Synthesis and anti-inflammatory activity of 1-acylthiosemicarbazides, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones

Article abstract of DOI:10.1016/S0014-827X(01)01176-4

Sixteen 1-(2-naphthyloxyacetyl)-4-substituted-3-thiosemicarbazide, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-oxadiazole, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-thiadiazole and 5-(2-naphthyloxymethyl)-4-substituted-1,2,4-triazole-3-thione derivatives have been prepared and evaluated as orally active anti-inflammatory agents with reduced side-effects. The structures of the compounds were confirmed by IR and ¹H NMR spectral data and microanalysis. The anti-inflammatory and ulcerogenic activities of the compounds were compared with naproxen, indomethacin and phenylbutazone. In carrageenan-induced foot pad edema assay, 2-(2-naphthyloxymethyl)-5-methylamino-1,3,4-oxadiazole, 5-(2-naphthyloxymethyl)-4-methyl-1,2,4-triazole-3-thione and 5-(2-naphthyloxymethyl)-4-ethyl-1,2,4-triazole-3-thione showed an interesting anti-inflammatory activity. In the air-pouch test, 1,3,4-oxadiazole and 1,2,4-triazole-3-thione derivatives reduced total number of leukocytes of the exudate that indicates excellent inhibition of prostaglandin production. Side effects of the compounds were examined on gastric mucosa, liver and stomach and none of the compounds showed significant side effects compared with reference nonsteroidal anti-inflammatory drugs (NSAIDs).

Full text of DOI:10.1016/S0014-827X(01)01176-4

Il Farmaco 57 (2002) 101–107
www.elsevier.com/locate/farmac
Synthesis and anti-inflammatory activity of
1-acylthiosemicarbazides, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and
1,2,4-triazole-3-thiones
Erhan Palaska a,*, Gu¨lay S¸ahin a, Pelin Kelicen b, N. Tug˘ba Durlu b, Gu¨lc¸in Altinok c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe Uni6ersity, 06100 Ankara, Turkey
b Department of Pharmacology, Faculty of Pharmacy, Hacettepe Uni6ersity, 06100 Ankara, Turkey
c Department of Pathology, Faculty of Medicine, Hacettepe Uni6ersity, 06100 Ankara, Turkey
Received in revised form 27 November 2001
Abstract
Sixteen 1-(2-naphthyloxyacetyl)-4-substituted-3-thiosemicarbazide, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-oxadia-
zole, 2-(2-naphthyloxymethyl)-5-substitutedamino-1,3,4-thiadiazole and 5-(2-naphthyloxymethyl)-4-substituted-1,2,4-triazole-3-
thione derivatives have been prepared and evaluated as orally active anti-inflammatory agents with reduced side-effects. The
1
structures of the compounds were confirmed by IR and H NMR spectral data and microanalysis. The anti-inflammatory and
ulcerogenic activities of the compounds were compared with naproxen, indomethacin and phenylbutazone. In carrageenan-in-
duced foot pad edema assay, 2-(2-naphthyloxymethyl)-5-methylamino-1,3,4-oxadiazole, 5-(2-naphthyloxymethyl)-4-methyl-1,2,4-
triazole-3-thione and 5-(2-naphthyloxymethyl)-4-ethyl-1,2,4-triazole-3-thione showed an interesting anti-inflammatory activity. In
the air-pouch test, 1,3,4-oxadiazole and 1,2,4-triazole-3-thione derivatives reduced total number of leukocytes of the exudate that
indicates excellent inhibition of prostaglandin production. Side effects of the compounds were examined on gastric mucosa, liver
and stomach and none of the compounds showed significant side effects compared with reference nonsteroidal anti-inflammatory
´
drugs (NSAIDs). © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Thiosemicarbazide; 1,3,4-Oxadiazole; 1,3,4-Thiadiazole; 1,2,4-Triazole-3-thione; Anti-inflammatory activity; Side effects
responses. Most notably, some PGs are cytoprotective
in the gastrointestinal tract, responsible for normal
renal function in the kidney and they allow platelet
aggregation. In addition, PGs sensitize peripheral nerve
endings to transmit pain signals to the brain and spinal
cord. Recent studies have shown that COX exists in
two isoforms which differ in their basal expression,
tissue localization, and induction during inflammation.
Differences in the pharmacological profiles of various
NSAIDs might be accounted for by varying degrees of
selectivity for COX-1 and COX-2. The potency and
selectivity of NSAIDs appear to be directly related to
their gastric, renal and hepatotoxicity.
