Preparation of some thiazolyl hydrazone derivatives and evaluation of their antibacterial activities

Article abstract of DOI:10.1080/10426500802534176

The increasing clinical importance of drug-resistant fungal and bacterial pathogens has provided additional urgency to microbiological research and to the development of new antibacterial compounds. For this purpose, new tert-butyl [1-aryl/alkyl-2[(4-aryl

Full text of DOI:10.1080/10426500802534176

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Preparation of Some Thiazolyl
Hydrazone Derivatives
and Evaluation of Their
Antibacterial Activities
Gülhan Turan-Zitouni a , Ahmet Özdemir a , Zafer
Asım Kaplancıklı a , Jean-Alain Fehrentz b , Jean
Martinez b , Pierre Chevallet b & Gislaine Dusart c
a Anadolu University, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Eskişehir,
Turkey
b Institut des Biomolécules Max Mousseron, CNRS-
Université Montpellier 1 et 2, Faculté de Pharmacie,
UMR 5247, Montpellier Cedex, France
c Laboratoire de Bactériologie, Faculté de
Pharmacie, Montpellier Cedex, France
Version of record first published: 21 Sep 2009.
To cite this article: Gülhan Turan-Zitouni, Ahmet Özdemir, Zafer Asım Kaplancıklı,
Jean-Alain Fehrentz, Jean Martinez, Pierre Chevallet & Gislaine Dusart (2009):
Preparation of Some Thiazolyl Hydrazone Derivatives and Evaluation of Their
Antibacterial Activities, Phosphorus, Sulfur, and Silicon and the Related Elements,
184:10, 2613-2623
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Phosphorus, Sulfur, and Silicon, 184:2613–2623, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802534176
Preparation of Some Thiazolyl Hydrazone Derivatives and
Evaluation of Their Antibacterial Activities
1
1
¨
¨
Gulhan Turan-Zitouni, Ahmet Ozdemir, Zafer Asım
Kaplancıklı,1 Jean-Alain Fehrentz,2 Jean Martinez,2
Pierre Chevallet,2 and Gislaine Dusart3
1Anadolu University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Eskis¸ehir, Turkey
2Institut des Biomole´cules Max Mousseron, UMR 5247,
CNRS-Universite´ Montpellier 1 et 2, Faculte´ de Pharmacie, Montpellier
Cedex, France
3Laboratoire de Bacte´riologie, Faculte´ de Pharmacie, Montpellier
Cedex, France
The increasing clinical importance of drug-resistant fungal and bacterial
pathogens has provided additional urgency to microbiological research and
to the development of new antibacterial compounds. For this purpose, new
tert-butyl [1-aryl/alkyl-2[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate deriva-
tives were synthesized and evaluated for antibacterial activity. The reaction of Boc-
L-phenylalaninal, Boc-D-phenylalaninal, Boc-L-leucinal, and Boc-L-tryptophanal
with thiosemicarbazide yielded the thiosemicarbazones, which furnished the title
compounds on reaction with phenacyl bromides. The new compounds were screened
for antibacterial activity. The results from the bioassay tests show that some of the
compounds have notable activity against Gram-positive bacteria.
Keywords Amino acid; antibacterial activity; thiazole
INTRODUCTION
The treatment of infectious diseases still remains an important and
challenging problem because of a combination of factors, including
emerging infectious diseases and the increasing number of multi-
drug–resistant microbial pathogens with particular relevance for
Gram-positive bacteria. The therapeutic problem has achieved increas-
ing importance in hospitalized patients, in immunosuppressed patients
Received 21 September 2008; accepted 7 October 2008.
Address correspondence to Gu¨lhan Turan-Zitouni, Anadolu University, Faculty of
Pharmacy, Department of Pharmaceutical Chemistry, Eskis¸ehir 26470, Turkey. E-mail:
gturan@anadolu.edu.tr
2613

