Design, synthesis and molecular modeling study of acylated 1,2,4-triazole-3-acetates with potential anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2008.03.017

The present investigation is concerned with the synthesis of different acylated 1,2,4-triazole-3-acetates with the objective of discovering novel and potent anti-inflammatory agents. Structures of the synthesized compounds were elucidated by spectral and

Full text of DOI:10.1016/j.ejmech.2008.03.017

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 117e123
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and molecular modeling study of acylated
1,2,4-triazole-3-acetates with potential anti-inflammatory activity
*
Ashraf M. Abdel-Megeed , Hamdy M. Abdel-Rahman, Gamal-Eldien S. Alkaramany,
Mahmoud A. El-Gendy
Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
Received 22 January 2008; received in revised form 9 March 2008; accepted 13 March 2008
Available online 1 April 2008
Abstract
The present investigation is concerned with the synthesis of different acylated 1,2,4-triazole-3-acetates with the objective of discovering
novel and potent anti-inflammatory agents. Structures of the synthesized compounds were elucidated by spectral and elemental analyses.
The obtained compounds were evaluated for their anti-inflammatory activites as well as gastric ulcerogenic effects and acute toxicity. Results
showed that 1-acylated-5-amino-1,2,4-triazole-3-acetates 3aee showed higher anti-inflammatory activity than the corresponding 5-acylamino
derivatives 4aee in carageenan-induced rat paw edema test with low gastric ulcerogenicity compared with indomethacin. Furthermore, molec-
ular modeling studies were performed in order to rationalize the obtained biological results.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Heteroarylacetic acids; 1,2,4-Triazole; Acylation; Anti-inflammatory; Docking; COX-2
1. Introduction
only in the reduction of the ulcerogenic effect but also in an
increased anti-inflammatory activity [5e7].
Non-steroidal anti-inflammatory drugs (NSAIDs), which
are widely used for reducing pain and swelling associated
with inflammation, represent a research area of continuous
and ever-growing development. Aryl- and heteroarylacetic
acids are well-established class of non-steroidal anti-inflam-
matory agents therapeutically useful in the treatment of acute
and chronic inflammatory conditions. This class had produced
many pharmacologically interesting compounds, several of
which had been in clinical use, e.g., indomethacin and tolme-
tin, Fig. 1.
Furthermore, it had been reported that many compounds
having a 1,2,4-triazole skeleton possessed significant anti-in-
flammatory activity [8e12].
On the basis of the abovementioned reports, the present
work is concerned with the synthesis of different 1,2,4-triazo-
lylacetic acid derivatives with the objective of discovering
novel and potent anti-inflammatory agents that might be de-
void of the gastrointestinal side effects.
In the past few years, research for new non-steroidal anti-
inflammatory agents had focused on numerous structural pat-
terns of aryl- and heteroarylacetic acids [1e4].
Besides, studies that described simple chemical derivatiza-
tion (esterification or amidation) of the carboxylic function of
representative NSAIDs showed that this approach resulted not
* Corresponding author. Tel.: þ20 101698023; fax: þ20 882332776.
E-mail address: ashraf.elruby@yahoo.com (A.M. Abdel-Megeed).
Fig. 1. Examples from heteroarylacetic acid class of NSAIDs.
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.017

