Schiff base clubbed benzothiazole: synthesis, potent antimicrobial and MCF-7 anticancer activity, DNA cleavage and computational study

Article abstract of DOI:10.1080/07391102.2019.1621213

Schiff bases containing benzothiazole unit are synthesized and characterized by ¹H NMR, ¹³C NMR, LC–MS, UV–visible and fluorescence spectroscopy methods. Synthesized compounds were evaluated for in vitro antibacterial activities on f

Full text of DOI:10.1080/07391102.2019.1621213

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Schiff base clubbed Benzothiazole: Synthesis,
Potent Antimicrobial and MCF-7 Anticancer
Activity, DNA cleavage and Computational Study
Virendra R. Mishra, Chaitannya W. Ghanavatkar, Suraj N. Mali, Dr.
Hemchandra K. Chaudhari & Nagaiyan Sekar
To cite this article: Virendra R. Mishra, Chaitannya W. Ghanavatkar, Suraj N. Mali, Dr.
Hemchandra K. Chaudhari & Nagaiyan Sekar (2019): Schiff base clubbed Benzothiazole:
Synthesis, Potent Antimicrobial and MCF-7 Anticancer Activity, DNA cleavage and Computational
Study, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2019.1621213
To link to this article: https://doi.org/10.1080/07391102.2019.1621213
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Schiff base clubbed Benzothiazole: Synthesis, Potent Antimicrobial and MCF-
7 Anticancer Activity, DNA cleavage and Computational Study
Virendra R. Mishraa, Chaitannya W. Ghanavatkara, Suraj N. Malib, Dr.
Hemchandra K. Chaudharib, Nagaiyan Sekara,*
aVirendra R. Mishra -ORCID ID- https://orcid.org/0000-0003-0070-4705
aChaitannya W. Ghanavatkar -ORCID ID- https://orcid.org/0000-0003-0821-1083
a,*Department of Dyestuff Technology, Institute of Chemical Technology, Matunga (E), Mumbai,
Maharashtra, India, PIN: 400 019.
E-mail: nethi.sekar@gmail.com; n.sekar@ictmumbai.edu.in
bSuraj Narayan Mali -ORCID ID- https://orcid.org/0000-0003-1995-136X
bDepartment of Pharmaceutical Sciences and Technology, Institute of Chemical Technology,
Matunga (E), Mumbai, Maharashtra, India, PIN: 400 019.
Abstract
Schiff bases containing benzothiazole unit are synthesized and characterized by 1H NMR, 13C NMR, LC-
MS, UV-visible and fluorescence spectroscopy methods. Synthesized compounds were evaluated for in-
vitro antibacterial activities on four different strains (S.aureus, Bacillus sps, E. coli, and K.pneumoniae)
and antifungal activity on one strain (C. albicans) by using “Micro-broth dilution method” (MIC in
µg/mL). All compounds showed excellent antibacterial activity for S.aureus, E. coli, and K.pneumoniae
strains, as well as an antifungal activity for C. albicans, compared to standard Ciprofloxacin and
Fluconazole respectively. Molecular docking studies were carried out to get more insight into the binding
mechanism. Reactivity of molecules was assessed using Molecular Electrostatic Potential (MEP).
HOMO-LUMO gap, chemical hardness, and global softness were calculated and correlated with
antimicrobial activity. VS5-e exhibits excellent antimicrobial activities and also shows more chemical
reactivity and less toxicity which were confirmed by smaller HOMO-LUMO gap, lesser chemical
hardness, higher global softness, and lesser electrophilicity index. Henceforth it was further assessed for
DNA cleavage and against MCF-7 breast cancer cell. VS5-e reveals 85.82% inhibition of cancer cells at a
concentration of 200 µg/mL. The compound VS5-e showed less toxicity to normal cells at the
concentration required to produce an anticancer effect (with high IC50 = 973 µg/mL).
Keywords: Schiff base, Benzothiazole, Antimicrobial and anticancer activities, DNA cleavage,
Molecular docking, DFT.
1

1. Introduction
Nitrogen and sulfur-containing heterocyclic compounds are attracting researchers due to their
wide range of pharmacological activities (Al-salihi et al., 2019; Parveen et al., 2017; Shahraki et
al., 2018, 2017a, 2017b). Benzothiazole derivatives were attracted researchers due to their
diverse biological activities (Keri et al., 2015; Sharma et al., 2013) including anticancer (Akhtar
et al., 2017; Arafath et al., 2017), antimicrobial (Berk et al., 2017; Mishra et al., 2019; Zehra et
al., 2018), antitubercular (Mir et al., 2014; Venugopala et al., 2018), anti-inflammatory (Jacob et
al., 2018; Savjani et al., 2017). Sharma et al (Sharma et al., 2010) synthesized benzothiazole
containing pyrimidine derivatives and evaluated their antimicrobial activity by using " Disk
diffusion method" and observed that all compounds show moderate to good antimicrobial
activity. Barkin Berk et al (Berk et al., 2017) synthesized benzothiazole-2-thiol derivatives and
evaluated their antimicrobial activities by using “Broth microdilution method”. But it was
observed that all synthesized compounds are inactive against "Gram-negative" bacteria and only
a few are active against "Gram-positive bacteria", however they displayed moderate to good
activities against antifungal strains. Therefore benzothiazole derivatives are one of the most
adaptable classes of compounds against microbes.
On the other hand, the Schiff base has great importance in a biological field due to their
structural similarities with natural biological compounds (Cleiton et al., 2011). Schiff base has
also attracted researchers due to their wide range of pharmacological activities(Kajal et al., 2013;
Shahraki et al., 2017c; Shahraki and Heydari, 2017) including antibacterial (Bagihalli and
Gouda, 2008; Fekri et al., 2019; Mounika et al., 2010), antifungal (Mohamed et al., 2019;
Mohapatra et al., 2019; Murat et al., 2019), anti-inflammatory (Alafeefy et al., 2015; Pandey et
al., 2011; Sondhi et al., 2006), antioxidant (Danyi et al., 2006; Kumar et al., 2017; Shahraki et
al., 2019) and anticancer (Fekri et al., 2019; Miri and Razzaghi-asl, 2013; Saedi et al., 2019;
Shabbir et al., 2016). Balram Soni et al (Soni et al., 2010) synthesized a series of Schiff bases
containing benzothiazole derivatives and evaluated antimicrobial activity by using “Nutrient agar
diffusion method”, and observed that all derivatives show moderate to good antibacterial
activity, while excellent to good antifungal activity. These activities are mainly due to the
presence of azomethine (-N=CH-) or imine group which will help in elucidating the mechanism
of some reactions in a biological system such as transformation reactions (Flora and Pachauri,
2

