An efficient one-pot synthesis of thiochromeno[3,4-d]pyrimidines derivatives: Inducing ROS dependent antibacterial and anti-biofilm activities

Article abstract of DOI:10.1016/j.bioorg.2016.08.006

An efficient synthesis of thiochromeno[3,4-d]pyrimidine derivatives has been achieved successfully via a one-pot three-component reaction of thiochrome-4-one, aromatic aldehyde and thiourea in the presence of 1-butyl-3-methyl imidazolium hydrogen sulphate

Full text of DOI:10.1016/j.bioorg.2016.08.006

Bioorganic Chemistry 68 (2016) 159–165
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
An efficient one-pot synthesis of thiochromeno[3,4-d]pyrimidines
derivatives: Inducing ROS dependent antibacterial and anti-biofilm
activities
a,
Lingala Suresh a, P. Sagar Vijay Kumar a, Y. Poornachandra b, C. Ganesh Kumar b, , G.V.P. Chandramouli
⇑
⇑
a Department of Chemistry, National Institute of Technology, Warangal 506 004, Telangana, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500 007, Telangana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of thiochromeno[3,4-d]pyrimidine derivatives has been achieved successfully via a
one-pot three-component reaction of thiochrome-4-one, aromatic aldehyde and thiourea in the presence
of 1-butyl-3-methyl imidazolium hydrogen sulphate [Bmim]HSO4. This new protocol has the advantages
of environmental friendliness, high yields, short reaction times, and convenient operation. Furthermore,
among all the tested derivatives, compounds 4b and 4c exhibited promising antibacterial, minimum bac-
tericidal concentration and anti-biofilm activities against Staphylococcus aureus MTCC 96, Staphylococcus
aureus MLS16 MTCC 2940 and Bacillus subtilis MTCC 121. The compound 4c also showed promising
intracellular ROS accumulation in Staphylococcus aureus MLS16 MTCC 2940 comparable to that of
ciprofloxacin resulting in apoptotic cell death of the bacterium.
Received 10 March 2016
Revised 4 August 2016
Accepted 8 August 2016
Available online 9 August 2016
Keywords:
Ionic liquid
Thiochromeno[3,4-d]pyrimidine
Antibacterial activity
Anti-biofilm
Ó 2016 Elsevier Inc. All rights reserved.
Reactive Oxygen Species (ROS)
1. Introduction
In the present circumstances, the pyrimidine derivatives play a
prominent role in organic and medicinal chemistry [6,7]. They
In the recent years, the treatment of bacterial infections has
become a major challenge in the realm of conventional antibiotic
therapy. In parallel, the disease causing microbes have gained
resistance to several antibiotics and are causing serious health care
problems [1]. It is reported that a high percentage of hospital-
acquired infections are caused by highly resistant bacteria such
as methicillin-resistant Staphylococcus aureus (MRSA), which is a
major nosocomial pathogen [2]. Thus, antimicrobial resistance
has gained serious attention as a global health threat [3]. According
to the World Health Organization (WHO), the infections caused by
resistant microorganisms often fail to respond to conventional
antibiotic therapy, resulting in prolonged illness and greater risk
of death. The primary reason is the inappropriate and irrational
use of currently available antimicrobial agents which has led to
the emergence of highly resistant pathogenic microbes [4]. The
increased threat from the drug-resistant Gram-positive and -
negative bacterial strains has emphasized the urgent need and
perusal to identify new antimicrobial agents with high efficiency,
low toxicity, broad spectrum and safety [5].
exist in a wide variety of natural products and in rationally
designed pharmaceutical agents [8,9]. Pyrimidines have become
integral units to a large number of drug substances with activities
including antimicrobial [10], antioxidants [11], anticonvulsant
[12], antidepressant [13] anticancer [14], and anti-biofilm [15]. In
view of the diverse pharmacological properties of these com-
pounds some of the well-known biologically potent thiochromen
pyrimidine scaffolds (I, II and III) [16–18] are shown in Fig. 1.
