Synthesis and antibacterial evaluation of some semicarbazides and 1,2,4-triazol-5-ones containing thiophene moieties

Article abstract of DOI:10.1002/jccs.201000040

In this study, some 3-(thiophen-2-ylmethyl)-4-substituted-4,5-dihydro-1H-1, 2,4-triazol-5-one derivatives were synthesized by the cyclization reaction of 1-(thiophen-2-ylacetyl)-4-substituted semicarbazide derivatives in alkaline medium or in the immediate reaction of thiophen-2-yl-acetic acid hydrazide with isocyanates. The structures of all new compounds were confirmed by analytical and spectroscopic methods. Selected derivatives were evaluated in vitro against several species of aerobic bacteria. Some of them showed activity against S. pyogenes, P. aeruginosa and S. aureus.

Full text of DOI:10.1002/jccs.201000040

260
Journal of the Chinese Chemical Society, 2010, 57, 260-265
Synthesis and Antibacterial Evaluation of Some Semicarbazides and
1,2,4-Triazol-5-ones Containing Thiophene Moieties#
Monika Pitucha,a* Alina Olender,b Monika Wujec,a Piotr Borowskic and Marek Mardarowiczd
aDepartment of Organic Chemistry, Medical University, Staszica 6, 20-081 Lublin, Poland
bChair and Department of Microbiology, Medical University, Chodzki 1, 20-093 Lublin, Poland
cFaculty of Chemistry, Maria Curie-Sklodowska University, pl. M. Curie-Sklodowskiej 3,
20-031 Lublin, Poland
dAnalytical Laboratory, Maria Curie-Sklodowska University, pl. M. Curie-Sklodowskiej 3,
20-031 Lublin, Poland
In this study, some 3-(thiophen-2-ylmethyl)-4-substituted-4,5-dihydro-1H-1,2,4-triazol-5-one deriv-
atives were synthesized by the cyclization reaction of 1-(thiophen-2-ylacetyl)-4-substituted semicar-
bazide derivatives in alkaline medium or in the immediate reaction of thiophen-2-yl-acetic acid hydrazide
with isocyanates. The structures of all new compounds were confirmed by analytical and spectroscopic
methods. Selected derivatives were evaluated in vitro against several species of aerobic bacteria. Some of
them showed activity against S. pyogenes, P. aeruginosa and S. aureus.
Keywords: Semicarbazide; 1,2,4-Triazol-5-one; Antibacterial activity.
INTRODUCTION
The chemistry of heterocyclic compounds has been
convulsant activity in mice and rats compared to that of
phenytoin.13 The 1-[(1-methylpyrrol-2-yl)acetyl]-4-substi-
tuted semicarbazide derivatives have shown some antibac-
terial activity against B. cereus, P. microbilis, M. luteus and
K. pneumonia.14 These results promoted us to design new
analogues with further modification of semicarbazide and
triazole ring.
an interesting field of study for a long time. The synthesis
of novel 1,2,4-triazole derivatives and investigation of
their chemical and biological behaviour have gained more
importance in recent decades for biological, medicinal, and
agricultural reason. 1,2,4-Triazole compounds possess im-
portant pharmacological properties. Examples of such com-
pounds bearing the 1,2,4-triazole residue are fluconazole,1
the powerful azole antifungal agent, and the potent antivi-
ral N-nucleoside ribavirin.2 Furthermore, various 1,2,4-tri-
azole derivatives have been reported as showing antican-
cer,3 antifungal4 and antimicrobial activity,5 as well as hav-
ing applications as antidepressants6 and anticonvulsant.7
Additionally, in many compounds, the thiophene system is
associated with high anticancer and antifungal activi-
ties.8,9
In the present work, we report the results of prelimi-
narily antibacterial investigation of newly synthesized
semicarbazides and 1,2,4-triazol-5-ones. It is known that
the biological activity of the molecules is related to their
structure and physicochemical properties. The obtained de-
rivatives may exist in both keto and enol tautomeric forms.