2010). But in recent decades, an impressive increase in human pathogenic bacteria was seen due
to resistance to a number of antimicrobial agents (Raja, A., Prabakaran, P., Gajalkshmi, 2010).
Therefore, the exploration of new antimicrobial drugs for better resistance has become the main
goal for scientists (Yusuf Özkay,Yagmur Tunalı, Hülya Karaca and Isıkdag, 2010). So, Schiff
base clubbed benzothiazole can be utilized as valuable substructures for further exploration to
improve the biological activities.
In this regard, we have selected and synthesized Schiff base containing benzothiazole moiety
which is not yet explored for their biological and comparative computational studies. In-vitro
antibacterial activities (S.aureus, Bacillus sps, E. coli, and K.pneumoniae) and antifungal
activities (C. albicans) were evaluated using micro-broth dilution method. Molecular docking
study has been performed to investigate the interaction and binding activity of the molecules
with the variety of target proteins of different bacterial strains. Antimicrobial activities of
molecules were correlated with the HOMO-LUMO energy gap, chemical hardness, and global
softness by using Density Functional Theory (DFT) calculation. Moreover, the DNA binding
interaction with pBR 322 plasmid DNA has been studied using the electrophoretic mobility shift
assay. The anticancer study was performed on MCF-7 cell line using the 3-[4, 5-dimethylthiazol-
2-yl]-2, 5-diphenyltratrazolium bromide (MTT) assay for future exploration of these
heterocycles as anticancer agents. The cytotoxicity on normal cells for compound VS5-e was
also calculated. UV-visible absorption and emission spectra in various solvents are also recorded.
2. Experimental Section
2.1 Materials and Equipment
All the required chemicals were obtained from SD Fine Chemical Ltd. (Mumbai, India) and used
as received without any further purification. The solvents used for synthesis and analytical
measurements were procured from SD Fine Chemical Ltd. (Mumbai, India) and were used
without further purification. The reactions were monitored by thin-layer chromatography (TLC)
using 0.25mm E-Merck silica gel 60 F254 pre-coated plates, which were visualized with UV
1
light. H NMR spectra were recorded on a 500MHz and 400MHz. 13CNMR spectra were
recorded on the 126MHz instrument of Agilent Technology in CDCl3 and DMSO-d6. The
absorption spectra of the compounds were recorded on a Perkin Elmer Lambda-25
spectrophotometer at room temperature. Fluorescence spectra were performed with a Varian
Cary Eclipse fluorescence spectrophotometer at room temperature.
3

2.2 Antimicrobial studies
All synthesized compounds were evaluated for antibacterial and antifungal screenings using the
micro-broth dilution method. The MIC values were calculated for all compounds on four
bacterial and one fungal strain. The totals of 9 dilutions of each compound were prepared using
the BHI (brain heart infusion). For facultative anaerobes, tubes were incubated at 37°C for 48-72
h in CO2 Jar. For strict anaerobes, tubes were incubated in anaerobic jars for 48-72 h. The details
of the whole assay protocol are in Supplementary information.
2.3 DNA cleavage Analysis (Electrophoretic mobility shift assay)
The DNA cleavage study was carried out with the plasmid pBR 322 plasmid DNA using the
Electrophoretic mobility shift assay by incorporating the well-known protocol (Bhat et al., 2017).
The details for electrophoretic mobility shift assay are shown in Supplementary information.
2.4 Anticancer studies
Benzothiazoles are well-known for activity against MCF-7 breast cancer cell lines. It is well
known that the epidermal growth factor receptor (EGFR) has high levels of expression in certain
types of cancers like breast, colon, and bladder. The molecular docking results (on pdb id-1m17)
clearly showed that compound VS5-e has high docking score (-9.002 kcal/mol) than its native
erlotinib (-7.053 kcal/mol) on EGFR. The in-silico cytotoxicity predictions for compound VS5-e,
also showed that this compound has high Pa (probability"to be active") on MCF-7 as predicted
by online web-tool CLC-Pred (http://www.way2drug.com/Cell-line/interpr.php) (See SI, Figure
29). This inspired us to screen VS5-e against MCF-7 breast cancer cell line using MTT assay. As
the MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble,
colored (dark purple) formazan product, we performed the colorimetric assay. The cancer cells
were revived and 0.1 million cells were seeded in 96 well plate. The cells were incubated in CO2
o
incubator at 37 C containing 5% CO2 for overnight. After the observation of fully confluent
cells under microscope, all cells were treated with the given sample at 7 different concentrations.
Further, the cells were incubated for overnight in presence of drug in 5% CO2 incubator. 10 µL
of 5mg/mL MTT reagent was added in the wells and incubated for 4 h. The results for
colorimetric changes, which were giving the end points to purple color, were observed at 595
nm. The cytotoxic effect of VS5-e on normal cells was also performed with the same protocol as
4

