Novel imidazole aldoximes with broad-spectrum antimicrobial potency against multidrug resistant gram-negative bacteria

Article abstract of DOI:10.3390/molecules23051212

In the search for a new class of potential antimicrobial agents, five novel N-substituted imidazole 2-aldoximes and their six quaternary salts were evaluated. The antimicrobial activity was assessed against a panel of representative Gram-positive and Gram-negative bacteria, including multidrug resistant bacteria. All compounds demonstrated potent in vitro activity against the tested microorganisms, with MIC values ranging from 6.25 to 50.0 μg/mL. Among the tested compounds, two quaternary compounds (N-but-3-enyl- and meta- (10) or para- N-chlorobenzyl (11) imidazolium 2-aldoximes) displayed the most potent and broad-spectrum activity against both Gram-positive and Gram-negative bacterial strains. The broth microdilution assay was also used to investigate the antiresistance efficacy of the both most active compounds against a set of Enterobacteriaceae isolates carried a multiple extended-spectrum β-lactamases (ESBLs) in comparison to eight clinically relevant antibiotics. N-but-3-enyl-N-meta-chlorobenzyl imidazolium 2-aldoxime was found to possess promising antiresistance efficacy against a wide range of β-lactamases producing strains (MIC 2.0 to 16.0 μg/mL). Best results for that compound were obtained against Escherichia coli and Enterobacter cloacae producing multiple β-lactamases form A and C molecular classes, which were 32- and 128-fold more potent than ceftazidime and cefotaxime, respectively. To visualize the results, principal component analysis was used as an additional classification tool. The mixture of ceftazidime and compound 10 (3 μg:2 μg) showed a strong activity and lower the necessary amount (up to 40-fold) of 10 against five of ESBL-producing isolates (MIC ≤ 1 μg/mL).

Full text of DOI:10.3390/molecules23051212

molecules
Article
Novel Imidazole Aldoximes with Broad-Spectrum
Antimicrobial Potency against Multidrug Resistant
Gram-Negative Bacteria
1,
3
3,†
Mirjana Skocˇibušic´
*
ID , Renata Odžak 2 ID , Alma Ramic´ ID , Tomislav Smolic´
,
Tomica Hrenar 3 and Ines Primožicˇ
*
3,
1
Department of Biology, Faculty of Science, University of Split, R. Boškovic´a 33, HR-21 000 Split, Croatia
Department of Chemistry, Faculty of Science, University of Split, R. Boškovic´a 33,
HR-21 000 Split, Croatia; rodzak@pmfst.hr
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a,
HR-10 000 Zagreb, Croatia; alma.ramic@gmail.com (A.R.); Tom_Smolic@cargill.com (T.S.);
tomica.hrenar@chem.pmf.hr (T.H.)
2
3
*
†
Correspondence: mirskoc@pmfst.hr (M.S.); ines.primozic@chem.pmf.hr (I.P.); Tel.: +385-1-460-6408 (I.P.)
Present Address: Cargill Food Ingredients & Bio-Industrial, Cargill 84 Havenstraat, Vilvoorde,
Vlaams-Brabant 1800, Belgium.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 April 2018; Accepted: 16 May 2018; Published: 18 May 2018
Abstract: In the search for a new class of potential antimicrobial agents, five novel N-substituted imidazole
2-aldoximes and their six quaternary salts were evaluated. The antimicrobial activity was assessed
against a panel of representative Gram-positive and Gram-negative bacteria, including multidrug
resistant bacteria. All compounds demonstrated potent in vitro activity against the tested
microorganisms, with MIC values ranging from 6.25 to 50.0 µg/mL. Among the tested compounds,
two quaternary compounds (N-but-3-enyl- and meta- (10) or para- N-chlorobenzyl (11) imidazolium
2-aldoximes) displayed the most potent and broad-spectrum activity against both Gram-positive
and Gram-negative bacterial strains. The broth microdilution assay was also used to investigate
the antiresistance efficacy of the both most active compounds against a set of Enterobacteriaceae
isolates carried a multiple extended-spectrum
relevant antibiotics. N-but-3-enyl-N-meta-chlorobenzyl imidazolium 2-aldoxime was found to possess
promising antiresistance efficacy against a wide range of -lactamases producing strains (MIC
2.0 to 16.0 g/mL). Best results for that compound were obtained against Escherichia coli and
Enterobacter cloacae producing multiple -lactamases form A and C molecular classes, which were
β-lactamases (ESBLs) in comparison to eight clinically
β
µ
β
32- and 128-fold more potent than ceftazidime and cefotaxime, respectively. To visualize the results,
principal component analysis was used as an additional classification tool. The mixture of ceftazidime
and compound 10 (3 µg:2 µg) showed a strong activity and lower the necessary amount (up to 40-fold)
of 10 against five of ESBL-producing isolates (MIC ≤ 1 µg/mL).
