Synthesis, characterization and antimicrobial evaluation of ethyl 2-arylhydrazono-3-oxobutyrates

Article abstract of DOI:10.1016/S0223-5234(99)80048-8

Ethyl 2-arylhydrazono-3-oxobutyrates 6a-f, 5-(4-aminophenyl)-2,4-dihydro-4-phenyl-3H-1,2,4-triazol-3-one 5d and 1-<4-(benzoylamino)benzoyl>-4-phenylsemicarbazide 4d were synthesized in order to determine their antimicrobial properties. Their structures were elucidated using spectral data. The synthesized compounds were tested in vitro against one Gram-positive and 2 Gram-negative bacterial strains, one Mycobacterial strain and a fungus Candida albicans. Compound 6d showed significant activity against Staphylococcus aureus whereas the others had no remarkable activity on this strain. Compound 6e was found to be more active than the others against Mycobacterium fortuitum at a MIC value of 32 μg/ml. The antibacterial and antifungal activities of 4d, 5d and 6a-f were also compared with various standard drugs. - Keywords: 1,2,4-triazolin-3-ones; 1,2,4-triazoline-3-thiones; semicarbazides; coupling products; microdilution method

Full text of DOI:10.1016/S0223-5234(99)80048-8

Eur. J. Med. Chem. 34 (1999) 153−160
© Elsevier, Paris
153
Short communication
Synthesis, characterization and antimicrobial evaluation
of ethyl 2-arylhydrazono-3-oxobutyrates
S. Güniz Küçükgüzela, Sevim Rollasa*, Habibe Erdenizb, Muammer Kirazb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Tibbiye cad. 81010, Haydarpasa, Istanbul, Turkey
bCenter for Research and Application of Culture Collections of Microorganisms, Faculty of Medicine, University of Istanbul,
Çapa, Istanbul, Turkey
(Received 2 January 1998; accepted 31 July 1998)
Abstract – Ethyl 2-arylhydrazono-3-oxobutyrates 6a–f, 5-(4-aminophenyl)-2,4-dihydro-4-phenyl-3H-1,2,4-triazole-3-one 5d and 1-[4-
(benzoylamino)benzoyl]-4-phenylsemicarbazide 4d were synthesized in order to determine their antimicrobial properties. Their structures
were elucidated using spectral data. The synthesized compounds were tested in vitro against one Gram-positive and 2 Gram-negative bacterial
strains, one Mycobacterial strain and a fungus Candida albicans. Compound 6d showed significant activity against Staphylococcus aureus
whereas the others had no remarkable activity on this strain. Compound 6e was found to be more active than the others against Mycobacterium
fortuitum at a MIC value of 32 µg/mL. The antibacterial and antifungal activities of 4d, 5d and 6a–f were also compared with various standard
drugs. © Elsevier, Paris
1,2,4-triazoline-3-ones / 1,2,4-triazoline-3-thiones / semicarbazides / coupling products / microdilution method
1. Introduction
zoylamino)benzoyl]-4-phenylsemicarbazide 4d and 5-(4-
aminophenyl)-2,4-dihydro-4-phenyl-3H-1,2,4-triazole-3-
one 5d. The structures of the synthesized compounds
A number of compounds derived from hydrazide-
hydrazones, 1,2,4-triazoline-3-thiones/1,2,4-triazoline-3-
ones and 1,3,4-oxadiazoline-2(3H)-thiones are known to
possess antibacterial, antifungal and antimycobacterial
activities [1–9]. The coupling products starting from
diazonium salts of 4-aminobenzoic acid hydrazide sub-
stituted benzalhydrazones with indole have also been
reported to possess promising antibacterial and antituber-
1
were elucidated by UV, IR, H-NMR, 13C-NMR and
EI-mass spectral data. These compounds were tested for
antimicrobial activities against the microorganisms used
in the present study.
2. Chemistry
cular
activities [10].
