Synthesis, characterization, and anticancer activity of Schiff bases

Article abstract of DOI:10.1080/07391102.2019.1654924

Five Schiff bases, 2-((3-chlorophenylimino)methyl)-5-(diethylamino)phenol (L1), 2-((2,4-dichlorophenylimino)methyl)-5-(diethylamino)phenol (L2), 5-(diethylamino)-2-((3,5-dimethylphenylimino)methyl)phenol (L3), 2-((2-chloro-4-methylphenylimino)methyl)-5-(d

Full text of DOI:10.1080/07391102.2019.1654924

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis, characterization, and anticancer activity
of Schiff bases
Noor Uddin, Faisal Rashid, Saqib Ali, Syed Ahmed Tirmizi, Iqbal Ahmad,
Sumera Zaib, Muhammad Zubir, Paula L. Diaconescu, Muhammad Nawaz
Tahir, Jamshed Iqbal & Ali Haider
To cite this article: Noor Uddin, Faisal Rashid, Saqib Ali, Syed Ahmed Tirmizi, Iqbal Ahmad,
Sumera Zaib, Muhammad Zubir, Paula L. Diaconescu, Muhammad Nawaz Tahir, Jamshed Iqbal
& Ali Haider (2019): Synthesis, characterization, and anticancer activity of Schiff bases, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2019.1654924
To link to this article: https://doi.org/10.1080/07391102.2019.1654924
Accepted author version posted online: 14
Aug 2019.
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Synthesis, characterization, and anticancer activity of Schiff bases
Noor Uddina, Faisal Rashidb, Saqib Alia, Syed Ahmed Tirmizia, Iqbal Ahmadc* Sumera Zaibb,
Muhammad Zubira, Paula L. Diaconescud, Muhammad Nawaz Tahire, Jamshed Iqbalb*, Ali
Haidera*
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
bCentre for Advanced Drug Research COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
cDepartment of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
dDepartment of Chemistry and Biochemistry, University of California, Los Angeles
607 Charles E. Young Drive East, Los Angeles CA 90095, USA
eDepartment of Physics, University of Sargodha, Pakistan
Key words: Schiff base, DFT, electrochemistry, DNA interaction studies, antitumor activity
Abstract
Five Schiff bases, 2-((3-chlorophenylimino)methyl)-5-(diethylamino)phenol (L1), 2-((2,4-
dichlorophenylimino)methyl)-5-(diethylamino)phenol
(L2),
5-(diethylamino)-2-((3,5-
dimethylphenylimino)methyl)phenol (L3), 2-((2-chloro-4-methylphenylimino)methyl)-5-
(diethylamino)phenol (L4), and 5-(diethylamino)-2-((2,6-diethylphenylimino)methyl)phenol
1
(L5) were synthesized and characterized by elemental analysis, FT-IR, H and 13C NMR
spectroscopy. Three of the compounds (L1, L2, and L4) were analyzed by single crystal X-ray
diffraction: L1 and L2 crystallized in orthorhombic P212121 and Pca21 space group, respectively,
while L4 crystallized in monoclinic P21/c space group. Theoretical investigations were
performed for all the synthesized compounds to evaluate the structural details. Drug-DNA
interaction studies results from UV-Vis spectroscopy and electrochemistry complement that the
compounds bind to DNA through electrostatic interactions. The cytotoxicity of the synthesized
compounds was studied against cancer cell lines (HeLa and MCF-7) and a normal cell line
(BHK-21) by means of an MTT assay compared to carboplatin, featuring IC50 values in the
micromolar range. The pro-apoptotic mechanism for the active compound L5 was evaluated by
fluorescence microscopy, cell cycle analysis, caspase-9 and -3 activity, reactive oxygen species

production, and DNA binding studies that further strengthen the results of that L5 is a potent
drug against cancer.
1. Introduction
Cancer is a multistage progressive disease, in which a group of cells start replicating in an
uncontrolled fashion after a major mutation occurs in the genetic makeup of normal cells(Amiri
et al., 2018; Zehra, Shavez Khan, Ahmad, & Arjmand, 2019). Genes regulating cellular growth,
reproduction, and cell cycle get mutated and turn normal cells into cancerous cells (Hyndman,
2016). Such cells bypass cell check points, evade from apoptosis, produce larger populations,
enlarge their mass, and start invasion into nearby and accessible tissues (Kim, 2015). Cancerous
disease affects almost all body systems, organs, and tissues, except dead cells like hair and nails,
and is being continuously detected in individuals of all age groups. After affecting a specific
body part or system, the disease progresses to other areas of the body as well. Cancer can be
managed and treated effectively if diagnosed early, but it can be fatal if it remains undiagnosed
and the disease progresses to an end stage (Virnig, Baxter, Habermann, Feldman, & Bradley,
2009). There are more than 270 types of cancer that have a great tendency to resist
chemotherapeutics, leading to a relapse after an initial cure. Therefore, the discovery of novel
molecules and strategies to combat this challenge is necessary (Cree & Charlton, 2017)-
(Hassanpour & Dehghani, 2017).
Apoptosis, a natural death process for injured, old, or
abnormal cells that prevents the development of cancer, is diminished or blocked in cancerous
cells, and can be triggered again by anticancer drugs. Thus, the induction of apoptosis in cancer
cells is a propitious target to be achieved by a novel molecule while carrying out anticancer
studies (Olsson & Zhivotovsky, 2011).
Schiff bases or azomethines are compounds formed by a condensation reaction between
primary amines and aldehydes, and have various biological, medicinal, clinical,
pharmacological, and analytical applications (Dehkhodaei, Sahihi, & Amiri Rudbari, 2018;
Deshmukh, Soni, Kankoriya, Halve, & Dixit, 2015; Goel, Kumar, & Chandra, 2014; Jeyaraman,
Alagarraj, & Natarajan, 2019; Kumaran, Priya, Gowsika, Jayachandramani, & Mahalakshmi,
2013). They are popular ligand precursors due to their versatility and ease of
preparation(Rambabu, Pradeep Kumar, Ganji, Daravath, & Shivaraj, 2019; Shahraki & Heydari,
2018; Shahraki, Shiri, & Saeidifar, 2018; Shokohi-Pour, Chiniforoshan, Sabzalian, Esmaeili, &