118
A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
Scheme 1. Synthetic pathway for intermediates 1 and 2.
2. Results and discussion
solvent, e.g., sulfolane, dimethylformamide, dimethylsulfox-
ide or without solvent at a temperature of 200e250 ꢀC to
give the 5-benzamido-1,2,4-triazole derivatives [15]. In the
present investigation, compounds 4aee were synthesized in
a quantitative yield by heating the corresponding 1-acyl deriv-
atives 3aee, without solvent, above their melting points,
Scheme 2.
2.1. Chemistry
Intramolecular condensation in alkaline medium of acyl de-
rivatives of aminoguanidine is a general method used for the
preparation of 3-amino-1,2,4-triazoles [13]. In the present in-
vestigation, the starting compound, 5-amino-1H-1,2,4-tria-
zole-3-acetic acid 1 was prepared by gradual addition of
aminoguanidine bicarbonate to a hot solution of malonic
acid to give the malonylaminoguanidine followed by cycliza-
tion in alkaline medium using potassium hydroxide. Upon
acidification of the formed potassium salt with concentrated
hydrochloric acid to pH 4e5, the desired 5-amino-1H-1,2,4-
triazole-3-acetic acid was obtained. Esterification of acid 1
was carried out by refluxing in ethanol saturated with hydro-
gen chloride gas to afford ethyl 5-amino-1H-1,2,4-triazole-3-
acetate 2, Scheme 1 [14].
Acylation of aminotriazoles with acid chlorides at room
temperature yielded a ring-acylated product rather than the
amino-acylated one [15]. Therefore, compound 2 was acylated
at room temperature with different (un)substituted benzoyl
chlorides in an organic aprotic solvent as dioxane, in the pres-
ence of an organic base as pyridine, to yield 1-benzoylated-
1,2,4-triazole derivatives 3aee, Scheme 2.
Acylation of 5-amino-3-substituted-1,2,4-triazoles with
acyl chlorides to yield either a ring-acylated product A or its
5-acylamino isomer B was intensively studied in the past 50
years by many authors [15e18], Fig. 2. Nevertheless, struc-
tural elucidation of the obtained compounds was in many
cases ambiguous. Reiter et al. [18] reported a detailed study
on the isomeric and tautomeric structures of the monoacylated
5-amino-1,2,4-triazole derivatives with the help of the spectro-
scopic data.
Studying the isomeric structures of type A and B monoacy-
lated 5-amino-1,2,4-triazole derivatives with the help of their
spectroscopic data resulted in the following conclusions.
In the IR spectra of the type A derivatives, the NH2 bands
(regardless on the type of R and R1) appeared in the region be-
tween 3400 and 3465 cmꢁ1. On the other hand, the corre-
sponding NH bands of the type B derivatives appeared as
broad bands never exceeding the value of 3300 cmꢁ1 giving
a good possibility for the differentiation between them. The
range of the carbonyl frequencies of type A derivatives
(1680e1700 cmꢁ1) practically overlapped the range of those
Interestingly, the 1-benzoylated-1,2,4-triazole derivatives
underwent thermal rearrangement either in an organic inert
Scheme 2. Synthetic pathway for target compounds 3aee and 4aee.