In
a
previous
study,
4-Aminobenzoic acid [(4-fluorophenyl) methylene]hy-
drazide 3; 5-(4-aminophenyl)-2,4-dihydro-4-ethyl/allyl/
phenyl-3H-1,2,4-triazole-3-thiones 5a–c; 5-(4-amino-
phenyl)-1,3,4-oxadiazole-2(3H)-thione 5e were prepared
as described previously [6, 12, 13] (figure 1). 1-[4-
(Benzoylamino) benzoyl]-4-phenylsemicarbazide 4d and
5-(4-aminophenyl)-2,4-dihydro-4-phenyl-3H-1,2,4-
triazole-3-one 5d were determined to be original com-
pounds (figure 1).
These products 3, 5a–e were then diazotized and
coupled with ethyl acetoacetate (figure 2). The coupling
products were formulated as ethyl 2-arylhydrazono-3-
oxobutyrates 6a–f.
4-(acetylacetonylidenehydrazono)-[(4-fluorophenyl)-me-
thylene]benzoic acid hydrazide were found to be active
against Mycobacterium fortuitum ATCC 6841 [11]. These
observations led us to synthesize some novel coupling
products.
In the present study, a new series of ethyl 2-aryl-
hydrazono-3-oxobutyrates 6a–f were obtained via cou-
pling of diazonium chlorides of the above-mentioned
heterocyclic rings with ethyl acetoacetate. In addition, the
present study includes the synthesis of 1-[4-(ben-
*Correspondence and reprints

154
Figure 1. Synthesis of aromatic primary amines.
3. Microbiological results
that of tobramycine which was 16–32 lg/mL against
medium resistant bacteria.
For the observation of the in vitro antifungal activity,
the synthesized compounds were screened against C.
albicans ATCC 2091. However, they were found to have
no activity except 5d, 6c, 6d, 6e and 6f which showed
only marginal effect at a MIC value of 62.5 lg/mL.
The synthesized compounds 4d, 5d, 6a–f were tested
for antibacterial, antifungal and antimycobacterial
activities against various strains by microdilution
method [14–18]. For the determination of antibacterial
activity, one Gram-positive and 2 Gram-negative bacte-
rial strains were utilized. Antimycobacterial and antifun-
gal assays of all compounds were also performed against
M. fortuitum ATCC 6841, a rapidly growing mycobacte-
rium and the yeast C. albicans ATCC 2091, respectively.
The results reported in table I indicate that, the
synthesized compounds were not active against S. aureus
ATCC 29213, Escherichia coli ATCC 25922, Pseudomo-
nas aeruginosa ATCC 27853 except 6d, which had a
significant growth inhibitory effect on S. aureus ATCC
29213 at a MIC value of 7.8 lg/mL. For comparison of
the antibacterial activity observed with the test com-
pounds, ceftriaxone was selected as standart drug. The
fact that this standart had a reported MIC value of
1–8 lg/mL against S. aureus ATCC 29213 showed the
significance of further studies on compound 6d.
4. Discussion
The novel compounds 4d, 5d, 6a–f gave satisfactory
elemental analyses. Their structures were established by
1
the use of spectral data (UV, IR, H-NMR, 13C-NMR,
EI-mass). UV absorption maxima [19], IR stretching and
1
bending vibrations [8] and H-NMR chemical shifts [7,
20] were all supported the proposed structures of 4d and
5d. 13C-NMR data obtained from 5d were also in line
with literature values [20].
The UV spectra of 6a–f showed two absorption bands
at 235–263 and 360–390 nm regions which were charac-
teristic for carbonyl and hydrazone groups, respectively.
