New tailored substituted benzothiazole Schiff base Cu(II)/Zn(II) antitumor drug entities: effect of substituents on DNA binding profile, antimicrobial and cytotoxic activity

Article abstract of DOI:10.1080/07391102.2018.1467794

New tailored Cu(II) & Zn(II) metal-based antitumor drug entities were synthesized from substituted benzothiazole o?vanillin Schiff base ligands. The complexes were thoroughly characterized by elemental analysis, spectroscopic studies {IR, ¹H & ¹³C NMR, ESI?MS, EPR} and magnetic susceptibility measurements. The structure activity relationship (SAR) studies of benzothiazole Cu(II) & Zn(II) complexes having molecular formulas [C₃₀H₂₂CuN₅O₇S₂], [C₃₀H₂₀Cl₂CuN₅O₇S₂], [C₃₀H₂₀CuF₂N₅O₇S₂], [C₃₀H₂₂N₄O₄S₂Zn], [C₃₀H₂₀Cl₂N₄O₄S₂Zn], and [C₃₀H₂₀F₂N₅O₇S₂Zn], with CT?DNA were performed by employing absorption, emission titrations, and hydrodynamic measurements. The DNA binding affinity was quantified by K _b and K _sv values which gave higher binding propensity for chloro-substituted Cu(II) [C₃₀H₂₀Cl₂CuN₅O₇S₂] complex, suggestive of groove binding mode with subtle partial intercalation. Molecular properties and drug likeness profile were assessed for the ligands and all the Lipinski’s rules were found to be obeyed. The antimicrobial potential of ligands and their Cu(II) & Zn(II) complexes were screened against some notably important pathogens viz., E. coli, S. aureus, P. aeruginosa, B. subtilis, and C. albicans. The cytotoxicity of the complexes [C₃₀H₂₀Cl₂CuN₅O₇S₂], [C₃₀H₂₀CuF₂N₅O₇S₂], [C₃₀H₂₀Cl₂N₄O₄S₂Zn], and [C₃₀H₂₀F₂N₅O₇S₂Zn] were evaluated against five human cancer cell lines viz., MCF?7 (breast), MIA?PA?CA?2 (pancreatic), HeLa (cervix) and Hep?G2 (Hepatoma) and A498 (Kidney) by SRB assay which revealed that chloro-substituted [C₃₀H₂₀Cl₂CuN₅O₇S₂] complex, exhibited pronounced specific cytotoxicity with GI₅₀ value of 4.8?μg/ml against HeLa cell line. Molecular docking studies were also performed to explore the binding modes and orientation of the complexes in the DNA helix.

Full text of DOI:10.1080/07391102.2018.1467794

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
New tailored substituted‒benzothiazole Schiff
base Cu(II)/Zn(II) antitumor drug entities: Effect of
substituents on DNA binding profile, antimicrobial
and cytotoxic activity
Siffeen Zehra, Mohammad Shavez Khan, Iqbal Ahmad & Farukh Arjmand
To cite this article: Siffeen Zehra, Mohammad Shavez Khan, Iqbal Ahmad & Farukh Arjmand
(2018): New tailored substituted‒benzothiazole Schiff base Cu(II)/Zn(II) antitumor drug entities:
Effect of substituents on DNA binding profile, antimicrobial and cytotoxic activity, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2018.1467794
To link to this article: https://doi.org/10.1080/07391102.2018.1467794
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Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://doi.org/10.1080/07391102.2018.1467794
New tailored substituted‒benzothiazole Schiff base Cu(II)/Zn(II) antitumor
drug entities: Effect of substituents on DNA binding profile, antimicrobial
and cytotoxic activity
a
b
b
a ,
Siffeen Zehra , Mohammad Shavez Khan , Iqbal Ahmad and Farukh Arjmand *
a
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
b
Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh 202002, Uttar
Pradesh, India
*
*
Corresponding author. E-mail address: farukh_arjmand@yahoo.co.in (F. Arjmand).
Corresponding author. Tel.: +91 5712703893.
Abstract
New tailored Cu(II) & Zn(II) metal−based antitumor drug entities were synthesized from
substituted benzothiazole o‒vanillin Schiff base ligands. The complexes were thoroughly
1
13
characterized by elemental analysis, spectroscopic studies {IR, H & C NMR, ESI−MS, EPR}
and magnetic susceptibility measurements. The structure activity relationship (SAR) studies of
benzothiazole Cu(II) & Zn(II) complexes having molecular formulas [C H CuN O S ],
3
0
22
5
7 2

[C H Cl CuN O S ], [C H CuF N O S ], [C H N O S Zn], [C H Cl N O S Zn] and
30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2
[
C H F N O S Zn], with CT‒DNA were performed by employing absorption, emission
3
0
20
2
5
7 2
titrations and hydrodynamic measurements. The DNA binding affinity was quantified by K and
b
K values which gave higher binding propensity for chloro substituted Cu(II)
sv
[
C H Cl CuN O S ] complex, suggestive of groove binding mode with subtle partial
30 20 2 5 7 2
intercalation. Molecular properties and drug likeness profile were assessed for the ligands and
all the Lipinski’s rules were found to be obeyed. The antimicrobial potential of ligands and their
Cu(II) & Zn(II) complexes were screened against some notably important pathogens viz., E.
coli, S. aureus, P. aeruginosa, B. subtilis and C. albicans. The cytotoxicity of the complexes
[
C H Cl CuN O S ], [C H CuF N O S ], [C H Cl N O S Zn] and [C H F N O S Zn]
30 20 2 5 7 2 30 20 2 5 7 2 30 20 2 4 4 2 30 20 2 5 7 2
were evaluated against five human cancer cell lines viz., MCF‒7 (breast), MIA‒PA‒CA‒2
pancreatic), HeLa (cervix) and Hep‒G2 (Hepatoma) and A498 (Kidney) by SRB assay which
revealed that chloro substituted [C H Cl CuN O S ] complex, exhibited pronounced specific
(
3
0
20
2
5
7 2
cytotoxicity with GI value of 4.8 μg/ml against HeLa cell line. Molecular docking studies
5
0
were also performed to explore the binding modes and orientation of the complexes in the DNA
helix.
Keywords: Benzothiazole Cu(II) & Zn(II) Schiff base complexes; structure activity
relationship; antimicrobial studies; cytotoxicity; molecular docking.