1. Introduction
Prostaglandins (PGs) are well known to be mediators
of inflammation, pain and swelling. They are produced
by the action of cyclooxygenase (COX) enzyme on
arachidonic acid. Metabolites of the COX pathway are
widely accepted as mediators of the inflammatory re-
sponse. COX is known to be the principal target of
nonsteroidal anti-inflammatory drugs (NSAIDs).
NSAIDs, block the formation of PGs and have anal-
gesic, antipyretic and anti-inflammatory activity. How-
ever, PGs are produced by most cells, and their
presence in tissues elicits a broad array of biological
A number of 1-acylthiosemicarbazides, 1,3,4-oxadia-
zoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones
were identified as potent anti-inflammatory compounds.
Carrageenan-induced foot paw edema (CPE) inhibitory
* Corresponding author.
E-mail address: epalaska@hacettepe.edu.tr (E. Palaska).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(01)01176-4

102
E. Palaska et al. / Il Farmaco 57 (2002) 101–107
activity of 1-aryloxyacetyl-4-alkyl/aryl-3-thiosemicar-
bazide and their corresponding cyclized 1,3,4-oxadia-
zole, 1,3,4-thiadiazole and 1,2,4-triazole-3-thione
derivatives were shown equipotent with naproxen,
phenylbutazone, hydrocortisone and other NSAIDs [1–
9]. As a part of our continuing efforts in this area,
2.1.3. 2-(2-Naphthyloxymethyl)-5-substitutedamino-
1,3,4-oxadiazoles 2a–d
The appropriate 1-(2-naphthyloxyacetyl)-4-substi-
tuted thiosemicarbazides (1 mmol) were suspended in
ethanol, 5 ml of 4N sodium hydroxide and iodine in
potassium iodide solution (5%) were added gradually
with stirring and cooling until the color of iodine
persisted. The precipitated product was filtered, dried
and crystallized from suitable solvents.
a
series of 1-(2-naphthyloxyacetyl)-4-substituted
thiosemicarbazides 1a–d and corresponding 2-(2-naph-
thyloxymethyl) - 5 - substitutedamino - 1,3,4 - oxadiazoles
2a–d,
2-(2-naphthyloxymethyl)-5-substitutedamino-
1,3,4-thiadiazoles 3a–d and 5-(2-naphthyloxymethyl)-4-
substituted-1,2,4-triazole-3-thiones 4a–d were syn-
thesized, and evaluated for their anti-inflammatory ac-
tivities and side effects.
2.1.4. 2-(2-Naphthyloxymethyl)-5-substitutedamino-
1,3,4-thiadiazoles 3a–d
The appropriate 1-(2-naphthyloxyacetyl)-4-substi-
tuted thiosemicarbazide derivatives (1 mmol) were dis-
solved in 10 ml toluene and methanesulfonic acid (15
mmol) was added dropwise and refluxed 45 min. The
reaction mixture was neutralized with 10% ammonium
hydroxide and the precipitated product was filtered and
crystallized from suitable solvents.
2. Experimental
2.1. Chemistry
2.1.5. 5-(2-Naphthyloxymethyl)-4-substituted-1,2,4-
triazole-3-thiones 4a–d
All chemicals used in this study were supplied from
Aldrich, Merck and Fluka. Melting points were taken
in a Thomas Hoover capillary melting point apparatus
and are uncorrected. UV and IR spectra were recorded
in Shimadzu UV-160A UV–Vis spectrophotometer
(2.5×10−5 mol/l, ethanol) and Perkin–Elmer FT-IR
1720x spectrophotometer (KBr disc) respectively. 1H
NMR spectra were run on a Bruker AC 80 MHz
FT-NMR spectrometer (DMSO-d6, TMS) and all
chemical shifts were reported as l (ppm) values. Ele-
mental analysis was provided by the Scientific and
Technical Research Council of Turkey. All new com-
pounds yielded spectral data consistent with the pro-
posed structure and microanalysis within90.4% of the
theoretical values.
1-(2-Naphthyloxyacetyl)-4-substitutedthiosemicarba-
zides (10 mmol) were refluxed for 8 h in 40 ml 1 N
aquoeus sodium hydroxide. The mixture was acidified
to pH 2 and the precipitated product was filtered off,
washed with water and crystallized from suitable sol-
vents.
