Synthesis and anti-biofilm activity of thiazole Schiff bases

Article abstract of DOI:10.1007/s00044-013-0672-7

A series of thiazole Schiff bases (SB-1 to SB-7) have been synthesized by reacting 4-(o-methoxyphenyl)-2-aminothiazole and R substituted salicylaldehyde (R = H, 3-Me, 4-Me, 5-Me, 3-OMe and 5-Br) or 2-hydroxy-1-naphthaldehyde under microwave irradiation (a green chemistry approach). The compounds were characterized by spectral (UV-Vis, IR, ¹H NMR, ¹³C NMR and GC-MS) and thermal analyses, and tested for the evaluation of anti-biofilm activity against Pseudomonas aeruginosa and anti-bacterial activity against Gram positive (Bacillus subtilis NCIM 2063) and Gram negative (Escherichia coli NCIM 2931) bacteria. The scanning electron microscopic images of the bacterial surfaces have shown that the Schiff bases have impeded the biofilm formation at 50-100 μg/mL concentration, without affecting the growth of the cells (and thus behave as antiquorum sensing agents). Confocal laser scanning microscopy has also confirmed the biofilm inhibition. The anti-biofilm and anti-bacterial activities of the Schiff bases are promising in the design and bio-fabrication of medical devices to combat the biofilm-forming pathogenic organisms. Graphical Abstract: Graphical abstract of the events occurring in present work [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0672-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0672-7
ORIGINAL RESEARCH
Synthesis and anti-biofilm activity of thiazole Schiff bases
•
Prakash G. More Netaji N. Karale
•
•
Anjana S. Lawand Naina Narang
•
Rajendra H. Patil
Received: 17 March 2013 / Accepted: 4 June 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of thiazole Schiff bases (SB-1 to SB-7)
have been synthesized by reacting 4-(o-methoxyphenyl)-2-
aminothiazole and R substituted salicylaldehyde (R = H,
3-Me, 4-Me, 5-Me, 3-OMe and 5-Br) or 2-hydroxy-1-naph-
thaldehyde under microwave irradiation (a green chemistry
approach). The compounds were characterized by spectral
(UV–Vis, IR, 1H NMR, 13C NMR and GC–MS) and thermal
analyses, and tested for the evaluation of anti-biofilm activity
against Pseudomonas aeruginosa and anti-bacterial activity
against Gram positive (Bacillus subtilis NCIM 2063) and
Gram negative (Escherichia coli NCIM 2931) bacteria. The
scanning electron microscopic images of the bacterial sur-
faces have shown that the Schiff bases have impeded the
biofilm formation at 50–100 lg/mL concentration, without
affecting the growth of the cells (and thus behave as an-
tiquorum sensing agents). Confocal laser scanning micros-
copy has also confirmed the biofilm inhibition. The anti-
biofilm and anti-bacterial activities of the Schiff bases are
promisinginthedesignandbio-fabricationofmedicaldevices
to combat the biofilm-forming pathogenic organisms.
Introduction
Treatment of emerging infectiousdiseases and the increasing
number of multidrug resistant microbial pathogens are the
taunting tasks in the medical community (Dessen et al.,
2001; Tenover and McDonald, 2005; Muroi et al., 2004;
Pfeltz and Wilkinson, 2004; Roberts, 2004). The search for
an effective compound to combat bacterial pathogenicity is
the need of an hour. Thiazoles are well known as biologically
active compounds and exhibit several biological activities
such as anti-hypertensive, anti-inflammatory, anti-bacterial,
anti-HIV, anti-tumor and cytotoxic activity (Kashyap et al.,
2012). Schiff bases derived from aminothiazoles have been
shown to possess anti-bacterial and anti-fungal activities
(More et al., 2001).