1
. Introduction
N‒heterocycles viz., benzimidazole, benzothiazole, indole, pyrazole and quinolines etc. have
emerged as an important distinct class of anticancer therapeutic agents (D. Kumar & Kumar,
2
016). These compounds possess N‒heterocyclic aromatic pharmacophore synthon which is
responsible for harnessing medicinal properties by multiple mechanisms which may be largely
responsible for inhibiting cell growth and inducing apoptosis (Solomon, Hu & Lee, 2009).
FDA’s Center for Drug Evaluation and Research (CDER) approves a wide range of new
molecular entities (NMEs) possessing bioactive moieties which may provide important new
therapies for treating patients with many chronic diseases. Many prominent N‒heterocyclic
compounds developed by reputed pharmaceutical companies (Pfizer, AstraZeneca, Novartis
etc.) have been approved by US FDA as anticancer agents for myriad phenotypes of cancers
(http://www.fda.gov). Unfortunately, many of these NMEs fail either in clinical trials or in the
developmental stages due to issues of systemic toxicity and low absorption at cellular levels.
Among the N‒heterocycles, benzothiazole derivatives have gained prominence due to diverse
biological activities viz., antimicrobial, antiviral and anticancer activities (Racané et al., 2013).
Besides this, benzothiazole is a key component of nucleic acids and therefore, can participate
directly in encoding of genetic information. Being an important drug synthon, it can be tethered
to other organic ancillary ligands for targeted therapy to yield more efficacious and potent
ligand scaffolds. Several benzothiazole derivatives are reported to possess excellent in vitro and
in vivo cytotoxicity at low nanomolar concentrations (Prota et al., 2014). Literature reports
reveal that 2‒aminobenzothiazole derivatives were synthesized and tested at National Cancer
Institute (NCI) against nine panels of cancer cell lines for antitumor activity. It was observed
that chloro derivative, 7‒chloro‒N‒(2,6‒dichlorophenyl)benzo[d]thiazol‒2‒amine was most
active against non‒small lung cancer cell line, HOP‒92 with significantly good GI value of
5
0

‒
8
7
.18 x10 M (Noolvi Patel & Kaur, 2012). Furthermore, modifications of the benzothiazole
scaffold could yield improved cytotoxicity profile which has been demonstrated in a series of
‒anilinopyridinyl‒benzothiazole Schiff base conjugates reported by A. Kamal et al. The
2
inhibitory effects of varied magnitude against the tested cell lines (>20 μM‒100 μM
concentration) were observed. Interestingly, these conjugates have displayed specificity toward
the prostate cancer cell line (DU145) and most of them have demonstrated significant cytotoxic
activity (Shaik et al., 2017).
Most of the chemotherapeutics in clinical use for the treatment of cancer show toxicity due to
off‒target uptake in the cells in biological system. Many researchers in the area of inorganic
medicinal chemistry are making considerable efforts to achieve the goal of antitumor drugs with
reduced toxicities. Among the rational therapeutic approaches to develop new metal‒based
biologically active compounds, strategies using transition metal complexes {Co(II),Cu(II) &
Zn(II)} are in frontline exhibiting improved pharmacological profile (Renfrew, 2014).
Furthermore, the synergy between the metal ions and ligand scaffold would result in a more
bioactive therapeutic agent capable of selective targeted activity at the site of action, leaving
normal or healthy cells unaffected. In this research work, we have opted for a rational synthetic
approach in which a benzothiazole active pharmacophore synthon was used to obtain Schiff
base complexes which could be predicted for antimicrobial chemotherapy which is newly
conceived concept as it can furnish therapeutic entities showing both superior antimicrobial
potency as well as pronounced anticancer activity.
Schiff−bases are privileged ligand scaffolds exhibiting a wide range of applications in the field
of medicinal inorganic chemistry (Hameed, Rashida, Uroos, Ali & Khan, 2017). They have
been categorized as versatile ligand moieties due to their structural stability, synthetic flexibility

and excellent chelating ability with metal ions (Shahraki & Heydari, 2017). They are endowed
with potential biological and pharmacological activities such as antimicrobial (Jana, Dalapati &
Guchhait, 2012; Biswas et al., 2012), anti‒malarial (Facchinetti, Reis, Gomes & Vasconcelos
2
012), antioxidant (Dehkhodaeia, Sahihia & Rudbari, 2017) and anticancer (Amiri et al., 2017).
In addition, Schiff bases are utilized as chemical and stereo selective DNA structural probes,
mimicking the active sites in the biological system and DNA dependent electron transfer
processes (Shokohi‒pour, Chiniforoshan, Sabzalian, Esmaeili, Abbas & Borojeni). Schiff base
transition complexes modulated with several heterocyclic moieties such as pyrazole,
1
,2,4‒triazoles, coumarins, benzothiazole and benzoxazoles are of remarkable interest due to
exclusive properties associated with the heterocyclic pharmacophore synthon, which finds
application in the treatment of chronic diseases viz., HIV‒AIDS, tuberculosis, Alzheimer’s and
cancer with multiple etiologies (Jadhao et al., 2017; Creaven et al., 2010; Kumar et al., 2017).
Heterocyclic Schiff base transition metal complexes are of prominent importance due to their
efficient and enhanced bioactivity against a wide range of bacterial and fungal strains
(Manjunath, Kulkarni, Bagihalli, Malladi & Patil, 2017; Hranjec et al., 2011). A series of novel
bioactive Schiff base transition metal complexes derived from 8–formyl–7hydoxy–4–
methylcoumarin and 2–hydrazino benzothiazole have been synthesized and thouroughly
characterized. The metal complexes are screened for their bacterial and fungal activity and were
found to be more active than the free ligand. The cytotoxic activity of the metal complexes was
evaluated against human ovarian cancer cell line (PA‒1) and they are found to be non‒toxic
against the tested cell line. Preliminary screening of the metal complexes against the
antitubercular activity displayed a significantly low MIC value of 0.8μg/ml as compared to the
standard drug and free ligand (Jawoor, Patil & Toragalmath, 2018).

