Novel 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines: Synthesis, characterization, and evaluation of analgesic / anti-inflammatory, antioxidant activities

Article abstract of DOI:10.1002/ardp.200800188

In this study, the synthesis of a new series of 3,6-disubstituted-7H-1,2,4- triazolo[3,4-b][1,3,4]thiadiazine 1a-4c compounds derived from 4-amino-3-substituted-1,2,4-triazole-5-thiones 1-4 is described. All of the synthesized compounds were screened for

Full text of DOI:10.1002/ardp.200800188

Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
B. Tozkoparan et al.
291
Full Paper
Novel 3,6-Disubstituted 7H-1,2,4-Triazolo[3,4-
b][1,3,4]thiadiazines: Synthesis, Characterization, and
Evaluation of Analgesic / Anti-inflammatory, Antioxidant
Activities
Birsen Tozkoparan1, Sevim Peri Aytaꢀ1 and Gꢁknur Aktay2
1 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Sihhiye, Ankara,
Turkey
2 Inonu University, Faculty of Pharmacy, Department of Pharmacology, Malatya, Turkey
In this study, the synthesis of a new series of 3,6-disubstituted-7H-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazine 1a–4c compounds derived from 4-amino-3-substituted-1,2,4-triazole-5-thiones 1–4 is
described. All of the synthesized compounds were screened for their possible analgesic / anti-
inflammatory, antioxidant activities and gastric toxicity. The compound 2c was found to have
both significant analgesic and consistent anti-inflammatory activity without inducing any gas-
tric lesions along with minimal lipid peroxidation. A deep insight into the structures of the
active compounds revealed that the compounds carrying an electron withdrawing group (a
chloride or fluoride) on the phenyl ring at 6-position of the condensed heterocyclic derivatives
exhibited noticeable higher activity.
Keywords: 4-Amino-3-mercapto-1,2,4-triazole / Analgesic activity / Anti-inflammatory activity / Antioxidant activity /
1,2,4-Triazolo[3,4-b][1,3,4]thiadiazines /
Received: October 17, 2008; accepted: February 18, 2009
DOI 10.1002/ardp.200800188
on alternative structural templates. Therefore, several
strategies have been reported on the modification of
well-known nonselective NSAIDs for the design and
development of novel analgesic / anti-inflammatory com-
pounds. Studies have described that derivatization of the
carboxylate function of representative NSAIDs resulted
in an increased anti-inflammatory activity with reduced
ulcerogenic effects [6–10]. Furthermore, certain com-
pounds bearing 1,3,4-oxadiazole / thiadiazole and 1,2,4-
triazole nucleus have emerged as a promising group of
substances [11–19].
Over the last fifteen years, we have prepared many
compounds containing a 1,2,4-triazole skeleton and
screened them for their analgesic / anti-inflammatory
activities [20–25]. Additionally, we have synthesized new
derivatives by combining condensed triazole with ibu-
profen, (S)-naproxen, and flurbiprofen which belong to
the class of 2-arylpropionic acid derivatives of NSAIDs to
improve its effectivenes. In the screening test, many of
the synthesized compounds were found to have a signifi-
Introduction
Currently available nonsteroidal anti-inflammatory
drugs (NSAIDs) exhibit unwanted adverse effects includ-
ing gastrointestinal PUB (perforation, ulceration, and
bleeding) and renal toxicity and, thus, their therapeutic
usefulness is limited when long-term treatment is neces-
sary. The gastrointestinal damage from NSAIDs is gener-
ally attributed to two factors: local irritation by the car-
boxylic acid moiety, common to most of NSAIDs (topical
effect) and decreased cytoprotective prostaglandin pro-
duction [1–5]. Side-effect limitations of current NSAIDs
illustrate a need for the design of new compounds based
Correspondence: Assoc. Prof. Birsen Tozkoparan, Hacettepe Univer-
sity, Faculty of Pharmacy, Dept. of Pharmaceutical Chemistry, 06100 Sih-
hiye, Ankara, Turkey.
E-mail: tbirsen@hacettepe.edu.tr
Fax: + 90 312 311 4777
Abbreviation: thiobarbituric acid-reactive substances (TBARS)
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292
B. Tozkoparan et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
cant and consistent analgesic / anti-inflammatory activ-
ity profile with reduced ulcerogenic risk when com-
paired to standart compounds [26–28]. Lastly, we have
synthesized a series of 4-amino-3-substituted-1,2,4-tria-
zole-5-thiones and their corresponding condensed deriv-
atives, 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazines, with possible anti-inflammatory / analgesic
properties. Interestingly, these compounds also showed
negligible ulcer risk unlike aspirin which was used as
positive control causing severe bleeding at 200 mg/kg
dose [29]. This condensed ring is still a large untapped
source of structurally novel compounds that might serve
as lead for the development of a novel class of analgesic /
anti-inflammatory agents. Hence, further structural
modification is planned as an extension of our ongoing
project towards the development of new molecules. In
the present investigation, a new aminomercaptotriazol
compounds carrying a 2/3-methoxyphenylmethyl or 2/3-
methoxyphenylethyl moiety at the 3rd position was
selected as the starting material, and all newly synthe-
sized compounds were screened for their analgesic / anti-
inflammatory activities as well as their gastric risk and
antioxidant properties. The results of this studies are dis-
cussed herein.