2.2. Pharmacology
Mice used in the present study were housed and
cared in accordance with the Hacettepe University—
Animal Care Unit, which applies the guidelines of
National Institutes of Health (NIH) on Laboratory
Animal Welfare. Male albino mice (Hacettepe Univer-
sity, Animal House, Ankara, Turkey), weighing 2292
g, were used (local breed). The animals were housed in
groups of six and acclimatized to room conditions for
at least 2 days before the experiments, with food and
water at libitum. The food was withdrawn on the day
before the experiment, but free access to water was
allowed.
All the compounds (100 mg/kg) and reference
NSAIDs (phenylbutazone (100 mg/kg), indomethacin
(10 mg/kg) and naproxen (30 mg/kg)) were suspended
in 0.5% carboxymethylcellulose (CMC) and adminis-
tered orally by animal feeding needle. The control
groups received appropriate volumes of the vehicle
(0.5% CMC, oral) only.
2.1.1. 1-(2-Naphthyloxyacetyl)hydrazine
2-Naphthol (1.44 g, 10 mmol), anhydrous potassium
carbonate and 1.67 g ethyl bromoacetate (10 mmol) in
50 ml anhydrous acetone were refluxed on oil bath for
6 h. The reaction mixture was filtered and the excess
solvent was removed by distillation under reduced pres-
sure. The residue and 1.00 g hydrazine monohydrate
(20 mmol) were dissolved in 50 ml absolute ethanol and
refluxed on a steam bath for 1 h. The solid mass was
filtered, dried and crystallized from ethanol.
2.1.2. 1-(2-Naphthyloxyacetyl)-4-substituted
thiosemicarbazides 1a–d
The mixture of 1-(2-naphthyloxyacetyl)hydrazine (10
mmol) and 10 mmol appropriate substituted isothio-
cyanate derivatives was dissolved in 25 ml of EtOH and
refluxed 4 h. The crude product which precipitated on
cooling was filtered, washed with diethyl ether, dried
and crystallized from dioxane–water.
2.2.1. Anti-inflammatory acti6ity
2.2.1.1. Carrageenan Paw Edema Test (CPE) [10,11].
One hour after oral administration of the compounds,
the thickness of right hind paw was measured by a

E. Palaska et al. / Il Farmaco 57 (2002) 101–107
103
peacok dial thickess gauge and 0.01 ml 2% carrageenan
was injected subcutaneously into the plantar surface of
the right hind paw. Two h later the volume of the
edema was measured again and the antiedematous ef-
fects of the drugs were estimated in terms of percent
inhibition. Percent inhibition effects of drugs was calcu-
lated according to the following equation:
hematoxilen eosine and examined under the light
microscope.
2.2.3. Statistical analysis
All data are expressed as mean value9SEM. The
data obtained from the control group were compared
with pretreated groups, by analysis of variance
(ANOVA) followed by post-hoc Bonferroni test. A
PB0.05 was considered significant.
Anti-inflammatory activity (%)=[(n−n%)/n]×100
n and n% indicate the difference in thickness between
first and second measurements of hind paws in control
and test groups, respectively.
3. Results and discussion
2.2.1.2. Air-pouch test [12–14]. The carrageenan pow-
der was dissolved in 0.9% saline to a concentration of
10 mg/ml. The solution was sterilized and homogenized
by storing in an oven at 90 °C for about 1 h and
maintained at 37 °C. Air-pouches were formed by sub-
cutaneous (s.c.) injection of 1 ml of air for 3 days. On
the third day, after initial injection of air, 1 ml of
carrageenan solution was injected into the air-pouch to
induce inflammation. Compounds were administered
orally 1 h before injection of the carrageenan into the
pouch. Four h later, mice were killed by ether exposure
and pouches washed thoroughly with 3 ml of phosphate
buffer solution (PBS) containing 50 u/ml heparin.
Lavage fluids were centrifugated at 2000 rpm for 15
min at 4 °C and pellet was resuspended in 1 ml of
PBS–heparin. The total number of polymorphonuclear
leukocytes (PMNL) infiltration was measured using a
Coulter Counter (Model S-Plus VI. Coulter Electronics,
Inc. Hielah, Florida).
The synthesis of thiosemicarbazides, 1,3,4-oxadia-
zoles, 1,3,4-thiadiazoles and 1,2,4-triazoles are outlined
in Scheme 1. 2-Naphthyloxyacetyl-4-substituted thio-
semicarbazides 1a–d were prepared by the condensa-
tion of 1-naphthyloxyacetylhydrazine with alkyl or aryl
isothiocyanates in yields varying from 45.8 to 75.4%.