Incorporating metal ions in bioactive ligand scaffolds can furnish ‘metallo-drug’
chemotherapeutics which are supposed to exert their effect by inhibition of proteins and
enzymes, and catalyze various intracellular processes, enhance lipophilicity and cell permeation,
alteration of cell membrane functions and arrest of cell cycle etc. (Tan, Lu, Ji & Mao, 2014).
Among the transition metals, copper complexes have emerged as a notably important class of
cancer therapeutics (Santini et al., 2014). In contrast to Pt drugs that are covalently bound to
DNA nucleobases, Cu(II) complexes primarily form noncovalent interactions with DNA either
by electrostatic surface binding to the oxygen atoms of negatively charged DNA
phosphodiester helix or groove binding (both major or minor) and intercalation (Gama et al.,
2
014). Advances in chemistry of copper anticancer therapeutic agents have shown that many
copper complexes with diverse ligand topology Schiff base, peptides, amino acids, terpyridines,
polypyridyls etc. demonstrate excellent cytotoxicity against a wide spectrum of cancer
phenotypes including the cisplatin resistant strains (Tardito et al., 2011; Manikandamathavan et
al., 2014; Yu et al., 2011; Nagababu et al., 2015; Goswami, Chakravarthi, Roy, Karande &
Chakravarty, 2011; Maity et al., 2010). Two copper complexes have entered the clinical trials
(Galindo‒Murillo, García‒Ramos, Ruiz‒Azuara, III Cheatham & Cortés‒Guzmán, 2015) and as
copper complexes exhibit a different mechanism of action as compared to classical Pt drugs, it
is expected that the major challenge of chemoresistance can be successfully overcome. Being a
bioessential less toxic element, copper (II) complexes are believed to overcome membrane
transportation, multi‒resistance and toxicity issues (McGivern, Afsharpour & Marmion, 2017).
A newly envisaged design of antimicrobial copper‒based therapeutic agent was reported by
Hassan et al. They prepared a water‒soluble, copper‒containing terpyridine carboxymethyl
cellulose complex which demonstrated good antimicrobial activity and showed inhibition of the

growth of the Gram‒positive bacteria and the fungus as well as 90% of the Gram‒negative
bacteria at 8 mg/mL. Such new antimicrobial metallo‒therapeutics are being developed to
prevent and treat these drug‒resistant infections (E. A. Hassan, Hassan, Moorefield &
Newkome, 2015). Another important potential transition metal of interest is Zn(II) which is
endowed by distinctive properties to undergo Lewis activation, generation of nucleophiles and
regulating apoptosis. Zinc plays an important role in the regulation of various cellular processes
viz., DNA synthesis, gene expression, enzyme synthesis and catalytic functions (Mendiguchia,
Pucci, Mastropietro, Ghedini & Crispini, 2013). Besides this Zn(II) complexes exhibit low in
vivo cytotoxicity and new multimodes of action and cellular targets (Tan Wang & Zhu, 2009).
The novelty of Zn(II) complexes as potent anticancer therapeutics could be due to structural
stability that may be induced by changing the coordination sphere in vivo.
In our laboratory, strenuous efforts are being made to achieve efficacious metal‒based
chemotherapeutic drug candidates for treating cancer. In continuation to our pursuit for the
design of smart metal−based drug entities, we have prepared a novel series of substituted
benzothiazole‒o‒vanillin Schiff base Cu(II) & Zn(II) complexes. Substituents of different
nature such as chloro, fluoro along with unsubstituted one were used to study their effect on
anticancer and antimicrobial activity. The structure activity relationship (SAR) is helpful to
access NMEs and such functionalization of the bioactive benzothiazole scaffolds can throw
light on mechanistic insight for specific targeted therapies. Due to multi‒drug resistance, there
is a substantial need for the development of newer potential antimicrobial chemotherapeutic
drug entities endowed with improved mechanism of action and multifold enhanced activity.
2
2
. Experimental Section
.1. Materials and methods

o−vanillin, 2−Aminobenzothiazole, 2−Amino−4−chlorobenzothiazole, and
−Amino−6−fluorobenzothiazole (Sigma Aldrich), Cu(NO ) .2H O and Zn(CH COO) .2H O
2
3
2
2
2
2
2
(Merck) were used as received. Calf thymus DNA (CT‒DNA) and
Tris(hydroxymethyl)aminomethane or Tris Buffer, were purchased from Sigma Chemicals Co.
All reagents and solvents were of best commercial grade and were used without further
purification.
Elemental analyses were performed on Carlo Erba Analyser Model 1106. Molar conductance
was measured at room temperature on a Digisun Electronic conductivity Bridge. Fourier
transforms IR (FTIR) spectra were recorded on an Interspec 2020 FTIR spectrometer.
Electronic spectra were recorded on UV−1700 PharmaSpec UV–vis spectrophotometer
(Shimadzu). Fluorescence measurements were made on Schimadzu RF−5201 fluorescence
spectrophotometer. The EPR spectra of the copper complex was obtained on Varian E 112 EPR
spectrometer using X band frequency (9.1 GHz) at room temperature in solid state. Magnetic
moment measurements were carried out using Sherwood scientific magnetic susceptibility
1
13
balance at 25ºC. The H and C NMR spectra were obtained on a Bruker Avance DRX−400
spectrometer operating at room temperature. Electrospray mass spectra were recorded on
Micromass Quattro II triple quadrupol mass spectrometer. The viscosity measurements were
carried out by using Ostwald capillary viscometer at 25ºC.
2
.2. Synthesis
2
.2.1. Synthesis of ligands
The ligand L1−L3 were synthesized by adding o−vanillin (0.152 g, 1 mmol) to a stirred
methanolic solution (10 ml) of 2−aminobenzothiazole (0.150 g, 1 mmol),
2
−amino−4−chlorobenzothiazole (0.184 g, 1 mmol) and 2−amino−6−fluorobenzothiazole

(0.168 g, 1 mmol), respectively in a 1:1 M ratio (Fig.1). The reaction mixtures were refluxed
for 2 h and the progress of reaction was monitored by TLC. The yellow precipitate which
formed was filtered off under vacuum, washed thoroughly with methanol and dried in vacuo.
2
.3.2. Synthesis of complexes 1 and 2 (a‒c).
The complexes 1 and 2 (a‒c) were synthesized by adding methanolic solution of
Cu(NO ) .2H O (0.241 g, 1 mmol) and Zn(CH COO) .2H O (0.241 g, 1 mmol) to a methanolic
3
2
2
2
2
2
solution of ligands L1 (0.284 g), L2 (0.318 g), L3 (0.303 g), (1 mmol) in a 1:2 molar ratio
which was stirred for 2 h at room temperature to obtain brown and orange colored precipitates,
respectively.
F
S
S
S
N
N
N
N
N
N
HO
HO
HO
Cl
OCH3
OCH3
OCH3
Fig.1. Structure of the substituted benzothiazole derived ligands L1−L3.
.3. DNA binding studies
2
DNA binding studies, including absorption, fluorescence, emission titrations and viscosity
measurements conformed to the standard methods previously reported by our laboratory
(Lakowicz & Webber, 1973; Wolfe, Shimer & Meehan, 1987; Arjmand, Muddassir, Zaidi &
Ray, 2013) were carried out.
Absorption titration were performed by maintaining the constant complex concentration (1.67 ×
−
4
−5
1
0 M) and varying the concentration of the CT‒DNA (0.00–5.0 × 10 M). Fluorescence
quenching experiments were carried out by concomitant addition of metal complexes to the
EB−DNA and DAPI‒DNA system such that [complex]/[DNA] = 1 : 1 to 8 : 1 in 5 mM Tris-
HCl/50 mM NaCl buffer (pH = 7.2). While measuring the spectra, an equal amount of DNA