Reagents and conditions: (i) heat; (ii) absolute ethanol, reflux.
Scheme 1. Synthesis of compounds 1a–4c.
signals of NH2 protons and the NH proton appeared as
two singlets around 5.52–5.54 and 13.46–13.93 ppm,
respectively. In the IR spectra of the triazolothiadiazines
derivatives 1a–4c, disappearance of the characteristic
peaks belonging to N-H streching indicated that the ring-
1
closure reaction occured. The H-NMR spectrum of the
condensed derivatives 1a–4c displayed a singlet integrat-
ing two protons due to SCH2 protons differing from the
starting compounds [29]. All other protons were seen at
the expected chemical shifts and integral values. Besides,
13C-NMR findings of compounds 3 and 3b are in good
agreement with the proposed structures (Table 3).
Results and discussion
Chemistry
Pharmacology
Continuing our studies on condensed triazole derivatives
that are attractive candidates as analgesic / anti-inflam-
matory agents, we have designed a new series of function-
alized triazolothiadiazine derivatives. The required start-
ing compounds, 4-amino-3-substituted-1,2,4-triazole-5-
thiones 1–4 were obtained by the reaction of correspond-
ing acetic or propionic acids with thiocarbohidrazide
[29–31]. The condensation of 4-amino-1,2,4-triazole-5-thi-
ones 1–4 with appropriate phenacyl bromides (or chlor-
ides) in anhydrous ethanol under reflux gave 1,2,4-tria-
zolo[3,4-b][1,3,4]thiadiazines 1a–4c in 36 to 64% yield
(Scheme 1) [29, 31–33]. The data characterizing the syn-
thesized novel 7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines
are given in Tables 1 and 2. Elementary analyses besides
spectral data confirmed the structures of the isolated
compounds.
In the pharmacological study, we investigated the anti-
inflammatory and analgesic activities as well as the
ulcerogenic risk and antioxidant activity on acute admin-
istration of both starting compounds 4-amino-1,2,4-tria-
zole-5-thiones and the corresponding triazolothiadiazine
derivatives. The analgesic activity of the compounds was
studied by using acetic acid-induced abdominal constric-
tion test in mice [34]. The animals were administered a
100 mg/kg (body weight) dose of the test drugs. The com-
pounds which were screened for analgesic activity, were
further screened for their gastric toxicity and antioxi-
dant activity on acute administration. Compounds 2, 1b,
1c, 2c, 3a, 4a, and 4b showed either high or similar anal-
gesic activity profiles compared to that of aspirin,
whereas the other compounds were inactive (Table 4). All
mentioned compounds except 4b, did not cause any sign
of gastric lesions with a dose of 100 mg/kg, p.o., which
exhibited a noticeable analgesic activity while aspirin
caused severe bleeding lesions with 3/5 score at the same
dose. It was found that all derivatives showing less ulcero-
genic risk, except 4b, also showed a lower lipid peroxida-
tion level (Table 4, Fig. 1). Thus, these studies suggested
In accordance with our previous study, no absorption
band in the IR spectra of compounds 1–4 was detected at
about 2600–2550 cm– 1, attributable to the S-H group,
and the presence of a band at about 1188–1176 cm– 1
–
indicative of C=S group – revealed that the thione form
of the starting compounds was obtained. In the 1H-NMR
spectrum of 4-amino-1,2,4-triazole-5-thiones (DMSO-d6),
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Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
Disubstituted 1,2,4-Triazolo[3,4-b][1,3,4]thiadiazines
293
Table 1. Melting points, reaction yields, and formulas of the synthesized compounds.
