Design, synthesis and mode of action of novel 2-(4-aminophenyl)benzothiazole derivatives bearing semicarbazone and thiosemicarbazone moiety as potent antimicrobial agents

Article abstract of DOI:10.1007/s00044-015-1479-5

A novel series of substituted benzothiazoles, bearing semicarbazone and thiosemicarbazone moieties, was designed, synthesized and evaluated for their antimicrobial activity and possible mode of action. Structures of the synthesized compounds were elucidated by ¹H NMR, ¹³C NMR, IR and Mass spectral data. The results revealed that compounds SC06, SC09, TS05 and TS07 have potent antibacterial activity against both Gram-positive and Gram-negative strains. Compound TS05 displayed most potent activity with MIC values of 3.91, 7.81 and 1.56 μg/ml against S. aureus, E. coli and P. aeruginosa, respectively. The results from cytoplasmic membrane permeabilization assay, FACS study as well as DNA-binding assays, evaluated against clinically relevant pathogens S. aureus and E. coli, suggest membrane perturbing as well as intracellular mode of action of this class of compounds. In addition, hemolytic activity of the compounds was measured which indicated their low cytotoxicity.

Full text of DOI:10.1007/s00044-015-1479-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1479-5
ORIGINAL RESEARCH
Design, synthesis and mode of action of novel 2-(4-
aminophenyl)benzothiazole derivatives bearing semicarbazone
and thiosemicarbazone moiety as potent antimicrobial agents
Meenakshi Singh1 Sudhir Kumar Singh2 Mayank Gangwar3
•
•
•
Gopal Nath3 Sushil K. Singh1
•
Received: 30 January 2015 / Accepted: 12 November 2015
Ó Springer Science+Business Media New York 2015
Abstract A novel series of substituted benzothiazoles,
bearing semicarbazone and thiosemicarbazone moieties,
was designed, synthesized and evaluated for their antimi-
crobial activity and possible mode of action. Structures of
Introduction
Treating infectious diseases, caused by bacteria or fungi,
remains an important and challenging public health
problem (Jones et al., 2008). Such infections most com-
monly affect patients with decreased immunity, neoplastic
disorders and undergoing organ transplantation (Nathan,
2004). This was mainly due to increase in the number of
immunocompromised hosts because of advances in med-
ical technology and the human immunodeficiency virus
pandemic (Wills et al., 2000). Moreover, common
pathogens and new pathogenic species that possess
intrinsic primary resistance are rapidly developing sec-
ondary resistance to the current antimicrobial agents
(White et al., 1998). The availability of complete
microbial genome sequence has led to devise concerted
strategy to look at novel antibacterials. Nevertheless, in
spite of the identification of many new potential drug
targets, novel antimicrobial agents have not yet emerged
from such efforts to the satisfaction of researchers
(Schimd, 2004).
1
the synthesized compounds were elucidated by H NMR,
13C NMR, IR and Mass spectral data. The results revealed
that compounds SC06, SC09, TS05 and TS07 have potent
antibacterial activity against both Gram-positive and
Gram-negative strains. Compound TS05 displayed most
potent activity with MIC values of 3.91, 7.81 and 1.56 lg/
ml against S. aureus, E. coli and P. aeruginosa, respec-
tively. The results from cytoplasmic membrane permeabi-
lization assay, FACS study as well as DNA-binding assays,
evaluated against clinically relevant pathogens S. aureus
and E. coli, suggest membrane perturbing as well as
intracellular mode of action of this class of compounds. In
addition, hemolytic activity of the compounds was mea-
sured which indicated their low cytotoxicity.
Keywords Antimicrobials Á Benzothiazole Á
DNA binding Á Membrane permeabilization Á
Semicarbazone/thiosemicarbazone
Nowadays, multidrug resistance (MDR) to therapeutic
antibiotics constitutes a serious public health threat. The
prevalence of extremely resistant bacteria such as methi-
cillin-resistant Stayphlococcus aureus (MRSA), fluoro-
quinolone-resistant Pseudomonas aeruginosa (FQRP),
fluoroquinolone-resistant Enterococcus faecalis (QREF)
and vancomycin-resistant Enterococci (VRE) has greatly
increased in some hospitals, resulting in higher rate of
mortality (Dessen et al., 2001; Tenovera, 2001; Leclercq,
2009). Development in biological evaluation of hetero-
cyclic molecules has undergone manifold changes, and the
advancement in molecular biology has eased the design of
new molecules based on their mechanism of action.
Therefore, for a more efficient control, the discovery of
& Sushil K. Singh
sksingh.phe@iitbhu.ac.in
1
Pharmaceutical Chemistry Research Laboratory, Department
of Pharmaceutics, Indian Institute of Technology (BHU),
Varanasi, UP 221005, India
2
Molecular and Structural Biology Division, CSIR-Central
Drug Research Institute, Lucknow, UP 226031, India
3
Department of Microbiology, Institute of Medical Sciences,
Banaras Hindu University, Varanasi, UP 221005, India
123

Med Chem Res
novel drugs with novel mode of action will be imperative
to meet the threats by the emergence of resistance.
addition to these, there has been a growing attention toward
thiosemicarbazones and semicarbazones related to their
wide range of biological properties, specifically as anti-
fungal (Pandeya et al., 2012), antibacterial (Laxmi and
Rajitha, 2012; Kumar et al., 2001), anticonvulsants (Sid-
diqui et al., 2007), anti-HIV (Mishra et al., 2002), antitu-
bercular (Cocco et al., 2002) and anticancer (Shukla et al.,
2013) agents. Keeping this in view, we thought to develop
newer analogs of the benzothiazole nucleus, i.e., semicar-
bazone and thiosemicarbazone derivatives expecting better
biological activity owing to rationale in design of the target
molecules.
The exploration of new heterocycles that can accom-
modate potency to multiple biological targets remains an
intriguing scientific endeavor. The benzothiazole ring
system in numerous biologically active molecules has been
recognized, which shows important pharmacological
activities. Various benzothiazole bearing compounds pos-
sess activities such as antitumor (Shi et al., 1996),
antimicrobial (Catalano et al., 2013), schistosomicidal
(Mahran et al., 2007), anti-inflammatory (Gupta and
Rawat, 2010), anticonvulsants (Siddiqui et al., 2007),
antidiabetic (Navarrete-Vazquez et al., 2009), antipsy-
chotic (Arora et al., 2010), neuroprotective (Anzini et al.,
2010) and diuretic (Yar and Ansari, 2009). 2-(4-Amino-
phenyl) benzothiazole moiety is present in various bioac-
tive molecules such as antitumor I, II (Singh and Singh,
2014), orexin receptor antagonist III (Bergman et al.,
2006) and the Gram-positive selective antibacterial IV (Ali
et al., 2001) (Fig. 1). Structure–activity relationship (SAR)
carried out on these heterocycles has shown that positions 2
and 6 are crucial for antibacterial activity against Gram-
positive and Gram-negative bacterial strains (Yildiz-Oren
et al., 2004). The chemistry of thiosemicarbazone and
semicarbazone has received considerable attention because
of their variable bonding modes, promising biological
implications, structural diversity and ion-sensing ability
(Casas et al., 2000; Mishra et al., 2006; Kizilcikli et al.,
2004). Semicarbazones and thiosemicarbazones have been
known to show significant pharmacological profiles against
important pathogens. Owing to this, a large number of
organic and metal–organic compounds derived from these
have been the subject of most structural and medicinal
studies. Semicarbazones are among the most relevant
nitrogen–oxygen donor ligands, which seem to be valuable
in the structure of antimicrobials (Gingrass et al., 1961). In
The main objective of our study was to investigate how
the potency and selectivity against different Gram-positive
(S. aureus, E. faecalis) and Gram-negative (E. coli, S.
typhi, K. pneumoniae, P. aeruginosa) bacteria can be
modulated by replacement of carbonyl oxygen of the
benzothiazole amide ring system with groups that can
generate electronic, electrostatic as well as different steric
properties. In addition, we also investigated the role of
lipophilicity on the antibacterial activity through the syn-
thesis of a series of benzothiazole bearing semicarbazone
and thiosemicarbazone moieties, SC01-10 and TS01-10,
respectively (Table 1).
Herein, we explored the possibilities of above-synthe-
sized compounds to bind with lipopolysaccharides (LPS),
to hyperpolarize both outer and cytoplasmic membranes, to
interact with lipid monolayers and to kill different bacterial
strains. To circumvent this mechanism, we have employed
the fluorescence polarization assay using the cationic
membrane potential-sensitive dye DiSC3(5) to monitor the
changes associated with membrane permeability of intact
bacteria E. coli and S. aureus. Furthermore, to understand
the hypothesis that induced uptake of propidium iodide (PI)
is responsible for membrane rupture and hence killing,
fluorescence-assisted cell cytometer (FACS) analysis was
Fig. 1 Chemical structures of
pharmacologically relevant
benzothiazoles
NH2
CH3
O
F
N
N
H
NH
NH
NH2
(CH2)4NH2
NH2.HCl
S
S
O
I
II
H2N
N
N
Cl
N
O
N
N
S
H3C
N
NH
NH
O
S
IV
III
123