Keywords: imidazole 2-aldoximes; quaternary imidazolium salts; antimicrobial activity; extended-
spectrum β-lactamase (ESBL); multivariate analysis; multidrug resistance
1. Introduction
Over the past decade, the widespread emergence of antibiotic drug resistance has resulted in
a worldwide health crisis of global dimensions. The continuous emergence and proliferation of
pathogenic bacteria resistant to many or all current antibiotics is a major public health concern and one
of the particular importance in clinical settings. The most recent World Economic Forum Global Risks
reports [1] have listed antibiotic resistance as one of the greatest threats to human health. Meanwhile,
Molecules 2018, 23, 1212; doi:10.3390/molecules23051212
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Molecules 2018, 23, 1212
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resistance rates around the world are rising, new resistance mechanisms are emerging, and infections
caused by multidrug-resistant Gram-negative bacteria are becoming particularly difficult to treat.
The
diseases. Among clinically important Gram-negative bacteria, the production of
most frequent factor contributing to -lactam resistance [ ]. The molecular classification of
is based on the amino acid sequence and divides -lactamases into class A, C and D enzymes which
employ a serine residue as the nucleophilic species to attack the lactam carbonyl group, forming an
acyl enzyme intermediate before hydrolysis [ ]. Almost invariably, however, mutations or new
enzymes have emerged that enable the recognition and hydrolysis of the latest generation of -lactam
antibiotics. A new group of class A extended-spectrum -lactamases (ESBLs), called CTX-M enzymes,
has emerged worldwide and it is now the most frequently observed extended-spectrum -lactamases
β
-lactam antibiotics are essential for the treatment of a wide range of human bacterial
-lactamases is the
-lactamase
β
β
2
β
β
3
β
β
β
in several areas. CTX-M enzymes hydrolyse not only the penicillin, but also the first-, second and
third-generation cephalosporins and the current lack of effective treatment options is the reason for
increasing number of invasive bacterial infections caused by ESBL-producing pathogens [
4]. Of great
concern is the increasing ability of the bacteria to frequently harbour multiple
β-lactamases with
different substrate profiles, making virtually all β-lactam antibiotics ineffective.
To restore their antibacterial activity against Gram-negative pathogens,
paired with inhibitors of -lactamases. The currently available, of the three classical
inhibitors such as clavulanic acid, sulbactam and tazobactam, are derived from -lactam scaffolds,
are now met with an increasingly prevalent panel of inhibitor-resistant bacterial strains [ ].
Unfortunately, -lactamases have also evolved resistance to inhibitors; point mutations in blaTEM
and blaSHV gave rise to amino acid substitutions in the parent enzyme that result in inhibitor resistant
-lactamases causing elevated minimum inhibitory concentration (MIC) to -lactam/ -lactamase
inhibitor combinations. These limitations, as well as the widespread bacterial production of multiple
-lactamases including class-A, class-C ESBLs has motivated several research groups to search for new
inhibitors with a non-lactam structural scaffold as well with a broad-spectrum inhibition profile [ ].
β-lactams have been
β
β-lactamase
β
5
β
β
β
β
β
6,7
Imidazole is a five-membered aromatic heterocycle which contains two nitrogen atoms and is
present in the structure of many different molecules and intermediates of biological significance,
including purines, many cofactors, and histidine and synthetic bioactive molecules [8]. The imidazole
ring is amphoteric and highly polar, can easily accept or donate protons, readily form different weak
interactions and has multiple binding sites which allows it to interact easily with the biopolymers
of living systems. Based on several literature surveys, imidazole-based small molecules have
been described as promising pharmaceutical agents in view of their unique bioactivity patterns,
ranging from antioxidant, anti-inflammatory, analgesic, and neuroprotective to chemotherapeutic
and anti-HIV activities [8–11]. Imidazoles and in particular their salts comprise a boundless and
emerging field, due to not only their favourable properties such as excellent bioavailability, good tissue
penetrability and permeability but also a relatively low incidence of adverse and toxic effects [12].
So far, many imidazole-based derivatives (Figure 1) have been successfully developed and employed
as clinical drugs [13].
Metronidazole, a nitroimidazole compound, is one of the most commonly used antimicrobial
agents, which was initially found to be effective particularly for the treatment of parasitic infections.
Since then, this compound has also played an important role for the treatment of infections caused
by a variety of anaerobic and microaerophilic bacteria, including Helicobacter pylori which has been
strongly associated with gastritis and duodenal ulcers [14]. In addition, it has also been recognized [15
]
that many imidazole-containing ligands could exhibit large chemical-shift variations when bound
to a molecular target, such as a protein, offering valuable information about changes in the local
structure of the ligand or target. Among others, imidazole core structures are found in different
carboxypeptidase, hemeoxygenase and β-lactamase inhibitors [16,17] as well as among compounds
with anti-inflammatory, anticancer, antibacterial, antifungal, antitubercular, antidiabetic and antiviral
activity [18–23].

Molecules 2018, 23, 1212
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Figure 1. Some pharmaceuticals having imidazole cores.