In the UV spectra of compounds 6a–f, absorptions arising
from –N=N– band at around 332–360 nm [21, 22] and
above 400 nm [23] were not observed. Based on this
finding, the structures of these compounds have been
Compound 6e was the most active derivative against
M. fortuitum ATCC 6841 having the MIC value of
32 lg/mL. This MIC value was also in accordance with

155
Figure 2. Synthetic route to coupling products 6a–f.
given in hydrazone form. The UV spectra of 6a, 6d and
6f exhibited the characteristic bands arising from chro-
mophoric –C=N, C=S groups at 319.80, 294.60 and
305.60 nm, respectively.
IR spectra of 6a–f showed characteristic bands at
1720–1685 and 1690–1660 cm–1 corresponding to ester
and ketone C=O groups, respectively [24]. N–H strech-
ings due to hydrazone, 1,2,4-triazoline-3-thiones/3-one
and 1,3,4-oxadiazoline-2(3H)-thione were detected
within the 3400–3150 cm–1 region. In addition, the pres-
ence of an absorption band arising from triazoline C=S at
1180 cm–1 and the lack of absorption bands at around
2590–2250 cm–1 supported the thione form of 6b, 6c, 6d
and 6f [6, 25, 26]. The hydrazide–hydrazone C=O and
Ar–F bands of 6a were observed at 1650 and 1170 cm–1,
respectively.
1
The H-NMR spectra of 6b in CDCl3 and 6a, 6c, 6d
and 6f in DMSO-d6 displayed the hydrazone N–H pro-
Table I. The in vitro antimicrobial activity results of the synthesized compounds (MIC in µg/mL).
Compound
S. aureus
ATCC 29213
E. coli
ATCC 25922
P. aeruginosa
ATCC 27853
M. fortuitum
ATCC 6841
C. albicans
ATCC 2091
4d
5d
6a
6b
6c
6d
6e
6f
250
250
250
500
250
7.8
250
125
4
125
250
250
250
250
250
250
250
0.03
1
250
125
125
125
250
125
125
125
8
> 128
64
64
64
128
64
32
64
–
250
62.5
125
125
62.5
62.5
62.5
62.5
–
Ceftriaxone
Tobramycine
Miconazole
–
–
2
–
16
–
–
0.05
–

156
5. Experimental protocols
5.1. Chemistry
Ethyl isothiocyanate, phenyl isothiocyanate and
4-fluorobenzaldehyde were purchased from Sigma. All
other chemicals were purchased from Merck.
All melting points were recorded on a Buchi-530
melting point apparatus and uncorrected. UV spectra
were recorded on a Shimadzu UV 2100S spectrophotom-
eter (1 mg/100 mL in ethanol). IR spectra were run on a
Shimadzu IR-470 spectrophotometer (1 mg/200 mg in
KBr). Nuclear Magnetic Resonance spectra (1H, 13C)
were recorded on a Bruker AVANC-DPX 400 spectrom-
eter. MS spectra were obtained on a Fisons Instruments
VG Platform II LC-MS in the electron impact (EI) mode.
Elemental analyses were performed on a Carlo Erba 1106
instrument.
Figure 3. 13C-NMR spectral data of 6b.
tons at 11.83, 11.84, 11.20, 10.07 and 12.67 ppm,
respectively [27]. The hydrazone and triazoline N–H
protons of 6e were observed to exchange with deuterium
1
in DMSO-d6. In addition, in the H-NMR spectra of
compounds 6a–f, signals arising from >CH–N=N– struc-
ture at 3.00–4.00 ppm [28, 29] were not observed. This
finding also supported that the structures of these com-
pounds might be given in hydrazone form.
5.1.1. Preparation of aromatic primary amines
These compounds 3, 5a–c, 5e were prepared as de-
scribed previously [6, 12, 13]. In addition, 5-(4-
aminophenyl)-2,4-dihydro-4-phenyl-3H-1,2,4-triazole-3-
one 5d having a primary aromatic amine function was
synthesized as a novel compound in this study.
13C-NMR spectral data of 6b and 6e are shown in
figure 3 and figure 4, respectively. These results were also
in accordance with literature values [26, 30, 31].