Since tautomerism plays a significant role in a process re-
lated to proton transfer and hydrogen bonding, it seems de-
sirable to investigate their relative stability. A part of our
studies is devoted to this problem.
On the other hand semicarbazides are an important
class of molecules with a large spectrum of biological prop-
erties. These compounds have been studied as anticon-
vulsant,10 antitubercular11 and antinociceptive12 agents.
Aryl semicarbazides are reported to display excellent anti-
RESULTS AND DISCUSSION
Thiophen-2-yl-acetic acid hydrazide15 1 is a starting
material for the synthesis of all derivatives. First, the hy-
drazide was subjected to reaction with isocyanate and the
# This work was partly presented during XLVII Meeting of the Polish Chemical Society and the Association of Energinees&Technicians
of Chemical Industry, 12-17 September 2004, Wroc»aw, Poland
* Corresponding author. E-mail: monika.pitucha@am.lublin.pl

Semicarbazide and 1,2,4-Triazole with Thiophene Ring
J. Chin. Chem. Soc., Vol. 57, No. 2, 2010 261
products of these reactions depended on the substituents in
isocyanates. In this case study compounds possessing,
phenyl, ethyl, 4-tolyl, 4-bromophenyl, 4-methoxyphenyl,
benzyl and benzoyl group semicarbazide derivatives 2b-2g
were obtained (Scheme I). The reaction medium was di-
ethyl ether (at room temperature) or both substrates were
heated at the melt for 10 h. Compound 2a was obtained by
Roman et al.16
line medium was quite different. Our investigation showed
that during the reaction semicarbazide 2g was decomposed
and benzoic acid was formed. The structure of this com-
pound was confirmed by 1H NMR and MS spectra. Identifi-
cation was performed with NIST mass library and SDBS
(Spectral Database for Organic Compounds) spectra li-
brary.18
Theoretically, semicarbazides may exist in three
(Scheme II), and 1,2,4-triazol-5-ones in two (Scheme I)
different tautomeric forms.
When we used cyclohexyl or 1-phenylethyl isocya-
nate, the reaction led to new derivatives 3h and 3i with con-
taining cyclohexyl or 1-phenylethyl group at the position 4
of the formed 1,2,4-triazol-5-one ring (Scheme I).
The calculations of total energies of structures 2c(1)-
2c(3), as well as 3c(1) and 3c(2) were carried out in the way
described in the Experimental section. These structures are
presented in Fig. 1.
Probably, the course of this reaction includes the for-
mation of intermediate semicarbazide derivatives which
cyclized spontaneously to the 1,2,4-triazole system. An-
other method for the synthesis of 1,2,4-triazol-5-ones is
cyclization reaction of 1-(thiophen-2-ylacetyl)-4-substi-
tuted semicarbazide derivatives in alkaline medium. In the
case of semicarbazide derivatives with aliphatic or aro-
matic group 2b-2f, the cyclization led to the formation of
3-(thiophen-2-ylmethyl)-4-substituted-4,5-dihydro-1H-
1,2,4-triazol-5-one derivatives 3b-3f (Scheme I). The com-
pound 3a was obtained the same method.17 The structure of
new obtained compounds was confirmed by elemental
analysis as well as IR and 1H NMR spectra.