Cyclization of 1a–d using iodine and alkali gave the
desired products 2-(2-naphthyloxymethyl)-5-substitu-
tedamino-1,3,4-oxadiazoles 2a–d in 24.0–60.6% yields.
2 - (2 - Naphthyloxymethyl) - 5 - substitutedamino - 1,3,4-
thiadiazoles 3a–d were obtained in 15.2–60.4% yields
by cyclization of 1a–d by heating with methanesulfonic
acid in toluene. Ring closure of acylthiosemicarbazides
in the alkaline medium is a well known method for the
synthesis of 1,2,4-triazoles. 5-(2-Naphthyloxymethyl)-4-
substituted-1,2,4-triazole-3-thiones 4a–d were obtained
in 19.8–43.8% yields from the respective 1a–d with this
method (Scheme 1).
The structure of the compounds was elucidated by
1
UV, IR, H NMR and microanalyses. Crystallization
2.2.2. Hystopathological examination
solvents, melting points, yields%, spectral data and
microanalyses of the compounds are given in Table 1.
All spectral data were in accordance with the assumed
structures. In UV spectra all compounds have two
strong absorption bands at 218.8–228.8 and 254.5–
272.6 nm. The IR spectra of the acylthiosemicarbazide
derivatives 1a–d have CꢀO stretching bands at 1703–
1677 cm−1. In 1,3,4-oxadiazole, 1,3,4-thiadiazole and
1,2,4-triazole-3-thione derivatives, the disappearance of
CꢀO stretching bands of the acylthiosemicarbazides
and detecting of strong CꢀN stretching band at 1666–
1598 cm−1 is an evidence for ring closure. The oxadia-
zoles 2a–d and thiadiazoles 3a–d showed NꢁH
Mice were sacrified 4 h after the paw edema experi-
ments and their liver, stomachs and kidneys were re-
moved and put into 10% formalin solution. The
sections taken from these specimens were stained with
stretching bands at 3362–3236 and 3467–3106 cm−1
,
respectively. 1,2,4-Triazole-5-thiones 4a–d may exist in
thiole and thione forms [15]. The IR spectra of 4a–d
showed NꢁH bands in the region of 3106–3030 cm−1
and CꢀS absorbtion bands at 1258–1249 cm−1 instead
1
of SꢁH bands at around 2600–2550 cm−1. In the H
NMR spectra, all protons were seen accordingly to the
expected chemical shift and integral values. Methylenic
and aromatic protons of 2-naphthyloxymethyl groups
were seen at 4.65–5.80 and 6.80–8.75 ppm. The NꢁH
Scheme 1. Synthesis of compounds.

104
E. Palaska et al. / Il Farmaco 57 (2002) 101–107
Table 1
Crystallization solvents, melting points, % yields, formula and microanalyses of the synthesized compounds
Comp.
R
Cryst. Solvent a
Melting point (°C)
Yield (%)
Formula (molecular weight)
Analysis
1a[18]
1b
1c[18]
1d[18]
2a
2b
2c
2d[19]
3a
3b
CH3
Dioxane–Water
Dioxane–Water
Dioxane–Water
Dioxane–Water
Ethanol
Acetone
EtOAc–Ethanol
Ethanol
194–195
148
157
46
54
75
60
54
50
61
24
25
31
15
61
35
42
44
20
C14H15N3O2S (289.36)
C15H17N3O2S (303.39)
C16H17N3O2S (315.40)
C19H17N3O2S (351.43)
C14H13N3O2 (255.28)
C15H15N3O2 (269.31)
C16H15N3O2 (281.32)
C19H15N3O2 (317.34)
C14H13N3OS (271.34)
C15H15N3OS (285.37)
C16H15N3OS (297.38)
C19H15N3OS (333.41)
C14H13N3OS (271.34)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
(C,H,N)
C2H5
C3H5
C6H5
CH3
C2H5
C3H5
C6H5
CH3
C2H5
C3H5
C6H5
CH3
C2H5
C3H5
C6H5
149
139–141
140–141
134–135
141–142
188
EtOAc
EtOAc–Ethanol
EtOAc–Ethanol
EtOAc
Ethanol
Ethanol
161
166
3c
3d[19]
4a[18]
4b
4c[18]
4d[18,20]
209–210
184
215–216
135–136
156–158
C15H15N3OS (285.37)
C16H15N3OS (297.38)
C19H15N3OS (333.41)
Ethanol
EtOAc
a EtOAc, ethylacetate.
Recent studies have clearly shown that, the anti-in-
flammatory effect of carragenan causes a massive influx
of predominantly neutrophilic leucocytes (predomi-
nantly PMNL) from blood circulation into the cavity.
