Design, synthesis and mode of action of some benzothiazole derivatives bearing an amide moiety as antibacterial agents

Article abstract of DOI:10.1039/c4ra02649g

In this study ten benzothiazole derivatives bearing the amide moiety were designed, synthesized and evaluated for their antibacterial activity and possible mode of action. Structures of the synthesized compounds were elucidated by spectral data. Four different Gram-negative and two different Gram-positive bacterial strains were used in antibacterial activity tests. Among all the synthesised compounds, compound A07 displayed the most potent inhibitory activity with minimum inhibitory concentration (MIC) values of 15.6, 7.81, 15.6, 3.91 μg ml^-1 against S. aureus, E. coli, S. typhi and K. pneumoniae respectively. Structure-activity relationship (SAR) studies revealed that electronic and lipophillic factors of the phenyl ring had a significant effect on the antimicrobial activity of the designed compounds. The benzothiazole bearing amide (A01-A10) series exhibited different modes of action based on aryl group substitution as revealed by studies on intact bacterial cells and plasmid DNA. The present study provides us two active compounds (A07 and A10) with a membrane perturbing mode of action, and an intracellular mode of action due to binding with DNA along with potent activity against clinically relevant pathogens E. coli and S. aureus.

Full text of DOI:10.1039/c4ra02649g

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PAPER
Design, synthesis and mode of action of some
benzothiazole derivatives bearing an amide moiety
as antibacterial agents
Cite this: RSC Adv., 2014, 4, 19013
Meenakshi Singh,a Sudhir K. Singh,b Mayank Gangwar,c Gopal Nathc
a
*
and Sushil K. Singh
In this study ten benzothiazole derivatives bearing the amide moiety were designed, synthesized and
evaluated for their antibacterial activity and possible mode of action. Structures of the synthesized
compounds were elucidated by spectral data. Four different Gram-negative and two different Gram-
positive bacterial strains were used in antibacterial activity tests. Among all the synthesised compounds,
compound A07 displayed the most potent inhibitory activity with minimum inhibitory concentration
(MIC) values of 15.6, 7.81, 15.6, 3.91 mg mlꢀ1 against S. aureus, E. coli, S. typhi and K. pneumoniae
respectively. Structure–activity relationship (SAR) studies revealed that electronic and lipophillic factors of
the phenyl ring had a significant effect on the antimicrobial activity of the designed compounds. The
benzothiazole bearing amide (A01–A10) series exhibited different modes of action based on aryl group
substitution as revealed by studies on intact bacterial cells and plasmid DNA. The present study provides
us two active compounds (A07 and A10) with a membrane perturbing mode of action, and an
intracellular mode of action due to binding with DNA along with potent activity against clinically relevant
pathogens E. coli and S. aureus.
Received 26th March 2014
Accepted 7th April 2014
DOI: 10.1039/c4ra02649g
www.rsc.org/advances
and drugs for the treatment of infectious diseases are at the
forefront.
1. Introduction
A review of the recent literature revealed that many effective
antimicrobial agents show a heterocyclic moiety within their
structure,4 in particular, substituted benzimidazole, benzox-
azole and benzothiazole derivatives received special attention as
they belong to a class of compounds with proven utility in
medicinal chemistry. They possess different biological proper-
ties such as chemotherapeutical, antibacterial, antifungal, and
antiviral activities, with low toxicity for the antimicrobial ther-
apeutic use in man.5–7 Structure–activity relationship (SAR)
studies carried out on these types of heterocycles have shown
that positions 2 and 6 are crucial for antibacterial activity
against Gram-positive and Gram-negative bacteria strains.8 Our
attention was focused on the benzothiazole nucleus. Benzo-
thiazole nucleus is present in a wide variety of therapeutically
interesting drugs including antitumor,9–12 antimicrobial,13–15
schistosomicidal,16 anti inammatory,17,18 anticonvulsants,19–21
antidiabetic,22,23 antipsychotic,24,25 neuroprotective,26 and
diuretic27 activities. Amides, RCONHR0 moiety are known to
play a pivitol role in molecular recognition, being important
component in supramolecular chemical anion sensor28–30 tech-
nology. Furthermore, in nature, the selective binding with
anion substrates such as DNA is achieved via the positional
alignment of the amide hydrogen bonds. Many investigations
indicated that the presence of hydrogen bonding domain e.g.,
amide (–CONH–) seems to be valuable in the structures of
The growing prevalence of multidrug resistance to therapeutic
antibiotics poses a challenge to the identication of novel
targets and drugs for the treatment of infectious diseases and
therefore, constitutes a serious public health threat. These
organisms possess the ability to withstand attack by currently
available antimicrobial drugs, and the uncontrolled rise in
resistant pathogens threaten lives. The major concern is the
emergence of methicillin resistant strains of Staphylococcus
aureus (MRSA), uoroquinolone-resistant Pseudomonas aerugi-
nosa (FQRP), uoroquinolone-resistant Enterococcus faecalis
(QREF) and vancomycin-resistant Enterococci (VRE) results in
increased rate of mortality.1–3 Among discovery strategies, the
empirical screening of chemical entities that are structurally
distinct from clinically established agents represents an effec-
tive approach to developing novel antibiotics. Therefore, infec-
tions caused by the multidrug resistance bacteria create a
pressing need to design and synthesis of more potent and novel
antimicrobial agents. Thus, the development of novel targets
aDepartment of Pharmaceutics, Indian Institute of Technology (BHU),
Varanasi-221005, U.P., India. E-mail: sksingh.phe@itbhu.ac.in
bMolecular and Structural Biology Division, CSIR-Central Drug Research Institute,
Lucknow-226031, U.P., India
cDepartment of Microbiology, Institute of Medical Sciences, Banaras Hindu University,
Varanasi-221005, U.P., India
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antimicrobials.31 Recently, Patel et al.32 identied an agent strains. To explore the hypothesis that permeabilization of the
(compound 3, Fig. 1), comprises of benzothiazole–acetamide cytoplasmic membrane is responsible for killing, we monitored
system which plays an essential role to furnish promising cell viability via FACS (uorescence assisted cell cytometer)
antimicrobial activities.