The macroscopic sample is most likely composed of
structures corresponding to the lowest energies. There are,
in fact, two effects responsible for the content of a given
species in the macroscopic sample: the enthalpic effect and
the entropic effect. The former is related to its total energy,
the latter is mostly due to rotations (hindered in liquid) and
low-energy vibrations. We believe, that the entropic effect
is similar for all 2c and 3c compounds - the proton transfer
and formation of the OH group in enol structure affects the
highest-energy vibration without changing the overall
shape of a molecule, i.e. without affecting the principal mo-
ments of inertia. Consequently, the enhtalpic effect should
be mostly responsible for the composition of a macroscopic
sample. Thus, the most stable out of all structures consid-
The cyclization reaction of 4-benzoyl-1-(thiophen-
2-ylacetyl)semicarbazide 2g, obtained from thiophen-2-
yl-acetic acid hydrazide 1 and benzoyl isocyanate, in alka-
Scheme I
R = C6H5 (a), C2H5 (b), 4-CH3C6H4 (c), 4-BrC6H4 (d), 4-CH3OC6H4 (e), CH2C6H5 (f), COC6H5 (g), C6H11 (h),
CH3CHC6H5 (i)

262 J. Chin. Chem. Soc., Vol. 57, No. 2, 2010
Pitucha et al.
Scheme II
ered is keto-form 2c(1). Both enol tautomers 2c(2) and
2c(3) are energetically equivalent; they are by more than 17
kcal/mol less stable. Thus we conclude that the equilibrium
of a macroscopic sample is shifted towards keto 2c(1)
form. This is in accord with the spectroscopic observations
- the band corresponding to C=O stretching close to 1650
cm-1 rather than O-H stretching is observed. Apparently,
the cyclization reaction 2c®3c + H2O proceeds with an in-
crease of total energy by approximately 9 kcal/mol. Note,
that keto tautomer is also favored in the case of cyclic com-
pounds - the energy difference amounts to more than 14
kcal/mol. All discussed energetic effects are depicted in
Fig. 2.
crodilution and the disc-diffusion method. The results ob-
tained by using disc diffusion method by Kirby-Bauer do
not reveal inhibitory action of tested derivatives on bacte-
rial growth. The inhibitory action of given compounds was
obtained by using microdilution method. The inhibitory ac-
tion on bacterial growth was found at the dilution 1:16 in
TSB and this concentration was admitted as cut off value.
The data revealed the highest MIC values (Table 1):
9.375 µg/mL for 2c, 2b, 3c, 2a in case of Klebsiella
pneumoniae ATCC 700600, Staphylococcus aureus MR-3,
Streptococcus pyogenes, Pseudomonas aeruginosa ATCC
27853.
Among tested compounds, the most effective were
derivatives 2c and 3c, which include 4-tolyl substituent on
semicarbazide and 1,2,4-triazole derivatives. For these
compounds the MIC value was 9.375 µg/mL for Klebsiella
pneumoniae ATCC 700600, Staphylococcus aureus MR-3,
Streptococcus pyogenes, Pseudomonas aeruginosa ATCC
27853. The highest susceptibility to tested derivatives were
detected in S. pyogenes, P. aeruginosa and S. aureus. All
compounds were inactive against four references species:
The in vitro antibacterial activities of selected com-
pounds (2a-2d, 3a-3d, 3h) was performed by means of mi-
Fig. 2. Energetic effects of keto and enol form for 2c
and 3c.
Fig. 1. The structures of 2c(1) and 3c(1).

Semicarbazide and 1,2,4-Triazole with Thiophene Ring
J. Chin. Chem. Soc., Vol. 57, No. 2, 2010 263
4-Phenyl-1-(thiophen-2-ylacetyl)semicarbazide (2a)16
Yield (2.20 g, 80%), m.p. 160-162 °C.
Table 1. Antibacterial activities in vitro of the tested compounds
(2a-2d, 3a-3d, 3h) at concentration 9.375 mg/mL
4-Ethyl-1-(thiophen-2-ylacetyl)semicarbazide (2b)
Yield (2.00 g, 88%), m.p. 182-184 ºC. IR (KBr) 3270
(NH), 3030 (CHarom), 2960, 1499 ( CHaliph), 1678 (C=O).
1H NMR (DMSO-d6): d 1.01 (t, 3H, J = 7.15 Hz, CH3),
3.03 (q, 2H, J = 7.13 Hz, CH2), 3.62 (s, 2H, CH2), 6.29-
7.48 (m, 3H, CHarom), 6.89, 7.74, 9.71 (3s, 3H, 3NH). Anal.
