1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones, 1,3,4-thiadiazoles and hydrazones containing 5-methyl-2-benzoxazolinones: Synthesis, analgesic-anti-inflammatory and antimicrobial activities

Article abstract of DOI:10.1016/j.bmc.2007.06.006

Acetic acid hydrazide containing 5-methyl-2-benzoxazolinone (4) was synthesized by the condensation of 2-(5-methyl-2-benzoxazolinone-3-yl)acetate with hydrazine hydrate. Thiosemicarbazide derivatives (5a-5d) were afforded by the reaction of corresponding compound 4 with substituted isothiocyanates. The cyclization of compounds 5a-5d in the presence of triethylamine resulted in the formation of compounds 6a-6d containing 1,2,4-triazole ring. On the other hand, the treatment of compounds 5a-5d with orthophosphoric acid caused the conversion of side chain of compounds 5a-5d into 1,3,4-thiadiazole ring: thus, compounds 7a-7c were obtained. The treatment of compound 4 with aromatic aldehydes resulted in the formation of arylidene hydrazides as cis-trans conformers (8a-8e). The structures of the compounds were elucidated by spectral and elemental analysis. While most compounds were exhibiting high activity in the analgesic-anti-inflammatory field, most of them were found to be inactive against bacteria and fungi.

Full text of DOI:10.1016/j.bmc.2007.06.006

Bioorganic & Medicinal Chemistry 15 (2007) 5738–5751
1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones,
1,3,4-thiadiazoles and hydrazones containing
5-methyl-2-benzoxazolinones: Synthesis,
analgesic-anti-inflammatory and antimicrobial activities
b
c
Umut Salgın-Go¨kßsen,a Nesrin Go¨khan-Kelekc¸i,a, Ozgur Go¨ktaßs, Yavuz Ko¨ysal,
*
¨
¨
d
c
b
d
¨
Ekrem Kılıc¸, Samil Isık, Goknur Aktay and Meral Ozalp
ß
ß
¨
aHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhıye, Ankara, Turkey
b
_
Ino¨nu¨ University, Faculty of Pharmacy, Department of Pharmacology, Malatya, Turkey
cOndokuz Mayıs University, Faculty of Arts and Sciences, Department of Physics, 55139 Kurupelit Samsun, Turkey
dHacettepe University, Faculty of Pharmacy, Department of Microbiology, 06100 Sıhhıye, Ankara, Turkey
Received 21 March 2007; revised 25 May 2007; accepted 5 June 2007
Available online 8 June 2007
Abstract—Acetic acid hydrazide containing 5-methyl-2-benzoxazolinone (4) was synthesized by the condensation of 2-(5-methyl-2-
benzoxazolinone-3-yl)acetate with hydrazine hydrate. Thiosemicarbazide derivatives (5a–5d) were afforded by the reaction of cor-
responding compound 4 with substituted isothiocyanates. The cyclization of compounds 5a–5d in the presence of triethylamine
resulted in the formation of compounds 6a–6d containing 1,2,4-triazole ring. On the other hand, the treatment of compounds
5a–5d with orthophosphoric acid caused the conversion of side chain of compounds 5a–5d into 1,3,4-thiadiazole ring: thus, com-
pounds 7a–7c were obtained. The treatment of compound 4 with aromatic aldehydes resulted in the formation of arylidene hydraz-
ides as cis–trans conformers (8a–8e). The structures of the compounds were elucidated by spectral and elemental analysis. While
most compounds were exhibiting high activity in the analgesic-anti-inflammatory field, most of them were found to be inactive
against bacteria and fungi.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
COX-2 while sparing COX-1 represented a new attrac-
tive therapeutic development, they also gave rise to some
of the side effects seen with traditional dual COX inhib-
itors (NSAIDs), namely, effects on the kidney that might
manifest as an increased incidence of hypertension, oe-
dema and associated clinical states.4–6 Therefore, selec-
tive COX-2 inhibitors may not be the proper strategy
to overcome the damaging effects of conventional NSA-
IDs which are used for the chronic inflammatory dis-
eases in a long term basis. The other strategy is the
inhibition of inducible nitric oxide synthase (iNOS),
which contributes to acute and chronic inflamma-
tion.7–9
Rheumatic diseases are the most prevalent causes of
disability in Western countries, and non-steroidal
anti-inflammatory drugs (NSAIDs) are stilll the most
commonly used remedies. NSAIDs cause several serious
adverse effects, the most important one is gastric injury
that might later cause gastric ulceration and renal
injury.1 Attempts to develop non-steroidal anti-inflam-
matory drugs that are devoid of classical NSAID toxic-
ity, especially gastrointestinal injury, follow several
strategies. One of which is selective cyclooxygenase-2
inhibition (COX-2).2,3 Although agents that inhibit
In addition, it is known that bacterial infections often
produce pain and inflammation. In normal practice,
two groups of agents (chemotherapeutic, analgesic and
anti-inflammatory) are prescribed simultaneously.
Unfortunately, none of the drugs possesses these three
activities in a single component. Therefore, our aim is
Keywords: Thiosemicarbazide; Triazol; Thiadiazole; Hydrazone;
2-Benzoxazolinone; Analgesic-anti-inflammatory and antimicrobial
activities.
*
Corresponding author. Tel.: +90 312 305 30 17; fax: +90 312 311 47
77; e-mail: onesrin@hacettepe.edu.tr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.006

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5739
to find a compound having dual effect both analgesic-
anti-inflammatory and antimicrobial activities. While
searching for such a compound, we have found that 2-
benzoxazolinone ring is one of the moieties on which
studies have been concentrated. In our laboratory, we
have designed and synthesized some 2-benzoxazolinone
derivatives in the search for new non-steroidal anti-
inflammatory agents (Fig. 1).10–19 A considerable num-
ber of the prepared compounds have been found to have
analgesic-anti-inflammatory activity comparable to or
higher than that of indomethacine. 2-Benzoxazolinone
(I) is also a cyclic isostere of coumarin (II) whose antimi-
crobial activities have been extensively investigated and
performed.20,21 All of these have made us think that 2-
benzoxazolinones are promising compounds for finding
a drug which has analgesic-anti-inflammatory and anti-
microbial action.
derivatives having both analgesic/-anti-inflammatory
and antimicrobial activities. One of the most interesting
characteristics of these novel compounds is their basic
nature, which differentiates them from the classical,
acidic nonsteroidal anti-inflammatory agents (NSAIDs).
It is of interest, therefore to study analgesic-anti-inflam-
matory properties of these novel compounds.
The synthesis pathway leading to the title compounds is
given in Scheme 1. 5-Methyl-2-benzoxazolinone, start-
ing material 2, was synthesized according to the litera-
ture method using 5-methyl-2-hydroxyaniline and
urea.53 Treatment of 5-methyl-2-benzoxazolinone with
ethyl chloroacetate in K2CO3/Acetone gave the N-alkyl-
ated product (5-methyl-2-benzoxazolinone-3-yl)-acetic
acid ethyl ester 3.54 The acid hydrazide 4 was prepared
by the reaction of ethyl 2-benzoxazolinone-3-yl acetate
and hydrazine hydrate in ethanol, which produced a
89% yield. The reaction of hydrazide 4 with various al-
kyl/aryl isothiocyanate gave compounds 5a–5d. On
heating with triethyl amine in ethanol the thiosemicar-
bazides 5a–5d underwent smooth cyclization through
dehydration to afford the 3-[(5-methyl-2-benzoxazoli-
none-3-yl)methyl]-4-substituted-1H-1,2,4-triazole-5(4H)-
thione 6a–6d.
1,2,4-Triazoles, 1,3,4-thiadiazoles and their condensed
derivatives constitute an important class of organic
compounds with analgesic-anti-inflammatory22–32 and
antimicrobial activities.33–43 In addition, hydrazone
derivatives also exhibit potent antimicrobial44–48 and
analgesic-anti-inflammatory49–52 activities. Prompted
by these findings and in continuation of our efforts in
synthesizing various bioactive molecules, we have com-
bined 2-benzoxazolinone with 1,2,4-triazole, 1,3,4-thi-
adiazoles and investigated the eventual role of the
triazole, thiadiazole moieties and the para-phenyl substi-
tuent of the hydrazone subunit on the analgesic-anti-
inflammatory and antimicrobial activities.
2-Substituted amino-5-[(5-methyl-2-benzoxazolinone-3-
yl)methyl]-1,3,4-thiadiazoles 7a–7c were obtained by
cyclization of 5a–5d by treating with orthophosphoric
acid. The preference formation of the thiadiazole ring
under such acidic conditions can be due to the loss of
nucleophilicity of N-4 as a result of its protonation lead-
ing to a comparable increase in the nucleophilicity of the
sulfur atom towards the attack of the carbonyl carbon.
On the other hand, when the cyclization of 6a–6d was
carried out under alkaline conditions, the nucleophilicity
of N-4 was enhanced and led to cyclization with the car-
bonyl carbon atom to give the 1,2,4-triazole-5(4H)-thi-
ones 6a–6d. The carbohydrazide 4 was condensed with
different aromatic aldehydes in ethanol to give the corre-
sponding Schiff’s bases 8a–8e in very good yields.
2. Results and discussion
2.1. Chemistry
One series of novel derivatives of 5-methyl-2-benzoxazo-
linones has been synthesized in an attempt to find new
The purity of the synthesized compounds was checked
by elemental analyses. The structures of the various syn-
thesized compounds were determined on the basis of
1
spectral data analysis; such as UV, IR, H NMR, 13C
NMR and MS. The structure of compound 6b was also
elucidated by the X-ray diffraction.
The IR spectra of acid hydrazide 4 showed a peak at
1702 cmꢀ1 due to exocyclic carbonyl function derived
from hydrazide structure beside the endocyclic carbonyl
peak (1766 cmꢀ1) at position 2 of 5-methyl-2-benzoxaz-
olinone ring. The IR spectra of compounds 5a–5d exhib-
ited a characteristic strong absorption at 1343 cmꢀ1
attributable to the C@S of the thiosemicarbazide
residue.
1
In the H NMR spectra of compound 4, singlet signals
derived from hydrazide structure appeared between
4.23 ppm (–NHNH2) and 9.54 ppm (–NHNH2) showing
the integration for two protons and one proton, respec-
tively. Methylene protons resonated as a singlet at
Figure 1. 2-Benzoxazolinone derivatives having analgesic-anti-iflam-
matory inhibitory activity.

