Synthesis, antimicrobial activity and cytotoxicity of novel oxadiazole derivatives

Article abstract of DOI:10.3109/14756366.2011.574132

In the present study, 5-substituted-1,3,4-oxadiazolin-2-thiones (1ab) were synthesized via the ring closure reactions of appropriate acid hydrazides with carbon disulphide. N-(Benzothiazol-2-yl)-2-[[5-substituted-1,3,4-oxadiazol-2-yl] sulfanyl]acetamide d

Full text of DOI:10.3109/14756366.2011.574132

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 51–57
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.574132
RESEARCH ARTICLE
Synthesis, antimicrobial activity and cytotoxicity of
novel oxadiazole derivatives
Zafer Asim Kaplancikli1, Mehlika Dilek Altintop1, Gulhan Turan-Zitouni1, Ahmet Ozdemir1,
Rasime Ozic2, and Gülşen Akalın3
1Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskişehir, Turkey, 2Faculty of
Science, Department of Biology, Anadolu University, Eskişehir, Turkey, and 3Faculty of Pharmacy, Department of
Biochemistry, Anadolu University, Eskişehir, Turkey
Abstract
In the present study, 5-substituted-1,3,4-oxadiazolin-2-thiones (1a–b) were synthesized via the ring closure reactions
of appropriate acid hydrazides with carbon disulphide. N-(Benzothiazol-2-yl)-2-[[5-substituted-1,3,4-oxadiazol-2-yl]
sulfanyl]acetamide derivatives (3a–j) were obtained by the nucleophilic substitution reactions of 5-substituted-
1,3,4-oxadiazolin-2-thiones (1a–b) with N-(benzothiazol-2-yl)-2-chloroacetamides. The chemical structures of
1
the compounds were elucidated by IR, H NMR, 13C NMR and FAB+-MS spectral data and elemental analyses. The
synthesized compounds were screened for their antimicrobial activities against Micrococcus luteus, Bacillus subtilis,
Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Candida albicans. All
compounds except compound 3h exhibited the highest antibacterial activity against P. aeruginosa. Among all
compounds (3a–j), the compounds bearing 4-methoxyphenoxymethyl moiety on oxadiazole ring (3a–e) exhibited
the highest inhibitory activity against C. albicans. Although compound 3j did not possess 4-methoxyphenoxymethyl
moiety on oxadiazole ring, this derivative also exhibited the same level of anti-candidal activity. The compounds were
also investigated for their cytotoxic effects using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. Compound 3a exhibited the highest cytotoxic activity, whereas compound 3g possessed the lowest cytotoxic
activity against NIH/3T3 cells.
Keywords: Oxadiazole, benzothiazole, amide, antimicrobial activity, cytotoxicity
Introduction
problem, which is considered as the inevitable conse-
Nosocomial infections have emerged as important causes
of morbidity and mortality due to the fact that hospital-
ized patients tend to be more susceptible to infections
because of their severe underlying disease conditions. If
the patient is immunocompromised, micro-organisms
that are not normally pathogenic are also capable of
causing disease. Furthermore, hospital conditions con-
tribute to the acquisition of drug resistance, which com-
plicates the treatment of infections due to drug-resistant
pathogens1.
quence of the widespread use of these drugs, the devel-
opment of new effective antimicrobial agents has gained
great importance1–7
.
Oxadiazoles are widely used and studied pharma-
cophores in medicinal chemistry due to their broad
spectrum and metabolic profile. Some studies have con-
firmed that oxadiazole derivatives possess antimicrobial
activity. Furamizole, which is a nitrofuran derivative
bearing oxadiazole nucleus, is an example of promising
antibacterial agents8–17
.
e development of resistance to antimicrobial agents
has led to the failure of the treatment of bacterial and
fungal infections, which are responsible for the bulk of
nosocomial infections. In order to overcome this serious
Among oxadiazole derivatives, 1,3,4-oxadiazolin-2-
thiones, which are synthesized by the ring closure reac-
tions of various acid hydrazides with carbon disulphide,
areversatilereactionintermediatesinsyntheticchemistry
Address for Correspondence: Dr. Zafer Asim Kaplancikli, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu
University, 26470 Eskişehir, Turkey. Tel.: 90 222 3350580/3776. Fax: 90 222 3350750. E-mail: zakaplan@anadolu.edu.tr
(Received 03 March 2011; revised 18 March 2011; accepted 18 March 2011)
51

52 Z.A. Kaplancikli et al.
owing to the fact that the thiol group on oxadiazole ring
N-(benzothiazol-2-yl)-2-[[5-substituted-1,3,4-oxadiazol-2-yl]
sulfanyl]acetamides (3a–j)
acts as a strong nucleophile18,19
.
