Synthesis and evaluation of antimicrobial activity of hydrazones derived from 3-oxido-1H-imidazole-4-carbohydrazides

Article abstract of DOI:10.1016/j.ejmech.2013.04.023

In this work we reported the synthesis and evaluation of in vitro antimicrobial activities of hydrazones 6 obtained from 3-oxido-1H-imidazole-4- carbohydrazides 4. All new compounds were characterized by spectroscopic methods. Hydrazones 6 were tested for their in vitro antimicrobial activity against four Gram-positive and four Gram-negative strains of bacteria as well as one fungal species. Three of the tested compounds appeared to be promising agents against reference strains of Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. They were also tested against twelve clinical isolates of S. aureus and their cytotoxic effect on murine fibroblasts and HeLa human tumor cell line was determined.

Full text of DOI:10.1016/j.ejmech.2013.04.023

European Journal of Medicinal Chemistry 64 (2013) 389e395
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of antimicrobial activity of hydrazones
derived from 3-oxido-1H-imidazole-4-carbohydrazides
,
,
1
c
ꢀ a
Adam M. Pieczonka a 1, Aleksandra Strzelczyk b , Beata Sadowska , Grzegorz Mloston ,
,
Pawe1 Sta˛czek b
*
a
ꢀ
ꢀ
Department of Organic and Applied Chemistry, Faculty of Chemistry, University of Łódz, Tamka 12, 91-403 Łódz, Poland
Department of Microbial Genetics, Faculty of Biology and Environmental Protection, University of Łódz, Banacha 12/16, 90-237 Łódz, Poland
Chair of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Łódz, Banacha 12/16, 90-237 Łódz, Poland
b
c
ꢀ
ꢀ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we reported the synthesis and evaluation of in vitro antimicrobial activities of hydrazones 6
obtained from 3-oxido-1H-imidazole-4-carbohydrazides 4. All new compounds were characterized by
spectroscopic methods. Hydrazones 6 were tested for their in vitro antimicrobial activity against four
Gram-positive and four Gram-negative strains of bacteria as well as one fungal species. Three of the
tested compounds appeared to be promising agents against reference strains of Escherichia coli, Staph-
ylococcus aureus and Staphylococcus epidermidis. They were also tested against twelve clinical isolates of
S. aureus and their cytotoxic effect on murine fibroblasts and HeLa human tumor cell line was
determined.
Received 11 January 2013
Received in revised form
29 March 2013
Accepted 8 April 2013
Available online 16 April 2013
Keywords:
Imidazole N-oxides
Carbohydrazide hydrazones
Antimicrobial activity
Cytotoxicity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
2. Results and discussion
Infectious diseases caused by microorganisms are one of the
main reasons of death in the world. The search for new antibacterial
and antifungal drugs is like never-ending story because of the
increasing resistance of microbial pathogens. It is desirable to find
drugs with improved potency and wide activity spectrum. Acid
hydrazides and the corresponding hydrazones are well known as a
class of compounds with diverse biological activities [1e3]. Hy-
drazides of imidazolecarboxylic acids and their derivatives, e.g.,
hydrazones, thiosemicarbazides and 1,2,4-triazoles have been also
reported to exhibit antibacterial, fungicidal, antiparasitic, anti-
inflammatory, antitumor, antinociceptive, anticonvulsant, antihy-
pertensive, and antidepressant properties [4e18]. Moreover,
nitroimidazoles such as metronidazole, tinidazole, ornidazole are
well known as antibacterial agents [19,20].
2.1. Chemistry
In a series of recent reports, methods of synthesis and reactivity of
new imidazole derivatives bearing 3-oxido function were described
[21e26]. General strategy applied for the construction of the central
imidazole ring is based on condensation of the corresponding a-
hydroxyiminoketones with methylideneamines. Thus, starting with
ethyl 2-hydroxyimino-3-oxobutanoate 1 and corresponding meth-
ylideneamines 2ae2c expected ethyl 3-oxidoimidazole-4-carboxy
late 3 can be smoothly obtained at room temperature in glacial
acetic acid [22] (Scheme 1). Upon treatment with hydrazine hydrate,
esters 3 were converted into carbohydrazides 4, which after isolation
and purification were used for reactions with aldehydes and ketones
5 [25].
The aim of the present study was the synthesis of new hydra-
zones derived from 3-oxido-1H-imidazole-4-carbohydrazides and
evaluation of their biological activity.
Reactions of 4 with p-nitrobenzaldehyde and 5-nitrofurfural
required heating in MeOH solution, whereas analogous reactions
with benzaldehyde and other aromatic aldehydes containing
electron donating substituent, were carried out at room tempera-
ture. In all cases expected hydrazones 6 were formed as single
isomers, and isolated as crystalline materials in good to excellent
yields (Table 1).
* Corresponding author. Tel.: þ48 426354772; fax: þ48 426655818.
E-mail address: pstaczek@biol.uni.lodz.pl (P. Sta˛czek).
Their structures were confirmed based on spectroscopic data
The authors have equally contributed to this work.
(1H NMR, 13C NMR, IR, HR-MS). For example, the 1H NMR spectrum
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.023

390
A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
atom in the imidazole ring. We selected small alcylic methyl group
(6j R1 ¼ Me) and sterically more crowded cyclohexyl group (6k
R1 ¼ cyclohexyl). It is worth mentioning that compounds bearing 5-
nitrofuryl substituent have limited solubility in DMSO, which was
increased by transferring compounds 6i and 6j into their hydro-
chloride salts. Such modification had no influence on their anti-
bacterial activity. Compounds 6j and 6k were similarly tested
against reference microorganisms and showed positive although
slightly lower antibacterial activity than the initial one.
The in vitro results of antibacterial activity of the three com-
pounds 6ie6k are presented in Table 2 as a minimal inhibitory
concentration (MIC) and a minimal bactericidal concentration
(MBC). Among the Gram-positive species, the most sensitive to all
of the active compounds was Staphylococcus epidermidis, and in
case of 6i and 6k (MIC ¼ 4
control antibiotic vancomycin (MIC ¼ 3
against both reference Staphylococcus aureus strains was compound
g/mL) although its activity was lower than that
mg/mL) the results were similar to the
mg/mL). The most effective
6i (MIC ¼ 11
m
exhibited by vancomycin and oxacillin, which are the antibiotics
commonly used in the therapy of staphylococcal infections. Among
three tested compounds, 6i was also the most effective against
Escherichia coli reference strain (MIC ¼ 11
mg/mL), which was
higher activity than in case of nitrofurantoin but lower then for
chloramphenicol (MIC ¼ 16, and 0.25, respectively) e two antibi-
otics used in a treatment of E. coli infections.
