Synthesis of 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines as novel analgesic/anti-inflammatory compounds

Article abstract of DOI:10.1016/j.ejmech.2009.06.026

In this study, a new class of 4-amino-3-substituted-1,2,4-triazole-5-thiones (1-4) and their corresponding condensed derivatives 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1a-4c) were synthesized and evaluated for their analgesic/anti-

Full text of DOI:10.1016/j.ejmech.2009.06.026

European Journal of Medicinal Chemistry 44 (2009) 4528–4538
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines as novel
analgesic/anti-inflammatory compounds
,
b
c
c
c
S. Peri Aytaç a, Birsen Tozkoparan a , F. Betu¨l Kaynak , Go¨knur Aktay , Ozgu¨r Go¨ktas¸ , Songu¨l Unu¨var
*
¨
¨
a Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhıye, Ankara, Turkey
b Hacettepe University, Faculty of Engineering, Department of Physics Engineering, 06800 Beytepe, Ankara, Turkey
c Inonu University, Faculty of Pharmacy, Department of Pharmacology, 44280 Malatya, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a new class of 4-amino-3-substituted-1,2,4-triazole-5-thiones (1–4) and their corre-
sponding condensed derivatives 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1a–4c)
were synthesized and evaluated for their analgesic/anti-inflammatory activities. All synthesized
compounds were also tested for their gastric toxicity and antioxidant activity on acute administration.
Most of the compounds showed significant activity in both carrageenan-induced oedema and acetic acid-
induced writhing tests besides negligible gastrointestinal toxicity. The compounds showing less
ulcerogenic effect also showed less lipid peroxidation (LPO) level. Most promising results were obtained
with the compounds that placed a fluoro or a chloride on the phenyl ring at the sixth position of the
fused ring.
Received 15 January 2009
Received in revised form
11 June 2009
Accepted 19 June 2009
Available online 1 July 2009
Keywords:
Aminomercaptotriazoles
Triazolothiadiazines
Ó 2009 Elsevier Masson SAS. All rights reserved.
Analgesic/anti-inflammatory activity
Antioxidant activity
1. Introduction
that some fused heterocyclic compounds prepared from amino-
triazolethiones (e.g., triazolothiadiazoles (I), triazolothiadiazines (II))
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most useful clinical therapies for the treatment of inflammation,
pain, and inflammation-related disorders. Unfortunately, their
therapeutic and side effects are closely related to their biochemical
action. The anti-inflammatory mechanism of NSAIDs is due to
have received much attention because they are widely used as
anti-inflammatory and analgesic agents [7–11]. Recently 5-amino-
6-(2,3,5-trichlorophenyl)-1,2,4-triazine (III), which is an isostere of
1,3,4-thiadiazine ring, has been reported as novel analgesic
compound [12] (Scheme 1). Moreover, our former studies [13–19]
have shown that certain compounds bearing a 1,2,4-triazole
nucleus possess significant analgesic/anti-inflammatory activity
with reduced gastrointestinal toxicity. We therefore considered it
worthwhile to design and synthesize a novel series of 4-amino-3-
substituted-1,2,4-triazole-5-thiones (1–4) and their corresponding
condensed 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thia-
diazine derivatives (1a–4c), which were expected to show anti-
inflammatory/analgesic properties. This paper discusses the most
common and useful procedure for synthesizing 4-amino-3-mer-
capto-1,2,4-triazoles, the utility of triazoles in the synthesis of tri-
azolotriazines, and the evaluation of anti-inflammatory/analgesic
activities of both derivatives.
a
reduction of prostaglandin synthesis by inhibiting cyclo-
oxygenase enzyme (COX) in arachidonic acid metabolism [1]. The
disruption of cytoprotective prostaglandins by all currently used
NSAIDs results in unwanted adverse effects, including gastroin-
testinal PUB (perforation, ulceration, and bleeding), and renal
toxicity. These results thus limit their therapeutic usefulness when
long-term treatment is necessary [2–5]. There is still a need to
synthesize novel, potent analgesic/anti-inflammatory compounds
that have reduced side effects when compared with the drugs
currently available in the market.
In recent decades, the literature has been enriched with
progressive findings about the synthesis and pharmacological
activities of 4-amino-1,2,4-triazole-5-thione ring, which is a core
structure in various synthetic pharmaceuticals displaying a wide
variety of biological activities [6]. A survey of literature revealed
2. Chemistry
The synthesis of 4-amino-3-substituted-1,2,4-triazole-5-thio-
nes can be achieved by various methods starting either from
carboxylic acid or its derivative esters and hydrazides. There are
* Corresponding author. Tel.: þ90 312 305 30 20; fax: þ90 312 311 47 77.
E-mail address: tbirsen@hacettepe.edu.tr (B. Tozkoparan).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.026

S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
4529
attributable to the S–H group and the presence of a band at about
1188–1176 cmꢁ1 indicative of C]S group was the evidence of thione
form. The 1H NMR spectrum of 4-amino-1,2,4-triazole-5-thiones
N
N
N
N
N
N
N
NH2
Cl
Ar
Ar
S
N
N
N
S
(DMSO-d6) exhibited NH2 protons as a singlet at
grating two protons and a NH signal as a singlet at
d
5.52–5.55, inte-
13.41–13.52
d
Ar
(these protons were more shielded in the spectrum of compound 3
Cl
Ar
taken in CDCl3). Other peaks were observed at appropriate chemical
Cl
shifts and integralvalues. The peak resonated at d
166 in the 13C NMR
I
II
III
spectrum of compound 1 assigned for C]S, confirming the thione
form of the triazole ring [25–28]. Compound 1 was reported in 1984
by Sattur et al. and the compound obtained was assigned as
4-amino-3-(4-methoxyphenylmethyl)-5-mercapto-1,2,4-triazole by
IR spectral data [29]. However, our compounds have different m.p.
Since this difference may be related with thione–thiol tautomers, an
X-ray analysis of compound 1 was carried out and the structure was
identified as 4-amino-3-(4-methoxyphenylmethyl)-1,2,4-triazole-
5-thione. Relevant crystal data and details of the structure deter-
minations are given in Table 1, and selected geometric parameters
are given in Table 2. ORTEP-3 drawings of structure with atomic
numbering are shown in Fig. 1 [30].
In compound 1, all of the bond lengths and angles in the
phenyl rings are in the normal range. It is observed that the
compound exists in the thione form (Fig. 1). The C]S bond length
is 1.679(3) Å, which is slightly longer than in 4-phenyl-3-[(1H-
1,2,4-triazol-1-yl)methyl]-1H-1,2,4-triazole-5-thione [1.669(2) Å]
[31]. In the triazole ring, the C]N bond length is in the same
range, but the N–N bond is longer than those found in similar
structures [C]N 1.295(2) Å and N–N 1.376(2) Å] and the bond
lengths of C–N correspond with those found in the above-cited
structures [C–N 1.335(3)–1.364(4) Å] [31]. As expected, all atoms
of the triazole and the phenyl rings are planar. The crystal packing
is governed by N–H/S and N–H/N hydrogen bonds (Fig. 2). The
N–H/S intermolecular hydrogen bonds form a dimer, whereby
both molecules are related by the center of inversion (Table 3,
symmetry operator i). In addition to these centrosymmetric
Scheme 1. Some compounds have been reported as anti-inflammatory/analgesic
agents.
several reports on the synthesis of aminotriazolethiones using
different multi-step synthetic routes [20,21]. It is worth noting that
4-amino-3-substituted-1,2,4-triazole-5-thiones were also obtained
by a one-step synthesis. This simple method was first applied in the
preparation of 3-alkyl-4-amino-5-mercapto-1,2,4-triazoles, start-
ing from thiocarbohydrazide and ethyl esters of ortho-acids [22].
Beyer and Kroger [23] later advanced this method by refluxing
thiocarbohydrazide with aliphatic monocarboxylic acids. In our
study, we employed this one-step method to obtain starting
compounds by combining carboxylic acids with thiocarbohy-
drazide at its melting temperature (160–170 ꢀC) [9,24].