5740
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
Scheme 1. Synthetic pathway of compounds.
1
4.86 ppm. The H NMR spectra of compounds 5a–5d
from aldehydes and substituted hydrazides are present
in solution in the E form. It has been reported that when
hydrazones are dissolved in dimethyl-d6 sulfoxide solu-
tion, the E geometrical isomers of these compounds un-
dergo a rapid cis/trans amide equilibrium, in which the
cis conformer predominates.55,56
displayed three singlets due to three different –NH
groups around 8.1, 9.4 and 10.30 ppm (exchangeable
with D2O) each showing the integration for one proton.
In the 1H NMR spectra of compounds 6a–6d, signals of
the NH of the 1,2,4-triazole-5(4H)-thione derivatives
were observed in the d 13.7–13.98 ppm range. X-ray
analysis confirmed that compound 6b exists in the thi-
1
The investigation of H NMR spectra of 8a–8e demon-
1
one form (Fig. 2). The H NMR spectra of 1,3,4-thi-
adiazole derivatives 7a–7c showed the proton signals
typical of the NH group in the d range 7.72–10.39 ppm.
strated that these hydrazones behaved similarly in this
solution and no signal belonging to Z isomer was ob-
served. This assignment was further confirmed by a
molecular minimization energy study using Molecular
Operating Environment (MOE, version 2005.06)) for
8a. This study indicated that the compound bearing E
configuration (E/65.6856 Kcal molꢀ1) has lower energy
than the isomeric compound having Z configuration
(Z/74.0366 Kcal molꢀ1) (Fig. 3).
According to the literature, the hydrazones may exist as
Z/E geometrical isomers about C@N double bonds and
cis/trans amide conformers. Besides hydrazones derived
1
In the H NMR of the compounds 8a–8e, the signals
belonging to benzylidene group were observed at aro-
matic region while the signals belonging to –NHNH2
disappeared. Two sets of signals each belonging to the
CH2 and @CH group of cis and trans conformers were
observed between 4.60–4.85/8.18–8.25 and 5.02–5.05/
8.00–8.07 ppm. The upfield lines of @CH protons were
assigned to cis-conformer of the amide structure and
downfield lines of the protons of the same group were
assigned to trans-conformer of the amide structure.55
Additional support for the structures of the synthesized
compounds was provided by 13C NMR spectra. In the
13C NMR spectra of the compounds 5a, 8c, lactam
and hydrazide C@O gave two peaks at around 154
and 166 ppm. The peak resonated at 168 ppm in the
13C NMR spectrum of compound 6c, assigned for
C@S, confirming thione form of the triazole ring.
Figure 2. The structure of 6b, showing 50% probability displacement
ellipsoids and the atom numbering scheme.

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5741
Figure 3. Representation of E and Z configuration of compound 8a.
The mass spectra of all the synthesized compounds
were studied under electron ionization. The molecular
ion peak was present in all the compounds, although
2.2. X-ray crystal analysis of 6b
The molecular structure of compound 6b was determined
by X-ray crystallographic analysis. The compound crys-
tallizes in the monoclinic system, space group P21/c
their relative intensities varied from
(Scheme 2).
1 to 100%
˚
˚
˚
with a = 11.9854(11) A b = 7.2021(4) A c = 17.8274(14) A,
˚
Further spectroscopic details of these compounds are
presented in the experimental part. In addition, it was
shown by X-ray crystallographic analysis that com-
pound 6b and compound 7a contain 1 mol H20 and
b = 107.363(7) A. Ortep depiction with atom numbering
of compound 6b is shown in Figure 2, respectively. Com-
pound 6b contains essentially planar benzoxazolinone
ring system, within which the C–N bond distances and
angles do not differ significantly from our related
structures, 3-[4-(4-fluorophenyl)piperazine-1-ylmethyl]-
57
1 mol HPO4 with X-ray crystallographic analysis,
respectively.
Scheme 2. Fragmentation pattern of the compounds.

5742
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
the coordinated and uncoordinated water molecules are
engaged in hydrogen bonding with O2 and S1 atoms of
the ring systems, resulting in the formation of extended
chains along the c axis. There are also p–p stacking inter-
actions between the parallel triazole systems. The closest
˚
perpendicular separation is 2.948 A between the ring sys-
tem at (x,y,z) and that at (ꢀx,ꢀ1/2 + y,ꢀ1/2ꢀz).
2.3. Pharmacology
Continuing our studies on 2-benzoxazolinones that are
attractive candidates as antinociceptive agents, we de-
signed a new series of functionalized 2-benzoxazolinone
derivatives. The rational design of these new derivatives
was planned by molecular hybridization of previously
described analgesic compounds benzoxazolinone, tria-
zole and thiadiazole. In the pharmacological study, we
investigated anti-inflammatory and analgesic activity
as well as the ulcerogenic risk of both thiosemicarbazide
intermediates (5a–5d) and corresponding triazole, thi-
adiazole and benzylidene hydrazine derivatives (6a–6d,
7a–7c, 8a–8e).
Figure 4. Crystal packing of 6b projected onto ac plane. The dashed
lines indicates the intermolecular hydrogen bonds.
5-methyl-1,3-benzoxazol-2(3H)-one and 3-[4-(2-fluoro-
phenyl)piperazine-1-ylmethyl]-5-methyl-1,3-benzoxazol-
M2(3H)-one.58,59 The maximum deviations from the plane
2.3.1. Analgesic activity. The analgesic activity of the
compounds was studied by using both the acetic acid-in-
61
duced writhing test60 and hot plate test in mice. The
˚
of the nine-membered ring system is 0.013(2) A for atom
C15. The benzoxazolinone and substituent triazole groups
are almost perpendicular to each other with a dihedral an-
gle of 82.25(4)ꢁ. Thestructureofthecompound 6bcontains
intermolecular, O–Hꢁ ꢁ ꢁO [for O3–H3Aꢁ ꢁ ꢁO2i, donor and
animals were first administered at 100 mg/kg (body
weight) dose of the test drugs in two of the screening
tests. These compounds presented an important analge-
sic profile measured by the classical acetic acid induced
constrictions. From the obtained results of acetic acid-
induced writhing test, it was noticed that all compounds
possess analgesic activity at 100 mg/kg dose comparable
with or higher than that of aspirin (ASA) (Table 1).
However, hot plate test results showed a relative de-
crease in the analgesic activity of the compounds (except
7a, 8c, 8d). The analgesic effects of 7a (54.3%, 73.8%), 8c
(50.7%, 62.5%) and 8d (52.9%, 77.8%) were higher than
those of both morphine and aspirin (50.7%, 65.7%)
(Fig. 5).
˚
˚
acceptor distance is 2.8359(17) A, O3–H3A = 0.82(3) A,
i
O3ꢁ ꢁ ꢁ O2 = 2.03(3) A, O3–H3Aꢁ ꢁ ꢁO2i = 169(2)ꢁ i)
˚
x,ꢀy+1/2,+z ꢀ 1/2], O–Hꢁ ꢁ ꢁS [for O3–H3Bꢁ ꢁ ꢁ S1ii, donor
˚
and acceptor distance is 3.2929(14) A, O3–
ii
˚
˚
H3B = 0.82(2) A, O3ꢁ ꢁ ꢁ S1 = 2.47(2) A, O3–H3Bꢁ ꢁ ꢁ
S1ii = 175(2)ꢁ, (ii) x,+y+1,+z] and intramolecular N–
Hꢁ ꢁ ꢁO [for N3–H3ꢁ ꢁ ꢁO3, donor and acceptor distance is
˚
˚
˚
2.7793(17) A, N3-H3 = 0.88(2) A, N3ꢁ ꢁ ꢁO3 = 1.91(2)A,
N3–H3ꢁ ꢁ ꢁO3 = 171.1(18)ꢁ] type hydrogen bonds. Crystal
packings of 6b are shown in Figure 4. The H atoms of
Table 1. Effects of the compounds on acetic acid induced abdominal writhing and hot plate tests in mice
Compound
Dose (mg/kg)
Hind paw lick SEM (Inhibition %) (A)
Writhing reflex SEM (Inhibition %) (B)
Control
Morphine
13.8 0.2
6.8 0.5** (50.7)
—
24.8 0.9
—
10
200
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
ASA
4
8.5 0.6** (65.7)
6.5 0.6** (73.8)
9.5 0.6** (61.7)
13.8 1.1** (44.4)
10.0 1.1** (59.7)
11.0 0.9** (55.6)
12.5 1.2** (49.6)
14.0 0.4** (43.5)
8.3 0.5** (66.5)
12.5 0.6** (49.6)
6.5 0.6** (73.8)
7.8 0.6** (68.5)
8.3 0.6** (66.5)
9.3 0.9** (62.5)
6.8 0.6** (72.6)
9.3 0.8** (62.5)
5.5 0.6** (77.8)
8.5 0.6** (65.7)
8.0 0.6** (42.0)
10.5 0.3** (23.9)
11.3 0.3** (18.1)
11.3 0.3** (18.1)
10.5 0.6** (23.9)
13.5 0.3n.s. (2.2)
8.8 0.5** (36.2)
9.5 0.6** (31.2)
12.0 0.4* (13.0)
6.3 0.3** (54.3)
10.3 0.3** (25.4)
9.5 0.3** (31.2)
10.5 0.3** (23.9)
11.0 0.4** (20.3)
6.8 0.3** (50.7)
6.5 0.3** (52.9)
10.0 0.4** (27.5)
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
8a
8b
8c
8d
8e
*p < 0.01; **p < 0.001 Significant from control (n = 5); n.s., not significant.