analysis and cytoplasmic membrane permeabilization at the
These ndings pave a way for research to be carried out in same time. Our data suggests that cytoplasmic membrane
this area and prompted us to continue our investigation depolarization did not correlate well with bacterial cell lethality.
towards synthesis of amide bearing benzothiazole ring system We further evaluated the interaction of lead molecules with
as antimicrobial agents.
plasmid DNA to probe into the molecular mode of action.
In continuation to our previous work on the synthesis, and
The main objective of our program was to investigate how
the potency and selectivity against different Gram-positive biological properties of various Schiff base-benzothiazole
(Staphylococcus aureus, Enterococcus faecalis) and Gram-negative hybrids34 and 4-thiazolidinones–benzothiazole conjugates,35
(Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Pseu- herein we report the design and synthesis of a small library of
domonas aeruginosa) bacteria belonging to American Type benzothiazole bearing amide moiety (A01–A10, some of them
Culture Collection (ATCC) can be modulated by replacement of are commercially available)36 to extend the research to achieve
hydrogen at position-2 of the heterocyclic nucleus with groups potential antibacterial agents by structural optimization, in
that can generate electronic and electrostatic effects as well as order to obtain comprehensive SAR indications.
different steric properties. In addition, we also investigated the
role of lipophilicity on the antibacterial activity through the
synthesis of a series of benzothiazole bearing amide moiety.
2. Results and discussion
2.1. Chemistry
Over the past few decades, a number of synthetic and semi
synthetic antimicrobial agents have been discovered and used
in clinical practice such as sulphonamides, nitrofurans, peni-
cillins, cephalosporins, oxazolidinones, uoroquinolones, u-
conazole, ketoconazole and micanazoles. In spite of availability
of potent antimicrobial agents, problems against microbial
infections caused by S. aureus, B. subtilis, E. coli, E. aerogenes, A.
niger and C. albicans remain unresolved due to their potency,
toxicity and resistance development.33 Although these antibi-
otics were discovered more than 50 years ago, their mode of
action is still not precisely known. We studied the ability of
antimicrobial inhibitors to bind with lipopolysaccharides (LPS),
to depolarize both the outer and cytoplasmic membranes, to
interact with lipid monolayers, and to kill different bacterial
The synthesis of compounds (A01–A10) followed the general
pathway outlined in Scheme 1 and the structures of these
compounds are shown in Table 1. The nal products benzo-
thiazole bearing amides (A01–A10) were prepared in two steps.
Firstly, we synthesized key intermediate from 2-amino thio-
phenol and para amino benzoic acid (PABA) as the starting
materials, in a satisfactory yield, according to published
procedure37 with minor modications. Finally, to a solution of
key intermediate i.e., 2-(40-aminophenyl)benzothiazole in ace-
tonotrile, different benzoic acid derivatives were added,
together with double equivalent of N,N0-dicyclohexyl-
carbodiimide (DCC). DCC is mostly used as a coupling–dehy-
drating agent in peptide synthesis but we have used it directly in
Fig. 1 Recently disclosed benzothiazole–carboxamido as antibacterial inhibitors.
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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.
Table 1 Structure of compounds A01–A10
(E. faecalis) clinical isolate; and four Gram-negative bacterial
strains: Escherichia coli (E. coli) ATCC 35218, Salmonella typhi
(S. typhi) MTCC 3216, Pseudomonas aeruginosa (P. aeruginosa)
ATCC 27893, and Klebsiella pneumonia (K. pneumoniae) ATCC
31488, using the method recommended by National Committee
for Clinical Laboratory Standards (NCCLS).38 The MIC
(minimum inhibitory concentration) values of these
Compound code
R, Ar
compounds against the above bacteria are presented in Table 2.
Penicillin and standard antibacterial agent ciprooxacin were
also screened under identical conditions for comparison.
The results revealed that most of the compounds exhibited
good to moderate antibacterial activity with MIC values ranging
between 3.91 and 125 mg mlꢀ1 in DMSO. Out of the 10 benzo-
thiazole derivatives, compound N-(4-(benzo[d]thiazol-2-yl)
phenyl)-2-methoxy-5-chloro-benzamides (A07) which is carrying
chloro and methoxy groups on aryl ring displayed broad-spec-
trum antimicrobial activity against all the tested bacterial
strains with MIC values of 3.91–62.5 mg mlꢀ1. It also displayed
the most potent activity with MIC values of 15.6, 7.81, 15.6, 3.91
mg mlꢀ1 against S. aureus, E. coli, S. typhi and K. pneumoniae
respectively which was comparable with penicillin and cipro-
oxacin with corresponding MIC values of 3.12, 1.56, 1.56, 1.56
mg mlꢀ1 and 6.25, 6.25, 6.25, 6.25 mg mlꢀ1, respectively.