EI-mass spectra of 6c, 6e and 6f showed the correct
molecular ion (M+) peaks of different intensity which
confirmed their molecular weights. The major fragmen-
tation pathway appeared by the cleavage of –NH–N=C<
bonds of hydrazone moiety [26, 32, 33]. Molecular ion
was not detected in the mass spectrum of compound 6d
although characteristic fragment ions such as the one at
m/z 367 (M+–42), which formed by the loss of
CH2=C=O, were observed. In addition, EI-mass spectra
of the synthesized compounds exhibited the expected
fragmentation pattern of triazoline and oxadiazoline
structures (figure 5).
5.1.2. Synthesis of 1-[4-(benzoylamino)benzoyl]-4-
phenylsemicarbazide 4d and 5-(4-aminophenyl)-2,4-
dihydro-4-phenyl-3H-1,2,4-triazole-3-one 5d
4-(Benzoylamino)benzoic acid hydrazide 1 was pre-
pared as described previously [34]. To an ethanolic solu-
tion of this compound (0.01 mol) was added phenyl
isocyanate (0.015 mol) and the resulting mixture was
refluxed for 2 h. The obtained product 4d was filtered and
washed with boiling ethanol. Yield: 83%, m.p. =
270–275 °C. UV (EtOH): λmax (log e) = 282 (4.43) nm,
235.50 (4.31) nm. IR (KBr): 3450, 3300, 3080, 3050,
1660, 1650, 1600, 1570, 1520, 1440, 1420, 1320, 840,
690 cm–1. 1H-NMR (400 MHz, DMSO-d6) δ ppm:
6.95–8.13 (m, 14H, Ar-H); 8.84 (s, 1H, –CONHNH–);
9.66 (s, 1H, –CONH–C6H5); 10.19 (s, 1H,
C6H5–CONH–C6H4–); 10.41 and 10.47 (2s, 1H,
–CONHNH–);
analysis:
C21H18N4O3
(%
calculated/found): 67.37/66.64 (C); 4.84/4.88 (H);
14.96/15.47 (N).
Compound 4d (0.0134 mol) was refluxed in 65 mL
NaOH (2 N) for 4 h. On cooling, it solidified (sodium
salt). The precipitate was dissolved in water and acidified
with hydrochloric acid (37%) to give compound 5d. The
crude product was washed with water and recrystallized
from ethanol. Yield: 30%, m.p. = 274–275 °C. UV
(EtOH): λmax (log e) = 283.50 (4.27) nm. IR (KBr): 3410,
3350, 3190, 3050, 1695, 1630, 1615, 1580, 1510, 1460,
Figure 4. 13C-NMR spectral data of 6e.

157
Figure 5. Mass spectral fragmentation pattern of compounds 6c–f.
1440, 1280, 830, 710 cm–1. 1H-NMR (400 MHz, DMSO-
d6) δ ppm: 5.36 (s, 2H, Ar–NH2); 6.42 (d, 2H, o-NH2, J
= 8 Hz); 6.91 (d, 2H, m-NH2, J = 8 Hz); 7.21–7.46 (m,
5H, Ar–H); 11.78 (s, 1H, triazoline N–H). 13C-NMR (100
MHz, DMSO-d6) δ ppm: 114.01 (C2 and C6); 114.64 (C8
and C12); 128.63 (C4); 129.10 (C10); 129.49 (C9 and
C11); 130.03 (C3 and C5); 135.08 (C7); 147.04 (C1);
151.02 (triazoline, C=N); 155.51 (oxolactam, C=O);
analysis C14H12N4O (% calculated/found): 66.65/66.93
(C); 4.80/4.74 (H); 22.21/22.26 (N).
(q, 2H, –O–CH2–CH3); 7.27–8.00 (m, 8H, Ar–H); 8.68
(s, 1H, –CH=N–); 11.41 (s, 1H,=N–NH–CO–); 11.84 (s,
1H, –NH–N=C<); analysis C20H19FN4O4 (%
calculated/found): 60.30/61.91 (C); 4.81/4.53 (H);
14.06/13.59 (N).