The
4-amino-3-substituted-1,2,4-triazole-5-thiones
thus
obtained were treated with phenacyl bromides (or chlorides) in
anhydrous ethanol under reflux to obtain the title compounds
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives (1a–4c) (Scheme
2) [8–10]. All compounds were characterized by their melting
points, elementary analysis, IR and 1H NMR and mass spectra. The
spectral data agree with the proposed structures.
While 4-amino-3-mercapto-1,2,4-triazoles may exist in thione–
thiol tautomeric forms, in our study, the thione structure dominated
in the solid state. Usual spectroscopic methods IR and NMR distin-
guished between these constitutional isomers. In the IR spectra, no
absorption band was detected at about 2600–2550 cmꢁ1
,
N
NH
O
S
i
Ar-C-OH + H2NNH-C-NHNH2
S
Ar
N
NH2
1-4
R
1: R= -H, n=1
2: R= -H, n=2
3: R= -OCH3, n=1
4: R= -OCH3, n=2
Ar:
CH3O
(CH2)n
R
N
NH
N
N
O
ii
S
+ XCH2-C
R'
Ar
Ar
N
N
N
S
NH2
1-4
R'
1a-4c
1a: R= -H, n=1, R'= -H; 1b: R= -H, n=1, R'= 4-Cl; 1c: R= -H, n=1, R'= 4-F
2a: R= -H, n=2, R'= -H; 2b: R= -H, n=2, R'= 4-Cl; 2c: R= -H, n= 2, R'= 4-F
3a: R= -OCH3, n=1, R'= -H; 3b: R= -OCH3, n=1, R'= 4-Cl; 3c: R= -OCH3, n= 1, R'= 4-F
4a: R= -OCH3, n=2, R'= -H; 4b: R= -OCH3, n=2, R'= 4-Cl; 4c: R= -OCH3, n= 2, R'= 4-F
Scheme 2. Synthetic pathway of compounds. Reagents and conditions: (i) heat; (ii) absolute ethanol, reflux.

4530
S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
Table 1
Table 2
Selected bond lengths and angles (Å, ꢀ) for compounds 1 and 3a.
Total data and details of the structure of the compounds 1 and 3a
Formula
C10H12N4O1S1
236.3
Monoclinic
P21/c
C20H20N4O3S1
396.46
Monoclinic
P21/c
Compound 1
Compound 3a
Formula weight
Crystal system
Space group
N1
C3
C3
N4
C5
C5
C5
C7
C6
C6
N1
N2
N1
N2
C3
N2
C3
C14
C7
N4
N5
C7
C5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
N2
N2
N4
N5
N4
N1
S8
1.381 (4)
1.292 (4)
1.364 (4)
1.394(4)
1.368 (4)
1.335 (5)
1.679 (3)
–
1.410 (4)
1.296 (4)
1.367 (4)
1.385 (3)
1.356 (4)
1.292 (4)
1.739 (3)
1.796 (3)
1.511 (4)
1.280 (3)
128.4 (2)
106.6 (2)
107.2 (2)
110.2 (2)
124.0 (2)
125.7 (2)
116.1 (2)
119.3 (2)
118.4 (2)
ꢁ3.3 (4)
33.6 (4)
Cell constants
a, [Å]
b, [Å]
7.2658(12)
6.1596(10)
25.289(6)
91.614(17)
4; 1.387
11.7489(13)
8.0413(13)
20.228(3)
104.430(10)
4; 1.423
c, [Å]
S8
b
, [ꢀ]
C7
N5
C5
N1
N2
C3
N4
C3
C14
C15
C6
N5
C6
S8
–
–
Z; Dcalc [g cmꢁ3
(MoKa) [mmꢁ1
]
m
]
0.271
0.205
–
–
–
–
–
–
–
–
–
–
–
–
–
S8
C5
C3
N4
N5
C14
C15
C16
C8
C6
C7
C5
C7
130.4 (2)
113.1 (3)
104.3 (3)
110.7 (3)
125.1 (3)
125.4 (3)
115.0 (3)
121.2 (3)
–
–
–
–
–
Crystal size [mm]
Radiation
0.27 ꢃ 0.30 ꢃ 0.48
0.30 ꢃ 0.48 ꢃ 0.78
MoKa
293(2)
(l
¼ 0.71073 Å)
MoKa
293(2)
(
l
¼ 0.71073 Å)
Temp [K]
q
limits [ꢀ]
2.8 - 26.29
2340
2.8 - 26.29
3840
Reflections collected
Reflections observed
Reflns. used in refinement
No. of refined parameters
Rint
R / Rw values
GOF
Final shift
1510 [I > 2
2284
s
(I)]
2490 [I > 2s(I)]
3724
253
0.034
0.0591 / 0.2178
0.769
149
0.0436
0.0563 / 0.1986
0.849
0.000
–
–
–
–
C7
S8
N4
C6
26.5 (3)
ꢁ39.2 (3)
0.000
-0.428, 0.532
S8
(Dr
)
min, (Dr
)
(e Åꢁ3
)
-0.549, 0.405
max
3. Pharmacology
dimers there are also short S/S contacts (3.38 Å). Together with
the N–H/S hydrogen bonds, these form a zig–zag paralel to the
b-axis. These S/S contacts are probably another indication for the
presence of the thione form. The N–H/N hydrogen bonds are
formed between molecules related by translation along the b-axis,
involving the NH2 amino-group and the C]N double bonded ring
In the pharmacological study, weinvestigated anti-inflammatory
and analgesic activities as well as the ulcerogenic risk and antioxi-
dant activity on acute administration. Both starting compounds 1–4
and their corresponding condensed derivatives (1a–4c) were eval-
uated. In order to screen the anti-inflammatory profile of the
synthesized compounds, the carrageenan-induced hind paw edema
model in mice was used [34]. The analgesic activity of the
compounds was studied by using the acetic acid-induced writhing
test in mice [35]. In both screening tests, 100 mg/kg (body weight)
doses of the test drugs were orally administered to the animals as
a first step. The compounds having more than 20% effectiveness in
relieving symptoms, even some of them are not be significant
statistically, were considered for further evaluation, and the exper-
iments were repeated in two different dose levels (50 and 200 mg/
kg) (Tables 4 and 5). The analgesic activity was expressed as ‘‘mean
increase in latency after drug administration ꢂ SEM’’ relative to
controls, whereas the anti-inflammatory activity was expressed as
‘‘mean increase in paw volume ꢂ SEM,’’ in terms of mm and
percentage inhibition in paw volume by different doses of the
compounds. Ulcerogenic effect on acute administration was con-
ducted to assess the safety of compounds at high dose levels
(200 mg/kg). For the purpose of comparison, a nonselective COX
inhibitor, acetylsalicylic acid, was used as a positive control. It caused
severe bleeding at a dose level of 200 mg/kg. All compounds
screened for ulcerogenic risk were also analyzed for lipid
peroxidation.
nitrogen. In addition there is a short
parallel oriented triazol rings (the centroid–centroid distance is
3.44 Å). It can be said that O–CH3/ interactions play only
p/p interaction between the
p
a minor role. The polarisation of the methyl hydrogen will be
rather small.
Formation of the triazolothiadiazines was also confirmed on the
basis of IR, 1H NMR, and mass spectroscopic techniques, in addition
to elementary analysis. In the 1H NMR spectrum of tri-
azolothiadiazines, disappearance of the characteristic peaks
belonging to primary amine and N1–H and also the presence of
SCH2 protons, resonated at d 3.90–4.39 ppm as a singlet integrating
two protons, clearly indicated that ring closure reaction occurred.
All other protons were seen at the expected chemical shifts and
integral values [32,33]. The mass spectroscopic fragmentation of
the compounds was studied under electron ionization. Molecular
ion peaks confirmed the molecular weights of examined
compounds. Either molecular ion peaks (compounds 1a–c and 3a–
c) or tropylium peaks, formed from the cleavage of
b-bond of
benzene ring (compounds 2a–c and 4a–c), were the base peaks. In
spectra, the peaks possibly attributed to the elimination of
a methyl, methoxy, aryl, arylnitrile, sulfur and the peak belonging
the arylalkylnitrile ion were seen at different intensities. Further
spectroscopic details of these compounds are presented in the
experimental part. In addition, the X-ray structure of compound 3a
was determined to confirm the assigned structure and to establish
conformations of the molecule (Fig. 3, Table 1). In the structure, the
six-membered thiadiazine ring, S8/C5/N4/N5/C6/C7, is distorted
from planarity. Atoms C17 and N5 deviate ꢁ0.434(3) and 0.161(2) Å,
respectively. Two phenyl rings are located nearly perpendicular to
each other and to the dihedral angle between C8–C13 and C15–C20
ring is 84.07(9)ꢀ (Table 2). In 3a, molecules are linked by a combi-
nation of weak C–H/O and C–H/N hydrogen bonds (Table 3,
Fig. 4).