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5743
Figure 5. Analgesic effect of compounds on acetic acid induced abdominal writhing and hot plate tests in mice.
Additionally, these studies showed that the most potent
analgesic agent 8d carries methyl substituent at the
phenyl ring of the hydrazone group. As shown in Table
1, compound 6a has analgesic activity in acetic acid-
induced writhing test, although it was almost inactive
in the hot-plate test.
(Fig. 7). When compared the effect of substituents on
the phenyl ring of hydrazone derivatives (compounds
8a–8e), methoxy substituent (8e) resulted in significant
anti-inflammatory activity. Whereas, this derivative
gave also rise to the ulcer risk. As shown in Table 2, sub-
stituent size did not produce noticeable differences in
activity of the compounds. However an increase in
anti-inflammatory activity was observed with replace-
ment of alkyl chain to phenyl ring (compounds 5d, 6d,
7c). Among these compounds, compound 6d which
has N-phenyltriazole exhibited the highest anti-inflam-
matory activity. Measurements were done in every
90 min along 6 h at 100 mg/kg dose level and percentage
inhibition values were 79.8%, 63.3% and 31.8%, 27.4%.
This compound was also safe in ulcer incidence.
However, the mechanism underlying their antinocicep-
tive activity remains to some degree unknown. Thus fur-
ther studies are essential to ascertain the mechanisms
involved in the analgesic properties of the ring system.
2.3.2. Anti-inflammatory activity. In order to screen the
anti-inflammatory profile of the synthesized com-
pounds, carrageenan-induced hind paw oedema model
in mice was used.62 At first, the anti-inflammatory
activity of the synthesized compounds was studied at
100 mg/kg dose. Test compounds which possessed more
than 20% effect, even some of them are not significant
statistically, were considered for further evaluation and
the experiments were repeated for additional two
different dose levels (50 and 200 mg/kg) (Table 2). The
anti-inflammatoy activity decreased dramatically when
compounds were administered at a half dose (50 mg/
kg, p.o.). The anti-inflammatory effects of the com-
pounds at 100 mg/kg dosage reached significant values
after 90 min (Fig. 6). We also screened all compounds
for ulcerogenic adverse effect at 200 mg/kg dose level.
After microscopic examination, no ulceration risk was
seen in compounds 6a–6d which have triazole moiety
in their structure.
Since our findings are preliminary results; further studies
need to be carried out to investigate the other specifica-
tions, such as in vitro assays, toxicological studies or
side effect-activity profiles of these compounds.
2.4. Microbiology
2.4.1. In vitro antimicrobial activity. To develop new
antimicrobial agents, we have synthesized benzoxazoli-
none derivatives having thiosemicarbazide ring (5a–
5d), 1,2,4-triazole (6a–6d) and 1,3,4-thiadiazole moiety
(7a–7c) and hydrazone derivatives (8a–8e). The assess-
ment of the antimicrobial activities of the synthesized
compounds was performed using the broth microdilu-
tion test in Mueller-Hinton Broth and RPMI 1640 (with
L-glutamine without sodium bicarbonate) medium for
the determination of antibacterial and antifungal activ-
ity, respectively.63,64 The results revealed that most of
the newly synthesized compounds exhibited poor anti-
bacterial activities whereas promising antifungal activi-
ties (Table 3). It is worth mentioning that compounds
4, 5a, 6d, 8a, 8d, 8e (MIC 128 lg/mL) showed moderate
inhibitory activities against Candida krusei, Candida
albicans and Candida parapsilosis.
In order to probe structural requirements for optimal
anti-inflammatory activity in this series, the substituents
attached to the nitrogen atom, size of these substituents
and cyclization of thiosemicarbazide unit were exam-
ined. According to the results of in vivo experiments,
we can conclude that the cyclization of the thiosemicar-
bazide moiety to thiadiazole produced more active com-
pounds at 200 mg/kg (compare 7c/5d; 7b/5b; 7a/5a)

5744
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
Table 2. Anti-inflammatory effect of compounds against carrageenan-induced hind paw oedema model in mice and ratio of ulceration (n = 5–6)
Compound
Ratio of ulceration Dose (mg/kg)
Swelling in thickness (·10ꢀ2 mm) SEM (Inhibition %)
90 min
180 min
270 min
360 min
Control
4
0/5
100
50
66.0 1.0
10.0 0.2*** (84.8)
92.10 2.5
79.0 2.0
23.3 1.7*** (70.5)
88.7 3.6
90.0 3.4
54.5 3.4*** (39.4)
108.4 2.5
80.0 4.6
58.1 2.6* (27.4)
101.1 1.1
3/5
0/5
0/5
2/5
0/5
0/5
0/5
0/5
0/5
2/5
4/5
4/5
2/5
5/5
2/5
3/5
5/5
200
100
50
23.4 1.3*** (65.1)
23.3 3.3*** (64.7)
118.2 1.8
27.7 3.1*** (58.0)
28.3 1.7*** (57.1)
116.7 5.7
53.2 2.3*** (19.4)
13.3 1.7*** (79.8)
147.3 6.1
46.8 2.0*** (29.1)
13.3 3.3*** (79.8)
118.2 1.8
39.4 2.1*** (40.3)
13.3 1.7*** (79.8)
67.5 2.9
38.3 2.0*** (42.0)
28.3 1.7*** (57.1)
82.9 6.1
28.7 2.8*** (56.5)
11.7 1.7*** (82.3)
58.3 3.1* (11.7)
38.3 3.1*** (41.9)
13.3 1.7*** (79.8)
122.8 0.3
30.9 2.0*** (53.2)
24.6 3.7*** (62.7)
66.0 2.5
24.1 3.5*** (63.5)
10.0 0.2*** (84.8)
60.8 1.6* (7.9)
9.7 1.7*** (85.3)
10.0 0.2*** (84.8)
53.1 2.5** (19.5)
8.0 2.5*** (87.9)
13.3 3.3*** (79.8)
98.3 3.2
24.1 2.5*** (63.5)
36.7 3.3*** (44.4)
64.7 2.1n.s. (1.9)
33.8 3.1*** (48.8)
23.3 3.3*** (64.7)
72.5 2.5
22.4 1.4*** (71.6)
52.3 5.8** (33.8)
125.2 5.9
35.9 2.0*** (54.6)
50.3 7.8** (36.3)
121.5 2.7
62.8 2.4*** (20.5)
42.6 2.0*** (46.1)
147.1 2.9
59.2 1.7*** (25.1)
54.3 3.9*** (31.3)
140.9 4.9
36.8 2.9*** (53.4)
69.7 5.8n.s. (11.8)
85.1 2.7
48.5 2.6*** (38.6)
95.9 2.9
91.1 5.5
53.9 2.0*** (31.8)
60.0 7.7* (24.1)
120.3 4.9
44.0 2.6*** (44.3)
29.0 3.4*** (63.3)
115.5 2.4
40.8 1.6*** (54.7)
90.3 4.5n.s.
187.2 1.2
58.3 2.2*** (35.2)
58.0 3.4*** (35.6)
139.1 3.8
65.0 2.5*** (27.8)
78.3 1.7* (13.0)
188.2 1.9
80.0 1.6* (11.1)
92.0 10.2n.s.
162.6 2.5
62.5 1.8*** (30.5)
76.7 3.0* (14.8)
120.7 2.1
75.8 2.7* (15.8)
117.6 3.0
147.3 2.9
38.7 1.7*** (51.6)
85.8 1.3n.s.
188.9 5.0
58.0 2.5** (27.5)
56.6 0.2** (29.3)
127.8 4.7
42.2 2.2*** (47.2)
80.6 1.3n.s.
176.7 2.1
74.7 1.4 (6.6)n.s.
79.2 0.2n.s.
153.3 4.1
52.7 2.4*** (34.1)
58.1 2.6* (27.4)
97.8 1.3
79.1 1.9n.s.
87.1 2.1
146.7 5.4
64.2 2.3* (19.7)
60.8 1.3* (24.0)
160.0 5.9
5a
200
100
50
5b
200
100
50
5c
200
100
50
5d
200
100
50
6a
200
100
50
6b
200
100
50
66.7 1.8*** (25.9)
78.4 1.7* (12.9)
164.7 2.6
65.0 3.1*** (27.8)
61.4 3.0** (31.8)
157.3 2.5
6c
200
100
50
62.4 3.2* (22.0)
58.1 1.3* (27.4)
147.2 1.6
6d
200
100
50
41.3 3.6*** (47.7)
62.1 2.3** (21.4) 110.4 3.1
75.0 2.9** (16.7)
75.6 2.5n.s. (5.5)
7a
106.7 2.7
101.3 2.1
106.8 3.7
96.0 4.0
200
100
50
41.9 4.2*** (46.9)
52.3 1.7*** (33.8)
90.1 2.8
22.7 2.2*** (71.3)
54.3 3.9*** (31.3)
73.4 2.8n.s. (7.1)
47.9 5.6*** (39.4)
56.1 3.9** (30.0)
100.1 3.6
49.1 4.4*** (37.8)
44.5 3.9*** (43.7)
94.6 1.8
41.9 1.9*** (46.9)
75.0 0.2n.s. (5.1)
89.0 3.0
60.0 2.7*** (33.3)
83.6 1.7n.s. (7.1)
102.8 5.0
49.1 1.7*** (45.4)
95.4 3.4
93.9 4.1
68.4 2.3n.s. (14.5)
80.6 1.3n.s.
94.0 2.5
7b
200
100
50
71.1 3.1n.s. (11.1)
81.8 2.6
110.0 3.2
7c
200
100
50
63.6 3.2*** (29.3)
95.4 1.7
87.1 3.0
84.5 2.6
8a
108.8 4.5
65.5 2.3*** (27.2)
115.0 1.6
81.8 3.0
64.7 1.3* (19.1)
83.0 2.0
200
100
50
8b
92.0 0
95.9 2.9
80.0 1.8* (11.1)
85.2 3.4n.s. (5.3)
111.8 4.5
74.5 2.3** (17.2)
92.0 0.2
103.8 1.6
89.1 3.1n.s.
95.4 3.4
200
100
50
84.4 3.9
76.6 2.6n.s. (4.3)
107.0 3.0
73.8 3.9n.s. (7.7)
69.9 2.6n.s. (12.6)
98.0 3.4
8c
200
100
50
54.7 3.1** (17.1)
20.0 0.2*** (69.7)
73.8 1.6
54.7 3.1** (17.1)
11.6 1.7*** (82.4) 100.8 7.8
53.1 2.5** (19.5)
8.0 2.5*** (87.9)
38.3 1.7*** (42.0)
61.0 4.4** (22.8)
71.6 3.9n.s. (9.4)
86.8 1.3
8d
200
100
50
52.7 3.5*** (33.3)
83.6 1.7
66.0 2.6n.s. (17.5)
87.2 3.0
8e
82.3 2.8
27.5 3.1*** (65.2)
45.0 2.9*** (43.0)
108.8 1.6
55.5 3.3*** (38.3)
48.3 1.7*** (46.3)
200
10
86.2 3.9
46.7 1.7*** (41.6)
Indomethacin
*p < 0.05; **p < 0.01; ***p < 0.001; Significant from control (n = 5); n.s., not significant.
3. Conclusion
and benzylidene hydrazine showed strong analgesia in
both tests. Among the synthesized compounds, com-
pounds 4 and 6d at 100 mg/kg dose and compounds 4,
7b and 8e at 200 mg/kg dose possessed the most promi-
nent and consistent antiinflammatory activity. There-
fore, we can conclude that compound 4 possessed
considerably high amount of analgesic activity and high
anti-inflammatory activity. These activities might be
Various substituted thiosemicarbazides, triazoles, thia-
diazoles and benzylidene hydrazine derivatives were
synthesized and screened for analgesic-anti-inflamma-
tory and antimicrobial activities. Most compounds
exhibited high analgesic-anti-inflammatory activity.
Compounds 7a, 8c, 8d which have thiadiazole moiety

