Synthesis, characterization and microbiocidal studies of novel ionic liquid tagged Schiff bases

Article abstract of DOI:10.1016/j.crci.2012.05.023

The synthesis of novel imidazolium ionic liquid, tagged Schiff, has been described. The synthesis was achieved in three steps from 2,4- dihydroxybenzaldehyde by selective alkylation with 1,3-dibromopropane, followed by reaction with 1-methylimidazole and Schiff base formation with aromatic amines. The compounds were evaluated for antibacterial and antifungal activities. The ionic liquid tagged Schiff base 4a showed the inhibition of both Gram positive and Gram negative bacteria. It also showed broad spectrum antifungal activity against all four tested fungi; however, 4f showed highest antifungal activity against A. niger.

Full text of DOI:10.1016/j.crci.2012.05.023

C. R. Chimie 15 (2012) 669–674
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´
Full paper/Memoire
Synthesis, characterization and microbiocidal studies of novel ionic liquid
tagged Schiff bases
a
a
Bharti Khungar a, , Madharam Sudershan Rao , Kasiviswanadharaju Pericherla ,
*
Pankaj Nehra a, Navin Jain b, Jitendra Panwar b, Anil Kumar a,
*
a Department of Chemistry, Birla Institute of Technology and Science, 333031 Pilani, Rajasthan, India
b Department of Biological Sciences, Birla Institute of Technology and Science, 333031 Pilani, Rajasthan, India
A B S T R A C T
A R T I C L E I N F O
Article history:
The synthesis of novel imidazolium ionic liquid, tagged Schiff, has been described. The
synthesis was achieved in three steps from 2,4-dihydroxybenzaldehyde by selective
alkylation with 1,3-dibromopropane, followed by reaction with 1-methylimidazole and
Schiff base formation with aromatic amines. The compounds were evaluated for
antibacterial and antifungal activities. The ionic liquid tagged Schiff base 4a showed
the inhibition of both Gram positive and Gram negative bacteria. It also showed broad
spectrum antifungal activity against all four tested fungi; however, 4f showed highest
antifungal activity against A. niger.
Received 23 February 2012
Accepted after revision 30 May 2012
Available online 12 July 2012
Keywords:
Ionic liquid
Functionalized ionic liquid
Schiff base
´
ß 2012 Published by Elsevier Masson SAS on behalf of Academie des sciences.
Antibacterial
Antifungal
1. Introduction
substituent on the anion or cation in an ionic liquid, leading
toionic liquidwithdifferent properties. Functionalizedionic
Ionic liquids (ILs), the molten salts with melting points
at or below ambient temperature, have attracted intense
focus owing to their remarkable chemical and physical
properties such as high thermal stability, [1–3] negligible
vapor pressure, [4] high ionic conductivity [5]. They have
been used as alternatives to volatile organic solvents for
organic synthesis in homogeneous as well as biphasic
processes [6–8].
Initially, the focus of research for ionic liquids was on
their role as alternative solvents in organic synthesis;
however, after synthesis of an ionic liquid derived from the
antifungal drug miconazole a new concept of ‘‘task specific
ionic liquids’’ (TSILs) was introduced [9,10]. There is vast
scope of chemical structural modifications that can be
introduced either in the anion, the cationic core, or
liquids (FILs) are prepared by introducing a specific
functionality covalently tethered to the cation, or the anion,
or a zwitterionic form of the salt [11–13]. The enduring
popularity of these TSIL or FILs lies in the fact that both the
cationic and anionic components can be varied and the ionic
liquid can be tailored to a specific application. Moreover, the
incorporation of functional groups can impart a particular
capability to the ionic liquids, enhancing catalytic stability
and reducing catalyst leaching [14–17].
Recently, understanding biological activity and toxicity
of ionic liquids has been of great interest due to their
increasing applications. They have been shown to have
significant toxic effects on different bacteria and fungi.