2614
G. Turan-Zitouni et al.
with AIDS or undergoing anticancer therapy, and in the case of or-
gan transplant patients. In spite of a large number of antibiotics and
chemotherapeutics available for medical use, the emergence of old and
new antibiotic resistance created in the last decades causes a substan-
tial medical need for new classes of antibacterial agents. A potential
approach to overcoming the resistance problem is to design innovative
agents with a different mode of action so that no cross-resistance with
the present therapeuticals can occur.1
Several natural products contain thiazole derived from precursor
peptides with Xaa-Ser/Cys sequences (where Xaa represents any amino
acid).1 These molecules often exhibit useful therapeutic properties,
ranging from the anticancer activity of the bis(thiazole)-containing
drug bleomycin A2 to the Gram-positive antibiotic activity of GE2270,3
which is assembled around a thiazole-pyridine-oxazoline scaffold. This
compound is thus structurally related to other thiazolylpeptide an-
tibiotics (thiostrepton, nosiheptide, etc.), which also consist of a highly
modified peptide with several thiazole rings. While all thiazolylpeptides
exert their action by inhibiting bacterial protein synthesis, GE2270, to-
gether with the closely related molecules, amithiamycin,4 is a potent
and selective inhibitor of bacterial elongation factor Tu.5,6 Thus it dif-
fers in its molecular target from thiostrepton and related compounds,
which bind to the L11 protein in the 23S ribosomal complex.7 The
biosynthesis of thiazolylpeptide antibiotics has been investigated in
several instances.8,9 These studies have shown that the thiazole rings
are labelled by serine, likely after its conversion to cysteine, and that
the pyridine ring, also present in the thiostrepton family of antibiotics,
derives from the head-to-head condensation of two serine residues.10
Previously we reported that some tert-butyl [1-benzyl-2-[(4-aryl-
2-thiazolyl)hydrazono]ethyl]carbamate derivatives can exhibit signif-
icant antibacterial activity.11 As the extension of our interest in
the search for antibacterial agents, a different series of tert-butyl
[1-aryl/alkyl-2-[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate deriva-
tives has been synthesized and investigated for antibacterial activity.
RESULTS AND DISCUSSION
Chemistry
In the present work, nine new compounds were synthesized. tert-
Butyl [1-aryl/alkyl-2-thiosemicarbazonoethyl]carbamates (3) were pre-
pared by reacting Boc-L-phenylalaninal, Boc-D-phenylalaninal, Boc-L-
leucinal, and Boc-L-tryptophanal with thiosemicarbazide in accordance

Thiazolyl Hydrazone Derivatives
2615
SCHEME 1
with the method described in the literature.11–16 The reaction
of tert-butyl [1-aryl/alkyl-2-thiosemicarbazonoethyl]carbamates (3)
with phenacyl bromides gave the tert-butyl [1-aryl/alkyl-2-[(4-aryl-
2-thiazolyl)hydrazono]ethyl] carbamate derivatives 4 as shown in
Scheme 1 and Table I.
The identity of compounds 3 and 4 was confirmed by elemental anal-
yses as well as by IR, 1H NMR, and FAB+-MS spectral data. The IR data
were very informative and provided evidence for the formation of the
expected structures. N−H, C=O, C=N, C=C, and C−O−C functions ab-
sorbed strongly in the expected regions: N−H at 3490–3410 cm−1, C=O
at 1697–1664 cm−1, C=N and C=C at 1564–1502 cm−1, and C−O−C at
1
1164–1135 cm−1. The H NMR spectra showed the signal for the tert-
butyl protons at 1.05–1.60 ppm for all nine compounds. The signals for
the CH2 protons of benzyl, isobutyl, and 3-indolylmethyl substituents
were observed at 2.70–3.15 ppm as a multiplet; for NH-CH a broad sig-
nal appeared at 4.05–4.50 ppm. The signals of the CO−NH and CH=N