Compounds
R
n
1
R9
Yield (%)
79
M.p. (8C)
Formula
1
2-OCH3
–
145–146
C10H12N4OS (236.29):
Calcd.: C, 50.83; H, 5.12; N, 23.71; S, 13.57
Found: C, 51.16; H, 5.32; N, 23.61; S, 13.47
C10H12N4OS (236.29):
Calcd.: C, 50.83; H, 5.12; N, 23.71; S, 13.57
Found: C, 51.08; H, 4.91; N, 23.61; S, 13.50
C11H14N4OS (250.32):
Calcd.: C, 52.78; H, 5.64; N, 22.38; S, 12.81
Found: C, 53.26; H, 5.64; N, 22.04; S, 12.66
C11H14N4OS (250.32):
Calcd.: C, 52.78; H, 5.64; N, 22.38; S, 12.81
Found: C, 52.43; H, 5.59; N, 22.12; S, 12.79
C18H16N4OS (336.41):
Calcd.: C, 64.27; H, 4.79; N, 16.65; S, 9.53
Found: C, 64.37; H, 4.93; N, 16.52; S, 9.52
C18H15ClN4OS (370.86):
Calcd.: C, 58.30; H, 4.08; N, 15.11; S, 8.64
Found: C, 57.99; H, 4.19; N, 15.06; S, 8.66
C18H15FN4OS (354.40):
Calcd.: C, 61.00; H, 4.27; N, 15.81; S, 9.05
Found: C, 60.95; H, 4.28; N, 15.83; S, 9.16
C18H16N4OS (336.41):
Calcd.: C, 64.27; H, 4.79; N, 16.65; S, 9.53
Found: C, 63.89; H, 4.88; N, 16.60; S, 9.48
C18H15ClN40S (370.86):
Calcd.: C, 58.30; H, 4.08; N, 15.11; S, 8.64
Found: C, 58.10; H, 4.22; N, 15.14; S, 8.71
C18H15FN4OS (354.40):
Calcd.: C, 61.00; H, 4.27; N, 15.81; S, 9.05
Found: C, 59.35; H, 4.24; N, 15.86; S, 8.85
C19H18N4OS (350.44):
Calcd.: C, 65.12; H, 5.18; N, 15.99; S, 9.15
Found: C, 65.14; H, 5.17; N, 16.01; S, 9.21
C19H17ClN40S (384.88):
Calcd.: C, 59.29; H, 4.45; N, 14.56; S, 8.33
Found: C, 59.10; H, 4.39; N, 14.56; S, 8.29
C19H17FN4OS (368.43):
Calcd.: C, 61.94; H, 4.65; N, 15.21; S, 8.70
Found: C, 61.54; H, 4.69; N, 15.22; S, 8.89
C19H18N4OS (350.44):
Calcd.: C, 65.12; H, 5.18; N, 15.99; S, 9.15
Found: C, 65.03; H, 4.88; N, 15.91; S, 9.20
C19H17ClN40S (384.88):
Calcd.: C, 59.29; H, 4.45; N, 14.56; S, 8.33
Found: C, 59.20; H, 4.51; N, 14.59; S, 8.45
C19H17FN4OS (368.43):
2a)
3
3-OCH3
2-OCH3
3-OCH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
3-OCH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
3-OCH3
1
2
2
1
1
1
1
1
1
2
2
2
2
2
2
–
85
83
82
36
56
61
62
56
62
54
53
50
47
60
64
132–133
159–160
165–166
124–126
202–203
186–187
153–154
165–166
178–180
196–197
186–187
164–166
175–176
186–187
167–168
–
4
–
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
-H
-Cl
-F
-H
-Cl
-F
-H
-Cl
-F
-H
-Cl
-F
Calcd.: C, 61.94; H, 4.65; N, 15.21; S, 8.70
Found: C, 62.00; H, 4.72; N, 15.15; S, 8.77
a)
Compound 2 (1038304-53-8) seems to be available as a commercial product in “Science Finder”. Since there is no any information
in the literature related to preparation and spectral characteristics, this compound has been included in our research program
and characterized by spectral analysis.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
Table 2. IR and 1H-NMR spectroscopic data of compounds 1–4 and 1a–4c.
Compound
IR (KBr) m (cm– 1
)
1H-NMR d (ppm)
1
3304, 3238 (N-H), 1600 (C=N), 1325 (C-N),
1238 (C-O), 1180 (C=S).
(DMSO-d6): 3.76 (s, 3H, -OCH3), 3.94 (s, 2H, -CH2-), 5.54 (s, 2H,
-NH2), 6.87 (d, 2H, arom. H), 7.08 (d, 2H, arom. H), 13.46 (s, 1H,
-NH)
2
3
3309, 3169 (N-H), 1608 (C=N), 1330 (C-N),
1226 (C-O), 1178 (C=S).
