Synthesis and antimicrobial profile of N-substituted imidazolium oximes and their monoquaternary salts against multidrug resistant bacteria

Article abstract of DOI:10.1016/j.bmc.2013.09.041

Two different series of N-substituted imidazolium oximes and their monoquaternary salts were synthesized and biologically tested with respect to their ability to inhibit growth a diverse panel of antibiotic susceptible Gram-positive and antibiotic resistant Gram-negative bacteria as well fungal strains. The newly synthesized compounds were analyzed by spectral studies to confirm their structure. The preliminary results showed that all compounds tested possess promising antimicrobial potential against both susceptible Gram-positive and antibiotic resistant Gram-negative isolates, exhibiting a wide range of MIC values from 0.14 to 100.0 μg/mL. The structure-activity relationship demonstrates that the p-methylphenyl and p-fluorophenyl groups in monoquaternary salts 6 and 7 attached directly to the imidazolium ring could be essential for observed remarkable inhibitory profiles against clinically important pathogens Pseudomonas aeruginosa (MIC = 0.14 μg/mL) and Klebsiella pneumoniae (MIC = 1.56 μg/mL). Furthermore, the broth microdilution assay was then used to investigate the antiresistance efficacy of compound 7 against fourteen extended-spectrum β-lactamase (ESBL)-producing strains in comparison to eight clinically relevant antibiotics. Compound 7 exhibited a remarkable antiresistance profiles ranging between 0.39 and 12.50 μg/mL against all of ESBL-producing strains, which leads to the suggestion that may be interesting candidate for development of new antimicrobials to combat multidrug resistant Gram-negative bacteria.

Full text of DOI:10.1016/j.bmc.2013.09.041

Bioorganic & Medicinal Chemistry 21 (2013) 7499–7506
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimicrobial profile of N-substituted imidazolium
oximes and their monoquaternary salts against multidrug resistant
bacteria
Renata Od zˇ ak , Mirjana Sko cˇ ibuši c´ , Ana Maravi c´ b
a
b,
⇑
a
Department of Chemistry, Faculty of Science, University of Split, Teslina 12, 21 000 Split, Croatia
Department of Biology, Faculty of Science, University of Split, Teslina 12, 21 000 Split, Croatia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2013
Accepted 16 September 2013
Available online 25 September 2013
Two different series of N-substituted imidazolium oximes and their monoquaternary salts were
synthesized and biologically tested with respect to their ability to inhibit growth a diverse panel of anti-
biotic susceptible Gram-positive and antibiotic resistant Gram-negative bacteria as well fungal strains.
The newly synthesized compounds were analyzed by spectral studies to confirm their structure. The pre-
liminary results showed that all compounds tested possess promising antimicrobial potential against
both susceptible Gram-positive and antibiotic resistant Gram-negative isolates, exhibiting a wide range
of MIC values from 0.14 to 100.0 lg/mL. The structure–activity relationship demonstrates that the
p-methylphenyl and p-fluorophenyl groups in monoquaternary salts 6 and 7 attached directly to the imi-
dazolium ring could be essential for observed remarkable inhibitory profiles against clinically important
pathogens Pseudomonas aeruginosa (MIC = 0.14 lg/mL) and Klebsiella pneumoniae (MIC = 1.56 lg/mL).
Keywords:
Imidazolium oximes
Monoquaternary salts
Synthesis
Antimicrobial activity
b-Lactamase
Multidrug resistance
ESBL
Furthermore, the broth microdilution assay was then used to investigate the antiresistance efficacy of
compound 7 against fourteen extended-spectrum b-lactamase (ESBL)-producing strains in comparison
to eight clinically relevant antibiotics. Compound 7 exhibited a remarkable antiresistance profiles ranging
between 0.39 and 12.50 lg/mL against all of ESBL-producing strains, which leads to the suggestion that
may be interesting candidate for development of new antimicrobials to combat multidrug resistant
Gram-negative bacteria.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
The therapeutic treatment of bacterial and fungal diseases with
extended-spectrum cephalosporins and b-lactamase inhibitors.1
Nonetheless, the recent increase and spread of acquired carbapen-
ems resistance due to the production of metallo-b-lactamases
antimicrobial drugs has had a remarkable and profound impact on
human health around the world. The current worldwide
emergence of resistance to extended spectrum cephalosporins,
monobactams, and carbapenems among Gram-negative patho-
genic bacteria constitutes an important growing public health
threat. Infections caused by extended-spectrum b-lactamase
(MBLs) such as SIM- and VIM-type enzymes already are starting
to limit the clinical use of carbapenems.
3
In addition to the infection control challenges that have arisen,
infections caused by these bacterial and fungal pathogens present
clinicians with serious treatment challenges, due to limited antibi-
4
otic options. New strategies are therefore needed to identify and
(
ESBL) producing pathogens have become a clinical and therapeu-
developed the next generation of drugs or agents with diverse
chemical structures and novel mechanisms of action to control
microbial infections. Thus, the search for effective molecules that
have the ability to restore the susceptibility of multi-drug-resistant
bacteria, such as clinical problematic ESBL- and MBL-containing
strains, to clinically available antibiotics are a promising alterna-
tive to the development of novel antimicrobials.
tic problem because these organisms are resistant not only to
b-lactam antibiotics but also to many other antimicrobial agents.
1
The dominant mechanisms for resistance to the Gram-negative
bacteria are the production of clinically relevant b-lactamases, par-
ticularly extended-spectrum b-lactamases (ESBLs), such as class A
TEM, SHV, and CTX-M b-lactamases.2 Single amino acid substitu-
tions in the SHV, TEM and CTX-M b-lactamases can drastically alter
the substrate profiles of the enzymes and confer resistance to
In this regard, the prevention and treatment of these infectious
diseases by applying heterocyclic imidazole derivatives as poten-
tial and promising sources of antimicrobial agents have been a fo-
cus of recent research for antibacterial drug discovery.5 Among
the numerous nitrogen heterocyclic compounds of biological and
,6
⇑
Corresponding author. Tel.: +385 21 385 133; fax: +385 21 384 086.