Med Chem Res
Table 1 Structure of compounds SC01-10 and TS01-10
S
N
S
NNHCONH2
Ar
NNHCONH2
NHC
NHC
N
R
SC01-09
SC10
S
S
NNHCSNH2
Ar
NNHCSNH2
R
NHC
NHC
N
N
TS01-09
TS10
Compound code
R, Ar
SC01, TS01
SC02, TS02
SC03, TS03
SC04, TS04
SC05, TS05
SC06, TS06
SC07, TS07
SC08, TS08
SC09, TS09
SC10, TS10
H
3,5-di NO2
2-OH
3-NO2
2-Cl
2-F
5-Cl, 2-OCH3
2,4-diOH
2-OCH3
HC
CH
performed. Outcome of both the data strongly suggested
that cytoplasmic membrane hyperpolarization did correlate
well with bacterial cell lethality. We further evaluated
interaction of the lead molecules with bacterial plasmid
DNA (pUC19) to probe the molecular mode of action. In
addition, hemolytic activity of the compounds measured
was used as an indicator of their cytotoxicities.
Our research group is working on this benzothiazole
scaffold (Singh et al., 2014a, b, 2015a, b). Recently, we
reported the synthesis and design of analogs of benzoth-
iazole amide system, their antimicrobial activity and the
possible mode of action (Singh et al., 2014b). In continu-
ation to our efforts, it was thought worthwhile to design
and synthesize thiosemicarbazone and semicarbazone
123

Med Chem Res
derivatives hoping to go a step forward in the field of
antimicrobial agents. We report here design, synthesis,
evaluation of antimicrobial activity and the possible mode
of action of novel benzothiazole–semicarbazone and
thiosemicarbazone as antimicrobial agents.
Semicarbazide/thiosemicarbazide hydrochloride reacts
with benzothiazole amide derivatives through nucleophilic
addition reaction. Amino group of semicarbazide/
thiosemicarbazide hydrochloride acts as a nucleophile, and
addition of nucleophilic nitrogen occurs on the active
carbonyl group of benzothiazole amide derivatives,
resulting in formation of target compounds as shown in
Fig. 2. Characterization data of the compounds are given
below.
Materials and methods
Chemistry
All the chemicals and solvents were of analytical grade and
procured from Merck, HiMedia (Mumbai) and Aldrich.
Plasmid DNA (pUC19) was purchased from Invitrogen.
DiSC3(5) and propidium iodide (PI) were from Aldrich.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
semicarbazone)benzamides (SC01)
IR (KBr, tmax cm-1): 3248.06 (–NH str.), 3367.04,
3343.63 (–NH2 str.), 1673.50 (C=O str. of semicarbazone),
1572.57 (C=N str.), 3078.12 (Ar–C–H str.); 1H NMR
(CDCl3, 300 MHz, d ppm): 7.21–7.88 (m, 13H, Ar–H),
13.56 (s, 1H, NH), 9.35 (s, 1H, –CNH), 8.95, 8.93 (2 s, 2H,
NH2); 13C NMR (DMSO–d6, 300 MHz, d ppm): 165.28
(C=O of semicarbazone), 151.32 (–C=N), 156.23 (ben-
zothiazole–C–2), 118.16–141.67 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 388 (M ? 1, 100).
1
The synthesized compounds were characterized by IR, H
NMR, 13C NMR, Mass spectral and elemental analysis.
Melting points were determined in open glass capillaries on
Electrothermal Stuart-SMP10 melting point apparatus and
were uncorrected. IR absorption spectra were recorded on
Shimadzu FTIR-8400 s, FTIR spectrometer. 1H NMR
spectra were recorded on the Bruker DRX-300; FT-NMR
and 13C NMR spectra were recorded on the JEOL AL300,
FT-NMR spectrometer. Both NMR instruments operate at
300 MHz/25 °C using DMSO-d6 as solvent and TMS as
internal standard. Chemical shifts were reported in parts
per million (ppm, d:0 units). ESI–MS spectra were recor-
ded on Micromass Quattro II spectrometer. Elemental
analysis was performed with CE-440 elemental analyzer
(Exeter Analytical Inc., USA). All the IR, Mass, 1H-NMR,
13C-NMR spectral data of compounds (SC01-10 and TS01-
10) were in accordance with the proposed molecular
structures. The purity of the synthesized compounds was
monitored by TLC and ascertained by elemental analysis.
Analytical and physicochemical properties of the com-
pounds are given in Table 2.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-
3,5-dinitro-benzamides (SC02)
IR (KBr, tmax cm-1): 3143.81 (–NH str.), 3214.09,
3358.10 (–NH2 str.), 1631.42 (C=O str. of semicarbazone),
1565.38 (C=N str.), 1320.31, 1555.28 (NO2 str.), 3079.23
(Ar–C–H str.); 1H NMR (CDCl3, 300 MHz, d ppm):
7.12–7.78 (m, 11H, Ar–H), 13.11 (s, 1H, NH), 9.41 (s, 1H,
–CNH), 8.42, 8.36 (2 s, 2H, NH2); 13C NMR (DMSO–d6,
300 MHz, d ppm): 162.97 (C=O of semicarbazone), 152.64
(–C=N), 157.06 (benzothiazole–C–2), 121.36–138.42
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
478 (M ? 1, 100).
General procedure for the synthesis of compounds
(SC01-10) and (TS01-10)
Semicarbazide/thiosemicarbazide hydrochloride (1.0 mM)
dissolved in 5 ml hot distilled water and catalyst NaOAc
were added to a suspension of N-(4-(benzo[d]thiazol-2-
yl)phenyl)-substituted benzamides A01-10 (1.0 mM)
reported earlier (Singh et al., 2014a, b) in 50.0 ml ethanol.
The reaction mixture was refluxed on a water bath for
4–10 h (Scheme 1). The progress of the reaction was
monitored by TLC at appropriate time intervals. After
completion of the reaction, the reaction mixture was cooled
to room temperature. The precipitate formed was filtered,
washed several times with warm ethanol and dried. Com-
pounds were purified by either re-crystallization or column
chromatography as required.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-
hydroxy-benzamides (SC03)
IR (KBr, tmax cm-1): 3231.06 (–NH str.), 3359.21,
3375.03 (–NH2 str.), 1655.85 (C=O str. of semicarbazone),
1571.18 (C=N str.), 3491.83 (–OH str.), 3047.33 (Ar–C–H
1
str.); H NMR (CDCl3, 300 MHz, d ppm): 7.18–7.80 (m,
12H, Ar–H), 13.42 (s, 1H, NH), 9.33 (s, 1H, –CNH), 8.85,
8.78 (2 s, 2H, NH2), 10.46 (br, s, 1H, –OH); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 166.08 (C=O of semicar-
bazone), 152.54 (–C=N), 155.13 (benzothiazole–C–2),
113.10–142.52 (Aromatic–C, C4–C9 C10–C60 C100–C600);
MS (m/z, %): 404 (M ? 1, 100).
123