Besides, leucocyte migration is induced locally in the
inflammatory process and leucocytes also intensify in-
flammation by releasing several inflammatory media-
tors. The method is presented for measuring the edema
induced by injection of carrageenan into the pouches in
mice back. Air-pouches were highly reactive to inflam-
matory stimulus. The enhanced inflammatory reactions
in the sites correlated with formation of lining tissue,
the type of cells constituting the inflammatory sites, the
activation of cells, and/or the reactivity of newly
formed blood cells [12,13,16,17]. Mean leukocyte num-
bers per ml of exudate for each drug compared with
control values obtained from similar group of animals
receiving vehicle alone and the degree of inflammatory
response produced in the air-pouch cavity was assessed
by measuring total cell number of the exudate. 1,3,4-
Oxadiazole 2a–d (except 2b) and 1,2,4-triazole-3-thione
4a–d derivatives reduced total number of leukocytes of
the exudate. These findings indicate a good inhibition
of prostaglandin production. 5-Methylamino analogue
of 1,3,4-oxadiazole 2a (30.7590.75×105/cm3) and 4-
methyl substituted-1,2,4-triazole-3-thione 4a (32.439
3.15×105/cm3) were extremely potent in inhibiting
PMNL production compared with control and refer-
ence compounds (control: 122.5094.10, phenylbuta-
zone: 53.2791.71, indomethacin: 68.8091.80×
105/cm3) (Table 3).
protons of acylthiosemicarbazides 1a–d were observed
at 8.05–8.20, 9.15–9.70 and 10.00–10.35 ppm. NꢁH
protons of the compounds having oxadiazole 2a–d,
thiadiazole 3a–d and triazole-3-thione 4a–d ring were
seen at about 7.55–10.55, 7.90–10.45 and 13.90–15.30
ppm, respectively (Table 2). The results of microanaly-
ses were within90.4% theoretical values.
All the synthesized compounds and reference
NSAIDs were screened for anti-inflammatory activities
by carrageenan hind-paw edema test (CPE) and by the
air-pouch test. The obtained pharmacological results
(Table 3) indicate that some of the title compounds
possess notable anti-inflammatory properties. Accord-
ing to the results of the in vivo experiments, it is
difficult to extract a definite structure–activity relation-
ship between the compounds and anti-inflammatory
properties. In CPE assay, 1,3,4-oxadiazole derivatives
2a, 2c and 2d showed notable anti-inflammatory ac-
tivity. Most effective compound was 2-(2-naph-
thyloxymethyl)-5-methylamino-1,3,4-oxadiazole
2a
(67.3299.26% inh.). All 1,3,4-thiazole derivatives 3a–d
showed weak anti-inflammatory activity whereas 5-(2-
naphthyloxymethyl)-4-methyl-1,2,4-triazole-3-thione 4a
and 5-(2-naphthyloxymethyl)-4-ethyl-1,2,4-triazole-3-
thione 4b showed promising activity with the 60.629
8.55 and 51.4193.98% CPE inhibition, respectively.
Additionally, allyl 4c (45.1899.89%) and phenyl
analogs 4d (42.5093.82% inh.) significantly inhibited
edema when compared with phenylbutazone (53.279
1.83% inh.). For the compounds having 1,3,4-oxadia-
zole, 1,3,4-thiadizole and 1,2,4-triazole-3-thione ring,
methyl substituted derivatives showed equal or higher
anti-inflammatory activity compared with their allyl
and phenyl analogs.
Ulcerogenic potential of anti-inflammatory com-
pounds can be demonstrated in animal models using
positive (naproxen, indomethacin) and negative
(phenylbutazone) controls. On microscopic examina-

E. Palaska et al. / Il Farmaco 57 (2002) 101–107
105
tion, lesions seen in stomach, kidney and liver tissues
are graded according to their severity. The grade of the
scale is designed as (+) for mild, (+ +) for moderate,
and (+ + +) for severe changes. Stomachs of the mice
are thoroughly sectioned and both corpus and antrum
are evaluated. Surface epithelium and the lamina pro-
pria of the gastric mucosa are all examined. Acute
gastritis may exist in an earlier or milder nonerosive
Table 2
UV, IR and 1H NMR spectral data of the compounds
ethanol
max
Comp.