E-mail address: Mirjana.Skocibusic@pmfst.hr (M. Sko cˇ ibuši c´ ).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.041

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R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
pharmacological interest, imidazole derivatives play very impor-
tant role in chemistry as mediators for synthetic reactions primar-
ily for drug discovery to a broad field of biological applications.
Moreover, imidazole derivatives are structural bioisosteres of nat-
urally occurring nucleotides, which allows them to interact easily
with the biopolymers of the living system, which is responsible
for their promising biological activities and functions. In recent
decades, research has indicated that the imidazole derivatives
exhibiting a wide range of pharmacological activities, which in-
translate the oxime in the usual manner by reaction with hydrox-
1
8
ylammonium chloride in ethanol. In the last step, the final mono-
quaternary imidazolium oximes 5–8 were prepared by the
addition benzyl bromide in equimolar quantities to the solution
of appropriate N-substituted imidazolium oximes in dry ace-
5
,6
1
9–21
tone,
Figure 1. The reaction mixtures were kept at room tem-
perature without stirring in the dark for 2–3 days. The
crystallization was spontaneous. Acetone was removed and white
crystals were washed with dry diethyl ether and obtained in very
good yields.
7
8
9
cluded antitumor, antimicrobial, antiviral, antitubercular activi-
1
0
ties. Additionally, imidazoles are found to possess antifungal
activity and form an essential part of the molecular structures of
some antifungal agents. A wide variety of imidazole containing
compounds having high cytostatic and low cytotoxic effects on
several cancer cell lines with different mechanisms of action have
All synthesized monoquaternary oximes from series B, 5–8,
1
13
where identified by IR, 1D ( H and C (APT) and 2D (HETCOR)
NMR spectroscopy. Analytical and spectral data of these oximes
1
1
1
are given in Section 4. The synthesis and H NMR spectra of com-
2
1–23
pounds from series A, 1–4 were previously reported.
5
been developed over years. They have also been utilized as a ver-
satile template for the synthesis of compounds with potential the
2.1.2. Structural properties
inhibition of b-lactamase enzymes.1
2
The structures of compounds from series B, 5–8 were deter-
On the other hand, oximes represent versatile group of organic
compounds that were extensively used as excellent ligands in phar-
maceutical and synthetic applications as important precursor in
new drug discovery, including some antibiotics.13 Beside its antibac-
terial activity, oximes have been firstly reported as compounds with
a great potential in the treatment of organophosphorus compounds
poisoning including insecticides and nerve agents for acetylcholin-
esterase (AChE) reactivation.14 From the view point of molecular de-
sign, more efficacious compounds can be designed by joining two or
more biologically active system together in a single molecular
framework. In the view of above mentioned facts we synthesized
some N-substituted imidazolium oximes (series A) with phenyl, p-
methyl, p-fluorophenyl or benzyl substituent on imadazolim ring.
The most commonly used antibacterial reagents, benzalkonium
chloride is monoquaternary ammonium salts containing benzyl
moiety, so we also synthesized newly benzyl substituted com-
pounds (series B) from N-substituted imidazolium oximes. It has
been hoped that combination of these active groups in the new
molecular design would lead to the development of adjuvants mol-
ecules that either directly target resistance mechanisms such as the
inhibition of b-lactamase enzymes. Initially we wish to identify for
the first time antimicrobial efficacy of each compounds (from series
A and B) by using a disc diffusion assay as well as brothmicrodilution
assay against a panel of both susceptible Gram-positive and clini-
cally relevant multidrug resistant Gram-negative strains. Antifungal
activity was also assessed against several fungal strains. In the final
part of this study, we investigated the efficacy of the most potent
newly synthesized antimicrobial compound to reverse antibiotic
resistance in the clinical problematic ESBLs- and MBLs-containing
strains and additionally explore its mechanism of action.
mined by spectral analyses and the spectroscopic properties were
in accord with the proposed structures. The IR spectra showed in-
À1
tense absorption bands within 2690–3350 cm
range,
À1
À1
1428–1512 cm range and 980–990 cm that were attributed
to O–H, C@N and N–O function vibrations, respectively. In the 1
H
NMR spectra for the monoquaternary salts, the proton signals
due to the N–OH group were recorded between 13.00 and
13.03 ppm as singlet. The proton signals belonging to the CH-
1
NOH group appeared as singlet at 8.01–8.09 ppm. H NMR data
confirmed the quaternization of imidazole ring as expected, while
the imidazole hydrogen at the 4 and 5 positions of compounds 1–4
showed peaks between 7.16 and 7.43 ppm, the imidazolium
hydrogen at these positions in the final compounds 5–8 showed
peaks to a higher frequency, between 8.19 and 8.21 ppm, which
indicated that these compounds were quaternised with benzyl
1
3
bromide. All the C NMR findings and elemental analysis con-
firmed the structures proposed as indicated in the Section 4.
