Synthesis, experimental and theoretical study on the structure of some semicarbazides with potential antibacterial activity

Article abstract of DOI:10.1515/znb-2011-0511

A series of 1,4-disubstituted semicarbazide and 4,4'-bis[1-substituted semicarbazide]diphenyl-methane derivatives were synthesized to explore their antibacterial activity. New compounds were characterized by elemental analysis and spectroscopic data. In order to find the tautomeric equilibrium for the molecules energy calculations for each possible tautomeric form of model compound 2, and for the most antibacterially active compound 7 in the investigated series, were calculated for the gas phase at the RHF/SCF/6-31G** level of theory.

Full text of DOI:10.1515/znb-2011-0511

Synthesis, Experimental and Theoretical Study on the Structure
of Some Semicarbazides with Potential Antibacterial Activity
Monika Pituchaa, Zbigniew Karczmarzykb, Urszula Kosikowskac, and Anna Malmc
a Department of Organic Chemistry, Medical University, Chodz´ki 4a, 20-093 Lublin, Poland
b Department of Chemistry, University of Podlasie, 3 Maja 54, 081-10 Siedlce, Poland
c Department of Pharmaceutical Microbiology, Medical University, Chodz´ki 1, 20-093 Lublin,
Poland
Reprint requests to Dr. Monika Pitucha. E-mail: monika.pitucha@umlub.pl
Z. Naturforsch. 2011, 66b, 505 – 511; received January 22, 2011
A series of 1,4-disubstituted semicarbazide and 4,4ꢀ-bis[1-substituted semicarbazide]diphenyl-
methane derivatives were synthesized to explore their antibacterial activity. New compounds were
characterized by elemental analysis and spectroscopic data. In order to find the tautomeric equilib-
rium for the molecules energy calculations for each possible tautomeric form of model compound 2,
and for the most antibacterially active compound 7 in the investigated series, were calculated for the
gas phase at the RHF/SCF/6-31G** level of theory.
Key words: Semicarbazides, Keto-Enol Tautomerism, Gas Phase, Antibacterial Activity,
RHF/SCF/6-31G** Calculations
Introduction
tomeric equilibrium for the molecules, the energy of
each possible tautomeric form in the gas phase was cal-
culated.
Semicarbazides are important building blocks for
the synthesis of five-membered heterocycles [1 – 3].
Biologically active semicarbazide derivatives were
studied as anticonvulsant [4] and antitubercular [5]
agents. Additionally, derivatives having the pyrrole
ring showed antibacterial activity, inhibiting growth
of some Gram-positive bacteria, including Bacillus
cereus and Micrococcus luteus [6]. Aryl semicar-
bazides have been reported to display excellent anti-
convulsant activity in mice and rats compared to that
of phenytoin [7]. It is known that the biological ac-
tivity of the molecules is related to their structure and
physicochemical properties. The electronic effects and
the position of substituents, the pH value and the sol-
vent polarity are major factors that influence this tau-
tomerism [8]. In particular, the proton transfer associ-
ated with keto-enol tautomerization has long been of
much interest because such interactions may catalyze
certain biochemical transformations [9]. Aliphatic car-
bonyl compounds that contain hydrogens alpha to the
carbonyl group undergo keto-enol tautomerism. There-
fore, semicarbazide derivatives may exist theoretically
in both keto and enol tautomeric forms.
Results and Discussion
The new semicarbazide derivatives 1 – 18 were
synthesized according to Scheme 1. The key hy-
drazide intermediates were obtained by addition of
the corresponding carboxylic acid ester to an ex-
cess of hydrazine hydrate in anhydrous ethanol, fol-
lowed by the method described earlier [10]. The
acid hydrazides were reacted with 4-ethoxyphenyl,
phenyl, 4-bromophenyl, ethyl, cyclohexyl, and 4-tolyl
isocyanate, and 1,4-disubstituted semicarbazides 1 –
8 were obtained. Another series of new 4,4ꢀ-bis[1-
substituted semicarbazide]diphenylmethane deriva-
tives 9 – 18 were synthesized in the reaction of car-
boxylic acid hydrazide with 4,4ꢀ-diphenylmethane di-
isocyanate. The reactions were carried out in anhy-
drous diethyl ether or N,N-dimethylacetamide at r. t.