Compound N-(4-(benzo[d]thiazol-2-yl)phenyl)-styrene-amides
(A10) appears to exhibit maximum antibacterial activity against
E. coli and P. aeruginosa (zone of inhibition up to 16–19 mm at
concentration of 15.6 mg mlꢀ1) while compounds A01, A02, A03,
A04, A05, A06, A08 and A09 showed moderate activity against
few bacterial strains. Among the potent compounds (A07 and
A10) against S. aureus, it can be clearly seen that the compound
A07 with optimum lipophilicity displayed higher activity. In
addition, the above mentioned two derivatives were also found
to contribute highest inhibition of E. coli at good MIC's.
A01
A02
A03
A04
A05
A06
A07
A08
A09
H
3,5-di NO2
2-OH
3-NO2
2-Cl
2-F
5-Cl, 2-OCH3
2,4-diOH
2-OCH3
A10
heterocyclic coupling for the amide preparation, inspite of
activating aromatic acids to acyl chlorides or esters. Moreover,
due to the direct reaction the yield of products is quite appre-
ciable (90–96%). DCC was used to enhance the electrophilicity
of carboxylate group by activating it and hence facilitating the
nucleophilic attack by the terminal amino group more effi-
ciently and therefore affords a better leaving group (Fig. 2).
The reaction mixtures were reuxed with stirring for 4–10 h
to obtain the desired compounds (A01–A10). All the IR, Mass,
1H-NMR, 13C-NMR spectral data of compounds (A01–A10) were
in accordance with the proposed molecular structures. The
purity of the synthesized compounds was monitored by TLC
and ascertained by elemental analysis.
Subsequently structure–activity relationship (SAR) studies
were performed to determine how the substituents on the
benzene ring affected the antimicrobial activity. Compounds
A02, A04, A05, A06 with electron withdrawing group (NO2) and
halogen substitutions (Cl, F) were found to be more active than
the compounds with electron releasing (OH, OCH3) groups A03,
2.2. Antibacterial activity
The antibacterial activity of the compounds were evaluated
against two Gram-positive bacterial strains: Staphylococcus
aureus (S. aureus) ATCC 25323 and Enterococcus faecalis
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Fig. 2 Proposed reaction mechanism of formation of benzothiazole–amide ring system.
A08, A09 against both the Gram-positive and negative strains. In group bearing compound A10 reaches an optimum electron
addition, compounds A05 with chlorine and A06 with uorine density which is important for its signicant antibacterial
on phenyl ring appeared with almost same potential inhibitory activity.
efficacy against E. faecalis and E. coli at 31.2 mg mlꢀ1 of MIC.
Hence, it can be inferred that chloro, uoro and methoxy
substituents bearing derivatives are the most suitable
2.3. Bactericidal kinetics
compounds for achieving the best antibacterial spectrum. The The bactericidal activity of designed compounds was deter-
results may be explained by electron density of the compounds. mined at regular time intervals so as to evaluate the potential
It has been reported that electron-donating groups increase the killing effect of bacterial cells within minutes at concentrations
electron density which makes the compounds effective against higher than MIC. In order to monitor the rapidity of mode of
microorganisms and enhances the antibacterial activity.39 action of designed compounds, we incubated compounds A07
However, high electron density causes more difficult diffusion and A10 with log-phase Gram-positive S. aureus and Gram-
through the bacteria cell and substantial activity loss may negative E. coli at 37 ꢁC and monitored the course of change in
occur.40 Thus, for a compound an optimum electron density is optical density (OD600) at different time intervals. The results
inevitable to gain signicant antibacterial activity. Hence, the showed a signicant reduction in numbers of gram-positive and
combined electron donating ability of chloro and methoxy negative bacteria (90 to 99%) within the rst hour aer addition
groups contribute to the activity of compound A07 and aryl of the compound A07 at 4ꢂ MIC (Fig. 3). Compound A10 was
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Table 2 Antibacterial activities of the synthesized benzothiazole bearing amide compounds (A01–A10)a
Bacteria
Gram-positive bacteria
Gram-negative bacteria
Compound
code
S. aureus
(ATCC 25323)
E. faecalis
(clinical isolate)
E. coli
(ATCC 35218)
S. typhi
(MTCC 3216)
K. pneumonia
(ATCC 31488)
P. aeruginosa
(ATCC 27893)
A01
A02
A03
A04
A05
A06
A07
A08
A09
A10
Penicillin
Ciprooxacin
12–14 (62.5)
11–14 (62.5)
<10 (125)
11–13 (62.5)
—
11–13 (62.5)
12–15 (31.2)
—
—
—
—
—
10–12 (62.5)
10–13 (62.5)
<10 (125)
—
—
—
28–31 (3.91)
<10 (125)
—
13–15 (31.2)
36–40 ($1.56)
29–30 ($6.25)
—
—
—
—
—
—
11–13 (62.5)
<10 (125)
—
12–13 (62.5)
14–16 (31.2)
13–15 (31.2)
24–25 (7.81)
11–13 (62.5)
10–13 (62.5)
16–19 (15.6)
36–40 ($1.56)
33–35 ($6.25)
12–15 (31.2)
—
—
16–18 (15.6)
<10 (125)
—
13–15 (31.2)
36–40 ($1.56)
34–35 ($6.25)
12–15 (31.2)
13–15 (31.2)
12–15 (31.2)
—
11–13 (62.5)
—
—
17–19 (15.6)
10–13 (62.5)
<10 (125)
14–17 (31.2)
33–35 ($3.12)
30–32 ($6.25)
10–12 (62.5)
—
—
16–19 (15.6)
36–40 ($1.56)
33–35 ($3.12)
33–35 ($3.12)
28–30 ($6.25)
a
The value of each compound consisted of ‘zone of inhibition range (MIC)’ of 03 replicates. Level of signicance p < 0.05.
