2
C8-H8…Cl1
C28-H28…O3
C32-H32…N3
C34-H34…Cl2
C37-H37…Cl1
C49-H49…Cl1
2.74
2.68
2.46
2.53
2.69
2.76
2.73
145
119
166
157
119
134
154
3
.3.
MTT assay
The well-established MTT assay was used to study the cytotoxicity of the complex 1 at the
varying concentrations. At first we have checked the cytotoxicity of the complex 1, which was
evaluated on the human colon cancer cell line (HCT116) and Human embryonic kidney 293 cells
(
HEK 293) by MTT assay. In case of both the cell line, the dose was selected 0-30 μM, for 12hr
Fig.3). The colorimetric data says that, in case of HCT116cell line at 20 μM concentration
(
almost all the cells were death after 12hr of treatment. The data implicated that 30%, 50% and
1
5

7
0% cells were death at 3.68 μM, 9.82 μM and 15.96 μM respectively after 12h of treatment. So
we have chosen the IC value (9.82 μM) for further biological experiments.
5
0
Fig. 3. MTT assay data of complex 1 after 12hr of treatment against A. HCT 116 and B. HEK
2
93 cell line.
3
.4.
Quantification of apoptosis and necrosis using flow cytometry
To explore whether the complex 1 was involved in apoptosis/necrosis, we conducted flow
cytometric assessment using Annexin-V-FITC/PI staining by studying the exposed level of
serine phosphatidyl in the outer membrane of cells [60]. The flow cytometric data of Annexin V-
FITC-PI assay confirmed that the cell death is occurring due to Apoptosis. The experimental
results showed that the cell death is increasing in time dependent manner after treatment of 9 μM
complex 1 (Fig. 4). The treatment was given for 12 and 24h duration. These results suggested
that complex 1 induced cell death was directly correlated with cytotoxicity followed by
apoptosis.
1
6

Fig. 4 Determination of apoptosis/necrosis after treatment of 9 µM complex 1 against human
colorectal HCT 116 cancer cell lines.
3
.5.
Determination of intracellular ROS (iROS)
There are several reports confirmed that Apoptosis is directly dependent on Reactive Oxygen
Species (ROS) [61,62]. The total ROS generation has been quantified after treatment of 9 μM
complex 1 (Fig. 5) by using a cell permeable dye, DCFH-DA.
Fig. 5 Determination of iROS after treatment of 9 µM complex 1 against human colorectal HCT
1
16 cancer cell lines.
In this flow cytometric data, complex 1 treated with HCT-116 cells showed an overproduction of
ROS by markedly increased DCF fluorescence reflecting the increased DCF +Ve cells when
1
7

treated for 12 and 24 h and compared with the control cells. This data confirmed that the cell
death of HCT 116 was occurring by ROS dependent apoptosis [63].
3
.6.
Assessment of protein expression
Bax acts upstream of increased Reactive Oxygen Species (ROS) production [64]. There are also
some reports which cleared that Bax activation are essential for cytochrome c release from
Mitochondria during apoptosis [65]. Bax and Cytochrome c are also two key pro-apoptotic
marker for apoptosis [66]. So, we have tried to establish the relation of Bax and Cytochrome c
release after treatment of 9 μM complex 1 in presence of MitoTracker Red and Nuclear stainer
DAPI (Fig. 6).
Fig. 6 Expression of Bax and Cytochrome c after treatment of 9 µM complex 1 in presence of
DAPI and MitoTracker-Red on human colorectal HCT 116 cancer cell lines.
The expression of Bax and Cytochrome c has been increased after treatment of complex 1 time
dependently. The final apoptotic fate was determined by the activity of caspase-9 and caspase-3
[67] (Fig. 7), which was simultaneously enhanced with the treatment of complex 1.
Simultaneously, scratch assay was performed for measuring cell proliferation after 12h of
1
8

complex 1 treatment. The microscopic images (Fig. S4) revealed that there was no such cell
proliferation after 12h of complex 1 treatment.
Fig.7 Expression of caspase 3 and 9 after treatment of 9 µM complex 1.
The result summarily confirms that complex 1 mediated apoptosis might be regulated through
ROS driven mitochondrial pathway of apoptosis. Thus, complex 1 induces mitochondrial
dependence apoptosis through the modulation of pro- and anti-apoptotic proteins (Scheme 2).
1
9