Momtazi-borojeni, 2018). Aromatic primary amines can also contain additional donor functional
groups like -Cl, -OH, -CH3, etc., which help enhancing and regulating biological activities
(Alaghaz & Bayoumi, 2013; Siddiqui, Iqbal, Ahmad, & Weaver, 2006). Schiff bases have been
shown to exhibit anticancer, antimycobacterial, antibacterial, antidepressant, and analgesic
properties (Kajal, Bala, Kamboj, Sharma, & Saini, 2013).
In the present study, azomethines were synthesized and explored for their antineoplastic
effect on HeLa and MCF-7 cancer cell lines, while cytotoxicity to normal cells was evaluated on
BHK-21 cell line by using an MTT viability assay. The pro-apoptotic mechanism for active
compounds were evaluated by fluorescence microscopy, cell cycle analysis, caspase-9 and -3
activity, reactive oxygen species production, and DNA binding studies.
2. Experimental
2.1. Materials and instrumentation
Reagents, 4-(N-diethylamino)salicylaldehyde (98%), 3-chloroaniline (99%), 2,4-dichloroaniline
(99%), 2,3-dimethylaniline (99%), 2-chloro-4-methylaniline (98%) and 2,6-diethylaniline (98%)
were purchased from Sigma Aldrich (USA) and were used without additional purification.
Reagent grade solvents were dried according to a literature procedure (Armarego, 2017). A
Gallenkamp (UK) electrothermal melting point apparatus was used for the determination of
melting points. CHN analysis was done using a CE-440 Elemental Analyzer (Exeter Analytical,
1
13
Inc.). FT-IR spectra were recorded on a Bruker Tensor II instrument. H and C NMR spectra
were obtained by using a Bruker-300 MHz FT-NMR spectrometer with DMSO-d6 as a solvent
13
[1H (DMSO) = 2.50 ppm and C (DMSO) = 40 ppm] (Gottlieb, Kotlyar, & Nudelman, 1997).
Crystal analysis was done by using a Bruker Smart Apex II CCD-Single X-ray diffractometer
(Mo X-ray source).
2.1.2. Synthesis of Schiff bases
Schiff bases were synthesized by the condensation reaction of 4-(diethylamino)-2-hydroxy
benzaldehyde with R-amines (R-amine = 3-chlorobenzenamine, 2,4-dichlorobenzenamine, 3,5-
dimethylbenzenamine, 2-chloro-4-mehtylbenzenamine, and 2,6-diethylbenzenamine, 20 mM
each) in dried ethanol at reflux conditions and stirred for 6-7 h (Scheme 1). The precipitates
obtained were filtered on Whatman filter paper and washed several times with ethanol, diethyl
ether, and dried in a vacuum desiccator.

Scheme 1. General synthesis of Schiff bases (L1-L5).

Chart 1. Structures and numbering of L1-L5.

2.2. Computational details
The computational calculations were accomplished by using the DFT approach to optimize the
structures in the gas phase (Alyar et al., 2019). The Gaussian 09 package was used to visualize
all the theoretical results (Frisch et al., 2008). The chemical shift (δ) values were calculated from
the optimized geometries by employing the Gauge Independent Atomic Orbital (GIAO) with
B3LYP functional and LANL2DZ basis set (Kutzelnigg, Fleischer, & Schindler, 1990).
Moreover, the fundamental vibrational frequencies were also evaluated for comparison with
experimentally determined values by using the Gauss view software package.
2.3. DNA binding studies
DNA interaction ponders of the foremost strong compounds were performed following a
published strategy (J. Iqbal, Ejaz, Saeed, & al-Rashida, 2018),(Sirajuddin, Ali, McKee, Zaib, &
Iqbal, 2014). Briefly, the concentration of mammalian DNA was estimated at 260 nm using a
FLUOstar Omega microplate reader (BMG Labtech, Germany). The test compound (100 µM)
was interacted with different concentrations of hs-DNA (herring sperm) from 0 µM to 392 µM
whereas the same concentrations of DNA were used in respective reference solutions. After an
incubation of 10 minutes, the absorption spectra were recorded in 96 well plates with the path
length of 5.5 mm.
2.4. Electrochemical studies
Electrochemical studies were performed using a Corrtest CS (Potentiostat/Galvanostat)
Electrochemical Workstation, China, in a DMSO solution containing 0.1 M TBAP
(tetrabutylammonium perchlorate) as a supporting electrolyte in a three-electrode cell under an
argon saturated environment. A saturated silver/silver chloride was used as a reference electrode,
Pt as a counter electrode and glassy carbon (GC) as a working electrode with a diameter of 0.03
cm2. Before starting the experiment, the GC electrode was washed with an aqueous suspension
of alumina (Al2O3) on a nylon buffing pad with deionized water at room temperature (25 oC).
2.5. Cell viability assay (MTT assay)
The cytotoxic potential of the test analogues was evaluated in the human breast adenocarcinoma
cells MCF-7, human cervical adenocarcinoma cells HeLa by an MTT (dimethyl–2–thiazolyl–