In the context of sustainable chemistry [19–21], ionic liquids
(ILs) have gained considerable attention in several branches of
the chemical industry as potential ‘‘green” substitutes for conven-
tional organic solvents [22,23]. The green aspect of ILs is mainly
due to non-flammability and reduced air pollution [24–26]. The
introduction of structural functionalities on the cationic or anionic
part has made it possible to design new ILs with targeted proper-
ties [27]. In the recent years, ionic liquids (ILs) especially the acidic
types have gained a renewed interest in the area of heterocyclic
synthesis [28,29]. Nowadays, ILs is being used in multicomponent
reactions (MCRs) and their properties have also been investigated
in organic synthesis [30–33]. Hammam et al. [34] have described
the reported methods of few compounds which suffer from
drawbacks such as harsh reaction conditions, unsatisfactory yields,
prolonged reaction times, and cumbersome product isolation
procedures. Hence, our goals to use a green, one-pot synthetic
⇑
Corresponding authors.
E-mail addresses: cgkumar5@gmail.com (C. Ganesh Kumar), gvpc2000@gmail.
com (G.V.P. Chandramouli).
http://dx.doi.org/10.1016/j.bioorg.2016.08.006
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

160
L. Suresh et al. / Bioorganic Chemistry 68 (2016) 159–165
NH2
O
r
A
r
A
O
O
S
N
N
N
S
R
N
NH2
HN
N
Ar
N
NH
R
S
N
N
N
H
Ar
S
R
Ar
I
II
III
IV
Fig. 1. Biologically potent thiochromen pyrimidine scaffolds.
approach of the above title compounds which are a welcome goal
due to the wide spectrum of biological activities.
not give the require products even after 24 h (Table 1 entry 1).
Later the reaction was carried out with organic solvents such as
MeOH, EtOH, MeCN and DMF which resulted in poor yields
(Table 1, entries 2–6). It was found that when acid was applied,
the acidic mixture of all the reactants under reflux conditions gave
4a in 35% yields (Table 1, entry 6). While, studying the scope and
efficiency of the reaction with different ILs in this MCR, it was
observed that almost all of the investigated ILs such as [Bmim]
BF4, [Bmim]Br, [Bmim]PF6 and [Bmim]HSO4 were capable of
promoting the synthesis of desired compound 4a. Good results
were achieved by carrying out the reaction using [Bmim]HSO4
ionic liquid and the derived product was obtained in good isolated
yields when compared to the results obtained using ionic liquid
analogous tetrafluoroborate, bromide and hexafluorophosphate
(Table 1, entries 7–9). The yield of product 4a was improved and
the reaction time was shortened as the temperature was increased
from room temperature to 70 °C (Table 1, entries 10–14). No
further improvement was observed at 80 °C (Table 1, entry 15).
Therefore, a temperature of 70 °C was considered as the most
suitable optimal reaction temperature for performing all these
reactions. It was inferred from the above results that the ionic
liquid medium is an essential and crucial factor for promoting
the reaction.
As a part of our ongoing research in the area of MCRs and ILs,
targeted towards the synthesis of novel heterocyclic compounds
[35–38], we herein report a three-component domino processes
for the synthesis of thiochromeno[3,4-d]pyrimidine derivatives
and were further screened for antibacterial, minimum bactericidal
concentration and anti-biofilm activities.
2. Results and discussion
2.1. Chemistry
Highly volatile and toxic organic solvents were replaced by a
green medium namely [Bmim]HSO4 in the present research
program. Thiochromeno[3,4-d]pyrimidine derivatives (4a-n) were
obtained in good yields. The conditions were optimized for the
designed protocol based on the reaction of thiochroman-4-one 1
(1 mmol), aromatic aldehyde 2a-n (1 mmol) and thiourea
3
(1 mmol) in the presence of [Bmim]HSO4. Initially, a one-pot three
component reaction with thiochroman-4-one 1, benzaldehyde 2a
and thiourea 3 was taken up as a model reaction to optimize the
reaction conditions and the results are summarized in Table 1.
When the reaction was carried out under neat conditions it did
To demonstrate the generality of this method, the reaction was
investigated under optimized conditions, and the results are
Table 1
Optimization of reaction parameters for the synthesis of compound 4a.
