Synthesis and biological evaluation of 2-mercapto-1,3-benzothiazole derivatives with potential antimicrobial activity

Article abstract of DOI:10.1002/ardp.200900092

The enhancement of bacterial resistance of pathogens to currently available antibiotics constitutes a serious public health threat. So, intensive efforts are underway worldwide to develop new antimicrobial agents. To identify compounds with a potent antimicrobial profile, we designed and synthesized low molecular weight 2-mercaptobenzothiazole derivatives 2a-2l and 3a-3l. Both series were screened for in-vitro antibacterial activity against the representative panel of Gram-positive and Gram-negative bacteria strains. The biological screening identified compounds 2e and 2l as the most active ones showing an interesting antibacterial activity with MIC values of 3.12 μg/mL against Staphylococcus aureus and 25 μg/mL against Escherichia coli, respectively. The replacement of the S-H by the S-Bn moiety resulted in considerable loss of the antibacterial action of the 3a-3l series. The antibiotic action of compounds 2e and 2l was also investigated by testing their activity against some clinical isolates with different antimicrobial resistance profile. Moreover, the involvement of the NorA efflux pump in the antibacterial activity of our molecules was evaluated. Finally, in this paper, we also describe the cytotoxic activity of the most interesting compounds by MTS assay against HeLa and MRC-5 cell lines.

Full text of DOI:10.1002/ardp.200900092

Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
C. Franchini et al.
605
Full Paper
Synthesis and Biological Evaluation of 2-Mercapto-1,3-
benzothiazole Derivatives with Potential Antimicrobial Activity
Carlo Franchini1, Marilena Muraglia1, Filomena Corbo1, Marco Antonio Florio1, Antonia Di Mola1,
Antonio Rosato1, Rosanna Matucci2, Marta Nesi2, Francoise van Bambeke3, Cesare Vitali1
1 Department of Pharmaceutical Chemistry, University of Bari, Bari, Italy
2 Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy
3 Department of Pharmaceutical Sciences, Cellular and Molecular Pharmacology Unit, Catholic University of
Louvain, Bruxelles, Belgium
The enhancement of bacterial resistance of pathogens to currently available antibiotics consti-
tutes a serious public health threat. So, intensive efforts are underway worldwide to develop
new antimicrobial agents. To identify compounds with a potent antimicrobial profile, we
designed and synthesized low molecular weight 2-mercaptobenzothiazole derivatives 2a–2l and
3a–3l. Both series were screened for in-vitro antibacterial activity against the representative
panel of Gram-positive and Gram-negative bacteria strains. The biological screening identified
compounds 2e and 2l as the most active ones showing an interesting antibacterial activity with
MIC values of 3.12 lg/mL against Staphylococcus aureus and 25 lg/mL against Escherichia coli,
respectively. The replacement of the S-H by the S-Bn moiety resulted in considerable loss of the
antibacterial action of the 3a–3l series. The antibiotic action of compounds 2e and 2l was also
investigated by testing their activity against some clinical isolates with different antimicrobial
resistance profile. Moreover, the involvement of the NorA efflux pump in the antibacterial activ-
ity of our molecules was evaluated. Finally, in this paper, we also describe the cytotoxic activity
of the most interesting compounds by MTS assay against HeLa and MRC-5 cell lines.
Keywords: Antibacterial activity / Benzothiazole / Efflux pump / MTS assay /
Received: April 26, 2009; accepted: June 15, 2009
DOI 10.1002/ardp.200900092
gence of Gram-negative pathogens is of major concern. In
fact, the most important cases of sepsis were caused by
virulent Gram-negative bacteria such as Pseudomonas aer-
uginosa, Klebsiella pneumoniae, Escherichia coli, and Entero-
bacter spp. [3, 4]. Furthermore, emerging resistance
among new pathogens such as Acinetobacter baumannii,
and also the appearance of multidrug resistant Gram-pos-
itive bacteria, in particular, methicillin-resistant Staphylo-
coccus aureus (MRSA) and vancomycin-resistant Enterococci,
are causing a serious menace to public health. Therefore,
the development of new and different antimicrobial
drugs is a very important goal, and most of the research
program efforts in this field are directed towards the
design of new agents. A review of the recent literature
revealed that many effective antimicrobial agents show a
heterocyclic moiety within their structure [5] and, in par-
ticular, that substituted benzimidazole, benzoxazole,
Introduction
The current interest in the development of new antimi-
crobial agents can be partially ascribed both to the
increasing emergence of bacterial resistance to antibiotic
therapy and to newly emerging pathogens [1, 2]. Despite
advances in antibacterial therapy, many problems
remain to be solved for most available antimicrobial
drugs. For example, in the hospital setting, the re-emer-
Correspondence: Prof. Carlo Franchini, Dipartimento Farmaco-Chimico,
Facoltꢀ di Farmacia, Universitꢀ di Bari, Via E. Orabona 4, 70125 Bari,
Italy.
E-mail: cfranc@farmchim.uniba.it
Fax: +39 080 544-2724
Abbreviations: National Committee on Clinical Laboratory Standards
(NCCLS); norfloxacin (NRF); oxacillin (OXA); vancomycin (VAN)
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Reagents and conditions: a) DMF, 1608C, 1 h; b) K2CO3, H2O/dioxane, 758C, 15
min; c) Ar-CH2-Br, Bu4NBr3, p-anisidine, CH2Cl2/CH3OH, r.t., 2 h.