mention above (SI Figure 30). The 96 well plate reader-BioRad was used for calculations of
colorimetric changes.
2.5 Computational Method
2.5.1 Molecular modeling studies
The molecular modeling studies were conducted with “Schrodinger’s Molecular Modeling”
package, 2017. The Qikprop module was incorporated for calculations of ADME. The molecular
dynamics was carried using the Desmond V.4 module. The molecular docking calculations were
performed using Glide module (Schrodinger LLC, NY, 2017). (Glide., 2017)
2.5.2 Density Functional Theory
“Density Functional Theory” (DFT) method was used to optimize the ground state of compounds
by using the Gaussian 09 package (Frisch et al., 2009) and the popular hybrid functional B3LYP.
The popular Pople’s split-valence basis sets were used for all atoms. The B3LYP functional is
used with the double zeta basis set 6-31G (d) for optimization of molecules in the gas phase.
2.6 Synthesis
Intermediates 3a (Piva, 1983), 3b (Holler et al., 2002), and VS5-a (Li et al., 2007) were
synthesized as reported literature.
2.6.1 General method for synthesized of VS5 (b-g)
In a stirred mixture of 5-amino-2-(benzo[d]thiazol-2-yl) phenol (3a) or 4-amino-2-
(benzo[d]thiazol-2-yl) phenol (3b) (1.0 mmol) and substituted salicylaldehyde (1.2 m/m of 3a
and 3b) in ethanol added few drops of acetic acid. The reaction mixture was stirred at room
temperature for 8 h. The precipitate was filtered and recrystallized with hot ethanol.
2.6.1.1 2-(benzo [d]thiazol-2-yl)-5-((2-hydroxybenzylidene) amino) phenol (VS5-a)
o
o
Yellow solid Yield: 88% m.p. = 242 C (Lit. (Li et al., 2007) m.p. = 244 C). 1H NMR (500
MHz, CDCl3) δ 13.02 (s, 1H), 12.75 (s, 1H), 8.69 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.93 (d, J =
8.0 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.43 (dd, J = 12.5, 6.1 Hz, 3H),
7.06 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.91 (dd, J = 8.3, 2.0
Hz, 1H). 13C NMR (126 MHz, CDCl3)δ 168.71, 163.71, 161.29, 159.001, 152.27, 151.79,
133.74, 132.65, 132.48, 129.45, 126.80, 125.60, 122.12, 121.54, 119.27, 119.00, 117.37, 115.51,
113.76, 109.38. LC-MS (m/z): (C20H14N2O2S) Calculated: 346.08 Found: 347.2 [M+H] +.
2.6.1.2 2-(benzo[d]thiazol-2-yl)-5-((4-(diethylamino)-2-hydroxybenzylidene) amino) phenol
(VS5-b)
5

1
Yellow solid Yield: 89% m.p. = 208 oC. H NMR (500 MHz, CDCl3) δ 13.65 (s, 1H), 12.64 (s,
1H), 8.44 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 7.3 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H),
7.48 (s, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.93 (s, 1H), 6.84 (d, J = 7.9 Hz,
1H), 6.25 (d, J = 8.5 Hz, 1H), 6.17 (s, 1H), 3.40 (m, J = 6.6 Hz, 4H), 1.20 (t,6H). 13C NMR (126
MHz, CDCl3) δ 168.97, 164.64, 160.82, 159.03, 152.66, 152.29, 151.85, 134.19, 132.40, 129.32,
126.63, 125.28, 121.90, 121.44, 114.25, 113.62, 109.06, 108.44, 104.16, 97.65, 44.65, 12.70.
LC-MS (m/z): (C24H23N3O2S) Calculated: 417.15 Found: 418.3 [M+H] +.
2.6.1.3 2-(((4-(benzo[d]thiazol-2-yl)-3-hydroxyphenyl) imino) methyl)-4-bromophenol (VS5-c)
Orange solid Yield: 90% m.p. = 230 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 11.86
(s, 1H), 8.95 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H),
7.90 (s, 1H), 7.53 (dd, J = 15.7, 7.8 Hz, 2H), 7.43 (d, J = 6.7 Hz, 1H), 7.04 (d, J = 12.9 Hz, 2H),
6.94 (d, J = 8.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 162.83, 159.77, 151.87, 151.86,
137.53, 136.39, 134.71, 134.70, 134.27, 130.12, 126.97, 125.53, 122.51, 122.47, 121.74, 121.72,
121.15, 119.59, 110.56, 109.98. LC-MS (m/z): (C20H13BrN2O2S) Calculated: 425.99 Found:
424.9 [M-H] -.
2.6.1.4 2-(benzo[d]thiazol-2-yl)-4-((2-hydroxybenzylidene) amino) phenol (VS5-d)
o
1
Green solid Yield: 88% m.p. = 216 C. H NMR (500 MHz, DMSO-d6) δ 13.20 (s, 1H), 11.74
(s, 1H), 9.03 (s, 1H), 8.25 (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 8.09 (s, 1H), 7.68 (d, J = 6.8 Hz, 1H),
7.55 (d, J = 7.2 Hz, 2H), 7.47 – 7.36 (m, 2H), 7.16 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 7.7 Hz, 2H).
13C NMR (126 MHz, DMSO-d6) δ 164.40, 162.22, 160.64, 155.81, 151.83, 140.44, 135.23,
133.38, 132.90, 126.92, 125.95, 125.56, 122.70, 122.46, 121.32, 119.87, 119.64, 119.55, 118.32,
116.99. LC-MS (m/z): (C20H14N2O2S) Calculated: 346.08 Found: 347.2 [M+H] +.
6