2.2. Biological evaluation
2.2.1. Antimicrobial activity
In this study, the antimicrobial efficacy of the imidazolium oxi-
mes from series A, 1–4 and newly synthesized monoquaternary
compounds from series B, 5–8 were preliminary screened against
a diverse panel of selected antibiotic susceptible Gram-positive
bacteria including Bacillus cereus ATCC 11778, Clostridium perfrin-
gens FNSST 4999, Enterococcus faecalis ATCC 29212, Staphylococcus
aureus ATCC 25923 and antibiotic resistant Gram-negative bacteria
namely Pseudomonas aeruginosa FNSST 014, Escherichia coli FNSST
982, Klebsiella pneumoniae FNSST 011 and Aeromonas hydrophila
FNSST 118. Antifungal activity was also assessed on the yeast Can-
dida albicans ATCC 6275 and filamentous fungi including Penicil-
lium funiculosum FNSST 3724 and Aspergillus fumigatus FNSST
2
2
2
. Results and discussion
.1. Chemistry
3
833 by disc diffusion assay. Cefotaxime, gentamicine and ampho-
tericin B were used as standard antibacterial as well as antifungal
agents.
.1.1. Design and synthesis
As shown in Table 1, the results of the disc diffusion assay indi-
cate that all of the compounds tested exhibit the most potent and
broad spectrum activity against bacterial and fungal strains tested.
The mean zones of inhibition of the target compounds against all
the bacterial strains tested were found in the range of 8.6 ± 1.1 to
N-Substituted imidazolium oximes 1–4 were prepared in three
steps according to the reported procedure. When the correspond-
ing N-substituted imidazole (except N-benzylimidazole) is not
commercially available, the first step in the synthesis is the aryla-
tion of nitrogen atoms in position 1 of imidazole ring with appro-
2
0.7 ± 0.4 mm. It is noteworthy that target compounds are not only
1
5
priate aryl halide with potassium carbonate as base.
The
active against antibiotic susceptible Gram-positive bacteria, but
also exhibit significant antibacterial effects against Gram-negative
isolates with various antibiotic resistance profiles. Among the
Gram-negative bacteria tested, two strains namely Pseudomonas
aeruginosa and Aeromonas hydrophila showed relative high
sensitivity towards the tested compounds with considerable zones
resulting N-substituted imidazoles can be translated into a 2-carb-
aldehyde by reacting with n-butyl lithium (n-BuLi), and then, with-
out isolating the unstable organolithium compounds addition of N,
16
N-dimethylformamide (DMF) reaction with formaldehyde and
17
then oxidation with SeO
2
.
Imidazole-2-carbaldehyde is easy to

R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
7501
+
R-X
N
N
H
a
R
N
N
N
N
N-benzylimidazole
R
is commercial material
c
H C
3
F
b
N
N
CH OH
2
d
N
N
R
HC
O
e
f
N
N
N
N
R
R
Br-
HC
HC
NOH
NOH
R
1
2
3
4
5
6
7
H C
3
F
8
.
Figure 1. Reagents and conditions: (a) K
2
3
CO , CuBr, nitrobenzen; (b) n-BuLi, DMF; (c) HCHO,
D
; (d) SeO
2
, dioxane; (e) NH
2
OH HCl, ethanol, pH 7–8; (f) C
6
H
5
CH
2
Br, acetone.
Table 1
Antimicrobial activity of substituted imidazolium oximes (1–4) and their monoquaternary salts (5–8) by disc diffusion assay
Microorganisms
No strains
Diameters of the inhibition zonea,c (mm)
No Compound
Standard antibioticb,c
CTX GEN
1
2
3
4
5
6
7
8
Gram-positive bacteria
Bacillus cereus
Enterococcus faecalis
Staphylococcus aureus
Clostridium perfringens
ATCC11778
15.4 ± 0.5 14.6 ± 0.5 14.0 ± 0.2 17.5 ± 0.1 15.0 ± 0.3 12.2 ± 1.3
18.3 ± 0.5 15.5 ± 0.3 26.8 ± 0.4 18.2 ± 0.3
19.6 ± 0.2 17.1 ± 0.8 23.5 ± 0.1 14.6 ± 1.4
19.9 ± 0.7 14.2 ± 0.2 21.7 ± 0.4 23.9 ± 0.1
11.4 ± 0.4 18.3 ± 0.6 28.5 ± 0.7 21.7 ± 0.4
ATCC 29212 15.7 ± 1.1 13.8 ± 0.7 12.2 ± 0.1 14.9 ± 0.3 13.3 ± 0.2 14.5 ± 0.2
ATCC 25923 10.1 ± 0.7 18.5 ± 0.1 12.4 ± 0.4 10.8 ± 0.9 12.5 ± 0.7 18.4 ± 0.6
FNSST 4999
16.5 ± 0.5 12.6 ± 0.3
8.6 ± 1.1 15.9 ± 0.5 16.6 ± 1.2 20.7 ± 0.4
Gram-negative bacteria
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa FNSST 982
Aeromonas hydrophilla
FNSST 982
FNSST 011
17.8 ± 0.3 14.9 ± 0.2 14.8 ± 0.9 17.3 ± 1.1 12.3 ± 0.4 17.7 ± 0.3
17.2 ± 1.5 9.3 ± 0.1 10.3 ± 0.0 15.8 ± 0.3 15.5 ± 0.1 16.3 ± 0.3
15.8 ± 1.5 10.9 ± 0.4 9.8 ± 0.3 17.9 ± 0.1 14.9 ± 0.3 20.6 ± 0.5
17.7 ± 1.5 15.7 ± 1.9 15.3 ± 0.1 10.1 ± 0.3 15.3 ± 0.8 16.6 ± 0.7
15.3 ± 0.5 14.2 ± 0.4 22.8 ± 0.3 11.5 ± 1.2
14.7 ± 0.6 18.6 ± 0.2 21.4 ± 1.2 18.2 ± 0.6
15.9 ± 0.4 12.4 ± 0.1 10.4 ± 0.9
9.2 ± 0.4
FNSST 014
16.5 ± 0.2 17.1 ± 0.3 14.7 ± 0.4 12.6 ± 0.1
Fungi
AMPHB
Candida albicans
Penicillium funiculosum
Aspergillus fumigatus
ATCC 10231 16.4 ± 1.5 20.2 ± 0.4 18.9 ± 0.2 17.2 ± 0.2 18.1 ± 0.2 20.8 ± 0.9
21.9 ± 0.6 20.9 ± 0.8
17.9 ± 2.2 17.6 ± 0.4
21.6 ± 0.0
18.3 ± 0.3
19.2 ± 0.4
FNSST 3724
FNSST 3833
17.9 ± 1.5 15.4 ± 0.2 18.6 ± 0.5 17.6 ± 0.4 17.4 ± 0.8 18.4 ± 0.0
19.5 ± 1.5 16.3 ± 0.1 15.0 ± 0.4 11.5 ± 0.1 20.8 ± 0.7 20.3 ± 0.5 19. 4 ± 1.2 20.9 ± 0.2
a
b
c
Diameter of inhibition zone (values in mm) around the disc: 250
l
g/disc.
