Design and synthesis of some thiazolotriazolyl esters as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1007/s00044-010-9508-x

In order to develop potent analgesic/antiinflammatory compounds with reduced ulcerogenic risk, a series of thiazolotriazolyl-carboxylic and acetic acid esters were synthesized and characterized by spectral and elementary analysis. All synthesized compounds were screened for in vivo anti-inflammatory activities in mice by carregeenan-induced paw edema model. The compounds showing 20% reduction in paw edema were also evaluated for their analgesic activities by acetic acid-induced writhing test and the gastric ulceration risk by determining the lipid peroxidation level in stomachs. Among the compounds tested, compounds 1, 4, 6, 7, 8, 1a, 2a, 3a, 4a, 7a, 2b, and 8b showed moderate-to-good anti-inflammatory activity at various doses in any of the measurement intervals. Compounds 7a, 2b, and 8b were the most actives of the series in analgesic activity test. Moreover, compounds 1, 4, and 8 were found to be safe in stomach in respect of free radical production. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9508-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:192–201
DOI 10.1007/s00044-010-9508-x
ORIGINAL RESEARCH
Design and synthesis of some thiazolotriazolyl esters
as anti-inflammatory and analgesic agents
•
Birsen Tozkoparan S. Peri Aytac¸
•
•
¨
S¸ule Gu¨rsoy Goknur Aktay
Received: 28 June 2010 / Accepted: 4 November 2010 / Published online: 12 December 2010
Ó Springer Science+Business Media, LLC 2010
Abstract In order to develop potent analgesic/anti-
inflammatory compounds with reduced ulcerogenic risk, a
series of thiazolotriazolyl-carboxylic and acetic acid esters
were synthesized and characterized by spectral and ele-
mentary analysis. All synthesized compounds were screened
for in vivo anti-inflammatory activities in mice by carre-
geenan-induced paw edema model. The compounds show-
ing 20% reduction in paw edema were also evaluated for
their analgesic activities by acetic acid-induced writhing test
and the gastric ulceration risk by determining the lipid per-
oxidation level in stomachs. Among the compounds tested,
compounds 1, 4, 6, 7, 8, 1a, 2a, 3a, 4a, 7a, 2b, and 8b showed
moderate-to-good anti-inflammatory activity at various
doses in any of the measurement intervals. Compounds 7a,
2b, and 8b were the most actives of the series in analgesic
activity test. Moreover, compounds 1, 4, and 8 were found to
be safe in stomach in respect of free radical production.
pharmacological activities of various condensed heterocy-
cles derived from symmetrical triazoles. The compounds
bearing both a symmetrical triazole and 1,3-thiazole moie-
ties represent an interesting class of compounds possessing a
wide spectrum of biological activities such as anticancer,
anti-inflammatory/analgesic, and antimicrobial properties
(Barbuceanu et al., 2009; El-Emam and Ibrahim, 1991;
Karthikeyan et al., 2008, 2009; Lesyk et al., 2007; Pignatello
et al., 1991).
Thiazolo[3,2-b]-1,2,4-triazole fused ring system (I and
II) having various substituents has become the subject of
our recent attention as new analgesic/anti-inflammatory
agent. We have prepared many compounds and screened
them for their anti-inflammatory/analgesic properties in
vivo. A considerable number of the prepared compounds
have anti-inflammatory/analgesic activities comparable to
or higher than the reference compounds besides lower
ulcerogenic risks in the stomach (Dogdas et al., 2007;
Tozkoparan et al., 1999, 2000, 2001, 2004, 2005). These
results have further promoted our interest to design and
synthesize some new products derived from thiazolo[3,2-
b]-1,2,4-triazoles and evaluate their anti-inflammatory and
analgesic activities.
Keywords Thiazolotriazolyl-carboxylic acid esters ꢀ
Thiazolotriazolyl acetic acid esters ꢀ
Analgesic/antiinflammatory activity
Introduction
In recent decades, the literature has been enriched with
progressive findings about the synthesis and
O
OH
S
H
C
N N
N
C H
N N
N
N
S
R
R
B. Tozkoparan (&) ꢀ S. P. Aytac¸
R'
R'
Department of Pharmaceutical Chemistry, Hacettepe University,
Faculty of Pharmacy, 06100 Sıhhıye, Ankara, Turkey
e-mail: tbirsen@hacettepe.edu.tr
II
I
S¸. Gu¨rsoy ꢀ G. Aktay
Some thiazole-4(5)acetic acid derivatives are also known
Department of Pharmacology, Inonu University,
Faculty of Pharmacy, 44280 Malatya, Turkey
to possess anti-inflammatory activity (Brown et al., 1968,
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Med Chem Res (2012) 21:192–201
193
1974; Sharma and Sawhney, 1997). For example, fenclozic
acid (III, I.C.I. 54,450; Myalex) is a synthetic thiazol-4-yl-
acetic acid derivative, which has been reported to have
potent anti-inflammatory, analgesic, and antipyretic prop-
erties (Hepworth et al., 1969).