Medicinal chemists have carried out considerable
research for novel antimicrobial agents that possess
amide moiety. e prominent drugs bearing amide
group are penicillins and cephalosporins, all of which
are widely used as antibiotics for the treatment of sys-
temic infections. Penicillins and cephalosporins, which
are also classified as β-lactam antibiotics, possess cyclic
amide as the main scaffold and acetamide moiety as the
A mixture of 1,3,4-oxadiazolin-2-thione (1) (2 mmol) and
appropriate N-(benzothiazol-2-yl)-2-chloroacetamide
(2) (2 mmol) in acetone was stirred at room tempera-
ture for 8 h in the presence of potassium carbonate and
filtered. e residue was washed with water and crystal-
lized from ethanol.
N-(benzothiazol-2-yl)-2-[[5-(4-methoxyphenoxymethyl)-
1,3,4-oxadiazol-2-yl]sulfanyl]acetamide (3a)
side chain20–24
.
IR (KBr) νmax (cm−1): 3206 (amide N–H), 1681 (amide
C=O), 1605, 1551, 1441 (C=N and C=C), 1206 (C–O).
1H NMR (400 MHz, DMSO-d6): 3.69 (3H, s), 4.41 (2H,
s), 5.32 (2H, s), 6.86 (2H, d, J= 8.9 Hz), 6.95 (2H, d, J= 8.9
Hz), 7.30 (1H, t, J= 7.1 Hz), 7.43 (1H, t, J= 7.1 Hz), 7.75
(1H, d, J= 7.9 Hz), 7.99 (1H, d, J= 7.8 Hz), 12.71 (1H, br).
13C NMR (100 MHz, DMSO-d6): 35.57 (CH2), 55.59
(CH3), 60.15 (CH2), 104.99 (CH), 113.95 (2CH), 115.25
(CH), 116.18 (2CH), 121.14 (CH), 131.31 (CH), 151.22 (C),
154.31 (C), 155.79 (C), 156.45 (C), 164.19 (C), 164.20 (C),
166.04 (C), 180.06 (C).
Compounds bearing benzothiazole moiety have also
been reported to exhibit a wide spectrum of biological
effects including antimicrobial activity25–27
.
In this study, we described the synthesis of some new
oxadiazole derivatives, which possess two important
functional moieties, namely amide and benzothiazole
and focused on their potential antimicrobial and cyto-
toxic effects.
Methods
For C19H16N4O4S2 calculated: 53.26% C, 3.76% H,
13.08% N; found: 53.30% C, 3.82% H, 13.10% N.
MS (FAB) [M+1]+: m/z 429.
Chemistry
All reagents were used as purchased from commer-
cial suppliers without further purification. Melting
points were determined using an Electrothermal 9100
digital melting point apparatus and were uncorrected
(Electrothermal, Essex, UK). e compounds were
checked for purity by thin-layer chromatography on
silica gel 60 F254. Spectroscopic data were recorded on
the following instruments: IR, Shimadzu 435 IR spectro-
N-(6-chlorobenzothiazol-2-yl)-2-[[5-(4-
methoxyphenoxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]
acetamide (3b)
IR (KBr) νmax (cm−1): 3210 (amide N–H), 1685 (amide
C=O), 1615, 1560, 1445 (C=N and C=C), 1211 (C–O).
1H NMR (400 MHz, DMSO-d6): 3.70 (3H, s), 4.40 (2H,
s), 5.35 (2H, s), 6.88 (2H, d, J= 9.0 Hz), 6.99 (2H, d, J= 9.0
Hz), 7.45 (1H, dd, J= 8.6, 2.4 Hz), 7.76 (1H, d, J= 8.6 Hz),
8.13 (1H, d, J= 2.4 Hz), 12.79 (1H, br).
1
photometer (Shimadzu, Tokyo, Japan); H NMR, Bruker
400 MHzNMRspectrometer(BrukerBioscience,Billerica,
MA) and 13C NMR, Bruker Avance II 100 MHz NMR spec-
trometer (Bruker Bioscience, Billerica, MA) in DMSO-d6
using tetramethylsilane as internal standard; MS-FAB,
VG Quattro mass spectrometer (Fisons Instruments
Vertriebs GmbH, Mainz, Germany), elemental analyses
were performed on a Perkin-Elmer EAL 240 elemental
analyser (Perkin-Elmer, Norwalk, CT).