Entrya
Solvent
Temp (°C)
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
Neat
MeOH
EtOH
CH3CN
DMF
AcOH
[Bmim]BF4
[Bmim]Br
[Bmim]PF6
[Bmim]HSO4
[Bmim]HSO4
[Bmim]HSO4
[Bmim]HSO4
[Bmim]HSO4
[Bmim]HSO4
70
70
70
70
70
70
70
70
70
70
rt
24
12
12
10
14
8
2
2
3
1
NR
08
12
06
0
35
70
68
52
92
38
48
62
84
92
9
10
11
12
13
14
15
3
3
3
2
40
50
60
80
1
a
Reaction conditions: thiochroman-4-one 1 (1 mmol), benzaldehyde 2a (1 mmol) and thiourea 3 (1 mmol) and solvent (2 mL).
Yields of the isolated products.
b

L. Suresh et al. / Bioorganic Chemistry 68 (2016) 159–165
161
Table 2
Synthesis of thiochromeno[3,4-d]pyrimidine derivatives (4a-n).a
Entry
R
Product
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
C6H5
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
1
1
1
1
1.5
1
1
1.5
1.5
1.5
2
92
90
92
92
88
90
86
84
86
90
84
84
84
84
4-Me-C6H4
4-OMe-C6H4
4-Cl-C6H4
4-F-C6H4
4-NO2-C6H4
3-NO2-C6H4
2-Cl-C6H4
C10H7
6-OMe-C10H6
Pyrazole
9
10
11
12
13
14
4-NO2-Pyrazole
4-Cl-Pyrazole
4-OCH3-Pyrazole
2
2
2
a
Reaction conditions: thiochroman-4-one 1 (1 mmol), aromatic aldehydes 2a-n (1 mmol) and thiourea 3 (1 mmol) and [Bmim]HSO4 (2 mL).
Yields of the isolated products.
b
Scheme 1. Proposed mechanism for the formation of compound 4a.

162
L. Suresh et al. / Bioorganic Chemistry 68 (2016) 159–165
Table 3
Antibacterial activity of the synthesized thiochromeno[3,4-d]pyrimidines (4a-n).
Test
Minimum inhibitory concentration (
lg/mL)
compound
Staphylococcus
aureus
MTCC 96
Bacillus
subtilis
MTCC 121
Staphylococcus aureus
MLS16
MTCC 2940
Micrococcus
luteus
MTCC 2470
Klebsiella
planticola
MTCC 530
Escherichia
coli
MTCC 739
Pseudomonas
aeruginosa
MTCC 2453
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
125
7.8
3.9
125
15.6
7.8
125
7.8
3.9
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
25
125
125
125
>125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
Ciprofloxacin
0.9
0.9
0.9
0.9
0.9
0.9
0.9
Note: Compounds (4a, 4d-4n) showed MIC values of 125 lg/mL.
illustrated in Table 2. It was observed that a wide range of aromatic
aldehydes (2a-n) bearing either electron-donating or electron-
withdrawing groups could be employed as coupling partners with
thiochroman-4-one 1 and thiourea 3 and they were smoothly
transformed to the corresponding thiochromeno[3,4-d]pyrimidi-
nes (4a-n) in good to moderate yields. All para, meta, and ortho-
substituted aldehydes were easily converted into the desired prod-
ucts, indicating that steric bulk did not have any significant impact
on the reactivity. Aldehydes bearing pyrazole group furnished the
corresponding product in good yields. The tolerance of functional
group such as chloro, fluoro, nitro, methyl, methoxy in this method
provides an opportunity for further chemical transformation. Sev-
eral pharmaceutically relevant scaffolds could be easily generated
with the help of this green protocol. Although the proposed mech-
anism of this reaction remains to be fully clarified, the formation of
compound 4 is explained by the reaction sequence in Scheme 1. In
the first step, a hydrogen bond formation between the hydrogen
atom of [Bmim]HSO4 and carbonyl group of benzaldehyde 2 pro-
duced a complex which upon condensation with thiochroman-4-
concentration (MBC), anti-biofilm and accumulation of intracellu-
lar ROS in Staphylococcus aureus MLS-16 MTCC 2940 biofilms.