derivatives (1–4) of series were prepared by the reaction of
benzoyl chlorides with the thiosemicarbazide, while the 3-
arylalkyl-1,2,4-triazole-5-thiones (5–8) were obtained via
dicyclohexylcarbodiimide (DCC)-promoted amide forma-
tion reaction starting from a carboxylic acid to the method
reported earlier (Dogdas et al., 2007; Tozkoparan et al.,
1999, 2000).
Scheme 3 reports the reactions which led to thiazolo-
triazole derivatives. It has been reported that thiazolo[3,2-
b]-1,2,4-triazole derivatives could be obtained either by a
direct reaction between mercaptotriazoles and appropriate
halogeno-b-ketoesters (A route) or through alternative
pathway involving the formation of S-alkyl products,
which were then cyclized by an acid-catalyzed reaction
(POCl3, HCl, H2SO4) (B route) (Mazzone et al., 1981;
Moskowitz et al., 1980; Pignatello et al., 1991; Simiti
et al., 1991). In our study, title compounds, thiazolo[3,2-b]-
1,2,4-triazole derivatives, were obtained by refluxing
3-substituted-1,2,4-triazole-5-thiones with either ethyl
4-chloro-3-oxobutyrate (a) or ethyl 2-chloroacetoacetate
(b) in anhydrous ethanol in one-step.
CH2COOH
N
S
Cl
III
Besides, the literature survey revealed that derivatization
(esterification or amidation) of carboxylic acid function of
representative NSAIDs, resulted in an increased anti-
inflammatory activity with reduced ulcerogenic effect
(Galanakis et al., 2004; Kalgutkar et al., 2000, 2002, 2005;
Khanna et al., 2006; Zhong et al., 2009).
Based on these findings and as part of our continuous
efforts in this area, it was thought to be interesting to
combine thiazolo[3,2-b]-1,2,4-triazole condensed ring with
an ester side chain to elucidate the essential structure
requirements in relation to activity. On this basis, herein we
report the synthesis and preliminary pharmacological
evaluation of a series of novel esters of thiazolotriazolyl
carboxylic and acetic acids in this study.
Assignment of new compounds was based on their
elemental analysis and spectroscopic data. Characterization
data of all new compounds are summarized in monographs.
The IR spectra of the compounds 1a–8a and 1b–8b
showed only one C=O stretching bands at
1
1737–1691 cm-1 because of ring closure. In 400 MHz H
NMR spectrum of compounds 1a–8a, the CH (6) proton of
the thiazolotriazole ring resonated as
a singlet at
6.90–7.02 ppm, in agreement with data reported for anal-
1
ogous compounds (Pignatello et al., 1991). The H NMR
Results and discussion
spectra of compounds 1b–8b showed a particular singlet
due to the methyl group 1.28–1.42 ppm. The protons
belonging to the aromatic ring and the other aliphatic
groups were observed with the expected chemical shifts
and integral values. All compounds gave satisfactory ele-
mental analysis.
Chemistry
The synthesis of starting compounds, 3-aryl-/3-arylalkyl-
1,2,4-triazole-5-thiones was, accomplished by various
methods as outlined in Schemes 1 and 2. 3-Aryl substituted
Scheme 1 Synthesis of 3-aryl-
1,2,4-triazole-5-thiones (1–4)
S
1. KOH 10%
2. HCl
S
N N H
Ar-COCl
H2NNH-C-NH2
Ar-CONHNH-C-NH2
Ar
S
N
H
1-4
Scheme 2 Synthesis of
3-arylalkyl-1,2,4-triazole-
5-thiones (5–8)
O
O
S
DCC, 0 oC
Ar-(CH2)n-COO
N
+ H2NNH-C-NH2
Ar-(CH2)n-COOH
Reflux
+
HO N
O
O
O
S
1. KOH 10%
2. HCl
N N H
Ar-(CH2)n-C-NHNH-C-NH2
Ar(CH2)n
S
N
H
5-8
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Med Chem Res (2012) 21:192–201
CH2COOCH2CH3
N N
A
B
1a-8a
S
Cl-CH2COCH2COOCH2CH3 (a)
R
R
N
B
N NH
S-CH2COCH2COOCH2CH3
N
N NH
N NH
N
CH3
R
R
SH
S
N
H
N N
COOCH2CH3
1-8
COCH3
S
1b-8b
R
R
A
N
Cl-CH-COOCH2CH3 (b)
B
B
N NH
COCH3
S-CH
N
COOCH2CH3
Scheme 3 The reactions leading to thiazolo[3,2-b]-1,2,4-triazole derivatives
Pharmacological studies
2b) or decreased activity (2a, 3a, and 8b). These results may
suggest that the compounds 1, 4, 6, 7, 8, 1a, 4a, 7a, and 2b
reach their maximum effects at 100 mg/kg. There was a
noticeable anti-inflammatory activity at 50 mg/kg dose only
in compounds 1, 6, 7, 8a, and 8b. Moreover, the activity of
the compound 8b at 50 mg/kg dose was more prominent
than the ones seen with other doses.