Standard antibiotics disc: CTX, cefotaxime (30
Values are expressed as mean ± SE.
lg/disc), GEN, gentamicine (15 lg/disc), AMPHB, amphotericin B (10 lg/disc).

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R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
Table 2
Minimum inhibitory concentration of N-substituted imidazolium oximes (1–4) and their monoquaternary salts (5–8)
Compound
MIC (l
g/mL)
Structure
Gram-positive bacteria
Gram-negative bacteria
Bacillus
cereus
Enterococcus
faecalis
Staphylococcus
aureus
Clostridium
perfringens
Escherichia
coli
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Aeromonas
hydrophilla
HO
N
H
1
2
3
4
50.00
50.00
50.00
6.25
6.25
100.0
32.00
100.0
6.25
100.0
50.00
25.00
25.00
25.00
100.0
25.00
50.00
50.00
12.50
50.00
25.00
25.00
25.00
N
N
N
N
N
HO
N
H
H
H
CH3
50.00
25.00
6.25
100.0
100.0
50.00
100.0
100.0
25.00
N
HO
N
F
N
HO
N
N
HO
H
N
5
6
7
8
N
-
25.00
12.50
6.25
25.00
25.00
6.25
12.50
25.00
25.00
25.00
25.00
3.12
31.20
12.50
1.56
25.00
25.00
12.50
12.50
50.00
N
Br
HO
HO
HO
H
CH3
N
N
N
-
6.25
3.12
0.14
N
Br
H
F
N
-
6.25
12.50
25.00
N
N
Br
H
N
N
-
75.00
12.50
12.50
100.0
42.0
62.50
Br
Gentamicin
Cefotaxime
4.00
0.25
4.00
0.50
1.00
0.50
0.50
0.10
32.00
0.50
8.00
0.50
64.00
16.00
8.00
8.00

R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
7503
of growth inhibition and were even more effective than the
cefotaxime and gentamicin which were used as positive controls.
Moreover, the antifungal profiles of the these compounds was
found to be the most potent in inhibiting fungal growth, with mean
diameter of inhibition zones ranging from 11.5 ± 0.1 to
(MIC = 0.14 lg/mL) it is known to have high level of intrinsic
resistance to virtually all known antibiotics. In comparison with
relevant antibiotic compound 6 is 450-fold better than the genta-
micin and 115-fold better than the cefotaxime. Methyl group have
been added incrementally to the phenyl ring with the intention of
increasing lipophilicity and as a consequence improving the activ-
ity. This compound and compound 5 also showed excellent in vitro
2
1.9 ± 0.6 mm. It was also worth noting that monoquaternary imi-
dazolium oximes were active against the fungal strains tested,
which indicates a promising antifungal potential on important
pathogen such as Candida albicans ATCC 6275 when compared with
standard antifungal agent amphotericin B.
activity against Escherichia coli at MIC values of 3.12 lg/mL, which
was 10-fold more potent than the gentamicin. It is interesting to
note that benzyl derivatives 7 with substitution of p-fluorophenyl
of imidazole ring exhibited excellent in vitro activity against a
broad spectrum of clinically important resistant Gram-negative
2
.2.2. Minimum inhibitory concentrations (MICs)
The antimicrobial activity was also investigated by a broth mic-
pathogens such as Klebsiella pneumoniae (MIC = 1.56
richia coli, Aeromonas hydrophila (MIC = 12.5 g/mL) and Pseudo-
monas aeruginosa (MIC = 25.00 g/mL) which is 2.5 to 5-fold
better antimicrobial activity than the gentamicin. Moreover, this
compound also displayed excellent activity against Gram-positive
bacteria Bacillus cereus, Enterococcus faecalis and Staphylococcus
aureus, and MICs values were fourfold decrease (MIC =
lg/mL) Esche-
rodilution method to determine the minimum inhibitory concen-
trations (MICs) of the series A of substituted imidazolium oximes
and their quaternary salts, series B, necessary to inhibit the bacte-
rial growth. As a comparison, we also tested the MICs of relevant
conventional antimicrobial agents that are currently used in
clinical treatment, such as gentamicin and cefotaxime. The results
presented in Table 2 revealed that the all compounds tested
showed remarkable selectivity and effective activities against
l
l
6.25–25.00
lg/mL) when compared to their analog 3 (MIC =
25.00–100.0
lg/mL). This result may be substantiated on the basis
tested microorganisms with MIC values from 0.14 to 100.0
l
g/
of previous publications which demonstrated that the substitution
of fluorine atom at the p-position of the phenyl ring also increases
the positive charge density on the imidazolium cation. Also, fluo-
rine atom with high electronegativity, relatively small size, and
low polarisability of the C–F bond and, in many cases, increased
lipophilicity of fluorine containing organic molecules can have
mL. In general, compounds of series A (1–4), N-substituted imi-
dazolium oximes bearing phenyl, p-methylphenyl, p-fluorophenyl
and benzyl substituent on the imidazolium ring, displayed more
potent activity against the Gram-positive (6.25–100.0 lg/mL), then
against the Gram-negative bacteria tested which is in according to
the previous literature.24 Differences in the composition of the cell
wall of Gram-positive and Gram-negative bacteria and a possible
relationship with the lipophilic characteristics of the target
molecules could be helpful to explain the difference of these anti-
bacterial compounds. Interestingly, compound 4 among these un-
charged compounds, with benzyl substitutent demonstrated
excellent in vitro activity against Gram-positive and common
Gram-negative pathogens. Compared to other compounds from
these series, compounds 4 we found that to be 5- to 16-fold more
potent than other N-substituted imidazolium oximes against clin-
ically relevant pathogens such as Bacillus cereus ATCC 11778,
Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC
26
considerable influence their therapeutic potency. The results
suggested that introduction of a substituent, either electron-
donating (6) or electron-withdrawing (7), to the phenyl ring could
significantly enhance the antimicrobial potential.