2.2.1. Antibacterial activity
Compounds 4a-n were screened for antibacterial activity [39]
in vitro against different Gram-positive and Gram-negative bacte-
rial strains such as Micrococcus luteus MTCC 2470, Staphylococcus
aureus MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940, Bacillus
subtilis MTCC 121, Escherichia coli MTCC 739, Pseudomonas aerugi-
nosa MTCC 2453 and Klebsiella planticola MTCC 530. Among all
the derivatives screened, compound 4c showed promising activity
(MIC value of 3.9
and Staphylococcus aureus MLS16 MTCC 2940 and 7.8
against Bacillus subtilis MTCC 121; while compound 4b exhibited
promising activity (MIC value 7.8 g/mL) specifically towards
Staphylococcus aureus MTCC 96 and Staphylococcus aureus MLS16
MTCC 2940 and MIC value of 15.6 g/mL was observed against
Bacillus subtilis MTCC 121. However, all the other tested com-
pounds (4a and 4d-4n) showed MIC values of >125 g/mL against
lg/mL against Staphylococcus aureus MTCC 96
lg/mL
l
l
one 1 gave the
a,b-unsaturated intermediate (A). The formation
l
of intermediate (B) took place by a condensation between
a,b-
all the tested bacterial strains. Based on the structure-activity
relationship of the synthesized derivatives, it was observed that
the compound 4c has a methoxy substituent attached to the basic
thiochromeno[3,4-d]pyrimidine scaffold, which has an electron
donating property which probably may be contributing to the
antibacterial activity. In case of compound 4b, a methyl substituent
is attached to the basic thiochromeno[3,4-d]pyrimidine scaffold,
having electron donating property is probably contributing to the
antibacterial activity. The antibacterial activity results to this
regard are tabulated in Table 3.
unsaturated intermediate A and thiourea 3. Subsequently, inter-
mediate B underwent intramolecular cyclization to form another
intermediate (C). In this last step, the intermediate C underwent
dehydration affording the final product 4.
Ionic liquid recyclability is one of the most necessary features
and thus makes it useful for commercial applications. Conse-
quently, we studied the recyclability of the [Bmim]HSO4 ionic liq-
uid in the above model reaction. After completion of the reaction,
the mixture was transferred into ice cold water and stirred
thoroughly. The solid product thus obtained was isolated by filtra-
tion, and the filtrate having ionic liquid was extracted with ethyl
acetate (2 ꢀ 20 mL) to eliminate the non-ionic organic impurities.
Then the water was evaporated under reduced pressure and the
recovered ionic liquid was dried under vacuum and recycled for
four times in consequent reactions without evident changes in
the product yields.
Table 4
Minimum Bactericidal Concentration (MBC) of the synthesized thiochromeno[3,4-d]
pyrimidines (4a-n).
Test compounds
Minimum bactericidal concentration (lg/ml)
Staphylococcus
aureus
Bacillus
subtilis
Staphylococcus aureus
MLS16
MTCC 96
MTCC 121 MTCC 2940
4b
4c
15.6
3.9
31.2
15.6
15.6
7.8
2.2. Biological evaluation
Ciprofloxacin
(Standard control)
1.9
0.9
0.9
All the synthesized compounds were evaluated for various
biological activities such as antibacterial, minimum bactericidal

L. Suresh et al. / Bioorganic Chemistry 68 (2016) 159–165
163
Table 5
formation and probably may be contributing to the anti-biofilm
activity. The activity data to this regard is shown in Table 5.