found to be less effective as compared to compound A07 in inhibition of growth compared to compound A10 in both the
E. coli and S. aureus even up to 5 h. At 4ꢂ MIC both compounds strains. Thus the designed compounds are capable of inhibiting
inhibited bacterial growth from 2 h onwards keeping growth bacterial growth within hours of initial interactions.
arrested till 5 h. However, complete eradication of bacterial
growth by any of the tested compounds was not observed up to
5 h. At 4ꢂ MIC compound A07 exhibited the most potent
2.4. Cytoplasmic membrane depolarization assay
To study the mode of action of designed analogs the assay for
depolarization of the cytoplasmic membrane was done with
both Gram-positive (S. aureus) and Gram-negative (E. coli)
bacteria. A family of uorescent cyanine dyes has been devel-
oped that can be used to monitor membrane depolarization.41
These dyes lose uorescence intensity in polarized membranes
and become highly uorescent once polarization is lost.
Therefore, the effects of the designed analogs on the membrane
of respective strains by using DiSC3(5) dye was determined. In
this experiment, if the analogs alter the membrane potential as
a result of pore formation/membrane destabilization, an
increase in uorescence intensity is observed. Fig. 4 shows
membrane depolarization, by an increase in uorescence units,
as a function of time. An increase of approximately 150 uo-
rescence unit was observed using triton 2% as a positive control.
Compounds A07 and A10 shows similar prole with respect to
positive control. At the highest concentration of designed
analogs A07 and A10 tested (i.e., 4ꢂ MIC), the level of depo-
larization appeared to reach a maximum at times from about 2
min to 5 min. All of the designed analogs studied here had the
ability to depolarize the cytoplasmic membrane of S. aureus and
E. coli; however, compounds with different structures had
different concentration–activity proles (data not shown here).
Comparisons were made between the maximum levels of
depolarization indicated by the uorophore to levels of depo-
larization reached at various time points and concentrations of
designed analogs. Compounds A07 and A10 completely depo-
larized the membrane at lower concentrations than those of the
other compounds studied.
Fig. 3 Time dependent killing of (A) S. aureus (B) E. coli upon treat-
ment with compounds A07 and A10 at 4ꢂ MIC.
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Fig. 4 Membrane depolarization ability of designed compounds on (A) E. coli and (B) S. aureus. Cultures were grown to log phase (OD600 ¼ 0.05)
and treated with 4ꢂ MIC concentration of compounds A07 and A10. Compounds are presented as (red curve) A07, (green curve) A10 and (black
curve) Triton 2% as positive control.
of the cells following the treatments of these compounds (Fig. 5).
However, the number of PI-stained cells in both strains
decreased signicantly in A10 analogs. Overall this suggests that
the mode of action of designed analogs A07 and A10 and related
compounds might result from disruption of the membrane
potential that is utilized for cellular energy production. However,
this is unlikely to be the sole mechanism, since compound A10
was slightly less active than A07. The results further indicated
that the ability of A10 analog to damage the organization of both
bacterial cell membranes decreased appreciably due its aryl-
substitution. Thus the results showed that the A10 analog
exhibited contrasting lytic activity towards the bacterial cells.
2.5. Flow cytometry analysis
The change in membrane potential may leads to rupture of
bacterial cells. So to further explore the permeabilization of the
cytoplasmic membrane responsible for cell killing, we monitored
cell viability via FACS (uorescence assisted cell cytometer) anal-
ysis by quantifying the amount of DNA released using propidium
iodide (PI) as DNA probe. Similar to the uorometric analysis, ow
cytometric assays were performed concurrently with cell viability
measurements. The large number of dots in lower right quadrant
uorescing with propidium iodide aer exposure to compounds
might indicate defective outer-membrane repair and consequent
leakiness of more DNA and RNA.42
Consistent with the permeabilization of bacteria, designed
analogs A07 and A10 induced damages to the membrane orga-
nization of S. aureus but induced signicantly lesser damage to
the membrane organization of E. coli as indicated by PI staining
2.6. DNA binding
Antimicrobial potency of various classes of DNA binding agents
is well reported in the literature.43,44 We determined the DNA
Fig. 5 Determination of compound-induced membrane damage of E. coli and S. aureus cells by flow cytometric studies. (A–H) PI staining of
E. coli (A) control without PI treatment (B) control with PI treatment (C) A07 (D) A10 and on S. aureus (E) control without PI treatment (F) control
with PI treatment (G) A07 (H) A10 respectively.
