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A. Boguszewska-Czubara et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
keratinocyte-like HaCaT cells, to evaluate the in vitro cytotoxicity Antibacterial and antifungal activity assessment
of our compounds of interest.
All the synthesized compounds were in vitro screened for their
antibacterial and antifungal activities using a broth microdilution
Materials and methods
method according to European Committee on Antimicrobial
Susceptibility Testing (EUCAST)19 and Clinical and Laboratory
Standards Institute guidelines (CLSI)20 against a panel of 20
reference microorganisms strains. All microorganisms were
obtained from American Type Culture Collection (ATCC,
Rockville, MD). Bacteria strains were subcultured onto nutrient
agar at 35 ꢁC for 18–24 h. Fungal cultures were carried out on
Analytical techniques
Knauer apparatus equipped with an UV-VIS detector and the
Lichrospher 100-3 RP18 column (4 ꢀ 250) was applied for
monitoring the reaction course. Methanol–water mixture (63:35,
v/v) was used as an eluent at the flow rate of 0.6 mL/min. Melting
points were determined on the Boetius apparatus and were not
corrected. IR spectra were recorded on Bio-Rad 175C spectro-
photometer in KBr pellets. 1H-NMR (500 MHz) spectra were
taken on a Bruker AMX 500 spectrometer using CDCl3 as a
solvent and TMS as an internal standard. Chemical shifts are
given in a ppm scale.
Sabouraud agar at 30 ꢁC for 24 – 48 h19–21
.
Antibacterial properties of tested compounds were evaluated
in both Gram-positive (Staphylococcus aureus ATCC 6538,
Staphylococcus aureus ATCC 25923, Staphylococcus aureus
ATCC 43300, Staphylococcus epidermidis ATCC 12228,
Streptococcus pyogenes ATCC 19615, Streptococcus pneumoniae
ATCC 49619, Streptococcus mutans ATCC 25175, Bacillus
subtilis ATCC 6633, Bacillus cereus ATCC 10876, and
Micrococcus luteus ATCC 10240) and Gram-negative bacteria
(Escherichia coli ATCC 3521, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC
12453, Bordetella bronchiseptica ATCC 4617, Salmonella
typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC
9027). Candida albicans ATCC 2091, Candida albicans ATCC
10231, and Candida parapsilosis ATCC 22019 were utilized in an
antifungal activity assessment.
Preparation of nitroalkenes
(Z)-2-phenyl-1-bromo-1-nitroethene (A1)
Firstly, 2-phenyl-1,2-dibromo-1-nitroethane was obtained
from commercially available (E)-2-phenyl-1-nitroethene in
reaction with bromine in methylene chloride solution.
Parham and Bleasdale methodology was applied15. Product
with 90% yield was obtained. Next, (Z)-2-phenyl-1-bromo-1-
nitroethene was prepared by dehydrobromination of 2-phenyl-
1,2-dibromo-1-nitroethane in the presence of pyridine accord-
ing to the previously described procedure16. Product with 80%
yield was obtained (m.p.: 65–66 ꢁC; reported: 64–66 ꢁC15;
63–64 ꢁC17).
The surface of Mueller–Hinton agar or Mueller–Hinton agar
with 5% sheep blood (for bacteria) and RPMI 1640 with MOPS
(for fungi) were inoculated with the suspensions of bacterial or
fungal species. Microbial suspensions were prepared in sterile
saline (0.85% NaCl) with an optical density of McFarland
standard scale 0.5 [approximately 1.5 ꢀ 108 CFU (Colony
Forming Units)/mL] for bacteria and 0.5 McFarland standard
Nitroacetonitril
scale [approximately 5 ꢀ 105 CFU/mL)] for fungi19–21
.
In 760 mL, four-neck glass equipped with a mechanic stirrer, a
dropping funnel, a thermometer and a reflux condenser, 60 g of
methazonic acid (prepared from nitromethane according to the
procedure described previously18) was dissolved in 300 mL of
diethyl ether. Next, reaction mixture was heated to boiling, and
dropped slowly 43 mL of tionyl chloride. During introduction of
tionyl chloride to reaction system, temperature should be higher
than 30 ꢁC. Solution was stirred for 120 min, filtered and
evaporated in argon atmosphere. Oil residue was treated by
240 mL of diethyl ether and 90 mL of water. Organic layer was
separated, dried over anhydrous calcium chloride and evapo-
rated in argon atmosphere. Oil residue was purified by column
chromatography. Silica gel was used as stationary phase, and
benzene as eluent. Fourteen grams (28%) of pure nitroacetoni-
trile was obtained as yellow liquid. Product was converted
immediately in reaction with aldehydes without storage, as its
long-term storage leads to the formation of explosive
substances.
Samples containing 5 mg, 1 mg or 0.5 mg of tested compounds
A1–A7 were dissolved in 1 mL of DMSO. Subsequently, 50 ml of
vehicle or the compounds of interest was applied into the wells
(d ¼ 6 mm) on the agar media mentioned above. The agar plates
were preincubated at room temperature for 1 h and incubated at
37 ꢁC for 24 h and at 30 ꢁC for 48 h for bacteria and fungi,
respectively. After the incubation period, the zones of growth
inhibition were measured and average values were calculated.
Vehicle-containing wells were used as controls. Furthermore,
bacterial and fungal suspensions were applied onto Petri dishes
with solid media containing 1 mg/mL of tested compounds
A1–A7 and they were incubated. The inhibition of microorgan-
isms growth was judged by comparing with a control culture
prepared without any sample tested. Ciprofloxacin, vancomycin
and fluconazole (Sigma-Aldrich, St. Louis, MO) were
used as a reference antibacterial or antifungal compounds,
respectively19–21
.
Selected spectral data: 1H NMR (CDCl3): 5.23 (s, 2H);
IR (cmꢂ1): 2963, 3015 (–CH2–), 2363 (–C ꢃ N), 1573, 1363
(–NO2).
Subsequently, minimal inhibitory concentration (MIC) of the
compounds was examined by the microdilution broth method,
using their two-fold dilutions in Mueller–Hinton broth or
Mueller–Hinton broth with 5% sheep blood (for bacteria) and
RPMI 1640 broth with MOPS (for fungi) prepared in 96-well
polystyrene plates. Final concentrations of the compounds ranged
from 1000 to 0.488 mg/mL. Microbial suspensions were prepared
in sterile saline (0.85% NaCl) with an optical density of 0.5
McFarland standard. Next, 2 ml of each bacterial or fungal
suspension was added per each well containing 200 ml of
broth and various concentrations of the examined compounds.
After incubation (37 ꢁC, 24 h), the MIC was assessed spectro-
fotometrically as the lowest concentration of the samples
showing complete bacterial or fungal growth inhibition.
(E)-2-aryl-1-cyano-1-nitroethenes (A2–A7) – general procedure
Appropriate aldehyde (0.1 mol) was dissolved in 2 mL of 1-butyl-
3-methylimidazolium chloride. Next, 0.2 g of A4 molecular sieves
and 0.1 mol of nitroacetonitrile were added. Reaction mixture was
stirred by 5 min. Product was separated by filtration and
recrystallized from ethanol, thus the series of 6 (E)-2-aryl-1-
cyano-1-nitroethenes was obtained. Yields and melting points for
all compounds are collected in Table 1. After washing the ionic
liquid with diethyl ether and dried in vacuo, it may be applied
again for next synthesis.