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5745
Figure 6. Effect of compounds against carrageenan-induced hind paw edema model at 100 mg/kg dose.
Figure 7. Effect of compounds against carrageenan-induced hind paw edema model at 200 mg/kg dose.
resulting from degradation of compound 4 to the corre-
sponding acid by hydrolysis during metabolism of it.
Most of the compounds were inactive against bacteria
but they showed promising antifungal activity. Further-
more, in order to understand the structure activity rela-
tionship and the mechanism of inhibition, the research
on modification of the title compounds is underway.
solvent. Chemical shifts are expressed in d (parts per mil-
lion) relative to tetramethylsilane. Splitting patterns are
as follows: s, singlet; d, doublet; m, multiplet; b, broad;
dd (doublet in doublet). The mass spectra were obtained
with electron impact technique using a Direct Insertion
Probe and Agilent 5973-Network Mass Selective Dedec-
tor at 70 eV. Elemental analyses (C, H, N) were per-
formed on Leco CHNS 932 analyzer.
4. Experimental
4.1. Chemistry
4.1.1. Synthesis of 5-methyl-2-benzoxazolinones. 5-Methyl-
2-benzoxazolinone was synthesized as a result of the reac-
tion of 5-methyl-2-hydroxyaniline with urea in oil bath
according to the method reported earlier.53
All chemicals were obtained from Aldrich Chemical Co.
(Steinheim, Germany). Melting points were determined
through a Thomas Hoover capillary melting point appa-
ratus and are uncorrected. Ultraviolet (UV) spectra were
taken with a Agilent 8453 UV–visible spectra spectrom-
eter in methanol at approximately 2 · 10ꢀ5 M concen-
tration. Infrared (IR) spectra were obtained with a
Bruker Vector 22 IR (Opus Spectroscopic Software Ver-
sion 2.0) spectrometer using potassium bromide plates
and the results were expressed in wave number (cmꢀ1).
Nuclear magnetic resonance (1H NMR and 13C NMR)
spectra were scanned on a Bruker 400 MHz UltraShield
spectrometer using dimethylsulfoxide (DMSO-d6) as
4.1.2. Synthesis of ethyl 2-(5-methyl-2-benzoxazolinone-
3-yl)acetate. Ethyl 2-(5-methyl-2-benzoxazolinone-3-
yl)acetate was synthesized as a result of the reaction of
5-methyl-2-benzoxazolinone and ethyl chloroacetate in
acetone under basic conditions as published before.54
4.1.3. Synthesis of 2-(5-methyl-2-benzoxazolinone-3-
yl)acetylhydrazine (4). About 1.46 mL (30 mmol)
Hydrazine hydrate (100%) was added to a 50 mL ethan-
olic solution of 3.525 g (15 mmol) ethyl 2-(5-methyl-2-
benzoxazolinone-3-yl)acetate and refluxed for 2 h. The
reaction mixture was then cooled and the solid precipi-