Scheme 1: Synthesis scheme for Schiff base.
2.6.1.5 2-(benzo[d]thiazol-2-yl)-4-((4-(diethylamino)-2-hydroxybenzylidene) amino) phenol
(VS5-e)
o
1
Green solid Yield: 91% m.p. = 210 C. H NMR (500 MHz, DMSO-d6) δ 13.61 (s, 1H), 11.56
(s, 1H), 8.74 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 2.6 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H),
7

7.54 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 7.36 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 8.6 Hz,
1H), 6.30 (dd, J = 8.8, 2.2 Hz, 1H), 6.08 (d, J = 2.2 Hz, 1H), 3.38 (dd, J = 14.1, 7.1 Hz, 4H), 1.11
13
(t, J = 7.0 Hz, 6H). C NMR (126 MHz, DMSO-d6) δ 164.70, 163.39, 160.78, 154.75, 151.86,
151.74, 141.09, 135.16, 134.47, 126.90, 125.51, 125.35, 122.66, 122.44, 120.40, 119.48, 118.27,
109.04, 104.18, 97.32, 44.35, 12.95. LC-MS (m/z): (C24H23N3O2S) Calculated: 417.15 Found:
418.3 [M+H] +.
2.6.1.6 2-(benzo[d]thiazol-2-yl)-4-((2-hydroxy-5-nitrobenzylidene) amino) phenol (VS5-f)
Green solid Yield: 85% m.p. = 264 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 2H), 9.23 (s,
1H), 8.68 (d, J = 2.7 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 9.2, 2.8 Hz, 1H), 8.13 (d, J
= 7.7 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.63 – 7.57 (m, 1H), 7.56 – 7.50 (m, 1H), 7.47 – 7.40
13
(m, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 9.2 Hz, 1H). C NMR (126 MHz, DMSO-d6) δ
167.28, 164.02, 160.26, 156.39, 151.79, 139.47, 138.79, 135.33, 128.79, 128.54, 126.95, 126.23,
125.60, 122.70, 122.49, 121.36, 119.86, 119.19, 118.68, 118.40. LC-MS (m/z): (C20H13N3O4S)
Calculated: 391.06 Found: 390.0 [M-H] -.
2.6.1.7 2-(benzo[d]thiazol-2-yl)-4-((5-bromo-2-hydroxybenzylidene) amino) phenol (VS5-g)
o
1
Green solid Yield: 88% m.p. = 226 C. H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 11.79
(s, 1H), 9.00 (s, 1H), 8.26 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.88 (s, 1H),
7.53 (br s, 3H), 7.46 – 7.41 (m, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H). 13C NMR
(126 MHz, DMSO-d6) δ 160.46, 159.66, 156.25, 151.81, 140.03, 138.92, 135.56, 135.31, 134.35,
126.92, 126.16, 125.55, 122.67, 122.47, 121.79, 121.32, 119.75, 119.42, 118.38, 110.33. LC-MS
(m/z): (C20H13BrN2O2S) Calculated: 425.99 Found: 424.9 [M-H] -.
3. Results and Discussion
3.1 Chemistry
The synthetic route to prepare Schiff base linked to benzothiazole is depicted in Scheme 1. 4-
aminosalicylic acid (1a) and 5-aminosalicylic acid (1b) were reacted with 2-aminothiophenol (2)
in presence of polyphosphoric acid to produce intermediates 3a and 3b. Finally, the
intermediates 3a and 3b were treated with different derivatives of aldehyde to produce the target
compound VS5 (a-g) in good yield. The structure of target compounds are well characterized by
13
1H NMR, C NMR, and mass spectroscopy. All the compounds obtained are yellow to green
solid. The 1H NMR spectra of all compound shown the aromatic protons in the range of 6.08 and
8.68 ppm. The singlet peak of hydroxyl proton was in the range of 11.74 to 13.65 ppm. The
8