2,5–diphenyl–2H–tetrazolium bromide) based cell viability assay as described previously
[(Mosmann, 1983; Niks, 1990)]. The impact of these derivatives
was moreover inspected against normal cells, i.e., child hamster kidney cells (BHK–21). Briefly,
the cells (1 × 104) were cultured in a volume of 90 µL in each well of a 96-well flat-bottom
culture plate and kept in a 5% CO2 incubator at 37 ˚C. After an overnight incubation, the cells
were treated with the test compounds (100 µM) and incubated thereafter for 24 h. The media was
aspirated and 10 µL MTT (2 mg/mL) along with 90 µL media was added to each well. The plate
was incubated for 4 h at 37 ˚C and 5% CO2, followed by the addition of 100 µL of the reagent
(1:1 solution of 50% isopropanol and 10% sodium dodecyl sulfate prepared in 0.1 N HCl). The
plate was further incubated for 30 minutes at 37 ˚C and the optical density was observed at 570
nm while a background reading was done at 630 nm using a microplate reader (Bio-Tek ELx
800TM, Winooski, USA). The experiment was carried out in triplicate and the results were
calculated as percent inhibition values with the mean of three independent values (± SEM). The
derivatives that exhibited equal to or more than 50% inhibition were further evaluated for their
inhibitory concentration (IC50) values.
2.6. Cell cycle analysis assay
The treated cells were subjected to a cell cycle analysis by flow cytometry, using the method as
reported earlier (Saito et al., 2010). Initially, the cells (2 × 105 cells/mL) were treated with the
most potent derivatives obtained from the MTT assay from the selected series and incubated
o
overnight at 37 C. Then, the pellets of cells were attained by centrifugation at 4000 g (for 5
minutes) and, after that, the pellets were resuspended in a 3% FBS solution (200 µL) that
contained 5 µL of propidium iodide (20 µg/mL), 0.1% (v/v) Triton X-100, and ribonuclease A
(10 µL, 10 mg/mL). The samples were analyzed by a BD Accuri flow cytometry as described
earlier (J. Iqbal et al., 2018).
2.7. Microscopic analysis of apoptosis
The microscopic analysis of the most potent derivative was carried out to support the flow
cytometry results. The analysis was done according to a previously reported method (J. Iqbal et
al., 2018) (G.-J. Lin et al., 2013). The confluent cells (2 × 105 cells/well) were treated with the

o
test compound (100 µM) and kept in a 5% CO2 incubator at 37 C. After 24 h, the culture
medium was removed, and cells were washed thrice with cold PBS (phosphate buffered saline).
Then, the cells were treated with 4% formalin and 0.1% Triton X–100. Finally, after a 5 min
room incubation, 10 µL (0.1 mg/mL) of 4′,6-diamidino-2-phenylindole (DAPI) or propidium
iodide (PI) dye was mixed to stain the nuclear material. The glass slides were kept in the dark for
10 min and images were captured by using a fluorescence microscope (Nikon ECLIPSE Ni–U)
at an excitation/emission wavelength of 350/460 and 493/632 nm for DAPI and PI, respectively.
2.8. Determination of intracellular reactive oxygen species (ROS) production
The initiation of the production of ROS within HeLa cells of the most potent compound L5 by
the different concentrations (IC50 value i.e., 21 µM and 2x IC50 i.e., 42 µM) was studied by using
dichlorofluorescin diacetate (H2DCF-DA) dye and observed by fluorescence microscopy. Fig 12
shows that treated HeLa cells with potent compound L5 exhibit disintegrated cell membranes
and condensed cellular protein (DNA) that may be due to oxidation of lipids and proteins as
reported previously (R.P. Rastogi et al., 2010).
2.9. Apoptosis assessment by caspase -9 and -3 activity
Caspase-9/3 activity was analyzed using a fluorometric assay kit (Abcam). Cells were plated at 1
× 106 cells per well per 3 mL in a 6 well plate for overnight incubation. Fresh media was
introduced after the removal of old media and cells were treated with the compound of interest.
After 18 h, cells were collected, centrifuged, and lysed using 50 μL of lysis buffer on ice for 10
min, and incubated with DEVD-AFC substrate for caspase-3 (S.-F. Lin, Lin, Hsueh, Chou, &
Wong, 2018) and with LEHD-AMC substrate for caspase-9 (Pan, Xu, & Yeung, 2001) and
reaction buffer at 37 °C for 3 h. The amount of fluorescent cleavage product was measured using
a fluorescence microplate reader (FLUOstar Omega, BMG Labtech, Germany). All these
experiments were performed in triplicate.
3. Results and discussion
The Schiff bases (L1–L5) synthesized as described above were dried and characterized by
1
13
elemental analysis, FT-IR, H and C NMR spectroscopic techniques. The elemental analysis
(C, H, N) results were in good agreement with those calculated for the suggested formula. The