5746
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
Table 3. In vitro antibacterial and antifungal activity of compounds 4,
5a–5d, 6a–6d, 7a–7c, and 8a–8e (MIC lg/ml)
(NH–CH3), 43.4 (N–CH2), 109.7, 110.5, 123.1, 131.6,
133.8, 140.4 (Arom-C.), 154.7 (C@O), 166.3 (C@S),
182.7 (CH2–CO–NH); MS (70 eV, EI): m/z (%): 294
(M+., 74%), 276 (M+-H2O, 29%), 221 (M+-
S@CNHCH3, 33%), 206 (M+–NHCSNHCH3, 7%),
190 (M+–NHNHCSNHCH3, 9%), 162 (M+–CON-
HNHCSNHCH3, 100%), 149 (5-methyl-2-benz., 46%),
134 (2-benz., 28%), 107 (C7H5NO2, 9%), 91 (C6H5N,
51%), 77 (C6H5, 16%) and 74 (S@CNHCH3, 37%). Cal-
culated for C12H14N4O3S: C, 48.97; H, 4.79; N, 19.04.
Found: C, 48.82; H, 4.88; N, 18.53.
Compound
M IC values (lμg/ml)
A
B
C
D
E
F
G
256
512
256
256
256
256
256
512
512
256
256
512
512
256 128 128 128
128 128 128 128
512 256 256 256
512 256 256 256
256 256 256 128
512 512 512 256
256 512 256 256
256 256 256 128
256 128 128 128
512 256 256 256
512 512 512 256
512 256 256 256
256 128 128 128
4
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
512
256
256
256
256
512
512
512
>512 >512
512
256
256
512
512 >512
512
256
512
512
256
512
4.1.4.2. 1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-4-ethylthiosemicarbazide (5b). Derivative 5a was ob-
tained by the reaction of 4 with ethylisothiocynate as a
white solid substance with a yield of 60% and recrystal-
lized from acetone–water (4/1). Mp 200–202 ꢁC. UV
(CH3OH) nm: 203 (loge: 4.73), 245 (log e: 4.2) and
278 (loge: 3.73); IR (KBr) cmꢀ1; 3307, 3171, 2982,
512 >512
512 256
8a
8b
>512 >512 >512 >512
64 256 128
8c
>512 >512 >512 >512 128 256 256
8d
8e
512
256
1
256
128
8
128
512
2
256 128 128 128
256 128 128 512
1
2933, 1763, 1683, 1343; H NMR (400 mHz, DMSO-
d6) d (ppm) (J in Hz): 1.1 (t, 3H, –CH2–CH3), 2.3 (s,
3H, –CH3), 3.5 (q, 2H, –NH–CH2–CH3), 4.6 (s, 2H,
–N–CH2–CO), 6.96 (d, 1H, 2-benz.-H6, J 8), 7.02 (s,
1H, 2-benz.-H4), 7.23 (d, 1H, 2-benz.-H7, J 8), 8.0 (t,
1H, –CS–NH–CH2), 9.3 (s, 1H, –NH–NH–CS), 10.2
(bs, 1H, –CO–NH–NH); MS (70 eV, EI): m/z (%):
308 (M+., 32%), 290 (M+-H2O, 72%), 221 (M+-
S@CNHCH2CH3, 28%), 206 (M+–NHCSNHCH2CH3,
17%), 190 (M+–NHNHCSNHCH2CH3, 8%), 162
(M+–CONHNHCSNHCH2CH3, 97%), 149 (5-methyl-
2-benz., 97%), 142 (M+–C8H8NO3, 100%), 134 (2-benz.,
26%), 107 (C7H5NO2, 10%), 91 (C6H5N, 67%), 88
(S@CNHCH2CH3, 22%), 77 (C6H5, 22%) and 44
(NHCH2CH3, 31%). Anal. Calcd for C13H16N4O3S: C,
50.64; H, 5.23; N, 18.17. Found: C, 50.17; H, 5.20; N,
17.99.
Ampicillin
Fluconazole
—
—
—
1
—
64
—
8
—
—
—
A, Staphylococcus aureus; B, Enterococcus faecalis; C, Escherichia coli;
D, Pseudomonas aeruginosa; E, Candida albicans; F, Candida crusei; G,
Candida parapsilosis.
tated was recrystallized from ethanol–water (3/1) to give
89% of 4 as a cream solid. Mp 215–217 ꢁC. UV
(CH3OH) nm: 202 (loge: 4.3), 284 (loge: 3.28); IR
1
(KBr) cmꢀ1: 3337, 3222, 2923, 1766, 1702; H NMR
(400 mHz, DMSO-d6) d (ppm) (J in Hz): 2.21 (s, 3H,
–CH3), 4.23 (s, 2H, –NH2), 4.86 (s, 2H, –N–CH2–CO),
6.82 (d, 1H, 2-benz.-H7, J 8), 6.98 (dd, 1H, 2-benz.-
H6, J 8, J 2), 7.08 (d, 1H, 2-benz.-H4, J 2), 9.54 (bs,
1H, –CO–NH); MS (70 eV, EI): m/z (%): 221 (M+.,
76%), 190 (M+–NHNH2, 15%), 162 (M+–CONHNH2,
83%), 149 (M+–CH2CONHNH2, 40%), 134 (2-benz.,
100%), 107 (C7H5NO2, 16%), 91 (C6H5N, 30%) and 77
(C6H5, 28%). Anal. Calcd for C10H11N3O3: C, 54.29;
H, 5.01; N, 19.00. Found: C, 53.88; H, 5.11; N, 18.71.
4.1.4.3. 1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-4-allylthiosemicarbazide (5c). Derivative 5c was ob-
tained by the reaction of 4 with allylisothiocynate as a
white solid substance with a yield of 36% and recrystal-
lized from ethanol–water (3/1). Mp 193–194 ꢁC. UV
(CH3OH) nm: 203 (loge: 4.66), 246 (log e: 4.06) and
278 (loge: 3.57); IR (KBr) cmꢀ1: 3305, 3162, 3015,
2989, 2925, 1759, 1681, 1343; 1H NMR (400 mHz,
DMSO-d6) d (ppm) (J in Hz): 2.34 (s, 3H, –CH3), 4.15
(t, 2H, –NH–CH2–CH@), 4.57 (s, 2H, –N–CH2–CO),
5.06 (d, 1H, –CHX@CHAHB, HA, JAX: 10), 5.13 (d, 1H,
4.1.4. General procedure for the preparation of thiosemi-
carbazide derivatives (5a–5d). About 1.547 g (7 mmol) of
4 and 7 mmol appropriate isothiocyanate derivative
were refluxed for 4 h in 5 mL DMF and 25 mL ethanol
mixture. The reaction mixture was then cooled and
poured into ice water. The solid precipitated was filtered
off and recrystallized from appropriate solvents.
–CHX@CHAHB, HB, JBX: 17), 5.8–5.94 (m, 1H,
4.1.4.1. 1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-4-methylthiosemicarbazide (5a). Derivative 5a was
obtained by the reaction of 4 with methylisothiocynate
as a white solid substance with a yield of 63% and
recrystallized from methanol–ether (4/1). Mp 206–
207 ꢁC. UV (CH3OH) nm: 203 (loge: 4.67), 241 (log e:
4.12) and 278 (loge: 3.58); IR (KBr) cmꢀ1: 3320, 3154,
–CH2–CHX@CHAHB), 6.96 (d, 1H, 2-benz.-H6, J 8),
7.0 (s, 1H, 2-benz.-H4), 7.23 (d, 1H, 2-benz.-H7, J 8),
8.2 (t, 1H, –CS–NH–CH2), 9.4 (s, 1H, –NH–NH–CS),
10.25 (bs, 1H, –CO–NH–NH); MS (70 eV, EI): m/z (%):
320 (M+., 0.9%), 302 (M+-H2O, 17%), 221 (M+-
S@CNHCH2CH@CH2, 38%), 206 (M+–NHCSNHCH2
CH@CH2, 35%), 190 (M+–NHNHCSNHCH2CH@CH2,
10%), 162 (M+–CONHNHCSNHCH2CH@CH2, 100%),
149 (5-methyl-2-benz., 43%), 134 (2-benz., 61%), 107
(C7H5NO2, 12%), 99 (S@C@NCH2CH@CH2, 20%), 91
(C6H5N, 53%), 77 (C6H5, 22%), 56 (NHCH2CH@CH2,
27%) and 41 (CH2CH@CH2, 46%). Anal. Calcd for
C14H16N4O3S: C, 52.49; H, 5.03; N, 17.49. Found: C,
52.32; H, 5.21; N, 17.28.
1
2940, 1760, 1681, 1344; H NMR (300 mHz, DMSO-
d6) d (ppm) (J in Hz): 2.3 (s, 3H, –CH3), 2.9 (s, 3H,
–NH–CH3), 4.5 (s, 2H, –N–CH2–CO), 6.9 (d, 1H,
2-benz.-H6, J 8), 6.99 (s, 1H, 2-benz.-H4), 7.2 (d, 1H,
2-benz.-H7, J 8), 8.1 (s, 1H, –CS–NH–CH3), 9.4 (s,
1H, –NH–NH–CS), 10.2 (bs, 1H, –CO–NH–NH); 13C
NMR (400 mHz, DMSO-d6) d (ppm); 21.5 (CH3), 31.3