(CDCl3): 3.80 (s, 3H, -OCH3), 4.07 (s, 2H, -CH2-), 4.54 (s, 2H, -NH2),
6.81-6.88 (m, 2H, arom. H), 7.25 (t, 2H, arom. H), 10.71 (s, 1H,
-NH)
(DMSO-d6): 2.88 (t, 4H, -CH2CH2-), 3.74 (s, 3H, -OCH3), 5.52 (s, 2H,
-NH2), 6.83 (t, 1H, arom. H), 6.92 (d, 1H, arom. H), 7.10 (d, 1H,
arom. H), 7.17 (t, 1H, arom. H), 13.93 (s, 1H, -NH)
(DMSO-d6 + D2O): 2.96 (s, 4H, -CH2CH2-), 3.73 (s, 3H, -OCH3), 6.81-
6.76 (m, 3H, arom. H), 7.20 (t, 1H, arom. H)
3261 (N-H), 1598 (C=N), 1305 (C-N), 1242 (C-O),
1178 (C=S).
4
3263, 3122 (N-H), 1608 (C=N), 1309 (C-N),
1249 (C-O), 1188 (C=S).
1a
1600 (C=N), 1313 (C-N), 1249 (C-O).
(CDCl3): 3.83 (s, 3H, -OCH3), 3.92 (s, 2H, -CH2-), 4.36 (s, 2H, -SCH2),
6.89 (t, 2H, arom. H), 7.23 (q, 2H, arom. H), 7.48–7.55 (m, 3H,
arom. H), 7.84 (d, 2H, arom. H)
1b
1c
2a
2b
2c
3a
1589 (C=N), 1328 (C-N), 1246 (C-O).
1600 (C=N), 1307 (C-N), 1244 (C-O).
1608 (C=N), 1309 (C-N), 1257 (C-O).
1598 (C=N), 1305 (C-N), 1257 (C-O).
1602 (C=N), 1309 (C-N), 1257 (C-O).
1600 (C=N), 1323 (C-N), 1249 (C-O).
(CDCl3): 3.82 (s, 3H, -OCH3), 3.89 (s, 2H, -CH2-), 4.34 (s, 2H, -SCH2-),
6.88 (t, 2H, arom. H), 7.22 (m, 2H, arom. H), 7.47 (d, 2H, arom. H),
7.76 (d, 2H, arom. H)
(CDCl3): 3.82 (s, 3H, -OCH3), 3.90 (s, 2H, -CH2-), 4.34 (s, 2H, -SCH2-),
6.88 (t, 2H, arom. H), 7.16–7.26 (m, 4H, arom. H), 7.84 (d, 2H,
arom. H)
(CDCl3): 3.74 (s, 3H, -OCH3), 3.92 (s, 2H, -CH2-), 4.31 (s, 2H, -SCH2-),
6.77 (d, H, arom. H), 6.95 (t, 2H, arom. H), 7.20 (t, H, arom. H),
7.50-7.59 (m, 3H, arom. H), 7.84 (d, 2H, arom. H)
(DMSO-d6): 3.75 (s, 3H, -OCH3), 3.89 (s, 2H, -CH2-), 4.30 (s, 2H,
-SCH2-), 6.77 (d, 1H, arom. H), 6.93 (t, 2H, arom. H), 7.20 (t, 1H,
arom. H), 7.49 (d, 2H, arom. H), 7.77 (d, 2H, arom. H)
(DMSO-d6): 3.75 (s, 3H, -OCH3), 3.90 (s, 2H, -CH2-), 4.30 (s, 2H,
-SCH2-), 6.77 (d, 1H, arom. H), 6.93 (t, 2H, arom. H), 7.20 (t, 3H,
arom. H), 7.85 (d, 2H, arom. H)
(CDCl3): 3.12 (t, 2H, -CH2CH2-), 3.28 (t, 2H, -CH2CH2-), 3.74 (s, 3H,
-OCH3), 3.81 (s, 2H, -SCH2-), 6.66 (d, 1H, arom. H), 6.78 (t, 1H,
arom. H), 7.01–7.11 (m, 2H, arom. H), 7.49–7.56 (m, 3H, arom.
H), 7.84 (d, 2H, arom. H)
3b
3c
1587 (C=N), 1327 (C-N), 1249 (C-O).
1600 (C=N), 1309 (C-N), 1242 (C-O).
(CDCl3): 3.11 (t, 2H, -CH2CH2-), 3.27 (t, 2H, -CH2CH2-), 3.75 (s, 3H,
-OCH3), 3.77 (s, 2H, -SCH2-), 6.67 (d, 1H, arom. H), 6.78 (t, 1H,
arom. H), 6.99–7.10 (m, 2H, arom. H), 7.48 (d, 2H, arom. H), 7.75
(d, 2H, arom. H)
(CDCl3): 3.11 (t, 2H, -CH2CH2-), 3.27 (t, 2H, -CH2CH2-), 3.74 (s, 3H,
-OCH3), 3.78 (s, 2H, -SCH2-), 6.66 (d, 1H, arom. H), 6.78 (t, 1H,
arom. H), 7.01 (d, 1H, arom. H), 7.09 (t, 1H, arom. H), 7.19 (t, 2H,
arom. H), 7.82 (q, 2H, arom. H)
4a
4b
4c
1602 (C=N), 1323 (C-N), 1249 (C-O).