Among quaternary oximes, compound 8 was less in vitro effec-
tive against growth of wide spectrum pathogens, with MIC values
ranging between 12.50 and 100.00 lg/mL. The compounds 8 is
symmetrical 1,3-disubstituted imidazolium salts containing two
benzyl groups exhibited 2- to 12-fold decreased antibacterial
efficacy then their uncharged analogue 4. That two hydrophobic
benzyl groups on imidazolium ring enhances not improve the anti-
bacterial activity indicate that the symmetry of the molecule or
other factors such as electronic properties or aromatic interac-
2
5923, with equal MIC values of 6.25 lg/mL. The structure activity
2
7
analysis suggested that the presence of a benzyl ring on the imi-
dazolium ring in the compound 4 could be significant for observed
promising inhibitory profiles against clinically important patho-
gens, including Gram-negative P. aeruginosa, with a MIC fivefold
lower than the MIC of standard antibiotic gentamicin.
tion could be contribute to reduced activity might be to play a
role in the antimicrobial efficacy of this compound. Whether differ-
ences in antimicrobial activities are due to differential uptake or
different
intramolecular
interactions
requires
further
investigation.
Furthermore, the data indicate that, in general, the monoqua-
ternary derivatives of substituted imidazolium oximes are more
potent with broad spectrum antimicrobial activity compared to
their uncharged analogs, with the exception of compound 8. These
results suggested that the quaternization nitrogen atom of the imi-
dazolium ring can yield differences in both potency and spectrum
of antimicrobial activity and that is in accordance with previous
studies that confirmed that the higher electron density, the higher
2.2.3. Antiresistance profiles
Among the synthesized imidazolium oximes, compound 7
demonstrated the strongest efficacy against growth of broad
spectrum clinically relevant Gram-negative bacterial strains,
with MICs values in the range from 6.25 to 25.00 lg/mL. There-
fore, it was selected to verify its ability to inhibit the growth of
fourteen clinically and environmental multidrug isolates resis-
tant to third-generation cephalosporins and carbapenems via
expression of molecular classes A and B b-lactamase enzymes.
The antiresistance profiles of compound 7 was investigated by
the determination of the minimum inhibitory concentrations
(MICs) in comparison with different classes of antibiotics includ-
ing ceftazidime, cefotaxime, ciprofloxacin, gentamicin, tetracy-
cline, tobramycin, imipenem and meropenem. As shown in
Table 3, compound 7 treatment significantly reduced resistance
level in a variety of ESBL and MBL-producing isolates, with MICs
2
5
the antimicrobial activity of quaternary ammonium compounds.
Minimum inhibitory concentrations demonstrated that benzyl
derivatives 5–7 were found to be the most superior compounds,
especially against Gram-negative bacteria exhibited range of MIC
values from 0.14 to 25.0 lg/mL, when compared to the their
uncharged analogs (1–3). All these compounds showed good
antimicrobial activities especially for Escherichia coli which is 2.5
to 10-fold better than gentamicin. Monoquaternary imidazolium
oximes 6 and 7 were found to be the most potent compounds in
both series, with MIC values ranging from 0.14 to 25.00
l
g/mL
values ranging from 0.39 to 12.50 lg/mL when compare to the
and 1.56 to 25.00 g/mL, respectively. However, compounds 6
l
activity of eight broad-spectrum antibiotics. The promising anti-
with p-methylphenyl substituent on imidazolium ring exhibited
resistance efficacy was obtained against Pseudomonas aeruginosa
promising antimicrobial effect on Pseudomonas aeruginosa
197 producing GIM-1 metallo b-lactamase (MIC = 0.39 lg/mL),

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R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
Table 3
Minimum inhibitory concentration of monoquaternary imidazolium oxime (7) (
l
g/mL) against multidrug resistant Gram-negative ESBL and MBL-producing isolates
Isolates
Compound 7 MIC (
lg/mL) ESBL genotype
MBL genotype
Standard antibiotics MICa
(
lg/mL)
CAZ
CTX
CIP
GEN TET TOB IMP
MEM
Stenotrophomonas maltophilia 125 12.50
CTX-15
128
128
32
64
0.25
2.00 32
2
128 8.0
256 2.0
0.12
0.12
0.25
0.25
8
128
Klebsiella pneumoniae 303
Enterobacter cloacae 62
Enterobacter cloacae 306
Enterobacter cloacae 51
Enterobacter intermedius 243
Aeromonas hydrophila 268
Aeromonas caviae 258
Aeromonas caviae 260
Escherichia coli 4024
12.50
6.25
6.25
6.25
6.25
12.50
12.50
12.50
1.56
3.12
6.25
0.78
0.39
TEM-1, SHV-12
TEM-1, CTX-M-15
TEM-1, SHV-12
CTX-15
TEM-1, SHV-12, CTX 15
TEM-1, SHV-12
CTX-15
TEM-1, CTX-M-15
CTX-15
TEM-1, SHV-12
CTX-15
0.12
0.50
0.25
0.25
0.12
0.06
0.125
1.00
0.12
>256 >256 0.25
>256 >128 0.12 256
16
8
64
4
128
16
8
4
16
16
64
16
8
32
16
32
8
256
258
256
256
1.00 128
0.50
2.00
0.50 12.8 64
1.00 32
0.25 64
>32
>32 0.12
16
32
2
4
0.06
0.25
2.00
0.25
0.125 1.00
0.06
8
128 32
16 32
128 64
Escherichia coli 237
>256 0.50
>256 0.50 16
8
Escherichia coli 235
64
32
64
>8
16
16
16
0.12
16
>8
Acinetobacter baumannii 170
Pseudomonas aeruginosa 197
SIM-1
GIM-1
>256 >256 0.50 16
256 256 >4 >8
>8
a
MIC values highlighted by shading represent resistance according to the breakpoints established by CLSI (M100-S17, 2010). Abbreviations of standard antibiotics: CAZ,
ceftazidime; CTX, cefotaxime; CIP, ciprofloxacin; GEN, gentamicin; TET, tetracycline; TOB, tobramycin; IMP, imipenem; MEM, meropenem.