Compounds that showed activity in this test were then exam-
ined using the disc diffusion method in order to determine growth
inhibition zones of susceptible species of pathogens (Table 3). These
results confirmed inhibitory activity of the tested compounds with
the largest growth inhibition zones obtained for S. epidermidis.
Considering good activity of the tested 6i, 6j, and 6k compounds
against microorganisms of Staphylococcus spp., a set of 12 S. aureus
clinical strains including the ones isolated from 2 typical sources
such as naso-pharynx (carrier state) and ulcers/furuncles (skin and
soft tissue infections), but also those from infected bones (invasive
infections) was tested against these agents. Similarly to the refer-
ence Staphylococcus spp. strains clinical isolates displayed high
level of susceptibility to the analyzed compounds (MIC ranging
Scheme 1. Reagents and conditions: (i) AcOH, r. t., 16 h; (ii) NH2NH2$H2O, r.t., 16 h; (iii)
MeOH, r.t., 16 h or heating, 2 h.
of hydrazone 6i reveals the presence of two characteristic signals at
8.76 and 8.41 ppm attributed to HC(2) of imidazole and HC]N
protons, correspondingly. In the 13C NMR spectra absorption signals
of the C]O and C]N groups were found at 156.8 and 136.9 ppm,
respectively. In all compounds typical signals of HC(2) protons of
imidazole ring appeared between 8.55 and 8.78 ppm.
2.2. Biological activities
from 10 to 100
strongest activity, with MIC being equal to 10
tested clinical isolates, which for most of the strains was a better
result than in case of vancomycin used as the positive control
antibiotic. On the other hand, the second common antibiotic e
oxacillin showed up to be the most active in almost all cases
m
g/mL) (Table 4). Compound 6i again showed the
The in vitro antimicrobial activities of the compounds 6ae6i, at
concentrations ranging from 1 to 600 mg/mL, were screened using
mg/mL for all of the
the microdilution method against four Gram-negative and four
Gram-positive reference species of bacteria and one fungal species.
The results showed that compounds 6ae6h were inactive against
all tested bacteria and yeast (data not shown). Only compound 6i
showed strong antibacterial activity against some Gram-negative
and Gram-positive bacteria. Due to the high activity of the tested
compound 6i an attempt was made to potentially improve its
antimicrobial effect by introducing some modifications. Thus, on
the basis of 6i two more compounds were prepared, bearing
5-nitrofuryl group, differing from each other by substituent at N(1)
(MIC ¼ 0.2e1.5
mg/mL), except for the two strains isolated from
bones (S. aureus D15, and D17, MIC ¼ 75
mg/mL) which were 7 times
more sensitive to 6i compound.
For all three tested compounds the MBC values were in the
similar range, suggesting their bactericidal activity (MBC/MIC ꢀ 4)
(Table 4).
S. aureus permanentlycolonizes the epithelium of more than 20%
of the population and also is one of the common human pathogens.
These bacteria cause wide range of diseases: from acute infections
involving staphylococcal toxins and enzymes (e.g. ulcers, furuncles,
food poisoning, bacteremia, septic shock) to chronic infections,
usually associated with biofilm formation (e.g. infections developed
after invasive medical procedures using biomaterial devices,
wounds infections, arthritis, osteomyelitis, endocarditis, broncho-
pulmonary infections in patients with cystic fibrosis) [27,28]. The
emergence of multidrug-resistant S. aureus strains, including those
Table 1
Yields and melting points of the new hydrazones 6.
R1
R2
R3
Yield [%]
mp [ꢁC]
6a
6b
6c
6d
6e
6f
6g
6h
6i
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
Ph
92
98
76
90
89
89
78
97
87
84
76
232e234
220e222
222e226
221e222
252e254
229e232
220e221
264e265
237e240
313e315
222e225
e(CH2)5e
Me
H
H
H
H
H
H
H
Ph
p-MeO-C6H4
p-(Me)2NeC6H4
o-HOeC6H4
Furyl
p-NO2eC6H4
5-NO2efuryl
5-NO2efuryl
5-NO2efuryl
resistant to b-lactams, glycopeptides, MLS-B (macrolides, lincosa-
mides, streptogramin B), aminoglycosides, tetracyclines, fluo-
roquinolones, and even linezolid generate significant problem for
the treatment of staphylococcal infections [29e31]. Moreover, these
bacteria very easily form biofilm, which confers them severe
Bn
Me
cHex
6j
6k
H

A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
391
Table 2
In vitro antibacterial activity of 6i, 6j, 6k expressed as a minimal inhibitory concentration (MIC) [
m
g/mL] and a minimal bactericidal concentration (MBC) [
mg/mL].
Compound
6i
6j
6k
Vancomycin Oxacillin Chloramphenikol Nitrofurantoin
Tested strain
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
E. coli NCTC 8192
S. aureus ATCC 6538
11
11
11
>600
>600
>600
4
>600
>600
11
11
11
nt
nt
nt
4
25
25
25
>600
>600
>600
25
>600
>600
100
100
100
nt
nt
nt
75
nt
nt
27
18
18
>600
>600
>600
4
>600
>600
27
18
18
nt
nt
nt
4
nab
1.5
1.5
nt
nt
nt
3
nt
nt
na
1.5
3
nt
nt
nt
3
na
0.2
0.2
nt
nt
nt
0.2
nt
nt
na
0.2
0.2
nt
nt
nt
0.2
nt
nt
0.25
1
1
nt
nt
nt
0.5
nt
nt
0.5
4
4
nt
nt
nt
2
nt
nt
16
16
16
nt
nt
nt
8
32
32
32
nt
nt
nt
32
nt
nt
S. aureus ATCC 29213
P. vulgaris ATCC 49990
P. mirabilis ATCC 29906
E. faecalis ATCC 29212
S. epidermidis ATCC 12228
P. aeruginosa NCTC 6249
C. albicans ATCC 10231
nt
nt
nt
nt
nt
nt
nt
nta
a
nt e not tested.
na e no activity.
b
resistance to antibiotics, primarily as the result of its complex
structure, unique physiology and facilitated gene transfer within
biofilm [28,32].
reactions with diverse electron-poor as well as electron-rich
substituted aromatic aldehydes, and ketones 5. The new synthe-
sized hydrazones 6 were evaluated in vitro against bacterial and
fungal species.