presence of characteristic singlet peak of imine was in the range of 8.69 to 9.23 ppm. The
antimicrobial activities of all the compounds were carried out by micro-broth dilution method.
The computational method was used to get more insight into experimental findings.
3.2 UV-Visible and Fluorescence study
UV-visible and fluorescence spectra of VS5 (a- g) are recorded in various organic solvents of
o
different polarity at 27 C. It is observed that VS5 (a-g) show absorption and fluorescence
maxima (λmax.) in the range of 340-440 nm and 440-600 nm respectively. There is no much effect
of solvent polarity on absorption maxima of all molecules studied. VS5-b shows a red shift in
absorption maxima compared to VS5-e due to extended conjugation from donor site i.e. N, N-
diethyl aniline to benzothiazole moiety, whereas in VS5-e the conjugation is reaching up to
phenolic benzene ring. The emission spectra of all molecules are recorded by exciting at their
absorption maxima wavelength. All the molecules show emission in a range of 450-600 nm with
a large Stokes shift ranging from 70-220 nm. The properties related to absorption and emission
for VS5 b and e are summarized in Table 1 and the remaining are given in supplementary
information (SI Table 1). The absorption and fluorescence spectra of VS5 b and e are shown in
Figure 1 and the remaining are given in supplementary information (SI Figure 15-19).
3.3 Biological Studies
3.3.1 Antimicrobial Activity
The Schiff base molecules VS5 (a-g) were tested against four different bacterial strains:
S.aureus, Bacillus sps, (Gram-positive bacteria) and E. coli, K.pneumoniae (Gram-negative
bacteria) and one antifungal strain C. albicans. The antibacterial and antifungal activities were
evaluated using minimum inhibitory concentration (MIC) values for all compound and compared
with their respective standard Ciprofloxacin and Fluconazole respectively. The minimum
inhibitory concentration (MIC) values of all compound against bacterial and fungal strains are
summarized in Table 2. The antimicrobial activities for all compound are shown in Figure 2.
In the case of bacteriological studies, it was observed that all compound are potentially active
against S.aureus and E. coli bacterial strains when compared with Ciprofloxacin. All compound
is also showing excellent antifungal activity when compared with Fluconazole.
9

3.3.2 DNA cleavage analysis
The electrophoretic mobility shift assay was performed for assessment of nuclease activity. The
whole assay protocol was performed using the plasmid pBR 322 DNA. When a circular plasmid
DNA is run using the agarose gel electrophoresis, the fastest migration will be for supercoiled
form (SC) i.e. (Form I). The slower moving open circular nicked DNA form i.e. NC form will be
produced if one of the strands cleaved. The condensed DNA form was also observed if the
complex was so heavy that it can't be moved during electrophoresis. Our results showed
concentration-dependent formation of NC (Form II) form from SC form (Form I) as well as
condensed DNA form (Figure 3). The VS5-e (one representative among the series) showed a
formation of condensed DNA at concentrations of 40 μM and 60 μM. This study indicated the
probable mechanism of inhibition of bacterial strains by means of interactions with their genome.
3.3.3 Anticancer and Cytotoxicity Evaluation
The anticancer study carried out on MCF-7 cell line for VS5-e showed that the IC50 value for
VS5-e was 150µg/mL (Table-3) as calculated from colorimetric estimations by MTT assay
method. The concentrations ranging from 1000 µg/mL to 100 µg/mL were allowed to check for
percent inhibition of MCF-7 cancer cells. The 85.82% inhibition of cancer cells was observed at
a concentration of 200 µg/mL. The standard drug used was tamoxifen. The IC50 for the standard
tamoxifen on MCF-7 cells was observed at 0.4 µM. The image of MCF-7 cancer cell before and
after addition of VS5-e was shown in Figure-4. The cytotoxic effect of VS5-e was also
calculated with the MTT assay on normal cells (Table 3). The IC50 value of VS5-e on normal
cell line was observed at 973 µg/mL. As the IC50 value on MCF-7 cancer cell (150 µg/mL) is far
less than the value on normal cell (973 µg/mL). Therefore, we can say that this compound is
more likely to act specifically on breast cancer cells and it is less toxic at the concentration
required to achieve 50 % anticancer effect (See SI Figure 30).
3.4 Computational study
3.4.1 Molecular docking, ADME predictions, in-silico drug-likeness, Virtual screening, and
Molecular Dynamics Simulation study.
3.4.1.1 Comparative molecular docking studies.
Molecular docking is a valuable tool in computer-aided drug design (Diab et al., 2016; El-
bindary et al., 2016; Mali and Chaudhari, 2018; Mali and Chaudhari, 2019; Mali et al. 2019). It
aims to achieve an optimized conformation for both protein and drug with relative orientation
10

between them such that the free energy of the overall system is minimized. For the development
and progression of many types of cancer, receptor protein tyrosine kinases (RPTKs) plays an
important role. The high over-activation of these receptors leads to carcinogenesis. So, molecular
docking study was employed to explore the probable interactions of our synthesized compounds
with the EGFR (pdb id-1m17 i.e. EGFR tyrosine kinase domain with 4-anilinoquinazoline
inhibitor erlotinib).
The molecular docking of all the compounds were done against eight different active sites
including targets for antimicrobials as well as anticancer targets with PDB IDs: 1kzn (E. coli
24kDa Domain in Complex with Clorobiocin), 1BAG (alpha-amylase from Bacillus subtilis
complex), 1D7U (complex of 2, 2-dialkylglycine decarboxylase with LCS), 2XCT (S. aureus
Gyrase complex with Ciprofloxacin), 1IYL (Candida albicans N-myristoyltransferase with non-
peptidic inhibitor), 1AI9 (candida albicans dihydrofolate reductase), 1EA1 (14 alpha-sterol
demethylase (CYP51) and 1M17 (Epidermal Growth Factor Receptor tyrosine kinase domain
with 4-anilinoquinazoline inhibitor erlotinib). The docking scores for each compound in
comparisons with the standard drugs like ciprofloxacin, fluconazole, and amoxicillin are given in
Table 4. The binding interactions of the most docked compound with the respective targeted
protein are displayed in Table-5. The binding interaction of VS5-g with 1IYL is shown in
Figure 5 and the remaining details are given in supplementary information (SI Figure 20-25).
3.4.1.2 Pharmacokinetics, drug-likeness, ADME, target prediction and in-silico lead-based
virtual screening:
From our antimicrobial study, we have screened out our compounds for drug-likeness, molecular
docking, and virtual screening. The results obtained from drug-likeness values; show that there is
no violation for popular Lipinski rule of five but some are violating the others. The oral
bioavailability score of 0.55 is obtained by using the popular web-based tool called Swiss
ADME (http://www.swissadme.ch/index.php) (“SwissADME: A free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci.
Rep. (2017) 7:42717. https://www.nature.com/articles/srep42717Title,” n.d.). The
pharmacokinetic profile data show that all the compounds are inhibitors of CYP2C19 and
CYPC9. The effect on the other cytochrome enzymes is displayed in Table 6. The current
compounds are non-Blood brain barrier (BBB) permeating. Calculated ADME properties for
synthesized compounds using the Qikprop utility (Schrodinger LLC.NY, release 2017)
11