In the present study, the biofilm formation in different bacterial
strains including Staphylococcus aureus MTCC 96, Staphylococcus
aureus MLS16 MTCC 2940 and Bacillus subtilis MTCC 121 was
examined in a 96 well microtitre plate. It was noticed that these
strains formed biofilms at either the bottom of the well or at the
air-liquid interface [44]. For performing the assay, the strains
needs to be cultured to attain the required biomass concentration
(1.5 ꢀ 106 cfu/mL) after 24 h incubation and when the test com-
pounds were added at the minimum inhibitory concentration
(MIC) they exhibited inhibitory effect on the biofilm formation
and also showed drastic reduction in the bacterial cell growth,
which was quantified based on the crystal violet assay [43]. From
a mechanistic perspective, the test compounds caused the disrup-
tion and detachment of the biofilm from the surface due to the
destabilization of the EPS, which results in the dispersal of the bac-
terial cells from the biofilm. The dispersed cells were more suscep-
tible to these test compounds resulting in microbial cell death
which was quantified as a decrease in the microbial population
as compared to the untreated biofilms. However, the addition of
test compound below the sub-inhibitory concentration (that is less
than MIC value), did not exhibit much reduction in the bacterial
cell growth as compared to the untreated cells. The results on
the cell biomass and cell growth measurements are depicted in
Table 6 and Fig. 2, respectively. Some of the published reports
illustrate the potential of few small molecule scaffolds such as
halogenated furanones [45], sponge-derived natural alkaloid
Biofilm inhibition assay of the synthesized compounds (4b-c).
Test compounds
IC50 values in (
l
g/mL)
Bacillus
subtilis
MTCC 121
Staphylococcus
aureus
MTCC 96
Staphylococcus
aureus
MLS16 MTCC 2940
4b
4c
8.8 0.32
2.1 0.44
10.5 0.24
8.1 0.29
11.3 0.18
4.5 0.22
Ciprofloxacin
(Standard control)
0.6 0.06
0.5 0.09
0.4 0.07
2.2.1.1. Minimum bactericidal concentration (MBC). Based on the
antibacterial activity results, the compounds 4b and 4c were
screened for the minimum bactericidal concentration [40] against
Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS16
MTCC 2940 and Bacillus subtilis MTCC 121 in comparison to cipro-
floxacin as standard. Compounds 4b and 4c consistently showed
promising minimum bactericidal concentration against all the
tested bacterial strains. The activity data to this regard is shown
in Table 4.
2.2.2. Biofilm inhibition assay
A biofilm is a structured consortium of bacteria embedded in a
self-produced polymeric matrix consisting of polysaccharides, pro-
tein and DNA. Bacterial biofilms cause chronic infections in
humans via hospital and community environments since they
show increased tolerance to antibiotics and disinfectant chemicals
as well as resisting phagocytosis and other components of the
body’s defense system [41]. In the medical sector, bacteria colonize
through adhesion mechanism and result in biofilm formation on
several biomedical implants such as stents, heart valves, vascular
grafts and catheters [42]. In this context, the novel compounds that
can specifically target and inhibit the biofilm formation would be
of significance in comparison to the rational use of antibiotics
and/or biocides. Considering these facts, a further step was under-
taken to investigate whether these compounds exhibit a specific
anti-biofilm activity or whether this observation was simply
related to a general toxic effect on the Gram-positive bacterial
strains. To this regard, the compounds 4b and 4c were screened
for anti-biofilm activity [43] against Staphylococcus aureus MTCC
96, Staphylococcus aureus MLS16 MTCC 2940 and Bacillus subtilis
MTCC 121, which are common and important nosocomial patho-
gens having biofilm forming ability. The results summarized in
Table 5, clearly reveal that not much information on the
structure-activity relationship (SAR) can be highlighted at this
stage; however, it is observed that compound 4c exhibited promis-
derivatives
like
oroidin
and
bromoageliferin
[46–49],
2-aminoimidazoles and imidazopyridinium salts [50] and dichloro-
carbazol derivative [51] which caused the disruption of bacterial
chemical signaling and biofilm formation in some pathogenic
bacteria. These molecules also structurally resembled bacterial
acyl-homoserine lactone (AHL) quorum-sensing molecules [52,53]
and effectively interfered with the quorum signaling, the subse-
quent gene expression and the swarming phenotype [54–56].