UV
(log m)
IR (KBr) w (/cm)
1H NMR (DMSO-d6) l ppm (J in Hz)
1a
228.0 (4.78); 259.8 3336, 3197 (N-H), 1713 (CꢀO), 1216 (CꢀS), 1057
(4.16) (ArꢁOꢁCH2)
2.85 (3H; d; -CH3), 4.65 (2H; s; ArꢁOꢁCH2ꢁ), 7.10–8.00
(7H; m; aromatic prot.), 8.15 (1H; d; CSꢁNHꢁCH3),
9.30 (1H; s; COꢁNHꢁNHꢁCS), 10.15 (1H; s;
COꢁNHꢁNHꢁCS)
1b
1c
228.0 (4.58), 260.6 3506, 3364 (NꢁH), 1677 (CꢀO), 1217 (CꢀS), 1066
(4.12) (ArꢁOꢁCH2)
1.10 (3H; t; CH2ꢁCH3), 3.40–3.60 (2H; q; CH2ꢁCH3),
4.70 (2H; s; ArꢁOꢁCH2ꢁ), 7.05–7.95 (7H; m; aromatic
prot.), 8.05 (1H; d; CSꢁNHꢁCH3), 9.15 (1H; s;
COꢁNHꢁNHꢁCS), 10.00 (1H; s; COꢁNHꢁNHꢁCS)
4.10 (2H; m; ꢁCH2ꢁCH=CH2), 4.75 (2H; s;
ArꢁOꢁCH2ꢁ), 5.05 (1H; dd; ꢁCH2ꢁCH=CH2), 5.20
(1H; dd; ꢁCH2ꢁCH=CH2), 5.60–6.10 (1H; m;
ꢁCH2ꢁCH=CH2), 7.15–7.95 (7H; m; aromatic prot.),
8.15 (1H; s; CSꢁNHꢁCH2), 9.35 (1H; s;
227.2 (4.74), 259.8 3523, 3186 (NꢁH), 1678 (CꢀO), 1215 (CꢀS),
(4.17) 1064(ArꢁOꢁCH2)
COꢁNHꢁNHCS), 10.15 (1H; s; CONHNHꢁCS)
4.75 (2H; s; ArꢁOꢁCH6 2ꢁ), 7.05–7.95 (12H; m; aromatic
prot.), 8.20 (1H; d; CSꢁNHꢁCH3), 9.70 (1H; s;
COꢁNHꢁNHꢁCS), 10.35 (1H; s; COꢁNHꢁNHꢁCS)
2.85 (3H; d; ꢁCH3), 5.15 (2H; s; ArꢁOꢁCH2ꢁ), 7.10–7.95
(7H; m; aromatic prot.), 7.55 (1H; s; NHꢁCH3)
1d
227.4 (4.82), 268.2 3370, 3328 (NꢁH), 1703 (CꢀO), 1257 (CꢀS), 1051
(4.13)
(ArꢁOꢁCH2)
2a
2b
220.4(5.15),
264.0(4.13)
3236 (NꢁH), 1666 (CꢀN), 1025 (CꢁOꢁC ring)
219.8 (5.09), 265.0 3236 (NꢁH), 1651, 1629 (CꢀN) 1029 (CꢁOꢁC ring) 1.10 (3H; t; ꢁCH2ꢁCH3), 3.15(2H; m; ꢁCH2ꢁCH3), 5.30
(4.19)
(2H; s; ArꢁOꢁCH2ꢁ), 7.10–7.95 (7H; m; aromatic prot.),
7.70 (1H; s; NHꢁC2H5)
3.65–3.95 (2H; m; ꢁCH6 2ꢁCH=CH2), 5.05 (1H; dd;
ꢁCH2ꢁCH=CH2 HA), 5.15 (1H; dd; ꢁCH2ꢁCH=CH2;
HB), 5.25 (2H; s; ArꢁOꢁCH2ꢁ), 5.6–5.95 (1H; m;
ꢁCH2ꢁCH=CH2), 7.10–7.95 (7H; m; aromatic prot.),
7.90 (1H; s; NHꢁCH2)
2c
219.8 (5.14), 264.0 3362 (NꢁH), 1628, 1600 (CꢀN), 1039 (CꢁOꢁC
(4.12)
ring)
2d
3a
3b
220.4 (5.14), 263.8 3253 (NꢁH), 1628,1600 (CꢀN), 1039 (CꢁOꢁC ring) 5.35 (2H; s; ArꢁOꢁCH2ꢁ), 6.80–8.10 (12H; m; aromatic
(4.31)
prot.) 10.55 (1H; s; NHꢁC6H5)
2.83 (3H; d; ꢁCH3), 4.75 (2H; s; ArꢁOꢁCH2ꢁ), 7.05–7.85
(7H; m; aromatic prot.), 7.90 (1H; s; NHꢁCH3)
228.0 (5.12), 266.5 3467 (NꢁH), 1601, 1629 (CꢀN)
(4.11)
227.8 (4.90), 267.5 3182 (NꢁH), 1626, 1599 (CꢀN), 1057 (ArꢁOꢁCH2) 1.15 (3H; t; ꢁCH2ꢁCH3), 3.35 (2H; m; ꢁCH2ꢁCH3), 5.45
(4.24)
(2H; s; ArꢁOꢁCH2ꢁ), 7.05–7.95 (7H; m; aromatic prot.),
8.05 (1H; s; NHꢁC2H5)
3c
226.8 (4.74), 266.0 3319, 3241 (NꢁH), 1645 (CꢀN)
(4.12)
4.15 (2H; t; ꢁCH6 2ꢁCH=CH2), 4.95 (1H; dd;
ꢁCH2ꢁCH=CH2; HA), 5.15 (1H; dd; ꢁCH2ꢁCH=CH2;
HB), 4.75 (2H; s; ArꢁOꢁCH2ꢁ), 5.55–6.00 (1H; m;
ꢁCH2ꢁCH=CH2), 6.95ꢁ7.95 (7H; m; aromatic prot.),
8.15 (1H; t; NHꢁCH2)
3d
4a
227.6 (4.77), 272.6 3436 (NꢁH), 1614, 1598 (CꢀN)
5.50 (2H; s; ArꢁOꢁCH2ꢁ), 6.95–8.15 (12H; m; aromatic
prot.), 10.45 (1H; s; NHꢁC6H5)