Only compound 18 was obtained in absolute ethanol.
Substituents for newly synthesized compounds are
shown in Table 1.
There are 10 possible tautomeric forms of the keto-
semicarbazide system (Fig. 1) in compounds 1 – 8 with
one semicarbazide spacer and respectively 102 tau-
tomeric forms for compounds 9 – 18 with two semi-
In this paper, we present results of preliminary stud-
ies of the antibacterial activity of newly synthesized
semicarbazide derivatives. In order to find the tau-
c
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M. Pitucha et al. · Semicarbazides with Potential Antibacterial Activity
O
O
R1 CH2
C
C2H5
ethanol
hydrazine
O
O
R1 CH2
C
diethyl ether
N,N-dimethylacetamide
R1
2
R NCO
CH2
C
+
NH NH
NH NH2
C
NH
R2
O
MDI
1-8
R1
O
NH
NH
C
C
NH
NH
R1
NH
C
O
C
C
NH
O
CH2
Scheme 1. General procedure for the syn-
thesis of title compounds 1 – 18; MDI =
4,4ꢀ-diphenylmethane diisocyanate.
9-18
Table 1. Substituents for compounds 1 – 18.
Compound R1
R2
Compound R1
1
4-(C2H5O)-C6H4
9
2
3
4
C6H5
10
11
12
CH3
4-BrC6H4
C2H5
CH2C6H5
5
6
7
8
4-(C2H5O)-C6H4 13
Fig. 1. Possible tautomeric forms of the keto-semicarbazide
chain in compounds 1 – 8.
C6H11
14
15
16
C6H5
carbazide chains. In order to find the relative stabili-
ties of the tautomeric forms of molecules 1 – 18, en-
ergy calculations for each of the 10 possible tautomeric
forms of model compound 2 and compound 7, antibac-
terially the most active in the investigated series, were
performed in the gas phase at the RHF/SCF/6-31G**
level of theory. The results of these calculations are
presented in Table 2. As can be seen from Table 2,
the energetically most stable form existing in the gas
phase is X 01 (X = 2, 7; di-keto) for both investigated
compounds 2 and 7. The population of the remaining
4-BrC6H4
4-CH3C6H4
C6H5OCH2CH2
17
18
H
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M. Pitucha et al. · Semicarbazides with Potential Antibacterial Activity
507
(a)
(b)
Fig. 2 (color online). A view of the molecule 7
with the atomic labelling adopted (a) in the low-
energy extended conformation, and (b) cyclic
conformation of the ketosemicarbazide chain
with N–H···O intramolecular hydrogen bond
(dashed line), calculated at the RHF/SCF/6-
31G** level.
Table 2. The stabilization energy (∆E) for tautomeric forms
of 2 and 7.