Within E. coli species, several strains are associated with serious
infections, usually manifesting as inflammatory lesions in the in-
testines, but also cause systemic infections like hemolytic uremic
syndrome with hemolytic anemia, thrombocytopenia and acute
renal failure [33]. Enterobacteria resistant to antibiotics, such as
extended-spectrum beta-lactamases (ESBL) producing E. coli, are
very important therapeutic problem, particularly in hospitals. It is
also worth mentioning that the long-term studies of trends in
antimicrobial susceptibility performed by the European Antimi-
crobial Resistance Surveillance Network (EARS-Net) collecting data
from 198 laboratories in 22 European countries in the period from
2002 to 2009, identified E. coli and S. aureus as two main causes of
bloodstream infections and revealed significant rise of drug resis-
tance in E. coli [34] In the light of increasing resistance of micro-
organisms to chemotherapeutic agents and the involvement of
their biofilms in the development of pathological changes, it seems
necessary to design new strategies for infection treatment.
The cytotoxic activities of 6ie6k agents were assessed using
L929 murine cell line (recommended by the International Standard
ISO 10993:2009 for evaluation of cytotoxic activities) as well as
HeLa human tumor cell line. The percentage of viability inhibition
compared to the negative control in which cells were grown in the
absence of tested compounds was estimated for concentrations
There are many known biological activities of hydrazones
derived from carbohydrazides, however among nine products
initially synthesized and tested in this study, only 6i bearing 5-
nitrofuryl group showed antibacterial activity. The evidence of
the importance of this group in biological activity was confirmed by
the fact that other tested compounds which differed only in the
type of substituent in R3 (Scheme 1) did not display any antibac-
terial activity. Among them, especially 6g containing non-
substituted furan ring proved that the presence of the nitro group
was important for acquiring antibacterial activity. At the same time
hydrazone 6h containing p-nitrophenyl group instead of 5-
nitrofuryl substituent did not inhibit growth of the tested mi-
crobes, what indicated that the nitro-substituted furan ring is
essential for the antibacterial activity of the described hydrazones
derived from 3-oxido-1H-imidazoles. In order to test this hypoth-
esis two other compounds, 6j and 6k were synthesized, which
similarly to the active 6i contained 5-nitrofuryl substituent in R3
and differed only in the type of R1 (Table 1). Well-defined anti-
bacterial activity of both compounds confirmed eventually the role
of this group in conferring antibacterial activity on this type of
imidazole N-oxides derivatives.
ranging from 5 to 300
m
g/mL of the compound (Figs.1 and 2). The 6i
g/mL for L929 and LC50 > 300 g/mL for HeLa) and 6j
g/mL for L929 and LC50 > 300 g/mL for HeLa) showed
(LC50 ¼ 277
m
m
m
m
(LC50 ¼ 168
Table 4
weak cytotoxic effect on both tested cell lines. The toxicity
In vitro activity of 6i, 6j, 6k against clinical isolates of S. aureus expressed as the
minimal inhibitory concentration (MIC) [mg/mL] and minimal bactericidal concen-
of compound 6k was more than four-fold higher for L929 line
tration (MBC) [
m
g/mL].
(LC50
¼
38
mg/mL) and three-fold higher for HeLa cells
(LC50 ¼ 111
mg/mL).
Compound
6i
6j
6k
Vancomycin Oxacillin
Tested strain MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
3. Conclusion
Naso-pharynx isolates
S. aureus C4
S. aureus C7
S. aureus C8
10
10
10
25
25
10
10
100 100 10
100 100 25
100 100 25
100 100 25
25 10
25 35
25 75
25 75
10
35
75
75
0.4
0.2
1.5
0.2
0.4
0.4
1.5
0.2
The presented results show that 3-oxido-1H-imidazole-4-
carbohydrazides 4 can be easily converted into hydrazones 6 via
S. aureus C19 10
Ulcers/furuncles isolates
Table 3
S. aureus D12 10
10
10
25
10
100 100 25
100 200 25
100 100 25
100 100 25
110 75
25 75
25 35
110 10
75
75
35
10
0.2
0.8
0.2
0.2
0.4
0.8
0.2
0.4
In vitro antibacterial activity of 6i, 6j, 6k expressed as the growth inhibition zones
(giz) [mm].
S. aureus F1
S. aureus F7
10
10
Tested strain
Compound
S. aureus F12 10
6i
6j
6k
Bone isolates
S. aureus D14 10
S. aureus D15 10
S. aureus D17 10
S. aureus D20 10
10
10
10
10
100 100 25
100 100 25
100 100 25
100 100 25
110 75
25 75
110 75
25 75
75
75
75
75
0.2
0.2
E. coli NCTC 8192
7
13
15
20
8
7
10
10
13
75
75
0.4
150
150
0.4
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
10
10
11

392
A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
Fig. 1. Concentration-dependent cytotoxic activity of compound 6i (black), 6j (gray) and 6k (white) for L929 cell line. * Denotes significant difference (P < 0.05) when compared
with control.
Diverse compounds containing nitrofuran moiety are known for
their antibacterial activity and are extensively reported in the liter-
ature. For example, nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-
aminohydantoin) is the common chemotherapeutic used in urinary
tract infections, showing activity against Gram-positive as well as
Gram-negative bacteria [35]. Similarly, nifuroxazide (4-hydroxy-N0-
[(4-nitrocyclopenta-1,3-dienyl)methylene]benzohydrazide) is used
in gastrointestinal tract infections. Biological activity of these drugs is
conferred by the presence of nitrofuryl group which is reduced inside
bacterial cells by nitroreductases and thereby generating hydroxy
radicals. These highly reactive forms of oxygen are believed to inhibit
protein biosynthesis [35,36]. Additionally, it has been shown that
N-oxides derivatives, such as antitumor [38], herbicidal [39] or
antiprotozoal [40e42]. Derivatives 6ie6k obtained from 5-nitro-2-
furylaldehyde showed promising antimicrobial activity which,
together with their low cytotoxicity, make them good candidates
in the search for effective antibacterial drugs to combat most
burdensome microorganisms, among which both S. aureus and
E. coli but also S. epidermidis pose a significant threat to public
health.