Additionally, oximes and their complexes caught our attention, as they appear to be due not
only amenable to biotransformation and conjugations with organic and inorganic molecules but also
serve as the important templates of the novel antimicrobial drugs for the treatment of infections
caused by wide range of multidrug-resistant Gram-negative pathogens [24]. Oximes have been
recognized as privileged structures for many important pharmaceuticals and synthetic chemistry
applications, and often act as chemical building blocks for the synthesis of some relevant antimicrobial
and antihypertensive agents as well as insecticides [25,26]. Recently, the oxime scaffold has emerged
as a pharmacophore of choice for designing antimicrobial agents active and has been successfully
used for functionalized carbapenems are one of the most potent of antibacterial agents as well as are
used as last resort against infections in the clinical field. Therefore, the structural modification of
imidazole by introducing a 2-aldoximes fragment should be a highly attractive topic in the antibacterial
drug discovery.
We have recently described the potent in vitro antimicrobial efficacy of a series of substituted
N-phenyl imidazole oximes and their quaternary salts against a panel of both clinically relevant
antibiotic susceptible Gram-positive and antibiotic resistant Gram-negative bacteria that carry diverse
resistance genes for the most commonly important antibiotics [24]. These results have encouraged
further structural optimization studies to search for more potent antimicrobial agents particularly
against Gram-negative pathogens producing a wide range of the serine β-lactamases based on the
imidazole oxime scaffold. In this study, we have prepared series of novel N-substituted imidazoles
having alkyl (propyl, butyl, but-3-enyl) or chlorobenzyl (meta-chlorobenzyl, para-chlorobenzyl)
substituents at position 1 of an imidazole ring bearing a 2-aldoxime group to yield effective new
antimicrobial agents. In addition, N-substituted imidazole 2-aldoximes were converted into their
corresponding quaternary salts, Figure 2. In a preliminary screening, antimicrobial efficacy of all
the synthesized imidazole 2-aldoximes was analysed by disc diffusion assay against the collections
of both antibiotic susceptible Gram-positive and resistant Gram-negative bacteria in order to obtain
their antimicrobial profiles. Additionally, their antimicrobial efficacy against Gram-negative multiple
β-lactamases producing isolates are reported in the present work.

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Figure 2. Structures of the tested imidazole 2-aldoximes.
2. Results and Discussion
2.1. Chemistry
N-substituted imidazole oximes were prepared according to reported procedures (Scheme 1) [27–29]
.
Briefly, commercially available imidazole was alkylated with appropriate alkyl or benzyl halide derivatives.
Desired 2-carbaldehydes were prepared by the reaction with n-butyl lithium and dimethylformamide.
Imidazole-2-carbaldehyde derivatives were transformed into oximes
1–
5
with hydroxylamine in
ethanol. Reaction of oximes 1–3 with the appropriate substituted benzyl bromides gave desired
quaternary salts 6–11.
Scheme 1. General procedure for the synthesis of imidazole derivatives.
2.2. Biological Evaluation
2.2.1. Antimicrobial Activity
Antimicrobial efficacy of the synthesized imidazole 2-aldoximes, 1–5 and their corresponding
quaternary compounds, 6–11 were evaluated by the disc diffusion assay against the collections of both
antibiotic susceptible Gram-positive and Gram-negative bacteria in order to obtain their antimicrobial
profiles. Activities of the target compounds were expressed as the mean diameter of the growth
inhibition zone (mm) against these microorganisms and are listed in Table 1, along with the activity of
the reference compound gentamicin (GEN).

Molecules 2018, 23, 1212
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Despite the structural differences, most of the imidazole 2-aldoximes derivatives demonstrated
potent and broad-spectrum activity against selected clinically important pathogens with varying
degrees of inhibition under that condition. The mean inhibition diameters of compounds were found
in the range from 10.7 ± 0.6 to 21.8 ± 0.9 mm.
It is noteworthy that the tested compounds are not only active against Gram-positive bacteria,
but also demonstrated significant antibacterial effects against Gram-negative bacteria as well.
Among the Gram-negative bacteria tested, two strains, namely Escherichia coli (from 12.4
21.8 ± 0.9 mm) and Pseudomonas aeruginosa (from 12.2 1.1 to 20.2 0.4 mm) showed relatively high
sensitivity toward all tested compounds with considerable zones of growth inhibition. Among them,
compound , which has an n-propyl substituent at the nitrogen ring atom has been found as
one of the most efficient imidazole 2-aldoxime derivative against Escherichia coli (20.7 0.5 mm),
Klebsiella pneumoniae (20.1 0.8 mm) and Pseudomonas aeruginosa (20.2 0.4 mm) strains. It is
±
0.71 to
±
±
6
±
±
±
particularly important that the imidazole 2-aldoximes derivatives demonstrated potent activity
against P. aeruginosa which is one of the most prevalent multidrug-resistant pathogen worldwide,
exhibiting increasing resistance to the latest antibiotic therapies and responsible for 10% of hospital
acquired infections resulting in significant morbidity and mortality [30].