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5747
4.1.4.4. 1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-4-phenylthiosemicarbazide (5d). Derivative 5d was
obtained by the reaction of 4 with phenylisothiocynate
as a white solid substance with a yield of 59% and
recrystallized from acetonitrile–water (4/1). Mp 201–
202 ꢁC.UV (CH3OH) nm: 203 (loge: 4.82) and 273
(log e: 4.22); IR (KBr) cmꢀ1: 3260, 3162, 3018, 2970,
1756, 1679, 1343; 1H NMR (400 mHz, DMSO-d6) d
(ppm) (J in Hz): 2.3 (s, 3H, –CH3), 4.6 (s, 2H, –N–
CH2–CO), 6.96 (d, 1H, 2-benz.-H6, J 8), 7.06 (s, 1H,
2-benz.-H4), 7.2–7.5 (m, 6H, 2-benz.-H7 and arom-H),
9.7 (s, 2H, –NH–NH–CS–NH), 10.5 (bs, 1H, –CO–
NH–NH); MS (70 eV, EI): m/z (%): 356 (M+., 0.9%),
338 (M+-H2O, 3%), 221 (M+-S@CNHC6H5, 55%), 206
(M+–NHCSNHC6H5, 4%), 190 (M+–NHNHCSNH
C6H5, 13%), 162 (M+–CONHNHCSNHC6H5, 93%),
149 (5-methyl-2-benz., 36%), 135 (S@C@NC6H5,
100%), 134 (2-benz., 21%), 107 (C7H5NO2, 5%), 91
(C6H5N, 61%) and 77 (C6H5, 73%). Anal. Calcd for
C17H16N4O3S: C, 57.29; H, 4.52; N, 15.72. Found C,
57.72; H, 4.73; N, 15.65.
benz., 8%), 91 (C6H5N, 10%) and 77 (C6H5, 7%). Calcu-
lated for C13H14N4O2S.H2O: C, 50.64; H, 5.23; N,
18.17. Found: C, 50.21; H, 5.38; N, 18.05.
4.1.5.3. 3-[(5-Methyl-2-benzoxazolinone-3-yl)methyl]-
4-allyl-1H-1,2,4-triazole-5(4H)-thione (6c). Derivative
6c was obtained by the reaction of 5c as a white solid
substance with a yield of 88% and recrystallized from
acetone–water (3/1). Mp 202–204 ꢁC. UV (CH3OH)
nm: 202 (loge : 4.58) and 257 (loge : 4.21); IR (KBr)
cmꢀ1
:
3087, 3036, 2943, 1787, 1351; 1H NMR
(400 mHz, DMSO-d6) d (ppm) (J in Hz): 2.33 (s, 3H,
–CH3), 4.70 (d, 2H, –N–CH2–CH@; J 4.88), 4.81 (dd,
1H, –CHX@CHAHB; HB; JAB: 1, JBX: 18), 5.06 (dd,
1H, –CHX@CHAHB; HA; JAB: 1, JAX: 10), 5.15 (s,
2H, –N–CH2–CO), 5.79–5.88 (m, 1H, –CH2–CHX@
CHAHB), 6.96 (d, 1H, 2-benz.-H6, J 8), 7.03 (s, 1H, 2-
benz.-H4), 7.24 (d, 1H, 2-benz.-H7, J 8), 13.85 (bs, 1H,
C NMR (400 mHz, DMSO-d6) d
13
@N–NH–CS);
(ppm); 21.5 (CH3), 37.4 (CH2–CH@CH2), 45.6 (N–
CH2-), 117.2 (–CH@CH2), 130.8 (–CH2–CH@CH2),
109.9, 110.3, 123.5, 131.7, 134.0, 140.4 (Arom-C.),
147.5 (CH in triazole ring), 154.2 (C@O), 168.3 (C@S);
MS (70 eV, EI): m/z (%): 302 (M+., 100%), 287 (M+
–CH3, 58%), 261 (M+–CH2CH@CH2, 6%), 162 (M+-4-
allyl-1H-1,2,4-triazole, 12%), 154 (M+-5-methyl-2-benz.,
20%), 149 (5-methyl-2-benz., 43%), 134 (2-benz., 12%)
91 (C6H5N, 13%), 77 (C6H5, 9%) and 41 (CH2CH@
CH2, 15%). Anal. Calcd for C14H14N4O2S: C, 55.61;
H, 4.67; N, 18.53. Found C, 55.49; H, 4.95; N, 18.47.
4.1.5. General procedure for the preparation of 1,2,4-
triazole derivatives (6a–6d). A 15 mL ethanolic solution
of 1 mmol 1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-4-substituted thiosemicarbazide was refluxed with
15 drops of triethylamine for 6 h. The solvent was evap-
orated and the precipitated product was recrystallized
from appropriate solvents.
4.1.5.1. 3-[(5-Methyl-2-benzoxazolinone-3-yl)methyl]-
4-methyl-1H-1,2,4-triazole- 5(4H)-thione (6a). Derivative
6a was obtained by the reaction of 5a as a yellow solid
substance with a yield of 82% and recrystallized from ace-
tone–water (3/1). Mp 222–224 ꢁC. UV (CH3OH) nm: 202
(loge: 4.55), 231 (loge: 4.16) and 254 (loge: 3.85); IR
4.1.5.4. 3-[(5-Methyl-2-benzoxazolinone-3-yl)methyl]-
4-phenyl-1H-1,2,4-triazole- 5(4H)-thione (6d). Derivative
6d was obtained by the reaction of 5d as a white solid
substance with a yield of 93% and recrystallized from
acetone–water (3/1). Mp 263–265 ꢁC. UV (CH3OH)
nm: 203 (loge: 4.64) and 263 (loge: 4.08); IR (KBr)
cmꢀ1: 3196, 3058, 2957, 2923, 1764, 1348; 1H NMR
(400 mHz, DMSO-d6) (ppm) (J in Hz): 2.31 (s, 3H,
–CH3), 4.96 (s, 2H, –N–CH2–CO), 6.86–7.51 (m, 8H,
Arom-H.), 13.98 (bs, 1H, @N–NH–CS); MS (70 eV,
EI): m/z (%): 338 (M+., 17%), 311 (M+-HCN, 43%),
268 (M+–CNCS, 40%), 190 (M+-5-methyl-2-benz.,
24%), 162 (M+-4-phenyl-1H-1,2,4-triazole, 88%), 149
(5-methyl-2-benz., 48%), 134 (2-benz., 100%), 91
(C6H5N, 34%) and 77 (C6H5, 43%). Anal. Calcd for
C17H14N4O2S: C, 60.34; H, 4.17; N, 16.56. Found C,
60.59; H, 4.44; N, 16.68.
1
(KBr) cmꢀ1: 3106, 3050, 2927, 1744, 1330; H NMR
(400 mHz, DMSO-d6) d (ppm) (J in Hz): 2.3 (s, 3H,
–CH3), 3.5 (s, 3H, N–CH3), 5.2 (s, 2H, –N–CH2–CO),
6.97 (d, 1H, 2-benz.-H6, J 8), 7.1 (s, 1H, 2-benz.-H4),
7.25 (d, 1H, 2-benz.-H7, J 8), 13.7 (bs, 1H, @N–NH–
CS); MS (70 eV, EI): m/z (%): 276 (M+., 73%), 162
(M+-4-methyl-1H-1,2,4-triazole, 4%), 149 (5-methyl-2-
benz., 100%), 134 (2-benz., 4%), 128 (M+-5-methyl-2-
benz., 54%), 91 (C6H5N, 10%) and 74 (S@CNHCH3,
15%). Calculated for C12H12N4O2S.H2O: C, 48.97; H,
4.79; N, 19.04. Found: C, 49.37; H, 4.54; N, 19.46.
4.1.5.2. 3-[(5-Methyl-2-benzoxazolinone-3-yl)methyl]-
4-ethyl-1H-1,2,4-triazole-5(4H)-thione (6b). Derivative
6b was obtained by the reaction of 5b as a white solid
substance with a yield of 97% and recrystallized from
acetone–water (3/1). Mp 215–216 ꢁC. UV (CH3OH)
nm: 202 (loge: 4.54) and 256 (loge: 4.17); IR (KBr)
4.1.6. General procedure for the preparation of 1,3,4-
thiadiazole derivatives (7a–7c). About 1.25 mmol 1-[2-(5-
Methyl-2-benzoxazolinone-3-yl)acetyl]-4-substitutedthi-
osemicarbazide derivative was added to 4 mL ortho-
phosphoric acid at 95–100 ꢁC by mixing for 30 min.
After addition is finished, the reaction mixture was
heated for 45 min and later poured into ice water. The
solid precipitated was filtered off and recrystallized from
appropriate solvents.
cmꢀ1
:
3483, 3383, 2937, 1740, 1350; 1H NMR
(400 mHz, DMSO-d6) d (ppm) (J in Hz): 1.19 (t, 3H,
–CH2–CH3), 2.33 (s, 3H, –CH3), 4.05 (q, 2H, N–CH2–
CH3), 5.24 (s, 2H, –N–CH2–CO), 6.97 (d, 1H, 2-benz.-
H6, J 8), 7.1 (s, 1H, 2-benz.-H4), 7.26 (d, 1H, 2-benz.-
H7, J 8), 13.75 (bs, 1H, @N–NH–CS); MS (70 eV, EI):
m/z (%): 290 (M+., 100%), 262 (M+–C2H5, 12%), 162
(M+-4-ethyl-1H-1,2,4-triazole, 5%), 149 (5-methyl-2-
benz., 88%), 142 (M+-5-methyl-2-benz., 36%), 134 (2-
4.1.6.1. 2-Methylamino-5-[(5-methyl-2-benzoxazolinone-
3-yl)methyl]-1,3,4-thiadiazole (7a). Derivative 7a was ob-
tained by the reaction of 5a as a yellow solid substance
with a yield of 47% and recrystallized from ethanol–water