Scheme1.Syntheticroutesofcompounds1d, 2a–2f,and3a–3l.
and benzothiazole derivatives bring different biological synthesis of the benzylated series 3a–3l. Although com-
properties such as chemotherapeutical, antibacterial, pounds of the 2a–2l series are commercially available,
antifungal, and antiviral activities, with a low toxicity for they have been included in our research program to
the antimicrobial therapeutic use in man [6–8]. Struc- screen their antimicrobial profile in order to have more
ture-activity relationship (SAR) studies carried out on data for SAR (structure-activity relationship) proposal.
these types of heterocycles have shown that positions 2 Because of the prohibitive price, we synthesized com-
and 6 are crucial for antibacterial activity against Gram- pounds 2a–2f ex novo in short reaction time, with very
positive and Gram-negative bacteria strains [9].
good yields and in a cheap synthetic procedure.
All these observations prompted us to start a research
In the present work, we report the synthesis of two ser-
program for the synthesis of small molecules potentially ies of 2-mercaptobenzothiazoles 2a–2f and 3a–3l and
useful as antimicrobial agents. After a careful screening their preliminary antibacterial profile against different
of various heteronuclei, we have chosen to focus our Gram-positive and Gram-negative bacterial strains
attention on benzothiazole derivatives. In particular, we belonging to American Type Culture Collection (ATCC).
synthesized 2-mercaptobenzothiazole derivatives 2a–2f The most active compounds of the series were also stud-
and 3a–3l in order to explore the effects of substituents ied by using clinical isolates S. aureus with different anti-
at positions 2 and 6 on the antibacterial activity. Espe- biotic resistance profile. Finally, the most interesting
cially, the main objective of our program was to investi- molecules were also characterized with regard to their
gate how the potency and selectivity against different cytotoxic effects by testing them against a human cervi-
Gram-positive (Staphylococcus aureus, Bacillus cereus, Bacil- cal cancer cell line (HeLa) and a normal human lung
lus subtilis, Enterococcus faecalis) and Gram-negative (Escher- fibroblasts cell line (MRC-5).
ichia coli, Acinetobacter baumannii, Klebsiella pneumoniae,
Pseudomonas aeruginosa) bacteria, can be modulated by
the replacement of the hydrogen at the 6-position of the
heterocyclic nucleus with groups that can generate elec-
Results and discussion
tronic and electrostatic effects as well as different steric Chemistry
properties. In addition, we also investigated the role of The synthetic routes of compounds 1d, 2a–2f, and 3a–3l
lipophilicity on the antibacterial activity through the are reported in Scheme 1. The 6-substituted-2-mercapto-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
Mercaptobenzothiazole Derivatives: Antibacterial Activity
607
Table 1. Antimicrobial activity resultsa) of 2-mercaptobenzothiazole derivatives 2a–2l (MIC in lg/mL).
Microorganism
Gram-positive
S. a. b) E. f.c)
Gram-negative
E. c.f) A. b.g)
Compd
R
S. a.b)
S. a. b)
E. f. c)
B. s.d)
B. c. e)
E. c.f)
K. p.h)
P. a.i)
6538P 25923 29213 19433 29212 6633
11778 8739
35218 19606 13883 27853
2a
2b
2c
2d
2e
2f
2g
2h
2i
CH(CH3)2
Cl
CH3
OCH3
CF3
F
12.5
25
25
12.5
6.25
50
50
12.5
25
12.5
50
25
50
50
25
12.5
50
50
3.12
25
50
25
50
50
50
100
50
100
R
100
50
R
50
100
100
50
100
R
100
50
R
25
50
50
25
50
100
50
25
50
50
25
50
25
25
50
100
50
25
50
50
R
R
100
50
R
R
50
R
R
25
R
R
50
100
R
R
50
R
R
R
R
100
R
100
R
100
R
R
100
4
R
R
100
R
R
R
100
R
R
50
R
R
R
R
R
R
R
R
R
R
2
6.25
100
100
25
100
25
H
NH2
OCH2CH3
NO2
2l
12.5
0.5
100
4
100
2
50
NRF+
0.25
0.5
0.125
0.25
0.06
0.06
0.25
a)
Antimicrobial activity was estimated by using NCCLS assay [12].
b)
c)
d)
e)
f)
g)
h)
Abbreviations: S. a., S. Aureus, E. f., E. Faecalis, B. s., B. Subtilis; B. c., B. Cereus, E. c., E. Coli,
Pneumoniae, i) P. a., P. aeruginosa.
A. b., A. Baumannii, K. p., K.
+ NRF, norfloxacin; R, resistant.
Acceptable quality-control range of MICs for reference strains [NCCLS M7-A4]: S. aureus ATCC 29213: 0.5-2; E. faecalis ATCC 29212: 2-
8; P. aeruginosa ATCC 27853: 1–4.
benzothiazole derivatives 2a–2f were prepared accord- Gram-negative bacteria belonging to the ATCC collec-
ing to the literature procedure [10]. Reaction of the tion. All MIC determinations were carried out using
appropriate anilines 1a–1c, 1e, 1f, commercially avail- NCCLS guidelines [12]. MIC values are given in lg/mL and
able, with potassium ethyl xanthate in DMF gave the cor- were compared to MIC values for the standard antibacte-
responding 6-substituted-1,3-benzothiazole-2-thiols 2a– rial drug norfloxacin. Screening results are summarized
2c, 2e, 2f, respectively. Compounds 2g–2l are commer- in Table 1.
cially available.
The combined data showed that compounds 2a–2l
The 3a–3l series was prepared by the treatment of 6- exerted inhibitory activity against the tested bacterial
substituted-1,3-benzothiazole-2-thiols 2a–2l with benzyl strains with MIC values between 3.12 and 100 lg/mL. The
bromide, K2CO3, dissolved in a mixture of dioxane/water, obtained results generally indicate that most of the
providing the corresponding 2-benzylthio-6-substituted- tested molecules are more active against Gram-positive
1,3-benzothiazoles derivatives.
than Gram-negative bacterial strains. Among the men-
The starting 2-bromo-4-methoxyaniline 1d was synthe- tioned derivatives 2a–2l, the most promising results
sized according to a reported methodology via a bromi- were obtained with compounds 2e and 2l. In particular
nation strategy by using tetrabuthylammonium tribro- 2e was the most active derivative giving the best antibac-
mide (Bu4NBr3) following a synthetic procedure described terial activity against S. aureus with a MIC value of 3.12
in the literature [11].