which was 20-fold more potent than the imipenem and
4. Experimental section
meropenem, as well as against Acinetobacter baumannii 170 pro-
ducing SIM-1 metallo b-lactamase (MIC = 0.78
l
g/mL). Moreover,
4.1. Materials and general methods
the significant antiresistance potential of compound 7 was also
seen against three isolates of Escherichia coli ESBL-producing
Reagents and solvents used for synthesis were purchased from
Aldrich, USA. Reactions were monitors by thin-layer chromatogra-
phy using DC-Alufolien Aluminiumoxide 60 F254 plates (Merck)
with 9:1 chloroform-methanol as the eluent. The detection of spots
was achieved by UV light and by the reversible absorption of io-
dine. Melting points were determined in open capillaries using a
Stuart Scientific digital melting point apparatus SMP30 and are
uncorrected. FTIR spectra were recorded on a Bruker VECTOR
with MIC values ranging from 1.56 to 6.25
lg/mL. Compound
7
also demonstrated good activity by inhibiting the b-lactamases
produced by strains of the Enterobacter species including Enter-
obacter intermedius 243, Enterobacter cloacae 62, 306, and 51 at
concentration of 6.25
. Conclusions
In summary, we have investigated antimicrobial activity of
lg/mL.
3
2
2FT/IR spectrometer. All samples were prepared by mixing
FTIR-grade KBr (Sigma–Aldrich) with 1% (w/w) salt and grinding
À1
some N-substituted imidazolium oxime 1–4 and their monoqua-
ternary salts 5–8. The final synthesized compounds characterized
by spectral data (IR, 1D and 2D NMR). Among uncharged com-
pounds (1–4), it was found that the benzyl group as substituent
on imidazolium ring had the specific activity against Gram-positive
bacteria, including Gram-negative Pseudomonas aeruginosa with
MIC fivefold lower than MIC of gentamicin. Further, the results of
the present study indicated that monoquaternary oximes showed
better antimicrobial activity than they uncharged analogs with
exception of symmetrical compounds 8 with two benzyl groups
on imidazolium ring. Remarkable activity was found in quaternary
compounds carrying a p-methylphenyl or p-fluorophenyl substitu-
ent on the imidazolium ring. The MICs of the most active deriva-
to a fine powder. Spectra were recorded over the 400–4000 cm
range without baseline corrections. Characteristic absorptions are
À1
1
13
given in cm
. H and C NMR spectra were recorded in DMSO-
d
6
solution on a Bruker AV 300 spectrometer at room temperature.
Chemical shifts were measured in with TMS as internal reference.
Chemical shifts are reported as d values in ppm using TMS as an
internal standard. Abbreviations for data quoted are: s, singlet; d,
doublet; m, multiplet. Imidazole hydrogen was indicated as Im,
phenyl as Ph while benzyl hydrogen indicated as Bnl. Elemental
analysis were performed with a Perkin-Elmer PE 2400 Series II
CHNS/O Analyzer.
4.2. Synthesis
tives (6 and 7) were shown to be law as 0.14
aeruginosa and 1.56 g/mL against Klebsiella pneumoniae. A simple
inspection of Table 2 indicates that the presence of electron-donat-
ing (CH ) or electron-withdrawing (F) groups at C-4 of the phenyl
ring is mandatory for the broad spectrum antimicrobial activity
and with exception of P. aeruginosa and K. pneumoniae, the result-
ing activity is superior of the standards gentamicin and cefotaxime,
for example compound 6 is 450-fold better than gentamicin and
lg/mL against P.
l
4.2.1. General procedure for synthesis of quaternary derivatives
5–8
N-substituted imidazolium oxime (1.00 mmol) and benzyl bro-
mide (1.00 mmol) were stirred in acetone (15 mL) at room temper-
ature for 24 or 36 h in the dark. The precipitate was filtered and
washed with ether.
3
1
15-fold better than cefotaxime.
These results clearly indicate that compound 7 possess the abil-
4.2.1.1. N-Benzyl-2-hydroxyiminomethyl-3-phenylimidazolium
À1
bromide (5).