4. Results and discussion
4.1. Anti-inflammatory activity
It is deduced from Table
4 that all compounds except
compounds 3 and 1a showed reduction in oedema volume when
compared with the control group at 100 mg/kg dose, p.o. in any of
the measurement intervals. Among the tested compounds, the
compounds 4, 1c, 2b and 4c showed the most activity, while others
were less active at 100 mg/kg/p.o dose level. Although generally all
of the compounds showed either increased (compounds 1c, 2a, 2c,

S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
4531
Table 3
Hydrogen bonding geometry (Å, ꢀ) of the compounds 1 and 3a.
D–H/A
D–H
H/A
D/A
D–H/A
Compound 1
Compound 3a
N1–H1/S8i
0.87(4)
0.86
0.93
0.96
0.93
0.96
0.97
0.93
0.93
0.96
2.49(4)
2.33
2.91
2.82
2.59
2.57
2.94
2.93
2.79
2.96
3.348 (3)
3.056 (4)
3.712 (4)
3.552 (5)
3.499 (4)
3.522 (4)
3.777 (3)
3.302 (3)
3.639 (3)
3.628 (4)
171(3)
142
146
133
166
174
144
105
153
127
N5–H52/N2ii
C17–H17/Cg(2)iii
C21–H21B/Cg(2)iv
C10–H10/O2i
C23–H23B/N1ii
C7–H7A/Cg(3)iii
C12–H12/Cg(1)iii
C13–H13/Cg(4)iii
C23–H23/Cg(4)iv
Symmetry code: for compound 1: (i) 3 ꢁ x, ꢁy, ꢁz, (ii) x, 1 þ y, z, (iii) 2 ꢁ x, ꢁ1/2 þ y,
1/2 ꢁ z, (iv) 1 ꢁ x,1/2 þ y, 1/2 ꢁ z; Cg(2): C15–C20 ring; for compound 3a: (i)
x, ꢁ 1 þ y, z, (ii) 1 ꢁ x, 1/2 þ y, 1/2 ꢁ z, (iii)1 ꢁ x, ꢁy, ꢁz, (iv) 2 ꢁ x, 1/2 þ y, 1/2 ꢁ z;
Cg(1): N1–N2–C3–N4–C5, Cg(3): C8–C13, Cg(4): C15–C20 rings.
Fig. 1. ORTEP-3 view of the compound 1, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
3a–c, 4a–c) or similar anti-inflammatory activity (compounds 1, 2,
4, 1b, 2b) to that of 100 mg/kg dose at 200 mg/kg dose level, few
(compounds 3 and 4c) exhibited activity at a dose level of 50 mg/kg.
In general, in cases of anti-inflammatory activity, compounds
carrying an ethylene bridge (n ¼ 2) at the 3rd position of the fused
ring had higher activity than their analogs with methyl (compare
compounds 4 > 3; 2a > 1a; 2b > 1b; 4b > 3b; 4c > 3c). Among the
condensed derivatives, compounds 2b and 4c having 4-methox-
yphenylethyl and 3,4,5-trimethoxyphenylethyl at the 3rd position
and 4-chlorophenyl and 4-fluorophenyl at the 6th position of the
fused ring, respectively, possessed the most prominent and
consistent activity.
were repeated in 200 mg/kg/p.o. dose, the compounds 2, 4, 3a and
4b exhibited similar activity profile to that of a 100 mg/kg dose
level, whereas the compounds 3 and 2b showed less percent
inhibition. As a general consideration, the compounds, except
compound 4a, when administered decreasing dose (50 mg/kg), the
activity was found to be decreased. The compounds 3 and 2b
displayed a higher percent inhibition than aspirin in both 100 and
200 mg/kg/p.o. doses among the set of compounds.
4.3. Acute ulcerogenesis
4.2. Analgesic activity
The compounds, which were screened for analgesic activity,
were further screened for their acute ulcerogenic risk. As a general
consideration, all compounds except 2a and 3b were found to be
safer as gastric lesion risks at high dose (200 mg/kg, p.o.) when
compared to aspirin. Among the compounds, compounds 1, 1b, 2,
and 3a induced no ulcerogenic effect at high dose while aspirin
caused severe bleeding lesions with 4/5 score at the same dose. It
could be hypothesized that the compounds causing fewer gastric
lesions at 200 mg/kg dose when compared to aspirin, may display
diminished ulcerogenic risk at lower doses.
As seen in Table 5, the compounds 2, 3, 4, 2b, 3a and 4b had
either higher or similar analgesic activity to that of aspirin at the
100 mg/kg dose level, whereas others showed less activity when
compared for percent inhibition values. When the experiments
Fig. 3. ORTEP-3 view of the compound 3a, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Fig. 2. Crystal packing of compound 1 showing the intermolecular interactions.

4532
S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
Fig. 4. Crystal packing of compound 3a showing the intermolecular interactions.
4.4. Antioxidant activity
on the phenyl ring at the 6th position of the heterocyclic
compound, showed better analgesic and anti-inflammatory activ-
ities, and less ulcerogenic risk, along with minimum lipid perox-
idation. These findings parallel to those of Prasad et al. [10]. Prasad
et al. synthesized similar 3,6-disubstituted 7H-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine derivatives and screened their antibacterial,
antifungal, and anti-inflammatory activities. They reported that
among the compounds tested for anti-inflammatory activity, the
compound having 6-(4-chlorophenyl)substituent on the
condensed ring showed notable anti-inflammatory activity.
The fact that the compounds with low ulcerogenic risk do not
cause any increase on stomach lipid peroxidation level leads us to
think that the antioxidant properties of these compounds
contributes to the anti-inflammatory activities. Further studies may
reveal the exact mechanism responsible both for the analgesic/anti-
inflammatory and gastric toxicity.
It has been reported in the literature that lower ulcerogenic
activity of compounds is combined with a reduced TBARS content
in the affected area of the gastrointestinal tract, byproducts of lipid
peroxidation [36,37]. Therefore, an attempt was made to correlate
the decrease in ulcerogenic effect of the compounds with that of
lipid peroxidation. The lipid peroxidation is measured as nmol of
TBARS/g wet weight of tissue. The aspirin showed the highest
TBARS level (118.2 ꢂ 2.1, p < 0.001), whereas the control group
showed 78.5 ꢂ 5.0. It was found that all derivatives showing less
ulcerogenic risk also showed less lipid peroxidation production
(Table 5). Thus, these studies showed that synthesized compounds
have inhibited the induction of gastric mucosal lesions. The results
further suggested that their diminished harmful effects on the
stomach might be related to their antioxidant properties.
These types of compounds might lead to further studies for
developing better candidates with potent analgesic and anti-
inflammatory activities, together with less ulcerogenic activity and
minimum peroxidative stomach injury. To probe structural
requirements for optimal activity in this series, the research on
modification of the title compounds is underway.
4.5. Acute toxicity
Test compounds used in the pharmacologic study did not show
any acute toxicity. Common side effects such as mild diarrhea and
depression were not recorded.