13C NMR (100 MHz, DMSO-d6): 35.56 (CH2), 55.69
(CH3), 60.05 (CH2), 104.86 (CH), 114.55 (2CH), 115.15
(CH), 116.10 (2CH), 121.04 (CH), 127.62 (C), 151.13 (C),
154.20 (C), 155.70 (C), 156.21 (C), 164.15 (C), 163.91 (C),
166.02 (C), 180.02 (C).
For C19H15ClN4O4S2 calculated: 49.30% C, 3.27% H,
12.10% N; found: 49.35% C, 3.29% H, 12.14% N.
MS (FAB) [M+1]+: m/z 463.
General procedure for the synthesis of the compounds
5-Substituted-1,3,4-oxadiazolin-2-thiones (1a–b)
e acid hydrazide (5 mmol) was dissolved in a solution
of potassium hydroxide (5 mmol) in ethanol (50 mL).
Carbon disulfide (5 mL) was then added while stirring
and the reaction mixture was heated under reflux for
10 h. e solution was cooled and acidified to pH 5–6
with hydrochloric acid solution and crystallised from
ethanol28.
N-(6-Methylbenzothiazol-2-yl)-2-[[5-(4-
methoxyphenoxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]
acetamide (3c)
IR (KBr) νmax (cm−1): 3201 (amide N–H), 1676 (amide
C=O), 1601, 1566, 1435 (C=N and C=C), 1228 (C–O).
1H NMR (400 MHz, DMSO-d6): 2.41 (3H, m), 3.71 (3H,
s), 4.42 (2H, s), 5.33 (2H, s), 6.87 (2H, d, J= 9.1 Hz), 6.97
(2H, d, J= 9.1 Hz), 7.25 (1H, d, J= 8.2 Hz), 7.68 (1H, d,
J= 8.2 Hz), 7.75 (1H, m), 12.64 (1H, br).
N-(benzothiazol-2-yl)-2-chloroacetamides (2a–e)
Chloroacetyl chloride (5 mmol) was added dropwise
with stirring to a mixture of 2-aminobenzothiazole (5
mmol) and triethylamine (2 mL) in toluene (50 mL) at
0–5°C. e solvent was evaporated under reduced pres-
sure. e residue was washed with water and crystallized
from ethanol29.
13C NMR (100 MHz, DMSO-d6): 20.95 (CH3), 35.56
(CH2), 55.60 (CH3), 60.17 (CH2), 104.75 (CH), 114.60
(2CH), 115.05 (CH), 116.18 (2CH), 121.20 (CH), 132.71
(C), 151.22 (C), 154.29 (C), 155.72 (C), 156.29 (C), 164.05
(C), 164.09 (C), 165.87 (C), 179.89 (C).
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel oxadiazole derivatives 53
For C20H18N4O4S2 calculated: 54.29% C, 4.10% H,
12.66% N; found: 54.32% C, 4.11% H, 12.69% N.
MS (FAB) [M+1]+: m/z 443.
For C19H22N4O2S2 calculated: 56.69% C, 5.51% H,
13.92% N; found: 56.73% C, 5.52% H, 13.96% N.
MS (FAB) [M+1]+: m/z 403.
N-(6-methoxybenzothiazol-2-yl)-2-[[5-(4-
methoxyphenoxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]
acetamide (3d)
N-(6-chlorobenzothiazol-2-yl)-2-[[5-(2-cyclohexylethyl)-1,3,4-
oxadiazol-2-yl]sulfanyl]acetamide (3g)
IR (KBr) νmax (cm−1): 3201 (amide N–H), 2922, 2850 (ali-
phatic C–H), 1683 (amide C=O), 1604, 1556, 1442 (C=N
and C=C).
IR (KBr) νmax (cm−1): 3221 (amide N–H), 1685 (amide
C=O), 1621, 1552, 1415 (C=N and C=C), 1230 (C–O).
1H NMR (400 MHz, DMSO-d6): 3.69 (3H, s), 3.81
(3H, s), 4.44 (2H, s), 5.30 (2H, s), 6.84 (2H, d, J = 9.0
Hz), 6.97 (2H, d, J = 9.0 Hz), 7.05 (1H, dd, J = 8.8, 2.6
Hz), 7.59 (1H, d, J = 2.6 Hz), 7.67 (1H, d, J = 8.8 Hz),
12.66 (1H, br).