Both starting compounds 1–8 and their corresponding con-
densed derivatives (1a–8a and 1b–8b) were subjected to
preliminary testing for anti-inflammatory and analgesic
activity as well as the ulcerogenic risk on acute administra-
tion. At first, the substances were screened for anti-inflam-
matory activity at 100 mg/kg, p.o. dose. The compounds
having more than 20% effectiveness in relieving symptoms,
even some of them are not significant statistically, were
considered both for further evaluation of anti-inflammatory
activity in two different dose levels (50 and 200 mg/kg)
and for screening their analgesic activities at the dose of
100 mg/kg. Besides, these compounds were also evaluated
for their gastric toxicity by determining the lipid peroxida-
tion level in stomachs. All pharmacological activity results
have been summarized in Table 1.
In case of anti-inflammatory activity, many of the
compounds carrying acetic acid ester at the 6th position of
the fused ring (1a, 2a, 3a, 4a, and 7a) exhibited some
degree of anti-inflammatory activity in any of the mea-
surement intervals when compared to their carboxylic acid
esters counterparts. It was observed that among the deriv-
atives with acetic acid ester (1a–8a) compound 3a, having
a 2,4-dichlorophenyl at the 2nd position of the fused ring
possess highest activity (42.8%). Among the compounds
carrying a carboxylic acid ester (1b–8b), compound 8b
having a (3,4,5-trimethoxyphenyl)ethyl group at the 2nd
position have attracted attention with almost equivalent
activity to indomethacin at 50 mg/kg dose.
Anti-inflammatory activity
The anti-inflammatory activity of derivatives was assessed
from their ability to inhibit the paw edema induced by
carrageenan in mice, and activity was expressed as ‘‘mean
increase in paw volume SEM,’’ in terms of mm and
percentage inhibition in paw volume by different doses of
the compounds (Kasahara et al., 1985). As seen in Table 1,
the compounds 1, 4, 6, 7, 8, 1a–4a, 7a, 2b, and 8b possessed
moderate-to-good anti-inflammatory activity at 100 mg/kg
dose/p.o. in any of the measurement intervals. Especially,
It is known that an edema produced by carrageenan is a
biphasic event and if the inhibition is more effective in the
first phase of carrageenan-induced edema, it means that the
reply is histamin and serotonin mediated. In addition, if the
inhibition is more effective in the second phase of carra-
geenan-induced edema, it means that the reply is prosta-
glandin mediated (Holsapple et al., 1980). According to
this literature information, it might be stated that our
compounds show their anti-inflammatory effect through the
PG-mediated mechanism (Table 1). In order to enlighten
the mechanism in completely, further studies made with
serotonin, histamine, and PG are also needed.
the compounds
6 and 8 having 3-[2-(4-methoxy-
phenyl)ethyl] and 3-[2-(3,4,5-trimethoxyphenyl)ethyl]
substituent, respectively, showed the most remarkable
activity at this dose. It was interesting to note that these two
compounds when condensed with the thiazole ring, the
activity was lost except 8b. However, the selected com-
pounds when administered increasing dose in a twofold
(200 mg/kg) showed no activity (1, 4, 6, 7, 8, 1a, 4a, 7a, and
Analgesic activity
The analgesic activity of the compounds was studied using
the acetic acid-induced writhing test in mice and expressed
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Med Chem Res (2012) 21:192–201
195
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Med Chem Res (2012) 21:192–201
as ‘‘mean increase in latency after drug administra-
tion SEM’’ relative to control and percentage inhibition
in writhing reflex (Koster et al., 1959). Although the
compounds 1, 6, 2a, 3a, and 4a showed some activity, it is
not significant statistically. Among the compounds, the
compounds 7a, 2b, and 8b attracted attention with higher
analgesic activity than aspirin with percentage inhibition
values 62.8, 89.7, and 85.9%, respectively, at 100 mg/kg
dose level.