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binding ability of designed analogs A07 and A10. Compound presence of methyl green (major groove binder), the cleavage
A07 showed excellent binding with DNA (200 ng) causing pattern was also not affected (Fig. 7). The electrophoretic
retardation at 31.2 mg mlꢀ1 whereas for A10 no retardation was pattern demonstrates that compound A07 shows non-specic
observed at all the concentrations (Fig. 6). However it was affinity towards both the major and the minor grooves, and this
intriguing that DNA binding was inuenced by overall structure is only possible if the compound extend through the DNA
of the compounds as chloro and methoxy containing double helix, as seen from an intercalation binding mode. Thus,
compound A07 showed the most potent DNA retardation ability in the present study upon comparing DNA binding and anti-
as compared to styrene analog A10.
microbial potency (compound A10 showed no DNA binding
Regardless to the actual mechanism of staining, the MG/ though was equipotent as A07) it was evident that there were no
DAPI technique is of great value to both research cytogeneticists direct correlations between DNA retardation and antimicrobial
and clinical investigators in the identication of major and potency/mode of action.
minor groove regions of DNA. To probe the interacting site of
A07 with pUC19, the plasmid DNA was treated with DAPI or MG
(methyl green)45 prior to the addition of A07. DAPI (minor
3. Conclusion
groove binder) was added to the reaction mixture, no signicant This work focused on the development of new potentially active
inhibition was observed in the cleavage pattern. Whereas in antibacterial agents based on benzothiazole–amides compact
system. In general, the results of the in vitro pharmacological
activity are encouraging, as out of 10 benzothiazole bearing amide
derivatives, A07 compound with chloro and methoxy groups on
aryl ring showed the most potent antibacterial activity with MIC
values of 3.91–62.5 mg mlꢀ1 against the tested bacterial strains,
while compound A10 with styrene moiety was found almost
comparable in few bacterial strains. Based on the results
described above, such compounds with benzamide ring can be
highlighted as new active leads that may have a multi-targeted
mode of action, resulting from its ability to target the bacterial
membrane. Such agents are increasingly becoming attractive
therapeutic options owing to their potent actions, likely multi-
target effects and limited potential for resistance development.
Fig.
6 Agarose gel electrophoresis patterns of pUC19 (200 ng)
cleaved by (A) A07 (31.2–3.91 mg mlꢀ1), after 1 h incubation time Compounds A07 and A10 showed excellent activity and
(concentration dependent) Lane 1: control; Lane 2: 31.2 mg mlꢀ1 A07 +
membrane perturbing mode of action at concentration higher
than MIC. These compounds were also able to alter the electro-
phoretic mobility of DNA, which was not directly related to
DNA; Lane 3: 15.6 mg mlꢀ1 A07 + DNA; Lane 4: 7.81 mg mlꢀ1 A07 +
DNA; Lane 5: 3.91 mg mlꢀ1 A07 + DNA, and (B) A10 (31.2–3.91 mg mlꢀ1),
after 1 h incubation time (concentration dependent) Lane 1: control;
Lane 2: 31.2 mg mlꢀ1 A10 + DNA; Lane 3: 15.6 mg mlꢀ1 A10 + DNA; Lane
activity, may as well be responsible for further enhanced potency
4: 7.81 mg mlꢀ1 A10 + DNA; Lane 5: 3.91 mg mlꢀ1 A10 + DNA, in buffer (5 of these compounds due to intracellular mode of action. The
mM Tris–HCl/50 mM NaCl, pH 7.2 at 25 ꢁC).
characterization of benzothiazole bearing amides as a membrane-
targeting molecule therefore presents a promising lead for further
optimization in an attempt to identify advanced experimental
candidates with antimicrobial therapeutic potential.
4. Experimental section
4.1. Materials and measurements
All chemicals (reagent grade) used were commercially available.
The reactions were monitored with the help of thin-layer chro-
matography using pre-coated aluminium sheets with GF254
silica gel, 0.2 mm layer thickness (E. Merck) and visualized
under ultraviolet (UV) light (254 nm). Melting points were
determined in open-glass capillaries on Stuart-SMP10 melting
point apparatus and were reported uncorrected. IR absorption
spectra were recorded on Shimadzu FTIR-8400s. 1H NMR
spectra were recorded on the Bruker DRX-300 FTNMR and 13C
NMR spectra were recorded on the JEOL AL300 FTNMR spec-
trometer operating at 300 MHz, with TMS and solvent signals
allotted as internal standard. Chemical shis were reported in
Fig.
7 Agarose gel electrophoresis pattern for the cleavage of
parts per million (ppm, d : 0 units). ESI-MS spectra were recor-
supercoiled DNA (200 ng) by A07 in presence of minor groove (DAPI)
and major groove binding agent (methyl green).
ded on Micromass Quattro II spectrometer. IR, 1H NMR, 13C
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NMR and Mass spectra were consistent with the assigned (benzothiazole–C–2), 116.20–148.07 (aromatic–C, C4–C9 C10–
structures. Elemental analyses (C, H, N) were performed on C60 C100–C600); MS (m/z, %): 376 (C20H13N3O3S, [M + H]+). Anal
Exeter Analytical Inc., USA, CE-440 elemental analyzer and were
within 0.03% of the theoretical values.
C20H13N3O3S. Calc. for: C, 63.99; H, 3.49; N, 11.19. Found: 63.97;
H, 3.47; N, 11.17%.