1H NMR (400 MHz, DMSO-d6): 0.73–0.89 (2H, m),
0.97–1.26 (4H, m), 1.46–1.74 (7H, m), 2.81 (2H, t, J= 7.5
Hz), 4.37 (2H, s), 7.47 (1H, dd, J= 8.7, 2.3 Hz), 7.76 (1H, d,
J= 8.7 Hz), 8.13 (1H, d, J= 2.3 Hz), 12.79 (1H, bs).
13C NMR (100 MHz, DMSO-d6): 21.95 (CH2), 24.43
(2CH2), 26.92 (CH2), 32.13 (2CH2), 34.01 (CH2), 35.22
(CH2), 36.92 (CH), 121.18 (CH), 126.91 (CH), 150.02 (CH),
153.99 (C), 155.63 (C), 156.91 (C), 163.15 (C), 163.91 (C),
165.90 (C), 181.04 (C).
13C NMR (100 MHz, DMSO-d6): 35.57 (CH2), 55.32
(CH3), 55.62 (CH3), 60.18 (CH2), 104.75 (CH), 114.62
(2CH), 115.04 (CH), 116.15 (2CH), 121.29 (CH), 132.77
(C), 151.15 (C), 154.25 (C), 155.51 (C), 156.27 (C), 164.01
(C), 164.04 (C), 165.86 (C), 179.88 (C).
For C20H18N4O5S2 calculated: 52.39% C, 3.96% H,
12.22% N; found: 52.41% C, 3.91% H, 12.15% N.
MS (FAB) [M+1]+: m/z 459.
For C19H21ClN4O2S2 calculated: 52.22% C, 4.84% H,
12.82% N; found: 52.25% C, 4.80% H, 12.83% N.
MS (FAB) [M+1]+: m/z 437.
N-(6-methylbenzothiazol-2-yl)-2-[[5-(2-cyclohexylethyl)-
1,3,4-oxadiazol-2-yl]sulfanyl]acetamide (3h)
N-(6-ethoxybenzothiazol-2-yl)-2-[[5-(4-
methoxyphenoxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]
acetamide (3e):
IR (KBr) νmax (cm−1): 3200 (amide N–H), 2920, 2850 (ali-
phatic C–H), 1683 (amide C=O), 1612, 1564, 1460 (C=N
and C=C).
IR (KBr) νmax (cm−1): 3218 (amide N–H), 1682 (amide
C=O), 1619, 1541, 1401 (C=N and C=C), 1218 (C–O).
1H NMR (400 MHz, DMSO-d6): 1.35 (3H, t, J= 7.0 Hz),
3.68 (3H, s), 4.07 (2H, q, J= 7.0 Hz), 4.44 (2H, s), 5.30 (2H,
s), 6.84 (2H, d, J= 9.1 Hz), 6.96 (2H, d, J= 9.1 Hz), 7.03 (1H,
dd, J= 8.8, 2.5 Hz), 7.56 (1H, d, J= 2.5 Hz), 7.66 (1H, d,
J= 8.8 Hz), 12.63 (1H, br).
1H NMR (400 MHz, DMSO-d6): 0.73–0.89 (2H, m),
0.97–1.26 (4H, m), 1.46-1.74 (7H, m), 2.41 (3H, m), 2.81
(2H, t, J= 7.5 Hz), 4.36 (2H, s), 7.27 (1H, d, J= 8.3 Hz), 7.65
(1H, d, J= 8.3 Hz), 7.77 (1H, m), 12.64 (1H, bs).
13C NMR (100 MHz, DMSO-d6): 21.51 (CH3), 22.32
(CH2), 23.93 (2CH2), 26.81 (CH2), 31.99 (2CH2), 33.96
(CH2), 35.41 (CH2), 37.10 (CH), 120.96 (CH), 132.11 (CH),
152.02 (CH), 153.91 (C), 155.42 (C), 156.26 (C), 163.88
(C), 164.11 (C), 164.99 (C), 180.12 (C).
13C NMR (100 MHz, DMSO-d6): 14.63 (CH3), 35.55
(CH2), 55.31 (CH3), 60.17 (CH2), 63.62 (CH2), 105.39 (CH),
114.62 (2CH), 115.41 (CH), 116.15 (2CH), 121.28 (CH),
132.75 (C), 148.94 (C), 151.13 (C), 154.25 (C), 155.49 (C),
164.01 (C), 164.04 (C), 165.86 (C), 179.02 (C).