retical calculations are in good agreement with the ex-
perimental data obtained from the solid-state structure
of the structurally related (R)-(+)-1-[(1-methylpyrrol-
2-yl)acetyl]-4-(1-phenylethyl)-semicarbazide [11], in
which the semicarbazide chain exists in the same di-
keto tautomeric form in the solid state. The IR spec-
tra revealed the absence of hydroxyl groups but the
presence of an NH absorption in the region 3268–
3311 cm−1. Additionally, the ν (C=O) band was ob-
served in the region 1683– 1651 cm−1. The 1H NMR
spectra revealed the absence of the signal of OH pro-
tons, but exhibited a hydrazine NH proton signal in the
region 8.00– 10.46 ppm which disappeared on addi-
tion of deuterium oxide. This is in agreement with the
results of quantum chemical calculations. A presen-
tation of the low-energy conformation of molecule 7
as calculated at the RHF/SCF/6-31G** level, with the
semicarbazide spacer in the di-keto tautomeric form, is
shown in Fig. 2. The ketosemicarbazide chain adopts
a gauche-gauche-trans-gauche-trans-trans-cis confor-
mation with torsion angles ω1 = 26C–1C–2C–3C =
120.4◦, ω2 = 1C–2C–3C–4N = −95.3◦, ω3 = 2C–3C–
4N–5N = −170.7◦, ω4 = 3C–4N–5N–6C = 70.8◦, ω5 =
4N–5N–6C–7N = −167.2◦, ω6 = 5N–6C–7N–8C =
−176.6◦, and ω7 = 6C–7N–8C–9C = −11.3◦. Bond
lengths and angles of 7 are listed in Table 3. The 3C–
∆E (kcal mol−1
)
Tautomeric form
X 01
X = 2
0
X = 7
0
X 02
X 03
X 04
X 05
X 06
X 07
X 08
X 09
16.34
28.00
55.38
42.44
35.96
52.36
19.37
21.28
50.36
12.85
22.69
52.42
36.93
33.17
49.75
18.17
19.25
46.74
X 10
˚
Table 3. Bond lengths and angles (A, deg) for 7 calculated at
the RHF/SCF/6-31G** level.
2C–1C
3C–2C
4N–3C
5N–4N
6C–5N
7N–6C
8C–7N
14O–3C
15O–6C
1.52
1.52
1.37
1.37
1.39
1.36
1.41
1.19
1.19
1C–2C–3C
2C–3C–4N
2C–3C–14O
4N–3C–14O
3C–4N–5N
4N–5N–6C
5N–6C–7N
5N–6C–15O
7N–6C–15O
6C–7N–8C
111.7
115.2
122.9
121.8
119.2
116.2
112.1
121.9
125.9
127.9
tautomeric forms in vacuo estimated using a non-
degenerated Boltzmann distribution is below the
threshold of the detectability of conventional analyti-
cal methods. The energy differences between X 01 and
the other remaining forms change in the relatively wide
range of 16.34– 55.38 kcal mol−1 for 2 and 12.85–
52.42 kcal mol−1 for 7 and do not depend on the type
of the substituents R1 and R2. The results of the theo-
˚
14O and 6C–15O bond lengths of 1.20 and 1.19 A,
respectively, are in good agreement with that of a C=O
double bond. In the central semicarbazide moiety the
˚
˚
bond lengths 5N–4N = 1.37 A, 6C–5N = 1.39 A and
˚
7N–6C = 1.36 A are typical for a conjugated π elec-
tron system.
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M. Pitucha et al. · Semicarbazides with Potential Antibacterial Activity
Table 4. Potential-derived (ESP) and Mulliken atomic
charges on the atoms in the semicarbazide part of
molecules 2 and 7 at the RHF/SCF/6-31G** level.
polarization of the bonds in the semicarbazide chain,
but the distribution of the partial bond dipoles results
in a relatively small dipole moment of 0.81 D for 2
and 1.44 D for 7. Similar values of dipole moments
in the range of 1.03– 1.89 D were calculated for
molecules of other compounds in the series 1 – 8.