5.1.5. Ethyl 2-[4-(2,4-dihydro-4-ethyl-3H-1,2,4-tria-
zole-3-thione-5-yl)phenylhydrazono]-3-oxobutyrate 6b
Yield: 95%; m.p. = 170–172 °C. UV (EtOH): λmax (log
e) = 362 (4.43) nm, 256.80 (4.33) nm. IR (KBr): 3190,
3000, 2950, 1685, 1660, 1610, 1520, 1480, 1460, 1440,
1320, 1280, 1180, 845 cm–1. 1H-NMR (400 MHz,
CDCl3) δ ppm: 1.41 (m, 6H, –O–CH2–CH3 and
–N–CH2–CH3); 2.54 (s, 3H, –CO–CH3); 4.20 (q, 2H,
–N–CH2–CH3); 4.42 (q, 2H, –O–CH2–CH3); 7.50 (d, 2H,
o-NH, J = 8.5 Hz); 7.65 (d, 2H, m-NH, J = 8.5 Hz); 11.83
(b, 1H, –NH–N=C<); 12.76 (s, 1H, triazoline N–H).
13C-NMR (100.6 MHz, CDCl3) δ ppm: 14.45 (m,
–O–CH2–CH3 and –N–CH2–CH3); 27.28 (–CO–CH3);
40.57 (–N–CH2–CH3); 62.19 (–O–CH2–CH3); 116.69
(C2 and C6); 122.14 (C4); 129.26 (C1); 130.32 (C3 and
C5); 144.19 (hydrazone, C=N); 151.79 (triazoline, C=N);
163.89 (thiolactam, C=S); 168.12 (ester, –COO–C2H5);
194.72 (ketone, –CO–CH3); analysis C16H19N5O3S (%
5.1.3. Synthesis of ethyl 2-arylhydrazono-3-oxobuty-
rates 6a–f
Aryldiazonium salts of the aromatic primary amines 3,
5a–e were formed by the action of nitrous acid on
compounds 3, 5a–e at 0–5 °C. These were then coupled
with ethyl acetoacetate in the presence of sodium acetate.
5.1.4. Ethyl 2-{4-[[(4-fluorophenyl)methylene]hydrazi-
nocarbonyl]phenylhydrazono}-3-oxobutyrate 6a
Yield: 58%; m.p. = 171–175 °C. UV (EtOH): λmax (log
e) = 319.80 (4.60) nm. IR (KBr) 3200, 3080, 3040, 2910,
1720, 1690, 1650, 1600, 1570, 1510, 1420, 1380, 1170,
1
850, 840 cm–1. H-NMR (400 MHz, DMSO-d6) δ ppm:
1.28 (t, 3H, –O–CH2–CH3); 2.42 (s, 3H, –COCH3); 4.33

158
calculated/found): 53.17/52.88 (C); 5.30/5.22 (H);
19.38/19.08 (N); 8.87/8.32 (S).
(% calculated/found): 53.69/53.21 (C); 5.63/4.74 (H);
15.65/15.35 (N).