2616
G. Turan-Zitouni et al.
TABLE I Some Analytical Data of the New Compounds
R
R1
—
Yield (%)
79
Mp (◦C)
M.W.
322
3a [L]11 and
3a [D]
Bz
136–138
3b [L]
i-Bu
—
—
Cl
76
74
61
170–172
122–124
230–232
288
361
472.5
3c [L]
1H-indol-3-ylmethyl
Bz
4a [L]11 and
4a [D]
4b [L]11 and
4b [D]
Bz
OCH3
62
168–170
468
4c [L]
4d [L]
4e [L]
4f [L]
i-Bu
i-Bu
Cl
OCH3
Cl
68
58
65
56
216–218
214–215
219–221
211–213
438
434
511
507
1H-indol-3-ylmethyl
1H-indol-3-ylmethyl
OCH3
protons appeared in the aromatic region together with those of the
aromatic protons. All the other aromatic and aliphatic protons were
observed in the expected regions. All compounds gave satisfactory el-
emental analyses. Mass spectra (MS (FAB)) of the compounds showed
an M+1 peak, in agreement with their molecular formula.
Microbiology
All of the synthesized compounds were preliminarily evaluated for their
in vitro antibacterial activity against the Gram-positive bacteria S. au-
reus (ATCC 6538), S. aureus n◦ 82, S. aureus n◦ 83, S. aureus n◦ 84,
S. faecalis (ATCC 10541), S. faecalis n◦ 2, S. faecalis n◦ 3, M. luteus
(ATCC 9341), B. cereus (ATCC 14893), and B. subtilis (ATCC 6633) as
well as against the Gram-negative bacteria E. coli (ATCC 8739), E. coli
n◦ 151, E. coli n◦ 152, E. coli n◦ 153, K. pneumoniae (ATCC 10031),
K. pneumoniae n◦ 178, E. cloacae (ATCC 23355), E. cloacae n◦ 209,
P. aeruginosa (ATCC 9027), P. aeruginosa n◦ 147, and P. aeruginosa
n◦ 148. Amoxicillin and chloroamphenicol were used as standard an-
tibacterial references. Dimethylsulfoxide (DMSO) was used as a blank
and showed no antibacterial activity. As revealed from Table II, four
compounds (4a [L], 4a [D], 4b [L], and 4d [L]) showed promising
antibacterial activity.
In comparing their MIC values with that of chloramphenicol, 4a[L]
and 4a[D] were effective against S. aureus (ATCC 6538), S. aureus n◦
82, S. aureus n◦ 83, and S. aureus n◦ 84. Compounds 4a[L] and 4a[D]
showed a similar level of activity as chloramphenicol.