In order to confirm the found antimicrobial efficacy, imidazole 2-aldoxime derivatives were then
tested for activity against a panel of clinically relevant pathogens to determine MIC values by a broth
microdilution method.
The antimicrobial bioassay results of the target compounds and the reference compound
gentamicin are summarized in Table 2. It can be seen that all target imidazole 2-aldoxime derivatives
demonstrated potent and broad spectrum activities against selected microorganisms with MIC values
in the range from 6.25 to 50.0
antimicrobial activities against a representative panel of the Gram-positive and Gram-negative bacteria
tested (MIC = 12.5–50.0 g/mL). Compounds and were found to possess not only excellent activity
against Gram-positive Enterococcus faecalis and Staphylococcus aureus, but also strong inhibitory activity
against Gram-negative Escherichia coli with equal MIC (12.50 g/mL) which is to three-fold more active
than the standard antibiotic gentamicin. Compounds and also showed promising inhibitory
g/mL) which is approximately
µg/mL. In general, N-substituted imidazoles 1–5 showed similar potent
µ
1
4
µ
1,
3
4
profiles against clinically important Pseudomonas aeruginosa (25
µ
three-fold lower than the positive control gentamicin.
Furthermore, the data showed that, in general, N-substituted imidazole 2-aldoximes showed
very similar activity in inhibiting bacterial growth as quaternary imidazolium salts for Gram-positive
and Gram-negative bacteria with exception of compounds 10 and 11. Although possessing similar
structural elements as those in compounds 3, 4 and 5, the presence of both electron rich groups are
required for better potency and spectrum of antimicrobial activities.
However, compared with other compounds minimum inhibitory concentrations demonstrated
that m-chlorobenzyl derivative 10 was found to be the most superior compound, especially for
Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and exhibited
of MIC values in the range from 12.5 even to 6.25 µg/mL. This compound also showed promising
antimicrobial activities especially against Escherichia coli and Klebsiella pneumoniae with equal MIC
values of 6.25 µg/mL, which was superior to gentamicin.
Most promising compounds 10 and 11 were therefore selected for further MIC experiments against
a panel of Gram-negative isolates producing a wide range of the extended-spectrum
(ESBLs).
β-lactamases

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Table 1. Antimicrobial activity of N-substituted imidazole 2-aldoximes (1–5) and their quaternary salts (6–11) by disc diffusion assay.
Diameters of the Inhibition Zone (mm)
Microorganisms
Compound No a
Antibiotic b
GEN
1
2
3
4
5
6
7
8
9
10
11
Gram-positive bacteria
Bacillus cereus
16.4 ± 0.3
17.7 ± 1.2
16.1 ± 0.9
15.5 ± 0.2
15.4 ± 0.5
19.2 ± 0.4
15.1 ± 0.2
11.7 ± 0.6
11.0 ± 0.5
16.2 ± 0.6
14.4 ± 0.2
17.6 ±1.4
13.3 ± 0.8
10.7 ± 0.6
12.3 ± 0.5
11.2 ± 0.8
13.6 ± 0.8
12.7 ± 0.5
16.3 ± 2.4
14.4 ± 1.1
19.2 ± 0.5
14.7 ± 0.7
16.1 ± 0.5
13.9 ± 1.2
16.2 ± 0.2
20.4 ± 0.6
15.6 ± 1.6
17.7 ± 0.9
19.1 ± 0.6
12.3 ± 0.7
19.7 ± 0.3
19.4 ± 0.4
12.1 ± 0.6
20.3 ± 0.4
20.8 ± 0.7
15.2 ± 0.3
21.2 ± 0.6
16.3 ± 0.2
16.7 ± 0.9
14.1 ± 1.0
13.6 ± 0.1
14.5 ± 0.7
12.1 ± 0.8
15.9 ± 0.2
18.2 ± 0.7
14.6 ± 1.4
23.9 ± 0.9
21.7 ± 0.4
Enterococcus faecalis
Staphylococcus aureus
Clostridium perfringens
Gram-negative bacteria
Escherichia coli
18.8 ± 0.3
15.2 ± 1.5
16.8 ± 1.9
13.7 ± 1.5
18.9 ± 0.2
16.3 ± 0.1
18.9 ± 0.4
15.7 ± 1.9
21.8 ± 0.9
17.3 ± 0.0
16.8 ± 0.3
18.3 ± 0.1
17.3 ± 1.1
14.8 ± 0.3
14.9 ± 0.1
13.1 ± 0.3
18.3 ± 0.4
16.5 ± 0.1
14.9 ± 0.3
12.3 ± 0.8
20.7 ± 0.5
20.1 ± 0.8
20.2 ± 0.4
13.6 ± 0.2
17.1 ± 0.9
17.5 ± 0.8
18.6 ± 0.4
13.7 ± 0.1
20.7 ± 0.8
19.4 ± 0.2
16.7 ± 04
16.1 ± 0.6
12.4 ± 0.7
14.1 ± 0.3
13.3 ± 0.7
12.4 ± 0.1
20.4 ± 1.1
18.2 ± 0.8
18.8 ± 1.6
16.4 ± 0.7
13.4 ± 0.7
16.8 ± 0.5
12.2 ± 1.1
12.1 ± 0.6
11.5 ± 0.9
18.8 ± 0.6
9.7 ± 1.4
13.6 ± 0.9
Klebsiella pneumoniae
Pseudomonas aeruginosa
Cronobacter sakazakii
a
b
Diameter of inhibition zone (values in mm) around the disc: 200
three parallel measurements.