1-Alkylquinolinium bromides have been studied for their
antimicrobial and antibiofilm activities [18]. Pyridinium,
imidazolium and quaternary ammonium salts have been
found to be toxic for variety of bacteria and fungi [19]. It
has been observed that toxicity increases with the length
of n-alkyl substituent in the methylimidazolium cation
whereas anion has minimal effect [20,21].
* Corresponding authors.
E-mail addresses: bhartikh@gmail.com (B. Khungar),
anilkadian@gmail.com (A. Kumar).
1631-0748/$ – see front matter ß 2012 Published by Elsevier Masson SAS on behalf of Acade´mie des sciences.
http://dx.doi.org/10.1016/j.crci.2012.05.023

670
B. Khungar et al. / C. R. Chimie 15 (2012) 669–674
H
H
H
O
O
N R
OH
H
O
RNH2
N
N
OH
OH
OH
BrCH2CH2CH2Br
MeOH,60oC
Acetone, NaHCO3
60oC
N
Br-
N
OH
O
Br
O
N
O
N
Br-
1
2
4a-f
3
Scheme 1. Synthesis of ionic liquid tagged Schiff bases.
Schiff bases constitute an important class of organic
and alkylation has occurred selectively at the para-
hydroxy group. In addition to this sharper peak at
3080 cmꢀ1 for OH stretching also indicates alkylation at
para-hydroxy group. In the subsequent step, 1-methyli-
midazole was reacted with 2 to form ionic liquid tagged
aldehyde (3). The ESI-MS spectrum of 3 displayed a peak at
m/z 261.2 for [M + H–Br]+ ion. The IH NMR spectrum
compounds because of their biological, analytical and
pharmaceutical applications [22]. They possess excellent
characteristics, structural similarities with natural biolog-
ical substances. They are well known for broad spectrum
biological activities such as antiviral, [23] anticancer, [24]
antifungal, [25] antibacterial [26] agents. The microbioci-
dal activities associated with them is due to the presence of
the toxophoric C = N linkage in them. Relatively simple
preparation procedures and the synthetic flexibility enable
design of compounds with desired structural properties. It
is expected that synthesis of ionic liquid tagged Schiff
bases can have synergetic effects on their microbial
inhibition. With this aim and in continuation of our
interest in ionic liquids, [27–31] in this article we report
synthesis of novel ionic liquid tagged Schiff bases and their
antibacterial and antifungal activities.
showed an additional peak at
d 3.86 ppm for N-CH3 and
three new peaks at 9.25, 7.84 and 7.74 ppm for
d
imidazolium ring protons in addition to other protons.
In the third step, 3 was reacted with different aromatic
amines to yield ionic liquid tagged Schiff base (4). The
yields of 4 were moderate and the nature of various
substituents on the aromatic amines had no obvious effect
on the yields. The details of yield and structure for different
ionic liquid tagged Schiff bases are given in Table 1.
Compound 4a exhibited absorption bands at 1620 cmꢀ1
(C = N str.), 3100 cmꢀ1 (H–C(=N) str.) and 3415 cmꢀ1 (OH
str.) in the IR spectrum. In 1H NMR azomethine proton
2. Results and discussion
appeared at
d 8.79 and hydroxyl proton at d 13.77 along
In most functionalized ionic liquids, quaternization of
ring nitrogen by alkyl halide to produce 3-alkylimidazo-
lium salt is done after attaching the functional group to
imidazole at the N-1 position. These reactions are generally
performed in a narrow region of temperature and solvent
parameters due to competing side reaction of the
alkylating reagent with the incorporated functional group.
We have designed a novel approach for the synthesis of
functionalized ionic liquids where this alkylation of
functional group can be avoided (Scheme 1).
The first step of the synthetic route involved the
synthesis of 4-(3-bromopropoxy)-2-hydroxybenzalde-
hyde (2) by selective O-alkylation of 2,4-dihydroxyben-
zaldehyde (1). The reaction gives a mixture of 4-(3-
bromopropoxy)-2-hydroxybenzaldehyde (2) and 4,40-
(propane-1,3-diylbis(oxy))bis(2-hydroxybenzaldehyde).