Calcd. for C9H13N3O2S (227.28): C 47.56; H 5.76; N 18.49.
Found: C 47.71; H 5.82; N 18.70.
Bacterial
2a 2b 2c 2d 3a 3b 3c 3d 3h
species
A
B
C
D
+/- +/-
+
+
+
+
+/-
+
-
-
+
-
-
+/-
+
+
+
+
+/- +/- +/- +/- +/-
+
+
+
+
+
+
+
+
+
+
+/-
+/- +/-
A - Klebsiella pneumoniae ATCC700600
B - Staphylococcus aureus MR-3
C - Streptococcus pyogenes
1-(Thiophen-2-ylacetyl)-4-(4-tolyl)semicarbazide (2c)
Yield (2.40 g, 85%), m.p. 195-197 ºC. IR (KBr) 3271
(NH), 3033 (CHarom), 2917, 1430 (CHaliph), 1685 (C=O). 1H
NMR (DMSO-d6): d 2.22 (s, 3H, CH3), 3.72 (s, 2H, CH2),
6.93-7.38 (m, 7H, CHarom), 8.05, 8.61, 9.94 (3s, 3H, 3NH).
Anal. Calcd. for C14H15N3O2S (289.35): C 58.11; H 5.23; N
14.52. Found: C 57.91; H 5.00; N 14.63.
D - Pseudomonas aeruginosa ATCC27853
Escherichia coli ATCC 35218, Escherichia coli ATCC
25922, Enterococcus faecalis ATCC 29912, Staphylococus
aureus ATCC 25923.
EXPERIMENTAL AND THEORETICAL
4-(4-Bromophenyl)-1-(thiophen-2-ylacetyl)semicarba-
zide (2d)
INVESTIGATIONS
Melting points were determined in a Fisher-Johns
block and presented without corrections. Elemental analy-
sis was made on a Perkin-Elmer 2400 CHN Analyzer. The
1H NMR spectra (d, ppm) were recorded on a Brucer
Avance 300 MHz spectrometer in solution noted and with
TMS as an internal standard. The IR spectra (n, cm-1) were
recorded in KBr on a Perkin-Elmer 1725X FTIR spectrom-
eter. MS analysis was carried out with GC/MS method us-
ing GCQ spectrometer (Thermo-Finnigan, USA) on Restek
RT-5 column (20 m ´ 0.18 mm and 0,2 µm stationary phase
film thickness). MS conditions: EI–70 eV, mass range 35-
500 amu. GC program: 50-280 ºC at 10 ºC/min. The nec-
essary chemicals were obtained from Merck and Lancas-
ter. The purity of obtained compounds was checked by
TLC on aluminium oxide 60 F254 plates (Merck) in a
CHCl3/C2H5OH (10:1 and 10:2) solvent system with UV or
iodine visualization.
Yield (2.83 g, 80%), m.p. 195-197 ºC. IR (KBr) 3274
(NH), 3031 (CHarom), 2922, 1488 (CHaliph), 1647 (C=O). 1H
NMR (DMSO-d6): d 3.72 (s, 2H, CH2), 6.97-7.92 (m, 7H,
CHarom), 8.27, 9.01, 10.29 (3s, 3H, 3NH). Anal. Calcd. for
C13H12N3O2SBr (354.22): C 44.08; H 3.42; N 11.86.
Found: C 44.10; H 3.45; N 11.84.
4-(4-Methoxyphenyl)-1-(thiophen-2-ylacetyl)semicar-
bazide (2e)
Yield (2.65 g, 87%), m.p. 160-161 ºC. IR (KBr) 3273
(NH), 3053 (CHarom), 2958, 1435 (CHaliph), 1688 (C=O). 1H
NMR (DMSO-d6): d 3.32 (s, 2H, CH3), 3.71 (s, 2H, CH2),
6.83-7.93 (m, 7H, CHaromat), 8.07, 9.01, 10.29 (3s, 3H,
3NH). Anal. Calcd. for C14H15N3O3S (305.35): C 55.06; H
4.95; N 13.76. Found: C 55.41; H 4.81; N 13.83.