5. Conclusion
6. Experimental protocols
Various 4-amino-3-substituted-1,2,4-triazole-5-thiones (1–4)
and their corresponding condensed derivatives 3,6-disubstituted
7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1a–4c) were synthe-
sized with the objective of developing better analgesic/anti-
inflammatory molecules with minimum ulcerogenic risk. Several
derivatives have been evaluated for potential analgesic/anti-
inflammatory activity devoid of ulcerogenic risk. The results are
conclusive in showing that there is no noticeable increase in
analgesic and anti-inflammatory activity when 3-substituted
4-amino-5-mercapto-1,2,4-triazoles are condensed with the 1,3,4-
thiadizine ring. Among the condensed derivatives, the compounds
2b, 4b and 4c, carrying either a 4-chloride or 4-fluoride substituent
6.1. Chemistry
Melting points were detected with a Thomas Hoover capillary
melting point apparatus and are uncorrected. IR spectra (KBr) were
recorded on a Perkin Elmer 1720X FT-IR spectrometer. Nuclear
magnetic resonance (1H and 13C NMR) spectra were taken on
a Varian Mercury 400, 400 MHz High Performance Digital FT-NMR
instrument in DMSO-d6 or CDCl3 using TMS as internal standard.
All chemical shift values were recorded as
patterns are as follows: s, singlet; d, doublet; m, multiplet; b, broad;
dd (doublet in doublet). Mass spectra were obtained with an
d (ppm). Splitting

S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
4533
Table 4
Dose-dependent anti-inflammatory effects of the compounds against carrageenan-induced hind paw edema model in mice in different doses.
Compounds
Dose mg/kg (per os)
Swelling in thickness (ꢃ10ꢁ2 mm) ꢂ SEM (percent inhibitory activity)
90 min
180 min
270 min
360 min
Control
42.0 ꢂ 5.1
65.0 ꢂ 7.4
69.0 ꢂ 7.6
48.0 ꢂ 4.9
1
100
50
200
44.9 ꢂ 5.2
47.0 ꢂ 2.6
37.6 ꢂ 3.6
53.0 ꢂ 9.0
44.9 ꢂ 8.7 (34.9)
58.7 ꢂ 4.7
32.0 ꢂ 3.0* (33.3)
44.7 ꢂ 2.5
60.5 ꢂ 4.1
49.7 ꢂ 3.7 (23.5)
46.8 ꢂ 4.2 (32.2)
32.0 ꢂ 2.6* (33.3)
2
3
4
100
50
200
41.5 ꢂ 6.0
55.1 ꢂ 5.1
54.1 ꢂ 3.4 (21.6)
63.9 ꢂ 2.7
30.5 ꢂ 2.8* (36.5)
48.0 ꢂ 4.7
43.0 ꢂ 3.4
60.5 ꢂ 2.1
30.8 ꢂ 1.7 (26.6)
47.2 ꢂ 1.5 *(27.4)
45.6 ꢂ 1.5* (33.9)
30.0 ꢂ 1.6* (37.5)
100
50
200
42.0 ꢂ 4.5
58.6 ꢂ 3.2
55.4 ꢂ 1.9
39.0 ꢂ 2.9 (18.7)
24.4 ꢂ 1.9** (49.2)
37.8 ꢂ 1.7 (21.2)
29.4 ꢂ 4.4 (30.0)
39.9 ꢂ 3.0
44.0 ꢂ 6.5 (32.3)
86.7 ꢂ 8.7
51.8 ꢂ 4.7 (24.9)
59.3 ꢂ 4.5
100
50
200
39.5 ꢂ 1.8
42.0 ꢂ 3.4
39.8 ꢂ 4.1
57.2 ꢂ 3.2
61.6 ꢂ 4.7
53.5 ꢂ 5.2
44.3 ꢂ 2.4* (35.8)
63.9 ꢂ 2.7
27.5 ꢂ 4.1** (42.7)
38.2 ꢂ 3.8 (20.4)
28.0 ꢂ 3.4* (41.7)
41.9 ꢂ 4.6* (39.3)
1a
1b
100
41.7 ꢂ 1.7
61.7 ꢂ 3.3
68.3 ꢂ 3.3
46.7 ꢂ 3.3
100
50
200
40.1 ꢂ 3.9
43.0 ꢂ 2.6
35.4 ꢂ 2.9
52.1 ꢂ 9.7
56.7 ꢂ 6.5
29.1 ꢂ 4.8* (39.4)
42.5 ꢂ 3.2
60.5 ꢂ 4.8
64.9 ꢂ 2.7
48.4 ꢂ 3.3 (25.5)
51.8 ꢂ 3.7 (25.0)
29.0 ꢂ 2.9* (38.9)
1c
100
50
200
38.7 ꢂ 3.2
42.7 ꢂ 7.2 (34.3)
62.8 ꢂ 3.3
40.5 ꢂ 3.9** (41.3)
60.8 ꢂ 2.0
38.6 ꢂ 1.8
42.0 ꢂ 2.6
49.0 ꢂ 2.4
32.1 ꢂ 3.2 (23.6)
31.9 ꢂ 2.8** (50.9)
32.0 ꢂ 3.6** (53.6)
28.0 ꢂ 2.6** (41.7)
2a
100
50
200
39.7 ꢂ 6.9
43.0 ꢂ 2.6
34.3 ꢂ 2.1
53.9 ꢂ 5.4
43.1 ꢂ 7.3* (37.5)
63.9 ꢂ 4.7
32.0 ꢂ 3.0* (33.3)
41.4 ꢂ 2.8
59.4 ꢂ 2.2
49.7 ꢂ 4.2 (23.5)
34.5 ꢂ 4.2** (50.0)
30.0 ꢂ 4.2* (37.5)
2b
2c
100
50
200
42.1 ꢂ 3.7
44.0 ꢂ 1.9
37.6 ꢂ 3.6
45.9 ꢂ 7.4 (29.4)
61.6 ꢂ 2.5
40.6 ꢂ 5.2* (41.2)
56.6 ꢂ 2.2
23.4 ꢂ 3.8** (51.2)
38.2 ꢂ 3.5 (20.4)
24.0 ꢂ 3.7** (50.0)
45.9 ꢂ 3.7* (29.4)
30.8 ꢂ 4.3** (55.4)
100
50
200
31.1 ꢂ 3.9 (26.0)
38.0 ꢂ 2.6
43.1 ꢂ 2.7* (33.7)
55.0 ꢂ 4.5
54.2 ꢂ 3.1 (21.4)
66.0 ꢂ 3.8
43.3 ꢂ 2.7
48.0 ꢂ 3.2
34.3 ꢂ 3.2
33.1 ꢂ 3.7** (49.0)
45.6 ꢂ 3.2* (34.0)
35.0 ꢂ 2.2 (27.1)
3a
100
50
200
31.9 ꢂ 3.6 (24.0)
37.0 ꢂ 3.4
42.3 ꢂ 5.0* (34.9)
51.6 ꢂ 5.5 (20.6)
32.1 ꢂ 3.7** (50.6)
54.2 ꢂ 4.3 (21.4)
58.7 ꢂ 2.7
38.8 ꢂ 4.6
52.4 ꢂ 2.8
32.1 ꢂ 4.1 (23.6)
46.8 ꢂ 4.2* (32.2)
32.0 ꢂ 4.1* (33.3)
3b
3c
100
50
200
38.8 ꢂ 5.2
41.0 ꢂ 1.9
36.5 ꢂ 5.1
52.1 ꢂ 4.9
46.1 ꢂ 2.5* (33.1)
69.0 ꢂ 2.0
38.3 ꢂ 2.7 (20.2)
48.0 ꢂ 3.2
62.8 ꢂ 6.7
44.6 ꢂ 5.9 (31.4)
40.7 ꢂ 6.9 * (41.0)
36.0 ꢂ 4.0 (25.0)
100
50
200
41.4 ꢂ 3.9
45.0 ꢂ 4.2
35.4 ꢂ 4.5
47.6 ꢂ 5.6 (26.8)
65.0 ꢂ 3.8
58.3 ꢂ 3.1
47.9 ꢂ 2.2
68.0 ꢂ 3.0
50.2 ꢂ 3.2
44.6 ꢂ 4.1* (31.4)
51.8 ꢂ 5.4 (24.9)
36.0 ꢂ 3.7 (25.0)
4a
100
50
200
28.7 ꢂ 3.4 (31.7)
38.0 ꢂ 4.1
40.0 ꢂ 3.3* (38.5)
52.7 ꢂ 4.1
55.0 ꢂ 2.2 (20.3)
66.9 ꢂ 1.6
44.2 ꢂ 2.8
44.7 ꢂ 3.2
23.2 ꢂ 4.8* (44.8)
33.1 ꢂ 5.1** (49.0)
37.0 ꢂ 4.3** (46.4)
34.0 ꢂ 2.5* (29.2)
4b
4c
100
50
200
29.7 ꢂ 3.5 (29.3)
39.0 ꢂ 3.3
41.0 ꢂ 4.1* (36.9)
57.1 ꢂ 5.1
51.7 ꢂ 4.5 (25.0)
63.8 ꢂ 3.5
42.7 ꢂ 3.2
43.6 ꢂ 3.5
27.6 ꢂ 3.9 (34.3)
35.7 ꢂ 5.6* (45.1)
42.0 ꢂ 5.3* (39.1)
36.0 ꢂ 2.9 (25.0)
100
50
200
24.9 ꢂ 2.2** (40.7)
33.0 ꢂ 4.1 (21.4)
17.7 ꢂ 3.2** (57.9)
39.7 ꢂ 4.3* (38.9)
42.6 ꢂ 2.2* (34.5)
31.9 ꢂ 4.5** (51.0)
48.9 ꢂ 2.8* (29.1)
62.8 ꢂ 3.4
41.2 ꢂ 3.2
43.6 ꢂ 2.4
37.0 ꢂ 5.2** (46.4)
37.0 ꢂ 2.6 (23.0)
INDO
100
24.4 ꢂ 1.1** (41.9)
37.0 ꢂ 2.4*** (43.1)
37.1 ꢂ 1.3** (46.2)
28.1 ꢂ 1.0** (41.5)
*p < 0.05; **p < 0.01; ***p < 0.001; significant from control (n ¼ 4–5).