Yield 93%; mp: 190.3–191.5 °C; IR (
m
, cm ):
1
ity to inhibit the growth of fourteen clinically and environmental
b-lactamases producing isolates resistant to third-generation
cephalosporins and carbapenems. However, further work is needed
to optimize these compounds for specificity; efficacy and
experimental validation which may serve as adjuvant for the treat-
ment of multidrug resistant bacterial infections.
990 (N–O), 1595 (C@N), 2700–3300 (O–H); H NMR d: 5.73 (s,
2H, CH Bnl), 7.73–7.75 (m, 5H, Ph), 8.08 (s, 1H, CH@NOH),
8.14–8.21 (m, 7H, 2H Im and 5H Bnl), 13.00 (s, 1H, CH@NOH);
2
1
3
2
C NMR d: 52.09 (CH Bnl), 124.35 (C-4 Im), 126.10 (C-2 and C-6
Ph), 126.37 (C-5 Im), 127.96 (C-4 Bnl), 128.51 (C-4 Ph), 129.89
(C-3 and C-5 Bnl), 130.70 (C-2 and C-6 Bnl), 134.35 (C-1 Bnl),

R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
7505
6
1
(
34.54 (C-3 and C-5 Ph), 134.60 (C-1 Ph), 140.23 (C-2 Im), 151.17
CH=NOH) ppm. Anal. Calcd for C17 OBr (M: 358.23) C, 56.99;
H, 4.50; N, 11.73. Found: C, 57.05; H, 4.54; N, 11.80.
to the CLSI guidelines. Briefly, 100
l
L of suspension containing 10 -
4
H
16
N
3
colony-forming units (cfu)/mL of bacterial cells and 10 cfu/mL
spore for fungal strains was spread on a Mueller Hinton agar (Becton
Dickinson, Sparks, MD) and Sabouraud dextrose agar (SDA; Becton
Dickinson, Sparks, MD). The stock solutions of synthesized com-
pounds were prepared by dissolving in 96% ethanol to a final con-
centration of 10 mg/mL. The sterile filter discs (6 mm) were
4
.2.1.2. N-Benzyl-2-hydroxyiminomethyl-3-(p-methyl)phenyl-
imidazolium bromide (6).
IR (
Yield 90%; mp: 211.5–212.6 °C;
, cm ): 990 (N–O), 1595 (C@N), 2700–3300 (O–H); H NMR
À1
1
m
d: 3.32 (s, 3H, CH
3
-Ph), 5.74 (s, 2H, CH
2
Bnl), 7.46–7.65 (m, 5H,
individually loaded with 25
lL of the stock solution, equivalent to
Bnl), 8.09 (s, 1H, CH@NOH), 8.15–8.16 (m, 4H, Ph), 8.19–8.20 (m,
a final concentration at 250
and then placed on the nutrient agar that had been previously inoc-
ulated with the target microbial strains. Additionally, 96% ethanol
was used as a negative control, ampicillin (30
l
g/disc of synthesized compounds
2
5
1
1
1
1
H, 2H, Im), 13.01 (s, 1H, CH@NOH); 13C NMR d: 21.20 (CH
3
-Ph),
2.53 (CH Bnl), 121.12 (C-4 Im), 126.61 (C-2 and C-6 Ph),
2
28.44(C-5 Im), 128.99 (C-4 Bnl), 129.35 (C-3 and C-5 Bnl),
30.75 (C-2 and C-6 Bnl), 132.62 (C-1 Ph), 134.89 (C-1 Bnl),
35.06 (C-3 and C-5 Ph), 137.60 (C-4 Ph), 141.12 (C-2 Im),
l
g) gentamicin
g) were used
(15 g) and commercial fungicide amphotericin B (10
l
l
as positive controls. The plates were incubated for 24 h at 37 °C for
bacterial strains and 48 h or 72 h at 30 °C for yeast and mold isolates,
respectively. Antibacterial activity was assessed by measuring the
diameter of the inhibition zone in millimeters, including disc diam-
eter for the test isolates, compared to the controls. Samples were as-
sayed in triplicate for each condition and the diameter of inhibition
zones were presented as mean ± SE values.
18 3
52.70 (CH@NOH) ppm. Anal. Calcd for C18H N OBr (M: 372.25)
C, 58.07; H, 4.87; N, 11.29. Found: C, 58.20; H, 4.90; N, 11.35.
4
.2.1.3. N-Benzyl-2-hydroyximinomethyl-3-(p-fluoro)phenyl-
imidazolium bromide (7).
Yield 91%; mp: 227.4–228.5 °C;
À1
1
IR (m, cm ): 990 (N–O), 1595 (C@N), 2700–3300 (O–H); H NMR
d: 5.73 (s, 2H, CH
2
Bnl), 7.38–7.54 (m, 5H, Bnl), 8.01 (s, 1H,
CH@NOH), 8.15–8.20 (m, 6H, 2H, Im and 4H, Ph), 13.01 (s, 1H,
4.3.2. Minimum inhibitory concentration assay
1
3
CH@NOH); C NMR d: 52.50 (CH
2
Bnl), 117.14 (C-4 Im), 117.45
In addition, antimicrobial activities of the synthesized com-
pounds were tested by a broth microdilution assay in 96 well plates.