A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
119
Table 1
Anti-inflammatory activity of 1,2,4-triazole-3-acetic acid derivatives 1e4 at
5 mg/kg dose level
Compound
Anti-inflammatory activity
(5 mg/kg, oral dose) % inhibition ꢂ S.E.
1h
2h
3h
Control
Indomethacin
0.00
0.00
0.00
31.5 ꢂ 2.32
22.3 ꢂ 6.28
25.0 ꢂ 4.11
31.6 ꢂ 1.54*
34.2 ꢂ 2.54**
34.8 ꢂ 2.87**
32.8 ꢂ 2.98**
36.1 ꢂ 1.87**
26.8 ꢂ 5.11*
31.1 ꢂ 3.60*
31.3 ꢂ 2.59*
30.2 ꢂ 4.11*
32.6 ꢂ 2.33**
40.1 ꢂ 2.91
29.5 ꢂ 6.11
33.6 ꢂ 3.94
38.5 ꢂ 2.54*
42.3 ꢂ 1.45**
43.0 ꢂ 2.63**
41.5 ꢂ 3.54**
46.3 ꢂ 2.45**
36.2 ꢂ 4.89*
39.1 ꢂ 3.13*
40.4 ꢂ 3.44*
38.2 ꢂ 4.60*
42.1 ꢂ 1.89**
60.8 ꢂ 3.15
48.1 ꢂ 6.34
51.8 ꢂ 3.93
57.5 ꢂ 2.90*
62.8 ꢂ 1.22**
64.1 ꢂ 1.45**
60.0 ꢂ 3.87**
68.2 ꢂ 2.91**
55.3 ꢂ 4.66*
58.5 ꢂ 2.48*
58.8 ꢂ 2.45*
56.0 ꢂ 3.85*
59.8 ꢂ 2.77**
1
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
Fig. 2. Isomeric structures of monoacylated 5-amino-1,2,4-triazole derivatives.
of the corresponding type B derivatives (1665e1685 cmꢁ1).
However, the carbonyl frequency of the type A derivatives
was in all cases higher than that of the corresponding type B
derivatives.
*Significant difference at P < 0.05; **Significant difference at P < 0.01.
In the 1H NMR spectra of type A derivatives, the NH2 pro-
tons appeared as singlets between 7.8 and 8.3 ppm. On the other
hand, the two broad well-separated NH signals of type B deriv-
atives (NHCOR1 and N1eH) appeared between 12.3e12.8 ppm
(NHCOR1) and 13.2e14.3 ppm (N1eH), respectively. This
again makes it possible to differentiate between ring-acylated
products A and their 5-acylamino isomers B.
4b. Results indicated that compounds 3b and 4b did not in-
duce any ulcerogenic effect at 10 mg/kg dose. At higher doses,
the tested compounds exhibited low gastric ulcerogenicity
compared with indomethacin, which caused severe ulceration
at all doses, Table 2.
2.4. Acute toxicity (LD50)
2.2. Biological evaluation
The acute toxicity of the most active compounds; 3b, 3c
and 3e was determined by calculating their LD50 values by us-
ing graphical method [21]. The LD50 of compounds 3b, 3c and
3e was found to be 130, 120, and 90 mg/kg, respectively, while
LD50 of indomethacin was found to be 50 mg/kg.
The preliminary anti-inflammatory activity of the synthe-
sized 1,2,4-triazole-3-acetic acid derivatives 1e4 was evalu-
ated against carrageenan-induced rat paw edema using the
method by Kasahara et al. [19].
Results of the anti-inflammatory activity of the tested com-
pounds as well as indomethacin are shown in Table 1. Results
showed that most of the tested compounds exhibited signifi-
cant (P < 0.05) inhibition against carrageenan-induced rat
paw edema and comparable anti-inflammatory activity relative
to indomethacin. Among these derivatives, compounds 3b, 3c
and 3e were found to be more potent than indomethacin.
From the biological activity studies we concluded that (i)
ethyl 5-amino-1H-1,2,4-triazole-3-acetate 2 showed higher
anti-inflammatory activity than the acid derivative 1. (ii) 1-Ac-
ylated-5-amino-1,2,4-triazole-3-acetates 3aee showed higher
anti-inflammatory activity than the corresponding 5-acylamino
derivatives 4aee. (iii) Among the acyl substituents in the
1-acylated and/or the 5-acylamino derivatives, the order
of the anti-inflammatory activity was 4-nitrobenzoyl >
4-bromobenzoyl > 4-chlorobenzoyl > 4-methoxybenzoyl >
unsubstituted benzoyl.
2.5. Molecular modeling study
Molecular dockings as well as conformational alignment
studies of compounds 3aee and 4aee were performed in or-
der to rationalize the obtained biological results. Besides, mo-
lecular docking studies helped in understanding the various
Table 2
Ulcerogenic effects of compounds 3b and 4b in comparison with
indomethacin
Compound
Dose mg/kg
Ratio of ulcerated animals
Ulcer index
(mean ꢂ S.E.)
2.5** ꢂ 0.18
1.9** ꢂ 0.15
2.1** ꢂ 0.17
0.00
Indomethacin
10
50
3/6
5/6
6/6
0/6
1/6
1/6
0/6
0/6
1/6
100
10
3b
4b
50
0.50 ꢂ 0.1
0.75* ꢂ 0.12
0.00
100
10
2.3. Gastric ulceration
50
100
0.00
0.75* ꢂ 0.1
Gastric ulcerogenic effects were determined in rats [20] for
representative examples of the synthesized compounds, 3b and
*Significant difference at P < 0.05; **Significant difference at P < 0.01.

120
A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
Fig. 3. Docking of the acetic acid analogue of compound 3b (ball and stick) in
the active site of murine COX-2. Hydrogen atoms of the amino acid residues
have been removed to improve clarity.
Fig. 5. Docking of the acetic acid analogue of compound 4b (ball and stick) in
the active site of murine COX-2. Hydrogen atoms of the amino acid residues
have been removed to improve clarity.
interactions between the ligands and enzyme active site in de-
tail. Since it is highly probable that the investigated com-
pounds 3aee and 4aee undergo in vivo enzymatic cleavage
of the ester moiety to afford the corresponding acetic acid an-
alogues, molecular docking studies for these acetic acid deriv-
atives were performed.
Docking studies of the inhibitors were performed by MOE
(Molecular Operating Environment) [22] using murine COX-2
co-crystallized with indomethacin (PDB ID: 4COX) as a tem-
plate. We performed 100 docking iterations for each ligand
and the top-scoring configuration of each of the ligandeen-
zyme complexes was selected on energetic grounds.
Docking of the acetic acid analogue of compound 3b,
Fig. 3, showed that the ligand was oriented so that the carbox-
ylate moiety was in the vicinity of Arg120 residue forming an
ionic interaction with the guanidinium side chain (dis-
˚
tance ¼ 2.45 A). A hydrogen bond interaction between the li-
gand carboxylate and the OH group of Tyr355 was also
˚
observed (distance ¼ 2.3 A). Furthermore, the carbonyl group
Fig. 4. Conformational alignment of indomethacin from the crystal structure of
indomethacinemurine COX-2 complex (red) and that of compound 3b acetic
acid analogue from the docking simulation (green). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web ver-
sion of this article.)
Fig. 6. Conformational alignment of indomethacin from the crystal structure of
indomethacinemurine COX-2 complex (red) and that of compound 4b acetic
acid analogue from the docking simulation (green). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web ver-
sion of this article.)