Antioxidant activity, lipid peroxidation
It is well established that reactive oxygen species have a
decisive role in inflammatory conditions (Halliwell and
Gutteridge, 1999). It has also been reported that oxidative
stress is an important component of gastrointestinal ulcer-
ation (Bragt et al., 1980; Halliwell, 1991). The compounds
having antioxidant activity besides anti-inflammatory/
analgesic activity may offer a viable route safer anti-
inflammatory/analgesic agents. Therefore, all compounds
having more or less analgesic activity were also analyzed
for their antioxidant properties by determining lipid per-
oxidation level. The lipid peroxidation is measured as nmol
of TBARS/g wet weight of tissue. The obtained lipid per-
oxidation values revealed that the ulcerogenic effect of
only the compounds 1, 4, and 8 was appreciably less than
aspirin. The compound 8b, exhibited both noteworthy anti-
inflammatory and analgesic activities, was found relatively
safer for ulcerogenic risk than the other compounds.
Acute toxicity
None of the test compounds used in the pharmacologic
study produced lethal effect and did not induce any sig-
nificant behavioral modification at the employed doses
during observation period.
Conclusions
The research study reports the synthesis and anti-inflam-
matory/analgesic activity and ulcerogenic risk of new
triazolothiazolyl ester derivatives. Several derivatives have
been found to possess analgesic and anti-inflammatory
activities. Few derivatives have been evaluated for
remarkable anti-inflammatory activity devoid of ulcero-
genic risk. Although findings of our pharmacological tests
did not allow to delineate an exact relationship between the
structure of the synthesized thiazolotriazole derivatives and
their analgesic/anti-inflammatory activity, the results are
conclusive in showing that there is no noticeable increase
in analgesic and anti-inflammatory activity when 3-aryl-/5-
arylalkyl-1,2,4-triazole-5-thiones are condensed with the
thiazole ring.
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Med Chem Res (2012) 21:192–201
197
According to the results of the present study, among the
synthesized compounds, a carboxylic acid ester derivative
8b, having a (3,4,5-trimethoxyphenyl)ethyl group at the
2nd position of the fused ring, was found to have both
significant analgesic and consistent anti-inflammatory
activity with relatively reduced lipid peroxidation and
would deserve further attention to develop new leadings.
commercial product in ‘‘Science Finder.’’ Since there is no
information in literature for the preparation and spectral
characteristics, the compound has been included in our
research program and characterized by spectral data.
3-[2-(4-Methoxyphenyl)ethyl]-1,2,4-triazole-5-thione
(6)
1
Yield 63%. mp 211–212°C. H NMR (CDCl3-d): d 2.71
(2H, t, –CH2–), 2.81 (2H, t, –CH2–), 3.63 (3H, s, –OCH3),
6.66 (2H, d, arom.H), 6.95 (2H, d, arom.H), 12.73 (1H, s,
NH). IR mmax cm-1 (KBr): 3352 (N–H), 1598 (C=N), 1252
(C=S).
Experimental part
Chemistry
Melting points (mp) were detected with a Thomas Hoover
capillary melting point apparatus (Philadelphia, PA, USA)
and were uncorrected. Infrared (IR) spectra were recorded
on Perkin Elmer 1720X FT-IR spectrometer (Beaconsfield,
UK) using potassium bromide pellets, and the result was
3-[(3,4,5-Trimethoxyphenyl)methyl]-1,2,4-triazole-5-
thione (7)
1
Yield 70%. mp 235–237°C. H NMR (CDCl3-d): d 3.66
(2H, s, –CH2–), 3.73 (9H, s, 3OCH3), 6.46 (2H, s, arom.H),
12.99 (1H, bs, NH). IR mmax cm-1 (KBr): 3361 (N–H),
1590 (C=N), 1241 (C=S).
1
expressed in wave number (cm-1). H nuclear magnetic
resonance (1H NMR) spectra were taken on Varian Mer-
cury 400, 400 MHz High Performance Digital FT-NMR
instrument (Palo Alto, CA, USA) in CDCl3 using TMS as
internal standard. All chemical shifts were recorded as d
(ppm) values. Splitting patterns are as follows: s, singlet;
bs, broad singlet; d, doublet; t, triplet; m, multiplet. The
purity of the compounds was checked on silicagel-coated
3-[2-(3,4,5-Trimethoxyphenyl)ethyl]-1,2,4-triazole-
5-thione (8)
1
Yield 70%. mp 228–229°C. H NMR (CDCl3-d): d 2.47
(2H, t, –CH2–), 2.55 (2H, t, –CH2–), 3.38 (3H, s, –OCH3),
3.43 (6H, s, 2OCH3), 6.01 (2H, s, arom.H), 12.60 (1H, bs,
NH). IR mmax cm-1 (KBr): 3355 (N–H), 1590 (C=N), 1242
(C=S).
aliminium sheets (Merck, 1.005554, silicagel HF254–361
,
Type 60, 0.25 mm) by thin layer chromatography. The
elementary analysis of the new compounds were performed
with Leco CHNS 932 analyzer at Ankara University,
Faculty of Pharmacy Central II Laboratory. Elementary
analysis for C, H, N, and S were within 0.4% of theo-
retical values. All chemicals were from Aldrich Chemical
Co. (Steinheim, Germany).