4. Experimental
4.1. Chemistry
nitro group at R2
a-position of the furan ring increases genotoxicity of
furan derivatives leading to double strand breaks in DNA helix [37].
Thus, it is very likely that the mechanism of antibacterial activityof 6i,
6j, and 6k compounds is similar.
Biological activities of hydrazones derived from 5-nitro-2-
furylaldehyde and carbohydrazides are well known [1e3], but
our data reports for the first time antimicrobial activities of
imidazole-4-carboxylic acid hydrazide hydrazones containing the
unique N-oxide function. Moreover, in the literature there are only
few examples known reporting biological activities of imidazole
General: Melting points were determined in a capillary using a
Melt-Temp. II apparatus (Aldrich) or STUART SMP30 and are un-
corrected. The IR Spectra were recorded on a NEXUS FT-IR spec-
trophotometer; absorptions (
{1H}- (150 MHz) spectra were measured on a Bruker Avance III
instrument using solvent signals as reference. Chemical shifts (
n
) in cmꢂ1. The 1H- (600 MHz), 13C
d
)
are given in ppm and coupling constants J in Hz. Assignments of
signals in 13C NMR spectra were made on the basis of HMQC ex-
periments. HR-ESI-MS: Bruker Esquire LC spectrometers.
Fig. 2. Concentration-dependent cytotoxic activity of compound 6i (black), 6j (gray) and 6k (white) for HeLa cell line. * Denotes significant difference (P < 0.05) when compared
with control.

A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
393
4.1.1. Starting materials
120.8 (C(4), C(5)); 145.8, 129.5, 128.6, 127.7, 114.7, 112.6 (8 arom.
CH); 127.0 (C(2)); 48.8 (CH2); 9.9 (Me). HR-ESI-MS: 325.1295
([M þ H]þ, C17H17N4Oþ3 ; calc. 325.1295).
All solvents are commercially available and they were used as
received. Ethyl 3-oxido-1H-imidazole-4-carboxylate 3 was ob-
tained according to published procedure [22]. Carbohydrazides 4
were obtained from 3 by treatment with hydrazine hydrate
following previously published protocol [25].
4.1.2.5. 1-Benzyl-5-methyl-N0-[(Z)-(4-nitrophenyl)methylidene]-1H-
imidazole-4-carbohydrazide 3-oxide (6h). Yellowish crystals; yield:
97%; mp 264e265 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3081m, 1665vs
4.1.2. General procedure for synthesis of hydrazones 6
(C]O), 1614m, 1519s, 1342s. 1H NMR (600 MHz, DMSO-d6,
d, ppm):
To a stirred solution of a hydrazide 4 (1 mmol) in MeOH (5 mL)
at 20 ꢁC, an equimolar quantity of the carbonyl component 5 was
added slowly. The mixture was stirred for 16 h at room temperature
(6ae6g), or refluxed for 2 h (6he6k), the solution was concen-
trated, the resulting solid was treated with Et2O, filtered, and
crystallized from MeOH.
14.63 (1H, br. s, NH); 8.78 (1H, s, HC(2)); 8.52 (1H, s, HC]N); 8.30,
7.99 (4H, AA0BB0, JAB ¼ 9.0, AreH); 7.42e7.27 (5H, m, AreH); 5.27
(2H, s, CH2); 2.50 (3H, s, Me). 13C NMR (150 MHz, DMSO-d6,
d, ppm):
156.8 (C]O); 146.6 (C]N); 141.2, 135.7, 122.0 (3 arom. C); 132.5,
120.7 (C(4), C(5)); 129.5, 128.7, 128.5, 127.7, 124.5 (9 arom. CH);
127.1 (C(2)); 48.9 (CH2); 9.9 (Me). HR-ESI-MS: 380.1350 ([M þ H]þ,
1-Benzyl-5-methyl-N0-[(Z)-phenylmethylidene]-1H-imidazole-
4-carbohydrazide 3-oxide (6a), 1-Benzyl-N0-cyclohexylidene-5-
methyl-1H-imidazole-4-carbohydrazide 3-oxide (6b), 1-Benzyl-5-
methyl-N0-[(1Z)-1-phenylethylidene]-1H-imidazole-4-carbohydra
zide 3-Oxide (6c), were described in our earlier paper [25].
C
19H18N5Oþ4 ; calc. 380.1353).
4.1.2.6. 1-Benzyl-5-methyl-N0-[(Z)-(5-nitro-2-furyl)methylidene]-
1H-imidazole-4-carbohydrazide 3-oxide (6i). Yellowish crystals;
yield: 89%; mp 237e240 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3161m,
1680vs (C]O), 1607s, 1474s, 1362m, 1254m. 1H NMR (600 MHz,
4.1.2.1. 1-Benzyl-5-methyl-N0-[(Z)-(4-methoxyphenyl)methylidene]-
1H-imidazole-4-carbohydrazide 3-oxide (6d). Colorless crystals;
yield: 90%; mp 221e222 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3083m,
1663vs (C]O), 1607vs, 1510m, 1254m, 1170m. 1H NMR (600 MHz,
DMSO-d6, d, ppm): 14.71 (1H, br. s, NH); 8.76 (1H, s, HC(2)); 8.41
(1H, s, HC]N); 7.77, 7.17 (2H, 2d, J ¼ 4.2, furyl AreH); 7.42e7.27
(5H, m, AreH); 5.27 (2H, s, CH2); 2.49 (3H, s, Me). 13C NMR
(150 MHz, DMSO-d6, d, ppm): 156.8 (C]O); 152.2, 135.7, 127.7 (3
DMSO-d6,
d
, ppm): 14.24 (1H, br. s, NH); 8.72 (1H, s, HC(2)); 8.28
arom. C); 136.9 (C]N); 132.7, 120.6 (C(4), C(5)); 129.5, 128.7,
(1H, s, HC]N); 7.69, 7.01 (4H, AA0BB0, JAB ¼ 9.0, AreH); 7.42e7.26
(5H, m, AreH); 5.25 (2H, s, CH2); 3.80 (3H, s, OMe); 2.48 (3H, s, Me).