Thiazolyl Hydrazone Derivatives
2617
TABLE II MIC Values (µg /mL) of the New Compounds
Microorganisms
4a [L]
4a [D]
4b [L]
4d [L]
A
B
S. aureus (ATCC 6538)
S. aureus n◦ 82
S. aureus n◦ 83
S. aureus n◦ 84
S. faecalis (ATCC
10541)
15.6
15.6
15.6
15.6
31.25
15.6
15.6
15.6
15.6
31.25
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
62.5
125
<0.24
125
31.25
31.25
1.95
15.6
15.6
7.8
15.6
3.9
S. faecalis n◦
2
3
31.25
31.25
31.25
31.25
31.25
31.25
31.25
31.25
125
125
125
62.5
125
125
125
62.5
1.95
0.49
<0.24
31.25
3.9
3.9
1.95
3.9
S. faecalis n◦
M. luteus (ATCC 9341)
B. cereus (ATCC
14893)
B. subtilis (ATCC
6633)
15.6
31.25
62.5
62.5
<0.24
7.8
E. coli (ATCC 8739)
250
250
250
250
31.25
250
250
250
250
31.25
500
500
500
500
125
500
500
500
500
125
7.8
7.8
7.8
7.8
7.8
3.9
E. coli n◦ 151
>500
>500
>500
>500
E. coli n◦ 152
E. coli n◦ 153
K. pneumoniae (ATCC
10031)
K. pneumoniae n◦ 178
E. cloacae (ATCC
23355)
500
250
500
250
500
250
500
250
125
250
7.8
15.6
E. cloacae n◦ 209
P. aeruginosa (ATCC
9027)
500
–
500
–
>500
–
>500
–
>500
>500
7.8
250
P. aeruginosa n◦ 147
P. aeruginosa n◦ 148
–
–
–
–
–
–
–
–
>500
>500
250
250
A: Amoxicillin, B: Chloroamphenicol.
S. aureus n◦ 82, 83, 84; S. faecalis n◦ 2, 3; E. coli n◦ 151, 152, 153; K. pneumoniae n◦
178; E. cloacae n◦ 209; and P. aeruginosa n◦ 147, 148 isolates were obtained from Centre
Hospitalier Universitaire of Montpellier, France.
When compared with chloramphenicol, the compound 4a[L] showed
moderate activity against B. subtilis (ATCC 6633), whereas all other
compounds were less active.
In comparing their MIC values with that of amoxicillin, all com-
pounds were effective against S. aureus n◦ 82, S. aureus n◦ 83, and S.
aureus n◦ 84. Compounds 4a[L] and 4a[D] showed a strong activity.
Compounds 4b[L] and 4d[L] showed moderate activity when compared
with amoxicillin.
From similar results obtained with E. coli n◦ 151, E. coli n◦ 152, and
E. coli n◦ 153, compounds 4a[L] and 4a[D] showed a strong activity,

2618
G. Turan-Zitouni et al.
while 4b[L] and 4d[L] showed approximately a similar level of activity
when compared with that of amoxicillin.
All compounds were effective against B. cereus (ATCC 14893). Com-
pounds 4a[L] and 4a[D] showed equal activity, and the other com-
pounds were found to be less active than amoxicillin.
When compared with amoxicillin, all compounds showed strong ac-
tivity against K. pneumoniae (ATCC 10031).
In comparing their MIC values with that of amoxicillin, all com-
pounds were active against E. cloacae (ATCC 23355) and E. cloacae n◦
209. Compounds 4a[L], 4a[D], 4b[L] and 4d[L] exhibited the same
level of activity as amoxicillin.
The new compounds were found to be inactive against S. faecalis
(ATCC 10541), S. faecalis n◦ 2, S. faecalis n◦ 3, M. luteus (ATCC 9341),
P. aeruginosa (ATCC 9027), P. aeruginosa n◦ 147, P. aeruginosa n◦ 148,
E. coli (ATCC 8739), and K. pneumoniae n◦ 178 when compared with
the reference agents.
Based on the limited number of compounds evaluated, it appears
that ortho hydroxy substitution at the phenyl ring attached to the thi-
azole moiety plays an important role for the antibacterial activity in
the series of L-phenylalanine-thiazole, D-phenylalanine-thiazole, and
L-leucine-thiazole combinations.
EXPERIMENTAL
Chemistry
All melting points (mp) were determined in open capillaries on a Gal-
lenkamp apparatus (Weiss-Gallenkamp) and are uncorrected. The pu-
rity of the compounds was routinely checked by thin layer chromatog-
raphy (TLC) using silica gel 60G (Merck). Spectroscopic data were
recorded with the following instruments: IR: Shimadzu IR-435 spec-
1
trophotometer (Shimadzu); H NMR: Bruker 250 MHz spectrometer
(Bruker) in DMSO-d6 using TMS as internal standard; MS-FAB: VG
Quattro Mass spectrometer (Agilent). Elemental analyses were per-
formed with a Leco CHNS-932 (LECO Corporation) instrument.
Synthesis of α-(tert-Butoxycarbonylamino)aldehydes12,14 (1):
General Procedure
The aldehydes 1 were obtained from N-methoxy-N-methyl-α-(tert-
butoxycarbonylamino)-carboxamides according to the literature.12,13