5748
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
(3/1). Mp 193–195 ꢁC. UV (CH3OH) nm: 202 (loge: 4.49)
lized from acetone–water (3/1). Mp 241–243 ꢁC. UV
(CH3OH) nm: 203 (loge: 4.62), 217 (log e: 4.38) and
281 (loge: 4.35; IR (KBr) cmꢀ1: 3337, 3086, 2982,
1
and 275 (loge: 3.89); IR (KBr) cmꢀ1: 2816, 1776; H
NMR (400 mHz, DMSO-d6) d (ppm) (J in Hz): 2.34 (s,
3H, –CH3), 2.84 (s, 3H, NH–CH3), 5.23 (s, 2H, –N–
CH2–CO), 6.97 (d, 1H, 2-benz.-H6, J 8), 7.11 (s, 1H,
2-benz.-H4), 7.25 (d, 1H, 2-benz.-H7, J 8), 7.72 (bs, 1H,
–NH–CH3); MS (70 eV, EI): m/z (%): 276 (M+., 79%),
162 (M+-2-methylamino-1,3,4-thiadiazole, 7%), 149 (5-
methyl-2-benz., 22%), 128 (M+-5-methyl-2-benz.,
100%), 91 (C6H5N, 11%) and 74 (S@CNHCH3, 24%).
Anal. Calcd for C12H12N4O2S.HPO4: C, 38.71; H, 3.52;
N, 15.05. Found: C, 38.58; H, 4.27; N, 14.93.
1
1774, 1682; H NMR (400 mHz, DMSO-d6) d (ppm)
(J in Hz): 2.33 (s, 3H, –CH3), 4.85 and 5.05 (s, 2H,
–N–CH2–CO), 6.81–7.77 (m, 8H, Arom-H.), 8.07 and
8.25 (s, 1H, N@CH), 11.80 (bs, 1H, –CO–NH–N@);
MS (70 eV, EI): m/z (%): 309 (M+., 68%), 206 (M+
–N„C–C6H5, 18%), 189 (M+–NHN@CH–C6H5, 10%),
162 (M+-O„CNHN@CH–C6H5, 50%), 161 (CH2CON-
HN@CH–C6H5, 23%), 147 (O„C–NHN@CH–C6H5,
100%), 134 (2-benz., 32%), 91 (C6H5N, 60%) and 77
(C6H5, 23%). Anal. Calcd for C17H15N3O3: C, 66.01; H,
4.89; N, 13.58. Found: C, 65.76; H, 4.80; N, 13.58.
4.1.6.2. 2-Ethylamino-5-[(5-methyl-2-benzoxazolinone-
3-yl)methyl]-1,3,4-thiadiazole (7b). Derivative 7b was ob-
tained by the reaction of 5b as a white solid substance
with a yield of 74% and recrystallized from acetone–water
(3/1). Mp 188–189 ꢁC. UV (CH3OH) nm: 202 (loge: 4.48)
and 275 (loge: 3.92; IR (KBr) cmꢀ1: 3195, 2976, 2883,
4.1.7.2. 2-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-
4-bromobenzylidenehydrazine (8b). Derivative 8b was
obtained by the reaction of 4 and 4-bromobenzaldehyde
as a white solid substance with a yield of 80% and recrys-
tallized from acetone–water (3/1). Mp 261–262 ꢁC (dec.).
UV (CH3OH) nm: 202 (loge: 4.60), 220 (log e: 4.44) and
286 (loge: 4.30); IR (KBr) cmꢀ1: 3095, 2980, 2948,
1
1781; H NMR (400 mHz, DMSO-d6) d (ppm) (J in
Hz): 1.14 (t, 3H, –CH2–CH3), 2.34 (s, 3H, –CH3), 3.26
(2H; q; –NH–CH2–CH3), 5.23 (s, 2H, –N–CH2–CO),
6.97 (d, 1H, 2-benz.-H6, J 8), 7.12 (s, 1H, 2-benz.-H4),
7.25 (d, 1H, 2-benz.-H7, J 8), 7.91 (bs, 1H, –NH–CH2–
CH3); 13C NMR (400 mHz, DMSO-d6) d (ppm); 14.6
(CH2–CH3), 21.5 (CH3), 109.9, 110.3, 123.5, 130.7,
134.0, 140.5 (Arom-C.), 151.5 (CH2–C@N in thiadiaozle
ring), 154.1 (C@O), 169.9 (N@C–NH); MS (70 eV, EI):
m/z (%): 290 (M+., 98%), 162 (M+-2-ethylamino-1,3,4-
thiadiazole, 12%), 149 (5-methyl-2-benz., 7%), 142 (M+-
5-methyl-2-benz., 100%), 91 (C6H5N, 18%), 88
(S@CNHCH2CH3, 24%) and 77 (C6H5, 9%). Anal. Calcd
for C13H14N4O2S: C, 53.78; H, 4.86; N, 19.30. Found: C,
53.00; H, 4.99; N, 19.19.
1
1774, 1683; H NMR (400 mHz, DMSO-d6) d (ppm) (J
in Hz): 2.33 (s, 3H, –CH3), 4.62 and 5.05 (s, 2H,
–N–CH2–CO), 6.94–7.73 (m, 7H, Arom-H.), 8.04 and
8.22 (s, 1H, N@CH), 11.87 (bs, 1H, –CO–NH–N@);
13C NMR (400 mHz, DMSO-d6) d (ppm); 21.5 (CH3),
43.5 (CH2), 109.7, 110.5, 123.0, 123.8, 129.4, 132.0,
132.3, 133.7, 133.9, 140.5 (Arom-C.), 143.7 (N@CH),
154.9 (C@O), 167.9 (CH2–CO–NH); MS (70 eV, EI):
m/z (%): 387 (M+., 10%), 389 (M+2, 11%), 225
(O„CNHN@CH–C6H4Br, 18%), 221 (M+–CH–
C6H4Br, 70%), 206 (M+–N„C–C6H4Br, 9%), 190 (M+
–NHN@CH–C6H4Br, 13%), 162 (M+-O„CNHN@CH–
C6H4Br, 88%), 149 (5-methyl-2-benz., 39%), 134 (2-benz.,
100%), 107 (C7H5NO2, 17%), 91 (C6H5N, 43%) and 77
(C6H5, 24%). Anal. Calcd for C17H14N3O3Br: C, 52.60;
H, 3.63; N, 10.82. Found C, 52.62; H, 3.93; N, 10.97.
4.1.6.3. 2-Phenylamino-5-[(5-methyl-2-benzoxazolinone-
3-yl)methyl]-1,3,4-thiadiazole (7c). Derivative 7c was
obtained by the reaction of 5d as a white solid substance
with a yield of 93% and recrystallized from acetone–water
(3/1). Mp 195–197 ꢁC. UV (CH3OH) nm: 203 (loge: 4.62),
230 (loge: 4.21) and 285 (loge: 3.94); IR (KBr) cmꢀ1: 3200,
3050, 2924, 2828, 1769; 1H NMR (400 mHz, DMSO-d6) d
(ppm) (J in Hz): 2.34 (s, 3H, –CH3), 5.35 (s, 2H, –N–CH2–
CO), 6.96–7.59 (m, 8H, Arom-H.), 10.39 (bs, 1H, –NH–
C6H5); MS (70 eV, EI): m/z (%): 338 (M+., 32%), 190
(M+-5-methyl-2-benz., 100%), 162 (M+-2-phenylamino-
1,3,4-thiadiazole, 50%), 149 (5-methyl-2-benz., 19%), 136
(S@CNHC6H5, 24%), 134 (2-benz., 14%), 91 (C6H5N,
63%) and 77 (C6H5, 55%). Anal. Calcd for C17H14N4O2S:
C, 60.34; H, 4.17; N, 16.56. Found C, 59.57; H, 4.39; N,
16.54.
4.1.7.3. 2-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-
4-chlorobenzylidenehydrazine (8c). Derivative 8c was
obtained by the reaction of 4 and 4-chlorobenzaldehyde
as a white solid substance with a yield of 82% and
recrystallized from acetone–water (3/1). Mp 264–
265 ꢁC (dec). UV (CH3OH) nm: 202 (loge: 4.64), 220
(log e: 4.49) and 285 (loge: 4.37); IR (KBr) cmꢀ1
:
3184, 3096, 2980, 1774, 1683; 1H NMR (400 mHz,
DMSO-d6) d (ppm) (J in Hz): 2.33 (s, 3H, –CH3), 4.63
and 5.05 (s, 2H, –N–CH2–CO), 6.94–7.80 (m, 7H,
Arom-H.), 8.06 and 8.24 (s, 1H, N@CH), 11.87 (bs,
1H, –CO–NH–N@); MS (70 eV, EI): m/z (%): 343
(M+., 27%), 345 (M+2, 9%), 221 (M+–CH–C6H4Cl,
38%), 206 (M+–N„C–C6H4Cl, 10%), 190 (M+
–NHN@CH–C6H4Cl, 9%), 181 (O„CNHN@CH–
C6H4Cl, 31%), 162 (M+-O„CNHN@CH–C6H4Cl,
44%), 149 (5-methyl-2-benz., 28%), 134 (2-benz.,
100%), 107 (C7H5NO2, 15%), 91 (C6H5N, 20%) and 77
(C6H5, 18%). Anal. Calcd for C17H14N3O3Cl: C, 59.40;
H, 4.10; N, 12.22. Found C, 58.98; H, 3.83; N, 12.33.
4.1.7. General procedure for the preparation of benz-
ylidenehydrazine derivatives (8a–8e). A 40 mL ethanolic
solution of 1 mmol 1 was refluxed with 1 mmol of
substituted benzaldehyde for 30 h. The reaction mixture
was then cooled and the solid precipitated was recrystal-
lized from appropriate solvents.
4.1.7.1. 2-[2-(5-Methyl-2-benzoxazolinone-3-yl)acet-
yl]-benzylidenehydrazine (8a). Derivative 8a was ob-
tained by the reaction of 4 and benzaldehyde as a
white solid substance with a yield of 80% and recrystal-
4.1.7.4. 2-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-
4-methylbenzylidenehydrazine (8d). Derivative 8d was
obtained by the reaction of 4 and 4-methylbenzaldehyde