Thiazolo[3,2-b]-1,2,4-triazoles, 2,6-disubstituted
and 2,5,6-trisubstituted (1a–8a and 1b–8b)
A solution of 0.01 mol 5-mercapto-1,2,4-triazole and
0.01 mol ethyl 4-chloro-3-oxobutyrate or 2-chloroace-
toacetate in 8 ml of absolute etanol was refluxed over a
period of 4 days. After cooling, the reaction mixture was
filtered, and the resulting solid was recrystallized from
appropriate solvents. Compounds 1a, 2-phenyl-thiazol-
o[3,2-b]-1,2,4-triazole-6-acetic acid ethyl ester, and 2a,
2-(4-chlorophenyl)-thiazolo[3,2-b]-1,2,4-triazole-6-acetic
acid ethyl ester, 1b 6-methyl-2-phenyl-thiazolo[3,2-b]-1,2,4-
triazole-5-carboxylic acid ethyl ester, and 2b, 2-(4-chloro-
phenyl)-6-methyl-thiazolo[3,2-b]-1,2,4-triazole-5-carboxylic
acid ethyl ester, and 4b, 2-(4-methoxyphenyl)-6-methyl-
thiazolo[3,2-b]-1,2,4-triazole-5-carboxylic acid ethyl ester,
have been reported previously (Moskowitz et al., 1980;
Simiti et al., 1991; Zaharia et al., 2000).
Synthesis of 3-aryl-1,2,4-triazole-5-thiones (1–4)
The synthesis of the compounds 3-phenyl-5-mercapto-1,2,
4-triazole (1), 3-(4-chlorophenyl)-5-mercapto-1,2,4-triazole
(2), 3-(3,5-dichlorophenyl)-5-mercapto-1,2,4-triazole (3),
and 3-(4-methoxyphenyl)-5-mercapto-1,2,4-triazole (4) were
performed according to the reported literature procedure
(Tozkoparan et al., 1999). The compounds had already been
reported previously (Diaz et al., 2006; Malbec et al., 1984).
Synthesis of 3-arylalkyl-1,2,4-triazole-5-thiones (5–8)
The compounds 5–8 were prepared to the method reported
˘
earlier (Tozkoparan et al., 2000; Dogdas¸ et al., 2007).
Since we are not able to reach literature belonging
Zaharia et al. (2000) in where compound 4b is reported, the
compound has been characterized by spectral and ele-
mental analysis.
Compound 5, 3-[(4-methoxyphenyl)methyl]-1,2,4-triazole-
5-thione, has been reported before (Ovsepyan et al., 1977).
Furthermore, compound 6 [1097795-00-0] is seemed as
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Med Chem Res (2012) 21:192–201
–fCH=). IR mmax cm-1 (KBr): 1720 (C=O), 1593 (C=N).
Anal. Calcd. for C18H21N3O5S: C, 55.23; H, 5.41; N,
10.73; S, 8.19. Found: C, 54.90; H: 5.35; N, 10.77; S, 8.29.
2-(3,5-Dichlorophenyl)-thiazolo[3,2-b]-1,2,4-triazole-
6-acetic acid ethyl ester (3a)
1
Yield 64%. mp 144–145°C. H NMR (CDCl3-d): d 1.32
(3H, t, –CH3), 4.01 (2H, s, –CH2C=O), 4.27 (2H, q, –CH2–
), 7.02 (1H, s, –CH=), 7.40 (1H, s, arom.H-4), 8.06 (2H, s,
arom.H-2 and H-6). IR mmax cm-1 (KBr): 1715 (C=O),
1594 (C=N). Anal. Calcd. for C14H11Cl2N3O2S: C, 47.20;
H, 5.54; N, 12.17; S, 9.28. Found: C, 46.66; H, 5.19; N,
12.07; S, 9.28.