(Qikprop, 2017) are presented in Table-7, it is clear that most of our compounds are having high
human oral absorption values. The PSA (polar surface area) values were found within range of
61.507-106.431 which are well correlated with the percent oral absorption values as predicted by
Qikprop. The caco-cell permeability values of all the compounds are good except for VS5-f.
Other properties obtained were found within the recommended ranges and are listed in Table-7.
Among all the compounds VS5-e is most active, therefore we have screened it against different
targets so as to explore future designing using the tool called Swiss Target Prediction (Gfeller
et al., 2014)( http://www.swisstargetprediction.ch/). From Figure 6 it was clear that VS5-e,
would likely to act on kinases having a percentage of prediction 60% and may be evolved as
future designing as anticancer agents. We further explored our study with respect to VS5-e and
used in-silico ligand-based virtual screening by using Swiss Similarity (Zoete et al., 2016). The
top 3 hits from each database are displayed in the supplementary information (SI Figure 26).
3.4.1.3 Molecular Dynamics Simulation study
The molecular dynamics simulation was carried out using Desmond. The molecular dynamics
was performed for best dock VS5-g: 1IYL complex using water as a solvent. The counter ions
used during a simulation period of 1.2 ns were Na+. The system was kept at 300 K temperature.
The totals of 263223 atoms were simulated. The molecular dynamics results retained the exact
interaction pattern as obtained from molecular docking. The complex was enough stable during
the simulation time of 1.2 ns (1200 ps). The corresponding RMSD and RMSF plots are
represented in Figure 7.
3.4.2 Density Functional Theory
The geometries of all the dyes were optimized at the B3LYP/6-31G (d) level of theory in the gas
phase. Optimized structure of VS5-b and VS5-e are shown in Figure 8 and remaining are given
in supplementary information (SI Figure 27). The dipole moments of all the dyes are tabulated
in Table 8, it was observed that VS5 (d-g) exhibit higher values of dipole moment than VS5 (a-
c) and hence it may favor more interaction with high dipole moment species (dipole-dipole
interactions) especially in biological systems and should give good antimicrobial activities which
are reflected in antimicrobial studies (Ekennia et al., 2017). The energy of the Highest Occupied
Molecular Orbital (EHOMO) and the Lowest Unoccupied Molecular Orbital (ELUMO) are used for
further studies.
12

3.4.2.1 Molecular Electrostatic Potential (MEP)
Molecular electrostatic potential (MEP) is an important method to understand the relative
polarity and reactivity of a molecule (Serdaro and Uluda, 2018; Soliman et al., 2015). It is a
valuable tool for qualitative elucidation of an electrophilic and nucleophilic site of reaction for
the study of a biological discovery process. MEP is a valuable tool for predicting and analyzing
molecular interaction such as hydrogen bonding, drug-receptor, and enzyme-substrate
interactions. The MEP plot for synthesized molecules is constructed from B3LYP/6-31g (d)
optimized geometry as shown in Figure 9. MEP simultaneously displays the molecular size,
shape, positive, negative and neutral electrostatics potential region in terms of color grading. The
different color at the surface illustrates the different values of electrostatic potential. The red,
yellow and orange region represent negative electrostatics potential and are related to
electrophilic reactivity. Green and blue region represent positive electrostatics potential and are
related to nucleophilic reactivity.
3.4.2.2 Frontier Molecular Orbitals
“Highest occupied molecular orbital” (HOMO) and “lowest unoccupied molecular orbital”
(LUMO) are the most important orbitals in a molecule. Ionization potential is directly related to
HOMO energy. So, when the molecule is energized it easily loses an electron. In the case of
LUMO, it is directly accomplice with electron affinity because it accepts an electron. The small
energy gap between frontier orbital of molecules indicates that it is chemically more active and
has low kinetic stability (Chauhan et al., 2018; Enoch et al., 2017). All molecules were optimized
at B3LYP/6-31g (d) level of theory in the gas phase. From Table 8, it is observed that VS5-e has
less energy gap i.e.3.3261 eV compared to all other molecules. Hence, it should reveal better
biological activity from all other molecules. The “HOMO-LUMO” energy gap of VS5-a and
VS5-e is shown in Figure 10.
3.4.2.3 Global Reactivity Descriptors
On the basis of Koopman’s theorem (De Proft and Geerlings, 2001), global reactivity descriptors
- chemical potential (μ), chemical hardness (η), global softness (S), and electrophilicity index (ω)
- are calculated using the energies of Frontier Molecular Orbitals EHOMO , ELUMO given by
equations (1-4) (Chattaraj et al., 2009; Parr et al., 1999).
μ = -½ (EHOMO+ ELUMO) (1)
η = ½ (ELUMO - EHOMO) (2)
13