U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
5749
as a white solid substance with a yield of 84% and
recrystallized from acetone–water (3/1). Mp 265–
266 ꢁC (dec.). UV (CH3OH) nm: 202 (loge: 4.63), 220
On the day before the treatments, the food was with-
drawn, but the animals were allowed free access to
water. A minimum of five animals was used in each
group, otherwise it is described in the procedure. Mice
used were cared in accordance with the directory of
Refiksaydam Hıfzıssıhha Institute’s Animal Care Unit,
where the Guidelines of National Institutes of Health
on Laboratory Animal Welfare were applied.
(log e: 4.43) and 285 (loge: 4.39); IR (KBr) cmꢀ1
:
3192, 3092, 2981, 1773, 1681; 1H NMR (400 mHz,
DMSO-d6) d (ppm) (J in Hz): 2.33 (s, 3H, –CH3), 4.60
and 5.03 (s, 2H, –N–CH2–CO), 6.94–7.66 (m, 7H,
Arom-H.), 8.03 and 8.20 (s, 1H, N@CH), 11.73 (bs, 1H,
–CO–NH–N@); MS (70 eV, EI): m/z (%): 323 (M+.,
40%), 206 (M+–N„C–C6H4CH3, 15%), 162 (M+-O„
CNHN@CH–C6H4CH3, 39%), 161 (O„CNHN@CH–
C6H4CH3, 100%), 134 (2-benz., 22%), 118 (N@CH–
C6H4CH3, 18%), 91 (C6H5N, 44%) and 77 (C6H5,
14%). Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30;
N, 13.00. Found: C, 66.41; H, 4.99; N, 13.11.
4.3.2. Preparation of test samples for bioassay. After sus-
pending in 0.5% sodium carboxymethyl cellulose (CMC)
and distilled water, test samples were given to test ani-
mals orally. The control group animals received the
same experimental handling as those of the test groups
except that the drug treatment was replaced with
appropriate volumes of the reference drug, either indo-
methacin (10 mg/kg), or acetyl salicylic acid (ASA)
(200 mg/kg).
4.1.7.5. 2-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-
4-methoxybenzylidenehydrazine (8e). Derivative 8e was
obtained by the reaction of 4 and 4-methoxybenzalde-
hyde as a white solid substance with a yield of 85%
and recrystallized from acetone–water (3/1). Mp 254–
256 ꢁC (dec.). UV (CH3OH) nm: 202 (loge: 4.55), 222
4.3.3. Analgesic activity
4.3.3.1. Koster test.60 One hour after the oral admin-
istration of test sample, each mouse was injected with
3% (w/v) acetic acid solution (0.1 ml/10 g body weight)
intraperitoneally. Starting 5 min after the acetic acid
injection, the number of muscular contractions on mice
was counted for a period of 10 min. A significant reduc-
tion in the number of writhings by any treatment as
compared to control animals was considered as a posi-
tive analgesic response. The antinociceptive activity
was expressed as percentage change from writhing con-
trols. Percent inhibitory effects were estimated according
to the following equation, where n was the average dif-
ference in thickness between the left and right hind
paw of control group and n0 was that of test group of
animals.
(log e: 4.34) and 287 (loge: 4.27); IR (KBr) cmꢀ1
:
3194, 3097, 2982, 1773, 1683; 1H NMR (400 mHz,
DMSO-d6) d (ppm) (J in Hz): 2.33 (s, 3H, –CH3), 3.82
(s, 3H, -OCH3), 4.60 and 5.02 (s, 2H, –N–CH2–CO),
6.94–7.71 (m, 7H, Arom-H.), 8.00 and 8.18 (s, 1H,
N@CH), 11.67 (bs, 1H, –CO–NH–N@); MS (70 eV,
EI): m/z (%): 339 (M+., 29%), 206 (M+–N„C–
C6H4OCH3, 12%), 177 (O„CNHN@CH–C6H4OCH3,
100%), 162 (M+-O„CNHN@CH–C6H4OCH3, 24%),
149 (5-methyl-2-benz., 8%), 134 (2-benz., 29%), 91
(C6H5N, 39%) and 77 (C6H5, 17%). Anal. Calcd for
C18H17N3O4: C, 63.71; H, 5.05; N, 12.38. Found C,
63.36; H, 5.05; N, 12.39.
Inhibitionð%Þ ¼ ½ðn ꢀ n0Þ=nꢂ ꢃ 100
4.2. Single crystal X-ray crystallographic data of com-
pound 6b
4.3.3.2. Constant temperature hot-plate test.61 An
HTC Inc. Mod. 35-D analgesiameter was set to give a
plate temperature of 54 0.5 ꢁC. One hour after oral
administration of the compounds (100 mg/kg), the ani-
mals were placed on the hot-plate and confined by a
lidded perspex box in a compartment measuring 13.8 x
13.8 cm, and the latency to the first hind-paw lick was
recorded. If no hind-paw lick occurred, the test was ter-
minated after 30 s. Morphine was used as a reference
analgesic and administered i.p. at 10 mg/kg.
The data collection was performed on a STOE IPDS 2
diffractometer employing graphite-monochromated
˚
MoKa radiation (k = 0.71073 A). H atoms were located
geometrically and treated using a riding model, with C-
˚
H distances of 0.93 A (aromatic), 0.97 A (CH2) and
˚
65
˚
0.96 A (CH3). Data collection: X-AREA ; cell refine-
ment: X-AREA; data reduction: X-RED3265; pro-
gram(s) used to solve structure: SHELXS9766;
program(s) used to refine structure: SHELXL9767; soft-
ware used to prepare material for publication: WinGX68
and PARST.69 Crystallographic data (excluding struc-
ture factors) for the title compound reported in this pa-
per have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publica-
tion number CCDC 635224. Copies of the data can be
obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44
(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
4.3.4. Anti-inflammatory activity
4.3.4.1. Carrageenan induced oedema.62 For the
determination of the effects of samples on carra-
geenan-induced paw oedema, the modified method of
Kasahara et al. was employed.40 One hour after the
oral administration of either test sample or the control,
the subplantar tissue of the right hind paw of each
mouse was injected with freshly prepared (0.5 mg/
25 ll) suspension of carrageenan (Sigma, St. Louis,
Missouri, USA.) in physiological saline (154 mM
NaCl). As controls, 25 ll saline solutions were used.
After induction of inflammation, paw oedema was mea-
sured in every 90 min during 6 h period. Afterwards,
4.3. Pharmacology
4.3.1. Animals. Before treatments, the animals were
acclimatized to animal lab conditions for two days and
were fed on standard pellet diet and water ad libitum.

5750
U. Salgın-Go¨kßsen et al. / Bioorg. Med. Chem. 15 (2007) 5738–5751
the difference in footpad thickness between the right
and left foot of each mouse was measured with a pair
of dial thickness gauge callipers (Ozaki Co., Tokyo, Ja-
pan). Mean values of treated groups were compared
with mean values of a control group and analyzed
using statistical methods.
References and notes
1. Wolfe, M. M.; Lichtenstein, D. R.; Singh, G. New Engl. J.
Med. 1999, 340, 1888.
2. Dannhardt, G.; Kiefer, W. Eur. J. Med. Chem. 2001, 36,
109.
3. Kiefer, W.; Dannhardt, G. Expert Rev. Clin. Immunol.
2005, 1, 431.
4.3.5. Gastric–ulcerogenic effect. Six hours after the
treatments of the mice with synthesized compounds at
a dose of 200 mg/kg b.w. for anti-inflammatory activity
tests they were killed under deep ether anaesthesia and
their stomachs were removed. Then the stomach of each
mouse was opened through great curvature and exam-
ined for lesions or bleedings under dissecting
microscope.
4. Burnier, M. Expert Opin. Drug Saf. 2005, 4, 491.
5. Wong, D.; Wang, M.; Cheng, Y.; FitzGerald, G. A. Curr.
Opin. Pharmacol. 2005, 5, 204.
6. Rudic, R. D.; Brinster, D.; Cheng, Y.; Fries, S.; Song, W.
L.; Austin, S.; Coffman, T. M.; FitzGerald, G. A. Circ.
Res. 2005, 96, 1240.
7. Nussler, A. K.; Billiar, T. R. J. Leukocyte Biol. 1993, 54,
171.
8. Ohshima, H.; Bartsch, H. Mutat. Res. 1994, 305, 253.
9. Cochran, F. R.; Selph, J.; Sherman, P. Med. Res. Rev.
1996, 16, 547.
4.3.6. Statistical analysis of data. Data obtained from
animal experiments were expressed as means standard
error (SEM). Statistical differences between the treat-
ment and the control group of animals were evaluated
by two tailed Student’s t test.
10. Erdog˘an, H.; Debaert, M.; Cazin, J. Arzneimittel-Forsch
1991, 41, 73.
11. Go¨khan, N.; Erdog˘an, H.; Tel, B. C.; Demirdamar, R.
Eur. J. Med. Chem. 1996, 31, 625.
12. Go¨khan, N.; Erdog˘an, H.; Durlu, N. T.; Demirdamar, R.
Arc. Pharm. Pharm. Med. Chem. 1999, 332, 43.
13. Go¨khan, N.; Erdog˘an, H.; Durlu, N. T.; Demirdamar, R.
Farmaco 1999, 54, 112.
4.4. Microbiology
4.4.1. In vitro antimicrobial activity. Minimal inhibitory
concentrations (MICs, ug/ml) were determined on dif-
ferent microorganisms using broth microdilution pro-
cedure according to the recommendations of the
National Committee for Clinical Laboratory Stan-
dards.63,64 Minimum inhibitory concentrations (MIC)
were defined as the lowest concentration of the com-
pounds that inhibited visible growth of microorganisms
after incubation at 35 ꢁC for 24 h for bacteria or 48 h
for yeast like fungi. Gram-positive (Staphylococcus aur-
eus ATCC 29213, Enterococcus faecalis ATCC 29212)
and Gram-negative (Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853) bacteria were
used as quality control strains. For testing antifungal
activities of the compounds, following reference strains
were tested: Candida albicans ATCC 90028, Candida
crusei ATCC 6258 and Candida parapsilosis ATCC
22019. Bacterial strains were grown in Mueller-Hinton
broth (Difco Laboratories, Detroit, MI, USA). The
inoculum densities were 5 · 105 colony forming units
(cfu) in 1 mL for bacteria and 0.5–2.5 · 103 cfu/mL
for fungi. Ampicillin anhydride and fluconazole were
used as standard antibiotic powders. Each of the test
compounds was dissolved in DMSO and further dilu-
tions were prepared in sterile distilled water. Ampicillin
anhydride and Fluconazole were diluted in sterile dis-
tilled water. Two fold dilutions of the compounds
and standards were prepared as 512–0.5 lg/mL and
64–0.0625 lg/mL concentrations, respectively. After
dilution was completed, microorganism suspensions
were inoculated into each well of the row. Minimum
inhibitory concentrations (MIC) were defined as the
lowest concentration of the compounds that inhibited
visible growth of microorganisms after incubation at
35 ꢁC for 24 h for bacteria or 48 h for yeast like fungi.
MIC values were given as lg/mL. It was determined
that the solvent had no antimicrobial activity against
any of the test microorganisms.
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