2-[2-(3,4,5-Trimethoxyphenyl)ethyl]-thiazolo[3,2-b]-1,2,4-
triazole-6-acetic acid ethyl ester (8a)
1
Yield 45%. mp 99–100°C. H NMR (CDCl3-d): d 1.29
(3H, t, –CH3), 3.07–3.16 (4H, m, –CH2CH2–), 3.83 (9H, s,
3OCH3), 3.94 (2H, s, –CH2C=O), 4.24 (2H, q, –CH2–),
6.49 (2H, s, arom.H-2 and H-6), 6.91 (1H, s, –CH=). IR
mmax cm-1 (KBr): 1720 (C=O), 1591 (C=N). Anal. Calcd.
for C19H23N3O5S: C, 56.28; H, 5.72; N, 10.36; S, 7.91.
Found: C, 56.14; H, 5.50; N, 10.52; S, 8.05.
2-(4-Methoxyphenyl)-thiazolo[3,2-b]-1,2,4-triazole-
6-acetic acid ethyl ester (4a)
1
Yield 81%. mp 115–116°C. H NMR (400 MHz, CDCl3-
d): d 1.31 (3H, t, –CH3), 3.87 (3H, s, –OCH3), 4.00 (2H, s,
–CH2C=O), 4.26 (2H, q, –CH2–), 6.92 (1H, s, –CH=), 6.98
(2H, d, arom.H-3 and H-5), 8.09 (2H, d, arom.H-2 and
H-6). IR mmax cm-1 (KBr): 1732 (C=O), 1616 (C=N). Anal.
Calcd. for C15H15N3O3S: C, 53.72; H, 5.11; N, 12.53; S,
9.56. Found: C, 54.08; H, 4.77; N, 12.66; S, 9.66.
2-(3,5-Dichlorophenyl)-6-methyl-thiazolo[3,2-b]-1,2,4-
triazole-5-carboxylic acid ethyl ester (3b)
1
Yield 53%. mp 213–215°C. H NMR (CDCl3-d): d 1.42
(3H, t, –CH3), 2.94 (3H, s, –CH3), 4.43 (2H, q, –CH2–),
7.43 (1H, t, arom.H-4), 8.09 (2H, d, arom.H-2 and H-6). IR
mmax cm-1 (KBr): 1711 (C=O), 1584 (C=N). Anal. Calcd.
for C14H11Cl2N3O2S: C, 47.20; H, 5.54; N, 12.17; S, 9.28.
Found: C, 46.23; H, 5.19; N, 11.63; S, 8.97.
2-[(4-Methoxyphenyl)methyl]-thiazolo[3,2-b]-1,2,4-
triazole-6-acetic acid ethyl ester (5a)
Yield 62%. mp 59–60°C. 1H NMR (CDCl3-d): d 1.28 (3H,
t, –CH3), 3.77 (3H, s, OCH3), 3.94 (2H, s, –CH2–), 4.11
(2H, s, –CH2C=O), 4.23 (2H, q, –CH2–), 6.85 (2H, d,
arom.H-3 and H-5), 6.88 (1H, s, –CH=), 7.29 (2H, d,
arom.H-2 and H-6). IR mmax cm-1 (KBr): 1719 (C=O),
1611 (C=N). Anal. Calcd. for C16H17N3O3S: C, 57.99; H,
5.17; N, 12.68; S, 9.67. Found: C, 57.90; H, 5.16; N, 12.74;
S, 9.80.
2-(4-Methoxyphenyl)-6-methyl-thiazolo[3,2-b]-1,2,4-
triazole-5-carboxylic acid ethyl ester (4b)
1
Yield 80%. mp 147–148°C. H NMR (CDCl3-d): d 1.42
(3H, t, –CH3), 2.93 (3H, s, –CH3), 3.88 (3H, s, –OCH3),
4.41 (2H, q, –CH2–), 7.00 (2H, d, arom.H-3 and H-5), 8.13
(2H, d, arom.H-2 and H-6). IR mmax cm-1 (KBr): 1704
(C=O), 1612 (C=N). Anal. Calcd. for C15H15N3O3S: C,
53.72; H, 5.11; N, 12.53; S, 9.56. Found: C, 56.45; H, 4.63;
N, 13.18; S, 10.16.
2-[2-(4-Methoxyphenyl)ethyl]-thiazolo[3,2-b]-1,2,
4-triazole-6-acetic acid ethyl ester (6a)
2-[(4-Methoxyphenyl)methyl]-6-methyl-thiazolo[3,2-b]-1,2,4-
triazole-5-carboxylic acid ethyl ester (5b)
Yield 53%. mp 61–62°C. 1H NMR (CDCl3-d): d 1.31 (3H,
t, –CH3), 3.08–3.12 (4H, m, –CH2CH2–), 3.79 (3H, s,
OCH3), 4.09 (2H, s, –CH2C=O), 4.21 (2H, q, –CH2–), 6.85
(2H, d, arom.H-3 and H-5), 6.90 (1H, s, –CH=), 7.31 (2H,
d, arom.H-2 and H-6). IR mmax cm-1 (KBr): 1720 (C=O),
1612 (C=N). Anal. Calcd. for C17H19N3O3S: C, 59.11; H,
5.54; N, 12.17; S, 9.28. Found: C, 59.46; H, 5.91; N, 12.30;
S, 9.19.