127.7, 116.3, 115.0 (7 arom. CH); 127.2 (C(2)); 48.9 (CH2); 10.0
(Me). HR-ESI-MS: 370.1145 ([M
370.1146).
þ
H]þ, C17H16N5Oþ5 ; calc.
13C NMR (150 MHz, ()DMSO-d6,
d, ppm): 161.4 (C]O); 156.3, 135.8,
127.4 (3 arom. C); 148.6 (C]N); 131.8, 120.9 (C(4), C(5)); 129.5,
129.3, 128.6, 127.7, 114.8 (9 arom. CH); 126.9 (C(2)); 55.8 (OMe);
48.8 (CH2); 9.9 (Me). HR-ESI-MS: 365.1608 ([M þ H]þ, C20H21N4O3þ;
calc. 365.1606).
4.1.2.7. 1,5-Dimethyl-N0-[(Z)-(5-nitro-2-furyl)methylidene]-1H-imid-
azole-4-carbohydrazide 3-oxide (6j). Yellowish crystals; yield: 84%;
mp 313e315 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3149m,1680vs (C]O),
1603m, 1477s, 1355s, 1269m. 1H NMR (600 MHz, DMSO-d6,
d, ppm):
4.1.2.2. 1-Benzyl-5-methyl-N0-[(Z)-(4-dimethyloaminophenyl)methyl-
idene]-1H-imidazole-4-carbohydrazide 3-oxide (6e). Colorless crys-
tals; yield: 89%; mp 252e254 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr):
3088m, 2906m, 1664vs (C]O), 1603vs, 1524m, 1368m, 1181m. 1H
14.77 (1H, br. s, NH); 8.55 (1H, s, HC(2)); 8.39 (1H, s, HC]N); 7.77,
7.16 (2H, 2d, J ¼ 4.2, furyl AreH); 3.59 (3H, s, NMe); 2.54 (3H, s, Me).
13C NMR (150 MHz, DMSO-d6,
d, ppm): 158.8 (C]O); 152.2, 136.7 (2
arom. C); 136.9 (C]N); 132.5, 120.7 (C(4), C(5)); 116.2, 115.1 (2
arom. CH); 127.2 (C(2)); 32.5 (NMe); 9.7 (Me). HR-ESI-MS:
294.0830 ([M þ H]þ, C11H12N5Oþ5 ; calc. 294.0833).
NMR (600 MHz, DMSO-d6, d, ppm): 14.10 (1H, br. s, NH); 8.71 (1H, s,
HC(2)); 8.15 (1H, s, HC]N); 7.56, 6.74 (4H, AA0BB0, JAB ¼ 9.0, AreH);
7.42e7.26 (5H, m, AreH); 5.25 (2H, s, CH2); 2.97 (6H, s, Me2N); 2.48
(3H, s, Me). 13C NMR (150 MHz, ()DMSO-d6,
d, ppm): 160.0 (C]O);
4.1.2.8. 1-Cyclohexyl-5-methyl-N0-[(Z)-(5-nitro-2-furyl)methylidene]-
1H-imidazole-4-carbohydrazide 3-oxide (6k). Yellowish crystals;
yield: 74%; mp 222e225 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3105m,
2939m, 2856m, 1675vs (C]O), 1590s, 1472s, 1350m, 1266m. 1H NMR
152.1, 135.8, 122.0 (3 arom. C); 149.4 (C]N); 131.5, 121.1 (C(4), C(5));
129.5,129.1,128.6,127.7,112.2 (9 arom. CH); 126.9 (C(2)); 48.7 (CH2);
40.5 (Me2N); 9.9 (Me). HR-ESI-MS: 378.1923 ([M
þ
H]þ,
C21H24N5Oþ2 ; calc. 378.1924).
(600 MHz, DMSO-d6, d, ppm): 14.81 (1H, br. s, NH); 8.80 (1H, s,
HC(2)); 8.40 (1H, s, HC]N); 7.77, 7.16 (2H, 2d, J ¼ 4.2, furyl AreH);
4.1.2.3. 1-Benzyl-5-methyl-N0-[(Z)-(2-hydroxyphenyl)methylidene]-
1H-imidazole-4-carbohydrazide 3-oxide (6f). Colorless crystals;
yield: 89%; mp 229e232 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3140m,
4.14e4.09 (1H, m, CH); 2.61 (3H, s, Me); 1.96e1.80 (4H, m); 1.67e
1.39 (5H, m); 1.21e1.13 (5H, m). 13C NMR (150 MHz, DMSO-d6,
d,
ppm): 157.1 (C]O); 152.2, 133.7 (2 arom. C); 136.7 (C]N); 132.1,
119.9 (C(4), C(5)); 116.2, 115.0 (2 arom. CH); 124.9 (C(2)); 55.3 (CH);
32.9, 25.3, 24.9 (5 cyclohexyl CH2); 9.7 (Me). HR-ESI-MS: 362.1455
([M þ H]þ, C16H20N5Oþ5 ; calc. 362.1459).
1670vs (C]O),1616s,1590m,1277m.1H NMR (600 MHz, DMSO-d6,
d,
ppm): 14.55 (1H, br. s, NH); 8.76 (1H, s, HC(2)); 8.58 (1H, s, HC]N);
7.54e6.90 (9H, m, AreH); 5.27 (2H, s, CH2); 2.49 (3H, s, Me).13C NMR
(150 MHz, DMSO-d6, d, ppm): 157.9 (C]O); 156.3, 135.7, 120.5 (3
arom. C); 149.6 (C]N); 132.0, 119.1 (C(4), C(5)); 132.2, 130.3, 128.7,
119.8, 116.9 (9 arom. CH); 127.1 (C(2)); 48.8 (CH2); 9.9 (Me). HR-ESI-
MS: 351.1449 ([M þ H]þ, C19H19N4O3þ; calc. 351.1452).