3
Control
2.5
7.8 µg/mL
2
3.9 µg/mL
1.5
A
1
0.5
0
12
0
3
6
9
15
18
21
24
ing activity (IC50 values ranged between 2.1 and 8.1 lg/mL)
towards all the tested bacterial species, while compound 4b
Time (h)
showed good activity (IC50 values ranged between 8.8 and
11.3 lg/mL) against all the tested bacterial strains. The basic
thiochromeno[3,4-d]pyrimidine scaffold of these compounds
possesses different substituents which exhibit electron donating
or electron withdrawing properties which antagonize the biofilm
2.5
2
Control
3.9 µg/mL
1.9 µg/mL
1.5
1
B
Table 6
Cell biomass measurement of the Staphylococcus aureus MTCC 96 biofilms treated
with the synthesized compounds (4b and 4c).
0.5
0
Time
Cell dry weight (g/mL)
0
3
6
9
12
15
18
21
24
Control
4b (MIC)
4b (<MIC)
4c (MIC)
4c (<MIC)
Time (h)
0
12
24
0.042
0.85
1.2
0.042
0.64
0.71
0.042
0.81
1.08
0.042
0.58
0.64
0.042
0.75
1.01
Fig. 2. Cell growth measurement of Staphylococcus aureus MTCC 96 treated with the
synthesized compounds (A: compound 4b) and (B: compound 4c).

164
L. Suresh et al. / Bioorganic Chemistry 68 (2016) 159–165
10000
1000
100
10
Staphylococcus aureus MLS16 MTCC 2940 and Bacillus subtilis MTCC
Control
4C
Ciprofloxacin
121. Compound 4c showed promising intracellular ROS accumula-
tion in Staphylococcus aureus MLS16 MTCC 2940 comparable to
that of ciprofloxacin suggesting that the bacterium underwent
apoptotic cell death.
Acknowledgments
The author LS is thankful to the Director, National Institute of
Technology, Warangal, for providing research facilities and also
PSVK is grateful to Council of Scientific and Industrial Research,
New Delhi, India [File 09/922 (0005) 2012/EMR-I], for financial
support.
1
0.5
1
2
4
Concentration (µg/mL)
Fig. 3. Intracellular Reactive Oxygen Species (ROS) accumulation in Staphylococcus
aureus MLS16 (MTCC 2940) biofilms.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.08.
006.
2.2.3. Accumulation of intracellular ROS in Staphylococcus aureus
MLS16 MTCC 2940 biofilms
In order to elucidate whether oxidative stress in involved in the
apoptotic cell death, the intracellular ROS accumulation within the
cells of mature biofilms was measured using the fluorescent probe
20,70-dichlorofluorescein-diacetate (DCFH-DA), a general ROS fluo-
rescent probe. The DCFH-DA dye conversion mainly depends on
the metabolically active cells in the biofilm. DCFH-DA is deacety-
lated in the cells where it can react quantitatively with intracellu-
lar radicals (mainly H2O2) to get converted to its fluorescent
product (DCF), which is retained within the cells and thus provides
an index of oxidation in the cell cytosol. In the present study, the
accumulation of intracellular Reactive Oxygen Species (ROS) in
mature Staphylococcus aureus MLS16 biofilms was measured using
DCFH-DA dye [57]. The accumulation of intracellular ROS plays a
major role in apoptotic mediated cell death [58]. After treatment
with compound 4c, the ROS levels were significantly accumulated
in the tested Staphylococcus aureus MLS16 strain (Fig. 3). At a con-
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increased levels of intracellular ROS accumulation which was equal
to the standard drug ciprofloxacin. The ROS measurements were
also carried out separately in both sessile cells and in the super-
natant. The ROS induced increase in fluorescence was observed
only for the sessile cells, suggesting that the ROS accumulation is
of intracellular origin. This accumulated ROS may be responsible
for the bactericidal activity. Oxygen-containing free radical mole-
cules and their precursors formed in biological systems are collec-
tively termed as ROS, which include superoxides (OÅ2–), peroxides
(H2O2 and ROOH) and free radicals (HOÅ and ROÅ). Some of the bac-
tericidal drugs stimulate free radical formation via the Fenton reac-
tion [59]. Recent studies suggest that when bacteria is under
oxidative stress, these free radicals contribute to arrest in the cell
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