1
Yield 77%. mp 135–136°C. H NMR (CDCl3-d): d 1.28
(3H, t, –CH3), 2.87 (3H, s, CH3), 3.78 (3H, s, OCH3), 4.11
(2H, s, –CH2–), 4.38 (2H, q, –CH2–), 6.86 (2H, d, arom.H-
3 and H-5), 7.30 (2H, d, arom.H-2 and H-6). IR mmax cm-1
(KBr): 1695 (C=O), 1606 (C=N). Anal. Calcd. for
C16H17N3O3S: C, 57.99; H, 5.17; N, 12.68; S,9.67. Found:
C, 57.88; H, 5.19; N, 12.71; S, 9.78.
2-[(3,4,5-Trimethoxyphenyl)methyl]-thiazolo[3,2-b]-
1,2,4-triazole-6-acetic acid ethyl ester (7a)
2-[2-(4-Methoxyphenyl)ethyl]-6-methyl-thiazolo[3,2-
b]-1,2,4-triazole-5-carboxylic acid ethyl ester (6b)
Yield 73%. mp 81–82°C. 1H NMR (CDCl3-d): d 1.29 (3H,
t, –CH3), 3.80 (3H, s, OCH3), 3.84 (6H, s, –OCH3), 3.96
(2H, s, –CH2–), 4.11 (2H, s, –CH2C=O), 4.24 (2H, q,
–CH2–), 6.61 (2H, s, arom.H-2 and H-6), 6.91 (1H, s,
Yield 69%. mp 91–92°C. 1H NMR (CDCl3-d): d 1.40 (3H,
t, –CH3), 2.87 (3H, s, –CH3), 3.10–3.14 (4H, m,
–CH2CH2–), 3.79 (3H, s, –OCH3), 4.39 (2H, s, –CH2–),
123

Med Chem Res (2012) 21:192–201
199
6.84 (2H, d, arom.H-3 and H-5), 7.18 (2H, d, arom.H-2 and
H-6). IR mmax cm-1 (KBr): 1709 (C=O), 1613 (C=N). Anal.
Calcd. for C17H19N3O3S: C, 59.11; H, 5.54; N, 12.17; S,
9.28. Found: C, 58.70; H, 5.79; N, 12.05; S, 9.23.
drugs were administered to mice at doses of 100 mg/kg
(body weight). Test compounds that possessed more than a
20% inhibitory effect in any of the measurement ranges
were selected for further evaluation of the activity–dose
relationship in two different doses (50 and 200 mg/kg).
Because of the daily changing circumstance, we used
another control group for each experiment. In the final
stage of the experiment, all the results were prepared and
showed on Table 1, considering only one control group.
2-[(3,4,5-Trimethoxyphenyl)methyl]-6-methyl-thiazolo[3,2
-b]-1,2,4-triazole-5-carboxylic acid ethyl ester (7b)
1
Yield 47%. mp 114–115°C. H NMR (CDCl3-d): d 1.39
(3H, t, –CH3), 2.88 (3H, s, –CH3), 3.78 (3H, s, OCH3), 3.82
(6H, s, 2OCH3), 3.96 (2H, s, –CH2–), 4.26 (2H, q, –CH2–),
6.90 (2H, s, arom.H-2 and H-6). IR mmax cm-1 (KBr): 1700
(C=O), 1591 (C=N). Anal. Calcd. for C18H21N3O5S: C,
55.23; H, 5.41; N, 10.73; S, 8.19. Found: C, 54.95; H, 5.27;
N, 10.69; S, 8.26.
Preparation of test samples for bioassay
Test samples, suspended in a mixture of distilled water and
0.5% sodium carboxymethyl cellulose (CMC), were given
orally to the animals. The control group animals received
the same experimental handling as those of the test groups,
except that the drug treatment was replaced with appro-
priate volumes of the dosing vehicle. Either indomethacin
(10 mg/kg) or acetylsalicylic acid (ASA, 200 mg/kg) in
0.5% CMC was used as reference drug.