Med Chem Res
Table 2 Antimicrobial activity and MIC values (lg/ml) against the tested bacteria and hemolytic activity of the synthesized compounds
Compound
code
Bacteria
% Hemolysis
Gram-positive bacteria
Gram-negative bacteria
S. aureus
(ATCC 25323) (clinical isolate) 35218)
E. faecalis
E. coli (ATCC S. typhi
K. pneumonia
(MTCC 3216) (ATCC 31488)
P. aeruginosa
(ATCC 27893)
SC01
SC02
SC03
SC04
SC05
SC06
SC07
SC08
SC09
SC10
TS01
TS02
TS03
TS04
TS05
TS06
TS07
TS08
TS09
TS10
Penicillin
–
13.33 0.57
(62.5)
12.45 0.47
(62.5)
–
–
–
–
–
11.28 0.66
(62.5)
11.96 0.30
8.71 0.45
13.09 0.18
12.14 0.67
12.03 0.33
7.20 0.28
12.32 0.50
15.02 0.27
4.36 0.82
32.01 0.33
10.62 0.28
8.11 0.39
14.09 0.54
6.94 0.14
3.21 0.67
7.90 0.39
5.38 0.45
13.59 0.20
12.83 0.50
9.42 0.58
–
19.43 0.46
(15.6)
12.63 1.16
(62.5)
15.16 0.68
(31.2)
14.20 0.41
(31.2)
–
13.30 0.48
(62.5)
–
12.73 0.66
(62.5)
–
14.38 0.40
(31.2)
11.20 0.61
(62.5)
–
13.36 0.97
(31.2)
18.34 0.27
(15.6)
–
14.23 0.44
(31.2)
11.33 0.60
(62.5)
14.53 0.37 15.23 0.69
(31.2) (31.2)
18.30 0.43 11.82 0.43 18.40 0.16
–
11.80 0.35
(62.5)
15.20 0.43
(31.2)
–
–
(15.6)
(62.5)
(15.6)
11.57 0.37
(62.5)
–
15.10 0.58
(31.2)
–
–
–
–
–
–
–
–
17.80 0.45
(15.6)
13.15 0.37
(31.2)
–
10.23 0.45
(125)
11.63 0.72
(62.5)
11.67 0.40
(62.5)
–
25.23 0.77
(7.81)
10.86 0.29
(62.5)
14.23 0.39
(31.2)
14.70 0.17
(31.2)
18.10 0.43
(15.6)
12.10 0.66
(62.5)
10.15 0.36
(125)
11.67 0.18
(62.5)
10.70 0.45 (125) –
–
13.40 0.50
(62.5)
12.35 0.44
(62.5)
13.67 0.17
(31.2)
11.44 0.38
(62.5)
15.20 0.75
(31.2)
14.73 0.48
(31.2)
15.48 0.28
(31.2)
14.56 0.46
(31.2)
–
–
–
14.6 0.46
(31.2)
–
–
–
11.60 0.37
(62.5)
–
13.76 0.82
(31.2)
–
29.26 0.63
(3.91)
18.19 0.40
(15.6)
24.21 0.55 15.29 0.19 14.29 0.50
(7.81)
35.30 0.33
(1.56)
(31.2)
(31.2)
12.03 0.61
(62.5)
–
11.72 0.45
(62.5)
–
14.23 0.25
(31.2)
–
25.06 0.34
(7.81)
18.33 0.19
(15.6)
18.34 0.47 15.16 0.50 17.17 0.40
(15.6)
13.33 0.61
(31.2)
(31.2)
(15.6)
–
10.26 0.34
(125)
11.39 0.18
(62.5)
–
–
–
–
11.50 0.16
(62.5)
15.15 0.86
(31.2)
12.30 0.31
(62.5)
11.62 0.33
(62.5)
15.27 0.40
(31.2)
–
12.51 0.26
(62.5)
11.93 0.28
(62.5)
14.82 0.72
(31.2)
14.30 0.26
(31.2)
18.39 0.94
(15.6)
34.38 0.63
(3.12)
34.21 0.39
(3.12)
38.68 0.25 37.40 0.30 38.27 0.90
(1.56) (1.56) (1.56)
34.61 0.27 34.15 0.72 29.30 0.30
(6.25) (6.25) (6.25)
38.20 0.45
(1.56)
Ciprofloxacin 31.60 0.40
(6.25)
29.27 0.77
(6.25)
34.52 0.45
(3.12)
–
The value of each compound calculated in triplicate, consisted of zone of inhibition (in mm) SEM with MIC values in brackets. Level of
significance P \ 0.05
123

Med Chem Res
COOH
NH2
NH2
N
S
[a]
NH2
SH
2-Aminothiophenol
2-(4'-Aminophenyl) benzothiazole
[b]
PABA
O
O
N
S
Ar
N
S
NH
R
NH
A10
A01-09
[c]
[d]
[c]
[d]
S
N
S
N
S
S
N
NNHCONH2
NHC
NNHCSNH2
NNHCONH2
Ar
NNHCSNH2
NHC
NHC
NHC
Ar
N
R
R
TS01-09
TS10
SC01-09
SC10
Scheme 1 Reagents and conditions: a polyphosphoric acid (PPA),
220 °C, 1–2 h reflux with stirring b ArCOOH, Acetonitrile, DCC,
reflux 4–10 h c Semicarbazide hydrochloride, CH3OH/C2H5OH,
NaOAc, reflux 6–8 h d Thiosemicarbazide hydrochloride, CH3OH/
C2H5OH, NaOAc, reflux 4–8 h
O
O
S
S
N
NH2NHCONH2.HCl / NH2NHCSNH2.HCl
NH
C
NH
C
HOH
N
Semicarbazide/Thiosemicarbazide hydrochloride
R
O
R
Benzothiazole amide ring system
H
H
H
O
O
OH
S
N
S
N
NH
C
N
N
C
NH
C
N
N
C
H
H
NH2
NH2
R
R
H
H
O
OH2
O
S
S
N
NH
N
C
NH
C
N
N
C
C
N
NH2
H
N
NH2
H
O
S
N
NH
HN
C
C
N
NH2
Benzothiazole semicarbazone ring system
Fig. 2 Proposed reaction mechanism of formation of benzothiazole–semicarbazone/thiosemicarbazone ring system
123

Med Chem Res
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-3-
156.49 (benzothiazole–C–2), 122.62–140.61 (Aromatic–C,
C4–C9 C10–C60 C100–C600); MS (m/z, %): 452 (M ? 1, 100).
nitro-benzamides (SC04)
IR (KBr, tmax cm-1): 3180.72 (–NH str.), 3267.52,
3327.32 (–NH2 str.), 1624.12 (C=O str. of semicarbazone),
1577.82 (C=N str.), 1313.57, 1533.46 (NO2 str.), 3037.99
(Ar–C–H str.); 1H NMR (CDCl3, 300 MHz, d ppm):
7.00–7.91 (m, 12H, Ar–H), 13.17 (s, 1H, NH), 9.36 (s, 1H,
–CNH), 8.43, 8.31 (2 s, 2H, NH2); 13C NMR (DMSO–d6,
300 MHz, d ppm): 163.27 (C=O of semicarbazone), 153.00
(–C=N), 156.56 (benzothiazole–C–2), 124.56–133.94
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
433 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-
2,4-dihydroxy-benzamides (SC08)
IR (KBr, tmax cm-1): 3242.16 (–NH str.), 3374.13,
3379.03 (–NH2 str.), 1645.08 (C=O str. of semicarbazone),
1562.78 (C=N str.), 3361.83 (–OH str.), 3054.63 (Ar–C–H
1
str.); H NMR (CDCl3, 300 MHz, d ppm): 7.16–7.70 (m,
11H, Ar–H), 13.23 (s, 1H, NH), 9.26 (s, 1H, –CNH), 8.71,
8.79 (2 s, 2H, NH2), 10.21, 10.34 (s, 2H, –OH); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 162.15 (C=O of semicar-
bazone), 153.64 (–C=N), 156.38 (benzothiazole–C–2),
111.54–138.02 (Aromatic–C, C4–C9 C10–C60 C100–C600);
MS (m/z, %): 420 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-
chloro-benzamides (SC05)
IR (KBr, tmax cm-1): 3226.81 (–NH str.), 3315.94,
3365.03 (–NH2 str.), 1678.30 (C=O str. of semicarbazone),
1566.32 (C=N str.), 1052.67 (–C–Cl str.), 3082.92 (Ar–C–
H str.); 1H NMR (CDCl3, 300 MHz, d ppm): 7.01–7.75 (m,
12H, Ar–H), 13.46 (s, 1H, NH), 9.43 (s, 1H, –CNH), 8.76,
8.85 (2 s, 2H, NH2); 13C NMR (DMSO–d6, 300 MHz, d
ppm): 163.88 (C=O of semicarbazone), 152.12 (–C=N),
156.06 (benzothiazole–C–2), 121.15–144.21 (Aromatic–C,
C4–C9 C10–C60 C100–C600; MS (m/z, %): 422 (M ? 1,
100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-
methoxy-benzamides (SC09)
IR (KBr, tmax cm-1): 3206.41 (–NH str.), 3295.14,
3342.83 (–NH2 str.), 1666.13 (C=O str. of semicarbazone),
1573.72 (C=N str.), 2948.30 (–C–H str. of OCH3), 3074.22
(Ar–C–H str.); 1H NMR (CDCl3, 300 MHz, d ppm):
7.13–7.69 (m, 12H, Ar–H), 13.28 (s, 1H, NH), 9.48 (s, 1H,
–CNH), 8.64, 8.82 (2 s, 2H, NH2), 3.80 (s, 3H, OCH3); 13
C
NMR (DMSO–d6, 300 MHz, d ppm): 55.62 (OCH3),
162.14 (C=O of semicarbazone), 153.23 (–C=N), 155.10
(benzothiazole–C–2), 118.35–148.10 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 418 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-
fluoro-benzamides (SC06)
IR (KBr, tmax cm-1): 3298.38 (–NH str.), 3421.83,
3506.70 (–NH2 str.), 1647.26 (C=O str. of semicarbazone),
1543.10 (C=N str.), 3107.43 (Ar–C–H str.); 1H NMR
(CDCl3, 300 MHz, d ppm): 7.07–7.88 (m, 12H, Ar–H),
13.32 (s, 1H, NH), 9.56 (s, 1H, –CNH), 8.53, 8.51 (2 s, 2H,
NH2); 13C NMR (DMSO–d6, 300 MHz, d ppm): 167.31
(C=O of semicarbazone), 153.17 (–C=N), 159.73 (ben-
zothiazole–C–2), 115.79–139.17 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 406 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-
styrene-amides (SC10)
IR (KBr, tmax cm-1): 3237.47 (–NH str.), 3335.14, 3371.39
(–NH2 str.), 1652.92 (C=O str. of semicarbazone), 1563.12
(C=N str.), 3083.02 (Ar–C–H str.); 1H NMR (CDCl3,
300 MHz, d ppm): 7.19–7.75 (m, 13H, Ar–H), 9.51 (s, 1H,
–CNH), 6.70, 6.73 (dd, –CH=CH); 13C NMR (DMSO–d6,
300 MHz, d ppm): 165.28 (C=O of amide), 153.13 (–C=N),
156.00 (benzothiazole–C–2), 117.32–138.75 (Aromatic–C,
C4–C9 C10–C60 C100–C600); MS (m/z, %): 414 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
methoxy-5-chloro-benzamides (SC07)
thiosemicarbazone)benzamides (TS01)
IR (KBr, tmax cm-1): 3253.12 (–NH str.), 3365.31, 3387.73
(–NH2 str.), 1673.30 (C=O str. of semicarbazone), 1532.92
(C=N str.), 1062.17 (–C–Cl str.), 2917.66 (–C–H str. of
OCH3), 3069.92 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz,
d ppm): 7.27–7.98 (m, 11H, Ar–H), 13.31 (s, 1H, NH), 9.54
(s, 1H, –CNH), 8.37, 8.74 (2 s, 2H, NH2), 3.81 (s, 3H,
OCH3); 13C NMR (DMSO–d6, 300 MHz, d ppm): 55.60
(OCH3), 162.38 (C=O of semicarbazone), 154.26 (–C=N),
IR (KBr, tmax cm-1): 3176.56 (–NH str.), 3326.12,
3210.73 (–NH2 str.), 1271.60 (C=S str.), 1542.17 (C=N
1
str.), 3048.15 (Ar–C–H str.); H NMR (CDCl3, 300 MHz,
d ppm): 7.32–7.98 (m, 13H, Ar–H), 13.61 (s, 1H, NH),
9.28 (s, 1H, –CNH), 9.15, 8.92 (2 s, 2H, NH2); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 181.08 (C=S), 149.02
(–C=N), 157.13 (benzothiazole–C–2), 121.16–141.63
123