µ
g/disc. Standard antibiotic disc: GEN, gentamicin (15
µ
g/disc). All values are expressed as mean
±
SD of
Table 2. The minimum inhibitory concentration of N-substituted imidazole 2-aldoximes (1–5), their quaternary salts (6–11) and gentamicin (GEN). Values in bold
indicate very strong bioactivityof compounds.
MIC
Compound No
6
Antibiotic
GEN
1
2
3
4
5
7
8
9
10
11
Microorganisms
µg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/Lµg/mL µmol/L
Gram-positive bacteria
25.00 163.2 50.00 299.0 50.00 302.7 25.00 106.1 25.00 106.1 12.50 34.9
12.50 81.6 50.00 299.0 50.00 302.7 25.00 106.1 25.00 106.1 25.00 69.7
Bacillus cereus
Enterococcus faecalis
25.00 69.7
25.00 69.7
12.50 34.9
25.00 67.1
25.00 67.1
25.00 67.1
25.00 67.1
50.00 134.2 25.00 67.5
50.00 134.2 25.00 67.5
25.00 67.5
12.50 33.7
25.00 67.5
50.00 134.9 1.00
4.00
4.00
8.4
8.4
2.1
Staphylococcus aureus 50.00 326.4 25.00 149.5 25.00 151.3 12.50 53.0
25.00 106.1 25.00 69.7
Clostridium
perfringens
25.00 163.2 12.50 74.8
25.00 151.3 25.00 106.1 12.50 53.0
50.00 139.4 12.50 34.9
25.00 67.1
50.00 134.2 25.00 67.5
12.50 33.7
0.50
1.1
Gram-negative bacteria
50.00 212.2 25.00 69.7
Klebsiella pneumoniae 25.00 163.2 25.00 149.5 50.00 302.7 25.00 106.1 25.00 106.1 25.00 69.7
Escherichia coli
12.50 81.6
25.00 149.5 50.00 302.7 12.50 53.0
25.00 69.7
25.00 69.7
50.00 134.2 50.00 134.2 6.25
25.00 67.1 50.00 134.2 6.25
16.9
16.9
25.00 67.5
25.00 67.5
32.00
8.00
67.0
16.8
Pseudomonas
aeruginosa
25.00 163.2 50.00 299.0 25.00 151.3 25.00 106.1 50.00 212.2 25.00 69.7
25.00 69.7
50.00 134.2 50.00 134.2 12.50 33.7
12.50 33.7
64.00 134.0
8.00 16.8
Cronobacter sakazakii 25.00 163.2 25.00 149.5 25.00 151.3 25.00 106.1 50.00 212.2 50.00 139.4 25.00 69.7
50.00 134.2 50.00 134.2 25.00 67.5
25.00 67.5

Molecules 2018, 23, 1212
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2.2.2. Activity toward Gram-Negative β-Lactamase Producing Strains
In the present study, we also performed an extensive in vitro screening of both quaternary
but-3-enyl compounds 10 and 11 against genetically diverse Gram-negative -lactamase producing
strains carried a wide variety of ESBLs to overcome -lactamase mediated resistance. For two
compounds, 10 and 11 antimicrobial profiles have been determined since they demonstrated the
strongest efficacy against a wide range of Gram-negative -lactamase-producing isolates with a MIC
β
β
β
values in the range from 6.25 to 25.00 µg/mL and 12.50 to 25.00 µg/mL, respectively (Table 2).
Antimicrobial activity of both tested compounds was investigated by the determination of the
minimum inhibitory concentrations (MICs) in comparison with the different classes of antibiotics with
diverse mechanisms of action (Table 3). The MICs of relevant antibacterial agents including ceftazidime,
cefotaxime, ciprofloxacin, gentamicin, tetracycline, tobramycin, imipenem and meropenem are shown
to illustrate the types of resistance associated with the each phenotype and genotype profiles and
indicates the levels and profiles of resistance in the isolates tested. The microdilution assays were
performed against ten Enterobacteriaceae strains (2 Klebsiella pneumoniae, 3 Escherichia coli, 1 Citrobacter
freundii, 2 Enterobacter cloacae, 1 Raultella terrigena and 1 Pantoea agglomerans), all with known resistance
mechanisms such as various combinations of extended-spectrum β-lactamases of both class A and C
phenotypically and molecularly determined.