4-Benzyl-1-(thiophen-2-ylacetyl)semicarbazide (2f)
Yield (2.37 g, 82%), m.p. 186-188 ºC. IR (KBr) 3279
(NH), 3027 (CHarom), 2944, 1692 (CHaliph), 1678 (C=O). 1H
NMR (DMSO-d6): d 3.68 (s, 2H, CH2), 4.23 (s, 2H, CH2),
6.93-7.37 (m, 8H, CHarom), 7.93, 9.05, 9.80 (3s, 3H, 3NH).
Anal. Calcd. for C14H15N3O2S (289.35): C 58.11; H 5.23; N
14.52. Found: C 58.32; H 5.11; N 14.50.
Compounds 4-phenyl-1-(thiophen-2-yl acetyl)semi-
carbazide 2a and 3-(thiophen-2-ylmethyl)-4-phenyl-4,5-
dihydro-1H-1,2,4-triazol-5-one 3a were synthesized and
characterized earlier.16,17
1-(Thiophen-2-ylacetyl)-4-substituted semicarbazide.
Procedure for (2a-f)
Procedure for 4-benzoyl-1-(thiophen-2-ylacetyl)semi-
carbazide (2g)
A mixture of hydrazide 1 (1.4 g, 0.01 mole) and iso-
cyanate (0.01 mole) was heated in an oil bath at 90-100 ºC
for 10 h. The product was washed with diethyl ether to re-
move the unreacted isocyanate, filtered off and dried and
crystallized from ethanol.
A mixture of hydrazide 1 (1.4 g, 0.01 mole) and ben-
zoyl isocyanate (0.01 mole) in 10 mL of diethyl ether was
kept at room temperature for 24 h. Then the formed com-
pound was filtered off, washed with diethyl ether and crys-
tallized from ethanol.

264 J. Chin. Chem. Soc., Vol. 57, No. 2, 2010
Pitucha et al.
Yield (1.79 g, 79%), m.p. 155-157 ºC. IR (KBr) 3270
(NH), 3019 (CHarom), 2945, 1402 (CHaliph), 1670 (C=O). 1H
NMR (DMSO-d6): d 3.33 (s, 2H, CH2), 6.78-8.11 (m, 8H,
CHarom), 10.14, 10.46, 11.10 (2s, 3H, 3NH). Anal. Calcd.
for C14H13N3O2S (227.28): C 55.43; H 4.32; N 13.85.
Found: C 55.51; H 4.41; N 13.77.
for C14H13N3O2S (287.33): C 58.52; H 4.56; N 14.62.
Found: C 58.51; H 4.43; N 14.70.
4-(4-Benzyl)-3-(thiophen-2-ylmethyl)-4,5-dihydro-1H-
1,2,4-triazol-5-one (3f)
Yield (1.87 g, 69%), m.p. 115-117 ºC. IR (KBr) 3230
(NH), 3038 (CHarom), 2921, 1490 (CHaliph), 1680 (C=O). 1H
NMR (DMSO-d6): d 4.05 (s, 2H, CH2), 4.75 (s, 2H, CH2),
6.86-7.38 (m, 8H, CHarom), 11.71 (s, 1H, NH). Anal. Calcd.
for C14H13N3OS (271.33): C 61.97; H 4.83; N 15.49.
Found: C 61.71; H 4.92; N 15.40.
3-(Thiophen-2-ylmethyl)-4-subtstituted 4,5-dihydro-
1H-1,2,4-triazol-5-one. Procedure for (3a-f)
A mixture of semicarbazide 2a-f (0.01 mole) and 40
mL of 2% solution of sodium hydroxide was boiled for 4 h.