Med Chem Res
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
404 (M ? 1, 100).
thiosemicarbazone)-2-fluoro-benzamides (TS06)
IR (KBr, tmax cm-1): 3168.13 (–NH str.), 3336.25,
3214.65 (–NH2 str.), 1258.12 (C=S str.), 1566.39 (C=N
1
str.), 3078.54 (Ar–C–H str.); H NMR (CDCl3, 300 MHz,
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
thiosemicarbazone)-3,5-dinitro-benzamides (TS02)
IR (KBr, tmax cm-1): 3122.86 (–NH str.), 3321.53,
3184.58 (–NH2 str.), 1242.20 (C=S str.), 1575.89 (C=N
str.), 3036.06 (Ar–C–H str.), 1311.64, 1348.29, 1533.46
(NO2 str.); 1H NMR (CDCl3, 300 MHz, d ppm): 7.26–8.23
(m, 11H, Ar–H), 13.68 (s, 1H, NH), 9.72 (s, 1H, –CNH),
9.21, 8.91 (2 s, 2H, NH2); 13C NMR (DMSO–d6,
300 MHz, d ppm): 182.49 (C=S), 149.53 (–C=N), 158.11
(benzothiazole–C–2), 124.07–139.42 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 494 (M ? 1, 100).
d ppm): 7.19–7.79 (m, 12H, Ar–H), 13.97 (s, 1H, NH),
9.42 (s, 1H, –CNH), 9.83, 8.78 (2 s, 2H, NH2); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 182.10 (C=S), 154.76
(–C=N), 158.14 (benzothiazole–C–2), 123.66–141.78
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
422 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
thiosemicarbazone)-2-methoxy-5-chloro-benzamides
(TS07)
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
IR (KBr, tmax cm-1): 3223.12 (–NH str.), 3358.41,
3327.73 (–NH2 str.), 1273.30 (C=S str.), 1562.02 (C=N
str.), 1065.15 (–C–Cl str.), 2932.56 (–C–H str. of OCH3),
3071.62 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz, d
ppm): 7.15–7.90 (m, 11H, Ar–H), 13.63 (s, 1H, NH), 9.47
(s, 1H, –CNH), 9.32, 8.90 (2 s, 2H, NH2), 3.82 (s, 3H,
OCH3); 13C NMR (DMSO–d6, 300 MHz, d ppm): 56.43
(OCH3), 182.08 (C=S), 153.06 (–C=N), 156.37 (benzoth-
iazole–C–2), 118.62–140.01 (Aromatic–C, C4–C9 C10–C60
C100–C600); MS (m/z, %): 469 (M ? 1, 100).
thiosemicarbazone)-2-hydroxy-benzamides (TS03)
IR (KBr, tmax cm-1): 3183.15 (–NH str.), 3363.13,
3217.80 (–NH2 str.), 1239.10 (C=S str.), 1562.19 (C=N
1
str.), 3079.16 (Ar–C–H str.); H NMR (CDCl3, 300 MHz,
d ppm): 7.12–8.02 (m, 12H, Ar–H), 13.93 (s, 1H, NH),
9.67 (s, 1H, –CNH), 9.10, 8.89 (2 s, 2H, NH2); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 183.45 (C=S), 145.72
(–C=N), 156.14 (benzothiazole–C–2), 121.37–138.83
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
420 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
thiosemicarbazone)-2,4-dihydroxy-benzamides (TS08)
thiosemicarbazone)-3-nitro-benzamides (TS04)
IR (KBr, tmax cm-1): 3147.46 (–NH str.), 3338.52,
3312.63 (–NH2 str.), 1245.75 (C=S str.), 1568.64 (C=N
IR (KBr, tmax cm-1): 3212.95 (–NH str.), 3381.15 (–NH2
str.), 1246.90 (C=S str.), 1573.29 (C=N str.), 3099.26 (Ar–
1
str.), 3376.53 (–OH str.), 3085.63 (Ar–C–H str.); H NMR
1
(CDCl3, 300 MHz, d ppm): 7.26–7.87 (m, 11H, Ar–H),
13.46 (s, 1H, NH), 9.62 (s, 1H, –CNH), 8.91, 8.73 (2 s, 2H,
NH2), 10.33, 10.13 (s, 2H, –OH); 13C NMR (DMSO–d6,
300 MHz, d ppm): 182.55 (C=S), 154.09 (–C=N), 156.68
(benzothiazole–C–2), 113.80–141.62 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 436 (M ? 1, 100).
C–H str.); H NMR (CDCl3, 300 MHz, d ppm): 7.14–7.87
(m, 12H, Ar–H), 13.72 (s, 1H, NH), 9.84 (s, 1H, –CNH),
9.21, 8.76 (2 s, 2H, NH2); 13C NMR (DMSO–d6,
300 MHz, d ppm): 181.44 (C=S), 147.84 (–C=N), 156.39
(benzothiazole–C–2), 122.46–139.03 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 449 (M ? 1, 100).
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
thiosemicarbazone)-2-chloro-benzamides (TS05)
thiosemicarbazone)-2-methoxy-benzamides (TS09)
IR (KBr, tmax cm-1): 3122.86 (–NH str.), 3329.25 (–NH2
str.), 1244.13 (C=S str.), 1575.89 (C=N str.), 3034.13 (Ar–
IR (KBr, tmax cm-1): 3134.96 (–NH str.), 3395.14,
3238.03 (–NH2 str.), 1256.13 (C=S str.), 1558.42 (C=N
str.), 2936.13 (–C–H str. of OCH3), 3075.32 (Ar–C–H str.);
1H NMR (CDCl3, 300 MHz, d ppm): 7.24–7.88 (m, 12H,
Ar–H), 13.58 (s, 1H, NH), 9.42 (s, 1H, –CNH), 8.97, 8.84
(2 s, 2H, NH2), 3.83 (s, 3H, OCH3); 13C NMR (DMSO–d6,
300 MHz, d ppm): 55.48 (OCH3), 182.54 (C=S), 151.23
(–C=N), 155.78 (benzothiazole–C–2), 121.65–144.32
1
C–H str.) 1087.89 (C–Cl); H NMR (CDCl3, 300 MHz, d
ppm): 7.31–7.88 (m, 12H, Ar–H), 13.87 (s, 1H, NH), 9.66
(s, 1H, –CNH), 8.93, 8.81 (2 s, 2H, NH2); 13C NMR
(DMSO–d6, 300 MHz, d ppm): 181.30 (C=S), 157.42
(–C=N), 164.24 (benzothiazole–C–2), 124.56–139.90
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
438 (M ? 1, 100).
123