Formation of dimeric 4,40-(propane-1,3-diylbis(oxy))-
bis(2-hydroxybenzaldehyde) was minimized by dilution
method. Usage of mild reaction conditions and presence of
hydrogen bonding resulted in O-alkylation selectively at
the para-positon. The 1H NMR spectrum of the compound
with other protons of the molecule. A peak appeared at
d
163.6 for azomethine carbon along with other carbons of
the molecule in 13C NMR spectra. Similarly, spectroscopic
data for all other compounds have been found to be
consistent with the structure.
Bactericidal assay of all the synthesized compounds
(4a-f) was performed against common food pathogenic
bacteria to evaluate their usability as efficient alternative
to well-established chemotherapeutic agents. The zone of
inhibition and minimum inhibitory concentration (MIC)
values were measured to determine the antibacterial
efficiency of the tested compounds. Gram positive and
Gram negative bacterial strains were selected on the basis
of their clinical importance.
The compounds (4a-f) exhibited moderate to good
antibacterial activity against Gram positive bacteria (Table
2). This may be due to the interactions between
peptidoglycan component of Gram positive cells and
compounds which leads to disruption of cell wall resulting
in cell death. Out of all the tested compounds, only 4a
showed inhibition of both Gram positive and Gram
negative bacteria, whereas 4d did not show any inhibition
for both bacterial groups. Except 4a, all the compounds
were found to be insensitive against the tested gram
negative bacterial strains.
showed singlet at
aldehydic proton, triplet at
4.10 for OCH2, multiplet at
aromatic protons at 6.49-7.64. The ESI-MS spectrum of 2
d
11.02 for OH, singlet at
3.66 for CH2Br, triplet at
2.28 for CH2 along with
d 10.02 for
d
d
d
d
displayed a peak at m/z 259.0 and 261.1 for [M]+ and
[M + 2]+ ions, respectively. In the IR spectra a peak at
1624 cmꢀ1 was observed for aldehydic C = O stretching and
a sharper peak at 3080 cmꢀ1 for OH stretching. There was
no significant shift in the C = O stretching frequency that
also indicates that intramolecular hydrogen bonding of
ortho-hydroxy group with carbonyl group is not disturbed
All the synthesized compounds (4a-f) were screened for
in vitro antifungal activity. Table 3 shows zone of inhibition
and MIC values for the tested pathogenic fungal species.
Similar to the antibacterial activity, compound 4a was
found to exhibit broad spectrum antifungal activity against
all the tested fungi. The highest MIC value was observed
with 4f followed by 4a and 4c against A. niger which was

B. Khungar et al. / C. R. Chimie 15 (2012) 669–674
671
Table 1
Synthesis of ionic liquid tagged Schiff bases 4a-f.
Sr. No.
R
Structure of 3
Yielda (%)
1
C6H5
4a
4b
4c
68
60
59
N
N
N
N
O
N
Br
Br
OH
2
3
4-BrC6H4
4-ClC6H4
O
N
Br
OH
N
N
N
N
O
N
Cl
Br
Br
OH
4
4-FC6H4
4d
64
O
N
F
OH
5
6
4-CH3OC6H4
4e
4f
68
50
N
N
N
N
O
O
N
OCH3
Br
Br
OH
C10H7
N
OH
a
Isolated yield.
Table 2
Zone of inhibition and minimum inhibitory concentration (MIC) values of compounds against Gram positive and Gram negative bacteria.
Compound Gram positive
Gram negative
B. cereus
S. aureus
P. putida
E. coli
K. pneumoniae
S. typhimurium
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
)
(
m
)
(
m
)
(
m
)
(
m
)
(
m
)
(
m
4a
4b
4c
4d
4e
4f
11
10
10
*
128
13
11
11
*
64
13
11
10
*
64
11
*
128
12
*
64
11
*
128
> 128
128
128
128
> 128
64
128
> 128
>128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
*
*
*
> 128
> 128
> 128
*
*
*
10
11
12
11
10
10
*
*
*
128
*
*
*
*: No activity was observed.
found to be the most sensitive fungal strain. Further
studies are required to characterize the mode of action of
these compounds against microbes.
evaluation of their antimicrobial activity are in progress in
our laboratory.