After cooling, the solution was neutralized with dilute hy-
drochloric acid. The precipitate was filtered off and then
crystallized from ethanol.
3-(Thiophen-2-ylmethyl)-4-subtstituted 4,5-dihydro-
1H-1,2,4-triazol-5-one. Procedure for 3h and 3i
A mixture of hydrazide 1 (1.4 g, 0.01 mole) and iso-
cyanate (0.01 mole) in 10 mL of diethyl ether was kept at
room temperature for 24 h. Then the formed compound was
filtered off, washed with diethyl ether and crystallized from
ethanol.
4-Phenyl-3-(thiophen-2-ylmethyl)-4,5-dihydro-1H-
1,2,4-triazol-5-one (3a)17
Yield (1.57 g, 61%), m.p. 186-187 °C.
4-Ethyl-3-(thiophen-2-ylmethyl)-4,5-dihydro-1H-1,2,4-
triazol-5-one (3b)
4-Cyclohexyl-3-(thiophen-2-ylmethyl)-4,5-dihydro-1H-
1,2,4-triazol-5-one (3h)
Yield (1.48 g, 71%), m.p. 153-155 ºC. IR (KBr) 3233
(NH), 3075 (CHarom), 2918, 1492 (CHaliph), 1685 (C=O). 1H
NMR (DMSO-d6): d 1.10 (t, 3H, J = 7.15, CH3), 3.69 (q,
2H, J = 7.18, CH2), 3.75 (s, 2H, CH2), 6.29 (s, 1H, NH),
7.14-7.65 (m, 3H, CHarom), 11.89 (s, 1H, NH). Anal. Calcd.
for C9H11N3OS (209.27): C 51.65; H 5.30; N 20.08. Found:
C 51.59; H 5.22; N 20.11.
Yield (1.71 g, 69%), m.p. 176-178 ºC. IR (KBr) 3228
(NH), 3056 (CHarom), 2927, 1448 (CHaliph), 1691 (C=O). 1H
NMR (DMSO-d6): d 1.01-1.95 (m, 10H, 5CH2), 6.29 (d,
1H, CH), 4.54 (s, 2H, CH2), 6.94-7.96 (m, 3H, CHarom),
10.09 (s, 1H, NH). Anal. Calcd. for C13H17N3OS (263.35):
C 59.28; H 6.51; N 15.96. Found: C 59.31; H 6.58; N
15.88.
3-(Thiophen-2-ylmethyl)-4-tolyl-4,5-dihydro-1H-1,2,4-
triazol-5-one (3c)
4-(1-Phenylethyl)-3-(thiophen-2-ylmethyl)-4,5-dihydro-
1H-1,2,4-triazol-5-one (3i)
Yield (1.95 g, 72%), m.p. 201-203 ºC. IR (KBr) 3226
(NH), 3063 (CHarom), 2913, 1426 (CHaliph), 1685 (C=O). 1H
NMR (DMSO-d6): d 2.34 (s, 3H, CH3), 4.01 (s, 2H, CH2),
6.62-7.40 (m, 7H, CHarom), 11.72 (s, 1H, NH). Anal. Calcd.
for C14H13N3OS (271.33): C 61.97; H 4.83; N 15.49.
Found: C 61.80; H 4.81; N 15.54.
Yield (1.99 g, 70%), m.p. 176-178 ºC. IR (KBr) 3232
(NH), 3057 (CHarom), 2970, 1446 (CHaliph), 1641 (C=O). 1H
NMR (DMSO-d6): d 1.34 (d, 3H, J = 7.01, CH3), 3.37 (s,
2H, CH2), 4.78 (q, 1H, J = 7.28, CH), 6.78-7.91 (m, 7H,
CHarom), 10.13 (s, 1H, NH). Anal. Calcd. for C15H15N3OS
(285.36): C 63.13; H 5.29; N 14.72. Found: C 63.08; H
5.19; N 14.77.