AsitisoutlinedinScheme1, compounds2a–2fand3a–
lg/mL. On the other hand, compound 2l showed a wide
antimicrobial activity toward Gram-positive such as S.
3l were synthesized in high yields. The structures of the aureus (MIC: 12.5 lg/mL) and Gram-negative such as E. coli
obtainedcompoundswereelucidatedbyspectraldata.
(MIC: 25 lg/mL). Furthermore, it is noteworthy that the
data registered in Table 1 reveal that 2a–2l derivatives
generally have a significant influence on the antibacte-
Biological evaluation
The antibacterial activity of compounds 2a–2l was eval- rial profile of S. aureus. In this series, compounds 2a, 2b,
uated in vitro against an assortment of Gram-positive and 2d, 2h, and 2l were found to inhibit S. aureus at a MIC
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Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
Table 2. Antimicrobial activity results (MIC in lg/mL) of com-
pounds 2e and 2l against clinical isolates of S. aureus.
value of 12.5 lg/mL. In addition, compounds 2c, 2d, and
2g revealed moderate antimicrobial activity against
Gram-negative bacteria such as E. coli strains.
2e
2l
NRFa)
VANb)
OXAc)
With regards to the antimicrobial activity, the behav-
iour of the 3a–3l series was very different compared with
the 2a–2l series: under the same experimental conditions
it was found that derivatives having a thiobenzyl group at
the 2-position of the heterocyclic nucleus did not inhibit
the bacterial growth in spite of biological results previ-
ouslyobservedonbenzothiazolederivatives[9].
S. a. 25923
VRS2
NRS52
4
8
8
16
32
16
32
32
32
32
1
128
256
256
256
1
n. e.
64
4
1
1
n. e.
128
0.5
N4120032 16
16
N4112910
NRS100
STA268
8
16
8
128
256
n. e.
1
1
1
a)
Due both to the small numbers of evaluated com-
pounds and the low diversity of the involved chemical
features of the series reported herein, an attempt to ana-
lyse the structure-activity relationships does not seem
reasonable. Several comparisons on the results could be
made. Initially, it should be noted that small structural
changes at the 6-position do not significantly alter the
antibacterial activity, except for compounds 2e and 2l
carrying a trifluoromethyl group and a nitro group at
the 6-position of the 2-mercapto 1,3-benzothiazole,
respectively. In detail, the biological results identified
compound 2e as a potent and selective inhibitor of S. aur-
eus strains (MIC: 3.12 lg/mL) and whereas compound 2l
was less active than 2e against Gram-positive bacteria, it
was able to inhibit the growth of Gram-negative micro-
organism such as E. coli (MIC: 25 lg/mL).
By evaluating the biological results, it is possible to
observe that the presence of a hydrogen atom (2g) or a
methyl group (2c) at the 6-position on the heterocyclic
nucleus, preserves a wide spectrum of action. In addition,
it is interesting to consider that an increase of steric hin-
drance, though an increment of lipophilicity, probably
could lead to a loss of activity against Gram-negative
strains, as observed for compound 2a.
Moreover, the displacement of the hydrogen atom (2g)
at the 6-position with chlorine (2b) and fluorine (2f)
atoms as well as with methoxy (2d) and ethoxy (2i) groups
generally produced an increment in the activity against
S. aureus with MIC values in the range of 12.5 to 100 lg/
mL. Furthermore, the introduction of a polar group such
as the aminic one (2h) led to an interesting antibacterial
activity against Gram-positive strains.
Because among the mentioned derivatives a remark-
able activity was registered against S. aureus strains, we
focused our attention on our best molecules, 2e and 2l, to
investigate their antimicrobial profile against seven clin-
ical isolates of S. aureus belonging both to NARSA and pri-
vate collections with a different antimicrobial resistance
profile as detailed in the experimental section. In Table 2
the microbiological data obtained according to NCCLS
protocol [12] are reported and compared with norfloxa-
cin (NRF), vancomycin (VAN), and oxacillin (OXA) used as
NRF, Norfloxacin.
VAN, Vancomycin.
OXA, Oxacillin.
b)
c)
standard drugs. From the results reported in Table 2, it
appears that compound 2e generally revealed better
growth inhibitory effects against the clinical isolates
than compound 2l. In particular, compound 2e was sur-
prisingly efficacious against two vancomycin-resistant
clinical isolate strains (VRS2 and NRS52) which were
close to VAN in the control experiments. In detail, com-
pound 2e displayed an interesting antibacterial activity
against VRS2, a vancomycin fully resistant strain, show-
ing a MIC value three-fold smaller than the MIC value reg-
istered for the commercially available drug VAN (MIC val-
ues: 8 lg/mL vs. 64 lg/mL, respectively). Additionally,
compound 2e was active against NRS52, a vancomycin
intermediate resistant strain (VISA), having a MIC value
of 8 lg/mL comparable to the reference drug VAN.
To further investigate the antibacterial effects in sev-
eral clinical isolates, molecules 2e and 2l were tested
against two different methicillin-resistant bacteria
strains (MRSA) such as N4120032 and N4112910. As
shown in Table 2, both compound 2e and 2l possessed
interesting antimicrobial effects against N4112910 bacte-
ria strain in comparison to the control drugs NRF and
VAN. The biological data reported in Table 2 indicate that
compound 2e was more active against N4112910 rather
than N4120032 when compared to OXA, showing MIC
values of 8 lg/mL and 16 lg/mL, respectively. Despite this
relevant data, it is worthy to note that compound 2e
inhibited the growth of the clinical isolate N4120032
with a MIC value similar to the reference drug OXA. Tak-
ing into account that the last-mentioned bacteria strain
belongs to the HA-MRSA family characterized by high
morbidity and mortality degrees, data registered for com-
pound 2e appear to be very significant.