Thiazolyl Hydrazone Derivatives
2619
Synthesis of tert-Butyl (1-Aryl/alkyl-2-
thiosemicarbazonoethyl) carbamates (3): General
Procedure
A mixture of the aldehyde12,14 1 and thiosemicarbazide 2 in ethanol
(50 mL) was refluxed for 1 h. The solid that formed upon cooling was
filtered and crystallized from ethanol.11
L-tert-Butyl (1-Benzyl-2-thiosemicarbazonoethyl)carbamate
(3a [L])11
D-tert-Butyl (1-Benzyl-2-thiosemicarbazonoethyl)carbamate
(3a [D])
IR [ν, cm−1, KBr]: 3488–3442 (N−H), 1695 (C=O), 1550–1525 (C=N,
C=C), 1162 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 1.25 (s, 9H,
tBu), 2.70–3.05 (m, 2H, CH2), 4.20–4.45 (br, 1H, NH-CH), 7.10–7.40
(m, 7H, CO−NH, CH=N, arom-H), 7.80 (s, 1H, NH2), 8.10 (s, 1H, NH2),
11.15 (s, 1H, N−NH). MS (FAB) [M+1]: m/z 323. For C15H22N4O2S
calcd: C 55.88, H 6.88, N 17.38%; Found: C 55.95, H 6.93, N 17.33%.
L-tert-Butyl (1-Isobutyl-2-thiosemicarbazonoethyl)carbamate
(3b [L])
IR [ν, cm−1, KBr]: 3442–3410 (N−H), 1670 (C=O), 1540–1505 (C=N,
C=C), 1145 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 0.8–0.9 (m,
6H, CH3), 1.30–1.40 (m, 11H, tBu, CH2), 1.50–1.60 (m, 1H, (CH3)2CH-),
4.05–4.15 (br, 1H, NH-CH), 7.05–7.35 (m, 2H, CO−NH, CH=N), 7.65
(s, 1H, NH2), 8.05 (s, 1H, NH2), 11.10 (s, 1H, N−NH). MS (FAB) [M+1]:
m/z 289. For C12H24N4O2S calcd: C 49.97, H 8.39, N 19.43%; Found: C
50.05, H 8.36, N 19.37%.
L-tert-Butyl (1-(3-Indolylmethyl)-2-
thiosemicarbazonoethyl)carbamate (3c [L])
IR [ν, cm−1, KBr]: 3489–3445 (N−H), 1679 (C=O), 1563–1510 (C=N,
C=C), 1157 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 1.10–1.60 (m,
9H, tBu), 2.90–3.15 (m, 2H, CH2), 4.40–4.50 (br, 1H, NH-CH), 6.95–7.60
(m, 7H, CO−NH, CH=N, arom-H), 7.95 (s, 1H, NH2), 8.05 (s, 1H, NH2),
10.85 (s, 1H, NH), 11.15 (s, 1H, N−NH). MS (FAB) [M+1]: m/z 362.
For C17H23N5O2S calcd: C 56.49, H 6.41, N 19.38%; Found: C 56.41, H
6.37, N 19.35%.