The anti-bacterial activity of different types of Schiff
bases is also well reported. Karthikeyan et al. (2006) have
synthesized Schiff bases (with a 2,4-dichloro-5-fluor-
ophenyl moiety) exhibiting anti-microbial activity against
Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, and Klebsiella pneumonia. Shi et al. (2007)
have synthesized a series of Schiff bases by reacting
5-chloro-salicylaldehyde and primary amines, and evalu-
ated their anti-bacterial activity against Bacillus subtilis,
E. coli, P. fluorescence and S. aureus. Sinha et al. (2008)
have synthesized eight novel heterocyclic Schiff bases
derived from the condensation reactions of indole 3-car-
boxaldehyde with different l-amino acids (histidine, glu-
tamic acid, aspartic acid, leucine, valine) as well as with
some aminophenols, and these compounds were found to
exhibit anti-bacterial activity against B. subtilis, P. fluo-
rescence, S. aureus. Kamaria et al, (2011) have reported a
series of Schiff bases (derived from indole-3-aldehyde by
microwave-assisted synthesis), which were active against
S. aureus and B. subtilis. However, synthesis of present
Keywords Quorum sensing ꢀ Biofilm ꢀ Schiff Bases ꢀ
Anti-bacterial
P. G. More (&) ꢀ A. S. Lawand
School of Chemical Sciences, Solapur University,
Solapur 413255, Maharashtra, India
e-mail: pramore01@yahoo.com
N. N. Karale
Vidya Pratishthan’s Arts, Science and Commerce College,
Baramati 413133, Maharashtra, India
N. Narang ꢀ R. H. Patil
Department of Biotechnology, University of Pune,
Pune 411007, Maharashtra, India
123

Med Chem Res
series of Schiff bases derived from aminothiazoles, and
evaluation of their bioactivities are not yet reported.
The biofilm synthesized by pathogenic organism has
been one of the reasons for the ineffectiveness of antibi-
otics and development of antibiotics resistance. The for-
mation of biofilm is QS mediated phenomenon. The QS is
an inter-cell communication system aided by released
chemical signals when cell density reaches a critical con-
centration. Bacteria utilize QS to control a wide variety of
processes, including the biofilm (Dickschat, 2010). A
number of pathogenic bacteria employ QS virulence
determinants, for example, biofilm formation in P. aeru-
ginosa (Stewart and Costerton, 2001; Irie and Parsek, 2008;
Hentzer et al., 2003; de Kievit, 2009; Bjarnsholt et al.,
2011). Biofilms are defined as conglomerations of bacterial
cells protected by a self-synthesized extrapolymeric sub-
stance (EPS) (Parsek and Singh, 2003). As microorganisms
are protected inside the biofilm, they show an increased
resistance to anti-microbial agents including antibiotics
compared to free-floating cells. In the medical sector,
biofilms have been implicated as the cause of serious
infections and up to 60 % of all the human infections are
caused by biofilms (Spoering and Lewis, 2001). Bacterial
biofilm formation also causes significant economic loss in
industrial sectors (VanHoudt and Michiels, 2010). In
industry, biofilms have been implicated in the contamina-
tion of installations in food industry, decreased passage
through pipelines by colonization of the interior of the
pipes, and resistance of vessels by initiation of ‘biofouling’
on the vessel hulls. The yearly economic loss caused by
‘biofouling’ in the marine industry is estimated at $ 6.5
billion (Yebra et al., 2004).
(Yamada, 1999). In view of the importance of Schiff bases,
and as part of our ongoing project for the synthesis of new
antimicrobial agents (Lawand et al., 2008, 2011), we report
herein microwave-assisted synthesis of thiazole Schiff bases,
their characterization (elemental, spectral and thermal anal-
yses) and exploration of novel functionality as anti-bacterial
and anti-biofilm agents.
Results and discussion
Chemistry
All the Schiff bases (SB-1 to SB-7) were yellow crystalline
solids having sharp melting points and gave satisfactory
data of elemental (C, H and N) analyses.
Two representatives Schiff bases, SB-1 and SB-5, were
selected for thermal studies. The thermogravimetric (TG)
curves of SB-1 and SB-5 are shown in Figs. 1 and 2,
respectively. No weight loss was observed upon heating till
190 °C, and thus, ruling out the presence of water molecules.
SB-1 undergoes decomposition in two stages (stage I:
213.05–317.89 °C, weight loss 34.90 % and stage II:
512.18–708.74 °C, weight loss 23.43 %), SB-5 also under-
goes decomposition in two stages (stage I: 226.50–
470.54 °C, weight loss 38.88 % and stage II 527.39–
732.14 °C, weight loss 32.53 %). The kinetic parameters,
viz., E (energy of activation), n (order of reaction), Z (pre
exponential factor), DS (entropy of activation) and G (free
energy change) have been calculated using Coats Redfern,
MacCallum–Tanner and Horowitz–Metzger methods
(Horowitz and Metzger, 1963; Coats and Redfern, 1964;
MacCallum and Tanner, 1970).
Antibiotics act by inhibiting the growth (microbiostatic)
or killing of the microorganisms (microbiocidal). They act
by inhibiting the bacterial functions (such as cell wall
synthesis, DNA replication, RNA transcription and protein
synthesis) that are essential for growth. However, the
actions of antibiotics have been shown to impose a selec-
tive pressure that fosters the growth of antibiotic-resistant
strains (Sharma et al., 2009). Therefore, antimicrobial
therapies that are neither bacteriostatic nor bacteriocidal
(i.e. that reduce selective pressure) need to be explored to
curb the menace of antibiotic-resistant pathogens.