Moreover, compound 2e showed a remarkable antimi-
crobial profile against the multi-drug resistant strain
NRS100, an oxacillin/tetracycline-resistant strain, with a
MIC value of 16 lg/mL in comparison to 256 lg/mL of
OXA.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
Mercaptobenzothiazole Derivatives: Antibacterial Activity
609
Table 3. Antimicrobial activity results (MIC in lg/mL) on NorA
over-expressing strain with and without reserpine.
ATCC 25923
Res +
SA-1 (NorA over-expr.)
Compound Res -
Res -
Res +
NRF
2e
1
4
1
4
16
64
4
64
2l
16
16
128
128
Finally, the antibacterial activity exhibited by both
compound 2e and 2l against STA268, a potentially lethal
strain producing the Panton–Valentine Leukocidin
(PVL+) [13], seems to be negligible in comparison to NRF
and VAN.
Results obtained during our preliminary investigation
caused us to carry out additional tests using multidrug-
resistant bacteria SA-1, a modified S. aureus strain that
overexpresses the NorA, the most studied efflux pump at
the present time [14].
Vehicle (DMSO 0.1%) was used as control. Each experiment was performed in quad-
ruplicate; data are expressed as mean € S.E.M of three to five experiments. * p a
0.0001 vs. respective control.
Figure 1. Cytotoxicity of compounds 2e and 2l (100 lM) in
MRC-5 and HeLa cells.
for the synthesis of antibacterial compounds acting on
resistant bacterial strains overexpressing NorA efflux
pump.
Finally, taking into account the well-known antimicro-
bial and anticancer activities of benzothiazole deriv-
atives, structurally related to our 2-mercaptobenzothia-
zole compounds but, of course, with different chemical
features, we also investigated the cytotoxic activity of the
molecules with most meaningful antibacterial activity,
2e and 2l. This study was realized by using the MTS assay
against HeLa and MRC-5 cell lines, following a 3-days
exposure [18].
In general, the cancer cells were more sensitive to the
tested agents. As shown in Fig. 1, the derivatives 2e and 2l
tested at the single dose of 100 lM, did not produce a rel-
evant change in cell viability in MRC-5 cells (maximal
inhibition of cell growth never exceeded 25%), whereas
their cytotoxic effect in HeLa cells was remarkable (about
80% of inhibition).
Generally, the overexpression of multidrug-resistance
(MDR) efflux pumps confer clinically relevant resistance
to antibiotics (e.g. fluoroquinolones), dyes, detergents,
and disinfectants. In particular, the typical substrate pro-
file of NorA includes quinolones, chloramphenicol, and
several unrelated substances [15].
The literature clearly describes a detailed biological
assay used to evaluate the NorA involvement in the mech-
anism of antibacterial multidrug resistance, by using
reserpine as NorA blocker [16, 17]. In fact, this alkaloid
seems to be able to inhibit multidrug transporters like
NorA, increasing the intracellular concentration of fluo-
rochinolones, thus potentially lowering MICs.
In light of this evidence, we investigated the involve-
ment of the NorA efflux pump in the mechanism of
action of our best molecules 2e and 2l by using the bio-
logical assay reported before and detailed in the experi-
mental section, herein. The antimicrobial results exhib-
ited by compounds 2e and 2l against both wild-type and
SA-1 bacteria strains were compared with the control
drug NRF as reported in Table 3. By reading Table 3, it is
clear that the standard drug NRF produced a lower SA-1
bacteria growth in the presence, rather than in absence,
of reserpine (MIC values: 4 lg/mL vs. 16 lg/mL, respec-
tively) suggesting that the latter is a good substrate for
the NorA efflux pump. Herein, we also noticed that mole-
cules 2e and 2l exhibited comparable inhibition of bacte-
ria growth in absence so as in presence of reserpine indi-
cating that these compounds are not involved in this
active efflux system.
Data obtained confirm that our tested molecules can
be considered moderately toxic for HeLa cells while they
lack of any toxicity for normal cells like MRC-5.
Conclusion
In summary, in the present study, we report the synthesis
and the antimicrobial studies of 2-mercapto 1,3-benzo-
thiazole derivatives. It was observed that the synthesized
compounds substituted with a S-H moiety at the 2-posi-
tion of the heterocyclic nucleus (2a–2l) favored the anti-
bacterial activity especially against the Gram-positive
strains. On the contrary, compounds bearing the S-Bn
moiety 3a–3l at the 2-position of the benzothiazole
nucleus did not show any antimicrobial profile.
This interesting result suggests that both compounds
2e and 2l could be considered as candidates for new tools
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Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
iodine chamber. All chemicals, compounds 2g–2l included,
were purchased from Aldrich Chemical Co. (Sigma-Aldrich, Ger-
many) in the highest quality commercially available. The struc-
tures of the compounds were confirmed by routine spectromet-
ric and spectroscopic analyses. Only spectra for compounds not
previously described are given.
Among the series 2a–2l, the most prominent and con-
sistent antimicrobial activity was obtained with com-
pound 2e (MIC: 3.12 lg/mL) carrying a trifluoromethyl
moiety at the 6-position of the heterocycle. Compound 2l
showed an appreciable broad spectrum of action against
both Gram-positive and Gram-negative bacteria. Its MIC
value (25 lg/mL) toward E. coli is very significant.