sharp melting points obtained indicate the purity of the synthesized compounds. The structures of
compounds (L1–L5) along with numbering are given in Chart 1.
3.1. Infrared spectroscopy
In the IR spectra of L1–L5 show a sharp band at 3340 cm-1 (L1), 3386 cm-1 (L2), 3346 cm-1 (L3),
3374 cm-1 (L4) and 3364 cm-1 (L5) that is assigned to free Ar-OH. A strong band attributable to
C=N is observed at 1605 cm-1 (L1), 1621 cm-1 (L2), 1600 cm-1 (L3), 1609 cm-1 (L4) and 1605
cm-1 (L5). The stretching vibration of medium intensity for N-C bond occurs at 1244 cm-1 (L1),
1235 cm-1 (L2), 1240 cm-1 (L3), 1241 cm-1 (L4) and 1236 cm-1 (L5), while a weak intensity peak
for the stretching vibration of C=C is observed at 1575 cm-1 (L1), 1593 cm-1 (L2), 1573 cm-1
(L3), 1581 cm-1 (L4) and 1584 cm-1 (L5).
3.2. NMR spectroscopy
The NMR spectra of compounds L1–L5 were recorded in dimethyl sulfoxide (DMSO). The
chemical shifts of the different types of protons and carbons are given in the experimental part
(Chart 1). The formation of L1–L5 was supported by the appearance of a sharp singlet
corresponding to the azomethine proton (-N=CH-) at 9.64, 8.73, 8.66, 8.71, 8.25 ppm,
respectively. The hydroxyl proton gives a broad singlet at 13.31, 13.50, 13.77, 13.73, 13.13 ppm,
respectively. This downfield shifting of the OH proton is due to strong intramolecular O-H-N
hydrogen bonding, to which the chemical shift of the proton is very sensitive (Sirajuddin, Ali,
Shah, Khan, & Tahir, 2012). In the 13C NMR spectra, the peak at 163.17, 162.96, 161.39,
161.79, and 163.28 ppm, respectively, belongs to the azomethine carbon (CH=N). The remaining
peaks are described in the same positions as calculated by incremental methods (Popovic, Roje,
& Pavlovic, 2001).
3.3. Crystal structures
The molecular structures of the compounds L1, L2, and L4 were authenticated by single crystal
X-ray diffraction analysis. The solid-state molecular structures along with selected distances and
angles of L1, L2, and L4 are shown in Fig. 1, 2, and 3, respectively. Compound L1 crystalized as
a monomer and L2 as a dimer in an orthorhombic crystal system having the space group P212121
and Pca21, while L4 also crystalized as a dimer in a monoclinic crystal system with the space
group P21/c. Unit cells and hydrogen bonding of compounds L1, L2 and L4 are shown in Fig. 1,

2 and 3 respectively. The phenyl ring of all the compounds having bond length ranging from
1.367 to 1.402 Å denoting aromatic character. All the compounds have one dimensional
polymeric chain through intermolecular H-bonding. These compounds are stable through
intramolecular hydrogen bonding between hydroxyl group and nitrogen (O1-H1…N1).
Table 1. Experimental data for crystallographic analysis for compounds L1, L2 and L4.
Compound
1
2
4
C17H18Cl2N2O
337.23
C18H21ClN2O
316.82
Empirical
formula
C17H19ClN2O
302.79
296(2)
100(2)
Formula weight
Temperature/K
Crystal system
Space group
a/Å
296(2)
orthorhombic
Pca21
monoclinic
P21/c
orthorhombic
P212121
22.0044(19)
6.4705(5)
23.861(2)
90
8.3773(4)
10.9189(5)
34.7609(15)
90
6.6285(4)
15.1724(9)
15.5972(13)
90
b/Å
c/Å
90
93.342(3)
90
α/°
90
90
β/°
90
3397.3(5)
8
3174.2(3)
8
γ/°
1568.61(19)
4
Volume/Å3
1.319
1.326
Z
1.282
0.385
2.148
ρcalcg/cm3
μ/mm-1
1408.0
1344.0
0.244
0.440 × 0.300 × 0.260
MoKα (λ = 0.71073)
3.702 to 55.906
-23 ≤ h ≤ 28, -8 ≤ k ≤
8, -31 ≤ l ≤ 26
16633
0.200 × 0.080 × 0.050
CuKα (λ = 1.54178)
5.092 to 139.854
-10 ≤ h ≤ 9, -13 ≤ k ≤
12, -37 ≤ l ≤ 41
25414
640.0
F(000)
0.400 × 0.340 × 0.300
MoKα (λ = 0.71073)
5.37 to 55.848
-8 ≤ h ≤ 6, -15 ≤ k ≤
19, -20 ≤ l ≤ 14
Crystal size/mm3
Radiation
2Θ range for data
collection/°
Index ranges