4-(4-Bromophenyl)-3-(thiophen-2-ylmethyl)-4,5-dihy-
dro-1H-1,2,4-triazol-5-one (3d)
Yield (2.27 g, 66%), m.p. 193-195 ºC. IR (KBr) 3233
(NH), 3055 (CHarom), 2934, 1448 (CHaliph), 1694 (C=O). 1H
NMR (DMSO-d6): d 3.78 (s, 2H, CH2), 6.94-7.46 (m, 7H,
CHarom), 12.09 (s, 1H, NH). Anal. Calcd. for C13H10N3OSBr
(336.21): C 46.44; H 3.00; N 12.50. Found: C 46.51; H
3.15; N 12.46.
THEORETICAL CALCULATIONS
The molecular modeling studies were carried out us-
ing molecular mechanic and quantum mechanic methods as
implemented in the HyperChem Version 7.5 program.19
The initial geometry optimization of 2c(1)-2c(3) and 3c(1),
3c(2) compounds, as well as water molecule was per-
formed with the MM+ molecular mechanics method. Con-
formations obtained in this way were further optimized at
AM1 semiempirical level. Default convergence criteria
were adopted. This step was followed by calculations of to-
tal energies of all final structures. This time calculations
4-(4-Methoxyphenyl)-3-(thiophen-2-ylmethyl)-4,5-dihy-
dro-1H-1,2,4-triazol-5-one (3e)
Yield (2.09 g, 73%), m.p. 216-218 ºC. IR (KBr) 3250
(NH), 3040 (CHarom), 2928, 1438 (CHaliph), 1680 (C=O). 1H
NMR (DMSO-d6): d 3.36 (s, 3H, CH3), 3.78 (s, 2H, CH2),
6.98-7.41 (m, 7H, CHarom), 12.10 (s, 1H, NH). Anal. Calcd.

Semicarbazide and 1,2,4-Triazole with Thiophene Ring
J. Chin. Chem. Soc., Vol. 57, No. 2, 2010 265
were carried out at DFT/B3LYP level20-22 with 6-31+G**
basis set.23 The main shortcoming of such approach is that
the AM1 structure does not correspond to the DFT mini-
mum on the potential energy hypersurface. However, we
believe that this does not constitute a serious problem since
AM1 is known to predict reasonable molecular geometries.
Thus, we managed to identify the most stable structure, as
well as determine the relative energies.
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ANTIMICROBIAL ACTIVITY
The in vitro antibacterial activity of newly synthe-
sized compounds was tested against selected bacterial spe-
cies: Escherichia coli ATCC 35218, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC 700600,
Enterococcus faecalis ATCC 29212, Staphylococcus
aureus MR-3, Staphylococcus aureus ATCC 25923, Strep-
tococcus pyogenes, Pseudomonas aeruginosa ATCC
27853. Susceptibility testing was performed on cultured
isolates using the agar disc diffusion method by Kirby-
Bauer. The sterile discs were soaked with antibiotics in the
concentration of 10 and 20 µg (starting concentration 300
µg/mL DMF). The bacterial cultures were incubated on
MH Agar+5% of sheep blood at 35 °C for 24 h. In order to
determine the Minimal Inhibitory Concentration (MIC) we
used microdilution method. The following concentrations
of tested compounds dissolved in DMF were used: 37.500;
18.750; 9.375; 4.687; 2.343; 1.172; 0.586; 0.293; 0.146;
0.073 µg/mL. The cultures of given bacterial strains (0.5
Mc Farland’s) were performed in TSB (Trypcase Soy
Broth) in 96-well plates (NUNC) (200 mL, at 35 °C, 24 h).
The controls were bacterial cultures performed at the same
conditions and in the same dilutions of DMF as used for
particular concentrations of tested compounds.
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REFERENCES AND NOTES
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