1587 (C=N), 1327 (C-N), 1249 (C-O).
1602 (C=N), 1309 (C-N), 1240 (C-O).
(DMSO-d6): 3.12 (t, 2H, -CH2CH2-), 3.28 (t, 2H, CH2CH2), 3.70 (s, 3H,
-OCH3), 3.86 (s, 2H, -SCH2-), 6.65–6.78 (m, 3H, arom. H), 7.12 (t,
1H, arom. H), 7.48–7.58 (m, 3H, arom. H), 7.81 (d, 2H, arom. H)
(DMSO-d6): 3.11 (t, 2H, -CH2CH2-), 3.27 (t, 2H, -CH2CH2-), 3.75 (s,
3H, -OCH3), 3.77 (s, 2H, -SCH2-), 6.65-6.76 (m, 3H, arom. H), 7.11
(t, 1H, arom. H), 7.48 (d, 2H, arom. H), 7.75 (d, 2H, arom. H)
(CDCl3): 3.12 (t, 2H, -CH2CH2-), 3.27 (t, 2H, -CH2CH2-), 3.70 (s, 3H,
OCH3), 3.82 (s, 2H, -SCH2-), 6.65-6.77 (m, 3H, arom. H), 7.10–7.22
(m, 3H, arom. H), 7.82 (q, 2H, arom. H)
that the diminished harmful effects of the synthesized
In order to screen the anti-inflammatory profile of the
compounds for the stomach might be related to their synthesized compounds, the carrageenan-induced hind
antioxidant properties. Among the mentioned com- paw oedema model in mice was used [35]. At first, the
pounds, compounds 2 and 2c which have a 3-methoxy- anti-inflammatory activity of the synthesized com-
phenylmethyl substituent at the 3rd position of the ring pounds was studied at 100 mg/kg, p.o. dose. Test com-
exhibited the highest analgesic activity. These com- pounds having more than 20% effect, even some of them
pounds were also safe from a viewpoint of ulcer inci- are not significant statistically, were further evaluated
dence.
and the experiments were repeated for two additional
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Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
Disubstituted 1,2,4-Triazolo[3,4-b][1,3,4]thiadiazines
295
Table 3. Characteristic 13C-NMR shifts of compounds 3 and 3b.
Compound
C19
C29
C39
C3
C5
C6
C7
C8a
3
3b
25.50
23.37
27.89
24.92
55.52
55.39
157.70
154.70
167.01
–
–
–
–
138.80
29.27
139.83
The TBARS data obtained were analyzed by one-way analysis of variance (ANOVA) and Tukey-Kramer posthoc tests for the significant interrelation between the various groups
using INSTAT computer software. p a 0.05 was considered to be significant from the control.
Figure 1. Effects of synthesized compounds on the TBARS level in stomach tissue (** p a 0.01).
dose levels (50 and 200 mg/kg; see Table 4). As a general into the phenyl ring at the 6th position of the condensed
consideration, all compounds with the exception of 1a, ring.
1c, 2a, 3a, and 4b possessed mild to moderate anti-inflam-
matory activity at 100 mg/kg, p.o. dose in any of the meas-
urement intervals. There was a noticeable activity at the
50 mg/kg dose only in compounds 1b, 2b, 2c, 3c, and 4b
Conclusion
carrying halogen on the phenyl ring at the 6th position We have reported on the synthesis and biological evalua-
of the condensed ring during the first 90 minutes. Com- tion some 4-amino-3-substituted-1,2,4-triazole-5-thiones
pound 2b having 3-methoxyphenylmethyl and 4-chloro- 1–4 and their corresponding condensed derivatives 3,6-
phenyl group at the 3rd and 6th position of the ring, disubstituted-7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines
respectively, exhibited the most prominent and consis- 1a–4c, which represent novel analgesic / anti-inflamma-
tent activity along six hours at three doses. Although no tory compounds. The results of the biological evaluation
lesions or bleedings in stomach were detected under the allowed us to get insight into some structural features
microscope, the TBARS level of compound 2b was similar which are critical in this series. In accordance with our
to that of aspirin.
previous results, we can conclude that a chlorine or fluo-
When we compare the results of this study with those rine substituent on the phenyl ring at the 6th position of
previously obtained [29], we conclude that better activity the condensed derivatives resulted in better analgesic
within this series was obtained by introducing an elec- and anti-inflammatory activities in this series. As sum-
tron-withdrawing substituents (a chlorine or fluorine) mary, among the synthesized compounds, compound 2c
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Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
Table 4. Anti-inflammatory and analgesic effects of the test compounds against carrageenan-induced hind paw oedema model and
acetic acid-induced abdominal constriction test in mice, respectively and ulcer scorea)
Compounds Dose
Ulcer Analgesic
Swelling in thickness (6 10– 2 mm) l S.E.M.b)
(mg/kg) score activity
(percent inhibitory activity)
per os
Mean l S.E.M.