Synthesis of 2-bromo-4-methoxyaniline 1d
Interesting, cytotoxicity against the MRC-5 cells was
not observed for compounds 2e and 2l. In light of the
results presented in this work and taking into account
that this preliminary study does not produce conclusive
evidence regarding a structure-antibacterial relation-
ship, we stopped our attention on the most promising
compounds 2e and 2l as an interesting starting point for
the development of a new class of antimicrobial agents.
Therefore, the synthesis of novel 2-mercaptobenzothia-
zole derivatives aimed to optimize the chemical features
involved in the antibacterial activity such as to investi-
gate the mechanism of action is currently going on our
laboratory.
Compound 1d was prepared according to the literature proce-
dure [11] starting from 4-methoxyaniline (commercially avail-
able). Column chromatography (silica gel, eluent: Et2O/petro-
leum ether, 4:6) of the reaction crude provided 2-bromo-4-
methoxyaniline 1d as purple oil (37% yield). 1H-NMR and MS
spectra were in agreement with those reported in the literature.
General procedure for the synthesis of 6-substituted-1,3-
benzothiazole-2-thiols 2a–2f
6-Substituted-1,3-benzothiazole-2-thiols 2a–2c, 2e, 2f were pre-
pared from the reaction of the corresponding aniline derivatives
1a–1c, 1e, 1f, commercially available, with potassium ethyl xan-
thate by using a literature procedure [10], while 2d was prepared
using aniline derivative 1d (see above).
6-(1-Methylethyl)-1,3-benzothiazole-2-thiol 2a
This work was accomplished thanks to the financial support of
the Ministero dell'Istruzione, dell'Universitꢀ e della Ricerca
(MIUR).
Yield: 88%; m.p.: 133–1358C (EtOAc); IR (CHCl3): 3092, 2911,
1
1496, 1257, 895 cm– 1; H-NMR was in agreement with the pub-
lished data [10]. MS (70 eV) m/z (%): 211 [M+ + 2] (8), 210 [M+ + 1]
(10), 209 [M+] (72), 194 (100), 161 (15). Anal. Calcd. for C10H11NS2:
C, 57.38; H, 5.30; N, 6.69; found: C, 57.17; H, 5.14; N, 6.71.
The authors have declared no conflict of interest.
6-Chloro-1,3-benzothiazole-2-thiol 2b
Yield: 83%; m.p.: >2458C (EtOAc/petroleum ether); IR (KBr): 3081,
2913, 1486, 1240, 874 cm– 1; 1H-NMR (300 MHz, DMSO-d6) d: 13.9
(br s, 1H), 7.81 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.8 Hz,
1H); MS (70 eV) m/z (%): 203 [M+ + 2] (47), 202 [M+ + 1] (12), 201 [M+]
(100), 166 (35). Anal. calcd. for C7H4ClNOS2: C, 41.69; H, 2.00; N,
6.94; found: C, 41.38; H, 2.15; N, 6.92.
Experimental
Chemistry
Melting points were recorded on Gallenkamp melting point
apparatus (Weiss-Gallenkamp, London, UK) in open glass capil-
lary tubes. The IR spectra were recorded on a Perkin-Elmer Spec-
trum One FT spectrophotometer (Perkin-Elmer, Norwalk, CT,
USA) and band positions are given in reciprocal centimeters
(cm– 1). 1H-NMR spectra were recorded on a FT Bruker Aspect
3000 spectrometer (Bruker Bioscience, USA) using CDCl3 as the
solvent, unless otherwise indicated. Chemical shifts are reported
in part per million (ppm) relative to the solvent resonance:
CDCl3, d = 7.26 (1H-NMR). Amino-proton assignments were con-
firmed by D2O exchange. J values are given in Hz. EIMS spectra
were recorded with a Hewlett-Packard 6890-5973 MSD gas chro-
matograph / mass spectrometer at low resolution (Hewlett-Pack-
ard, Palo Alto, CA, USA). Elemental analyses were performed on
a Eurovector Euro EA 3000 elemental analyzer (EuroVector,
Milan, Italy). The data for C, H, and N were within € 0.4 of the
theoretical values for all final compounds. Silica gel chromato-
graphic separations were performed by chromatography with
silica gel (Kieselgel 60, 40–63 lm; Merck, Germany) packed in
glass columns, using the technique described in the literature
[19]. The weight of the silica gel was approximately 100-times
that of the substance. The eluting solvent indicated in parenthe-
ses for each purification was determined by TLC performed on
6-Methyl-1,3-benzothiazole-2-thiol 2c
Yield: 77%; m.p.: 180–1818C (EtOAc/petroleum ether); IR (CHCl3):
3094, 2914, 1494, 1253, 893 cm– 1;1H-NMR and MS were in agree-
ment with the published data [10]. Anal. calcd. for C8H7NS2 N 0.25
H2O: C, 51.72; H, 4.07; N, 7.54; found: C, 51.76; H, 3.73; N, 7.48.
6-Methoxy-1,3-benzothiazole-2-thiol 2d
Yield: 90%; m.p.: 206–2078C (EtOAc/petroleum ether); IR (KBr):
3036, 2936, 1495, 1296, 893 cm– 1; 1H-NMR (300 MHz) d 13.2–
13.9 (br s, 1H), 7.26 (s, 1H), 7.02–6.97 (m, 2H), 3.82 (s, 3H); MS (70
eV) m/z (%): 199 [M+ + 2] (12), 198 [M+ + 1] (13), 197 [M+] (100), 182
(78), 154 (17). Anal. calcd. for C8H7NOS2 N 0.12 H2O: C, 48.16; H,
3.66; N, 7.02; found: C, 48.02; H, 3.35; N, 6.85.