2620
G. Turan-Zitouni et al.
tert-Butyl [1-Aryl/alkyl-2-[(4-aryl-2-thiazolyl)hydrazono]ethyl]
carbamates (4): General Procedure11
Equimolar amounts of the tert-butyl (1-aryl/alkyl-2-thiosemicar-
bazonoethyl)carbamate 3 (0.01 mol) and the phenacyl bromide (0.01
mol) were stirred in ethanol at room temperature for 8 h. The solid
that formed was filtered and crystallized from methanol.
L-tert-Butyl [1-Benzyl-2[[4-[(2-hydroxy-5-chloro)phenyl)]-2-
thiazolyl]hydrazono]ethyl] Carbamate (4a [L])11
D-tert-Butyl [1-Benzyl-2-[[4-[(2-hydroxy-5-chloro)phenyl)]-2-
thiazolyl]hydrazono]ethyl] Carbamate (4a [D])
IR [ν, cm−1, KBr]: 3490–3439 (N−H), 1689 (C=O), 1548–1521 (C=N,
C=C), 1160 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 1.30 (s, 9H,
tBu), 2.75–3.10 (m, 2H, CH2), 4.30–4.45 (br, 1H, NH-CH), 6.90–7.90
(m, 11H, CO−NH, CH=N, arom-H), 11.20 (s, 1H, N−NH), 11.90 (s, 1H,
OH). MS (FAB) [M+1]: m/z 473. For C23H25ClN4O3S calcd: C 58.40, H
5.33, N 11.85%; Found: C 58.44, H 5.31, N 11.82%.
L-tert-Butyl [1-Benzyl-2[[4-[(2-hydroxy-5-methoxy)phenyl)]-2-
thiazolyl]hydrazono]ethyl] Carbamate (4b [L])11
D-tert-Butyl [1-Benzyl-2[[4-[(2-hydroxy-5-methoxy)phenyl)]-2-
thiazolyl]hydrazono]ethyl] Carbamate (4b [D])
IR [ν, cm−1, KBr]: 3472–3447 (N−H), 1697 (C=O), 1563–1513 (C=N,
1
C=C), 1164 (C−O−C). H NMR (250 MHz, DMSO-d6): δ = 1.30 (s,
9H, tBu), 2.75–3.05 (m, 2H, CH2), 3.90 (s, 3H, OCH3), 4.25–4.40 (br,
1H, NH-CH), 6.85–7.60 (m, 11H, CO−NH, CH=N, arom-H), 10.60
(s, 1H, N−NH), 11.80 (s, 1H, OH). MS (FAB) [M+1]: m/z 469. For
C24H28N4O4S calcd: C 61.52, H 6.02, N 11.96%; Found: C 61.50, H 6.00,
N 11.99%.
L-tert-Butyl (1-Isobutyl-2[[4-[(2-hydroxy-5-chloro)phenyl)]-2-
thiazolyl]hydrazono]ethyl] Carbamate (4c [L])
IR [ν, cm−1, KBr]: 3448–3419 (N−H), 1664 (C=O), 1544–1510 (C=N,
C=C), 1135 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 0.70–0.85 (m,
6H, CH3), 1.25–1.45 (m, 11H, t-Bu, CH2), 1.50–1.60 (m, 1H, (CH3)2CH-),
4.05–4.15 (br, 1H, NH-CH), 6.85–7.90 (m, 6H, CO−NH, CH=N arom-
H), 11.25 (s, 1H, N−NH), 11.85 (s, 1H, OH). MS (FAB) [M+1]: m/z 439.
For C20H27ClN4O3S calcd: C 54.72, H 6.20, N 12.76%; Found: C 54.81,
H 6.29, N 12.72%.