3.75 (3H; d; NꢁCH3), 5.80 (2H; s; ArꢁOꢁCH2ꢁ),
7.80–8.75 (7H; m; aromatic prot.), 15.30 (1H; bs;
NHꢁCH3)
(4.18)
227.0 (4.79), 254.5 3106 (NꢁH), 1626 (CꢀN), 254 (CꢀS)
(4.28)
4b
4c
218.0 (4.53), 259.5 3054 (NꢁH), 1633 (CꢀN), 1258 (CꢀS), 1048
1.30 (3H; t; NꢁCH2ꢁCH3), 4.15 (2H; q; ꢁCH2ꢁCH3),
5.40 (2H; s; ArꢁOꢁCH2ꢁ), 7.05–8.25 (7H; m; aromatic
prot.), 13.93 (1H; s; NHꢁCH2)
4.75 (2H; d; NꢁCH2ꢁCH=CH2), 4.95 (1H; dd;
ꢁCH2ꢁCH=CH2), 5.10 (1H; dd; ꢁCH2ꢁCH=CH2), 5.25
(4.27)
(ArꢁOꢁCH2)
227.0 (4.82) 260.0 3055 (NꢁH), 1632, 1601 (CꢀN), 1249 (CꢀS)
(4.34)
(2H; s; ArꢁOꢁCH6 2ꢁ), 5.70–6.25 (1H; m;
ꢁCH2ꢁCH=CH2), 7.10–7.95 (7H; m; aromatic prot.)
13.95 (1H; bs; NHꢁCH3)
4.95 (2H; s; ArꢁOꢁCH2ꢁ), 6.95–7.90 (12H; m; aromatic
prot.), 13.90 (1H; bs; NHꢁC6H5)
4d
227.8 (4.74), 261.0 3030 (NꢁH), 1630, 1600 (CꢀN), 1258 (CꢀS)
(4.41)
s, Singlet; bs, broad singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quarted; m, multiplet.

106
E. Palaska et al. / Il Farmaco 57 (2002) 101–107
Table 3
form with merely mucosal congestion and edema, and
Anti-inflammatory activities of the compounds
histologic evidence of inflammation. These earlier
changes are known to be transient and completely
reversible within few days, but the development of
erosions and hemorrhages is more serious and related
to an increased risk of major upper gastrointestinal
bleeding. In our study, the animals were sacrified 4 h
later than the ingestion of the drugs. None of the
sections displayed the morphology of the ulceration but
non-erosive form of acute gastritis was observed in
various degrees of severity with compounds 1a–d and
4b (Table 4). On microscopic examination of the kidney
sections, the morphologic findings such as pronounced
edema and interstitial mononuclear cell infiltration,
were characteristic for tubulointerstitial nephritis. The
presence of some scattered eosinophil leucocytes also
provided evidence of the drug effect. Focal areas dis-
playing variable but generally mild degree of tubular
regeneration were also present. Acute pyelonephritis
should be considered in the differential diagnosis of the
tubulointerstitial nephritis, but none of our cases
showed interstitial suppurative (inflammation with
polymorphonuclear leucocytes) inflammation with mi-
croabscesses. Therefore, the presented renal morpho-
logic findings were accepted as the reflection of the
effects of the drugs. Liver tissues were also examined
thoroughly and the integrity of the basic structure,
degree of lobular and portal inflammation, and the
presence or absence of necrosis, fatty change, or
cholestasis were evaluated. Many NSAIDs, like
Comp. (100
mg/kg)
CPE (%
PMNL (×105/cm3)
(n=6, after 4 h) b
inhibition9SE) a
Control
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
122.5094.10
n.t.