All of the tested compounds were evaluated in vitro
for their antibacterial activity using the agar dilution
method, which is used as a standard procedure for
preliminary checking and helps to select compounds
showing promising activity. According to our results
none of the tested compounds, except compound 7,
inhibited the growth of the used reference strains of
Gram-positive or Gram-negative bacteria completely
even at 1000 µg mL−1 concentration. Only partial
inhibitory effects (about 60 – 80 % inhibition) were
observed for two reference species of Staphylococ-
cus spp. (Staphylococcus epidermidis ATCC 12228,
Staphylococccus aureus ATCC 6538) with high con-
2
7
Atom
ESP
Mulliken
ESP
Mulliken
2C
3C
4N
5N
6C
7N
14O
15O
23Br
−0.20
+0.82
−0.43
−0.69
+1.23
−0.96
−0.66
−0.70
–
−0.34
+0.75
−0.52
−0.53
+1.04
−0.84
−0.61
−0.62
–
−0.28
+0.81
−0.47
−0.64
+1.18
−0.91
−0.62
−0.68
−0.18
−0.32
+0.76
−0.51
−0.53
+1.04
−0.85
−0.59
−0.62
−0.04
It is worthwhile to note that the possibility of
forming cyclic structures of the semicarbazide via
the 7N–H···14O intramolecular hydrogen bond ex-
ists in the energetically lowest X 01 tautomeric form.
The theoretical calculations at the RHF/SCF/6-31G**
level for this kind of cyclic structure of 7 showed
that the ketosemicarbazide chain adopts a gauche-cis-
trans-gauche-cis-trans-cis conformation with torsion
angles ω1 = 78.6◦, ω2 = 22.01◦, ω3 = −176.3◦, ω4 =
−90.6◦, ω5 = 38.1◦, ω6 = 179.0◦, ω7 = 23.7◦ and
centrations of compound 8 (500 or 1000 µg mL−1
)
or for Staphylococcus epidermidis ATCC 12228 and
Bacillus subtilis ATCC 6633 with compounds 5 and 6
at 1000 µg mL−1. Compound 7 showed an inhibitory
effect on Gram-positive bacteria with MIC values
ranging from 31.25– 250 µg mL−1 (Table 5). Using
the broth microdilution method, it was found that com-
pound 7 inhibited the growth of Gram-positive bacte-
ria with MIC values from 15.63 to 125 µg mL−1 (Ta-
ble 5). The difference among MIC values determined
by two kinds of methods (agar dilution procedure and
broth microdilution test) is in accordance with results
communicated by other authors [12, 13]. It should be
noted that in our experiments the activity of gentam-
icin, widely used for the treatment of serious infec-
tions caused by Gram-negative and Gram-positive bac-
teria, for Staphylococcus species was very high (MIC =
0.12 µg mL−1). According to our previous data [6],
only a few of the tested compounds possessed selec-
tive and concentration-dependent antibacterial activity
against the reference strains of Gram-positive (beside
Staphylococcus spp.) or Gram-negative bacteria which
was revealed as complete or partial reduction (20 –
90 %) of the growth depending on the strains and the
concentration of the compound.
˚
an H···14O distance of 2.22 A and a 7N–H···14O
angle of 145.5◦ typical for intramolecular hydrogen
bonds (Fig. 2b). However, its energy is higher than
that of the non-cyclic low-energy conformation of 7
by about 2.11 kcal mol−1, resulting in a 3 % contri-
bution of the cyclic conformation to the equilibrium
of conformers of X 01 in the gas phase as estimated
using a non-generated Boltzman distribution. The rel-
atively large contribution of the cyclic form may be
one reason for the amorphous (non-crystalline) state
of the investigated compounds. The N–H···O and O–
H···O intramolecular hydrogen bonds may exist in
one or the other of the analyzed tautomeric forms.
However, the relatively wide energy range between
the lowest-energy tautomeric form and other remain-
ing forms (12– 50 kcal mol−1) causes us to believe that
intramolecular hydrogen bonds associated with an en-
ergy of (4 – 5 kcal mol−1) should not affect much the
tautomeric equilibrium.