For C21H20N4O5S2 calculated: 53.38% C, 4.27% H,
11.86% N; found: 53.43% C, 4.35% H, 11.90% N.
MS (FAB) [M+1]+: m/z 473.
For C20H24N4O2S2 calculated: 57.67% C, 5.81% H,
13.45% N; found: 57.70% C, 5.85% H, 13.49% N.
MS (FAB) [M+1]+: m/z 417.
N-(6-methoxybenzothiazol-2-yl)-2-[[5-(2-cyclohexylethyl)-
1,3,4-oxadiazol-2-yl]sulfanyl]acetamide (3i)
N-(benzothiazol-2-yl)-2-[[5-(2-cyclohexylethyl)-1,3,4-
oxadiazol-2-yl]sulfanyl]acetamide (3f):
IR (KBr) νmax (cm−1): 3200 (amide N–H), 2920, 2847 (ali-
phatic C–H), 1681 (amide C=O), 1610, 1564, 1465 (C=N
and C=C).
IR (KBr) νmax (cm−1): 3217 (amide N–H), 2922, 2848 (ali-
phatic C–H), 1683 (amide C=O), 1600, 1554, 1446 (C=N
and C=C).
1H NMR (400 MHz, DMSO-d6): 0.73–0.89 (2H, m),
0.97–1.26 (4H, m), 1.46–1.74 (7H, m), 2.81 (2H, t, J= 7.5
Hz), 3.81 (3H, s), 4.36 (2H, s), 7.27 (1H, d, J= 8.3 Hz), 7.65
(1H, d, J= 8.3 Hz), 7.77 (1H, m), 12.64 (1H, bs).
13C NMR (100 MHz, DMSO-d6): 22.06 (CH2), 23.72
(2CH2), 26.65 (CH2), 32.41 (2CH2), 34.11 (CH2), 35.82
(CH2), 37.20 (CH), 55.22 (CH3), 121.11 (CH), 131.99 (CH),
152.12 (CH), 153.88 (C), 156.10 (C), 156.38 (C), 163.48
(C), 164.05 (C), 165.16 (C), 181.01 (C).
1H NMR (400MHz, DMSO-d6): 0.73–0.89 (2H, m),
0.97–1.26 (4H, m), 1.46–1.74 (7H, m), 2.81 (2H, t, J=7.5
Hz), 4.38 (2H, s), 7.32 (1H, t, J=7.0 Hz), 7.45 (1H, t, J=7.0
Hz), 7.77 (1H, d, J=8.0 Hz), 7.98 (1H, d, J=7.8 Hz), 12.71
(1H, bs).
13C NMR (100 MHz, DMSO-d6): 21.90 (CH2), 24.60
(2CH2), 27.10 (CH2), 32.05 (2CH2), 33.61 (CH2), 35.18
(CH2), 38.27 (CH), 122.06 (CH), 132.61 (CH), 152.21 (CH),
154.68 (CH), 155.96 (C), 157.12 (C), 163.98 (C), 164.11
(C), 166.54 (C), 182.11 (C).
For C20H24N4O3S2 calculated: 55.53% C, 5.59% H,
12.95% N; found: 55.57% C, 5.61% H, 12.96% N.
MS (FAB) [M+1]+: m/z 433.
© 2012 Informa UK, Ltd.

54 Z.A. Kaplancikli et al.
in Dulbecco’s modified Eagle’s medium supple-
mented with 10% foetal calf serum (Life Technologies,
UK), 100 IU/mL penicillin (Gibco, Paisley, Scotland)
and 100 mg/mL streptomycin (Gibco) at 37°C in
a humidified atmosphere of 95% air and 5% CO2.
Exponentially growing cells were plated at 2 × 104 cells/
mL into 96-well microtitre tissue culture plates (Nunc,
Denmark) and incubated for 24 h before the addition
of the drugs (the optimum cell number for cytotox-
icity assays was determined in preliminary experi-
ments). Stock solutions of compounds were prepared
in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Poole,
UK) and further dilutions were made with fresh cul-
ture medium (the concentration of DMSO in the final
culture medium was <0.1% which had no effect on the
cell viability).