6979 [Rint = 0.0378,
Rsigma = 0.0593]
6979/1/403
0.998
5880 [Rint = 0.0501,
Rsigma = 0.0409]
5880/6/419
Reflections
collected
4985
3249 [Rint = 0.0380,
Rsigma = 0.0699]
3249/0/195
1.025
Independent
reflections
1.059
R1 = 0.0454, wR2 =
0.1026
R1 = 0.0399, wR2 =
0.1000
Data/restraints/pa
rameters
R1 = 0.0425, wR2 =
0.0934
R1 = 0.0991, wR2 =
0.1258
R1 = 0.0541, wR2 =
0.1058
Goodness-of-fit
on F2
R1 = 0.0590, wR2 =
0.1015
0.23/-0.23
0.34/-0.43
Final R index
[I>=2σ (I)]
0.02(4)
0.20/-0.17
Final R index [all
data]
0.11(7)
Largest diff.
peak/hole / e Å-3
Flack parameter

(a)
(b)
Fig. 1 (a) Crystal structure of L1; (b) unit cell showing H-bonding.

(a)
(b)
Fig. 2 (a) Crystal structure of L2; (b) unit cell showing H-bonding.

(a)
(b)
Fig. 3 (a) Crystal structure of L4 (b) Unit cell showing H-bonding.
3.4. Electronic structure studies
The L5 solution was freshly prepared just before the measurements. The absorption study of L5
was performed in a phosphate buffer (pH = 7) at 298 K. In typical experiments, 2.5 mL of an L5
solution (100 μM) was placed in the cuvette and the absorbance spectra were recorded at 258–
800 nm, after the addition of the stock solutions (70 μM). In these experiments, the observed
absorbance was corrected for the dilutions. Prior to recording the absorbance, the sample was

incubated for 10 minutes. The absorption spectrum of L5 shows a maximum absorption band at
355 nm assigned to π → π * transitions of the azomethine group.
3.5. Density functional theory
Quantum mechanical calculations were also carried out to support the experimental results. The
computed molecular structures of the studied Schiff bases are shown in Fig. 4. The distances and
angles calculated from DFT studies (Table 3) are in close agreement with the experimentally
determined data obtained from single crystal X-ray analysis (L1, L2 and L4).
Fig. 4 Optimized molecular geometries of the Schiff bases L1 – L5.
3.5.1. Frontier molecular orbital analysis
The HOMOs (highest occupied molecular orbitals) and LUMOs (lowest unoccupied molecular
orbitals) are collectively called frontier molecular orbitals and can be used to understand the
course of some reactions. The frontier molecular orbitals and the energies of the HOMO and
LUMO, which were computed with the B3LYP functional and using the LANL2DZ basis set of
the newly synthesized Schiff bases, are depicted in Fig. 5.

Fig. 5 Frontier molecular orbitals of the Schiff bases L1 -L5, along with their respective energy
gap in eV.
3.5.2. Molecular electrostatic potential
The molecular electrostatic potential (MEP) of investigated compounds (L1-L5) was predicted at
the same level of theory as above to find the reactive site for electrophilic as well as nucleophilic
attack. ESP analysis of the synthesized compounds (L1-L5) was done using DFT/B3LYP/6-
31G(d,p). The MEP surface portrays the reactive sites in the molecules as depicted in Fig. 6. The
red color shows the maximum negative charge region, which indicates that an electrophilic
attack is possible at this site. The site prone to nucleophilic attack is represented by the blue color
indicating the maximum positive charge region, while the green color shows the zero potential
region (Uddin, Siddique, Mase, Uzzaman, & Shumi, 2019). It is clear from the MEP map that the

negative potential region is located over the oxygen atom (electronegative) and the positive
potential region is located over the hydrogen atoms. For the oxygen atom, the negative potential
represented by the deepest red color is -0.058 au (L1), -0.069 au (L2), -0.072 au (L3), -0.070 au
(L4), and -0.074 au (L5), respectively, while for hydrogen atoms the positive potential
represented by the deepest blue color has the same value with a positive sign.
Fig. 6 The molecular electrostatic potential (MEP) surfaces of the Schiff bases L1-L5.
3.6. DNA binding studies
Cancerous cells have mutated DNA and targeting it is a promising way for achieving an anti-
oncogenic effect (Gurova, 2009). Small molecules can influence DNA either directly or
indirectly by interacting with a protein (such as polymerases or RNA). DNA binding species can
either bind covalently or non-covalently. The extent of DNA damage depends on the type of
binding with DNA. Covalent binding is irreversible and most damaging. However, molecules
binding through a non-covalent interaction are less toxic (Sirajuddin, Ali, & Badshah, 2013;
Zhang, Bi, Fan, Wang, & Bao, 2015). Compounds showing a DNA interaction are thus of great
importance in the search of new antineoplastic molecules. The ability of L5 to interact with hs-
DNA (herring sperm) was studied in the absence and presence of increasing concentrations of
DNA. By increasing the DNA concentration, it is quite clear that a hyperchromicity with no red
or blue shift was observed, showing a non-covalent interaction with a binding constant of K =
2.236 x 103 M-1 and Gibbs free energy ΔG = -19.11 KJ/mol (Fig. 7). The binding constant was