4.3.5. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2-chloro-benza-
mides (A05). White solid, mp: 136–139 ꢁC, yield: 92%. IR (KBr,
nmax cmꢀ1): 3333.10 (–NH str.), 1647.26 (C]O str. of amide),
4.2. General procedure for the synthesis of compounds [N-(4-
(benzo[d]thiazol-2-yl)phenyl)-substituted-benzamides (A01–10)]
1
3066.92 (Ar–C–H str.), 1070.63 (C–Cl str.); H NMR (CDCl3, 300
A reaction mixture in equimolar quantities of 2-(40-amino-
phenyl) benzothiazole (0.01 mol) and the appropriate
substituted aromatic acids (0.01 mol) in acetonitrile (ACN,
20 ml) was added together with DCC (2 equiv.). The mixture was
reuxed under stirring for 4–10 h. The completion of reaction
was monitored by TLC at appropriate time intervals. Following
completion, the reaction mixture was cooled to room temper-
ature. The reaction mixture was ltered through a medium frit.
The solution was then given aqueous wash several times, solid
thus separated was ltered and dried and the rest was ashed
away. The residue was crystallized from ethanol to obtain the
target compounds.
MHz) d (ppm): 7.26–7.61 (m, 12H, Ar–H), 9.49 (s, 1H, –CONH);
13C NMR (DMSO-d6) d (ppm): 162.71 (C]O of amide), 156.57
(benzothiazole–C–2), 119.64–145.85 (aromatic–C, C4–C9 C10–
C60 C100–C600); MS (m/z, %): 365 (C20H13ClN2OS, [M + H]+). Anal
C
20H13ClN2OS. Calc. for: C, 65.84; H, 3.59; N, 7.68. Found: C,
65.82; H, 3.60; N, 7.67%.
4.3.6. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2-uro-benza-
mides (A06). White solid, mp: 140–143 ꢁC, yield: 90%. IR (KBr,
nmax cmꢀ1): 3309.96 (–NH str.), 1653.05 (C]O str. of amide),
3061.13 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz) d (ppm): 7.08–
7.42 (m, 12H, Ar–H), 9.18 (s, 1H, –CONH); 13C NMR (DMSO-d6) d
(ppm): 162.88 (C]O of amide), 156.78 (benzothiazole–C–2),
115.83–143.86 (aromatic–C, C4–C9 C10–C60 C100–C600); MS
(m/z, %): 349 (C20H13FN2OS, [M + H]+). Anal C20H13FN2OS. Calc.
4.3. Characterization data of the compounds are given below
4.3.1. N-(4-(Benzo[d]thiazol-2-yl)phenyl)benzamides (A01). for: C, 68.95; H, 3.76; N, 8.04. Found: C, 68.93; H, 3.75; N, 8.05%.
White solid, mp: 126–128 C, yield: 91%. IR (KBr, nmax cmꢀ1):
4.3.7. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2-ꢁmethoxy-5-chloro-
ꢁ
3309.02 (–NH str.), 1668.12 (C]O str. of amide), 3048.14 (Ar–C– benzamides (A07). White solid, mp: 150–143 C, yield: 96%. IR
H str.); 1H NMR (CDCl3, 300 MHz) d (ppm): 7.02–7.98 (m, 13H, (KBr, nmax cmꢀ1): 3319.60 (–NH str.), 1637.62 (C]O str. of amide),
1
Ar–H), 9.48 (s, 1H, –CONH); 13C NMR (DMSO-d6) d (ppm): 3061.13 (Ar–C–H str.), 2852.81 (C–H str., OCH3 group); H NMR
163.34 (C]O of amide), 155.09 (benzothiazole–C–2), 118.98– (CDCl3, 300 MHz) d (ppm): 7.11–7.78 (m, 11H, Ar–H), 9.39 (s, 1H,
138.17 (aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %): 331 –CONH); 13C NMR (DMSO-d6) d (ppm): 162.49 (C]O of amide),
(C20H14N2OS, [M + H]+). Anal C20H14N2OS. Calc. for: C, 72.70; H, 58.11 (OCH3), 153.01 (benzothiazole–C–2), 122.36–136.75
4.27; N, 8.48. Found: C, 72.68; H, 4.25; N, 8.48%.
(aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %): 379
4.3.2. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-3,5-dinitro-benza- (C21H15ClN2OS, [M + H]+). Anal C21H15ClN2OS. Calc. for: C, 66.57;
mides (A02). White solid, mp: 122–124 ꢁC, yield: 90%. IR (KBr, H, 3.99; N, 7.39. Found: C, 66.54; H, 3.96; N, 7.37%.
nmax cmꢀ1): 3327.32 (–NH str.), 1627.92 (C]O str. of amide),
4.3.8. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2,4-dihydroxy-
3066.92, 3109.35 (Ar–C–H str.) 1342.50 (–NO2 str.); 1H NMR benzamides (A08). White solid, mp: 150–143 ꢁC, yield: 96%. IR
(CDCl3, 300 MHz) d (ppm): 7.31–7.87 (m, 11H, Ar–H), 9.13 (KBr, nmax cmꢀ1): 3353.18 (–NH str.), 1664.73 (C]O str. of
(s, 1H, –CONH); 13C NMR (DMSO-d6) d (ppm): 162.48 (C]O of amide), 3085.10 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz) d
amide), 156.42 (benzothiazole–C–2), 121.67–140.17 (aromatic– (ppm): 7.22–7.86 (m, 11H, Ar–H), 9.56 (s, 1H, –CONH), 10.48,
C, C4–C9 C10–C60 C100–C600); MS (m/z, %): 421 (C20H12N4O5S, [M 9.86 (s, 2H, –OH); 13C NMR (DMSO-d6) d (ppm): 162.79 (C]O of
+ H]+). Anal C20H12N4O5S. Calc. for: C, 57.14; H, 2.88; N, 13.33. amide), 155.44 (benzothiazole–C–2), 121.78–145.26 (aromatic–
Found: C, 57.12; H, 2.85; N, 13.31%.