was added to both the complex and the reference solution to eliminate the absorbance of the
CT‒DNA, and Tris−HCl buffer was subtracted through base line correction.
Viscosity measurements were carried out using Ostwald viscometer. The viscosity of CT‒DNA
in buffer solution was measured in the presence of increasing amounts of the complexes (up to
the value of r = 1.0) at room temperature. The flow time was measured using a digital
1
/3
stopwatch. Data are presented as (η/η ) vs. the ratio of the concentration of complex to DNA,
o
where η is the viscosity of CT‒DNA in the presence of compound and η is the viscosity of
o
CT‒DNA alone. Viscosity values were calculated according to the relation η = (t – t )/ t , where
o
o
t was the flow time of the CT‒DNA solution in the presence or absence of the complex and t of
o
the buffer alone. The average values were replicated thrice to evaluate the viscosity of the
complexes.
2
.5. Antimicrobial studies
All the synthesized ligands L1‒L3 and their complexes 1 and 2 (a‒c) were screened for their in
vitro antibacterial activity against two Gram‒negative [Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853)] and two Gram‒positive [Staphylococcus aureus
(ATCC 25923) and Bacillus subtilis (MTCC 121)] bacterial strains. The agar well diffusion
method of Perez et al., also described earlier by Ahmad et al. was adopted for measuring the
antibacterial assays. Briefly, all cultures were routinely maintained on NA (nutrient agar) and
incubated at 27 ºC for overnight in NB (Nutrient broth). After incubation, 0.1 ml aliquot of
bacterial culture was spread uniformly with the help of spreader on NA plates. Wells of 8 mm
diameter were punched into the agar medium, sealed with soft agar and loaded with different
concentrations of the metal complexes (0.1 ml). Antibiotic disc, Doxycycline (30 mcg/disc) and
solvent were used as positive and negative control, respectively. The plates were then incubated

for 24 h at 37 ºC and the resulting zones of inhibition (mm) were measured. The inoculums of
non‒spore forming fungi, Candida albicans was obtained by growing in SD (Sabouraud
Dextrose) broth at 28 ºC for 48 h. Volume of 0.1 ml of diluted fungal cell suspension was
uniformly spread on SDA (Sabouraud Dextrose Agar) plates and test compound were loaded
into wells (describe above). C. albicans plates were incubated at 27 ºC for 18−48 h and
antifungal activity was determined by measuring the diameters of the inhibition zone (mm).
2
.6. In vitro cytotoxic assay
The in vitro anticancer activity of 1 and 2 (b & c) was carried out against five human cancer cell
lines of different histological origin viz; MCF−7 (breast), MIA−PA−CA−2 (pancreatic), HeLa
(cervix), Hep−G2 (Hepatoma) and A498 (Kidney). G−Adriamycin, a standard anticancer drug,
was taken as the control. The cell lines were procured and grown in RPMI−1640 medium
supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics to study growth pattern of
these cells. The proliferation of the cells upon treatment with chemotherapy was determined by
means of the Sulphorhodamine–B (SRB) assay. All cell lines were seeded into 96 well plates
and cells were counted and cell counts was adjusted according to the titration readings so as to
give optical density in the linear range (0.5 to 1.8) and were incubated at 27 ºC in CO incubator
2
for 24 h. The stock solution of the complexes were prepared as 100 mg/ ml in DMSO and four
dilutions i.e. 10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ ml, in triplicates were tested, each well
receiving 90 μl of cell suspension. The plates were labeled and were incubated for 48 h.
Termination of experiment was done by gently layering the cells with 50 μl of chilled 20% TCA
(in case of adherent cells) and 50% TCA (in case of suspension cell lines) for cell fixation and
kept at 4ºC for 1 h. Plates stained with 50 μl of 0.4% SRB for 20 min. All experiments were
made in triplicate.

2
.7. Structure‒activity relationship studies
In order to demonstrate the structure‒activity relationship (SAR) and quantitatively estimate the
molecular properties of the ligands and their respective metal complexes, the molinspiration
calculations were carried out (www.molinspiration.com).
2
.8. Molecular docking studies
Molecular docking studies were performed by using HEX 8.0 software (Macindoe, Mavridis,
Venkatraman, Devignes & Ritchie, 2010), a molecular graphics program for calculating and
displaying feasible docking modes of DNA molecule. Structure of the complexes was sketched
by Chemdraw, energy minimized and converted into PDB format using Mercury software
(http://www.ccdc.cam.ac.uk/). The crystal structure of the B−DNA dodecamer
d(CGCGAATTCGCG) (PDB ID: 1BNA) were downloaded from the protein data bank
2
(
http://www.rcsb.org./pdb).Visualization of the docked pose was done by using Discovery
Studio molecular graphics program.
. Results and discussions
3
All the ligands L1−L3 and their respective metal complexes 1 and 2 (a‒c) are found stable at
room temperature and are soluble in common organic solvents viz., methanol, ethanol, DMF
‒
1
2
‒1
and DMSO. The molar conductance values (9.5–12.8 Ω cm mol ) are too low to account for
any dissociation of the complexes, indicating that the complexes are non‒electrolytes (Geary,
1
971). On the basis of spectral and magnetic studies, the coordination geometry of copper(II)
[
[
C H CuN O S ], [C H Cl CuN O S ] and [C H CuF N O S ] and zinc(II)
3
0
22
5
7
2
30 20
2
5
7
2
30 20
2
5
7 2
C H N O S Zn], [C H Cl N O S Zn] and [C H F N O S Zn] complexes were proposed
3
0
22
4
4
2
30 20
2
4
4
2
30 20
2
5
7 2
to be square pyramidal and tetrahedral, respectively (Fig. S1).
‒
1
IR spectra of ligands L1‒L3 showed a characteristic band at 3434‒3435 cm assigned to
phenolic (OH) group which was found absent in the spectra of complexes [C H CuN O S ],
3
0
22
5
7 2