90 min.
180 min.
270 min.
360 min.
Control
1
0/5
0/5
30.5 l 3.4
38.3 l 1.5
60.4 l 5.5
29.5 l 1.3** (51.1)
84.7 l 11.0
42.0 l 5.8
33.2 l 1.9 (20.9)
48.1 l 2.5
57.0 l 5.6
37.0 l 3.8* (35.1)
59.0 l 2.6
73.0 l 5.4
59.3 l 1.7
74.4 l 8.1
100
50
200
100
50
200
100
50
200
100
50
200
100
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
44.0 l 5.3* (27.2)
34.7 l 6.7* (42.5)
47.9 l 7.1 (20.7)
44.2 l 5.2* (26.8)
32.2 l 11.1* (46.7)
77.3 l 3.7
52.0 l 4.9
36.0 l 6.8* (40.4)
53.7 l 6.2
54.0 l 4.8
41.0 l 7.4 (2.4)
33.9 l 3.8
39.4 l 2.5
26.4 l 6.7 (37.1)
45.9 l 4.2
73.0 l 6.4
29.3 l 5.9 (30.2)
61.2 l 7.5
30.6 l 2.5 (27.1)
59.6 l 7.6
34.2 l 5.5
32.0 l 6.4** (43.8)
42.7 l 7.4 (25.1)
39.6 l 2.5* (30.5)
71.2 l 2.0
39.9 l 11.6 (32.9)
46.8 l 3.9 (19.2)
49.0 l 6.2
47.0 l 11.3
105.0 l 2.9
37.6 l 3.5** (34)
78.4 l 12.1
41.3 l 1.5 (27.5)
64.0 l 5.8
67.5 l 6.1
58.4 l 8.6
65.7 l 7.8
65.7 l 7.7
49.8 l 6.2
34.6 l 3.9** (39.3)
56.2 l 3.7
37.0 l 3.7* (35.1)
18.6 l 3.7*** (67.4)
36.6 l 2.3** (35.8)
41.3 l 3.8 (27.5)
57.0 l 6.3
92.0 l 10.7
49.2 l 11.9 (32.6)
64.8 l 4.6
64.8 l 2.4
26.4 l 6.1** (63.8)
60.0 l 6.0
2
3
4
0/5
0/5
0/5
13.7 l 3.8* (55.1)
24.5 l 2.1 (19.7)
21.7 l 6.1 (28.8)
96.0 l 11.2
57.7 l 10.3 (20.9)
145.0 l 11.9
52.8 l 6.1* (27.7)
89.9 l 9.5
40.7 l 2.9** (44.2)
92.0 l 9.7
62.6 l 4.7
73.3 l 3.8
1a
1b
–
1/5
–
17.8 l 1.9* (41.6)
47.6 l 2.5 (21.2)
39.0 l 6.6* (35.4)
29.5 l 5.2** (51.1)
42.4 l 2.2 (29.8)
56.8 l 6.0
56.8 l 7.9
42.4 l 2.2 (29.8)
49.6 l 5.4
36.0 l 5.8
24.1 l 2.2* (42.6)
41.0 l 5.9
65.9 l 1.2
74.6 l 11.9
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
0/5
0/5
0/5
0/5
0/5
0/5
1/5
0/5
2/5
2/5
18.0 l 2.5* (41.0)
27.5 l 2.7 (9.8)
35.3 l 3.0
42.0 l 4.2
109.5 l 11.3
129.4 l 10.9
64.4 l 6.1
57.6 l 7.9 (21.1)
97.5 l 7.5
40.7 l 5.1** (44.2)
94.0 l 9.3
49.2 l 6.3* (32.6)
50.9 l 10.6 (30.3)
92.9 l 11.3
32.4 l 3.0 (22.8)
33.2 l 3.9 (20.9)
30.6 l 2.5 (27.1)
55.0 l 2.9
26.4 l 4.4 (37.1)
15.0 l 1.6** (64.3)
32.8 l 2.2 (21.9)
27.4 l 4.2 (34.8)
33.6 l 6.1 (20)
38.4 l 4.9
36.1 l 2.6
36.0 l 2.7
43.2 l 5.2
30.3 l 3.5 (27.8)
38.9 l 3.2
34.8 l 5.5
31.2 l 2.6 (25.7)
37.5 l 5.2
43.7 l 3.6
25.4 l 5.2 (39.5)
36.7 l 2.9
40.8 l 4.4
26.4 l 6.1 (37.1)
37.0 l 2.0
50.3 l 6.6
26.4 l 6.1 (37.1)
49.2 l 7.7
50.0 l 4.1
41.1 l 3.4* (31.9)
24.2 l 3.8*** (59.9)
34.9 l 3.5** (42.2)
14.2 l 1.8** (53.4) 32.2 l 7.8* (46.7)
33.7 l 7.0** (44.2)
47.9 l 8.1 (20.7)
54.0 l 2.2
53.0 l 3.4
76.4 l 9.5
57.8 l 13.3
62.4 l 2.3
79.9 l 8.9
38.6 l 3.9* (36.0)
30.0 l 4.1** (50.3)
36.8 l 9.4* (39.1)
48.2 l 8.8
45.5 l 5.1 (20.2)
48.0 l 5.3
86.3 l 13.2
66.2 l 2.9
62.0 l 2.9
17.5 l 1.8**(42.6)
19.6 l 4.2 (35.7)
20.8 l 5.4 (31.8)
72.3 l 11.2
106.2 l 16.9
41.9 l 4.6** (42.6)
66.7 l 3.4
132.7 l 11.8
52.6 l 3.4* (27.9)
80.0 l 6.8
26.4 l 9.9 ** (63.8)
45.8 l 8.8* (37.3)
56.9 l 6.7* (22)
102.9 l 9.6
64.4 l 6.1