4. Experimental
3. Conclusions
4.1. General
In conclusion, a new approach for the synthesis of
imidazolium ionic liquid tagged Schiff bases has been
developed. Synthesis of ionic liquid tagged Schiff bases is
achieved in three steps from 2,4-dihydroxybenzaldehyde
by selective alkylation with 1,3-dibromopropane, followed
by reaction with 1-methylimidazole and Schiff base
formation with aromatic amines. The resulting compounds
have been tested for antibacterial and antifungal activities.
The present series of compounds exhibited significant
antimicrobial activity against both bacteria and fungi
which opens the avenue to utilize the compounds as broad
spectrum chemotherapeutic agents. Further studies on the
synthesis of a library of ionic liquid tagged Schiff bases and
All the solvents and reagents were purchased from
Sigma-Aldrich, India and Spectrochem Pvt. Ltd., India and
were used without further purification. Melting points
were determined in open capillary tubes on a MPA120-
Automated Melting Point apparatus and are uncorrected.
The 1H and 13C NMR spectra were recorded on a Bruker
Heaven 11400 (400 MHz) and Varian (500 MHz) spectro-
meters using CDCl3 and DMSO-d6 as solvents and the
chemical shifts were expressed in ppm. The bacterial
cultures used were Bacillus cereus (MTCC 430), Staphylo-
coccus aureus (MTCC 96), Pseudomonas putida (MTCC 102),
Escherichia coli (MTCC 1652), Klebsiella pneumoniae (MTCC

672
B. Khungar et al. / C. R. Chimie 15 (2012) 669–674
Table 3
Zone of inhibition values of compounds against various tested fungi.
Compound
A. flavus
A. niger
F. oxysporum
C. acutatum
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
Zone of
inhibition
(mm)
MIC
g mLꢀ1
)
(
m
)
(
m
)
(
m
)
(
m
4a
4b
4c
4d
4e
4f
15
14
12
13
13
14
64
16
13
16
13
14
18
64
15
13
15
14
14
14
64
15
14
14
13
13
15
64
128
128
64
> 128
64
128
> 128
128
128
> 128
128
32
128
128
128
> 128
> 128
64
> 128
128
432) and Salmonella typhimurium (MTCC 98). Aspergillus
niger (MTCC 282), Aspergillus flavus (MTCC 6674), Fusarium
oxysporum (MTCC 6045) and Colletotrichum acutatum
(MTCC 2247) were used for antifungal studies. The axenic
cultures of all strains were procured from MTCC, IMTECH,
India and revived as per suggested guidelines using
standard microbiological methods.
(s, 1H), 8.77 (s, 1H), 7.80 (t, J = 1.8 Hz, 1H), 7.70 (t, J = 1.7 Hz,
1H), 7.55 (td, J = 8.7, 2.04 Hz, 2H), 7.46 (d, J = 8.5 Hz, 1H),
7.27 (td, J = 8.7, 2.04 Hz, 2H), 6.49–6.45 (m, 2H), 4.45 (t,
J = 6.9 Hz, 2H), 4.12 (t, J = 5.8 Hz, 2H), 3.93 (s, 3H), 2.40–2.34
(m, 2H); 13C NMR (126 MHz, DMSO-d6)
d 163.7, 163.4,
163.1, 147.7, 137.2, 134.7, 132.7, 124.1, 123.8, 122.9, 119.4,
113.6, 107.7, 101.8, 65.4, 46.8, 36.2, 31.2, 29.3; ESI-MS (m/
z): 414.1 [M - Br]+, 416.1 [M + 2 - Br]+.