4.2. Antibacterial assay
The in vitro antimicrobial activity of newly synthesized com-
pounds was evaluated against the reference strains of Gram-
negative (E. coli NCTC 8192, Proteus vulgaris ATCC 49990, Proteus
mirabilis ATCC 29906, Pseudomonas aeruginosa NCTC 6249), Gram-
positive (S. aureus ATCC 6538, S. aureus ATCC 29213, Enterococcus
faecalis ATCC 29212, S. epidermidis ATCC 12228) bacterial and fungal
(Candida albicans ATCC 10231) species. Moreover, the most active
(against the reference strains) compounds were examined against a
set of twelve clinical isolates of S. aureus from the collection
belonging to the Chair of Immunology and Infectious Biology,
4.1.2.4. 1-Benzyl-5-methyl-N0-[(Z)-2-furylmethylidene]-1H-imid-
azole-4-carbohydrazide 3-oxide (6g). Colorless crystals; yield:
78%; mp 220e221 ꢁC (MeOH). IR (nmax, cmꢂ1, KBr): 3138m, 1656vs
(C]O), 1627s, 1593s, 1276m. 1H NMR (600 MHz, DMSO-d6,
d, ppm):
14.30 (1H, br. s, NH); 8.73 (1H, s, HC(2)); 8.25 (1H, s, HC]N); 7.84,
6.89, 6.62 (3H, 3d, J ¼ 3.6, furyl AreH); 7.42e7.25 (5H, m, AreH);
5.25 (2H, s, CH2); 2.48 (3H, s, Me). 13C NMR (150 MHz, DMSO-d6,
d,
ppm): 156.5 (C]O); 149.9, 135.8, (2 arom. C); 138.6 (C]N); 132.0,

394
A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
ꢀ
University of qódz. These strains were isolated from 3 sources:
naso-pharynx of young patients hospitalized at Children’s Hospital
Acknowledgments
ꢀ
in qódz (n ¼ 4), ulcers and furuncles from adult patients of
The authors are grateful to Dr. Katarzyna Dzitko from the
ꢀ
ꢀ
Dermatological Clinic in qódz (n ¼ 4), and from infected bones of
Department of Immunoparasitology, University of qódz, for her
ꢀ
patients hospitalized at Oncological Hospital in qódz (n ¼ 4). All
advice on cytotoxicity analyses, to Prof. H. Heimgartner and PD Dr.
L. Bigler, University of Zürich, for recording of the HR-ESI mass
spectra. A.M.P. is grateful for financial support within the project
European Social Fund ‘HUMAN e BEST INVESTMENT!’, co-funded
by the European Union. This research was partially supported by
the funds provided by the Faculty of Biology and Environmental
strains were kept frozen at ꢂ80 ꢁC on Tryptic Soy Broth with 15% of
glycerol until testing. Before using, S. aureus strains were sub-
cultured on blood agar and identified by routine methods (catalase,
coagulase and clumping factor). Minimal inhibitory concentration
(MIC) was determined as the lowest concentration of the com-
pound preventing growth of the tested microorganism using
microdilution method. The inoculum density was adjusted to 0.5
McFarland standard. All of the tested compounds were dissolved in
dimethyl sulfoxide (DMSO). Concentration of the agents evaluated
ꢀ
Protection, University of qódz.
References
in MuellereHinton broth ranged from 1 to 600 mg/mL. DMSO at the
[1] C.A.M. Fraga, E.J. Barreiro, Medicinal chemistry of N-acylhydrazones: new
lead-compounds of analgesic, antiinflammatory and antithrombotic drugs,
Curr. Med. Chem. 13 (2006) 167e198.
[2] S. Rollas, S¸ .G. Küçükgüzel, Biological activities of hydrazone derivatives,
Molecules 12 (2007) 1910e1939.
[3] B. Narasimhan, P. Kumar, D. Sharma, Biological activities of hydrazide de-
rivatives in the new millennium, Acta Pharm. Sci. 52 (2010) 169e180.
[4] J.H.M. Lange, H.H. Van Stuivenberg, H.K.A.C. Hein, T.J.P. Adolfs, A.C. McCreary,
H.G. Keizer, H.C. Wals, W. Veerman, A.J.M.W. De Looff, P.C. Verveer, C.G. Kruse,
Bioisosteric replacements of the pyrazole moiety of rimonabant: synthesis,
biological properties, and molecular modeling investigations of thiazoles,
triazoles, and imidazoles as potent and selective CB1 cannabinoid receptor
antagonists, J. Med. Chem. 48 (2005) 1823e1838.
final concentration in the medium had no influence on growth of
the tested microorganisms. The incubation was carried out at 37 ꢁC
for 18 h and optical density (OD600) measurements were deter-
mined for bacterial cultures in the presence and absence of the
tested compounds. Vancomycin and oxacillin, widely used in the
treatment of infectious diseases, were used as positive control
antimicrobial agents for Gram-positive strains. Chloramphenicol
was used as positive control for E. coli. Moreover, nitrofurantoin was
used because of bearing 5-nitrofuryl group as our 6ie6k com-
pound. Minimal bactericidal concentration (MBC), defined as the
lowest concentration of a compound that resulted in >99.9%
reduction in CFU of the initial inocula (2 ꢃ 108 cfu) was assessed
only for compounds 6ie6k. MBC was determined by a broth
microdilution technique followed by plating out the contents of the
wells that showed no visible growth of bacteria onto Muellere
Hinton agar plates and incubating at 35 ꢁC for 18 h. Both MIC and
[5] J.M. Figueiredo, C.A. Camara, E.G. Amarante, A.L.P. Miranda, F.M. Santos,
C.R. Rodrigues, C.A.M. Fraga, E.J. Barreiro, Design and synthesis of novel potent
antinociceptive agents: methyl-imidazolyl N-acylhydrazone derivatives, Bio-
org. Med. Chem. 8 (2000) 2243e2248.
ꢀ
[6] A. Siwek, P. Sta˛czek, J. Stefanska, Synthesis and structureeactivity rela-
tionship studies of 4-arylthiosemicarbazides as topoisomerase IV inhibitors
with Gram-positive antibacterial activity. Search for molecular basis of
antibacterial activity of thiosemicarbazides, Eur. J. Med. Chem. 46 (2011)
5717e5726.
MBC values were performed in triplicates and are given in
according to CLSI reference [43].
mg/mL,
[7] A.P. Liesen, T.M. de Aguino, C.S. Carvalho, V.T. Lima, J.M. de Araújo, J.G. de
Lima, A.R. de Faria, E.J.T. de Melo, A.J. Alves, E.W. Alves, A.Q. Alves, A.J.S. Góes,
Synthesis and evaluation of anti-toxoplasma gondii and antimicrobial activ-
ities of thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles, Eur. J.