C, C4–C9 C10–C60 C100–C600); MS (m/z, %): 363 (C20H14N2O3S,
4.3.3. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2-hydroxy-benza- [M + H]+). Anal C20H14N2O3S. Calc. for: C, 66.28; H, 3.89; N, 7.73.
mides (A03). White solid, mp: 132–134 ꢁC, yield: 94%. IR (KBr, Found: C, 66.24; H, 3.86; N, 7.71%.
nmax cmꢀ1): 3312.68 (–NH str.), 1681.42 (C]O str. of amide),
4.3.9. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-2-methoxy-benza-
3096.10 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz) d (ppm): 7.18– mides (A09). White solid, mp: 168–170 ꢁC, yield: 95%. IR (KBr,
7.79 (m, 12H, Ar–H), 9.32 (s, 1H, –CONH), 10.48 (br, s, 1H, –OH); nmax cmꢀ1): 3322.68 (–NH str.), 1647.23 (C]O str. of amide),
13C NMR (DMSO-d6) d (ppm): 163.90 (C]O of amide), 154.17 3054.13 (Ar–C–H str.), 2850.11 (C–H str., OCH3 group); 1H NMR
(benzothiazole–C–2), 110.28–135.46 (aromatic–C, C4–C9 C10– (CDCl3, 300 MHz) d (ppm): 7.28–7.91 (m, 12H, Ar–H), 9.26 (s,
C60 C100–C600); MS (m/z, %): 347 (C20H14N2O2S, [M + H]+). Anal 1H, –CONH); 13C NMR (DMSO-d6) d (ppm): 163.25 (C]O of
C
20H14N2O2S. Calc. for: C, 69.35; H, 4.07; N, 8.09. Found: 69.33; amide), 56.15 (OCH3), 156.23 (benzothiazole–C–2), 122.46–
H, 4.08; N, 8.10%.
146.35 (aromatic–C, C4–C9 C10–C60 C100–C600); MS (m/z, %): 361
4.3.4. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-3-nitro-benza- (C21H16N2O2S, [M + H]+). Anal C21H16N2O2S. Calc. for: C, 69.98;
mides (A04). White solid, mp: 136–139 ꢁC, yield: 91%. IR (KBr, H, 4.47; N, 7.77. Found: C, 69.96; H, 4.46; N, 7.75%.
nmax cmꢀ1): 3325.82 (–NH str.), 1648.12 (C]O str. of amide),
4.3.10. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-styrene-amides
1
3037.77 (Ar–C–H str.) 1356.50 (–NO2 str.); H NMR (CDCl3, 300 (A10). White solid, mp: 147–149 ꢁC, yield: 92%. IR (KBr, nmax
MHz) d (ppm): 7.24–7.69 (m, 12H, Ar–H), 9.58 (s, 1H, –CONH); cmꢀ1): 3317.40 (–NH str.), 1653.82 (C]O str. of amide), 3087.42
13C NMR (DMSO-d6) d (ppm): 164.04 (C]O of amide), 157.23 (Ar–C–H str.); 1H NMR (CDCl3, 300 MHz) d (ppm): 7.26–7.71
19020 | RSC Adv., 2014, 4, 19013–19023
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(m, 14H, Ar–H), 9.58 (s, 1H, –CONH), 6.72, 6.77 (dd, –CH]CH); 20 mM glucose buffer (pH 7.2) to an optical density of 0.06. Aer
13C NMR (DMSO-d6) d (ppm): 164.18 (C]O of amide), 156.30 three washings, pellets were resuspended in the same buffer.
(benzothiazole–C–2), 116.36–138.15 (aromatic–C, C4–C9 C10– This cell suspension was incubated with 100 mM KCl (to
C60 C100–C600); MS (m/z, %): 357 (C22H16N2OS, [M + H]+). Anal equilibrate cytoplasmic and external K+ concentration) and
C
22H16N2OS. Calc. for: C, 74.13; H, 4.52; N, 7.86. Found: C, DiSC3(5) at a concentration of 1 mM. The dye was allowed to
74.12; H, 4.50; N, 7.85%.
incorporate for 1 h at room temperature, when the uorescence
level (excitation and emission wavelength set at 622 and 670
nm, respectively) of the bacterial suspension became stable,
(approximately 90% reduction in uorescence due to DiSC3(5)
uptake and quenching in the cell in response to an intact
membrane potential), different amounts of each of the
compounds were added to these suspensions in order to record
the membrane depolarization of bacterial cell membrane. A 400
ml aliquot of cell suspension was placed in a cuvette, and the
desired concentration of compounds was added. Fluorescence
was monitored with a Perkin Elmer Life Sciences LS 50-B
spectrouorimeter (Perkin-Elmer Corp., Norwalk, Conn.) in a 5
mm path length quartz cell at 25 ꢁC. Samples were stirred
4.4. In vitro antibacterial activity
Antimicrobial activity was determined by agar disc diffusion
method and was evaluated according to the guidelines of
National Committee for Clinical Laboratory Standards (NCCLS,
1997).38 In vitro activity of the compounds was tested in nutrient
broth (NB) for bacteria by serial dilution method. Seeded broth
(broth containing microbial spores) was prepared in NB from 24
h old bacterial cultures on nutrient agar (Hi-media) at 37 ꢃ 1 ꢁC.