88.0 l 16.0
50.4 l 2.4** (30.9)
54.3 l 6.1 (25.6)
89.6 l 8.4
39.9 l 9.9 (30.0)
42.9 l 3.2* (24.7)
65.7 l 10.9
37.0 l 5.1 (35.1)
57.5 l 4.8
28.5 l 2.4*** (50)
41.3 l 6.2 (27.5)
63.4 l 3.6
48.2 l 8.1
38.5 l 9.8 (32.4)
55.0 l 5.5
44.8 l 7.6 (21.4)
34.2 l 4.3* (40.0)
74.5 l 5.3
15.7 l 2.3** (48.5) 39.9 l 6.8 (33.9)
54.9 l 6.3
60.4 l 4.3
17.6 l 1.7* (42.3)
29.3 l 5.7 (3.9)
38.6 l 7.7 (36)
33.0 l 3.1** (45.4)
47.9 l 11.0 (20.7)
47.6 l 3.4 (21.2)
53.3 l 14.0
200
100
10
88.8 l 20.2
–
25.7 l 8.4** (57.5)
69.6 l 9.6
–
21.5 l 5.9* (48.8)
54.8 l 4.8
–
31.4 l 8.6* (44.9)
79.6 l 23.4
–
28.8 l 11.0** (60.5)
ASA
INDO
3/5
–
17.8 l 1.3** (41.6)
–
* p a 0.05 (significant).
** p a 0.01 (very significant).
***p a 0.001 (extremely significant).
a)
To avoid wasting animals, groups composed of three or four mice were employed for the preliminary testing using the carrageenan-
induced paw edema model and writhing test, respectively. For preliminary activity screening, all test drugs were administered to mice at
doses of 100 mg/kg (body weight). Test compounds, which possessed more than 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) using groups consisting of
four to fife animals.
Data obtained from animal experiments were expressed as means l standard error (SEM). Statistical differences between the treatment
and the control group of animals were evaluated by two tailed Student's t test for evaluation the analgesic and anti-inflammatory activ-
ity.
b)
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
Disubstituted 1,2,4-Triazolo[3,4-b][1,3,4]thiadiazines
297
sity Animal Care Unit, which applies the guidelines of National
Institutes of Health on laboratory animal welfare. (The locally
bred BALB/c mice were purchased from the animal breeding lab-
oratories of Inonu University, Malatya, Turkey). Procedures
involving animals and their care were conducted in conformity
with international laws and policies and the studies on animals
accepted by Inonu University Ethical Council (2007/48).
exhibited both a significant and consistent analgesic /
anti-inflammatory effect in mice without inducing any
gastric lesions along with minimal lipid peroxidation,
and deserves further attention in order to develop new
leads.
This study is supported by a grant from Hacettepe University
Research Center (Project no: 0701301001). Additionally, the
authors gratefully acknowledge Prof. Dr. M. Ertan (Hacettepe
University, Faculty of Pharmacy, Dept of Pharmaceutical
Chemistry, Ankara-Turkey) for his financial support.
Preparation of test samples for bioassay
Test samples, suspended in a mixture of distilled water and 0.5%
sodium carboxymethyl cellulose (CMC), were administered
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 appropriate volumes
of the dosing vehicle.