Med. Chem. 45 (2010) 3685e3691.
The disc diffusion method was used to determine activity of
compound 6ie6k. Sterile filter paper discs (6 mm in diameter) were
soaked in compound solution to load 500 mM of compound per disc
[8] L. Savini, L. Chiasserini, C. Pellerano, W. Filippelli, G. Falcone, Synthesis and
pharmacological activity of 1,2,4-triazolo[4,3-a]quinolines, Farmaco 56 (2001)
939e945.
[9] B. Tozkoparam, E. Yesilada, M. Ertan, Preparation of 5-aryl-3-alkylthio-l,2,4-
triazoles and corresponding sulfones with antiinflammatory-analgesic activ-
ity, Bioorg. Med. Chem. 15 (2007) 1808e1814.
and dried. Dry discs were placed on the surface of MuellereHinton
agar medium. Diameter of the growth inhibition zones was read
after 18 h of incubation at 35 ꢁC.
[10] B.S. Holla, B.K. Sarojini, B.S. Rao, P.M. Akberali, N.S. Kumari, V. Shetty, Syn-
thesis of some new biologically active bis-(thiadiazolotriazines) and bis-
(thiadiazolotriazinyl) alkanes, Farmaco 56 (2001) 899e903.
[11] A.K. Singh, G. Mishra, K. Jyoti, Review on biological activities of 1,3,4-
thiadiazole, derivatives, J. Appl. Pharm. Sci. 01 (2011) 44e49.
[12] G. Capan, N. Ulusoy, N. Ergenç, A. Cevdet Ekinci, A. Vidin, Synthesis and
anticonvulsant activity of new 3-[(2-furyl)carbonyl]amino-4-thiazolidinone
and 2-[(2-furyl)carbonyl]hydrazono-4-thiazoline derivatives, Farmaco 51
(1996) 729e732.
4.2.1. Cytotoxicity assay
Cytotoxic effect of compounds 6ie6k on host cells was detected
by determining cellular viability using MTT reduction assay. Mouse
fibroblast cells L929 (ATTCÒ catalog no. CCL-1, mouse fibroblasts)
or human tumor cells HeLa (ATTCÒ catalog no. CCL-2Ô, human
epithelial cells) were plated in 96-well microplates at a density of
1 ꢃ 104 cells/mL (100
mL per well) and cultivated in Iscove’s
[13] Y.A. Al-Soud, M.N. Al-Dweri, N.A. Al-Masoudi, Synthesis, antitumor and
antiviral properties of some 1,2,4-triazole derivatives, Farmaco 59 (2004)
775e783.
modified Dulbecco’s medium (IMDM), supplemented with
100.0 U/mL penicillin and streptomycin, 5 ꢃ 10ꢂ5 M 2-mercapto
ethanol and enriched with 10% fetal bovine serum (FBS). After
overnight incubation at 37 ꢁC, the growth medium was removed
[14] X. Collin, A. Sauleau, J. Coulon, 1,2,4-Triazolo mercapto and aminonitriles as
potent antifungal agents, Bioorg. Med. Chem. Lett. 13 (2003) 2601e2605.
[15] T. Plech, M. Wujec, A. Siwek, U. Kosikowska, A. Malm, Synthesis and antimi-
crobial activity of thiosemicarbazides, s-triazoles and their Mannich bases
bearing 3-chlorophenyl moiety, Eur. J. Med. Chem. 46 (2011) 241e248.
[16] M.A. Iradyan, N.S. Iradyan, G.M. Stepanyan, F.G. Arsenyan, G.M. Paronikyan,
G.A. Darbinyan, É.V. Kazaryan, B.T. Garibdzhanyan, Imidazole derivatives.
XXIX. Synthesis and biological activity of thiosemicarbazides and hydrazo-
nohydrazides of 4-nitroimidazole-5-thioacetic acids, Pharm. Chem. J. 37
(2003) 67e70.
[17] A.A. Bekhit, H.T.Y. Fahmy, S.A.F. Rostom, A.A. Bekhit, Synthesis and biological
evaluation of some thiazolylpyrazole derivatives as dual anti-inflammatory
antimicrobial agents, Eur. J. Med. Chem. 45 (2010) 6027e6038.
[18] W. Rzeski, J. Matysiak, M. Kandefer-Szerszen, Anticancer, neuroprotective
activities and computational studies of 2-amino-1,3,4-thiadiazole based
compound, Bioorg. Med. Chem. 15 (2007) 3201e3297.
and 100
m
L of medium supplemented with different concentra-
g/mL were
tions of synthetic compounds in the range of 1e300
m
added. Cells were further incubated for 24 h with tested agents. At
the end of the incubation time, the medium was removed and MTT
was added to each well at a final concentration of 0.5 mg/mL and
plates were incubated for another 2 h at 37 ꢁC. Then, formazan
crystals were solubilized in 150 mL DMSO. The optical density was
measured at 550 nm. The results of experiments were shown as
mean arithmetic values of eight repeats (two experiments), the
percentage of viability inhibition compared to control wells was
calculated for each concentration of the tested compounds, and
LC50 values were determined. Analysis of statistical significance
was performed using ManneWhitney U test. During statistical
verification, significance levels of p < 0.05* were considered.
[19] A. Mital, Synthetic nitroimidazoles: biological activities and mutagenicity
relationships, Sci. Pharm. 77 (2009) 497e520.
[20] M.D. Crozet, V. Rémusat, C. Curti, P. Vanelle, Rapid synthesis of new 5-
nitroimidazoles as potential antibacterial drugs via VNS procedure, Synth.
Commun. 36 (2006) 3639e3646.

A.M. Pieczonka et al. / European Journal of Medicinal Chemistry 64 (2013) 389e395
395
ꢀ
[21] G. Mloston, T. Gendek, H. Heimgartner, First examples of reactions of azole N-
O. Heuer, The EARS-Net participants (Disease Specific Contact Points for AMR),
Escherichia coli and Staphylococcus aureus: bad news and good news from the
European Antimicrobial Resistance Surveillance Network (EARS-Net, formerly
EARSS), 2002 to 2009, Euro. Surveill. 16 (2011) 19819.
oxides with thioketones: a novel type of sulfur-transfer reaction, Helv. Chim.