Potential-derived (ESP) and Mulliken atomic
charges on the atoms in the ketosemicarbazide part
of molecules 2 and 7 calculated at the RHF/SCF/6-
In conclusion, we have described the synthesis
31G** level of theory (Table 4) are very similar of 1,4-disubstituted semicarbazide and 4,4ꢀ-bis[1-sub-
and independent on the type of substituent on both stituted semicarbazide]diphenylmethane derivatives
sides of the semicarbazide spacer. Large positive and and investigated their biological activity. The theoret-
large negative charges are observed at the carbon and ical calculations at the RHF/SCF/6-31G** level pro-
heteroatoms, respectively. This effect causes a strong vided the geometrical, conformational and electronic
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M. Pitucha et al. · Semicarbazides with Potential Antibacterial Activity
509
Table 5. Influence of compound 7 on the growth of Gram-positive reference strains of bacteriaa.
MIC (µg mL−1
)
Sa 6538
bdm
62.5
Sa 25923
bdm
31.25
Se 12228
adm
125
Bs 6633
bdm
15.63
Bc 10876
Ml 10240
adm
250
a
adm
125
bdm
62.5
adm
31.25
adm
250
bdm
125
adm
250
bdm
15.63
adm – agar dilution method; bdm – broth microdilution method; Sa – Staphylococcus aureus; Se – Staphylococcus epidermidis; Bs –
Bacillus subtilis; Bc – Bacillus cereus; Ml – Micrococcus luteus.
parameters of molecules 1 – 8, which can be correlated Compound 8
M. p. 240 – 241 ◦C. – Yield 79 %. – IR (KBr) ν =
3271, 3033, 2931, 1681 (C=O), 1428 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 2.22 (s, 3H, CH3), 3.39 (s,
2H, CH2), 7.04 – 7.40 (m, 4H, Ar-H), 8.41 – 8.66 (m, 4H,
Ar-H), 8.86, 9.28, 9.98 (3× s, 3H, 3NH). – 13C NMR
(300 MHz, [D6]DMSO): δ = 17.16 (CH3), 54.85 (CH2),
116.20, 123.17, 123.53, 123.96, 126.31, 129.66, 136.95,
137.08, 143.02, 147.86 (all C-Ar), 167.64 (C=O), 179.80
(C=O). – C15H16N4O2 (284.3): calcd. C 63.36, H 5.67,
N 19.71; found C 63.42, H 5.73, N 19.68.
with their biological activity. Compound 7 described in
the present paper could be regarded as a leading struc-
ture with increased antibacterial activity against Gram-
positive bacteria, including staphylococci (coagulase-
positive S. aureus and coagulase-negative S. epider-
midis).
Experimental Section
All chemicals were purchased from Merck Co. or Alfa-
Aesar (Gdan´sk, Poland) and used withouth further purifi-
cation. Melting points were determined in a Fisher-Johns
block and were not corrected. IR spectra were recorded from
KBr discs using a Specord IR-75 spectrophotometer. 1H and
13C NMR spectra were recorded on a Bruker AC 200F instru-
ment in [D6]DMSO with TMS as internal standard. The pu-
rity of the obtained compounds was checked by TLC on alu-
minum oxide 60 F254 plates (Merck) in a CHCl3-C2H5OH
(10 : 1 and 10 : 2) solvent system with UV or iodine visual-
ization.
Compound 13
M. p. 260 – 262 ◦C. – Yield 71 %. – 1H NMR (300 MHz,
[D6]DMSO): δ = 3.71 (s, 4H, 2CH2), 3.79 (s, 2H, CH2),
6.90 – 7.90 (m, 14H, Ar-H), 8.05, 8.78, 9.93 (3s, 6H, 6NH). –
13C NMR (300 MHz, [D6]DMSO): δ = 33.05, 39.12, 115.72,
117.36, 123.63, 125.02, 125.27, 127.46, 128.30, 133.78,
135.37, 136.09, 153.92, 167.73. – C27H26N6O4S2 (562.6):
calcd. C 57.63, H 4.66, N 14.93; found C 57.71, H 4.57,
N 14.86.
The hydrazides used for the reactions were obtained by
published methods [10]. Compounds 1 – 6 and 9 – 14 were
obtained and characterized earlier [6, 14 – 16].