4.2. Synthesis of ionic liquid tagged aldehyde (3)
4c: Yield 797 mg (59%); mp 150-155 8C; IR (cmꢀ1) 1612,
3325; 1H NMR (400 MHz, DMSO-d6)
d 13.45 (s, 1H), 9.33 (s,
In the first step, reaction of 1,3-dibromopropane
(8.0 mmol), 2,4-dihydroxybenzaldehyde (6.0 mmol) and
sodium bicarbonate (6.0 mmol) in acetone (50 mL) at 60 8C
for 60 hours gave mixture of 4-(3-bromopropoxy)-2-
hydroxybenzaldehyde (2) and 4,40-(propane-1,3-diylbi-
s(oxy))bis(2-hydroxybenzaldehyde). Column chromatog-
raphy over silica gel gave pure 2 in 65% yield. Reaction of 1-
methylimidazole (3.5 mmol) with 2 (3.5 mol) at 80 8C for
48 hours gave a viscous liquid which was washed with
diethyl ether-ethyl acetate mixture to give ionic liquid
tagged aldehyde 3 in almost quantitative yield.
1H), 8.74 (s, 1H), 7.77 (t, J = 1.8 Hz, 1H), 7.67 (t, J = 1.7 Hz,
1H), 7.44 (d, J =8.2 2H), 7.41 (td, J = 8.8, 2.08 Hz, 2H), 7.31
(td, J = 8.8, 2.16 Hz, 2H), 6.50–6.42 (m, 2H), 4.44 (t,
J = 6.9 Hz, 1H), 4.12 (t, J = 5.8 Hz, 1H), 3.93 (s, 3H), 2.40–
2.34 (m, 2H); 13C NMR (126 MHz, DMSO-d6)
d 163.6, 163.3,
163.1, 147.3, 137.3, 134.7, 131.1, 129.8, 128.9, 124.1, 123.5,
122.9, 115.9, 113.6, 107.6, 101.8, 65.4, 46.8, 36.2, 29.4; ESI-
MS (m/z): 370.1 [M - Br]+, 372.1 [M + 2 - Br]+, 373.1 [M + 3 -
Br]+.
4d: Yield 833 mg (64%); mp 148-152 8C; IR (cmꢀ1
1609, 3378; 1H NMR (400 MHz, DMSO-d6)
13.52 (s, 1H),
)
d
9.36 (s, 1H), 8.74 (s, 1H), 7.80 (t, J = 1.8 Hz, 1H), 7.70 (t,
J = 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.37–7.34 (m, 2H),
7.18–7.14 (m, 2H), 6.53–6.28 (m, 2H), 4.45 (t, J = 6.8 Hz,
2H), 4.12 (t, J = 5.8 Hz, 2H), 3.93 (s, 3H), 2.40–2.34 (m, 2H);
4.3. General procedure for synthesis of ionic liquid supported
Schiff bases (4a-f)
A mixture containing ionic liquid tagged aldehyde
(3.0 mmol) and amine (4.0 mmol) in ethanol was refluxed
for 4 hours. On completion of the reaction, ethanol (30 mL)
was added to the reaction mixture; the solid product
formed was filtered off and washed with cold ethanol. The
crude product was purified by recrystallization from
ethanol/ethyl acetate (3: 1 v/v). All the compounds were
characterized by ESI MS and 1H NMR and 13CNMR
spectroscopic data.
13C NMR (126 MHz, DMSO-d6)
d 163.2, 163.1, 162.9, 162.1,
160.2, 144.8, 137.3, 134.6, 124.0, 123.5, 123.4, 122.9, 116.7,
116.5, 113.6, 107.5, 101.8, 65.4, 46.8, 36.2, 29.4; ESI-MS (m/
z): 354.17 [M - Br]+.