The values of n, E, Z, DS and G calculated by the three
methods are in accordance with each other (Table 1). The
values of E for SB-1 (lying in the range 33–39 kcal mol-1
Microwave-assisted synthesis of organic compounds is an
efficient and eco-friendly synthetic strategy and has now
become a powerful tool in green chemistry. Microwave-
assisted organic reaction is advantageous over the conven-
tional heating owing to shorter reaction time, experimental
simplicity, selectivity of products, and easy working up pro-
cedures (Caddick, 1995; Verma, 1999; Mavandadi and
¨
Lidstrom, 2004). Schiff bases have been widely explored in
the field of coordinated chemistry because of their synthetic
flexibility, selectivity and sensitivity towardstransition metals
Fig. 1 Thermogrametric curve of SB-1
123

Med Chem Res
concentration (MBC) by counting the number of viable
organisms [colony forming units per millilitre (CFU/mL)],
which respectively are the lowest concentrations of a com-
pound that can inhibit bacterial growth or kill 99.9 % of
organisms (3log), in comparison to the control. We also
have performed antimicrobial activities by agar diffusion
test. The MIC determination by dye reduction method uses a
redox dye, resazurin, which shows a deep blue colour in its
oxidized state and a purple colour in its reduced state. When
the bacterial cells are viable and metabolically active, i.e. in
growing stage, oxidative enzymes present inside the cells
reduced the dye to pink colour, however, when the cells are
metabolically inactive, the deep blue colour of dye remains
unchanged. During the determination of MIC, the cells of
the test organism were subjected to Schiff bases in Muller
Hinton (MH) broth and incubated for 12 h. Schiff bases SB-
6 and SB-7 exhibited anti-bacterial activity whereas other
Schiff bases found to be inactive. SB-6 has shown anti-
bacterial activity against B. subtilis, whereas SB-7 has
shown anti-bacterial activity against B. subtilis and E. coli.
The MIC values of SB-6 and SB-7 lie in the range 25–50 lg/
mL for B. subtilis and 100 lg/mL for E. coli respectively.
The MIC determination gives information of the compound
as a bacteriostatic agent; however, it does not give any idea
about the compound as bactericidal agent, as well. The
bactericidal property of compounds is far better over bac-
teriostatic as former property irreversibly inhibit the growth
of bacteria. To know the bactericidal property of the Schiff
base, the determination of the minimum bactericidal con-
centration (MBC) was carried out by broth dilution and agar
diffusion methods. When an aliquot from all MIC-wells
were spread on the MH agar plate, and incubated for 24 h to
develop colony, if any, it was found that SB-6 and SB-7
showed a reduction in the number of viable cells, and the
rate of reduction was concentration dependent (Fig. 3). The
MBC value of SB-6 was found to be 50 lg/mL against B.
subtilis and that of SB-7 was found to be 100 lg/mL against
E. coli (Fig. 3). Agar diffusion test has confirmed the MBC
Fig. 2 Thermogravemetric curve of SB-5
for stage I and 34–45 kcal mol-1 for stage II) and for SB-5
(20–25 kcal mol-1 for stage I and 37–45 kcal mol-1 for
stage-II) are sufficiently high and indicate that the com-
pounds are thermally stable.
The reaction time, percentage yields and melting points
data are given in Table 2. The microwave irradiated syn-
thesis of Schiff bases is convenient and rapid, and resulting in
good yield of the expected product. The conventional
method of the synthesis of Schiff bases require 20–30 min
heating on water bath, whereas the microwave method in the
present study requires only 30 s heating in microwave oven
(180 times faster than the conventional method of synthesis).
Biological applications
Anti-bacterial studies
We have studied anti-bacterial action of Schiff base by
determining the minimum inhibitory concentration (MIC)
by dye reduction method and minimum bactericidal
Table 1 Calculation of kinetic
Schiff bases
SB-1
K.E
Stage-I
C.R
Stage-II
parameters by Coats-Redfern
(C.R.), MacCallum-Tanner
(M.T.) and Horowitz-Metzger
(H.M.) methods
M.T
1.77
H.M
1.38
C.R
M.T
1.285
H.M
1.65
n
1.17
33.13
9.81
1.30
E
37.57
7.64
39.02
12.34
-1.60
39.89
3.0
34.60
4.45
40.30
2.62
44.45
8.99
Z
DS
G
n
-7.46
37.19
2.35
-12.46
44.35
2.23
-20.32
53.13
1.35
-24.52
62.66
1.32
-14.46
57.63
1.52
SB-5
E
20.80
4.14
24.45
2.07
25.03
6.07
37.82
5.19
43.35
3.23
45.27
7.08
Z
E = kcal mol-1, Z = s-1
,
DS
G
-20.56
32.45
-25.33
38.40
-16.17
34.19
-18.66
54.96
-23.14
64.60
-14.26
58.36
DS = J K-1 mol-1
G = kcal mol-1
,
123

Med Chem Res
Table 2 Comparative account
of synthesis of Schiff bases by
conventional and microwave
methods
Sr No.