electron impact technique using a Direct Insertion Probe and Agi-
lent 5973-Network Mass Selective Dedector at 70 eV. The purity of
the compounds was checked on silicagel-coated aliminium sheets
(Merck, 1.005554, silicagel HF254–361, Type 60, 0.25 mm, Darmstadt,
Germany) by thin-layer chromatography. The elementary analysis
of the result compounds was performed with a Leco CHNS 932
analyzer at the Scientific and Technical Research Council of Turkey,
Instrumental Analysis Laboratory in Ankara. All chemicals were
from Aldrich Chemical Co. (Steinheim, Germany).
6.1.1. Synthesis of 4-amino-3-substituted-1,2,4-triazole-5-thiones
(1–4) [9,24]
A mixture of equimolar amount of thiocarbohydrazide and
aralkyl carboxylic acid was heated in an oil bath at 160–170 ꢀC for
30 min. The resulting melted mass was triturated with hot water to
obtain the compounds. Two methods were used to purify the
compounds. Compound 2 was purified with column chromatog-
raphy (CHCl3:CH3OH); the others were purified through crystalli-
zation. Compound 1 was previously synthesized [29].

4534
S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
Table 5
Dose-dependent analgesic effects of the test compounds on acetic acid-induced writhing response, ulcer score and lipid peroxidation (as TBARS level) in mice.
Compounds
Dose (mg/kg) per os
TBARS nmol/g wet w
Ulcer score
0/4
Analgesic activity mean ꢂ SEM
(% inhibition)
Control
78.5 ꢂ 5.0
88 ꢂ 1.08
1
100
50
200
61.4 ꢂ 0.8*** (30.2)
67.4 ꢂ 0.4* (23.4)
46.9 ꢂ 1.7*** (46.7)
93.0 ꢂ 5.3
105.9 ꢂ 10.8
96.2 ꢂ 2.0*
104.3 ꢂ 4.2**
112.3 ꢂ 3.1**
97.9 ꢂ 7.2
0/4
0/4
1/4
2/4
1/4
0/4
1/4
3/4
1/4
2/4
0/4
3/4
1/4
2/4
1/4
2
100
50
200
38.2 ꢂ 0.8*** (56.6)
79.6 ꢂ 3.2 (9.5)
38.6 ꢂ 1.4 *** (56.1)
3
100
50
200
12.5 ꢂ 0.5*** (85.8)
69 ꢂ 11.4 (21.6)
34.3 ꢂ 1.08*** (61.0)
4
100
50
200
41.9 ꢂ 2.3*** (52.4)
67.7 ꢂ 1.5* (23.1)
40.3 ꢂ 1.4*** (54.2)
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
ASA
100
50
200
53.2 ꢂ 2.2*** (39.5)
75.4 ꢂ 2.3 (14.3)
47.2 ꢂ 1.5*** (46.4)
100
50
200
59 ꢂ 0.8*** (33.0)
79.3 ꢂ 0.8 (9.9)
53.7 ꢂ 3.2*** (39.0)
100
50
200
64.9 ꢂ 1.6** (26.3)
70 ꢂ 7.0* (20.5)
95.4 ꢂ 4.7*
110.8 ꢂ 2.0***
108.4 ꢂ 5.7**
103.5 ꢂ 7.7*
92.2 ꢂ 5.7
60.3 ꢂ 2.9** (31.5)
100
50
200
50.3 ꢂ 1.8*** (42.8)
82.4 ꢂ 3.2 (6.4)
49.1 ꢂ 3.2*** (44.2)
100
50
200
25.1 ꢂ 0.5*** (71.5)
70.2 ꢂ 0.6 (20.2)
33.1 ꢂ 1.5*** (62.4)
100
50
200
62.3 ꢂ 1.2*** (29.2)
66 ꢂ 3.4*** (25.0)
55.4 ꢂ 0.7*** (37.0)
100
50
200
41.4 ꢂ 1.0*** (53.0)
59.5 ꢂ 5.4** (32.4)
36.6 ꢂ 1.3*** (58.4)
100
50
200
59.3 ꢂ 0.8*** (32.6)
76.1 ꢂ 2.4 (13.5)
67.4 ꢂ 4.5* (23.4)
101.1 ꢂ 6.2*
107.6 ꢂ 12.0
100.3 ꢂ 3.4*
101.9 ꢂ 9.2
100
50
200
57.2 ꢂ 0.4*** (35.0)
68.5 ꢂ 6.3* (22.1)
42.1 ꢂ 1.9*** (52.2)
100
50
200
73 ꢂ 0.4** (17.1)
59.5 ꢂ 5.4** (32.4)
63.8 ꢂ 3.3** (27.5)
100
50
200
43.8 ꢂ 1.8*** (50.2)
62.9 ꢂ 2.5** (28.5)
39.8 ꢂ 1.3*** (54.8)
100
50
200
50.1 ꢂ 1.4*** (43.1)
57 ꢂ 2.9*** (35.2)
38.6 ꢂ 0.9*** (56.1)
105.1 ꢂ 6.7*
1/4
4/5
200
118.2 ꢂ 2.1***
40.1 ꢂ .9***(54.4)
*p < 0.05; **p < 0.01; ***p < 0.001; significant from control (n ¼ 4–5).
6.1.1.1. 4-Amino-3-(4-methoxyphenylmethyl)-1,2,4-triazole-5-thione
(1). Yield 79%; mp 203–204 ꢀC (methanol) [(mp 164 ꢀC) [29]; IR
(KBr): 3292, 3203 (N–H), 1647 (C]N), 1315 (C–N), 1249 (C–O), 1180
74%),146 (CH3OC6H4CHCN,17%),121 (CH3OC6H4CH2,100%), 91 (C7H7,
12.50%), 77 (C6H5, 20.83%). Anal. Calcd. for C10H12N4OS: C, 50.83; H,
5.12; N, 23.71; S, 13.57. Found: C, 50.76; H, 4.93; N, 23.64; S, 13.56.
(C]S) cmꢁ1; 1H NMR
d
(ppm): 3.72 (s, 3H, OCH3), 3.95 (s, 2H, CH2-),
5.55 (s, 2H, NH2), 6.87 (d, 2H, arom. H-3, H-5), 7.20 (d, 2H, arom. H-2,
H-6), 13.52 (s, 1H, NH); 13C NMR
(ppm): 30.03 (–CH2–), 55.74
6.1.1.2. 4-Amino-3-[2-(4-methoxyphenyl)ethyl]-1,2,4-triazole-5-thi-
one (2). Yield 83%; mp 202–203 ꢀC (chloroform-methanol); IR
(KBr): 3250 (N-H), 1627 (C]N), 1307 (C–N), 1246 (C–O), 1176 (C]S)
d
(–OCH3), 114.57 (arom. C-3, C-5), 127.96 (arom. C-1), 130.61
(arom. C-2, C-6), 152.32 (arom. C-4), 158.83 (triazole C-3), 166.61
(triazole C-5); MS (70 eV, EI): m/z (%): 236 (Mþ, 93%), 220 (Mþ, –NH2,
cmꢁ1
;
1H NMR
d
(ppm): 2.87 (t, 4H, CH2CH2), 3.69 (s, 3H, OCH3),
5.55 (s, 2H, NH2), 6.82 (d, 2H, arom. H-3, H-5), 7.12 (d, 2H, arom.