calculated from the intercept to slope ratios of Ao/A-Ao vs 1/[DNA] plot, while the Gibbs free
energy was determined using the equation ∆G = -RTlnK.
Fig. 7 Absorption spectra of 100 μM of L5 in the absence (0 μM) and presence (66 μM, 131 μM,
196 μM, 261 μM, 327 μM and 392 μM) of DNA. The arrow direction shows increasing
concentration of DNA. The graph is the plot of Ao/(A-Ao) versus 1/[DNA] for the determination
of the binding constant and Gibbs free energy of the L5 -DNA product.
3.7. Electrochemical studies
3.7.1. Cyclic voltammetry
The cyclic voltammetry (CV) of the compounds L1-L5 was performed using glassy carbon as a
working electrode (GCE), Pt as a counter electrode and Ag/AgCl as a reference electrode, in
DMSO as a solvent. Initially, the controlled CV was performed in the absence of DNA for

comparison for all the Schiff bases (L1-L5) as shown in Fig. 8. As clear from the CV curves, an
oxidation peak for L1 - L4 around 1.10 V was found. This oxidation peak is attributed to the
hydroxyl moiety present in these compounds. L5 shows two oxidation peaks at 0.9 and 1.2 V
along with a reduction peak at -1.2 V.
These Schiff bases were employed for further DNA binding studies as shown in Fig. 9. In
the presence of different concentrations of DNA, L1-L5 show a decrease in the peak currents as
well as a shift in the peak potentials. The oxidation peak potential of all the compounds shifts to
a more positive value. The decrease in peak current values suggests that the compounds bind to
the bulky DNA molecule. The decrease in peak current and shifting of the peak potential towards
a more positive potential shows the association of DNA with the Schiff bases. Due to the
presence of DNA, certain changes occurred in the electrochemical response and respective
current-potential parameters indicating the compound-DNA presence in the system. These
changes, along with a shift toward a more positive potential, denote the electrostatic mode of
interaction between the compounds and DNA (M. Iqbal et al., 2013; Shabbir et al., 2015).
To further evaluate the interaction between DNA and Schiff bases, CV studies were done
at different scan rates. The plots of anodic peak current vs the square root of scan rate (v1/2) for
all the compounds were found linear before and after the addition of DNA with a decrease in
slope for the latter case compared to the former. The diffusion of the redox species is slower in
the presence of DNA and is evident from the values of the diffusion coefficient (Table 2). To
support the above argument, the stability constant (K) was calculated by using the following
equation (Shabbir et al., 2017):
1/[DNA] = K (1-A)/(1-I/Io) – K,
where A is an empirical constant. The K values are given in Table 2. Significantly, a large K
compared to the values reported for different compounds (Arshad & Farooqi, 2018; M. Iqbal et
al., 2013) suggests the potential ability of these Schiff base to interact with DNA. The number of
binding sites (s) in terms of the concentration of base pairs is shown in Table 2 and was
calculated using the following equation:
Cb /Cf = K [DNA] / 2s
where Cf and Cb are the concentrations of free and DNA-compound bound species respectively.
Also, Cb/Cf can be represented by the equation Cb/Cf = (Io-I)/I as reported in the literature

(Aslanoglu & Ayne, 2004). The values of binding site size show that L1 and L5 integrate more
than one base pair of the DNA resulting in strong interactions for these compounds.
Fig. 8 Cyclic voltammograms of compounds L1- L5 (1 mM each) recorded at GCE in an argon
saturated DMSO + 0.1M TBAP solution using a 100 mV s-1 scan rate at 25 oC.

Fig. 9 Cyclic voltammograms of 1 mM DMSO solutions of L1, L2, L3, L4 and L5 (─) without
DNA, (─) in the presence of 1.86 µM DNA, (─) 3.68 µM DNA on glassy carbon electrode at a
scan rate of 100 mV/s.
2.7.2. Differential pulse voltammetry
Differential pulse voltammograms of the Schiff bases are presented in Fig. 10. Peak currents are
in the order of L5 > L1 >L4 > L3 > L2 as observed by CV studies. The order is in accordance to
the Do values. The facile electron transfer (ET) process in L5 is understandable from its simple
and planar structure. The observed W1/2 values (≈200 mV) of all the five Schiff bases suggest
one electron transfer process. These values are larger than the theoretical value of 90 mV for an
ET process, which may be due to an uncompensated solution resistance.