Med Chem Res
(Aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %):
434 (M ? 1, 100).
(Wayne, 1997). Equal volume of test compounds with
different dilutions and nutrient broth was mixed in wells of
microtiter plate, which were serially twofold diluted to
determine the MIC. Specifically 0.1 ml with approximately
5 9 105 CFU/ml of actively dividing bacterial cells was
inoculated in each well. The standard antibiotic, cipro-
floxacin (10 lg/ml) for bacteria was used as positive con-
trols, and 100 ll of DMSO was used as a negative control.
All the inoculated plates were incubated at 37 °C, and the
results were evaluated by visible turbidity in each well after
24 h. All determinations were done in triplicate, and the
average was taken as final MIC value.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-(10-
thiosemicarbazone)-styrene-amides (TS10)
IR (KBr, tmax cm-1): 3175.87 (–NH str.), 3373.74,
3277.39 (–NH2 str.), 1252.64 (C=S str.), 1558.49 (C=N
1
str.), 3068.32 (Ar–C–H str.); H NMR (CDCl3, 300 MHz,
d ppm): 7.14–7.65 (m, 13H, Ar–H), 9.70 (s, 1H, –CNH),
6.71, 6.69 (dd, –CH=CH); 13C NMR (DMSO–d6,
300 MHz, d ppm): 184.27 (C=S), 154.16 (–C=N), 156.95
(benzothiazole–C–2), 119.13–135.67 (Aromatic–C, C4–C9
C10–C60 C100–C600); MS (m/z, %): 430 (M ? 1, 100).
Bactericidal kinetics
Antimicrobial assay
Overnight cultures of S. aureus ATCC 25323 and E. coli
ATCC 35218 were diluted 10-2 times in fresh MHB and
allowed to grow to exponential phase (optical density at
600 nm of 0.6). The time-course killing activity of synthe-
sized compounds was determined using the 96-well plates of
plate reader as described previously (Joshi et al., 2012). The
lead compounds were added at 4 times their MICs, and this
suspension was incubated at 37 °C at 200 rpm. After addi-
tion of the compounds, the absorbance of the plates was
recorded at 600 nm at regular time intervals, i.e., 0, 1, 2, 3, 4
and 5 h. The experiment was repeated on three different
days, and values are plotted as mean SD.
Disc diffusion method
Antimicrobial activities of newly synthesized compounds
were evaluated on different Gram-positive and Gram-nega-
tive human pathogens viz. Staphylococcus aureus (ATCC
25323), Escherichia coli (ATCC 35218), Pseudomonas
aeruginosa (ATCC 27893), Klebsiella pneumonia (ATCC
31488), Enterococcus faecalis (clinical isolate) and Sal-
monella typhi (MTCC 3216) according to the guidelines of
National Committee for Clinical Laboratory Standards
(Wayne, 1997) using the agar disc diffusion method (Bharti
et al., 2010a, b). Briefly, a 24-/48-h-old culture of selected
bacteria was mixed with sterile physiological saline
(0.85 %), and the turbidity was adjusted to the standard
inoculums of MacFarland scale 0.5 [*106 colony-forming
units (CFUs) per milliliter]. Petri plates containing 20 ml of
Mueller–Hinton Agar (MHA, HiMedia) were used for all the
bacteria tested. The inoculums were spread on the surface of
the solidified media, and Whatman no. 1 filter paper discs
(6 mm in diameter) impregnated with the test compound
(20 ll/disc) were placed on the plates. Ciprofloxacin (5 lg/
disc, HiMedia) was used as positive control for bacteria. A
paper disc impregnated with dimethyl sulfoxide (DMSO)
was used as negative control. Plates inoculated with the
bacteria were incubated for 24 h at 37 °C. The inhibition
zone diameters were measured in millimeters. All the tests
were performed in triplicate.
Hemolytic activity
Hemolytic activity assay was carried out according to the
procedure described previously (Singh et al., 2013).
Briefly, freshly collected 100 lL hRBC was washed thrice
in sterile phosphate-buffered saline (PBS) solution. The
hRBC suspension was re-suspended in 4 % v/v PBS. The
final concentration was adjusted to 5 9 108 cells/ml at pH
7.4. Compound solution containing 100 lM of test com-
pound (100 ll) was mixed with 100 ll of hRBC suspen-
sion, and final volume was made to 1 ml with buffer and
was placed in a 96-well plate. The cell suspensions were
incubated for 1 h at 37 °C with constant shaking. The
samples were then centrifuged for 5 min at 1300 rpm, and
the release of hemoglobin was monitored by measuring the
absorbance (Asample) of the supernatant at 540 nm. For
negative and positive controls, hRBC in PBS (Ablank) and in
0.2 % (final concentration v/v) Triton X-100 (Atriton) were
used, respectively. The percentage of hemolysis was cal-
culated according to the following equation.
Determination of MIC
Minimum inhibitory concentration (MIC) of any com-
pound is defined as the lowest concentration, which com-
pletely inhibits visible growth (turbidity on liquid media).
MIC values were determined by broth micro-dilution
method, according to NCCLS guidelines document M27-A
ÂÀ
Á
Percentage of hemolysis ¼ Asample À Ablank
=
ðAtriton À Ablankފ  100
123