S = 1/ 2η (3)
ω = μ 2/2η (4)
The stability and reactivity of molecules are significantly related to chemical hardness and global
softness. Smallest (greater) the value of hardness (softness) more will be the reactivity (Khan et
al., 2019). From Table 8, it was observed that VS5-e exhibit low value of chemical hardness and
high value of global softness, therefore, it is chemically more reactive and less stable than all
other compounds.
The predicted toxicity can be correlated with electrophilicity index (ω), From Table 8, it was
observed that VS5-e exhibit low value of electrophilicity index (ω) which indicates that it should
have low toxicity than all the other compounds (Bharanidharan et al., 2018; Roy et al., 2005).
4. Conclusion:
In the present work, we have successfully synthesized Schiff base containing benzothiazole unit.
1
All the compounds are well characterized by H NMR, 13C NMR, LC-MS, UV-visible and
fluorescence spectroscopy methods. The synthesized compounds were evaluated for four
antibacterial strains (S.aureus, Bacillus sps, E. coli, and K.pneumoniae) and one antifungal strain
(C. albicans) by using micro-broth dilution method. All the compounds exhibit excellent
antibacterial and antifungal activity against S.aureus, E. coli, and K.pneumoniae and C. albicans
respectively. Molecular docking studies of synthesized compounds were done on eight different
protein sites with PDB IDs: 1KZN, 1BAG, 1D7U, 2XCT, 1IYL, 1AI9 , 1M17and 1EA1 and
compared with standard drugs. Molecular Electrostatic Potential (MEP plot) were assessed to
identify the reactivity of the molecules. HOMO-LUMO gap and global reactivity descriptors i.e.
chemical potential, chemical hardness, global softness and electrophilicity index was calculated
from frontier molecular orbitals and correlated with antimicrobial activity and toxicity. VS5-e is
chemically more reactive, best dock on EGFR target (docking score:-9.002kcal/mol) and less
toxic in comparison of all other compounds and moreover experimentally exhibits excellent
antimicrobial activity and anticancer activity also. VS5-e was further assessed for DNA cleavage
and against MCF-7 breast cancer cell. It displayed the formation of condensed DNA at a
concentration of 40 µM. 85.82% inhibition of MCF-7 cancer cell observed at a concentration of
200 µg/mL. Moreover, the in-silico predictions for VS5-e also showed that this compound has
more probability to act on MCF-7 cancer cells as predicted by CLC-Pred tool. The compound
VS5-e also showed less toxicity at the concentration required to produce 50% inhibition i.e.
14

anticancer effect with high IC50 value(IC50 = 973 µg/mL).This compound upon optimization into
lead may leads to a good drug candidate in the future.
Acknowledgment
Authors (VRM and CWG) are thankful to the office of the Principal Scientific Adviser to Government of
India for financial support by way of Junior Research Fellowship, Maratha Mandal’s Central Research
Laboratory for Antimicrobial and DNA cleavage study and National Facility for Biopharmaceuticals
(NFB) for anticancer study.
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22

Figure 1: UV visible Absorption and Fluorescence spectra of VS5-b (λex. = 419 nm) and VS5-e
(λex. = 379 nm) in different solvents at 27oC.
23

Figure 2: Graph a) antibacterial activities against S.aureus (Gram-positive bacteria), E. coli, and
K.pneumoniae (Gram-negative bacteria) b) antibacterial activity against Bacillus sps (Gram-
positive bacteria) c) antifungal activity against C. albicans of VS5 (a-g) along with their
respective standard Ciprofloxacin and Fluconazole.
Figure 3: Electrophoretic mobility shift assay (DNA cleavage) using Agarose gel (1%)
electrophoresis of pBR322 DNA [complex] =5–60 μM, C=Control pBR322 [DNA] = 200 ng,
incubation 30 min at 37 °C, TAE buffer (40 mM Tris-Acetate, 1 mM EDTA) for VS5-e.
24

Figure 4: A] Untreated MCF-7 cancer cells and B] VS5-e treated MCF-7 cancer cells.
Figure 5: 2D and 3D interactions of VS5-g with target proteins 1IYL.
Figure 6: Swiss Target Prediction approach graph for most active VS5-e.
25

Figure 7: A] RMSD plot and B] RMSF plot obtained for VS5-g: 1IYL complex after a
simulation period of 1.2 ns.
Figure 8: Optimized geometry of VS5-b and VS5-e in the gas phase at the ground state.
Figure 9: Molecular Electrostatic Potential of VS5 (a-g).
26

Figure 10: Frontier Molecular orbitals of VS5-a and VS5-e.
Table 1: Experimental electronic absorption and emission data of VS5 b and e in different
solvents at 27oC.
Stokes shift
Compound
Solvents
Absorption
Emission
λabs
λemi
(nm)
(cm)-1
(nm)
(cm)-1
(nm)
(cm)-1
Toluene
EtOAc
DCM
MeOH
DMF
Toluene
EtOAc
DCM
MeOH
DMF
419
416
420
433
421
379
376
379
382
379
23866.35
24038.46
23809.52
23094.69
23752.97
26385.22
26595.74
26385.22
26178.01
26385.22
497
506
507
507
521
592
596
596
592
595
20120.72
19762.85
19723.87
19723.87
19193.86 100
16891.89 213
16778.52 220
16778.52 217
16891.89 210
16806.72 216
78
90
87
74
3745.62
4275.62
4085.66
3370.82
4559.11
9493.33
9817.22
9606.70
9286.12
9578.50
VS5-b
VS5-e
Table 2: In-vitro antibacterial and antifungal activities of VS5 (a-g).
Compound Antibacterial activities
Minimum inhibitory concentration* in µg/mL
Antifungal activity
27