Comp
Conventional method
Microwave method
M P (°C)
Yield (%)
Time (Min)
Yield (%)
Time (Sec)
1
2
3
4
5
6
7
SB-1
SB-2
SB-3
SB-4
SB-5
SB-6
SB-7
95
90
92
90
89
88
91
30
30
30
30
30
30
30
98
95
99
98
95
95
97
10
20
10
10
10
10
10
133
156
139
157
168
209
184
25.0 ug/ml
10.0
7.5
5.0
2.5
0.0
50.0 ug/ml
100.0 ug/ml
1
2
3
4
5
6
7
Sample Number
12.5 ug/ml
25.0 ug/ml
50.0 ug/ml
10.0
7.5
5.0
2.5
0.0
Fig. 4 Determination of minimum bactericidal concentration (MIC)
of Schiff bases against B. subtilis and E. coli. A zone of inhibition was
observed around disc against B. subtilis for sample 6 and 7, while
SB-7 only showed a zone of inhibition in E. coli
bases synthesized by microwave-assisted methods are rare
(Kamaria et al., 2011; Pandey et al., 2012; Venkatesan et al.,
2012) and that too by reacting amine with salicylaldehyde
are very rare (Pandey et al., 2012). Shi et al. 2007 have
evaluated the anti-microbial activity of a series of Schiff
bases derived from the condensation of 5-chloro-salicylal-
dehyde and primary amines, and showed that the com-
pounds with aromatic rings were more active than
compounds with aliphatic chains. Infact, it seems that the
order of anti-bacterial activity against bacterial strains
increases with an increase in the complexities of the
R-substituent of salicylaldehyde.
1
2
3
4
5
6
7
Sample Number
Fig. 3 Determination of minimum bactericidal concentration (MIC)
of Schiff bases (SB1–SB7) against B. subtilis and E. coli. Cells of test
organisms subjected to various conc. of Schiff bases showed a
reduction in number of viable cells. Only SB-6 and SB-7 at 25 lg/mL
showed a 3log reduction against B. subtilis (upper) while SB-7 at
100 lg/mL showed 2log reductions against E. coli (lower). Note very
less reduction of cells (1log reduction) in the presence of SB-5 against
B. subtilis. Error bars represents the standard deviation (n = 3)
Anti-biofilm
values (Fig. 4). A zone of inhibition was observed around
the disc of SB-6 and SB-7 against B. subtilis (25 and 50 lg/
mL), while a similar zone of inhibition was observed around
the disc of SB-7 against E. coli (100 lg/mL). Thus, the
characterized Schiff bases showed an anti-bacterial prop-
erty. Literature survey reveals that Schiff bases have been
shown to possess antimicrobial activities (Walsh et al.,
1996; Sridhar et al., 2001; Mladenova et al., 2002; Pan-
neerselvam et al., 2005; Bharti et al., 2010; Desai et al.
2013; Pandey et al., 2012), however, evaluation of Schiff
Most of the bacteria acquire the resistance to antibiotics by
virtue of the synthesis of an extracellular matrix (biofilm), a
QS trait. So, an anti-biofilm (anti QS) approach is the
promising to tackle the aforementioned problem (Hammer
and Bassler, 2003; Cvitkovitch et al., 2003). Current efforts
towards small molecule-based strategies to control biofilm
formation have focused almost exclusively on inhibiting QS,
a signalling cascade that is critical for bacterial communi-
cation (Smith et al., 2003; Geske et al., 2005). However,
123

Med Chem Res
Schiff bases were not yet explored as anti-biofilm agents. So,
we have taken up a study to explore a possibility role of
Schiff bases in inhibiting/impeding the formation of biofilm
in P. aeruginosa.
Biofilm and SEM
As we were interested to study the QS mediated biofilm
inhibition in P. aeruginosa, we have selected only those
Schiff bases that showed an anti-bacterial activity. Only
SB-5 and SB-6 showed an anti-bacterial activity against P.
aeruginosa. When the cells of P. aeruginosa were subjected
to sub-inhibitory concentration of Schiff bases, there was a
prominent decrease in the biofilm formation; the enmeshed
uncovered cells were observed i.e. cells without the biofilm.