As shown in Table 3, both tested compounds 10 and 11 showed significant antibacterial potency
against a wide variety of ESBL-producing strains when compared to eight broad spectrum clinically
relevant antibiotics, with MICs values ranging from 2 to 32
possess promising antimicrobial efficacy against Escherichia coli and Enterobacter cloacae producing
multiple -lactamases from A and C molecular classes which was 32-fold and 128-fold more potent
than ceftazidime and cefotaxime, respectively. Compound 10 also demonstrated potent activity
by inhibiting the multiple -lactamases in isolates of Klebsiella pneumoniae, Escherichia coli and
Raultella terrigena (MIC = 4 g/mL), produced clinically relevant class A and C -lactamases such as
TEM-1, CTX-15 and AmpC enzymes that are poorly inhibited by clavulanic acid with a MIC value of
g/mL. Moreover, the promising inhibitory activity toward a wide range of -lactamase-producing
µg/mL. Compound 10 was found to
β
β
µ
β
8
µ
β
Gram-negative strains of compound 10 was also seen against TEM-1, CTX-M-15 producing K.
pneumoniae, and against TEM-1, CTX-M-15, AmpC-overproducing Citrobacter freundii, with a MIC
of 8
activity against Escherichia coli producing TEM-1, SHV-12 and Enterobacter cloacae producing
TEM-1, SHV-12, CTX-15 -lactamases (MIC = 2 g/mL) than ceftazidime, cefotaxime, gentamicin,
tetracycline and tobramycin (Table 3). Those results demonstrated the antimicrobial efficacy of both
10 and 11 compounds differed depending on the isolates and their -lactamase expression profiles and
µg/mL. It is however interesting to note that compound 10 displayed potent and board spectrum
β
µ
β
that the compounds operate by a different mode of action and have not been affected by the resistance
mutations present in tested strains.
2.3. Principal Component Analysis
To classify compounds 10 and 11 and tested standard antibiotics according to their activities
(Table 3), we performed principal component analysis (PCA) on the mean-centered covariance matrix of
MIC values. PCA was used exclusively for classification purpose and visualization of the investigated
set of data presented in Table 3. The first three principal components explained more than 96%
of the total variance among the investigated data. Therefore, we used 3D classification model
presented in Figure 3 (together with all 2D projections). It was shown that compounds 10 and
11 have the most similar antimicrobial activity in comparison to imiperem and meropenem, which are
the broad-spectrum β-lactam antibiotics (subgroup of carbapenems).

Molecules 2018, 23, 1212
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Table 3. Minimum inhibitory concentration of quaternary imidazolium oximes 10 and 11 (µg/mL) against multidrug resistant Gram-negative ESBL-producing
isolates. Values in bold indicate very strong bioactivity (MIC < 10 µg/mL) of compounds 10 and 11.
MIC
Compounds
Standard Antibiotics a
GEN TET
µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L µg/mL µmol/L
Species, ESBL
10
11
CAZ
CTX
CIP
TOB
IMP
MEM
Klebsiella pneumoniae, TEM-1,
4
10.8
12
32
32.4
86.3
128
234.2
64
140.5
2.00
6.0
0.8
32
4
67.0
8.4
256
16
576.0
36.0
2.0
32
4.3
0.12
0.25
0.4
0.8
0.12
0.50
0.3
1.3
SHV-11, CTX-15
Klebsiella pneumoniae, TEM-1,
12
32.4
>128 >234.2 >256 >562.1 0.25
68.4
CTX-15, AmpC
Escherichia coli, CTX-15
Escherichia coli, SHV-12, CTX-15
Escherichia coli, TEM-1, SHV-12
Enterobacter cloacae, SHV-12
8
4
21.6
10.8
5.4
16
12
12
4
43.2
32.4
32.4
10.8
8
64
16
8
14.6
>256 >562.1 0.50
1.5
1.5
3.0
1.5
8
16
128
2
16.8
33.5
268.0
4.2
128
32
288.0
72.0
64
16
136.9
34.2
0.12
0.06
8
0.4
0.2
1.00
0.12
0.25
0.12
2.6
0.3
0.7
0.3
117.1 >256 >562.1 0.50
2
29.3
14.6
32
8
70.3
17.6
1.00
0.50
64
144.0
36.0
>32
>32
>68.4
>68.4
26.7
0.4
12
32.4
16
0.12
Enterobacter cloacae, TEM-1,
2
4
5.4
8
21.6
64
4
117.1
7.3
256
256
562.1
562.1
2.00
0.50
6.0
1.5
4
8.4
8
18.0
16
32
34.2
68.4
0.06
0.25
0.2
0.8
0.06
0.2
0.3
SHV-12, CTX-15
Raultella terrigena, TEM-1,
10.8
32
86.3
128
268.0
64
144.0
0.125
SHV-11, CTX-15
Citrobacter freundii, TEM-1,
SHV-12, CTX-15, AmpC
8
21.6
12
32.4
128
16
234.2
29.3
256
256
562.1
562.1
1.00
0.25
3.0
0.8
32
64
67.0
128
16
288.0
36.0
32
32
68.4
68.4
2.00
0.25
6.7
0.8
1.00
0.12
2.6
0.3
Pantoea agglomerans, CTX-15
16
43.2
4
10.8
134.0
a
Abbreviations of standard antibiotics: ceftazidime (CAZ), cefotaxime (CTX), ciprofloxacin (CIP), gentamicin (GEN), tetracycline (TET), tobramycin (TOB), imipenem (IMP) and
meropenem (MEM).