Acta 81 (1998) 1585e1595.
[22] M. Jasinski, G. Mloston, A. Linden, H. Heimgartner, Synthesis and selected
transformations of 1H-imidazole 3-oxides derived from amino acid esters,
Helv. Chim. Acta 91 (2008) 1916e1933.
ꢀ
ꢀ
[35] C.C. McOsker, P.M. Fitzpatrick, Nitrofurantoin: mechanism of action and im-
plications for resistance development in common uropathogens,
J. Antimicrob. Chemother. 33 (Suppl. A) (1994) 23e30.
[36] Y. Wang, J.P. Gray, V. Mishin, D.E. Heck, D.L. Laskin, J.D. Laskin, Role of cyto-
chrome P450 reductase in nitrofurantoin-induced redox cycling and cyto-
toxicity, Free Radic. Biol. Med. 44 (2008) 1169e1179.
[37] M. Hofnung, P. Quillardet, V. Michel, E. Touati, Genotoxicity of 2-nitro-7-
methoxy-naphtho[2,1-b]furan (R7000): a case study with some consid-
erations on nitrofurantoin and nifuroxazide, Res. Microbiol. 153 (2002)
427e434.
[38] Y. Mizuno, T. Endo, Y. Inoue, H. Tampo, A. Takahashi, M. Iigo, A. Hoshi,
K. Kuretani, Studies on the chemical synthesis of potential antimetabolites.
XXIV. The synthesis and antitumor activities of p-tosylhydrazones of 2-
formylpyridine 1-oxides and 2-formylazoles 3-oxides, Chem. Pharm. Bull.
28 (1980) 1584e1589.
[39] M. Witschel, Design, synthesis and herbicidal activity of new iron chelating
motifs for HPPD-inhibitors, Bioorg. Med. Chem. 17 (2009) 4221e4229.
[40] G. Aguirre, M. Boiani, H. Cerecetto, A. Gerpe, M. González, Y. Fernández-
Sainz, A. Denicola, C. Ochoa de Ocáriz, J.J. Nogal, D. Montero, J.A. Escario,
Novel antiprotozoal products: imidazole and benzimidazole N-oxide de-
rivatives and related compounds, Arch. Pharm. Pharm. Med. Chem. 337
(2004) 259e270.
[41] M. Boiani, L. Boiani, A. Denicola, S. Torres de Ortiz, E. Serna, N. Vera de Bilbao,
L. Sanabria, G. Yaluff, H. Nakayama, A. Rojas de Arias, C. Vega, M. Rolan,
A. Gómez-Barrio, H. Cerecetto, M. González, 2H-Benzimidazole 1,3-dioxide
ꢀ
ꢀ
[23] M. Jasinski, G. Mloston, Synthesis and selected transformations of 3-oxido-1H-
imidazole-4-carboxamides, Coll. Czech. Chem. Commun. 75 (2010) 871e885.
ꢀ
ꢀ
[24] G. Mloston, D. Rygielska, M. Jasinski, H. Heimgartner, Optically active imid-
azoles derived from enantiomerically pure trans-1,2-diaminocyclohexane,
Tetrahedron Asymmetry 22 (2011) 669e674.
ꢀ
[25] G. Mloston, A.M. Pieczonka, E. Kowalczyk, A. Linden, H. Heimgartner, Syn-
thesis and selected reactions of hydrazides containing an imidazole moiety,
Helv. Chim. Acta 94 (2011) 1764e1777.
[26] A.M. Pieczonka, G. Mloston, H. Heimgartner, Synthesis of bis-heterocyclic 1H-
imidazole 3-oxides from 3-oxido-1H imidazole-4-carbohydrazides, Helv.
Chim. Acta 95 (2012) 404e414.
[27] M. Wie˛ckowska-Szakiel, B. Sadowska, B. Rózalska, Staphylokinase production
by clinical Staphylococcus aureus strains, Polish J. Microbiol. 56 (2007) 97e102.
[28] B. Sadowska, E. Walencka, M. Wie˛ckowska-Szakiel, B. Rózalska, Bacteria
competing with the adhesion and biofilm formation by Staphylococcus aureus,
Folia Microbiol. 55 (2010) 497e501.
[29] S. Boyle-Vavra, R. Daum, Community-acquired MRSA: the role of Panton-
Valentine leukocidin, Lab. Invest. 87 (2007) 3e9.
[30] S.F. Graves, S.D. Kobayashi, Community-associated methicillin-resistant
Staphylococcus aureus immune evasion and virulence, J. Mol. Med. 88 (2010)
109e114.
ꢀ
_
_
[31] A. M1ynarczyk, G. M1ynarczyk, Molecular mechanisms of Staphylococcus
aureus antibiotic resistance, Post. Mikrobiol. 47 (2008) 423e429.
ꢀ
_
[32] A. Budzynska, M. Wie˛ckowska-Szakiel, B. Sadowska, D. Kalemba, B. Rózalska,
Antibiofilm activity of selected plant essential oils and their major compo-
nents, Polish J. Microbiol. 60 (2011) 35e41.
derivatives: a new family of water-soluble anti-trypanosomatid agents,
J. Med. Chem. 49 (2006) 3215e3224.
[42] A. Gerpe, L. Boiani, P. Hernández, M. Sortino, S. Zacchino, M. González,
H. Cerecetto, Naftifine-analogues as anti-Trypanosoma cruzi agents, Eur. J.
Med. Chem. 45 (2010) 2154e2164.
[43] CLSI, Performance Standards for Antimicrobial Susceptibility Testing; Eigh-
teenth International Supplement. CLSI Document M7-MIC, Clinical Laboratory
Standards Institute, Wayne, PA, 2008.
[33] D. Westra, J.F.M. Wetzels, E.B. Volokhina, L.P. van den Heuvel, N.C.A.J. van de
Kar, A new era in the diagnosis and treatment of atypical haemolytic uraemic
syndrome, Neth. J. Med. 70 (2012) 121e129.
[34] C. Gagliotti, A. Balode, F. Baquero, J. Degener, H. Grundmann, D. Gür, V. Jarlier,
G. Kahlmeter, J. Monen, D.L. Monnet, G.M. Rossolini, C. Suetens, K. Weist,