However, in absence of Schiff bases, cells of P. aeruginosa
were seen as enmeshed-covered structures, and cells were
enclosed, a typical of biofilm structures (Fig. 5). An enmesh
observed under SEM is a complex of polysaccharides,
synthesized by growing cells in response to QS; however, in
presence of Schiff bases QS was disturbed, and hence an
enmesh of the structures was not observed under SEM.
More importantly, the numbers of the planktonic cells (cells
in suspension) were not affected, suggesting that the inhi-
bition of the biofilm in P. aeruginosa is QS mediated.
Biofilm and CLSM
The biofilm is a complex mixture of DNAs, proteins and
majorly polysaccharides. The Concanavalin A is a fluorescent
molecule with a high affinity towards glucosyl-mannosyl
residue of polysaccharide of biofilm. When the biofilm of the
P. aeruginosa was treated with Con A alexafluor 488 fluo-
rescent dye and observed under CLSM at 10 X magnification,
a continuous and green fluorescence was observed. However,
in presence of the Schiff base, while the green fluorescence
was diminished in presence of SB-5, SB-6, it decreased in
presence of SB-7. The fluorescence intensity of the biofilm in
presence of SB-1, SB-2, SB3 and SB-4 was unaffected
(Fig. 6), thus further confirming the results of SEM.
Fig. 5 Scanning electron microscope analysis of Biofilm P. aeru-
ginosa shows dense mass of cells surrounded by biofilm. However, in
presence of Schiff bases, 5, 6 and 7, individual cells were observed,
indicating an inhibition of biofilm formation
2009; Steenackers et al., 2010; More et al. 2012; Rane
et al., 2012; Tello et al., 2013). Thus, vast reportier of the
chemical scaffolds are yet unexplored as an anti-biofilm
agent. Because expression of many virulence factors and
establishment of chronic infections take an advantage of
biofilm (regulated by QS) [Van Delden and Iglewski,
1998], the disruption of biofilm is expected to reduce the
pathogenicity of many biofilm forming pathogens.
Most of the anti-biofilm studies explored natural prod-
ucts or its derivatives. The best-studied examples of the
natural products as anti-biofilm agents are (1) halogenated
furanones, which were originally isolated from the seaweed
Deliseapulchra, (2) analogues of the homoserine lactone
signalling molecules and (3) analogues of the sponge-
derived marine natural alkaloids oroidin and bromoagelif-
erin (Stowe et al., 2011). However, examples of synthetic
compounds as anti-biofilm agents are few. The scaffolds of
the chemical structures explored till date to study the anti-
biofilm activities are aminothiazoles, substituted pyrimid-
inium salts, substituted imidazoles, TAGE-triazole conju-
gates and 4-Thiazolidinones derivatives (Huigens et al.,
Conclusion
The microwave-assisted method successfully synthesized
4-(o-methoxyphenyl)-2-aminothiazole by green chemistry
approach. The as-synthesized compound shows a thermal
123

Med Chem Res
resistance with antibiotics because QS is not directly
involved in the processes essential for growth of bacteria.
This is important because when the growth is not affected,
there is no selective pressure for the development of
resistant bacteria. Inhibition of bacterial QS, rather than
bactericidal or bacteriostatic strategies, may find applica-
tion in many different fields, such as medicine, agriculture
and food technology.
Experimental
All the chemicals used were of A. R. Grade. 4-(o-
methoxyphenyl)-2-aminothiazole was synthesized accord-
ing to the procedure available in literature (Patai, 1970).
The solvents were dried according to the standard proce-
dures and distilled before use. UV–Vis spectra were
recorded in ethanol on Shimadzu A 600 UV–Vis spec-
trometer. IR spectra were recorded in KBr pellets on Shi-
1
madzu FT-IR 8400 spectrometer. H NMR spectra were
recorded in CDCl3 using TMS as the standard on Varian
300 MHz spectrometer. 13C NMR spectra were recorded in
CDCl3 using TMS as the standard on Varian 300 MHz
spectrometer. GC–MS were recorded on Shimadzu GC–
MS QP 5050 mass spectrometer. Microwave mediated
reactions were carried out in Onida-power conventional 25
DLX microwave oven.
Microwave-assisted synthesis of Schiff bases (SB-1
to SB-7)
4-(o-Methoxyphenyl)-2-aminothiazole (1 mol) and o-hy-
droxyaldehyde (1 mol) were mixed with each other in mortar-
pestle, and the reaction mixture was placed in small conical
flask at room temperature. The mixture was then added with
1 mL alcohol exposed to microwave irradiation at 10 %
power for 10–20 s. (Fig. 7). Completion of the reaction was
tested by TLC. The reaction mixture was cooled to room
temperature. The yellow coloured Schiff base was further
recrystallized from ethanol and dried under reduced pressure.