Gram-positive
S.aureus Bacillus sps.
Gram-negative
E. coli K.pneumoniae
C. albicans
0.8
0.8
0.8
0.8
0.4
0.8
0.8
2
50
50
50
50
50
50
50
2
0.8
0.8
0.4
0.4
0.4
0.4
0.8
1
1.6
3.12
0.4
0.4
0.8
0.4
0.8
1
0.8
1.6
1.6
0.4
1.6
1.6
0.4
-
VS5-a
VS5-b
VS5-c
VS5-d
VS5-e
VS5-f
VS5-g
Ciprofloxacin
Fluconazole
*Standard
Deviations(±) for
VS5-a –VS5-g
-
-
-
-
16
SD=0.5421
SD=0.1399
SD=0
SD=0.1979
SD=0.92
Table 3: Anticancer effect and cytotoxic effect for compound VS5-e on MCF-7 cancer cells and
normal cells respectively.
Concentrations (µg/mL)
% inhibition of MCF-7
% inhibition of normal cells
cancer cells
1000
500
400
200
100
95.73
85.75
85.55
>50 (IC50=973 µg/mL)
26.39
22.56
21.45
18.63
85.82 (IC50=150 µg/mL)
12.21
Table 4- The Docking scores for corresponding targets selected for molecular docking.
Docking scores
(Kcal/mol)
1KZN
1BAG
1D7U
2XCT
S.
1IYL
Candida Candida
1AI9
1EA1
General
target for
Antifungals
1M17
Epidermal
Growth
Receptor tyrosine
kinase (As
Comp.ID/Std.
VS5-a
E.coli B.subtilis Common
Factor
bacterial aureus albicans albicans
target
Anticancer target)
-6.193
-5.289
-3.964
-5.838
-8.183
-7.642
(most
dock)
-5.283
-7.688
-5.482
-5.753
-5.39
-4.996
-5.141
-5.157
-
-4.47
-5.195
-5.079
-8.406
-9.293
-8.64
-5.392
-7.185
-6.057
-3.086
-5.021
-7.078
-6.073
-8.22
VS5-b
VS5-c
VS5-d
-3.363
-3.375
-8.059
(most dock)
-6.793
(most
dock)
-6.577
(most
dock)
-0.194
-5.964
-6.701
-5.599
-4.13
-9.002
(most dock)
VS5-e
28

-6.325
-4.878
-5.921
-6.551
-2.847
-7.551
(most
dock)
-7.487
-6.872
-6.168
-5.767
-7.584
-7.569
-7.189
VS5-f
VS5-g
-4.218
(most
dock)
-4.022
-9.344
(most
dock)
Ciprofloxacin
Amoxycillin
Fluconazole
Erlotinib
-5.282
-6.58
-3.005
-8.75
-
-
-
-
-4.342
-6.668
-5.145
-5.959
-
-7.673
-
-
-6.401
-
-
-7.648
-
-
-
-
-
-
-
-
-
-
-
-7.053
Table 5- The binding residues for the most docked compound for corresponding targets selected
for molecular docking.
PDB ID
MOST DOCKED
Comp.ID
VS5-e
BINDING INTERACTIONS
1KZN
1BAG
1D7U
2XCT
ALA86,VAL89,ILE90(NON-POLAR),ASN46,THR165(POLAR)
ASP274(SALT BRIDGE),TYR59 & HIS180(PI-PI STACKING)
SER215(H-BOND SIDECHAIN)
DA H:13,DG G:9(PI-PI STACKING),DG G:9(H-BOND),DC
H:15(H-BOND SIDECHAIN),MN G:2000(SALT BRIDGE)
HIE227,PHE240,PHE117 &TYR354(PI-PI
VS5-e
VS5-g
VS5-f
1IYL
VS5-g
STACKING),LEU451(H-BOND SIDECHAIN),GLU109(H-BOND)
1AI9
1EA1
1M17
VS5-a
VS5-d
VS5-e
ALA11(H-BOND)
THR260(H-BOND SIDECHAIN),TYR76(PI-PI STACKING)
ASP831(H-BOND,SALT BRIDGE),THR830(POLAR)
Table 6- In-silico pharmacokinetic profile of the compounds as obtained from Swiss ADME
server.
COMP.ID
BBB
permeant
NO
P-gp
substrate
NO
CYP1A2
inhibitor
YES
YES
YES
YES
NO
NO
YES
CYP2C19
inhibitor
YES
CYP2C9
inhibitor
YES
YES
YES
YES
YES
YES
YES
CYP2D6
inhibitor
YES
YES
NO
YES
YES
NO
NO
CYP3A4
inhibitor
YES
NO
NO
YES
NO
VS5-a
VS5-b
VS5-c
VS5-d
VS5-e
VS5-f
VS5-g
NO
NO
NO
NO
NO
NO
YES
NO
NO
YES
NO
NO
YES
YES
YES
YES
YES
YES
YES
NO
Table 7- ADME predictions for VS5 (a-g) by Qikprop
COMP.ID
volume
QPlogPo/w QPPCaco #metab
%Human
PSA
dipole
#stars
Oral
Absorption
29