4e: Yield 910 mg (68%); mp 140-145 8C; IR (cmꢀ1) 1614,
3406; 1H NMR (400 MHz, DMSO-d6)
d 13.85 (s, 1H), 9.38 (s,
1H), 8.70 (s, 1H), 7.78 (t, J =1.8, 1H), 7.68 (t, J = 1.7, 1H), 7.40
(d, J = 8.3, 1H) 7.30 (td, J = 8.9, 2.16 Hz, 2H), 6.95 (td, J = 8.9,
2.16 Hz, 2H), 6.45–6.43 (m, 2H), 4.47 (t, J = 6.9 Hz, 2H), 4.11
(t, J = 5.8 Hz, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 2.40-2.34 (m,
Physical and spectral data for 4a-f:
4a: Yield 849 mg (68%); mp 81- 83 8C; IR cmꢀ11620,
2H); 13C NMR (126 MHz, DMSO-d6)
d 163.3, 162.5, 161.0,
3416; 1H NMR (400 MHz, DMSO-d6)
d
13.77 (s, 1H), 9.48 (s,
158.6, 141.0, 137.3, 134.3, 124.0, 122.9, 122.8, 115.9, 115.1,
114.9, 113.7, 107.3, 101.8, 65.3, 55.8, 55.7, 46.8, 36.2, 29.4;
ESI-MS (m/z): 366.2 [M - Br]+.
1H), 8.79 (s, 1H), 8.20 (t, J = 1.8, 1H), 7.90 (t, J = 1.8 Hz, 1H),
7.79–7.78 (m, 1H), 7.69 (s, 1H), 7.57–7.48 (m, 3H), 7.29 (d,
J = 7.2 Hz, 1H), 6.58–6.47 (m, 2H), 4.50 (t, J = 6.5 Hz, 2H),
4f: Yield 699 mg (50%); viscous liquid; IR (cmꢀ1) 1613,
4.15 (t, J = 5.1 Hz, 2H), 3.97 (s, 3H), 2.50–2.31 (m, 2H); 13
C
3389; 1H NMR (400 MHz, DMSO-d6)
d 13.70 (s, 1H), 9.43 (s,
NMR (126 MHz, DMSO-d6)
d
163.6, 162.9, 148.2, 148.7,
1H), 8.73 (s, 1H), 7.79 (t, J 1.9 Hz, 1H), 7.69 (t, J = 1.9 Hz, 1H),
7.74 (d, –J = 1H) 7.44–7.40 (m, 3H), 7.31–7.24 (m, 3H),
6.65–6.58 (m, 1H), 6.48–6.40 (m, 2H), 4.47 (t, J = 6.9 Hz,
2H), 4.12 (t, J = 5.8 Hz, 2H), 3.95 (s, 3H), 2.42–2.35 (m, 2H);
137.3, 134.7, 129.9, 129.3, 126.9, 124.0, 122.9, 121.6, 116.4,
114.6, 113.6, 107.5, 101.8, 65.4, 46.8, 36.2, 31.2, 29.4; ESI-
MS (m/z): 336.3 [M - Br]+.
4b: Yield 891 mg (60%); mp 180-184 8C; IR (cmꢀ1) 1612,
13C NMR (126 MHz, DMSO-d6)
137.3, 134.7, 134.1, 128.5, 128.2, 127.0, 126.8, 124.1, 122.9,
d 163.9, 163.4, 163.1, 145.7,
3425; 1H NMR (400 MHz, DMSO-d6)
d 13.42 (s, 1H), 9.35

B. Khungar et al. / C. R. Chimie 15 (2012) 669–674
673
122.8, 114.8, 114.1, 107.7, 101.9, 65.5, 46.8, 36.3, 29.4; ESI-
MS (m/z): 386.2 [M - Br]+.
determine the magnitude of antifungal activity. The
experiment was performed in three replicates.
MIC assay was performed to determine the lowest
concentration of compound that prevents any discernible
growth which can be detected visually. Assay was carried
out for all the compounds at 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0,
5. In vitro antibacterial assay
In vitro antibacterial activities were studied using the
64.0, 128.0
ing 10 mL of sterilized Czapek’s dox broth medium
(HiMedia, India) were inoculated with 100 l of 3 days
m
g mLꢀ1 concentrations. A set of tubes contain-
disc diffusion method [32] at
a concentration of
128
m
g mLꢀ1 for all the tested compounds with DMSO
m
as a solvent. For this purpose, Mueller–Hinton agar
medium (HiMedia, India) was prepared and sterilized by
autoclaving at 121 8C at 15 psi for 15 min. Medium was
poured into sterile petri dishes (90 mm diameter) under
aseptic conditions using laminar air flow chamber. After
solidification of medium, the suspension of test organism
(106 cfu mLꢀ1) was swabbed on to individual media plates
old fungal culture. Appropriate amount of compounds
were added to achieve the desired concentrations. The
tubes were incubated at 28 8C for 4 days and carefully
observed for the presence of turbidity.