The bacterial suspension was adjusted with sterile saline to a
concentration of 105 CFU mlꢀ1 46 The inoculums were spread on
.
the surface of the solidied media and Whatman no. 1 lter
paper discs (6 mm in diameter) impregnated with the test
compounds (20 ml per disc) placed on the plates. Ciprooxacin
(5 mg per disc, Hi-Media) was used as positive control and
dimethyl sulfoxide (DMSO) was used as negative control. All the
inoculated plates were incubated at 37 ꢁC and results were
evaluated aer 24 h for bacteria. The zones of inhibition were
measured in millimetres. The tested compounds and reference
drugs were prepared by two-fold serial dilutions to obtain the
required concentrations of 125, 62.5, 31.2, 15.6, 7.81, 3.91 mg
mlꢀ1. All the dilutions were made with distilled water. All
determinations were done in triplicates and the average was
taken as nal reading. At the end of the incubation period, the
MIC values were determined. The lowest concentration of the
compound that prevented visible growth (turbidity on liquid
media) was considered as MIC.
ꢁ
during the experiment at a constant temperature of 37 C.
4.7. Flow cytometry analysis
The membrane damage of S. aureus ATCC 25323 and E. coli
ATCC 35218 was determined by staining the cells with propi-
dium iodide aer the treatment with the designed compounds
A07 and A10 at 37 ꢁC for 1 h. These cells were then analyzed by
ow cytometry in the form of dots plot with respect to the
control cells, not treated with any compounds and cells treated
with propidium iodide without preincubation with tested
compounds. In order to check the membrane integrity of
bacteria, the cells at mid-log phase were incubated with
designed compounds A07 and A10 for 1 h at 37 ꢁC with constant
shaking. The cells were collected by centrifugation, washed two
times with PBS and incubated further with propidium iodide at
4
ꢁC for 30 min, followed by removal of the unbound dye
4.5. Bactericidal kinetics
Bactericidal kinetics were performed as described previously.47
Overnight cultures of S. aureus ATCC 25323 and E. coli ATCC
35218 were grown in fresh MHB up to log phase. For deter-
mining the time course of killing activity 100 ml of fresh MHB
was added to all wells of the 96 well-plate. Then 90 ml of
approximately 105 CFU per ml were added to the wells of the 96-
through washing with an excess of PBS and re-suspended in
buffer. Compound-induced damage of bacterial cells was then
analyzed by ow cytometry (excitation and emission wavelength
set at 488 and 617 nm, respectively for propidium iodide).
well plates of plate reader (4 wells for a single concentration). 4.8. DNA binding assay
Then 10 ml of appropriate concentrations of test compounds
Gel retardation experiments were performed by agarose gel
A07 and A10 corresponding to 4ꢂ MIC were added to the wells.
electrophoresis as described previously.43,44 Briey, 200 ng of
pUC19 plasmid DNA was mixed with increasing amounts of test
compounds A07 and A10 in 20 ml of binding buffer (5% glycerol,
10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 20
mM KCl, and 50 mg mlꢀ1 bovine serum albumin). The samples
were incubated for 1 h at 37 ꢁC. A loading buffer containing 25%
bromophenol blue, 0.25% xylene cyanol, 30% glycerol was
ꢁ
The plates were incubated at 37 C at 200 rpm. Absorbance of
the plates was read at 600 nm at various time points at 0, 1, 2, 3,
4 and 5 h. The experiment was repeated on three different days
and values are plotted as mean ꢃ SD.
4.6. Cytoplasmic-membrane depolarization assay
The depolarization of the cytoplasmic membrane of S. aureus added and electrophoresis was carried out at 60 V for 1 h in
ATCC 25323 and E. coli ATCC 35218 by the designed Tris–HCl buffer using 1% agarose gel containing 1.0 mg mlꢀ1
compounds A07 and A10 was determined using the membrane ethidium bromide (EB).48 The illuminated gel was photo-
potential-sensitive cyanine dye DiSC3(5). Bacterial cultures were graphed by Alpha Innotech Corporation Instrument. The reac-
ꢁ
grown overnight in LB media at 37 C. Cells were harvested by tion was also monitored upon addition of various groove
centrifugation and washed in a buffer containing 5 mM HEPES– binders-methyl green, and DAPI.
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´
22 G. Navarrete-Vazquez, P. Paoli, I. Leon-Rivera, R. Villalobos-
Acknowledgements
Molina, J. L. Medina-Franco, R. Ortiz-Andrade, S. Estrada-
˜
Soto, G. Camici, D. Diaz-Coutino, I. Gallardo-Ortiz,
The authors are grateful to The Head, Department of Chemistry,
Faculty of Science, Banaras Hindu University (BHU), Varanasi,
India for 13C NMR spectroscopy and SAIF, Central Drug
Research Institute (CDRI), Lucknow for 1H NMR and Mass
spectroscopy. One of the author, Meenakshi Singh gratefully
acknowledge Indian Council of Medical Research (ICMR), New
Delhi for the award of senior research fellowship.
´
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