N-(6-ethoxybenzothiazol-2-yl)-2-[[5-(2-cyclohexylethyl)-
1,3,4-oxadiazol-2-yl]sulfanyl]acetamide (3j)
IR (KBr) νmax (cm−1): 3221 (amide N–H), 2922, 2850 (ali-
phatic C–H), 1676 (amide C=O), 1602, 1581, 1458 (C=N
and C=C).
1H NMR (400 MHz, DMSO-d6): 0.73–0.89 (2H, m),
0.97–1.26 (4H, m), 1.38 (3H, t, J= 7.1 Hz), 1.46–1.74 (7H,
m), 2.81 (2H, t, J= 7.5 Hz), 4.02 (2H, q, J= 7.1 Hz), 4.36
(2H, s), 7.27 (1H, d, J= 8.3 Hz), 7.65 (1H, d, J= 8.3 Hz), 7.77
(1H, m), 12.64 (1H, bs).
13C NMR (100 MHz, DMSO-d6): 14.55 (CH3), 21.88
(CH2), 23.46 (2CH2), 26.12 (CH2), 32.15 (2CH2), 33.98
(CH2), 35.61 (CH2), 36.95 (CH), 62.51 (CH2), 121.21
(CH), 132.09 (CH), 152.28 (CH), 153.68 (C), 155.81 (C),
156.63 (C), 162.11 (C), 164.78 (C), 165.06 (C), 182.15
(C).
For C21H26N4O3S2 calculated: 56.48% C, 5.87% H,
12.55% N; found: 56.51% C, 5.90% H, 12.56% N.
MS (FAB) [M+1]+: m/z 447.
MTT assay for cytotoxicity of the compounds
It is widely used as a measure of cytotoxicity. After 24 h
of preincubation, the tested compounds were added
to give final concentration in the range 0.5–500 µg/
mL and the cells were incubated for 24 h. At the end of
this period, MTT was added to a final concentration of
0.5 mg/mL and the cells were incubated for 4 h at 37 °C.
After the medium was removed, the formazan crystals
formed by MTT metabolism were solubilised by addi-
tion of 200 µl DMSO to each well and absorbance was
read at 540 nm with a microtitre plate spectrophotom-
eter (Bio-Tek plate reader). Every concentration was
repeated in three wells and IC50 values were defined as
the drug concentrations that reduced absorbance to
50% of control values.
Microbiology
Antimicrobial activity
e in vitro antimicrobial activities of the compounds
(3a–j) were tested using the microbroth dilution
method30. e tested micro-organism strains were
Micrococcus luteus (NRLL B-4375), Bacillus subtilis
(NRS-744), Pseudomonas aeruginosa (ATCC-254992),
Staphylococcus aureus (NRRL B-767), Escherichia coli
(ATCC-25922), Listeriamonocytogenes(ATCC-7644)and
Candida albicans (ATCC-22019). Microbroth dilution-
susceptibility assay was used for antimicrobial evalua-
tion of the compounds. Stock solutions of the samples
were prepared in dimethyl sulfoxide. Dilution series
using sterile distilled water were prepared from 1–4 mg/
mL to 0.001–0.007 mg/mL in micro-test tubes that were
transferred to 96-well microtitre plates. Overnight-
grown bacterial and C. albicans suspensions in double-
strength Mueller–Hinton broth were standardized to
108 colony-forming unit/mL using McFarland No: 0.5
standard solutions. Hundred microlitre of each micro-
organism suspension was then added into the wells. e
last well-chain without a micro-organism was used as a
negative control. Sterile 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 minimum inhibitory concentra-
tion. Streptomycin was used as an antibacterial agent,
whereas ketoconazole was used as an antifungal agent.
Results and discussion
Initially, the compounds which have oxadiazoline-2-
thione structure (1a–b) were obtained by the ring closure
reactions of acid hydrazides with carbon disulphide.
2-Chloro-N-(benzothiazol-2-yl)acetamides (2a–e) were
synthesized via the nucleophilic acyl substitution reac-
tions of 2-aminobenzothiazoles with chloroacetyl
chloride in the presence of triethylamine. e desired
compounds (3a–j) were prepared by reacting 2-chloro-
N-(benzothiazol-2-yl)acetamides (2a–e) with oxadi-
azoline-2-thiones (1a–b) under basic conditions. e
presence of a base favours the thiol form, which is prone
to nucleophilic substitution reactions. ese reactions
are summarized in Scheme 1 and some properties of the
compounds are given in Table 1.
e structures of the compounds (3a–j) were con-
Toxicity
1
firmed by IR, H NMR, 13C NMR and FAB+-MS spectral
e level of cellular MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; Sigma) reduction was
quantified as previously described in the literature with
small modifications31,32
data and elemental analyses.