Compound 15
◦
M. p. 244 – 246 C. – Yield 68 %. – IR (KBr) ν = 3311,
3037, 2923, 1651 (C=O), 1497 cm−1. – 1H NMR (300 MHz,
[D6]DMSO): δ = 2.63 (t, J = 6.1, 4H, 2CH2), 3.78 (s, 2H,
CH2), 4.20 (t, J = 6.1, 4H, 2CH2), 6.65 – 7.39 (m, 18H,
Ar-H), 8.05, 8.58, 9.79 (3× s, 6H, 6NH). – C33H34N6O6
(610.7): calcd. C 64.90, H 5.61, N 13.76; found C 65.12,
H 5.84, N 13.84.
Preparation of 1,4-disubstituted semicarbazides 7, 8, 13,
15 – 17
A mixture of the appropriate hydrazide (10 or 20 mmol)
and isocyanate (10 mmol) in 10 mL of diethyl ether was kept
for 48 h at r. t. Then the product was filtered off, washed with
diethyl ether and crystallized from ethanol.
Compound 16
M. p. 211 – 212 ◦C. – Yield 67 %. – 1H NMR (300 MHz,
[D6]DMSO): δ = 2.50 (s, 6H, 2 CH3), 3.73 (s, 2H, CH2),
6.80 – 7.62 (m, 12H, Ar-H), 8.00, 8.75, 9.79 (3× s, 6H,
6NH). – 13C NMR (300 MHz, [D6]DMSO): δ = 11.85
(CH3), 39.00 (CH2), 107.70, 112.35, 115.73, 117.25, 127.45,
128.28, 130.44, 133.81, 136.05, 136.14, 139.48, 154.33,
155.50 (all Ar-C), 161.77 (C=O). – C27H26N6O6 (530.5):
calcd. C 61.12, H 4.94, N 15.84; found C 61.23, H 4.84,
N 15.78.
Compound 7
◦
M. p. 127 – 129 C. – Yield 82 %. – IR (KBr) ν = 3268,
3027, 2928, 1683 (C=O), 1431 cm−1. – 1H NMR (300 MHz,
[D6]DMSO): δ = 3.58 (s, 2H, CH2), 7.25 – 7.82 (m, 4H,
Ar-H), 8.40 – 8.81 (m, 4H, Ar-H), 9.94, 9.78, 10.27 (3× s,
3H, 3NH). – 13C NMR (300 MHz, [D6]DMSO): δ = 18.92
(CH2), 117.05, 117.38, 123.16, 123.27, 127.80, 129.29,
129.61, 135.51, 135.84, 143.25, 147.99, 151.47, 154.11 (all
C-Ar), 166.70 (C=O), 167.84 (C=O). – C14H13N4O2Br
Compound 17
M. p. 256 – 258 ◦C. – Yield 70 %. – 1H NMR (300 MHz,
(349.2): calcd. C 48.15, H 3.75, N 16.05; found C 48.08, [D6]DMSO): δ = 3.85 (s, 2H, CH2), 4.27 (s, 1H, CH), 7.07 –
H 3.84, N 16.18.
7.46 (m, 10H, Ar-H), 8.83, 9.75, 10.06 (3× s, 3H, 3NH). –
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M. Pitucha et al. · Semicarbazides with Potential Antibacterial Activity
13C NMR (300 MHz, [D6]DMSO): δ = 38.88 (CH2), 117.13, bic bacteria, including Gram-positive (Staphylococcus au-
117.54, 117.73, 127.44, 133.97, 135.81, 135.90, 142.80, reus ATCC 6538, Staphylococcus aureus ATCC 25923,
153.72, 154.83, 157.85, 158.88, 159.49, 160.29 (all Ar-C), Staphylococcus epidermidis ATCC 12228, Bacillus subtilis
165.57 (C=O), 166.54 (C=O). – C17H18N6O4 (370.4): calcd. ATCC 6633, Bacillus cereus ATCC 10876, Micrococcus
C 55.13, H 3.75, N 22.69; found C 55.09, H 3.84, N 22.58.