n.a.
n.a.
15.4591.67
n.a.
n.t.
104.7690.80
n.t.
67.3299.26 c
28.6995.06 c
40.4295.42 c
40.4295.42 c
20.2094.14
35.5191.21 d
22.4493.08 d
n.a.
30.7590.75
91.0091.00
60.7590.75
63.3090.33
98.7592.88
69.4290.96
95.2591.56
n.t.
32.4393.15
56.2590.38
50.0091.32
46.3090.68
50.2091.30
60.6298.55 c
51.4193.98 c
45.1899.89 c
42.5093.82 c
67.5091.70 c
4d
Naproxen (30
mg/kg)
Phenylbutazone
(100 mg/kg)
Indomethacin (10
mg/kg)
53.2791.83 c
32.1091.38 c
53.2791.71
68.8091.80
PMNL, polymorphonuclear leukocytes; n.a.: no activity; n.t.: not
tested.
a Results are expressed as their mean values (n=6).
b Mean9SEM (n=6).
c PB0.01.
d PB0.05.
Table 4
Histopathological examination results of the compounds
Comp. (100 mg/kg)
Kidney
Stomach
Gastritis
Liver
Edema Infected cell (MNL)
Fatty change
Acute hepatitis/spotty necrosis
Cholestasis
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
−
−
−
−
−
−
−
−
−
−
−
−
++
−
++
−
−
++
−
++
−
+
+
−
−
+
++
++
++
−
−
−
−
−
+
++
+
+
−
+
+
++
+
+
+
−
−
++
+
+
−
−
−
+
−
++
−
−
−
−
−
−
+
+
−
−
−
++
++
++
−
−
−
−
−
−
−
−
−
++
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
Indomethacin (10
mg/kg)
Naproxen (30 mg/kg)
++
+
+
+
+++
+
++
+
++
−
−
+++
n.t., not tested; −, no; +, mild; ++, moderate; +++, severe side effects.

E. Palaska et al. / Il Farmaco 57 (2002) 101–107
107
dan, K.M. Imre, M.E. Lesch, M.D. Mullican, G.C.N. Okonkwo,
M.C. Conroy, The pharmacologic effects of 5-[3,5-bis(1,1-
dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)-thione
choline salt (CI-986), a novel inhibitor of arachidonic acid
metabolism in models of inflammation, Prostaglandins 47 (1994)
17–30.
phenylbutazone and naproxen, are known to cause
acute or chronic hepatitis, confluent or spotty necrosis,
cholestatic hepatitis and/or fatty change. This shows
the importance of examining the liver tissues to better
assess the safety of NSAIDs. In some cases like 1c, 1d,
2a, 2b, and 4a (moderate, ++) macro and microvesicu-
lar fatty change and several scattered mild (+) focal
spotty necrosis were observed. Multiple foci of spotty
necrosis (moderate, + +) was seen with compounds 2a
and 3c. Cholestatic hepatitis was observed with the
compounds 1c, 2a, 3d, and 4a (Table 4).
In conclusion, a new series of compounds showing
anti-inflammatory properties was synthesized. Among
them in particular compounds 2-(2-naphthyloxy-
methyl)-5-methylamino-1,3,4-oxadiazole 2a and 5-(2-
naphthyloxymethyl)-4-methyl-1,2,4-triazole-3-thione 4a
were found to have a superior anti-inflammatory profile
with low gastric ulceration incidence with similar toxic
profiles of reference NSAIDs in the liver.
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Acknowledgements
This work is supported by Hacettepe University Re-
search Fund (Project: 98.01.301.002). Procedures in-
volving animals and their care were conducted in
conformity with international laws and policies and the
studies on animals accepted by Hacettepe University
Ethic Council (99/15).
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