Fig.. 6 Confocal laser scanning microscope analysis of biofilm.
Pseudomonas aeruginosa shows the patches of fluorescence in
presence of Schiff bases, SB-5, SB-6 and SB-7, indicated a truncated
formation of biofilm. However, in absence of Schiff bases (control),
fluorescence was regular and entire, thus indicating an intact biofilm
stability and is bioactive. The reduction in the number of
viable cells in comparison to the control shows an anti-
bacterial nature of the compound. Reduction of the poly-
saccharides (a matrix) in presence of Schiff bases shows
impedance in the QS mediated biofilm formation. The anti-
QS approach is highly attractive because it does not
impose harsh selective pressure for the development of
Spectral analysis
UV–Vis spectra of the Schiff bases exhibit an intense band at
*400 nm. The UV–Vis spectrum of 2-aminothiazole
OCH3
OCH3
HO
OHC
HO
Microwave Irradiation
N
N
S
N
NH2
R
S
R
Fig. 7 Thiazole Schiff base SB-1: (R = H), SB-2: (R = 3-CH3), SB-3: (R = 4-CH3), SB-4: (R = 5-CH3), SB-5: (R = 3-OCH3), SB-6:
(R = 5-Br), SB-7: (R = 2-H1-N)
123

Med Chem Res
exhibits an intense band at *275 nm and other aromatic
amino compounds with comparable structures exhibit
absorption (Conover and Tarbell, 1950; Mathes and Gregory,
1952; Wilson and Woodger, 1955) at *300 nm. The shifting
of absorption band towards higher wavelength (*400 nm) in
thepresentSchiffbasesmaybedue toextendedconjugationin
the molecule (Patai, 1970). The IR spectra of the Schiff bases
exhibit m(O–H), m(C=N), m(C–O) and m(C–S–C) modes at
*3,430 to *3,460, *1,640, *1,280 and *660 cm-1
respectively, and these values are in accordance with the
earlier reports (Silverstein et al., 2005). The m(OH) mode is
broad and weak and this may due to hydrogen bonding
between phenolic OH and nitrogen of the azomethine group
forming a six membered ring (Kovacic, 1967). The 1H NMR
spectra of the Schiff bases were recorded. The assignments of
NMR signals show close resemblance with the earlier results
(Silverstein et al., 2005). The spectral analyses data confirm
the structure of Schiff bases as represented in Fig. 1. The
spectral data is represented below.
ring vibrations 1591, 1485, 1157, 1130; 1H NMR: (CDCl3,
TMS, dppm) 2.37 (3H, s, Ar–CH3), 3.95 (3H, s, Ar–
OCH3), 6.85 (1H, s, H-thiazole), 7.01–8.27 (7H, m, Ar–H),
9.24 (1H, s, benzylidenimin), 12.35 (1H, s, Ar–OH). GC–
MS: m/z (relative intensity, %): 324 (86.73) (M? peak)
(Molecular formula: C18H16N2O2S), 307 (34.69), 291
(51.02), 191 (23.46), 178 (16.32), 161 (23.46), 146 (29.59),
132 (100), 121 (37.75), 91 (38.77), 77 (30.61), 65 (22.44),
51 (13.26.)
Sb-4
UV–Vis: kmax 383 nm; IR(cm-1): m(O–H) 3455, m(C=N)
1626, m(C–O) 1271, m(C–S–C) 667 and phenyl and thiazole
ring vibrations 1570, 1356, 1182, 1146; 1H NMR: (CDCl3,
TMS, dppm) 2.30 (3H, s, Ar–CH3), 3.94 (3H, s, Ar–OCH3),
6.94 (1H, s, H-thiazole), 7.04–8.20 (7H, m, Ar–H), 9.21 (1H,
s, benzylidenimin), 12.20 (1H, s, Ar–OH). GC–MS: m/z
(relative intensity, %): 324 (97.02) (M? peak) (Molecular
formula: C18H16N2O2S), 307 (65.34), 291(94.35), 192
(18.81), 161 (21.78), 146 (23.74), 132 (100), 121 (42.57), 91
(33.66), 77 (39.60), 65 (21.78), 51 (17.82).