,
[
C H Cl CuN O S ] [C H CuF N O S ] [C H N O S Zn], [C H Cl N O S Zn] and
30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2
[
C H F N O S Zn], indicating its coordination to metal ion via deprotonation (Daravath et al.,
30 20 2 5 7 2
2
017). A significant shift was observed in the absorption frequency of azomethine group
‒
1
‒1
ν(C=N) in ligands at 1632−1635 cm as compared to the metal complexes 1645−1640 cm ,
thereby validating its coordination via the nitrogen atom (Bhowmik et al., 2016). In addition,
medium intensity bands were also observed for complexes 1 and 2 (a‒c) in the region 542−468
‒
1
and 438−420 cm attributed to the stretching frequency of ν(Cu/Zn‒O) and ν(Cu/Zn‒N)
vibrations, respectively (Deacon & Phillips, 1980; Nakamoto, 1986).
1
The H NMR spectra of ligands L1‒L3 and complexes [C H N O S Zn],
3
0
22
4
4 2
[
C H Cl N O S Zn] and [C H F N O S Zn] revealed signals in the range 6.90−7.99 ppm
30 20 2 4 4 2 30 20 2 5 7 2
range assigned to aromatic protons. A singlet corresponding to the azomethine group was
observed in the ∼9.43−9.26 ppm range which was shifted to 8.13−8.17 ppm in the complexes
suggesting coordination of the N‒atom to the metal ion (Kalaivani et al., 2012). The ligands
displayed singlets at ∼12.44−11.12 ppm attributed to the OH group (Mandegani, Asadi, Asadi,
Karbalaei‒Heidari & Rastegari, 2016) which was found absent in the spectra of complexes
1
3
indicating coordination to Zn(II) metal ion upon deprotonation. In the C NMR spectra, the
azomethine carbon signatures were observed at 167.49−166.51 ppm which was shifted to
1
68.35−167.34 ppm in the complexes while aromatic carbon signatures were observed
103.26−135.33 ppm (Fig.S2).
The ESI mass spectra of ligands L1−L3 exhibited molecular ion peak [M ] at m/z = 283, 318
∼
+
+
+
+
and 303, corresponding to [C H N O S] , [C H ClN O S] and [C H FN O S] moieties,
1
5
12
2
2
15 11
2
2
15 11
2
2
respectively. The mass spectra of metal complexes 1 (a‒c) showed m/z molecular ion peak at
+
+
6
91.08, 758.75 and 730.77, corresponding to [C H CuN O S ] , [C H Cl CuN O S ] and
30 22 5 7 2 30 20 2 5 7 2

+
[
C H CuF N O S ] moieties. The mass spectra for complexes 2 (a‒c) displayed molecular ion
30 20 2 5 7 2
+
peaks at m/z = 632.11, 700.79 and 667.1, associated with [C H N O S Zn] ,
3
0
22
4
4 2
+
+
[
C H Cl N O S Zn] and [C H F N O S Zn] moieties, respectively. The spectra of metal
30 20 2 4 4 2 30 20 2 5 7 2
complexes also displayed fragmentation peaks corresponding to their respective ligand moieties
Fig.S3)
(
To further deduce structural information, the electronic spectra of the complexes 1 and 2 (a‒c)
were measured in the UV‒Vis region (200−700 nm). The electronic absorption spectra of the
ligands displayed peaks at 260−264 and 348−341 nm corresponding to π–π* and low energy n–
π* ligand centered (IL) transitions. However, these bands were shifted to 270−275 and 354−367
nm suggesting coordination of the ligand to Zn(II) ion in complexes [C H N O S Zn],
3
0
22
4
4 2
[
C H Cl N O S Zn] and [C H F N O S Zn], respectively (Enamullah et al., 2016). The
30 20 2 4 4 2 30 20 2 5 7 2
electronic spectra of complexes [C H CuN O S ], [C H Cl CuN O S ] and
3
0
22
5
7
2
30 20
2
5
7 2
[
C H CuF N O S ] also showed low intensity broad bands centered at 521, 529 and 526 nm in
30 20 2 5 7 2
the visible region attributed to d‒d transitions, consistent with the pentacoordinated geometry of
the complexes (Bhowmik, Das, Chattopadhyay & Ghosh, 2015).
The magnetic susceptibility measurement of complexes [C H CuN O S ],
3
0
22
5
7 2
[
C H Cl CuN O S ] and [C H CuF N O S ] were carried out at room temperature to
30 20 2 5 7 2 30 20 2 5 7 2
provide insights into the geometry around the Cu(II) ion and to ascertain its paramagnetic
nature. The observed magnetic moment value for complexes 1 (a‒c) were found to be 1.75, 1.77
and 1.76 BM which is slightly higher than the spin only value due to one unpaired electron (µeff
=
1.73 BM), suggesting a possible penta coordinated geometry (Latif & Mohamed 2018).
However, complexes [C H N O S Zn], [C H Cl N O S Zn] and [C H F N O S Zn] were
3
0
22
4
4
2
30 20
2
4
4
2
30 20
2
5
7 2

found to be diamagnetic in nature, consistent with the tetrahedral geometry, respectively
Orojloo, Mohamed, Zolgharnein, Solimannejad & Amani., 2017).
The solid state X‒band EPR spectra of complexes [C H CuN O S ], [C H Cl CuN O S ]
(
3
0
22
5
7
2
30 20
2
5
7 2
and [C H CuF N O S ] were recorded at room temperature under the magnetic field strength
3
0
20
2
5
7 2
3
000 ± 1000 G using tetracyanoethylene as a field marker (Fig.S4). All the complexes 1 (a‒c)
displayed an isotropic EPR spectra exhibiting axial symmetry with g =2.212, g =2.214, g =
||
||
||
2
.213, g = 2.031, g = 2.064, g = 2.051and g = 2.10, g = 2.12, g = 2.11, respectively
⊥ ⊥ ⊥ av av av
computed from the formula gav2 = g + 2g /3. These parameters were consistent with the penta
2
2
||
⊥
coordinated geometry of the copper(II) metal ion (Arruda et al., 2017). The g ≤ 2.2 is in
||
agreement with the covalent character of the metal ligand bond (Kumar, Singh, Garg & Singh,
2
003).
3
3
.5. DNA binding studies
.5.1. Absorption titrations
Since benzothiazole is an important bioactive heterocyclic pharmacophore, its relevance in
metallo‒pharmaceuticals as a metal anchoring scaffold is enhanced by forming Schiff base with
other ancillary ligands. The structure‒activity relationship (SAR) of benzothiazoles viz.,
unsubstituted, chloro and fluoro derivatives, [C H CuN O S ], [C H Cl CuN O S ],
3
0
22
5
7
2
30 20
2
5
7 2
[
C H CuF N O S ], [C H N O S Zn], [C H Cl N O S Zn] and [C H F N O S Zn] with
30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2 30 20 2 5 7 2
CT‒DNA can lay a platform for the design of rational newer antitumor drug candidates. The
binding propensity of complexes 1 and 2 (a‒c) with CT‒DNA was studied by carrying out
−
5
absorption titrations with concomitant addition of CT‒DNA (0.00–5.5 x 10 M). Strong
interactions between the metal complexes and the DNA base pairs results in hypochromism in
the absorption intensity with bathochromic shift due to stacking interactions of the aromatic
moiety inside the DNA helix resulting in conformational changes. However, minor groove