The standard two fold serial microdilution assay described by the
Clinical and Laboratory Standards Institute was performed for the
assessment of the minimum inhibitory concentrations (MICs). Bac-
teria and fungi were grown overnightin Mueller-Hilton broth(MHB)
at 37 °C and Sabouraud dextrose broth (SDB) at 30 °C to the station-
ary phase. The microbial cultures were diluted in fresh MHB and SDB
(
C-3 and C-5 Ph), 128.42 (C-5 Im), 129.01 (C-2 and C-6 Ph),
1
29.37 (C-4 Bnl), 129.48 (C-3 and C-5 Bnl), 129.60 (C-2 and C-6
Bnl), 134.83 (C-1 Ph), 135.07 (C-1 Bnl), 141.82 (C-2 Im), 152.20
(
CH@NOH), 163.12 (C-4 Ph) ppm. Anal. Calcd for C17
15 3
H N OBrF
(
M: 376.22) C, 54.27; H, 4.02; N, 11.17. Found: C, 54.30; H, 4.10;
N, 11.31.
6
4
4
.2.1.4. N-Benzyl-2-hydroxyiminomethyl-3-benzylimidazolium
to a final concentration of 10 CFU/mL for bacteria and 10 CFU/mL
for fungal strains. The synthesized compounds was first dissolved
in DMSO and incorporated into aqueous nutrient medium to obtain
a concentration of 10 mg/mL. The stock solution was then serially
two fold diluted to obtain concentrations ranging from 400 to
À1
bromide (8).
Yield 94%; mp: 215.2–216.8 °C; IR (
m, cm ):
9
4
90 (N–O), 1595 (C@N), 2700–3300 (O–H); 1H NMR d: 5.66 (s,
H, CH Bnl), 7.32–7.43 (m, 12H, 2H Im and 10H Bnl) 8.02 (s, 1H,
2
13
CH@NOH), 13.03 (s, 1H, CH@NOH); C NMR d: 52.23 (2Â CH
2
Bnl), 124.61 (C-4 and C-5 Im), 128.14 (2Â C-4 Bnl), 129.55 (2Â
C-3 and C-5 Bnl), 130.05 (2Â C-2 and C-6 Bnl), 138.22 (2Â C-1
Bnl), 143.12 (C-2 Im), 151.70 (CH@NOH) ppm. Anal. Calcd for C18-
0.09 lg/mL in sterile plates containing Mueller Hinton broth
(MHB) for bacterial as well as Sabouraud dextrose broth (SDB) for
the fungi. Serial dilutions of the synthesized compounds were added
H
18
N
3
OBr (M: 372.25) C, 58.07; H, 4.87; N, 11.29. Found: C, 58.11;
to the microtiter plates in a volume of 100
lL. Each well was addi-
H, 4.93; N, 11.37.
tionally inoculated with 10 L of inoculums of the target microor-
l
ganism and incubated at 37 °C for 18–24 h for bacterial and at
30 °C for 48 h for fungal strains. The MIC value was determined as
the lowest concentration of the sample at which the tested microor-
ganisms did not demonstrate any visible growth after incubation. As
4
.3. Biological methods
All of the newly synthesized target compounds were evaluated
for their in vitro antibacterial activity. The tested microorganisms
were obtained from the culture collection at the American Type
Culture Collection (ATCC) (Rockville, MD, USA) and at the Microbi-
ology laboratory, Department of Biology, Faculty of Natural Sci-
ence, University of Split, Croatia (FNSST). The assayed collection
included four Gram-positive bacteria Bacillus cereus (ATTC
an indicator of bacterial growth, 50 lL of 0.2 mg/mL p-iodonitrotet-
razoliumchloride (INT; Sigma-Aldrich Co. Ltd, Poole, UK) was added
to the wells and incubated at 37 °C for 30 min. Following addition of
INT and incubation, the MIC was determined as the lowest sample
concentration at which no pink color appeared. Cefotaxime, genta-
micin and amphotericin B were used as positive controls. Minimum
inhibitory concentrations of the commercial antibiotics were deter-
mined by the E-test (AB Biodisk, Solna, Sweden). Each assay in this
experiment was replicated three times. The MIC was interpreted
as the point of intersection of the inhibition ellipse with the E-test
strip edge. In this study, no bioactivity was defined as a MIC
1
1778), Enterococcus faecalis (ATCC 29212), and Staphylococcus aur-
eus (ATCC 25923) Clostridium perfringens (FNSST 4999) and four
Gram-negative ampicillin-resistant bacterial strains Escherichia coli
(
FNSST 982), Klebsiella pneumoniae (FNSST 011), Pseudomonas aeru-
ginosa (FNSST 982) and Aeromonas hydrophilla FNSST 014. Anti-
fungal activity was assessed on the yeast Candida albicans (ATCC
>1000
mL, moderate bioactivity as a MIC in the range 128–512
bioactivity as a MIC in the range 32–128 g/mL, strong bioactivity as
a MIC in the range 10–32 g/mL and very strong bioactivity as a MIC
<10 g/mL.
lg/mL, mild bioactivity as a MIC in the range 512–1000
lg/
1
0231) and fungal strains Penicillium funiculosum (FNSST 3724)
l
g/mL, good
and Aspergillus fumigatus (FNSST 3833). Bacterial strains were cul-
tured overnight at 37 °C in tryptic soy broth (TSB) and fungi were
cultured overnight at 30 °C in Sabouraud dextrose broth (SDB) to
l
l
l
6
achieve optical densities corresponding to 10 colony forming
4
units (cfu/mL) for bacteria and 10 CFU/mL for fungal strains.
Acknowledgments
-
4
.3.1. Disc diffusion assay
In order to investigate the antimicrobial activities of the synthe-
The authors thank Professor Srdanka Tomi c´ for general support
and for discussion throughout the study. This work was financially
supported by the Ministry of Science, Education and Sport of the
sized compounds, a disc diffusion assay was employed according

7
506
R. Od zˇ ak et al. / Bioorg. Med. Chem. 21 (2013) 7499–7506
Republic of Croatia, Research Project No. 177-0000000-3182 and
19-1191344-3121.
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4. Dawson, R. M. J. Appl. Toxicol. 1994, 14, 317.
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