In the IR spectra of all compounds (3a–j), all deriva-
tives have a strong, characteristic band in the region
1685–1676 cm−1 due to the amide C=O stretching vibra-
tion. e amide N–H stretching vibration of the com-
pounds (3a–j) gives rise to a band at 3221–3200 cm−1. e
bands due to the C=C and C=N stretching vibrations are
.
Cell culture and drug treatment
NIH/3T3 cells were obtained from the American Type
Culture Collection (ATCC). The cells were incubated
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel oxadiazole derivatives 55
O
H
O
1. CS2 / KOH
2. HCl
N
N
H2N-NH2
NH2
R
N
H
R
O
R
O
S
1a-b
H
R'
R'
S
N
O
S
N
N(C2H5)3
N
NH2
+
Cl
Cl
Cl
O
2a-e
H
N
N
N
R
R'
S
N
K2CO3
+
2
1
S
O
O
3a-j
Scheme 1. e synthetic protocol of the compounds (3a–j).
observed in the region 1621–1401 cm−1. In the IR spectra
of the compounds (3a–e), the stretching bands for C–O
group occur at 1230–1206 cm−1.
In the H NMR spectra of the compounds (3a–j), the
signal due to the amide proton appears at 12–13 ppm. e
oxadiazole ring has an important impact on antibacterial
activity against L. monocytogenes and E. coli.
All compounds except compound 3hare equally active
against P. aeruginosa. Compounds 3b and 3c exhibit the
lowest antibacterial activity against M. luteus.
1
signal due to the−S–CH2– protons gives rise to a singlet
peak at 4.0–5.5 ppm. In the H NMR spectra of the com-
The compounds possessing 2-cyclohexylethyl
moiety on oxadiazole ring (3f–j) except 3j are
more effective than the compounds possessing
4-methoxyphenoxymethyl moiety on oxadiazole ring
(3a–e) against B. subtilis.
e compounds that possess 4-methoxyphenoxym-
ethyl moiety on oxadiazole ring (3a–e) exhibit the high-
est inhibitory activity against C. albicans. is outcome
confirms that the 4-methoxyphenoxymethyl group on
oxadiazole ring may have a considerable influence on
antifungal activity. Although compound 3j does not
possess 4-methoxyphenoxymethyl moiety on oxadiazole
ring, this derivative also exhibits the same level of antifun-
gal activity.
All compounds were also evaluated for their cyto-
toxic properties using MTT assay. e biological study
indicated that compound 3a possessed the highest
cytotoxicity, whereas compound 3g exhibited the lowest
cytotoxicity against NIH/3T3 cells among the title com-
pounds (Table 3).
1
pounds 3a–e, the O–CH2 protons are observed at 4.42–
4.44 ppm as a singlet peak. e benzothiazole protons of
all derivatives are observed in the region 6.8–8.2 ppm. All
other aromatic and aliphatic protons were observed at
expected regions.
In their 13C NMR spectra, the signal due to the−S–CH2–
carbon appears at 35–36 ppm. e other aromatic and
aliphatic carbons were observed at expected regions.
In the mass spectra of all compounds (3a–j), the
M+1 peak is observed. All compounds gave satisfactory
elemental analysis.
All compounds were tested in vitro against S. aureus,
L. monocytogenes, E. coli, P. aeruginosa, M. luteus, B.
subtilis and C. albicans and compared with streptomycin
and ketoconazole, respectively (Table 2).
All compounds (3a–j) showed less antimicrobial
potency than streptomycin and ketoconazole. e
results indicate that the compounds (3a–j) exhibit the
highest antimicrobial effect on P. aeruginosa and C.
albicans.
Among these compounds (3a–j), the compounds bear-
ing 2-cyclohexylethyl moiety on oxadiazole ring (3f–j)
exhibit the highest inhibitory activity against S. aureus. It
is clear that there is a positive correlation between anti-
bacterial activity against S. aureus and aliphatic group on
oxadiazole ring.
e compounds that possess 4-methoxyphenoxym-
ethyl moiety on oxadiazole ring (3a–e) are the most
effective derivatives against L. monocytogenes and E. coli.