Scheme 2. Schematic representation of the overall events of apoptotic pathway induced by
complex 1. The ↑ arrows indicate the up-regulation of that particular marker induces apoptosis in
cancer cells.
3
.7.
Catalytic activity
The catecholase-like activity of complex 1 was monitored using catechol as the substrates for the
identification of complex 1 as functional model for metalloenzymes. Copper(II) complex
containing 8‐hydroxyquinoline and pyrazine‐2‐carboxylic acid also serves as an efficient
heterogeneous catalysts for the elimination of dye in aqueous solution with H O in a short time
2
2
[68]. To date, many mono or binuclear copper complexes derived from oxygen and nitrogen
donating ligands which exhibited potential catalytic activity towards the conversion of 3,5-
DTBC to 3,5-DTBQ in presence of molecular oxygen [69]. The catecholase mimicking activity
of the complex 1 (0.0001M) was evaluated using 3,5-DTBC (3,5-di-tert-butylcatechol) (0.01M)
as a substrate under aerobic conditions. The reactions were carried out in dioxygen saturated
°
ethanol at 25 C under aerobic conditions and their absorption spectra were recorded at time
2
0

interval of every five minutes. The substrate 3,5-DTBC has low redox potential that facilitated its
oxidation, and the resulting product, 3,5-DTBQ has exhibited a characteristics strong absorption
-1
-1
maximum at ca. 393 nm ( = 1900 M cm ) [69]. The time dependent spectral changes of the
complex 1 was followed spectrophotometrically at 393 nm for about 100 min at regular time
interval of 5 min upon the addition of 3,5-DTBC (Fig. 8). The increase in absorbance at
monitoring wavelength clearly demonstrated that the complex 1 was efficiently and effectively
oxidizing substrate 3,5-DTBC to 3,5-DTBQ.
Fig. 8. Uv-vis spectra recorded in the presence of complex 1 upon addition of 3,5-DTBC at a
regular time interval of 5min.
3
.8.
Kinetic study of catechol oxidation
From the above experimental results, it was observed that complex 1 has ability to catalyze the
oxidation of subtrate 3,5-DTBC. Therefore, a detailed quantitative kinetic study model of
complex 1 was conducted to further elucidate its catalytic efficiency towards 3,5-DTBC in
dioxygen saturated ethanol under experimental conditions. Solutions of the complex 1 (0.0001
M) was treated with incremental addition of 3,5-DTBC (0.0001 M to 0.001 M), and their
2
1

absorption spectra were recorded. An analysis of the data based on the method of initial rate,
kinetics determination was applied following the conversion of 3,5-DTBC to the increase in
oxidized product 3, 5-DTBQ at 393 nm every five minutes. The average value of rate constant of
reaction for the complex 1 showed zero order at high concentration of 3,5-DTBC substrate but at
low concentration, average rate constant follows the first order reaction. The Michaelis-Menten
model, originally developed for enzyme kinetics may be appropriate for this conversion also.
V푚푎푥[푆]
푉 = 퐾푀 + [푆]
Where V = initial rate, [S] = concentration of 3,5-DTBC substrate; K = (k +k /K ), Michaelis-
M
2
3
1
Menten constant and Vmax = maximum initial rate accomplished for a particular concentration of
complex 1 in the presence of excess amount of 3,5-DTBC (Fig. 9a). Mathematically, the
Michaelis-Menten equation can be transformed into another form which is more useful in data
plotting. Taking the reciprocal of both sides, the above equation transformed to the well-known
Lineweaver-Burk equation;
1
퐾푀
1
1
=
×
+
푉
푉푚푎푥 [푆] 푉푚푎푥
The value of Michaelis-Menten binding constant (K ), maximum velocity (Vmax), and rate
M
constant for dissociation of substrate (i.e. turnover number K ) were calculated from the plot of
cat
1
/V vs 1/[S] (Fig. 9b), also known as Lineweaver-Burk plot. A linear relation between the
concentration of the complex 1 and its initial rate of the oxidation, confirming a first-order
dependence on the complex concentration and the calculated kinetic parameters obtained were
2
-1
-2
-1
k =1.03×10 h , K = 0.0209 M and V = 2.87×10 M min . Moreover, the catecholase
cat
M
max
mimics activity of the complex 1 was compared to previously reported metal complexes (Table
) [70–75].
4
2
2

(a)
(
b)
(
b)
Fig.9 Plot of (a) rate vs substrate concentration and (b) Lineweaver-Burk plot of complex 1.
Table 4. Catalytic turnover number (k ) for the oxidation of DTBC by complex 1 and
cat
previously known metal(II) complexes.
-
1
Metal(II) complexes
k (h )
Ref.
[70]
[70]
[70]
[70]
[70]
[71]
[72]
[73]
[74]
[74]
[74]
[75]
cat
2
3.24 × 104
1.44 × 104
1.08 × 104
1.44 × 104
2.88 × 104
0.76 ×102
102×104
1.68×102
64.1 ± 4.1
51.1 ± 6.2
81.7 ± 4.7
1.88×103
1.03×102
[
[
[
[
[
[
[
[
[
[
[
{
Cu (H L2 )(OH)(H O)(NO )](NO ) ·2H O
2
2
2
3
3 3
2
4
Cu(HL1 )(H O)(NO )] ·(NO ) ·2H O
2
3
2
3 2
2
1
Cu(L1 )(H O)(NO )]
3 2
2
3
Cu (L2 )(OH)(H O) ](NO )
3 2
2
2
2
1
Cu (L2 )(N ) ]
2
3 3
Cu (L)(μ-OH)(H O)(ClO ) ]
2
2
4 2
Cu (L2) (OAc) ]·H O
2
2
2
2
Co(L) (H O) ]
2
2
2
1
Ni (L ) (NCS) ]
2
2
2
2
Ni (L ) (NCS) ]
2
2
2
3
Ni (L ) (NCS) ]
2
2
2
I
II
[Cu (CN)(phen) Cu (CN) (phen)]·5H O}
2
2
2
[Cu(qmbn)(q)(Cl)]
this work
2
3