6-Trifluoromethyl-1,3-benzothiazole-2-thiol 2e
Yield: 99%; m.p.: A2408C (EtOAc/petroleum ether); IR (KBr): 3106,
2931, 1487, 1262, 882 cm– 1; 1H-NMR was in agreement with the
published data [20]. MS (70 eV) m/z (%): 237 [M+ + 2] (12), 236 [M+ +
1] (13), 235 [M+] (100), 157 (10). Anal. calcd. for C8H4 F3NS2: C,
40.84; H, 1.71; N, 5.95; found: C, 41.08; H, 1.71; N, 5.86.
precoated silica gel on aluminum sheets (Kieselgel 60, F254
Merck). TLC plates were visualized with UV light and/or in an
,
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Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
Mercaptobenzothiazole Derivatives: Antibacterial Activity
611
6-Fluoro-1,3-benzothiazole-2-thiol 2f
2-(Benzylsulphanyl)-6-trifluoromethyl-1,3-benzothiazole
Yield: 98%; m.p.: 227–2288C (EtOAc/petroleum ether); IR (KBr):
3442, 2938, 1946, 1287, 911 cm– 1; 1H-NMR (300 MHz) d: 10.5 (br
s, 1H), 7.21–7.03 (m, 3H).; MS (70 eV) m/z (%): 187 [M+ + 2] (1), 186
[M+ + 1] (4), 185 [M+] (100), 153 (27), 126 (16). Anal. calcd. for
C7H4FNS2 N 0.75 H2O: C, 42.30; H, 2.79; N, 7.05; found: C, 42.19; H,
2.38; N, 6.70.
3e
Yield: 98%; m.p.: 102-1048C (EtOAc); IR (KBr): 2925, 1566, 1437,
1308, 1235, 993 cm– 1; H-NMR (300 MHz) d: 8.15–7.92 (m, 2H),
1
7.65 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 7.65 Hz, 2H), 7.38–7.22 (m,
3H), 4.62 (s, 2H); MS (70 eV) m/z (%): 327 [M+ + 2] (6), 326 [M+ + 1]
(10), 325 [M+] (52), 292 (19), 91 (100). Anal. calcd. for
C15H10F3NS2: C, 55.37; H, 3.10; N, 4.30; found: C, 55.40; H, 3.09;
N, 4.32.
General procedure for the synthesis of 2-(benzylthio)-6-
substituted-1,3-benzothiazoles 3a–3l
The preparation of 2-(benzylthio)-6-isopropyl-1,3-benzothiazole
3a can be taken as the reference for the synthesis of 2-(ben-
zylthio)-6-substituted-1,3-benzothiazoles 3a–3l. 6-Isopropyl-1,3-
benzothiazole-2-thiol 2a (1.91 mmol) in dioxane (16 mL), was
added to a solution of K2CO3 (2.87 mmol) in water (5 mL). The
reaction mixture was stirred at 758C. Then, a solution of benzyl
bromide (2.1 mmol) in dioxane (16 mL) was added dropwise over
15 min. When the addition was completed, the resulting mix-
ture was stirred for another 15 min. The dioxane was evaporated
under reduced pressure and the residue was extracted with
EtOAc and washed with 2 N NaOH (3620 mL) up to pH 11. The
organic phase was dried over Na2SO4, filtered, and evaporated to
dryness. Column chromatography (silica gel, eluent: EtOAc/
petroleum ether, 3:7) of the reaction crude afforded the desired
compound 3a.
2-(Benzylsulphanyl)-6-fluoromethyl-1,3-benzothiazole 3f
Yield: 71%; m.p.: 69–708C (EtOAc); IR (KBr): 2923, 1565, 1437,
1307, 1238, 991 cm– 1; 1H-NMR (300 MHz) d: 7.86–7.79 (m, 1H),
7.47–7.41 (m, 3H), 7.38–7.27 (m, 3H), 7.18–7.11 (m, 1H), 4.59 (s,
2H); MS (70 eV) m/z (%): 277 [M+ + 2] (7), 276 [M+ + 1] (10), 275 [M+]
(61), 242 (33), 91 (100). Anal. calcd. for C14H10FNS2: C, 61.37; H,
3.66; N, 5.09; found: C, 61.45; H, 3.87; N, 4.91.
2-(Benzylsulphanyl)-benzothiazole 3g
Purified by chromatography (acetone/petroleum ether, 3:7).
Yield: 91%; m.p.: 46–478C (EtOAc/petroleum ether) (lit. 38–408C,
[21]) IR, 1H-NMR, and MS were in agreement with the published
data [21]. Anal. calcd. for C14H11NS2: C, 65.34; H, 4.31; N, 5.44;
found: C, 65.24; H, 4.40; N, 5.44.
2-(Benzylsulphanyl)-6-(1-methylethyl)-1,3-benzothiazole
3a
2-(Benzylsulphanyl)-6-amino-1,3-benzothiazole 3h
Yield: 69%; MS (70 eV) m/z (%): 274 [M+ + 2] (7), 273 [M+ + 1] (13), 272
[M+] (70), 239 (100), 181 (47), 91 (83). The biological data were car-
ried out on 3h N HCl. Yield: 74%; m.p.: 219–2218C; IR (KBr): 2850,
1494, 1275 cm– 1; 1H-NMR (300 MHz, DMSO-d6) d: 10.6 (br s, 1H),
8.21–7.90 (m, 2H), 7.55–7.21 (m, 6H), 5.60 (br s, 2H, exchange-
able with D2O), 4.61 (s, 2H). Anal. calcd. for C14H12NO2S2 N 0.16
HCl: C, 53.40; H, 4.21; N, 8.90; found: C, 53.74; H, 3.81; N, 8.51.