Sb-1
UV–Vis: kmax 381 nm; IR(cm-1): m(O–H) 3454, m(C=N)
1622, m(C–O) 1276, m(C–S–C) 665 and phenyl and thiazole
ring vibrations 1597, 1487, 1159; 1H NMR: (CDCl3, TMS,
dppm), 3.93 (3H, s, Ar-OCH3), 6.93 (1H, s, H-thiazole),
6.95–7.86 (8H, m, Ar–H), 8.25 (1H, s, benzylidenimin),
12.38 (1H, s, Ar–OH). GC–MS: m/z (relative intensity,
%): 310 (99.21) (M? peak) (Molecular formula:
C17H14N2O2S), 293 (30.69), 277 (70.29), 191 (27.72), 164
(29.70), 147 (56.43), 132(100), 121 (54.45), 104 (24.75),
91 (33.66), 77 (72.27), 63 (19.80), 51 (44.55).
Sb-5
UV–Vis: kmax 382 nm; IR(cm-1): m(O–H) 3435, m(C=N)
1627, m(C–O) 1258, m(C–S–C) 661 and phenyl and thiazole
ring vibrations 1588, 1485, 1465, 1175; 1H NMR: (CDCl3,
TMS, dppm) 3.94 (3H, s, Ar–OCH3 of thiazole), 3.96 (3H,
s, Ar–OCH3 of aldehyde), 6.92 (1H, s, H-thiazole),
6.99–8.28 (7H, m, Ar–H), 9.32 (1H, s, benzylidenimin),
12.63 (1H, s, Ar-OH); 13C NMR: (CDCl3, TMS, dppm)
167.2, 164.5,156.9, 151.5, 149.5, 148.3, 130.0, 129.0,
124.9, 122.7, 120.7, 119.0, 118.4, 117.1, 115.8, 111.0,
56.1, 55.3. GC–MS: m/z (relative intensity, %): 340 (100)
(M? peak) (Molecular formula: C18H16N2O3S), 323 (5.88),
307 (16.66), 282 (6.82), 264 (18.63), 205 (23.52), 192
(31.37), 177 (27.45), 162 (46.07), 150 (44.11), 132
(88.23),121 (46.07), 105 (21.56), 91 (34.31), 77 (58.82), 63
(48.03), 45 (48.03), 40 (70.58).
Sb-2
UV–Vis: kmax 384 nm; IR(cm-1): m(O–H) 3455, m(C=N)
1640, m(C–O) 1235, m(C–S–C) 665 and phenyl and thiazole
ring vibrations 1590, 1560, 1400, 1100; 1H NMR: (CDCl3,
TMS, dppm) 2.30 (3H, s, Ar–CH3), 3.92 (3H, s, Ar–
OCH3), 6.88 (1H, s, H-thiazole), 7.04–8.27 (7H, m, Ar–H),
9.22 (1H, s, benzylidenimin), 12.60 (1H, s, Ar–OH); 13C
NMR: (CDCl3, TMS, dppm) 167.4, 164.6, 159.8, 156.9,
149.5, 135.4, 131.2, 130.0, 129.0, 126.3, 122.8, 120.8,
119.0, 117.7, 116.9, 111.0, 55.3, 15.4. GC–MS: m/z (rel-
ative intensity, %): 324 (100) (M? peak) (Molecular for-
mula: C18H16N2O2S), 291 (50.49), 191 (20.79), 161
(17.82), 146 (21.78), 132 (80.19), 121 (43.56), 91 (33.66),
77 (40.59), 65 (22.77), 51 (17.82).
Sb-6
UV–Vis: kmax 390 nm; IR(cm-1): m(O–H) 3455, m(C=N)
1614, m(C–O) 1271, m(C–S–C) 679 and phenyl and thiazole
ring vibrations 1550, 1487, 1350, 1155; 1H NMR: (CDCl3,
TMS, dppm) 3.97 (3H, s, Ar–OCH3), 6.95 (1H, s, H-thia-
zole), 7.00–8.27 (7H, m, Ar–H), 9.24 (1H, s, benzylideni-
min), 12.39 (1H, s, Ar–OH); 13C NMR: (CDCl3, TMS,
dppm) 166.8, 163.0, 160.4, 157.0, 149.8, 136.9, 135.3, 130.1,
129.2, 122.7, 120.9, 119.1, 119.4, 117.6, 116.1, 111.0, 55.4.