Thiazolyl Hydrazone Derivatives
2621
L-tert-Butyl (1-Isobutyl-2-[[4-[(2-hydroxy-5-methoxy)phenyl)]-
2-thiazolyl]hydrazono]-ethyl] Carbamate (4d [L])
IR [ν, cm−1, KBr]: 3438–3410 (N−H), 1665 (C=O), 1535–1515 (C=N,
C=C), 1139 (C−O−C). 1H NMR (250 MHz, DMSO-d6): δ = 0.80–0.90 (m,
6H, CH3), 1.15–1.50 (m, 11H, t-Bu, CH2), 1.55–1.65 (m, 1H, (CH3)2CH-),
3.70 (s, 3H, OCH3), 4.10–4.20 (br, 1H, NH-CH), 6.70–7.45 (m, 6H,
CO−NH, CH=N, arom-H), 10.65 (s, 1H, N−NH), 11.75 (s, 1H, OH).
MS (FAB) [M+1]: m/z 435. For C21H30N4O4S calcd: C 58.04, H 6.96, N
12.89%; Found: C 58.09, H 6.91, N 12.83%.
L-tert-Butyl (1-(3-Indolylmethyl)-2-[[4-[(2-hydroxy-5-chloro)
phenyl)]-2-thiazolyl]-hydrazono]ethyl] Carbamate (4e [L])
IR [ν, cm−1, KBr]: 3487–3435 (N−H), 1677 (C=O), 1564–1532 (C=N,
1
C=C), 1158 (C−O−C). H NMR (250 MHz, DMSO-d6): δ = 1.05–1.50
(m, 9H, tBu), 2.75–3.15 (m, 2H, CH2), 4.40–4.50 (br, 1H, NH-CH),
6.90–7.90 (m, 11H, CO−NH, CH=N, arom-H), 10.85 (s, 1H, NH), 11.35
(s, 1H, N−NH), 11.95 (s, 1H, OH). MS (FAB) [M+1]: m/z 512. For
C25H26ClN5O3S calcd: C 58.64, H 5.12, N 13.68%; Found: C 58.68, H
5.09, N 13.73%.
L-tert-Butyl (1-(3-Indolylmethyl)-2-[[4-[(2-hydroxy-5-methoxy)
phenyl)]-2-thiazolyl]-hydrazono]ethyl] Carbamate (4f [L])
IR [ν, cm−1, KBr]: 3480–3433 (N−H), 1690 (C=O), 1553–1502 (C=N,
1
C=C), 1161 (C−O−C). H NMR (250 MHz, DMSO-d6): δ = 1.15–1.55
(m, 9H, tBu), 2.90–3.10 (m, 2H, CH2), 3.75 (s, 3H, OCH3), 4.30–4.40
(br, 1H, NH-CH), 6.70–7.60 (m, 11H, CO−NH, CH=N, arom-H), 10.65
(s, 1H, NH), 10.75 (s, 1H, N−NH), 11.85 (s, 1H, OH). MS (FAB) [M+1]:
m/z 508. For C26H29N5O4S calcd: C 61.52, H 5.76, N 13.80%; Found: C
61.57, H 5.79, N 13.73%.
Microbiology
Antibacterial activities of the new compounds were tested using the mi-
crobroth dilution method.17,18 Stock solutions of the samples were pre-
pared in dimethylsulfoxide. DMSO solutions of the tested compounds
and the standard agent chloramphenicol were prepared within the
concentration range of 0.24–1000 µg/mL. Dilution series using ster-
ile distilled water were prepared in micro-test tubes and were trans-
ferred to 96-well microtiter plates. Overnight grown bacterial suspen-
sions in double-strength Mueller-Hinton broth were standardized to
108 CFU/mL using McFarland No: 0.5 standard solution. 100 µL of
each microorganism suspension was then added into the wells. The last

2622
G. Turan-Zitouni et al.
well-chain without microorganism was used as a negative control. Ster-
ile distilled water and the medium served as a positive growth control.
After incubation at 37◦C for 18–24 h, the first well without turbidity
was determined as the minimal inhibitory concentration (MIC). Tested
microorganism strains were E. coli (ATCC 8739), E. coli n◦ 151, E. coli
n◦ 152, E. coli n◦ 153, K. pneumoniae (ATCC 10031), K. pneumoniae n◦
178, E. cloacae (ATCC 23355), E. cloacae n◦ 209, P. aeruginosa (ATCC
9027), P. aeruginosa n◦ 147, P. aeruginosa n◦ 148, S. aureus (ATCC
6538), S. aureus n◦ 82, S. aureus n◦ 83, S. aureus n◦ 84, S. faecalis (ATCC
10541), S. faecalis n◦ 2, S. faecalis n◦ 3, M. luteus (ATCC 9341), B. cereus
(ATCC 14893), and B. subtilis (ATCC 6633). The obtained data on the
antibacterial activity of the compounds and control drugs are given in
Table II.
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