luteus ATCC 10240) and Gram-negative microoorganisms
(Escherichia coli ATCC 25922, Proteus mirabilis ATCC
12453, Klebsiella pneumoniae ATCC 13883, Pseudomonas
aeruginosa ATCC 9027). In the first step, the agar dillution
method was used with Mueller-Hinton medium containing
from 31.25 to 1000 µg mL−1 of the tested compounds per
plate. All stock solutions of the tested compounds were dis-
solved in dimethyl sulfoxide (DMSO) mixed with distilled
water (1 : 1). It was found that DMSO at the final concen-
tration had no influence on the growth of the tested mi-
croorganisms. Bacterial suspensions were prepared in sterile
saline (0.85 % NaCl) with an optical density of McFarland
standard 0.5 (150×106 CFUmL−1; CFU = colony forming
units), and then diluted (1 : 100). 20 µL of each suspension
was put onto Mueller-Hinton agar containing the tested com-
Preparation of 4,4ꢀ-bis[1-(isoquinolin-3-yl)semicarbazide]
diphenylmethane 18
Isoquinoline carboxylic acid hydrazide (20 mmol) was
dissolved in absolute ethanol (10 mL) and 4,4ꢀ-methylene-
diphenylisocyanate (10 mmol) was added. The solution was
warmed for 3 h on a water bath, allowed to cool to r. t. and
then filtered to give a colorless solid.
M. p. 250 – 251 ◦C. – Yield 71 %. – 1H NMR (300 MHz,
[D6]DMSO): δ = 3.86 (s, 2H), 7.08 – 9.42 (m, 20H), 9.50
(2× s, 2H), 9.99 (2× s, 2H), 10.46 (2× s, 2H). – 13C NMR
(300 MHz, [D6]DMSO): δ = 39.06 (CH2), 116.92, 117.21,
119.01, 126.46, 126.64, 127.45, 127.55, 128.02, 130.08,
133.74, 133.85, 136.07, 141.64, 150.33 (all Ar-C), 162.73
(C=O). – C35H28N8O4 (624.6): calcd. C 67.29, H 4.52,
N 17.94; found C 67.31, H 4.44, N 17.88.
◦
pounds. The plates were incubated at 37 C for 18 h. The
MIC (minimal inhibitory concentration) was defined as the
lowest concentration preventing visible growth of the tested
bacteria.
Theoretical calculations
Energies, geometrical parameters (bond lengths, angles
and torsion angles) and charge distribution on the atoms for
structures 1 – 8 were calculated with NWCHEM 5.0 [17, 18]
at the RHF/SCF/6-31G** level of theory. The structures were
fully optimized without any symmetry constraints, and the
initial geometries were built de novo using the AM1 semi-
empirical SCF-MO method [19] implemented in the pro-
gram package HYPERCHEM (version 4.5) [20]. The program
ECCE [21, 22] was used for graphical visualization of the
molecular structure of compound 7.
In the next step, the susceptibility of reference strains
to compound 7 was assayed spectrophotometrically by the
broth microdilution method; the MIC was again defined as
the lowest concentration of a compound at which there is
no visible growth of the tested bacteria. After incubation
◦
(37 C for 18 h), the optical density (OD600) was measured
for the bacterial culture in the broth medium, and the MIC
values were determined by comparison with the growth of
a control (compound-free) medium. Gentamycin, a broad-
spectrum aminoglicoside, was used as a antibacterial refer-
ence compound. The same volume of DMSO without any
test compound was used as a control, and no inhibition was
observed.
Antimicrobial activity
All synthesized compounds were screened for in-vitro
antibacterial activity against 10 reference strains of aero-
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