GC–MS: m/z (relative intensity, %): 388/390* (35.29/
32.35) (M? peak) (Molecular formula: C17H13 Br N2O2S),
Sb-3
UV–Vis: kmax 383 nm; IR(cm-1): m(O–H) 3470, m(C=N)
1630, m(C–O) 1236, m(C–S–C) 662 and phenyl and thiazole
123

Med Chem Res
371/373* (11.76/12 .74), 355/357* (13.23/13.72), 199
(10.29), 191 (26.47), 164 (19.60), 146 (26.47), 132 (100),
121 (47.05), 102 (22.54), 89 (27.45), 77 (50.00), 63 (35.29),
51 (29.41). The peaks marked with (*) are isotopic peaks.
was diluted with LB medium (OD600 0.02), and 50 lL of
the diluted culture was added to 950 lL of LB medium
supplemented with 25 lg/mL of Schiff bases and were
incubated statically for 18 h at 37 °C in 8-well glass
chamber slide. After incubation, planktonic bacteria were
discarded, and the biofilms were washed three times with
cocdylate buffer (0.1 M, pH 7.4). Biofilms formed on glass
plates were fixed in 2 % glutaraldehyde in 0.1 M cocdylate
buffer (pH 7.4) for 4 h at 4 °C. After thorough washing
with cocadylate buffer, samples were dehydrated in a series
of ethanol solutions (10–100 %). The samples were dried,
mounted on aluminium stubs with conductive carbon
cement and then coated with a gold film. Samples were
observed with a Joel, JSM, 6760A SEM at 20 kV accel-
erating voltage.
Sb-7
UV–Vis: kmax 395 nm; IR(cm-1): m(O–H) 3410, m(C=N)
1620, m(C–O) 1242, m(C–S–C) 661 and phenyl and thiazole
ring vibrations 1554, 1456, 1155; 1H NMR: (CDCl3, TMS,
dppm) 3.97 (3H, s, Ar–OCH3), 7.00 (1H, s, H-thiazole),
7.10–8.35 (10H, m, Ar–H), 10.18 (1H, s, benzylidenimin),
14.42 (1H, s, Ar–OH). GC–MS: m/z (relative intensity, %):
360 (100) (M? peak) (Molecular formula: C21H16N2O2S),
343 (59.80), 327 (19.60), 192 (55.88), 169 (27.45), 164
(21.56), 149 (19.60), 132 (99.01), 121 (45.09), 89 (23.52),
77 (51.96), 63 (21.56), 45 (28.43).
CLSM analysis of biofilm
Anti-bacterial study
Pseudomonas aeruginosa was grown overnight in LB
medium at 37 °C with agitation. After growth, the culture
was diluted with LB medium (OD600 0.02), and 50 lL of
the diluted culture was added to 950 lL of LB medium
supplemented with 25 lg/mL of Schiff bases (SB-1 to SB-
7) and were incubated statically for 18 h at 37 °C in 8-well
glass chamber slide. After incubation, planktonic bacteria
were discarded, and the biofilms were washed three times
with cocdylate buffer (0.1 M, pH 7.4). Biofilms formed on
glass plates was covered with 50 lL of 10 lg/mL of dye,
Concanavalin A Alexafluor 488 and incubated for 30 min
at 4 °C. The biofilm was finally washed with cocdylate
buffer of 0.1 M, pH 7.4 and observed under fluorescence
microscopy (Zeiss, Germany) with an excitation of wave-
length of 488 nm and emission at 519 nm.
Determination of MIC and MBC values
An anti-bacterial study was carried out by broth dilution
method using two different organisms, Gram positive,
B. subtilis NCIM 2063 and Gram negative, E. coli NCIM
2931. The anti-bacterial study was carried out by dye
reduction method. In short, a 24-well microtitre plates
containing 1 mL MH broth, with various Schiff bases, in
the concentration range of 12.5–100 lg/mL, were inocu-
lated with the test strains (approximate final cell density of
1 9 106 CFU/mL), and incubated at 37 °C for 15 h. The
lowest concentration of compound showing no change in
colour (no growth) was considered as MIC. The MBC was
measured by preparing serial dilutions from the MIC assay
and plating the dilutions on MH agar plates. The MBC is
defined as the minimum concentration at which there was a
3 log reduction in the CFU. The data was recorded as
survival rates (CFU/mL), based on 100 % survival for the
untreated control. All MIC and MBC values reported were
based on three experimental repeats. The anti-bacterial
activity was also studied by disc diffusion methods. In
short, a 100 lL of overnight culture adjusted to 1.0 O.D
(600 nm) was spread on 90 mm MH gar plate and antibi-
otics discs with 2.5–100 lg of the respective compound
were impregnated and incubated at 37 °C for 15 h, and
observed for zone of inhibition around discs.
Acknowledgments Authors are thankful to the authorities of the
Solapur University-Solapur and the University of Pune for providing
research facilities. RHP is thankful to the Departmental research and
development programme Grant (Department of Biotechnology, Uni-
versity of Pune) for financial help.
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