It is apparent that 4-methoxyphenoxymethyl moiety on
Conclusions
In conclusion, we described the synthesis of new oxadi-
azole derivatives and evaluated their in vitro antimicro-
bial activities against various pathogenic bacteria and C.
albicans.
5-Substituted-1,3,4-oxadiazolin-2-thiones(1a–b)were
synthesized via the ring closure reactions of appropriate
acidhydrazideswithcarbondisulphide.N-(Benzothiazol-
2-yl)-2-[[5-Substituted-1,3,4-oxadiazol-2-yl]sulfanyl]
acetamide derivatives (3a–j) were prepared by the
© 2012 Informa UK, Ltd.

56 Z.A. Kaplancikli et al.
Table 1. Some properties of the synthesized compounds (3a–j).
Compound
R
Rʹ
H
Yield (%)
M.p. (°C)
206–207
224–226
215–217
207–208
196–200
202–204
237–238
219–220
184
Molecular formula
C19H16N4O4S2
C19H15ClN4O4S2
C20H18N4O4S2
C20H18N4O5S2
C21H20N4O5S2
C19H22N4O2S2
C19H21ClN4O2S2
C20H24N4O2S2
C20H24N4O3S2
C21H26N4O3S2
Molecular weight
3a
3b
3c
3d
3e
3f
4-Methoxyphenoxymethyl
4-Methoxyphenoxymethyl
4-Methoxyphenoxymethyl
4-Methoxyphenoxymethyl
4-Methoxyphenoxymethyl
2-Cyclohexylethyl
80
72
75
78
81
75
85
80
82
84
428
Cl
462,5
442
CH3
OCH3
OC2H5
H
458
472
402
3g
3h
3i
2-Cyclohexylethyl
Cl
436,5
416
2-Cyclohexylethyl
CH3
OCH3
OC2H5
2-Cyclohexylethyl
432
3j
2-Cyclohexylethyl
194–197
446
Table 2. Antimicrobial activities of the compounds (3a–j) (μg/mL).
Compound
A
500
500
500
500
500
250
250
250
250
250
31.25
—
B
C
250
250
250
250
250
500
500
500
500
500
31.25
—
D
E
250
500
500
250
250
250
250
250
250
250
15.625
—
F
500
500
500
500
500
250
250
250
250
500
15.625
—
G
3a
3b
3c
3d
3e
3f
3g
3h
250
250
250
250
250
500
500
500
500
500
7.81
—
250
250
250
250
250
250
250
500
250
250
125
—
500
500
500
500
500
1000
1000
1000
1000
500
—
3i
3j
Reference 1
Reference 2
250
Reference 1, streptomycin; reference 2, ketoconazole.
A: Staphylococcus aureus (NRRL B-767), B, Listeria monocytogenes (ATCC-7644), C: Escherichia coli (ATCC-25922), D: Pseudomonas
aeruginosa (ATCC-254992), E: Micrococcus luteus (NRLL B-4375), F: Bacillus subtilis (NRS-744), G: Candida albicans (ATCC-22019).
active against P. aeruginosa. e compounds which pos-
sess 4-methoxyphenoxymethyl moiety on oxadiazole
ring (3a–e) exhibit the highest inhibitory activity against
C. albicans. is outcome confirms that 4-methoxyphe-
noxymethyl group on oxadiazole ring may have a con-
siderable influence on anti-candidal activity. Compound
3j, which does not possess 4-methoxyphenoxymethyl
moiety on oxadiazole ring, also exhibits the same level of
antifungal activity.
e cytotoxic effects of the compounds were also
investigated and compound 3a possessed the highest
cytotoxicity, whereas compound 3g exhibited the lowest
cytotoxicity against NIH/3T3 cells.
Table 3. In vitro cytotoxicity of the compounds (3a–j)
Compound
IC50 (µg/mL)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
25.4 0.8
101.6 16.1
116.7 15.3
56.8 7.6
103.3 15.3
126.7 25.2
203.3 32.1
125 25
106 10.4
95 13.3
3j
aCytotoxicity of the compounds to mouse fibroblast (NIH/3T3)
cell line. Incubation for 24h. IC50 is the drug concentration
required to inhibit 50% of the cell growth. e values represent
mean standard deviation of triplicate determinations.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
nucleophilic substitution reactions of 5-substituted-1-
,3,4-oxadiazolin-2-thiones (1a–b) with N-(benzothiazol-
2-yl)-2-chloroacetamides in the presence of potassium
carbonate.
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