Molecules 2018, 23, 1212
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Figure 3. Classification of compounds 10 and 11 and tested standard antibiotics calculated by PCA
performed on the mean-centered covariance matrix of their MIC values.
2.4. Combination Study of Compound 10 with Ceftazidime
In order to evaluate effects of the most active compound 10 to
against a variety of ESBL-producing isolates, the various combinations of compound CAZ and 10 were
investigated. Among tested combinations, the ratio 3:2 (CAZ 10) was found as the one with the best
β-lactam antibiotic ceftazidime
:
activity which is greater than the sum of the effects produced by the components alone. As shown in
Table 4, compound 10 significantly improved the activity of ceftazidime (for Escherichia coli, SHV-12,
CTX-15 more then 200-fold) against a variety of ESBL-producing isolates, restoring susceptibility of
even high level resistance isolates with multiple β-lactamases. The present data also demonstrated
the strong effect of ceftazidime on compound 10, lowering the amount of 10 essential to obtain strong
antibiotic response. The addition of ceftazidime to the compound 10 resulted in a decrease of necessary
amount of 10 from 5- to 40-fold, the most reduced amount observed for Pantoea agglomerans, CTX-15.
Table 4. Combination study of antibiotic ceftazidime (CAZ) with compound 10 (CAZ:10 mass ratio = 3:2,
molar concentration ratio 1:1) against multidrug resistant Gram-negative ESBL-producing isolates.
Values in bold indicate very strong synergy and bioactivity (MIC ≤ 1 µg/mL) of 10 + CAZ mixture.
MIC CAZ Alone
MIC 10 Alone
MIC Combination CAZ, 10
Species, ESBL
(µg/mL) (µmol/L) (µg/mL) (µmol/L)
(µg/mL)
(µmol/L)
Klebsiella pneumoniae, TEM-1, SHV-11, CTX-15
Klebsiella pneumoniae, TEM-1, CTX-15, AmpC
Escherichia coli, CTX-15
128
>128
8
64
16
8
64
4
128
16
234
234
15
117
29
15
117
7
4
12
8
4
2
12
2
4
8
16
11
32
22
11
5
32
5
11
22
43
4.8, 3.2
9.6, 6.4
0.6, 0.4
0.3, 0.2
0.6, 0.4
0.3, 0.2
1.2, 0.8
2.4, 1.6
4.8, 3.2
0.6, 0.4
8.8, 8.6
17.6, 17.3
1.1, 1.1
0.5, 0.5
1.1, 1.1
0.5, 0.5
2.2, 2.2
4.4, 4.3
8.8, 8.6
1.1, 1.1
Escherichia coli, SHV-12, CTX-15
Escherichia coli, TEM-1, SHV-12
Enterobacter cloacae, SHV-12
Enterobacter cloacae, TEM-1, SHV-12, CTX-15
Raultella terrigena, TEM-1, SHV-11, CTX-15
Citrobacter freundii, TEM-1, SHV-12, CTX-15, AmpC
Pantoea agglomerans, CTX-15
234
29

Molecules 2018, 23, 1212
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3. Materials and Methods
3.1. General Information
All the synthesized compounds [29] 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 Microbiology laboratory, Department of Biology, Faculty of Natural
Science, University of Split, Croatia (FNSST). The assayed collection included four Gram-positive
bacteria Bacillus cereus (ATTC 11778), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus
(ATCC 25923) Clostridium perfringens (FNSST 4999) and four Gram-negative ampicillin-resistant
bacterial strains Escherichia coli (FNSST 982), Klebsiella pneumoniae (FNSST 011), Pseudomonas aeruginosa
(FNSST 982) and Chronobacter sakazakii (FNSST 014). Bacterial strains were cultured overnight at
◦
37 C in tryptic soy broth (TSB) to achieve optical densities corresponding to 106 colony forming units
(cfu/mL) for bacterial strains.