Scheme 3. A plausible mechanism for catecholase like activity promoted by complex 1
involving catalytic oxidation of 3,5-DTBC to 3,5-DTBQ.
A plausible mechanism for the aerobic oxidation of catechol to quinone promoted by
complex 1 is depicted in Scheme 3. In the first step of the catalytic cycle, the complex 1
successfully reacted with the catechol leads to the generation of radical intermediate Cu(I)-
semiquinoate complex followed by reduction of Cu(II)-Cu(I). Subsequently, Cu(I)-semiquinoate
radical intermediate get oxidized in the presence of dioxygen leading to the re-oxidation of Cu(I)
to Cu(II) and the reduction of dioxygen. Notably, it may be attributed that Cu(II)-semiquinone
radical species either get oxidized by O to quinone or disproportionated to quinone followed by
2
further oxidation of the copper ions.
3
.9.
Molecular docking
To explore the potential binding modes of the complex 1 and protein kinase (Akt), molecular
docking was performed to understand the experimental findings as described above. The target
2
4

receptor human Akt kinase (PDB: 3MVH) has been chosen to probe the possible molecular
mechanism of interactions with complex 1. It is well-known that Akt kinases are among the most
thoroughly studied targets for targeted cancer therapeutic agents, due to their close resemblance
with multiple cellular signaling pathways such as glucose metabolism, apoptosis, cell
proliferation, transcription, and cell migration. Based on in silico results, the docked pose
analysis results showed that complex 1 recognizes the binding pocket that was provided by the
target receptor (Fig. 10a). Furthermore, several amino acid residues directly interacted with
molecule, as lowest energy docked pose of the complex 1 displayed that planar aromatic part of
the complex develops arene-arene interactions with aromatic ring of Tysrosine residues (Tyr175
&
Tyr-176) as indicated by 2D ligplot (Fig. 10b).
Fig. 10(a) Molecular docking pose of complex 1 with human Akt kinase (PDB ID: 3MVH) and
b) 2D ligplots which show interactions of complex 1 with base pairs of Akt receptor.
(
The H-bonding interactions develop between –N atom of complex with H atom of Glutamic acid
Glu228) and Lysine (Lys284). Other two interactions include electrostatic interaction of Cl-
atom of complex with H-atom of Tyr-175. Dotted lines around molecule indicate hydrophobic
(
2
5

interactions with Akt receptor. Greater number of interactions are responsible for high binding
4
-1
constant (K = 2.52×10 M ) and higher spontaneity of complex formation with greater negative
b
-1
value of free energy (ΔG = -25.11 k⸱ ⸱⸱ mol ). The model might serve as a rational basis for
possible design of novel anticancer drugs targeting active site of human Akt kinase.
4
. Conclusion
Herein, we have designed and synthesized a new copper(II) complex [Cu(qmbn)(q)(Cl)] (1) as
potential chemotherapeutic agent. Structural elucidation of the complex 1 was done by various
analytical (elemental analysis, TGA and XRD) and spectroscopic techniques (IR and Uv-vis). In
vitro cytotoxicity of the complex 1 was evaluated on the human colon cancer cell line (HCT116)
which implicated that more than 50% cells were viable at 9.82 μM after 12h of treatment. The
outcomes of flow cytometric and fluorescence microscopic analysis were in concordance with
the results of MTT assay which showed a time-dependent increase in the number of apoptotic
cells. The mitochondrial pathway plays an important role in inducing cell apoptosis based on the
results of up-regulated expression of Bax and Cytochrome-c which ultimately triggers the
caspase 3/9 protein activation and ultimately lead to apoptosis. Interestingly, complex 1
2
-1
exhibited significant catecholase like activity k =1.03×10 h . Furthermore, molecular docking
cat
results revealed that the complex 1 binds to the active site of protein kinase (Akt), which plays a
key role in multiple cellular processes such as apoptosis, cell proliferation, and cell migration.
Acknowledgements
The Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim
University, UP, India, and Researchers Supporting Project number (RSP-2020/288), King Saud
University, Riyadh, Saudi Arabia are gratefully acknowledged for financial support and
2
6

laboratory facilities.UGC Start-up grant and TEQIP-III, ZHCET, Aligarh Muslim University, is
thanked for extensive help to procure chemicals.
Supporting Information
CCDC 2003694 contains the supplementary crystallographic data for complex1. It data can be
obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
1
223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Table for selected bond lengths and bond
angles, IR, TGA and PXRD and scratch assay are appended.
Authorship Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
There is no conflict of interest to declare.
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