Yield: 98%; m.p.: 80–818C (EtOAc); IR (KBr): 3058, 2950, 1438,
1237, 830 cm– 1; 1H-NMR (300 MHz, DMSO-d6) d: 7.85 (s, 1H), 7.76
(d, J = 8.5 Hz, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.38–7.25 (m, 4H), 4.61
(s, 2H), 2.92 (m, J = 7.2 Hz, 1H), 1.21 (d, J = 7.2 Hz, 6H); MS (70 eV)
m/z (%): 301 [M+ + 2] (11), 300 [M+ + 1] (21), 299 [M+] (100), 267 (17),
266 (85), 91 (63.4). Anal. calcd. for C17H17NS2: C, 68.19; H, 5.72; N,
4.68; found: C, 68.47; H, 5.84; N, 4.62.
2-(Benzylsulphanyl)-6-ethoxy-1,3-benzothiazole 3i
Yield: 96%; m.p.: 63–648C (EtOAc); IR (KBr): 2929, 1443, 998 cm–
1; 1H-NMR (300 MHz, CDCl3) d: 7.78 (d, J = 9.05 Hz, 1H), 7.41–7.40
2-(Benzylsulphanyl)-6-chloro-1,3-benzothiazole 3b
Purified by chromatography (EtOAc/petroleum ether, 3:7). Yield:
92%; m.p.: 80–828C (EtOAc/petroleum ether); IR (KBr): 3068,
2922, 1431, 1297, 861 cm– 1; 1H-NMR (300 MHz) d: 7.78 (d, J = 8.5
Hz, 1H), 7.72 (s, 1H), (m, 6H), 4.59 (s, 2H); MS (70 eV) m/z (%): 293
[M+ + 2] (26), 292 [M+ + 1] (11), 291 [M+] (62), 258 (38), 91 (100). Anal.
calcd. for C14H10ClNS2: C, 57.62; H, 3.45; N, 4.80; found: C, 57.86;
H, 3.51; N, 4.83.
(m, 2H), 7.36–7.25 (m, 3H), 7.20 (d, J = 2.47 Hz, 1H), 7.02 (dd, J1-2
=
3.22 Hz, J1-3 = 9.07 Hz, 1H), 4.56 (s, 2H), 4.06 (q, J = 6.8 Hz, 2H), 1.44
(t, J = 6.8 Hz, 3H); MS (70 eV) m/z (%): 303 [M+ + 2] (13), 302 [M+ + 1]
(22), 301 [M+] (100), 268 (97), 240 (18), 210 (69), 182 (22), 91 (80).
Anal. calcd. for C16H15NOS2: C, 63.76; H, 5.02; N, 4.65; found: C,
63.94; H, 4.83; N, 4.93.
2-(Benzylsulphanyl)-6-methyl-1,3-benzothiazole 3c
2-(Benzylsulphanyl)-6-nitro-1,3-benzothiazole 3l
Yield: 75%; m.p.: 54–558C (EtOAc); IR (KBr): 3053, 2915, 1436,
1
Yield: 77%; m.p.: 116.2–117.48C (EtOAc); IR (KBr): 3072, 1494,
1240, 873 cm– 1; H-NMR (300 MHz, DMSO-d6) d: 7.79–7.71 (m,
1
1265, 892 cm– 1; H-NMR (300 MHz) d: 8.67 (d, J = 2.20 Hz, 1H),
2H), 7.47 (d, J = 5.9 Hz, 2H), 7.36–7.23 (m, 4H), 4.61 (s, 2H), 2.39 (s,
3H); MS (70 eV) m/z (%): 273 [M+ + 2] (11), 272 [M+ + 1] (19), 271 [M+]
(100), 238 (82), 91 (85.1). Anal. calcd. forC15H10NS2: C, 66.38; H,
4.83; N, 5.16; found: C, 66.56; H, 4.87; N, 5.16.
8.31 (dd, J1-2 = 2.20 Hz, J1-3 = 9.07 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H),
7.59–7.43 (m, 2H), 7.39–7.30 (m, 3H), 4.65 (s, 2H); MS (70 eV) m/z
(%): 304 [M+ + 2] (9), 303 [M+ + 1] (14), 302 [M+] (73), 91 (100), 65 (13).
Anal. calcd. for C14H10NO2S2: C, 55.61; H, 3.33; N, 9.26; found: C,
56.00; H, 3.34; N, 9.21.
2-(Benzylsulphanyl)-6-methoxy-1,3-benzothiazole 3d
Yield: 76%; m.p.: 93–958C (EtOAc); IR (KBr): 3444, 2922, 2851,
Biology
1
1702, 1641, 1600, 1435, 1059, 999 cm– 1; H-NMR (300 MHz) d:
Test organisms
7.80 (d, J = 9.07 Hz, 1H), 7.46–7.42 (m, 1H), 7.36-7.21 (m, 5H), 7.03
(dd, J1-2 = 2.74 Hz, J1 – 3 = 8.35 Hz, 1H), 4.59 (s, 2H), 3.89 (s, 3H); MS
(70 eV) m/z (%): 289 [M+ + 2] (11), 288 [M+ + 1] (19), 287 [M+] (90), 254
(100), 91 (73.3). Anal. calcd. for C15H13NOS2: C, 62.69; H, 4.56; N,
4.87; found: C, 63.01; H, 4.72; N, 4.74.
Twelve bacteria strains belonging to the ATCC collection were
used: Gram-positive such as Staphylococcus aureus ATCC 6538P,
ATCC 25923, and ATCC 29213, Enterococcus faecalis ATCC 19433
and ATCC 29212, Bacillus subtilis ATCC 6633, Bacillus cereus
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C. Franchini et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 605–613
ATCC 11778, and Gram-negative Escherichia coli ATCC 8739 and
ATCC 35218, Acinetobacter baumannii ATCC 19606, Klebsiella
pneumoniae ATCC 13883 and Pseudomonas aeruginosa ATCC
27853.
mL. The MIC were determined by using an antibacterial assay
repeated twice in triplicates.