S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
4535
H-2, H-6), 13.41 (s, 1H, NH); MS (70 eV, EI): m/z (%): 250 (Mþ,
86.36%), 234 (Mþ –NH2, 2.61%), 134 (CH3OC6H4CHCH2, 50.44%), 121
(CH3OC6H4CH2, 100%), 91 (C7H7, 17.39%), 77 (C6H5, 20.87%). Anal.
Calcd. for C11H14N4OS: C, 52.78; H, 5.64; N, 22.38; S, 12.81. Found: C,
52.46; H, 5.57; N, 22.38; S, 12.71.
6.1.2.2. 3-(4-Methoxyphenylmethyl)-6-(4-chlorophenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (1b). Yield 56%; m.p. 188–190 ꢀC (It
is precipitated with H2NOH sol.); IR (KBr): 1610 (C]N), 1301 (C–N),
1249 (C–O) cmꢁ1; 1H NMR
d (ppm): 3.72 (s, 3H, OCH3), 3.95 (s, 2H,
CH2), 4.30 (s, 2H, SCH2), 6.84 (d, 2H arom. H-3, H-5), 7.29 (d, 2H,
arom. H-2, H-6), 7.52 (d, 2H, arom. H0-2, H0-6), 7.81 (d, 2H, arom. H-
30, H-50); MS (70 eV, EI): m/z (%): 374 (Mþ þ4, 2.42%), 372 (Mþ þ2,
35.76%), 370 (Mþ, 100%), 355 (Mþ –CH3, 15.76%), 338 (Mþ –S,
12.73%), 337 (Mþ –SH, 10.30%), 335 (Mþ –Cl, 13.33%), 259 (Mþ
–C6H4Cl, 26.67%), 233 (Mþ –ClC6H4CN, 7.27%), 218 ([Mþ
–(ClC6H4CN þ CH3)], 27.88%), 147 (CH3OC6H4CH2CN, 75.44%), 132
([CH3OC6H4CH2CN-CH3], 48.49%), 121 (CH3OC6H4CH2, 79.39%), 111
($C6H4Cl, 30.45%). Anal. Calcd. for C18H15ClN40S: C, 58.30; H, 4.08;
N, 15.11; S, 8.64. Found: C, 57.98; H, 3.92; N, 14.99; S, 8.54.
6.1.1.3. 4-Amino-3-(3,4,5-trimethoxyphenylmethyl)-1,2,4-triazole-5-
thione (3). Yield 47%; mp 173–175 ꢀC (benzene-methanol); IR
(KBr): 3300, 3234 (N–H), 1591 (C]N), 1311 (C–N), 1244 (C–O), 1182
(C]S) cmꢁ1 1H NMR
; d (ppm): 3.83 (s, 3H, OCH3), 3.85 (s, 6H,
OCH3), 4.02 (s, 2H, CH2), 4.57 (s, 2H, NH2), 6.52 (s, 2H, arom. H-2, H-
6), 10.56 (s, 1H, NH); MS (70 eV, EI): m/z (%): 296 (Mþ, 100%), 281
(Mþ –CH3, 32,14%), 280 (Mþ –NH2, 57.86%), 264 (Mþ –S, 4.29%), 192
((H3CO)3C6H2CH2CN–CH3, 12.14%), 181 ((CH3O)3C6H2CH2, 8.57%).
Anal. Calcd. for C12H16N4O3S: C, 48.64; H, 5.44; N, 18.91; S, 10.82.
Found: C, 48.60; H, 5.44; N, 18.72; S, 10.80.
6.1.2.3. 3-(4-Methoxyphenylmethyl)-6-(4-fluorophenyl)-7H-1,2,4-tri-
azolo[3,4-b]-1,3,4-thiadiazine (1c). Yield 47%; m.p. 192–193 ꢀC
(ethanol); IR (KBr): 1600 (C]N), 1307 (C–N), 1249 (C–O) cmꢁ1 1H
;
6.1.1.4. 4-Amino-3-[2-(3,4,5-trimethoxyphenyl)ethyl]-1,2,4-triazole-
NMR
d (ppm): 3.78 (s, 3H, OCH3), 3.94 (s, 2H, CH2), 4.29 (s, 2H,
5-thione (4). Yield 70%; m.p. 139–140 ꢀC (chloroform); IR (KBr):
SCH2), 6.84 (d, 2H, arom. H-3, H-5), 7.20–7.22 (m, 2H, arom. H-30, H-
50), 7.29 (d, 2H, arom. H-2, H-6) 7.86–7.91 (m, 2H, arom. H-20, H-60);
MS (70 eV, EI): m/z (%): 356 (Mþ þ2, 6.88%), 354 (Mþ, 100%), 339
(Mþ –CH3, 15.63%), 322 (Mþ –S, 1.88%), 321 (Mþ –SH, 10.00%), 259
(Mþ –C6H4F, 10.00%), 233 (Mþ –C6H5CN, 8.13%), 218 ([Mþ
–(FC6H4CN þ CH3)], 21.88%), 147 (CH3OC6H4CH2CN, 63.13%), 132
(CH3OC6H4CH2CN–CH3, 28.75%), 121 (CH3OC6H4CH2, 72.50%), 95
(C6H4F, 23.75%). Anal. Calcd. for C18H15FN4OS: C, 61.00; H, 4.27; N,
15.81; S, 9.05. Found: C, 61.32; H, 4.30; N, 15.87; S, 9.08.
3294 (N–H), 1587 (C]N),1311 (C–N), 1242 (C–O), 1188 (C]S) cmꢁ1
;
1H NMR
d (ppm): 2.93 (t, 4H, CH2CH2), 3.62 (s, 3H, OCH3), 3.74 (s,
6H, OCH3), 5.52 (s, 2H, NH2), 6.52 (s, 2H, arom. H-2, H-6), 13.44 (s,
1H, NH); MS (70 eV, EI): m/z (%): 310 (Mþ, 50.87%), 181
((CH3O)3C6H2CH2, 100%). Anal. Calcd. for C13H18N4O3S: C, 50.31; H,
5.85; N, 18.05; S, 10.33. Found: C, 49.96; H, 5.81; N, 17.88; S, 10.34.
6.1.2. Synthesis of 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazines (1a–4c) [8–10]
A mixture of the corresponding triazoles 1–4 (1 mmol) and
phenacyl halogenes (1 mmol) in anhydrous ethanol (20 mL) was
heated under reflux. Reactions were ended under control of TLC. The
mixture was then cooled to the room temperature and neutralized
with 20% ammonium hydroxide solution. The separated precipitate
was collected by filtration and purified with crystallization.
Compound 1a [38] has already been reported. Since there are
only NMR findings describing compound 1a in literature, all spectral
analysis was performed. Single crystal X-ray crystallographic and
spectral data of the compounds 2a and 4a have been submitted
elsewhere for publication. Compounds 1b (886949-15-1) and 1c
(933228-88-7) are seen as commercial products in ‘‘Science Finder.’’
Because there is no information in the literature for the preparation
and spectral characteristics, these two compounds have been
included in our researchprogram and characterized byspectral data.
Yields, melting points, spectral and analytical data of all
synthesized compounds are given below.
6.1.2.4. 3-[2-(4-Methoxyphenyl)ethyl]-6-phenyl-7H-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine (2a). Yield 64%; m.p. 142–143 ꢀC (ethanol). All
spectral data have been submitted for publication.