Acknowledgements
using
a sterile glass spreader. A sterile disc (9 mm
This work was financially supported by the Department
of Science and Technology, New Delhi, India. We thank
Regional Sophisticated Instrumentation Centre (RSIC),
Chandigarh and Advanced Instrumentation Research
Facility (AIRF), JNU, New Delhi for spectral analysis.
diameter) impregnated with compound was placed over
media surface and the plates were incubated at 37 8C for 18
to 24 h under dark condition. Solvent (DMSO) was used as a
negative control. Broad spectrum antibiotic chloramphen-
icol at a concentration of 30 m
g mLꢀ1 was used as a positive
control to confirm the susceptibility of test organisms.
Three replicates were maintained for each compound and
their mean values were calculated.
Appendix A. Supplementary data
MIC values were evaluated for all the compounds (4a-f)
using broth macrodilution method as per the standard
guidelines [33]. Experiments were carried out for all the
compounds at 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0,
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.crci.2012.05.023.
128.0 m
g mLꢀ1 concentrations. A set of tubes containing
Mueller–Hinton broth medium with different concentra-
tions of compounds were prepared. The tubes were
inoculated with bacterial cultures (106 cfu mLꢀ1) and
incubated on a rotary shaker at 37 8C for 18 to 24 hours
under dark conditions. MIC value was defined as lowest
concentration of compound that prevented the visible
growth of bacteria after the incubation period. All the
experiments were performed in three replicates.
References
[1] R.D. Rogers, K.R. Seddon, Science 302 (2003) 792.
[2] R. Sheldon, Green Chem. 7 (2005) 267.
[3] P. Wasserscheid, W. Keim, Angew. Chem. Int. Ed. 39 (2000) 3773.
[4] M.J. Earle, J.M.S.S. Esperanc, M.A. Gilea, J.N.C. Lopes, L.P.N. Rebelo, J.W.
Magee, K.R. Seddon, J.A. Widegren, Nature 439 (2006) 831.
[5] H. Sakaebe, H. Matsumoto, Electrochem. Commun. 5 (2003) 594.
[6] A.J. Carmichael, M.J. Earle, J.D. Holbrey, P.B. McCormac, K.R. Seddon,
Org. Lett. 1 (1999) 997.
[7] N.V. Plechkova, K.R. Seddon, Chem. Soc. Rev. 37 (2008) 123.
[8] T. Welton, Chem. Rev. 99 (1999) 2071.
[9] A.V. Visser, R.P. Swatloski, R.D. Rogers, Green Chem. 2 (2000) 1.
[10] A.V. Visser, R.P. Swatloski, W.M. Reichert, S.T. Griffin, R.D. Rogers, Ind.
Eng. Chem. Res. 39 (2000) 3596.
6. In vitro antifungal assay
In vitro antifungal activities were determined by
[11] T.L. Greaves, C.J. Drummond, Chem. Rev. 108 (2008) 206.
[12] R. Giernoth, Angew. Chem. Int. Ed. 49 (2010) 2834.
[13] R.S. Timofte, S. Woodward, Tetrahedron Lett. 45 (2004) 39.
[14] A.E. Visser, R.P. Swatloski, W.M. Reichert, R. Mayton, S. Sheff, A.
Wierzbicki, J.H. Davis Jr., R.D. Rogers, Chem. Commun. (2001) 135.