The authors have declared no conflict of interest.
Analgesic activity – Koster test [34]
Experimental
The method described by Koster et al. [34] was employed. One
hour after the oral administration of the test sample, each
mouse was injected intraperitoneally with 3% (w/v) acetic-acid
solution (0.1 mL/10 g body weight). A significant reduction in
the number of writhings by any treatment as compared to
vehicle-treated animals was considered as a positive analgesic
response. Aspirin (ASA) (100 mg/kg, p.o.) was used as a reference
drug.
Chemistry
Melting points were detected with a Thomas Hoover capillary
melting point apparatus (Philadelphia, PA, USA) and are uncor-
rected. IR spectra (KBr) were recorded on Perkin Elmer 1720X FT-
IR spectrometer (Perkin Elmer, Beaconsfield, UK) using potas-
sium bromide pellets and the result are expressed in wave num-
1
bers (cm– 1). H-Nuclear magnetic resonance spectra were taken
on Varian Mercury 400, 400 MHz High Performance Digital FT-
NMR instrument (Varian, Palo Alto, CA, USA) in DMSO-d6 or
CDCl3 using TMS as internal standard. All chemical shift values
were recorded as d (ppm). Splitting patterns are as follows: s, sin-
glet; d, doublet; t, triplet; m, multiplet. The purity of the com-
pounds were checked by thin layer chromatography on silicagel-
Anti-inflammatory activity: Carrageenan-induced oedema
[35]
For the determination of the effects on carrageenan-induced
paw oedema, the modified method of Kasahara et al. [35] was
employed. One hour after the oral administration of either test
sample or dosing vehicle, each mouse was injected with a freshly
prepared suspension of carrageenan (0.5 mg/25 lL; Sigma, St.
Louis, MO, USA) in physiological saline (154 mM NaCl) into the
subplantar tissue of the right hind paw. As the control, 25 lL sal-
ine solution was injected into that of the left hind paw. Paw
oedema was measured in every 90 min during a 6-h period after
induction of inflammation. The difference in footpad thickness
between the right and left foot was measured with a pair of dial
thickness gauge callipers (Ozaki Co., Tokyo, Japan). Indometha-
cin was used as a reference compound and was administered at
10 mg/kg.
coated aliminium sheets (Merck, 1.005554, silicagel HF254 – 361
,
Type 60, 0.25 mm; Merck, Darmstadt, Germany). The elementary
analyses of the resulting compounds were performed with Leco
CHNS 932 analyzer (Leco, St. Joseph, MI, USA) at the Scientific
and Technical Research Council of Turkey, Instrumental Anal-
ysis Laboratory in Ankara. All chemicals were from Aldrich
Chemical Co. (Steinheim, Germany).
Syntheses of 4-amino-3-substituted-1,2,4-triazole-5-
thiones 1–4
The syntheses of the compounds 1–4 were performed according
to a procedure reported in the literature [29–31]. The resulting
compounds were purified by column chromatography (chloro-
form or a chloroform / methanol mixture).
Gastric ulceration studies
Only the animals who were administered 100 mg/kg (body
weight) of test samples were subjected to this experimental proc-
ess. Eight hours after the analgesic activity experiment, mice
under deep ether anesthesia were killed and their stomachs
were removed. The abdomen of each mouse was opened through
the great curvature and examined for lesions or bleedings using
a dissecting microscope.
Syntheses of 3,6-disubstituted 7H-1,2,4-triazolo[3,4-
b][1,3,4]thiadiazines 1a–4c [29, 31–33]
A solution of the corresponding triazoles 1–4 (1 mmol) in anhy-
drous ethanol (20 mL) was heated under reflux with appropriate
phenacyl halogenes (1 mmol). The reaction mixture was allowed
to attain room temperature and was then neutralized with 20%
ammonium hydroxide solution. The separating precipitate was
collected by filtration and was purified by crystallization.
Lipid peroxidation
All the compounds screened for ulcerogenic activity were also
analyzed for lipid peroxidation (LPO) by usig the method of
Ohkawa et al. [36] modified by Jamall and Smith [37]. The LPO is
measured as nmol of thiobarbituric acid (TBA)-reactive substan-
ces (TBARS)/g wet weight of tissue. 1,1,3,3-Tetraethoxypropane
was used as standard for the calibration curve.
Pharmacology
Animals
BALB/c mice of both sexes (30 to 35 g) used in the present study
were cared for in accordance with the directory of Inonu Univer-
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

298
B. Tozkoparan et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 291–298
[19] U. Salgin-Gꢀksen, N. Gꢀkhan-Kelekꢁi, O. Gꢀktas, Y. Kꢀysal,
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www.archpharm.com