6.1.2.5. 3-[2-(4-Methoxyphenyl)ethyl]-6-(4-chlorophenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (2b). Yield 53%; m.p. 141–142 ꢀC
(ethanol); IR (KBr): 1616 (C]N), 1300 (C–N), 1249 (C–O) cmꢁ1 1H
;
NMR d (ppm): 3.12 (t, 2H, CH2CH2), 3.31 (t, 2H, CH2CH2), 3.70 (s, 3H,
OCH3), 3.91 (s, 2H, SCH2), 6.74 (d, 2H, arom. H-3, H-5), 7.09 (d, 2H,
arom. H-2, H-6), 7.52 (d, 2H, arom. H-20, H-60), 7.78 (d, 2H, arom. H-
30, H-50); MS (70 eV, EI): m/z (%): 388 (Mþ þ4, 0.97%), 386 (Mþ þ2,
17.10%), 384 (Mþ 45.81%), 369 (Mþ –CH3, 1.61%), 351 (Mþ –SH,
1.29%), 247 (Mþ, –ClC6H4CN, 2.58%), 232 ([Mþ –(ClC6H4CN þ CH3)],
36.13%), 134 (CH3OC6H4CHCH2, 9.68%), 121 (CH3OC6H4CH2, 100%),
111 (C6H4Cl, 6.45%). Anal. Calcd. for C19H17ClN40S: C, 59.29; H, 4.45;
N, 14.56; S, 8.33. Found: C, 59.13; H, 4.44; N, 14.70; S, 8.37.
6.1.2.6. 3-[2-(4-Methoxyphenyl)ethyl]-6-(4-fluorophenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (2c). Yield 61%; m.p. 155–156 ꢀC
6.1.2.1. 3-(4-Methoxyphenylmethyl)-6-phenyl-7H-1,2,4-triazolo[3,4-
(ethanol); IR (KBr): 1598 (C]N), 1300 (C–N), 1247 (C–O) cmꢁ1 1H
;
b]-1,3,4-thiadiazine (1a) [38]. Yield 54%; m.p. 153–154 ꢀC (ethanol);
IR (KBr): 1610 (C]N), 1301 (C–N), 1246 (C–O) cmꢁ1 1H NMR
;
NMR d (ppm): 3.14 (t, 2H, CH2CH2), 3.35 (t, 2H, CH2CH2), 3.70 (s, 3H,
d
(ppm): 3.79 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 4.25 (s, 2H, SCH2), 6.85
OCH3), 3.95 (s, 2H, SCH2), 6.74 (d, 2H, arom. H-3, H-5), 7.10 (d, 2H,
arom. H-2, H-6), 7.22–7.26 (m, 2H, arom. H-30, H-50), 7.85–7.88 (m,
2H, arom. H-20, H-60); MS (70 eV, EI): m/z (%): 370 (Mþ þ2, 6.00%),
368 (Mþ, 79.33%), 353 (Mþ –CH3, 3.00%), 335 (Mþ –SH, 1.67%), 247
(Mþ –FC6H4CN, 3.33%), 232 ([Mþ –(FC6H4CN þ CH3)], 41.33%), 134
(CH3OC6H4CHCH2, 10.67%), 121 (CH3OC6H4CH2, 100%), 95 (C6H4F,
12.00%). Anal. Calcd. for C19H17FN4OS: C, 61.94; H, 4.65; N, 15.21; S,
8.70. Found: C, 61.82; H, 4.74; N, 15.08; S, 8.80.
(d, 2H, arom. H-3, H-5), 7.32 (d, 2H, arom. H-2, H-6), 7.52–7.61 (m,
3H, arom. H-30, H-40, H-50), 7.87 (d, 2H, arom. H-20, H-60); 13C NMR
d
(ppm): 23.45 (–CH2–), 29.85 (-SCH2-), 55.71 (–OCH3), 114.60
(arom. C-3, C-5), 128.12 (arom. C-30, C-50), 128.61 (arom. C-40),
129.76 (arom. C-20, C-60), 130.54 (arom. C-2, C-6), 132.59 (arom. C-
1), 134.18 (arom. C-10), 140.93 (C-8a), 153.52 (arom. C-4), 155.36 (C-
3), 158.75 (C-6); MS (70 eV, EI): m/z (%): 338 (Mþ þ2, 7.90%), 336
(Mþ, 100%), 321 (Mþ –CH3, 14.91%), 304 (Mþ –S, 2.63%), 303 (Mþ
–SH, 11.40%), 259 (Mþ –C6H5, 18.42%), 233 (Mþ –C6H5CN, 10.53%),
218 (Mþ –[(C6H5CN) þ CH3], 20.18%), 147 (CH3OC6H4CH2CN,
75.44%), 132 ([CH3OC6H4CH2CN-CH3], 33.33%), 121 (CH3OC6H4CH2,
50.0%), 77 (C6H5, 61.40%). Anal. Calcd. for C18H16N4OS: C, 64.27; H,
4.79; N, 16.65; S, 9.53. Found: C, 64.12; H, 4.89; N, 16.75; S, 9.45.
6.1.2.7. 3-(3,4,5-Trimethoxyphenylmethyl)-6-phenyl-7H-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazine (3a). Yield 70%; m.p. 170–171.5 ꢀC
(ethanol); IR (KBr): 1587 (C]N), 1330 (C–N), 1240 (C–O) cmꢁ1 1H
;
NMR
d (ppm): 3.77 (s, 6H, OCH3), 3.81 (s, 3H, OCH3), 3.98 (s, 2H,

4536
S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
CH2), 4.30 (s, 2H, SCH2), 6.65 (s, 2H, arom. H-2, H-6), 7.51–7.59 (m,
3H, arom. H-30, H-40, H-50), 7.89 (d, 2H, arom. H-20, H-60); MS (70 eV,
EI): m/z (%): 398 (Mþ þ2, 7.86%), 396 (Mþ, 100%), 381 (Mþ –CH3,
12.14%), 365 (Mþ –OCH3,1.07%), 293 (Mþ –C6H5CN, 1.43%), 278 ([Mþ
–(C6H5CN þ CH3)], 38.57%), 207 ((CH3O)3C6H2CH2CN, 11.43%), 192
((H3CO)3C6H2CH2CN–CH3, 30.0%), 181 ((CH3O)3C6H2CH2, 13.57%),
77 (C6H5, 27.14%). Anal. Calcd. for C20H20N4O3S: C, 60.59; H, 5.08; N,
14.13; S, 8.09. Found: C, 60.57; H, 5.22; N, 14.11; S, 8.11.
6.2. Single crystal X-ray crystallographic data of compounds
1 and 3a
Single crystals suitable for X-ray diffraction of compounds 1 and
3a were mounted on the ends of glass and used for the intensity
data collection. The X-ray diffraction data on 1 and 3a were
collected using an Enraf-Nonius CAD-4 diffractometer employing
graphite-monochromated MoKa radiation
datasets were corrected for Lorentz and polarization effect. An
empirical -scan absorption correction was applied.
(l
¼ 0.71073 Å). The
6.1.2.8. 3-(3,4,5-Trimethoxyphenylmethyl)-6-(4-chlorophenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (3b). Yield 48%; m.p. 165–166 ꢀC
(ethanol); IR (KBr): 1589(C]N),1317(C–N),1246(C–O) cmꢁ1;1HNMR
j
Both structures were solved by the direct method programs
SHELXS-97 [39] and refined by the full-matrix least squares
method, based on F2 using SHELXL-97 [40]. All non-hydrogen
atoms were refined with anisotropic displacement factors. In
compound 1, H1 atom was found from difference map and all other
hydrogen atoms were positioned geometrically and refined using
a riding model. The geometric calculations were carried out with
the Platon program [41].