Fig. 10 Differential pulse voltammograms of the Schiff base compounds L1(▬), L2 (▬), L3
(▬), L4(▬) and L5 (▬) in 1 mM (15 mL) DMSO solutions, each recorded under argon in the
presence of 0.1 M TBAP at 25 °C.
Table 2. The compound-DNA interaction electrochemical parameters of compounds on glassy
carbon vs. Ag/AgCl in a DMSO solution, at a 100 mVs-1 scan rate, at 25 oC.
Compound Do (cm2s-1)(without DNA Do (cm2s-1)(with DNA)
K(M-1)
s (bp)
2.96
0.27
0.73
L1
L2
L3
5.56 x 10-6
1.34 x 10-6
1.90 x 10-6
3.42 x 10-6
1.90 x 10-7
2.25 x 10-7
9.76 x 104
9.02 x 104
4.43 x 104

L4
L5
4.58 x 10-6
5.88 x 10-5
2.71 x 10-6
1.43 x 10-5
3.49 x 104
8.37 x 104
0.28
1.56
3.8 Antitumor activity assay
3.8.1 Cell viability assay (MTT assay)
The cytotoxic potential of all the compounds was evaluated in human breast adenocarcinoma
cells MCF-7, human cervical adenocarcinoma cells HeLa by MTT (Dimethyl–2–thiazolyl–2,5–
diphenyl–2H–tetrazolium bromide)–based cell viability assay. Later, the effect of inhibitors
showing more than 50% inhibition was also examined against normal cells, i.e., baby hamster
kidney cells (BHK–21). Cells were initially treated with 100 µM end concentration, and
derivatives which exhibited more than 50% inhibition were further tested in 8 dilutions to
evaluate the inhibitory concentration (IC50 values). None of the compounds showed an inhibition
equal or above 50% against the MCF-7 cell line. However, L2 and L5 exhibited more than 50%
inhibition against HeLa cells, therefore, the dilution was carried out and IC50 values were
calculated. Moreover, these compounds were tested against normal cells i.e., baby hamster
kidney cells (BHK–21) and their IC50 values were reported in Table 3 as well. Data is presented
as the mean values (SEM) for three independent experiments. These results were revealed after a
24 hours treatment of compounds with cell lines. Carboplatin used as reference control showed
5.13 ± 0.45 µM against HeLa cells, however, the compound L2 and L5 showed 56.7 ± 1.45 µM
and 20.8 ± 0.46 µM, respectively.
Table 3. Cytotoxic efficacy of the compounds L1-L5 against HeLa, MCF-7, and BHK-21 cell lines at 100
µM.
Sample Code
HeLa
MCF-7
BHK-21
IC50 (µM) ± SEM/%age inhibition
L1
2.40%
56.7 ± 1.45
36.6%
1.08%
18.0%
2.57%
29.3%
31.0%
89.3%
-
L2
32.2 ± 1.78
L3
-
L4
4.50%
-
L5
20.8 ± 0.46
5.13 ± 0.45
60.2 ± 1.33
-
Carboplatin

3.8.2 Cell cycle analysis assay
Cell cycle analysis is a method to determine the DNA content in cells with a great accuracy. The
assay involves the use of a fluorescent dye, propidium iodide, which enters the cells whose
membranes are ruptured after treating with a cytotoxic molecule. Propidium iodide (PI) binds to
genetic material such as DNA, RNA, and mitochondrial DNA. Mitochondrial DNA has a
negligible amount in cytosol. To acquire only a DNA quantity in such cells, generally, RNase is
added to remove RNA so that PI could not bind to it. A difference in DNA content in G0/1 (gap 0
and gap1), S (synthesis), and G2/M (gap2/checkpoint and mitosis) cell cycle phases by using this
univariate analysis can help understanding the phase at which a certain molecule is causing an
arrest to show its cytotoxic potential when compared to control or untreated cells (Crissman &
Tobey, 1974; Darzynkiewicz, Halicka, & Zhao, 2010; Nunez, 2001). The most effective
cytotoxic analogue L5 was selected for flow cytometry analysis to further investigate its effect
on cell cycle progression and apoptosis in HeLa cells. Fig. 11 shows the DNA content within the
dividing cells as assessed by propidium iodide (PI) staining and the percent cell population
within the cell cycle phases, i.e., Go – G1, S, and G2/ M phases. For this purpose, the compound
was used in three different concentrations (i.e., 20, 50 and 100 µM) keeping in mind the IC50
values of the compound.

Fig. 11 Cell cycle analysis using propidium iodide staining and flow cytometry. Results
showing cell population in G0/G1, S and G2/M phases of the cell against HeLa cells at 20 µM
(a), 50 µM (b), and 100 µM (c) of L5, negative control (d), 5 µM (e) and 10 µM (f) of
carboplatin.
3.8.3 Microscopic analysis of apoptosis
Propidium iodide is a hydrophilic, light sensitive, and membrane impermeable fluorescent dye
that specifically binds to nucleic acids (DNA, RNA, mitochondrial DNA) in dead or apoptotic
cells, which possess a ruptured or permeable membrane, by intercalation with an almost 20 to
30-fold increase in fluorescence intensity and gives information about the integrity of cells
(Crowley et al., 2016; Dengler, Schulte, Berger, Mertelsmann, & Fiebig, 1995; Sträuber &
Müller, 2010). On the other hand, 4′,6-diamidino-2-phenylindole (DAPI) is a nuclear staining
dye that binds to adenine-thymine rich sequences of the DNA minor groove to form a fluorescent
complex while a non-fluorescent complex is formed through intercalation. However, binding at
the major groove of DNA is also observed for DAPI. An intercalative binding is observed when