Med Chem Res
described previously (Bharti et al., 2000). Briefly, 200 ng
of plasmid DNA (pUC19) was mixed with increasing
amounts of lead compounds in 20 lL of binding buffer
(5 % glycerol, 10 mM Tris–HCl, pH 8.0, 1 mM EDTA,
1 mM dithiothreitol, 20 mM KCl, and 50 lg/ml bovine
serum albumin). The reaction mixtures were incubated for
1 h at 37 °C. Consequently, loading buffer containing
25 % bromophenol blue, 0.25 % xylene cyanol and 30 %
glycerol was added and then subjected to 1 % agarose gel
electrophoresis at 60 V for 1 h in 10 mM Tris–HCl buffer.
The migration of DNA was detected by the fluorescence of
ethidium bromide. The illuminated gel was photographed
by Alpha Innotech Corporation Instrument. Besides, the
reaction was also monitored upon addition of various
groove binders—methyl green (MG) and DAPI.
Cytoplasmic membrane permeabilization assay
Compound-induced polarization of S. aureus ATCC 25323
and E. coli ATCC 35218 membrane measures the efficacy to
dissipate the potential across these cell membranes and was
determined using the membrane potential-sensitive cyanine
dye DiSC3(5) described previously (Friedrich et al., 2000).
Briefly, exponential-phase bacteria were harvested by cen-
trifugation, washed and re-suspended in 5 mM HEPES–
20 mM glucose buffer (pH 7.2) to an optical density of 0.06.
This cell suspension was incubated with 100 mM KCl (to
equilibrate cytoplasmic and external K1 concentration) and
DiSC3(5) at a concentration of 1 lM for 1 h at room tem-
perature. When the fluorescence level (excitation at 622 nm
and emission at 670 nm wavelength) of the bacterial sus-
pension became stable (approximately 90 % reduction in
fluorescence due to DiSC3(5) uptake and quenching in the cell
in response to an intact membrane potential), a 400 ll aliquot
of cell suspension and the 4 9 MIC concentration of lead
compounds were added in the cuvette in order to record the
membrane hyperpolarization of bacterial cell membrane. All
samples were then measured on PerkinElmer Life Sciences
LS 50-B spectrofluorimeter (PerkinElmer Corp., Norwalk,
Conn.) in a 5-mm path length quartz cell at 25 °C. Samples
werestirredduringtheexperimentataconstanttemperatureof
37 °C. An increase in fluorescence due to partitioning of
samples into the membrane was recorded as a function of time
until no further increase in intensity was observed.
Statistical analysis
Each experiment was repeated three times, and the results
were expressed as mean standard deviations. The data
were analyzed statistically using Prism 5.1 and Origin 6.0
software. Statistical analysis was performed using analysis
of variance with the Dunnett’s test. A P value of\0.05 was
considered statistically significant, whereas a P value of
[0.05 was insignificant.
Results and discussion
Antimicrobial activity
Flow cytometry analysis
The structures of the compounds are given in Table 1. The
antibacterial activity of the synthesized compounds was
determined using the method recommended by National
Committee for Clinical Laboratory Standards (NCCLS)
(Wayne, 1997) for a panel of two Gram-positive and four
Gram-negative bacteria. The antimicrobial results are pre-
sented in Table 3. Standard antibacterial agent penicillin and
ciprofloxacin were also screened under identical conditions
for comparison. The results revealed that most of the com-
pounds exhibited good-to-moderate MIC values ranging
between 1.56 and 125 lg/ml in DMSO. Out of the 20 ben-
zothiazole derivatives, compound N-(4-(benzo[d]thiazol-2-
yl)phenyl)-(10-semicarbazone)-2-floro-benzamides (SC06)
exhibited maximum antibacterial activity against E. coli and
K. pneumoniae (zone of inhibition up to 18–19 mm at con-
centration of 15.6 lg/ml). Similarly, compound N-(4-
(benzo[d]thiazol-2-yl)phenyl)-(10-semicarbazone)-2-meth-
oxy-benzamides (SC09) showed maximum activity against
S. aureus (zone of inhibition up to 24–26 mm at concentra-
tion of 7.81 lg/ml) and P. aeruginosa (zone of inhibition up
to 17–19 mm at concentration of 15.6 lg/ml). Compound N-
(4-(benzo[d]thiazol-2-yl)phenyl)-(10-thiosemicarbazone)-2-
The membrane damage of S. aureus ATCC 25323 and
E. coli ATCC 35218 was examined by flow cytometer after
employing suitable fluorescent probes (Novo et al., 2000).
Briefly, the cells at exponential phase were stained with
propidium iodide after the treatment with the lead com-
pounds at 37 °C for 1 h with constant shaking. The cells
were centrifuged, washed two times with PBS and incu-
bated further with propidium iodide (PI) at 4 °C for
30 min, followed by removal of the unbound dye through
washing with an excess of PBS and re-suspended in buffer.
These cells were then analyzed by flow cytometry in the
form of dots (excitation and emission wavelength set at 488
and 617 nm, respectively, for propidium iodide) and plots
with respect to the control without PI treated, with PI
treated cells without preincubation with test compounds
and with test compound treated cells.
DNA-binding assay
Gel retardation experiments were performed on synthe-
sized compounds by agarose gel electrophoresis as
123

Med Chem Res
Table 3 Analytical and physicochemical data of the synthesized compounds
Sr. No.
Compound code
Molecular formula
M.W.a
M.p.(°C)b
Yield (%)
% Analysis of C, H,N found (calc.)c
C
H
N
1
SC01
SC02
SC03
SC04
SC05
SC06
SC07
SC08
SC09
SC10
TS01
TS02
TS03
TS04
TS05
TS06
TS07
TS08
TS09
TS10
C21H17N5OS
387.46
477.45
403.46
432.46
421.92
405.45
451.93
419.46
417.48
413.49
403.52
493.52
419.52
448.52
437.97
421.51
467.99
435.52
433.55
429.56
198–200
222–224
218–220
192–194
248–250
240–242
259–260
210–212
227–229
201–203
189–190
187–189
211–213
224–225
261–263
236–238
239–240
227–229
206–208
215–218
76
56
78
46
81
67
84
72
66
52
70
59
84
68
85
79
91
86
72
82
65.10 (65.10)
52.81 (52.83)
62.51 (62.52)
58.30 (58.32)
59.76 (59.78)
62.20 (62.21)
58.45 (58.47)
60.12 (60.13)
63.27 (63.29)
66.80 (66.81)
62.49 (62.51)
51.10 (51.11)
60.12 (60.12)
56.22 (56.23)
57.57 (57.59)
59.84 (59.84)
56.43 (56.46)
57.91 (57.91)
60.93 (60.95)
64.30 (64.31)
4.40 (4.42)
3.15 (3.17)
4.23 (4.25)
3.72 (3.73)
3.80 (3.82)
3.96 (3.98)
4.00 (4.01)
4.07 (4.09)
4.57 (4.59)
4.61 (4.63)
4.24 (4.25)
3.04 (3.06)
4.06 (4.08)
3.60 (3.60)
3.67 (3.68)
3.81 (3.83)
3.86 (3.88)
3.90 (3.93)
4.40 (4.42)
4.44 (4.46)
18.06 (18.08)
20.54 (20.54)
17.35 (17.36)
19.43 (19.43)
16.60 (16.60)
17.26 (17.27)
15.50 (15.50)
16.70 (16.70)
16.76 (16.78)
16.92 (16.94)
17.34 (17.36)
19.88 (19.87)
16.68 (16.69)
18.72 (18.74)
15.97 (15.99)
16.60 (16.61)
14.95 (14.96)
16.06 (16.08)
16.14 (16.15)
16.29 (16.30)
2
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
21H15N7O5S
21H17N5O2S
21H16N6O3S
21H16ClN5OS
21H16FN5OS
22H18ClN5O2S
21H17N5O3S
22H19N5O2S
23H19N5OS
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
a
21H17N5S2
21H15N7O4S2
21H17N5OS2
21H16N6O2S2
21H16ClN5S2
21H16FN5S2
22H18ClN5OS2
21H17N5O2S2
22H19N5OS2
23H19N5S2
Molecular weight of the compound
b
c
Melting point of the compound at their decomposition
Elemental analyses for C, H and N were within 0.03 % of the theoretical value
chloro-benzamides (TS05) and N-(4-(benzo[d]thiazol-2-yl)
phenyl)-(10-thiosemicarbazone)-5-chloro-2-methoxy-ben-
zamides (TS07) displayed broad-spectrum antimicrobial
activity against all tested bacterial strains with MIC values in
the range of 1.56–31.2 lg/ml. Compound TS05 displayed the
most potent activity with MIC values of 3.91, 15.6, 7.81 and
1.56 lg/ml against S. aureus, E. faecalis, E. coli and
P. aeruginosa, respectively, and compound TS07 displayed
MIC values of 7.81, 15.6, 15.6 and 15.6 lg/ml against
S. aureus, E. faecalis, E. coli andK. pneumoniae, respectively,
which was comparable with penicillin and ciprofloxacin with
corresponding MIC values of 3.12, 3.12, 1.56, 1.56 lg/ml and
6.25, 6.25, 6.25, 6.25 lg/ml, respectively, used as standards.
Some of the compounds SC02, SC04, SC07 and SC10
exhibited good activity against few bacterial strains having
MIC value of 15.6 lg/ml, while rest of the compounds
showed moderateactivity. Inaddition, itwas observedthatthe
potent compounds (SC06, SC09, TS05 and TS07) displayed
greater activity against S. aureus and E. coli.
the procedure described previously (Singh et al., 2013) on
human hRBC at a fixed concentration of 100 lM. The
results showed that all the tested compounds caused
3–15 % hemolysis, which signifies non-toxicity of the
series. Compounds SC02, SC06, SC09, TS02, TS04,
TS05, TS06, TS07 and TS10 showed less than 10 %
hemolysis, while rest of the compounds except compound
SC10 showed maximum of 15 % hemolysis. The highest
hemolysis (32 %) of compound SC10 owes its greater
toxicity. Compound TS05 showed only 3.21 % hemolysis
at very high concentration, i.e., 100 lM. The results of the
in vitro hemolytic study of the compounds are shown in
Table 2 along with its antibacterial activity.
Bactericidal kinetics
The bactericidal activity of lead compounds SC06, SC09,
TS05 and TS07 was directed in confirming its potency
toward the specific S. aureus and E. coli strains to be used
in the evaluation. It was determined at regular time inter-
vals so as to see the potential killing effects of bacterial
cells within minutes at concentrations fourfold MICs.
Time–kill studies demonstrated that compounds SC06 and
TS05 were rapid with 90–99 % lethality within 2–5 h
Hemolytic activity
To assess toxicity of synthesized compounds SC01-10 and
TS01-10, hemolytic activity was carried out according to
123