Crystallographic data (excluding structure factors) for
compounds 1 and 3a reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC-700921, 700922. Copies of the data can
be obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: þ44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
d
(ppm): 3.57 (s, 3H, OCH3), 3.66 (s, 6H, OCH3), 4.19 (s, 2H, CH2), 4.39 (s,
2H, SCH2), 6.63 (s, 2H, arom. H-2, H-6), 7.63 (d, 2H, arom. H-20, H-60),
8.04 (d, 2H, arom. H-30, H-50); MS (70 eV, EI): m/z (%): 434 (Mþ þ4,
2.76%), 432 (Mþ þ2, 41.38%), 430 (Mþ 100%), 415 (Mþ –CH3, 13.10%),
399 (Mþ –OCH3, 1.72%), 293 (Mþ –ClC6H4CN, 2.07%), 278 ([Mþ
–(ClC6H4CN þ CH3)], 51.72%), 207 ((CH3O)3C6H2CH2CN, 17.93%), 192
(((H3CO)3C6H2CH2CN)–CH3, 40.69%), 181 ((CH3O)3C6H2CH2, 21.38%),
111 (C6H4Cl, 6.25%). Anal. Calcd. for C20H19ClN403S.H2O: C, 53.51; H,
4.72; N, 12.48; S, 7.14. Found: C, 53.42; H, 4.49; N, 12.62; S, 7.20.
6.1.2.9. 3-(3,4,5-Trimethoxyphenylmethyl)-6-(4-fluorophenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (3c). Yield 71%; m.p. 182–183 ꢀC (it is
precipitated with H2NOH sol.); IR (KBr): 1598 (C]N), 1317 (C–N),
1236 (C–O) cmꢁ1; 1H NMR
d (ppm): 3.57 (s, 3H, OCH3), 3.66 (s, 6H,
OCH3), 4.19 (s, 2H, CH2), 4.39 (s, 2H, SCH2), 6.63 (s, 2H, arom. H-2, H-
6), 7.39–7.43 (m, 2H, arom. H-30, H-50), 8.08–8.12 (m, 2H, arom.
H-20, H-60); MS (70 eV, EI): m/z (%): 416 (Mþ þ2, 6.92%), 414
(Mþ, 100%), 399 (Mþ –CH3, 12.31%), 383 (Mþ –OCH3, 0.77%), 293
(Mþ –FC6H4CN, 1.54%), 278 ([Mþ –(FC6H4CN þ CH3)], 33.85%), 207
((CH3O)3C6H2CH2CN, 12.31%), 192 (((H3CO)3C6H2CH2CN)–CH3,
30.39%), 181 ((H3CO)3C6H2CH2, 14.62%), 95 (C6H4F, 13.08%). Anal.
Calcd. for C20H19FN4O3S: C, 57.96; H, 4.62; N, 13.52; S, 7.74. Found:
C, 57.65; H, 4.44; N, 13.63; S, 7.78.
6.3. Pharmacology
6.3.1. Animals
Locally bred BALB/c mice of both sexes (30–35 g) were
purchased from the animal breeding laboratories of Inonu Univer-
sity (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 conducted in conformity with international
laws and policies and animal studies accepted by Inonu University
Ethical Council (2007/48).
To avoid wasting animals, groups composed of 3 or 4 mice were
employed for 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 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 mg/kg and 200 mg/kg) using groups con-
sisting of 4–5 animals.
6.1.2.10. 3-[2-(3,4,5-Trimethoxyphenyl)ethyl]-6-phenyl-7H-1,2,4-tri-
azolo[3,4-b]-1,3,4-thiadiazine (4a). Yield 39%; m.p. 158–159 ꢀC
(ethanol). All spectral data have been submitted for publication.
6.1.2.11. 3-[2-(3,4,5-Trimethoxyphenyl)ethyl]-6-(4-chlorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (4b). Yield 68%; m.p. 168–
169 ꢀC (ethanol); IR (KBr): 1595 (C]N), 1309 (C–N), 1232
(C–O) cmꢁ1
;
1H NMR
d
(ppm): 2.96 (t, 2H, CH2CH2), 3.15 (t, 2H,
CH2CH2), 3.50 (s, 3H, OCH3), 3.68 (s, 6H, OCH3), 4.28 (s, 2H, SCH2),
6.46 (s, 2H, arom. H-2, H-6), 7.61 (d, 2H, arom. H0-2, H0-6), 7.97 (d,
2H, arom. H-30, H-50); MS (70 eV, EI): m/z (%): 448 (Mþ þ4, 0.69%),
446 (Mþ þ2, 9.66%), 444 (Mþ, 27.24%), 429 (Mþ –CH3, 4.83%), 379
(Mþ –OCH3, 1.38%), 292 ([Mþ –(ClC6H4CN þ CH3)], 7.86%), 181
((CH3O)3C6H2CH2, 100%), 111 ($C6H4Cl, 3.96%). Anal. Calcd. for
C21H21ClN4O3S: C, 56.69; H, 4.76; N, 12.59; S, 7.21. Found: C, 56.46;
H, 4.59; N, 12.69; S, 7.25.
6.3.2. Preparation of test samples for bioassay
6.1.2.12. 3-[2-(3,4,5-Trimethoxyphenyl)ethyl]-6-(4-fluorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (4c). Yield 56%; m.p. 183–
184 ꢀC (ethanol); IR (KBr): 1589 (C]N), 1309 (C–N), 1238
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 experi-
mental handling as those of the test groups, except that the drug
treatment was replaced with appropriate 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.
(C–O) cmꢁ1 1H NMR
; d (ppm): 3.14 (t, 2H, CH2CH2), 3.39 (t, 2H,
CH2CH2), 3.73 (s, 6H, OCH3), 3.74 (s, 3H, OCH3), 3.88 (s, 2H, SCH2),
6.32 (s, 2H, arom. H-2, H-6), 7.20–7.24 (m, 2H, arom. H-30, H-50),
7.81–7.84 (m, 2H, arom. H-20, H-60); MS (70 eV, EI): m/z (%): 430
(Mþ þ2, 2.50%), 428 (Mþ, 31.43%), 413 (Mþ –CH3, 6.07%), 397 (Mþ
–OCH3, 1.79%), 292 ([Mþ –(FC6H4CN þ CH3)], 6.43%), 181
((CH3O)3C6H2CH2, 100%), 95 (C6H4F, 7.14%). Anal. Calcd. for
C21H21FN4O3S: C, 58.87; H, 4.94; N, 13.08; S, 7.48. Found: C, 58.72;
H, 4.91; N, 13.05; S, 7.58.
6.3.3. Anti-inflammatory activity
6.3.3.1. Carrageenan induced oedema [34]. For the determination of
the effects on carrageenan-induced paw oedema, the modified

S.P. Aytaç et al. / European Journal of Medicinal Chemistry 44 (2009) 4528–4538
4537
method of Kasahara et al. was employed [34]. One hour after the
oral administration of either test sample or dosing vehicle, each
same experimental procedure, except that the TBA solution was
replaced with distilled water. 1,1,3,3-Tetraethoxypropane was used
as a standard for the calibration curve.
mouse was injected with
a freshly prepared (0.5 mg/25 ml)
suspension of carrageenan (Sigma, St. Louis, Missouri, U.S.A.) in
physiological saline (154 mM NaCl). The subplantar tissue of the
right hind paw was the injection site for all mice. As the control,
6.3.7. Acute toxicity
Animals employed in the carrageenan-induced paw oedema
experiment were observed for 72 h, and the mortality rate was
recorded for each group at the end of the observation period.
25 ml saline solution was injected into the left hind paw. Paw
oedema was measured every 90 min for 6 h 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). Mean values of treated groups
were compared with mean values of a control group and analyzed
using statistical methods. 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.
6.3.8. Statistical analysis of data
Data obtained from animal experiments were expressed as
means ꢂ standard error (SEM). Statistical differences between the
treatment and the control group of animals were evaluated by two-
tailed Student’s t test.
Acknowledgements
Anti-inflammatory activityð%Þ ¼ ½ðn ꢁ n0Þ=nꢄ ꢃ 100
The authors express deep thanks to Prof. Dr. M. Ertan (Hacettepe
University, Faculty of Pharmacy, Department of Pharmaceutical
Chemistry, Ankara, Turkey) and Hacettepe University Research
Center for their financial supports (P.N.: 0701301001 and
0302602001).
Indomethacin was used as a reference compound and admin-
istered at 10 mg/kg.
6.3.4. Analgesic activity
6.3.4.1. Koster test [35]. 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 was 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 control animals was considered a positive
analgesic response. The antinociceptive activity was expressed as
a percentage change from writhing controls. Aspirin (ASA) was
used as a reference compound and administered at 200 mg/kg.
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