Cytotoxicity assay
We also used clinical isolates of S. aureus belonging both to
NARSA (Network on Antimicrobial Resistance in Staphylococcus
aureus) collection (NRS52 and VRS2 with glycopeptide inter-
mediate and fully resistant profile, respectively) and Prof. Glupc-
zynski's private collection with different antimicrobial resist-
ance profiles such as N4112910 a methicillin-resistant S. aureus
(MRSA) and N4120032 a healthcare-associated methicillin-resist-
ant S. aureus (HA-MRSA). Moreover, we used S. aureus STA268
such as Panton–Valentine Leukocidin (PVL+) [13] isolated by Y. C.
Huang in the Chang Gung Children's Hospital of Taiwan and S.
aureus SA-1, a NorA overexpressing strain, selected from S. aureus
ATCC 25923 [14].
The tested compounds were assayed against cell lines HeLa
(human epithelial cervical cancer cells) and MRC-5 (normal
human lung fibroblasts) for their cytotoxic effect, by using the
MTS cytotoxicity assay a variant of the widely used MTT assay
[18].
Cell culture
HeLa and MRC-5 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum (Gibco, Grand Island, NY, USA), 50 units/mL of penicillin
G and 0.05 mg/mL streptomycin (Sigma–Aldrich), 2 mM gluta-
mine (Sigma–Aldrich), 0.25 lg/mL amphotericin B (Sigma–
Aldrich) in a humidified atmosphere consisting of 5% CO2 and
95% air. HeLa cells were subcultured three times weekly to main-
tain continuous logarithmic growth. MRC-5 cells were grown to
confluence without subculturing; the medium was renewed
twice a week. Confluent cells were harvested after trypsinization
using Trypsin-EDTA solution 1X (Sigma–Aldrich).
Antimicrobial assay
The in-vitro Minimal Inhibitory Concentrations (MICs, lg/mL) of
the prepared compounds were determined by the broth micro-
dilution method according to the National Committee for Clini-
cal Laboratory Standards (NCCLS, 2003).
Drug dilutions
Agent treatment
For antibacterial assays the microdilution method, encoded by
CLSI, formerly NCCLS, using 96-wells plates (Microtiter1), was
used [12]. Stock solutions of the tested compounds 2a–2l, 3a–3l,
were obtained in absolute ethanol at a concentration of 800 lg/
mL. Stock solutions of lower concentrations were prepared for
those substances which did not dissolve well. Further two-fold
serial dilutions in the test medium between 100 and 0.78 lg/mL
were plated. In each well, 200 lL of these solutions was added.
To be sure that the solvent had no an adverse effect on bacterial
growth, a control test was carried out by using ethanol at its
maximum concentration along with the medium. Norfloxacin
(NRF) was used as the standard drug.
To evaluate the antimicrobial profile towards the clinical iso-
lates, the microdilution method [12] was used. Stock solutions of
the tested compounds 2e and 2l, were obtained in absolute etha-
nol at a concentration of 1024 lg/mL. NRF, VAN, and OXA were
used as standard drugs. The antibacterial activity against SA-1
strain was registered in the presence as well as in absence of
reserpine added at 10 lg/mL following the experimental proce-
dure suggested by Brenwald et al. [16].
The MTS cytotoxicity assay (Cell Titer 96m Aq One Solution Cell
Proliferation Assay, Promega), was used to screen the viability of
the cells incubated with the test compounds. This assay meas-
ures cell viability and is based on the bioreduction of MTS tetra-
zolium into formazan by NADH and NADPH produced by dehy-
drogenase enzymes only in metabolically active, viable cells.
Compounds were prepared as 100 mM top stock solutions, dis-
solved in DMSO, and stored at 48C. For each cytotoxicity assay
with HeLa cells, cells were seeded into 96-well microtiter plates
at a density of 200 cells per well and allowed 24 h to adhere
before drugs were introduced (final concentration 100 lM). For
the cytotoxicity assay with MRC-5, 600 cells per well were seeded
in 96-well microplates and allowed 24 h to adhere before drug
addition (final concentration 100 lM). Compounds that exhib-
ited activity in single-dose testing were further evaluated to gen-
erate dose-response curves, which were used to determine CC50
(cytotoxic concentration able to destroy 50% of the initial cell
amount). To perform dose-response curves, serial drug dilutions
(final concentration 1 to 100 lM) were prepared in medium
immediately prior to each assay. Cells were incubated for 72 h at
378C in a humidified atmosphere and 5% CO2.
The reagent (16% total well volume) was added to each well.
Plates were incubated at 378C until sufficient colour develop-
ment had occurred (usually 3–4 h). The purple formazan prod-
uct was then measured spectrophotometrically at 490 nm. The
optical density (O.D.) value of each culture is a function of the
amount of formazan produced and is proportional to the num-
ber of viable cells [18].
Inoculum preparation
Cultures were grown on Petri dishes with Mꢀller–Hinton agar
(Merck, Darmstadt, Germany) for 24 h at 37 l 18C. A number of
colonies were drawn using a sterile metal loop. They were then
dissolved in MHB (Mꢀller–Hinton broth) and were incubated for
approximately 3 to 4 h. The absorbance of these cellular suspen-
sions calibrated at a wavelength of 625 nm using spectrophoto-
metric method (Thermo Spectronic, Genesis 20), should be 0.08
to 0.10 for the 0.5 McFarland standard, corresponding approxi-
mately to 108 CFU (Colony Forming Unit)/mL. The inocula were
diluted to a ratio of 1:40 (100 lL of inocula in 3.9 mL of MHB) and
20 lL of this dilution were pipetted into each well. The final
inoculum value was 2.56106 CFU/mL. A number of wells con-
taining only inoculated broth as control growth were prepared.
After incubation for 24 h at 37 l 18C, the last well containing no
microbial growth was recorded to show the MIC, in values of lg/
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