Med Chem Res
(Fig. 3). Compound SC09 was found to be less effective as
compared to compounds SC06, TS05 and TS07 in both
strains even up to 5 h. At 4 9 MIC, compounds inhibited
bacterial growth from 2 h onwards keeping growth arrested
till 5 h. However, complete eradication of bacterial growth
by any of the tested compounds was not observed up to 5 h.
Compound TS05 exhibited the most potent inhibition of
growth in both the strains at 4 9 MIC. Based on these data,
studies were designed to learn the bactericidal mechanism
of test compounds against S. aureus and E. coli.
periphery medium is dependent on the cytoplasmic mem-
brane potential gradient (Wu et al., 1999). This cationic
dye readily partitions into the bacterial cell membrane and
aggregates within the membrane, causing self-quenching.
If the antimicrobial compounds perturb the cell membrane,
it can lead to the loss of the membrane potential gradient,
causing the dye to release into the medium. As a result,
membrane depolarization occurs and the fluorescence
intensity of the dye increases (Sims et al., 1974). Upon
hyperpolarization of the plasma membrane, the dye enters
the cell, binds to intracellular proteins and undergoes
quenching of its fluorescent emission with a decrease in its
fluorescent signal (Plasek et al., 1994). Triton (2 %) and
valinomycin (0.1 lM) were found to depolarize and
hyperpolarize the bacterial membrane, respectively. All
compounds showed similar profile with respect to positive
control proton ionophore valinomycin, which was found to
hyperpolarize the membrane by specifically binding to
potassium ions (K? ions) and thereby acts as K? ions
carrier molecule. However, none of the above-mentioned
compounds shows depolarization of the bacterial mem-
brane as observed in case of negative control Triton (Singh
Outer membrane permeabilization assay
To understand the basis of antimicrobial activity of
designed analogs, compound-induced permeability of S.
aureus and E. coli membrane was examined by determin-
ing the efficacy to dissipate the diffusion potential across
the LPS membrane. The ability of designed analogs to
depolarize the cytoplasmic membrane was determined by
using cationic membrane potential-sensitive cyanine dye
[3, 30-dipropyl- thiadicarbocyanine iodide DiSC3(5)]. The
distribution of DiSC3(5) between cell membrane and
Fig. 3 Time-dependent killing
of a S. aureus b E. coli upon
treatment with compounds
SC06, SC09, TS05 and TS07 at
4 9 MIC concentrations
123

Med Chem Res
et al., 2014b). At the highest concentration of designed
analogs SC06, SC09, TS05 and TS07 tested (i.e.,
4 9 MIC), the level of hyper polarization appeared to
reach a maximum at times from about 1–5 min (Fig. 4).
Consistent with their cytotoxic and antimicrobial proper-
ties, SC09 showed the minimum, while TS05 showed
maximum relative percentage fluorescence with respect to
valinomycin as described in Fig. 5. Unusual pattern of
emitted fluorescence was observed in the both strains. All
the designed analogs studied had the ability to hyperpo-
larize the cytoplasmic membrane of S. aureus and E. coli;
however, compounds with different structures had different
concentration-activity profiles (data not shown here).
Compounds SC06 and TS05 completely hyperpolarized
the membrane at lower concentrations than those of the
other compounds studied.
Permeabilization of membrane of viable bacteria
To determine the effect of test compounds to the integrity
of the bacterial membrane, PI a DNA-intercalating fluo-
rescent dye was used to incubate with S. aureus and E. coli
in the presence or absence of test compounds. PI could pass
through the damaged membrane and intercalate into DNA.
Cell viability was examined by FACS (fluorescence-as-
sisted cell cytometer) by quantifying the amount of DNA
released in terms of PI. The large number of dots in lower
right quadrant fluorescing with propidium iodide after
exposure to compounds might indicate the defective outer
membrane repair and consequent leakiness of more DNA
and RNA (Riccardi, 2006). As shown in Fig. 6, test com-
pounds SC06, SC09, TS05 and TS07 induced maximum
damages to the membrane organization of E. coli as
compared to significantly lesser damage to the membrane
of S. aureus. Further, the number of PI-stained cells
Fig. 4 Effects of designed analogs on a S. aureus and b E. coli,
membrane potential at 4 9 MIC lg/ml. Compounds are presented as
(cyan curve) TS05, (green curve) SC06, (blue curve) TS07, (pink
curve) SC09. Triton 2 % (black curve) and valinomycin 0.1 lM (red
curve) were used as negative and positive controls, respectively (Color
figure online)
Fig. 5 Bar diagram showing relative percentage fluorescence of a S. aureus and b E. coli
123

Med Chem Res
decreased significantly in SC09 and TS07 in S. aureus
(19.09 and 15.97 %, respectively) as compared to E. coli
(60.61 and 58.29 %, respectively), implicating lesser
membrane damage of S. aureus. However, maximum
damage of bacterial membrane of both strains was
observed in compounds SC06 and TS05 (Fig. 6).
demonstrated that compound SC09 showed preferential
affinity toward the major groove and partial affinity toward
minor groove of the DNA.
Conventional antibiotics target important mechanisms
or pathways of bacterial survival to inhibit their growth.
However, frequent emergence of resistance to these
molecules resulted in the emergence of ‘‘superbacteria,’’
which put people in potentially dangerous situations of
having no available antibiotics. Benzothiazole–semicar-
bazone and thiosemicarbazone as antimicrobial precursors
are considered as essential lead candidates for the devel-
opment of novel antimicrobial therapeutic agents. The
results presented in this study showed that designed com-
pounds have promising antimicrobial activity against both
Gram-positive and Gram-negative bacteria. Structure–ac-
tivity relationship (SAR) demonstrated the effect of sub-
stituents on the antimicrobial activity. Compounds SC06,
SC09, TS05 and TS07 with electronegative groups (F, Cl)
and electron-releasing (OCH3) groups were found to be
more active than the compounds with electron-withdraw-
ing (NO2) groups. However, compounds SC03, TS03,
SC08 and TS08 bearing electron-releasing OH group were
moderately active. Hence, it can be contingent that chloro,
fluoro and methoxy substituents bearing derivatives offer
the most suitable compounds for achieving the best
antibacterial spectrum. The results may be explained by
electron density of the compounds. It has been reported in
the literature that increased electron density makes the
compounds effective against microorganisms and enhances
the antibacterial activity (Kumar et al., 2009). Conversely,
soaring electron density makes diffusion through the bac-
terial cell more difficult, and considerable activity loss may
occur (Hania, 2009). Thus, for a compound an optimum
electron density is essential for significant gain in
antibacterial activity. In order to have insight into the
toxicity determination of designed compounds, hemolysis
on hRBCs was carried out. Almost all the tested com-
pounds exhibited moderate-to-very low hemolysis, which
indicated their low toxicity. In general, compounds with
better antimicrobial activity showed negligible or very low
toxicity profile (Table 2). These results further support the
significance of the study. Overall, the present study pro-
vides us compounds SC06, SC09, TS05 and TS07 with
potent activity against a broad range of bacterial strains
which also accounts for very low hemolysis in the range of
3–7 %. The antibacterial activity data of compounds
(SC05, SC07; MIC: 15.6–62.5 lg/ml) and (TS05, TS07;
MIC: 1.56–31.2 lg/ml) showed that thiosemicarbazone
derivatives exhibited more pronounced activity than
semicarbazones. However, both semicarbazones and
thiosemicarbazones exhibited much better activity in
comparison with amides, earlier reported in our previous
work (Singh et al., 2014a, b).
DNA-binding activity
Together with the results of membrane permeabilization
assay, FACS analysis, we can conclude that series of
benzothiazole bearing semicarbazone and thiosemicar-
bazone moiety could disrupt the integrity of membrane,
leading to the leakage of cell contents of bacteria. Simul-
taneously, during the interaction process with membrane,
test compounds unavoidably could enter into cytoplasm,
but the effect of test compounds in the cytoplasm is not
certainly known. Thus, we determined the DNA-binding
ability of test compounds SC06, SC09, TS05 and TS07 to
determine the mode of action. The DNA-binding affinities
of the test compounds were examined by analyzing the
electrophoretic mobility of plasmid DNA bands at various
compound concentrations. Compared with the test com-
pounds SC06, SC09, TS05 and TS07, compound SC06
could not inhibit the migration of plasmid DNA at con-
centration even up to 31.2 lg/ml, while compounds SC09,
TS05 and TS07 showed DNA retardation at 15.6, 7.81 and
7.81 lg/ml, respectively (Fig. 7). However, at higher
concentration compounds SC09 and TS05 cleaved the
DNA. Therefore, it can be concluded that test compounds
bind and cleaved the bacterial DNA, and thus, both
membrane and DNA may be its target.
To explore the actual mechanism of binding, the MG/
DAPI dyes were used because of their preferential binding
with major and minor groove regions of DNA. Therefore, to
probe the interacting site of compounds TS05, TS07 and
SC09 with plasmid DNA, the DNA was treated with DAPI
or MG prior to the addition of compounds. DAPI (minor
groove binder) was added to the reaction mixture containing
compounds TS05 and TS07; no significant inhibition was
observed in the cleavage pattern. Similarly, in the presence
of methyl green (major groove binder), the cleavage was
also not suppressed (Fig. 8). Thus, electrophoretic pattern
demonstrated that compounds TS05 and TS07 showed
nonspecific affinity toward both the major and the minor
grooves, and this was only possible if the compound
extended through the DNA double helix, as seen from an
intercalation binding mode. Further, in compound SC09
significant inhibition was observed in the cleavage pattern
when added with DAPI, whereas in the presence of methyl
green (major groove binder), the cleavage was partially
suppressed (Fig. 8). Thus, electrophoretic pattern
123