5.1.6. Ethyl 2-[4-(4-allyl-2,4-dihydro-3H-1,2,4-tria-
zole-3-thione-5-yl)phenylhydrazono]-3-oxobutyrate 6c
Yield: 98%; m.p. = 85–90 °C. UV (EtOH): λmax (log e)
= 378.60 (4.26) nm, 258.60 (4.17) nm. IR (KBr):
3450–3350, 3150, 2900, 1710, 1660, 1610, 1590, 1540,
1480, 1250, 1180, 840 cm–1. EI-MS (m/z): 373 (M+), 341,
332, 331, 326, 286, 260, 258, 246, 232, 231, 217, 216,
157, 133, 132, 119, 118, 92. 1H-NMR (400 MHz,
DMSO-d6) δ ppm: 1.18 (t, 3H, –O–CH2–CH3); 2.19 (s,
3H, –CO–CH3); 4.18 (q, 2H, –O–CH2–CH3); 4.90 (d, 1H,
allyl, =C<H trans, J = 16 Hz); 5.15 (d, 1H, allyl, =C<H cis,
J = 10 Hz); 5.82–6.10 (m, 1H, allyl, –CH =); 7.56 (d, 2H,
o-NH, J = 8.6 Hz); 7.73 (d, 2H, m-NH, J = 8.6 Hz); 11.20
(s, 1H, –NH–N=C<); 13.98 (s, 1H, triazoline N–H):
analysis for C17H19N5O3S•H2O (% calculated/found):
52.16/51.85 (C); 5.41/4.85 (H); 8.19/8.05 (S).
5.1.9. Ethyl 2-[4-(1,3,4-oxadiazole-2(3H)-thione-5-yl)
phenylhydrazono]-3-oxobutyrate 6f
Yield: 80%; m.p. = 178–180 °C. UV (EtOH): λmax (log
e) = 383.60 (4.40) nm; 305.60 (4.16) nm; 235 (4.17) nm.
IR (KBr): 3450–3350, 3100, 2950, 2800, 1680, 1610,
1580, 1510, 1500, 1180, 840 cm–1. EI-MS (m/z): 336,
335, 334 (M+), 290, 289, 262, 261, 246, 219, 194, 193,
192, 187, 158, 157, 133, 132 (100%), 92, 65, 63.
1H-NMR (400 MHz, CDCl3) δ ppm: 1.38 (t, 3H,
–O–CH2–CH3); 2.49 (s, 3H, –CO–CH3); 4.36 (m, 2H,
–O–CH2–CH3); 7.40 (d, 2H, o-NH, J = 8.6 Hz); 7.93 (d,
2H, m-NH, J = 8.6 Hz); 12.67 (s, 1H, –NH–N=C<);
14.60 (s, 1/2H, oxadiazoline N–H): analysis
C14H14N4O4S•H2O (% calculated/found): 47.72/47.52
(C); 4.58/3.85 (H); 15.90/16.03 (N); 9.10/10.01 (S).
5.2. Microbiology
5.1.7. Ethyl 2-[4-(2,4-dihydro-4-phenyl-3H-1,2,4-tria-
zole-3-thione-5-yl)phenylhydrazono]-3-oxobutyrate 6d
Yield: 89%; m.p. 205–210 °C. UV (EtOH): λmax (log
e) = 390 (3.65) nm, 294.60 (4.45) nm, 263 (4.37) nm. IR
(KBr): 3400, 3150, 3080, 2900, 1720, 1650, 1610, 1590,
1550, 1500, 1420, 1320, 1250, 1180, 840, 700 cm–1.
EI-MS (m/z): 367 (M+–42), 337, 269, 268 (100%), 267,
Compounds 4d, 5d and 6a–f were examined for their
in vitro growth inhibitory activity against different bac-
terial strains in addition to M. fortuitum ATCC 6841, a
rapidly growing mycobacterium and a yeast-like fungus,
C. albicans ATCC 2091. Bacterial strains utilized were S.
aureus ATCC 29213 as Gram-positive; E. coli ATCC
25922 and P. aeruginosa ATCC 27853 as Gram-negative
bacteria. Antibacterial, antifungal and antimycobacterial
assays were all performed by the use of the two-fold
serial microdilution technique [14–18].
1
150, 133, 132, 119, 118, 92, 77, 65, 63. H-NMR (400
MHz, DMSO-d6) δ ppm: 1.89 (m, 3H, –O–CH2–CH3);
2.03 (s, 3H, –CO–CH3); 4.11 (q, 2H, –O–CH2–CH3);
7.29–7.51 (m, 9H, Ar–H); 10.07 (s, 1/2H, –NH–N=C<);
13.36 (b, 1H, triazoline N–H): analysis C20H19N5O3S (%
calculated/found): 58.67/58.05 (C); 4.68/4.70 (H);
17.11/17.83 (N).