DAPI binds to RNA (Kapuscinski, 1995; Tanious, Veal, Buczak, Ratmeyer, & Wilson, 1992;
Trotta et al., 2003). As reported, DAPI is permeable to dead cells while having a slight
permeability in live cells (Gomes et al., 2013). Cell shrinkage and nuclear condensation are
salient features of apoptotic cells that can be visualized by DAPI staining and a difference can be
assessed as cells undergoing apoptosis possess a slightly smaller nucleus as compared to normal
cells (Doonan & Cotter, 2008). The HeLa cells were treated with L5 in 2 different
concentrations, i.e., IC50 values and 2 x IC50 values. The images were apprehended after 24
hours, demonstrating the cell death mechanism. 4′,6-diamidino-2-phenylindole (DAPI) and its
counter stain propidium iodide (PI) were used for staining the nuclear material (Fig. 12).
(b)
(a)
(c)
(d)
Fig. 12 Changes in nuclear morphology induced by IC50 21 µM of L5 (a), 2x IC50 42 µM of
L5 (b), after treatment with DAPI. Changes in nuclear morphology induced by IC50 21 µM of
L5 (c), 2x IC50 42 µM of L5 (d) after treatment with PI. The changes in nuclear morphology

were investigated under a fluorescence microscope (Nikon ECLIPSE Ni–U with the
magnification of 10X) against a cervical cancer cell line (HeLa).
3.8.4 Analysis of intracellular reactive oxygen species (ROS) production
2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a fluorescent and cell permeable dye
that is intracellularly hydrolyzed to polar form 2´,7´-dichlorodihydrofluroescein (DCFH) by
cellular esterase and preserved within the cell (R.P. Rastogi et al., 2010). The HeLa cells were
treated with the most potent compound L5 in different concentrations (IC50 value i.e., 21 µM and
2x IC50 i.e., 42 µM) and the cells were visualized by fluorescence microscope (Nikon ECLIPSE
Ni–U with the magnification of 20X) using an excitation wavelength of 488 nm and emission
was detected in the range of 500-600 nm. Fig. 13 shows that treated HeLa cells with the potent
compound L5 exhibit disintegrated cell membranes and condensed cellular protein (DNA) that
may be due to oxidation of lipids and proteins.
A
B

C
D
Fig. 13 Oxidized DCF fluorescence in HeLa cells induced by IC50 21 µM of L5 (A), 2x IC50 42
µM of L5 (B), carboplatin (C) and untreated cells (D), after treatment with 2',7'-
dichlorodihydrofluorescein diacetate. The images were taken under a fluorescence microscope
(Nikon ECLIPSE Ni–U with the magnification of 20X) against a cervical cancer cell line
(HeLa).
3.8.5 Apoptosis assessment by caspase -9 and -3 activity
In three pathways of apoptosis, the role of caspases is crucial for apoptosis induction. Initiator
caspases (e.g., caspases 8 and 9) cause activation of executioner caspases (e.g., caspases 3, 6 and
7), which play their role in triggering apoptosis (Elmore, 2007; Szczepaniak et al., 2018). A
slight decrease in HeLa cells was found in caspase 9 activity by both the concentrations of
compound, i.e., at IC50 value (21 µM) and at 2 x IC50 value (42 µM), as compared to control after
an 18 hours treatment. However, about 1.8-fold increase in caspase -3 was observed in HeLa
cells as compared to control after an 18 hours treatment (Fig. 14).
Fig. 14 Enhancement of caspase-9 activities after treatment with compound L5 for 18 h (A).
Compound L5 stimulates caspase-3 activity and causes apoptosis in HeLa cells (B).
Conclusion
1
Five Schiff base compounds (L1-L5) were synthesized and characterized by FT-IR, H
13
and C NMR spectroscopy, and X-ray crystallography. DNA interaction studies were analyzed
by UV-vis spectroscopy and cyclic voltammetry. The binding constants obtained for all

compounds from both UV-vis spectroscopy and cyclic voltammetry are in the order of 103-104
M-1 and indicate an electrostatic mode of interaction with DNA. The synthesized azomethines
were explored for their antineoplastic effect on HeLa and MCF-7 cancer cell lines, while their
cytotoxicity to normal cells was evaluated on a BHK-21 cell line by using the MTT viability
assay. The pro-apoptotic mechanism for an active compound was examined by fluorescence
microscopy, cell cycle analysis, caspase 9 and 3 activity, reactive oxygen species (ROS)
production, and DNA binding studies. Therefore, we suggest that the potent compound L5 could
be significant for drug discovery and development in the future having anticancer effects.
Acknowledgments
Noor Uddin is thankful to HEC for providing financial support through IRSIP program. Paula L.
Diaconescu acknowledges support from NSF Grant CHE-1809116. Saqib Ali is thankful to
Quaid-i-Azam University, Islamabad for financial support in the form of URF-2019. Jamshed
Iqbal is thankful to the Higher Education Commission of Pakistan for the financial support
through Project No.Ph-V-MG-3/Peridot/R&D/HEC/2019 and 6927/NRPU/R&D/17.
.
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