Med Chem Res
Fig. 6 Determination of
compound-induced membrane
damage of (I) S. aureus and (II)
E. coli cells by flow cytometric
studies. a–f PI staining of both
strains a control without PI
treatment b control with PI
treatment c SC06 d SC09
e TS05 and f TS07. Lower left
quadrant of each panel depicts
unstained cells, whereas the
lower right quadrant depicts the
stained cells. Concentrations of
the compounds were 4 9 MIC.
A total of 10,000 events were
recorded for each sample, and
control panels show staining of
the cells in the absence of any
compound
Our previous observations regarding the mode of action
of benzothiazole bearing amide moiety demonstrated that
these compounds disrupted the integrity of cell membrane
and exhibited bactericidal effects (Singh et al., 2014b).
Further, we wanted to know whether the designed ben-
zothiazole–semicarbazone
and
thiosemicarbazone
123

Med Chem Res
Fig. 6 continued
compounds, that are extension of the earlier reported series
(Singh et al., 2014b), also targets the plasma membrane
and disrupts its integrity, resulted in the killing of bacteria.
In this study, we examined the effect of designed
compounds on the S. aureus and E. coli bacterial mem-
brane to establish the mode of action. All the designed
123

Med Chem Res
Fig. 7 Agarose gel
electrophoresis patterns of
pUC19 (200 ng) cleaved by
a SC06 b SC09 c TS05 d TS07
in the range of 31.2–3.91 lg/ml,
after 1-h incubation time in
buffer (5 mM Tris–HCl/50 mM
NaCl, pH 7.2 at 25 °C)
analogs were found to rapidly hyperpolarize the Entero-
coccus and Staphylococcal membrane, in a manner similar
to ionophore valinomycin (Fig. 4) (Orlova et al., 1994).
However, it seems that these compounds create further
negative potential, which is observed with ionophore
valinomycin. Nevertheless, it is noteworthy that the com-
pounds TS05 and SC06 with higher bactericidal property
create more negative potential in the bacterial membrane
(Fig. 4). Valinomycin at the concentration 0.1 lM used in
the study exerts its effect at the cell membrane, possibly by
altering electrical properties. It selectively translocates
potassium ion across the cell membrane. The increase in
the K? permeability (Pk) may result in hyperpolarization
or an impairment of the electrical behavior of the cell
membrane (Spector et al., 1975). Alternatively, valino-
mycin may act by exchanging intracellular K? for extra-
cellular Na? or H?. However, in the absence of clear
understanding of the relative concentrations and perme-
abilities of sodium and potassium, as well as chloride, in
the bacteria, and relative contribution to the membrane
potential made by electrogenic pumps (i.e., Na?, K?
-ATPase), the present observations can be considered only
indirect evidence implicating membrane potential in the
events leading to bactericidal effect. Furthermore, changes
123

Med Chem Res
bactericidal kinetics, membrane-disrupting ability and
DNA-binding ability of designed compounds. For com-
pound SC06, good level of membrane hyperpolarization
and bactericidal kinetics and high level of PI uptake as well
as reduced DNA-binding ability make membrane destabi-
lization and membrane disruption, a probable mode of
action. A slower bactericidal kinetics of compound SC09
might be due to different modes of action as analyzed by
lower level of membrane hyperpolarization, low level of PI
uptake and preferential DNA binding toward major groove.
Compound TS05 showed high level of membrane hyper-
polarization, rapid killing kinetics, high level of PI uptake
and excellent DNA-binding ability. Therefore, this com-
pound displayed membrane perturbing as well as intracel-
lular mode of action. Similarly, compound TS07 gave
significant level of hyperpolarization, good bactericidal
kinetics, low level of PI uptake and excellent DNA-binding
ability, supporting its membrane and genomic DNA tar-
geting mode of action.
Fig. 8 Agarose gel electrophoresis pattern for the cleavage of
supercoiled DNA (200 ng) by TS05, TS07 and SC09 in the presence
of minor groove (DAPI) and major groove (methyl green) binding
agent
Conclusion
in membrane potential status were not associated with
structural damage to the membrane integrity, since hyper-
polarized cells in a similar fashion to valinomycin act not
by forming channels or pores through which potassium can
travel, but by forming one-to-one complexes with K? ions
(Tosteson et al., 1968), which can then diffuse across the
hydrophobic lipid membranes directly. Overall, these
observations suggest that the mode of action of benzoth-
iazole–semicarbazone and thiosemicarbazone compounds
might result from disruption of the membrane potential,
which is utilized for cellular energy production and hence
killing of bacterial cells.
This study was focused on the development of new ther-
apeutically active antibacterial agents containing semicar-
bazone and thiosemicarbazone building blocks. We
obtained four lead compounds having broad range of
activity with MIC values between 1.56 and 31.2 lg/ml
against Gram-positive as well as Gram-negative bacterial
strains. The variation in activity of the compounds can be
correlated with their lipophilic and electronic behavior.
The presence of a benzothiazole ring with semicarbazone/
thiosemicarbazone groups along with different R substitu-
tions in compounds enhanced their electronic and lipo-
philic nature, respectively, resulting in higher capability to
penetrate the microorganisms through the lipid layer of the
cell membrane. Benzothiazole–semicarbazone and
thiosemicarbazone derivatives showed excellent cell
selectivity and killed the bacteria by disrupting the mem-
brane. These compounds were also able to alter the elec-
trophoretic mobility of DNA, which was not directly
related to activity but may as well be responsible for
enhanced potency of these compounds due to intracellular
mode of action. Thus, the designed compounds serve as
promising leads for further optimization and are advanced
experimental candidates with antimicrobial therapeutic
potential with respect to earlier reported amides (Singh
et al., 2014b).
However, this is unlikely to be the sole mechanism,
since designed compounds induced the uptake of PI as
shown by FACS data (Fig. 6), which is consistent with the
findings for agents targeting the membrane (Singh et al.,
2014b). Since lead compounds showed potent activity
against E. coli and S. aureus, therefore to have better
insight into the mode of action of such compounds we
performed gel electrophoresis experiment, to evaluate
whether designed compounds could kill the bacteria
through disrupting DNA genome of bacteria or not? The
results showed that benzothiazole–semicarbazone and
thiosemicarbazone derivatives have significant effect on
the plasmid DNA. Considering this in conjunction with the
above results, we concluded that the plasma membrane as
well as genomic DNA of bacteria was the target of
designed analogs.
Acknowledgments The authors are grateful to The Head, Depart-
ment of Chemistry, Faculty of Science, Banaras Hindu University
(BHU), Varanasi, India, for 13C NMR spectroscopy and SAIF, Cen-
tral Drug Research Institute (CDRI), Lucknow for 1H NMR and Mass
spectroscopy. One of the authors, Meenakshi Singh, gratefully
Furthermore, these studies also revealed a predominant
role of the electron-releasing groups in initial binding,
123

Med Chem Res
acknowledges Indian Council of Medical Research (ICMR), New
Delhi for the award of Senior Research Fellowship.
Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL,
Daszak P (2008) Global trends in emerging infectious diseases.
Nature 451:990–993
Joshi S, Dewangan RP, Yadav S, Rawat DS, Pasha S (2012)
Synthesis, antibacterial activity and mode of action of novel
linoleic acid–dipeptide–spermidine conjugates. Org Biomol
Chem 10:8326–8335
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
¨
Kizilcikli I, Ulku¨seven B, Das¸demir Y, Akkurt B (2004) Zn(II) and
Pd(II) complexes of thiosemicarbazone-S-alkyl esters derived
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