3.2. Disc Diffusion Assay
In order to investigate the antimicrobial activities of the synthesized compounds, a disc diffusion
assay was employed according to the CLSI guidelines. Briefly, 100 µL of suspension containing
106 colony-forming units (cfu/mL) of bacterial cells was spread on a Mueller Hinton agar (Becton
Dickinson, Sparks, MD, USA). The stock solutions of synthesized compounds were prepared by
dissolving in DMSO to a final concentration of 10 mg/mL. The sterile filter discs (6 mm) were
individually loaded with 20 µL of the stock solution, equivalent to a final concentration at 200 µg/disc
of synthesized compounds and then placed on the nutrient agar that had been previously inoculated
with the target microbial strains. Additionally, DMSO was used as a negative control, and gentamicin
(15 µ
g) was used as positive controls. The plates were incubated for overnight at 37 ◦C for bacterial
strains. Antibacterial activity was assessed by measuring the diameter of the inhibition zone in
millimetres, including disc diameter for the test isolates, compared to the controls. Samples were
assayed in triplicate for each condition and the diameter of inhibition zones were presented as mean
± SE values.
3.3. Minimum Inhibitory Concentration Assay
In addition, antimicrobial activities of the synthesized compounds were also 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). Bacteria were grown overnight in Mueller-Hilton broth (MHB) at
37 ◦C. The microbial cultures were diluted in fresh MHB to a final concentration of 106 CFU/mL for
bacteria. Initially the target compounds were dissolved in DMSO to prepare the stock solutions of
10 mg/mL, then the tested compounds and reference drugs were diluted to obtain concentrations
ranging from 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 to 0.78 µg/mL in Mueller-Hinton broth (MHB).
Serial dilutions of the synthesized compounds and reference drug were added to the microtiter plates
in a volume of 100
µ
L. Each well was additionally inoculated with 10
µ
L of the target microorganism and
◦
incubated at 37 C for 18–24 h. The MIC value was determined as the lowest concentration of the sample
at which the tested microorganisms did not demonstrate any visible growth after incubation. As an
indicator of bacterial growth, 50 µL of 0.2 mg/mL p-iodonitrotetrazoliumchloride (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
colour appeared. In this study, no bioactivity was defined as a MIC > 1000
as a MIC in the range 512–1000 g/mL, moderate bioactivity as a MIC in the range 128–512
good bioactivity as a MIC in the range 32–128 g/mL, strong bioactivity as a MIC in the range 10–32
µg/mL and very strong bioactivity as a MIC < 10 µg/mL.
µg/mL, mild bioactivity
µ
µg/mL,
µ

Molecules 2018, 23, 1212
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3.4. Principal Component Analysis
Statistical analysis were performed using the second order tensor analysis tool principal
component analysis (PCA) where data matrix (or two-way array) of rank r with mean centered
X
columns that consists of i rows (compunds) and j variables (MIC values) is decomposed as a sum of
r matrices tipτi with rank 1
r
X =
tipτ
i
∑
i=1
where ti stand for score and pτi for loading vectors. PCA finds the best linear projections for a
high dimensional set of data in the least squares sense. Scores represent projections of the original
points on the principal component direction and can be used for classification, whereas loadings
represent eigenvectors of data covariance (or correlation) matrix and can be used for the identification
of variability among the data. Ideas of PCA go back to Beltrami [31] and Pearson [32] while the name
was introduced by Hotelling [33]. More details can be found in the recent literature [34,35].
Minimum inhibitory concentration data were exported to the ASCII format and arranged
in the matrix
X (numbers written in a free format). Data were mean-centered and PCA on the
covariance matrix was carried out using our own multivariate analysis code [36] based on the NIPALS
algorithm [37].
4. Conclusions
Although the imidazole aldoxime scaffold is not common in the antimicrobial agent literature,
our previous study [24] and the novel library of imidazole 2-aldoxime derivatives presented in
this paper show that there is a huge potential for the design of a compounds effective against a
wide range of clinically relevant Gram-negative pathogens carrying multiple
β-lactamases from
the A and C molecular classes. All compounds showed good to strong broad-spectrum activity
against a representative panel of the Gram-positive and Gram-negative bacteria, among which
compound 10 demonstrated strong and very strong bioactivity toward the tested Gram-negative
bacteria. Furthermore, compound 10 was found to possess very strong efficacy against a wide range
of β-lactamases producing strains, especially when combined with standard antibiotic ceftazidime.
Our research revealed that further optimization of imidazole 2-aldoxime derivatives can result with
greater specificity and efficacy for the treatment of multidrug resistant bacterial infections.
Author Contributions: I.P. designed and supervised the synthesis of compounds. M.S. conceived and designed all
the biological experiments. T.H. and T.S. conceived and designed multivariate analysis and molecular modeling.
M.S., R.O. and A.R performed experiments and analyzed the data. M.S., I.P., R.O., and T.H. contributed to writing
of the paper.
Acknowledgments: This work was supported by the Croatian Science Foundation, Project No: IP-2016-06-3775
ADESIRE. Compounds used in this study were synthesized by S. Morasi Pipercˇic´ and S. Makaric´ within their
diploma thesis. I.P. would like to acknowledge networking contribution by the COST Action CM1407 “Challenging
organic syntheses inspired by nature - from natural products chemistry to drug discovery.”
Conflicts of Interest: The authors declare no conflict of interest.
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