binding interaction of the complexes with the DNA usually results in hyperchromism with no
red/blue shift (Banerjee et al., 2018). An increase in the absorbance of the complexes 1 and 2
(a‒c) was observed at 265 nm indicating “hyperchromic” effect and “hypochromic” shift was
observed at the intraligand absorption band centered at 397 nm (Fig.2). These changes in the
absorbance were attributed to groove binding mode of complexes with partial intercalation (Liu
et al., 2018; Zhang et al., 2017). In order to quantify the binding strength the intrinsic binding
constant, K values were obtained by using Wolfe−Shimer equation (1);
b
[
DNA] [DNA]
1
(1)


a   f b   f K (   )
b
a
f
where  ,  and  are the apparent extinction coefficient (A /[M]), the extinction coefficient
a
f
b
obs
for free metal complex (M), and the extinction coefficient for the metal complex (M) in the fully
bound form, respectively. The K values of complexes 1 and 2 (a‒c) are found to be 4.02(±
b
5
5
5
5
5
0
3
.15) x 10 , 4.40(± 0.08) x 10 , 4.26 (± 0.11) x 10 , 3.07(± 0.12) x 10 , 3.67(± 0.09) x 10 and
5
‒1
.31(±0.12) x 10 M , respectively.
The binding constant values suggested that Cu(II) complexes [C H CuN O S ],
3
0
22
5
7 2
[
C H Cl CuN O S ] and [C H CuF N O S ], demonstrated higher binding propensity for
30 20 2 5 7 2 30 20 2 5 7 2
CT‒DNA than corresponding zinc complexes [C H N O S Zn], [C H Cl N O S Zn] and
3
0
22
4
4
2
30 20
2
4
4 2
[
C H F N O S Zn] and followed the trend 1b > 1c > 1a > 2b > 2c > 2a which was further
30 20 2 5 7 2
substantiated by other spectroscopic studies. The rationale for higher binding strength of
complexes is due to the presence of electron withdrawing group which increases π‒stacking
ability of the benzothiazole moiety and further induces positive charge for electrostatic
interaction (Lu et al., 2008).

Fig.2. UV‒vis titration spectra of complexes 1 and 2 (a‒c) in presence of CT‒DNA.
.5.2. Fluorescence titrations
3
Another more sensitive optical fluorescence titration technique was employed to quantify the
magnitude of binding of complexes 1 and 2 (a‒c) corresponding to [C H CuN O S ],
3
0
22
5
7 2
[C H Cl CuN O S ], [C H CuF N O S ], [C H N O S Zn], [C H Cl N O S Zn] and
30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2
[
C H F N O S Zn], respectively with CT‒DNA. In the absence of CT‒DNA, complexes 1
3
0
20
2
5
7 2
and 2 (a‒c) emit strong luminescence (Fig.S5) with a maxima appearing at 290–310 nm
excited at 265 nm). There was an apparent increase in the fluorescence intensity upon
(

increasing concentration of CT‒DNA which implicates strong interactions between complex
and the DNA base pairs. The results showed that the increase in emission intensities upon
binding to CT‒DNA support strong interaction of complexes 1 and 2 (a‒c) with CT‒DNA
(Chan, Ma, Yang & Che, 2003; Bhat, Kumbhar, Lönnecke & HeyHawkins, 2010). The binding
strength of complexes 1 and 2 (a‒c) were ascertained by the binding constant (K) values derived
5
from Scatchard equation (Cui et al., 2009) and was found to be 3.09(± 0.03) x 10 , 3.21(± 0.19)
5
5
5
5
5
‒1
x 10 , 3.11(± 0.08) x 10 , 2.01(± 0.11) x 10 , 2.25(± 0.06) x 10 and 2.11(± 0.05) x 10 M ,
respectively.
3
.5.3. Competitive binding assay
In order to get further insight into the binding affinities of complexes competitive binding
experiments were carried out on EB−DNA system by varying the concentration of the complex.
EB is a planar cationic dye that emits intense fluorescence in presence of CT‒DNA at ca. 600
nm (λ ) due to its strong intercalation between the adjacent base pairs. The enhanced
em
fluorescence was quenched upon addition of the second molecule by replacing the bound EB or
accepting the excited electron from EB.
Fluorescence quenching of the EB−DNA system in the presence of complexes
[C H CuN O S ], [C H Cl CuN O S ], [C H CuF N O S ], [C H N O S Zn],
30 22 5 7 2 30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2
[
C H Cl N O S Zn] and [C H F N O S Zn] was carried out to characterize the binding
3
0
20
2
4
4
2
30 20
2
5
7 2
mode of these complexes (Fig. 3). Since EB was not completely displaced, partial intercalation
mode of interaction cannot be ruled out (Gou et al., 2017). Furthermore, the extent of quenching
was determined by using the Stern‒Volmer equation and the K value for complexes 1 and 2
sv
(a‒c) were found to be 2.21, 2.42, 2.22, 1.12, 1.54, and 1.28 respectively.

Fig.3. Emission titration spectra of the EB‒ CT‒DNA system of complexes 1 and 2 (a‒c).
To validate the groove binding preference of the complexes 1 and 2 (a‒c) fluorescence
competitive binding studies were carried out by employing DAPI
(4',6‒diamidino‒2‒phenylindole) as an external fluorophore. DAPI is a minor groove binding