[15] S.G. Lee, Chem. Commun. (2006) 1049.
standard agar well diffusion method at a concentration
of 128
Autoclaved Potato Dextrose Agar (PDA) medium (HiMedia,
India) was poured into sterile petri dishes (90 mm
diameter) under aseptic conditions in a laminar air flow
chamber. The medium was allowed to solidify and then
m
g mLꢀ1 for all the synthesized compounds [34].
[16] Z. Fei, T.J. Geldbach, D. Zhao, P.J. Dyson, Chem. Eur. J. 12 (2006) 2122.
[17] J.H. Davis Jr., Chem. Lett. 33 (2004) 1072.
[18] A. Busetti, D.E. Crawford, M.J. Earle, M.A. Gilea, B.F. Gilmore, S.P. Gor-
man, G. Laverty, A.F. Lowry, M. McLaughlin, K.R. Seddon, Green Chem.
12 (2010) 420.
[19] D. Kelman, Y. Kashman, E. Rosenberg, M. Ilan, I. Ifrach, Y. Loya, Aquat.
Microb. Ecol. 24 (2001) 9.
[20] J. Pernak, P. Chwala, Eur. J. Med. Chem. 38 (2003) 1035.
[21] J. Pernak, I. Goc, I. Mirska, Green Chem. 6 (2004) 323.
[22] S.K. Bharti, G. Nath, R. Tilak, S.K. Singh, Eur. J. Med. Chem. 45 (2010) 651.
[23] P. Vicini, A. Geronikaki, M. Incerti, B. Busonera, G. Poni, C.A. Cabras, P. la
Colla, Bioorg. Med. Chem. 11 (2003) 4785.
inoculated with 100 ml of 3 days old fungal broth culture.
After inoculation, wells of 9 mm diameter were prepared in
petri dishes using sterile metallic borer. A drop of the soft
agar was dropped into the well to seal the bottom. After
allowing agar solidification for 10 minutes, 100
ml of
compound solutions were added in respective wells and
the plates were incubated at 28 8C for 4 days under dark
conditions. DMSO and carbendazim (Methyl 1H-benzimi-
dazol-2-ylcarbamate, a standard antifungal antibiotic)
were used as negative and positive controls, respectively.
Mean diameter of inhibition zone was measured to
[24] S.M. Sondhi, N. Singh, A. Kumar, O. Lozach, L. Meijer, Bioorg. Med.
Chem. 14 (2006) 3758.
[25] D. Sinha, A.K. Tiwari, S. Singh, S. Gauri, G. Shukla, P. Mishra, H. Chandra,
A.K. Mishra, Eur. J. Med. Chem. 43 (2008) 160.

674
B. Khungar et al. / C. R. Chimie 15 (2012) 669–674
[26] K. Cheng, Q.Z. Zheng, Y. Qian, L. Shi, J. Zhao, H.L. Zhu, Bioorg. Med. Chem.
17 (2009) 7861.
[27] A. Kumar, M.K. Muthyala, Tetrahedron Lett. 56 (2011) 5368.
[28] M.K. Muthyala, A. Kumar, ACS Comb. Sci. 12 (2011) 5.
[29] M.K. Muthyala, B.S. Chhikara, K. Parang, A. Kumar, ACS Comb. Sci. 12
(2011) 60.
[32] Clinical and laboratory standards institute (NCCLS) 2006, Performance
standards for antimicrobial disk susceptibility tests; approved stan-
dard. 9th ed. M2-A9, Wayne, PA, USA.
[33] Clinical and laboratory standards institute (NCCLS) 2006, Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aero-
bically; approved standard. 7th ed. M7-A7, Wayne, PA, USA.
´
[30] M.S. Rao, I. Ahmad, B. Khungar, A. Kumar, Can. J. Chem. 87 (2009)
714.
[31] A. Kumar, M.S. Rao, I. Ahmad, B. Khungar, Aus. J. Chem. 62 (2009) 322.
[34] A. Espinel-Ingroff, E. Canton, in: R. Schwalbe, L. Steele-Moore, A.C.
Goodwin (Eds.), Antimicrobial susceptibility testing protocols, CRC
Press Taylor & Francis Group, 2007, 209.