5.2.1. Antibacterial and antifungal activity
Standardized bacterial and fungal inocula were pre-
pared by touching the top of four or five colonies of a
single type and inoculating them into a tube containing
5 mL of Mueller–Hinton broth (Difco) at pH 7.3 for
bacteria and buffered Yeast Nitrogen Base (YNB) at pH 7
for C. albicans. Incubations of these microorganism
suspensions were carried out at 35 °C for bacteria and the
yeast C. albicans until a visible turbidity was obtained.
The density of these cultures were then adjusted to a
turbidity equivalent to that of a 0.5 Mc Farland standard
and finally, the adjusted culture was diluted so that, after
inoculation, each microplate well had an inoculum size of
5 × 105 cfu/mL for bacteria and 0.5 × 103 to 2.5 × 103
cells per mL for C. albicans.
Antibacterial and antifungal assays were performed in
Mueller–Hinton broth at pH 7.3 and buffered Yeast
Nitrogen Base at pH 7, respectively. Ceftriaxone for
bacteria and Miconazole for the yeast C. albicans were
used as standard drugs. All the test compounds were
dissolved in DMSO. Further dilutions of the compounds
5.1.8. Ethyl 2-[4-(2,4-dihydro-4-phenyl-3H-1,2,4-tria-
zole-3-one-5-yl)phenylhydrazono]-3-oxobutyrate 6e
Yield: 80%; m.p. 235–238 °C. UV (EtOH): λmax (log
e) = 360 (4.05) nm; 239.40 (4.36) nm. IR (KBr):
3500–3400, 3300, 3200, 3100, 2900, 1680, 1600, 1550,
1500, 1440, 1280, 840, 700 cm–1. EI-MS (m/z): 393 (M+)
306, 278, 261, 259, 253, 252, 251, 177, 175, 134, 133,
1
120, 119, 118, 92, 77, 63. H-NMR (400 MHz, DMSO-
d6) δ ppm: 1.28 (t, 3H, –O–CH2–CH3); 1.80 (s, 3H,
–CO–CH3); 4.27 (m, 2H, –O–CH2–CH3); 7.24–7.98 (m,
9H, Ar–H).13C-NMR (100.6 MHz, DMSO-d6): δ ppm
14.81 (–O–CH2–CH3); 24.04 (–CO–CH3); 61.80
(–O–CH2–CH3); 119.47 (C2 and C6); 122.31 (C8 and
C12); 128.57 (C4); 129.39 (C10); 129.64 (C9 and
C11);129.94 (C1); 130.23 (C3 and C5); 131.35 (C7);
141.06 (hydrazone, C=N); 149.42 (triazoline, C=N);
157.25 (oxolactam, C=O); 166.32 (ester, –COO–C2H5);
174.85 (ketone, –CO–CH3): analysis C20H19N5O4•3H2O

159
and the standard drugs in the test medium were furnished
at the required quantities of the broth used. The concen-
tration range was 32–0.015 lg/mL for Ceftriaxone,
100–0.05 lg/mL for Miconazole and 128–1 lg/mL for
the test compounds. After inclusion of 100 lL of the
broth containing the standard drugs or the test com-
pounds, 100 lL of bacterial or fungal suspension were
inoculated into microplate wells. After incubation for
16–20 h at 35 °C (for bacteria) or 46–50 h at 35 °C (for
C. albicans), the well, containing the lowest concentra-
tion of the standard drugs or the test compounds that
inhibits microorganism growth as detected by the unaided
eye, was recorded to represent the MIC expressed in
lg/mL.