fluorescent dye that binds preferentially in the AT‒rich regions of B‒DNA (Larsen, Goodsell,
Cascio, Grzeskowiak & Dickerson, 1989; Trotta & Paci, 1998; Banerjee & Pal, 2008). Upon
binding to CT-DNA, 1:1 complex of DNA and dye exhibited an enhancement in the emission
maxima (λex = 358 nm, λem = 461 nm). In the groove binder displacement assay a significant
decrease was observed upon concomitant addition of metal complexes [C H CuN O S ],
3
0
22
5
7 2
[C H Cl CuN O S ], [C H CuF N O S ], [C H N O S Zn], [C H Cl N O S Zn] and
30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2
[
C H F N O S Zn] to the DAPI‒DNA system (Fig. 4). The observed decrease in fluorescence
3
0
20
2
5
7 2
intensity is suggestive of the gradual displacement of the DAPI by the metal complexes,
indicative of the preferential groove binding mode of the complexes (Yun et al., 2003). The
quantitative determination of the extent of quenching was done by employing Stern Volmer
equation to calculate the quenching constants for the complexes 1 and 2 (a−c) and were found to
be 2.54, 2.95, 2.76, 1.56, 1.89, and 1.62, respectively.
Table.1. Intrinsic binding constant K , Binding constant K and Stern ‒ Volmer quenching
b
constant K values for the complexes 1 and 2 (a‒c) with CT‒DNA.
sv
‒
1
K (M‒1)
Complexes
K (M )
Ksv
b
5
1
1
1
2
2
2
a
b
c
a
b
c
4.02(± 0.15) x 10 3.09(± 0.03) x 105
2.21
2.42
2.22
1.12
1.54
1.28
5
5
4.40(± 0.08) x 10 3.21(± 0.19) x 10
5
5
4.26(± 0.11) x 10 3.11(± 0.08) x 10
3.07(± 0.12) x 105 2.01(± 0.11) x 105
3.67(± 0.09) x 105 2.25(± 0.06) x 105
3.31(± 0.12) x 105 2.11(± 0.05) x 105

Fig.4. Emission titration spectra of the DAPI‒ CT‒DNA system of complexes 1 and 2 (a‒c).

3
.5.4. Hydrodynamic measurements
To further investigate the binding mode between the complexes 1 and 2 (a‒c) with CT‒DNA,
hydrodynamic measurements were carried out. Intercalation results in the lengthening of the
DNA helix, leading to an increase in the DNA viscosity. In contrast, complexes that binds
exclusively in DNA grooves by partial or non−classical intercalation may bend or kink DNA
helix, causing less pronounced (positive or negative) change in the effective length of DNA
thereby decreasing its viscosity (Satyanarayana, Dabrowiak & Chaires, 1993). The effect of
each complex on the viscosity of CT‒DNA was investigated in order to assess the binding mode
1
/3
and strength of these complexes. The plots of (η/η ) vs R (where R = [complex]/[DNA]; η
o
and η are the relative viscosities of CT‒DNA in presence and absence of complexes,
o
respectively) gives a measure of the viscosity changes (Fig. 5). In our experiments it was
observed that the complexes [C H CuN O S ], [C H Cl CuN O S ], [C H CuF N O S ],
3
0
22
5
7
2
30 20
2
5
7
2
30 20
2
5
7 2
[
C H N O S Zn], [C H Cl N O S Zn] and [C H F N O S Zn] exhibited a decrease in the
30 22 4 4 2 30 20 2 4 4 2 30 20 2 5 7 2
relative viscosity of CT‒DNA and follows the trend 1b > 1c > 1a > 2b > 2c > and 2a. Moreover,
the results of hydrodynamic measurements are in close concordance with other biophysical
studies and support the groove binding mode of the complexes (Lakshmiprabaa et al., 2015;
Arif et al., 2018).

Fig. 5. Effect of complexes 1 and 2 (a‒c) on the relative viscosities (η/η ) of CT‒DNA.
ο
3
.6. Structure‒activity relationship (SAR) studies
To rationalize the DNA binding constants and to analyze the drug‒likeness of the complexes,
structure‒activity relationship (SAR) studies were carried out. There are several basic features
which affect a molecule to be a drug, and they are described as a ‘‘Lipinski’s rule of five”: (i)
Log P should not exceed 5; (ii) Molecular weight not exceeding 500 Da; (iii) Number of
hydrogen bond acceptors not exceeding 10; (iv) Number of hydrogen bond donors not
exceeding 5 (Maass, Schulz‒Gasch, Stahl & Rarey 2007). Various molecular properties (TPSA,
miLogP, molecular weight, OH‒NH interaction, O‒N interaction, nrotb and number violations
from Lipinski rule) are calculated using molinspiration software (Table 2). Topological polar
surface area (TPSA) and lipophilicity (LogP) are two important parameters for predicting oral
bioavailability of drug molecules (Parvez et al., 2010; Jarrahpour et al., 2012). Molecules
2
having TPSA value of 140 Å or more are expected to show poor absorption (Veber et al.,
2
002).
Molinspiration calculations for the ligands indicated that all the compounds have TPSA values
2
<
140 Å and they are expected to have good intestinal absorption. As the cytotoxicity of

anticancer agents is linked with their ability to accumulate within the cells, lipophilicity plays a
major role in their bioactivity. In general, more the lipophilicity of the ligand more will be its
potency to act as cytotoxic agent when coordinated to the metal centre and vice versa. The
results have shown that chloro derived ligand, L2 was found to be more lipophilic which is
responsible for its better uptake in the cell membranes. This is also evidenced in the chloro
substituted synthesized complex [C H Cl CuN O S ], which has demonstrated higher binding
3
0
20
2
5
7 2
affinity with CT‒DNA and good cytotoxic activity as compared to other complexes. None of
the ligand skeletons L1‒L3 (Fig.6) tested showed violation of drug‒likeness rule.
Table.2. Molinspiration calculated properties of substituted benzothiazole derived ligands
L1‒L3.
Properties
miLogP
TPSA
L1
L2
L3
3.57
4.20
3.71
54.72
54.72
54.72
n atoms
MW
20
21
21
284.24
318.79
302.22
nON
4
4
4
nOHNH
n violations
nrotb
1
1
1
0
0
0
3
3
3
Volume
242.82
256.26
247.75
miLogP – theoretically calculated Log P; TPSA – Total Polar Surface Area; MW – Molecular
Weight; nON – number of hydrogen bond acceptors; nOHNH – number of hydrogen bond
donors; n violations – number of violated drug-likeness rules; nrotb – number of rotating bonds,
Volume – molecular volume.
Fig.6. Molinspiration generated 3D structure of substituted benzothiazole derived ligands
L1‒L3.