2-[2-(3,4,5-Trimethoxyphenyl)ethyl]-6-methyl-thiazolo[3,2
-b]-1,2,4-triazole-5-carboxylic acid ethyl ester (8b)
1
Yield 32%. mp 103–104°C. H NMR (400 MHz, CDCl3-
d): d 1.41 (3H, t, –CH3), 2.88 (3H, s, –CH3), 3.09–3.14
(4H, m, –CH2CH2–), 3.81 (3H, s, OCH3), 3.85 (6H, s,
2OCH3), 4.25 (2H, q, –CH2–), 6.89 (2H, s, arom.H-2 and
H-6). IR mmax cm-1 (KBr): 1691 (C=O), 1588 (C=N). Anal.
Calcd for C19H23N3O5S: C, 56.28; H, 5.72; N, 10.36; S,
7.91. Found: C, 55.85; H, 6.06; N, 10.17; S, 7.95.
Anti-inflammatory activity, carrageenan-induced
edema
For the determination of the effects on carrageenan-
induced paw edema, the modified method of Kasahara
et al. was employed (Kasahara et al., 1985). One hour
after the oral administration of either test sample or
dosing vehicle, each mouse was injected with a freshly
prepared (0.5 mg/25 ll) suspension of carrageenan
(Sigma, St. Louis, MO, USA) in physiological saline
(154 mM NaCl). The subplantar tissue of the right hind
paw was the injection site for all mice. The contralateral
paw (left hind paw) was injected with 25 ll saline solu-
tion as the internal control. Paw edema was measured
every 90 min for 6 h after induction of inflammation with
a pair of dial thickness gauge callipers (Ozaki Co., Tokyo,
Japan). The difference in footpad thickness between the
right and left foot indicated the degree of inflammation
for each mouse. The change in paw volume, either
increase or decrease, was calculated and compared with
the control group (dosing vehicle) and analyzed using
statistical methods.
Pharmacology
Animals
Locally bred BALB/c mice of both sexes (30–35 g) were
purchased from the animal breeding laboratories of Inonu
University (Malatya, Turkey). The animals were fed a
standard pellet diet and water ad libitum in a temperature-
controlled room. On the day before the treatments, food
was withdrawn, but the animals were allowed free access
of water. The allocation of animals to different groups was
randomized, and the experiments were carried out under
blind conditions. Mice used in the present study were cared
for in accordance with the directory of the Inonu University
Animal Care Unit, which applies the guidelines of the
National Institutes of Health on laboratory animal welfare.
Procedures involving animals and their care were con-
ducted in conformity with international laws and policies
and animal studies accepted by Inonu University Ethical
Council (2007/48).
Percent inhibitory effects were estimated according to
the following equation, where n was the average difference
in thickness between the left and right hind paw of the
control group and n0 was that of test group of animals.
All of the experimental groups composed of five mice
were employed for preliminary testing using the carra-
geenan-induced paw edema model and writhing test,
respectively. For preliminary activity screening, all test
Anti-inflammatory activityð%Þ ¼ ½ðn ꢁ n0Þ=nꢂ ꢃ 100:
Indomethacin was used as a reference compound and
administered at 10 mg/kg.
123

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Med Chem Res (2012) 21:192–201
Analgesic activity, Koster test (Koster et al., 1959)
was recorded for each group at the end of the observation
period.
One hour after oral administration of test sample, each
mouse was injected intraperitoneally with 3% (w/v) acetic
acid solution (0.1 ml/10 g body weight). Starting 5 min
after the acetic acid injection, the number of muscular
contractions on mice were counted for a period of 10 min.
A significant reduction in the number of writhings by any
treatment as compared to the number of writhings in con-
trol animals was considered a positive analgesic response.
The analgesic activity was expressed as a percentage
change from writhing controls. Aspirin (ASA) was used as
a reference compound and administered at 200 mg/kg.
Statistical analysis of data
Data obtained from animal experiments were expressed as
means standard error (SEM). Statistical differences
between the treatment and the control group of animals
were evaluated by two-tailed Student’s t test.
Acknowledgments We are grateful Hacettepe University Research
Center for its financial supports (P. N.: 0701301001).
Conflict of interest The authors have declared no conflict of
interest.
Antioxidant activity, lipid peroxidation
Only the animals who were administered 100 mg/kg (body
weight) of the test samples were subjected to this experi-
mental process. Eight hours after the analgesic activity
experiment, mice under deep ether anesthesia were killed,
and their stomachs were removed. The stomach of each
mouse was opened through great curvature and was
examined for antioxidant activity. Lipid peroxidation was
assessed by using the method of Ohkawa et al. (1979) as
modified by Jammal and Smith (1985) and was expressed
nmol of thiobarbituric acid (TBA)-reactive substances
(TBARS)/g wet weight of tissue. ASA was used as a
standard drug (200 mg/kg).
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