Acknowledgements
This work was supported by The Scientific and Tech-
nical Research Council of Turkey (TÜBITAK), Research
Fund Project Number: TBAG-AY/72.
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Chem. Soc. 61 (1984) 718–720.
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21 (1984) 1225–1229.
5.2.2. Antimycobacterial activity
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Pharmazie 48 (1993) 308–309.
Preparation of Mycobacterial inoculum required a few
modifications due to the difficulty on obtaining a homog-
enous suspension of M. fortuitum in the broth used. Four
or five colonies of M. fortuitum which were previously
grown in Tryptic Soy Agar (TSA) after 72 h of incubation
at 30 °C were collected by means of a swab and sus-
pended in 4.5 mL of Mueller–Hinton broth enriched with
Tween 80 (0.2%). Following the inclusion of 4–5 glass
beads, this mixture was whirled using a vortex-mixer to
ensure a good suspension. The density of this culture was
then adjusted to a turbidity equivalent to that of a 0.5 Mc
Farland standard and finally, the adjusted culture was
diluted with sterile water so that, after inoculation, each
microplate well had an inoculum size of 1.5 × 105
cfu/mL.
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(1976) 2871–2876.
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International Symposium on Medicinal Chemistry, Maastricht, The Neth-
erlands, 1996, Abstract P-10.13.
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Boll. Chim. Farmac. 134 (1995) 375–379.
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Mariollet M., Parot J.L., Ros A., Maugein J.T., Volle P.J., J. Clin. Microbiol.
26 (1988) 2307.
Antimycobacterial testing of all compounds was car-
ried out in Mueller–Hinton broth enriched with Tween 80
(0.2%) at pH 7.3. Tobramycine which is an active
antibiotic against rapidly growing mycobacteria was
selected as standard drug. Quality control strains used in
the present study were E. coli and S. aureus. Tobramycine
was used at a concentration range of 4–0.03 lg/mL
against the quality control strains. The standard drug and
test compounds were dissolved in water and DMSO,
respectively, and were diluted with the broth used. The
concentration intervals were 32–0.5 lg/mL and
128–1 lg/mL for the standard drug and the test com-
pounds, respectively. Microplate wells, added with
100 lL of broth containing Tobramycine or the test
compounds, were then inoculated with 10 lL of M.
fortuitum suspension whose preparation were mentioned
above. Sheep-blooded agar was used for the purity
control. After incubation for 72 h at 30 °C, the last
microplate well with no growth of microorganism was
recorded to represent MIC expressed in lg/mL.
[15] Hawkins J.E., Wallace R.J., Brown B.A., In: Balows A., Hausler
W.J., Hermann K.L., Isenberg H.O., Shadomy H.I. (Eds.), Manual of
Clinical Microbiology (5th ed.), American Society for Microbiology,
Washington DC, 1991, pp. 1138-1152.
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Laboratory Medicine III, Williams and Wilkins, London, Los Angeles,
1991, p. 198.
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for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically, 4th Informational Supplement, Approved Standard, NCCLS
document M 100 S4, Villanova, 1992.
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Chemother. 22 (1992) 186–192.
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of Drug Substances, Vol. 16, Academic Press, New York, 1987, p. 124.
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1738–1740.
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(1966) 2981–2989.
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[25] Rollas S., J. Fac. Pharm. Istanbul 17 (1981) 155–163.

160
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39–49.
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Analytical Profiles of Drug Subtances, Vol. 15, Academic Press, New York,
1986, pp. 90-91.
(1990) 346–347.
[32] Ergenç N., Özçekiç H., Pharmazie 43 (1988) 832–834.
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[33] Mazzone G., Pignatello R., Panico A., Mazzone S., Puglisi G.,
Pennisi G., Raciti G., Mazzone P., Matera M., Pharmazie 47 (1992)
902–910.
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53–58.
[34] Durgun B.B., Rollas S., J. Pharm. Univ. Mar. 7 (1991) 107–111.