3
.7. Antimicrobial activity
To overcome the resistance due to microbial infections, Schiff base derived transition metal
complexes are developed to evaluate their efficient bioactivity against various pathogens. The
ligands L1‒L3 and their complexes 1 and 2 (a‒c) were screened for the in vitro antibacterial
activity against Gram‒positive B. subtilis, S. aureus and Gram‒negative E.coli, P. aeruginosa,
and in vitro antifungal activity against C. albicans and the relevant data are presented in Table
3
.The antibacterial activity of ligands L1‒L3 and their complexes [C H CuN O S ],
30 22 5 7 2
[C H Cl CuN O S ], [C H CuF N O S ], [C H N O S Zn], [C H Cl N O S Zn] and
30 20 2 5 7 2 30 20 2 5 7 2 30 22 4 4 2 30 20 2 4 4 2
[
C H F N O S Zn] were evaluated by measuring the diameter of the zone of inhibition (mm)
3
0
20
2
5
7 2
to compare with the commercial drug (doxycycline). A comparative study indicates that metal
complexes exhibit higher antibacterial and antifungal activity than the parent Schiff base ligands
(Fig.7). It has been reported previously in the literature that the bioactivity of a ligand is
enhanced upon metal complexation which can be explained on the basis of Overtone’s concept
and Chelation theory (Joseph, Nagashri & Janaki, 2012). In case of transition metal complexes,
chelation considerably reduces the polarity of the metal ion due to (a) overlap of ligand orbital,
(b) partial sharing of positive charge with donor groups and (c) possible π‒electron
delocalization over the entire chelate ring. Such chelation increases the lipophilic character of
the complexes, thereby favouring easy penetration into the lipid membrane and inhibiting the
growth of the microorganisms (Khosravi & Torshizi, 2018).
The complexes [C H Cl CuN O S ] and [C H Cl N O S Zn], showed maximum zone of
3
0
20
2
5
7
2
30 20
2
4
4 2
inhibition, 23 and 21 mm against Gram‒negative E. coli as compared to the positive control,
doxycycline (19mm). The complexes also showed remarkably good anti‒bacterial activity
against S. aureus while moderate activity against B.subtilis and P. aeruginosa. The synthesized

complexes were also screened for their antifungal activity against C. albicans exhibiting
promising antifungal activities with a broad spectrum effect. The complexes
[
C H Cl CuN O S ] and [C H Cl N O S Zn], exhibited prominent antifungal activity
30 20 2 5 7 2 30 20 2 4 4 2
against the C. albicans with zone diameter of 27 and 25 mm, respectively. The screening results
indicated that complexes [C H Cl CuN O S ] and [C H Cl N O S Zn] exhibited promising
3
0
20
2
5
7
2
30 20
2
4
4 2
activity against these pathogens as compared with other metal complexes. The disparity
observed in the activity of complexes against different microorganisms depends upon the nature
of the metal ions, their coordination modes and impermeability in the microbial cells.
Table.3. In vitro antimicrobial activity of ligands L1‒L3 and complexes 1 and 2 (a‒c).
Complexes =1000 μg/ml, Doxycycline 30 μg/disc, --- No zone of inhibition, NA = Not defined
Complexes Zone of inhibition (mm)
B. subtilis
S. aureus
E. coli
P. aeruginosa
C. albicans
L1
L2
L3
---
---
11±0.57
15±0.67
13±1.00
12±1.33
21±2.01
19±1.76
11±1.33
20±1.08
17±1.91
22±2.12
11±1.08
14±.90
14±0.50
13±1.00
23±2.18
20±1.21
---
21±1.9
18±2.03
19±1.25
---
---
---
---
15±0.90
14±1.12
---
14±1.01
14±0.50
NA
11±1.00
13±0.67
12±0.80
16±1.00
27±2.09
24±1.5
14±0.33
25±1.78
23±1.00
----
12±0.33
---
17±1.15
15±0.86
---
16±1.01
14±0.91
22±2.90
1
1
1
2
2
2
a
b
c
a
b
c
Positive
Control
(Dx)

3
2
2
1
1
0
5
0
5
0
5
0
1
2
3
4
5
6
7
8
9
Complexes
B. subtilis
S. aureus
E. coli
P. aeruginosa
C. albicans
Fig.7. The antimicrobial activity of ligands L1‒L3 and complexes 1 and 2 (a‒c).
.8. In vitro cytotoxic assay
3
In vitro cytotoxicity of benzothiazole–derived chemotherapeutic agents 1 and 2 (b & c) viz.,
C H Cl CuN O S ], [C H CuF N O S ], [C H Cl N O S Zn] and [C H F N O S Zn]
[
3
0
20
2
5
7
2
30 20
2
5
7
2
30 20
2
4
4
2
30 20
2
5
7 2
were evaluated on five different human cancer cell lines viz., MCF−7 (breast), MIA−PA−CA−2
pancreatic), HeLa (cervix) and Hep‒G2 (Hepatoma) and A498 (Kidney) by SRB assay (Fig.8).
The cytotoxic activity was evaluated in terms of GI (concentration of drug that produces 50%
(
5
0
inhibition of the cell growth), TGI (concentration of the drug that produces total inhibition of
the cell growth) and LC (concentration of the drug that kills 50% of the cell kill) values in
5
0
μg/ml (Table 4 and S1). Comparing the cytotoxic potential of these antitumor drug entities, it
was found that chloro substituted copper complex [C H Cl CuN O S ], exhibited
3
0
20
2
5
7 2
pronounced cytotoxicity only against HeLa (cervix) cell line which was evidenced from its
surprisingly low GI value of 4.8 μg/ml as compared to the standard drug Adriamycin (GI <
5
0
50
1
0).

Table.4. Cytotoxic activity of the complexes 1 and 2 (b & c) (in μg/mL). GI = Concentration
5
0
of drug producing a 50% inhibition of cell growth.
GI values (μg/ml)
5
0
Complexes
MCF−7
31.2
MIA−PA−CA−2 HeLa
Hep‒G2
59.2
A498
61.7
1
2
1
2
b
b
c
31.2
49.38
55.2
48.2
4.8
˃80
˂80
32.6
58.3
˃80
49.38
70.3
46.6
74.2
c
24.5
˃80
58.0
The cytotoxicity profile of complexes reveals the preferential selective nature of complexes
against different cancer lines with moderate to high GI values. However, complex
5
0
[C H Cl CuN O S ], was selective for HeLa cell line while its zinc analogue was inactive
30 20 2 5 7 2
(
GI value > 80) for the identical cell line. From these studies, it can be inferred that chloro
5
0
substituted complex [C H Cl CuN O S ], is a potent antitumor drug entity which is quite in
3
0
20
2
5
7 2
agreement with previous literature reports on cytotoxic effect of chloro substituted copper (II)
Schiff base complexes (Qiao et al., 2011).

Fig.8. Growth curves showing % control growth vs. drug concentration (μg/ml) against different
human cancer cell lines.
3
.9. Molecular docking studies
Molecular docking studies were performed with DNA duplex of sequence
d(CGCGAATTCGCG) dodecamer (PDB ID: 1BNA) in order to envisage the potential binding
2
mode of the complexes inside the DNA helix thereby, validating our spectroscopic studies