A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
121
of the ligand’s benzoyl moiety formed a hydrogen bond inter-
˚
4. Experimental
action with the Ser530 side chain (distance ¼ 3.03 A). The p-
chlorophenyl moiety was located in the hydrophobic pocket
near Tyr385 forming hydrophobic interactions with Leu352,
Phe381, Leu384, Tyr385, and Trp387 residues. The 1,2,4-tria-
zole ring is surrounded by Val349, Met522, and Ala527 resi-
dues. This arrangement is also common to all acetic acid
analogues of 1-acyl-5-amino-1,2,4-triazole-3-acetates 3aee
with small differences in contacts with the hydrophobic pocket.
Conformational superposition of indomethacin (from the
X-ray crystal structure of indomethacin-COX-2 complex)
and the acetic acid analogue of compound 3b (from the dock-
ing simulation) are shown in Fig. 4. The superposition showed
their hydrophilic and hydrophobic groups overlapped with
each other.
From the abovementioned data, the molecular modeling
studies of the examined compounds 3a,b and their acetic
acid analogues showed that they bound to the COX-2 active
site with position and orientation very close to that resulting
from the crystal structure of indomethacin complex with
COX-2 [23]. Consequently, these observations provided
a good explanation for the observed potent inhibitory activity
of compounds 3aee.
4.1. Chemistry
Melting points were determined using an electrothermal ap-
paratus (Stuart Scientific, England) and were uncorrected. IR
spectra were recorded as KBr disk using Shimadzu IR 200-
91527 spectrophotometer (Schimadzu Corp., Kyoto, Japan)
and the data are given in ymax (cmꢁ1). H NMR (60 MHz)
1
spectra were carried out on Varian EM-360L, 60 MHz, (Var-
ian, palo Alto, CA, USA) using DMSO-d6 as a solvent and
the chemical shifts are given in d (ppm). All NH and NH2 pro-
tons were exchangeable with D2O. Mass spectra were per-
formed on Joel, JMS-600 spectrometer at an ionization
voltage of 70 eV (Joel, Tokyo, Japan). Elemental analyses
were performed on ‘‘Analytischer Funktionstest vario EL
Fab.-Nr. 11982027’’ (Germany). Analyses indicated by the
symbols of the elements functions were within ꢂ0.4% of the
theoretical values. All reactions were monitored by thin-layer
chromatography (TLC) using silica gel 60 GF245 precoated
sheets 20 ꢃ 20 cm, layer thickness 0.2 mm (E-Merck, Ger-
many) and were visualized by UV-lamp at wave length (l)
254 nm. All chemicals and solvents were of reagent grade
and the latter were distilled and dried before use.
On the other hand, docking of the acetic acid analogue of
4b into the COX-2 active site, Fig. 5, showed almost similar
contacts as observed for the corresponding 1-acyl derivative
with one missing feature. Unfortunately, the interaction be-
tween the benzoyl carbonyl group of the ligand and the
Ser530 side chain was lost. Accordingly, the parent triazole
ring was oriented so that the carboxylate moiety was posi-
tioned near the mouth of COX-2 active site forming ionic
4.1.1. Synthesis of 5-amino-1H-1,2,4-triazole-3-acetic
acid (1) [14]
Aminoguanidine bicarbonate (13.6 g, 0.1 mol) was added
gradually with stirring to a hot solution (60e70 ꢀC) of malonic
acid (10.4 g, 0.1 mol) in water (30 mL). The mixture was
heated on a boiling water bath for 4 h and cooled to room tem-
perature. Potassium hydroxide (10 g) was then added in por-
tions and the mixture was heated to 100 ꢀC for 2 h. The
mixture was then cooled and acidified with concentrated hy-
drochloric acid to pH 3e4. The precipitated product was fil-
tered off and crystallized from hot water to yield 9.3 g
(65%), mp 190e192 ꢀC (as reported) [14].
˚
bond with the guanidino group of Arg120 (distance ¼ 2.51 A).
The p-chlorophenyl moiety was located in the vicinity of the
aromatic pocket forming hydrophobic interactions with
Val349, Leu352, Phe381, Tyr385, Trp387, Phe518, and
Met522. This arrangement is also common to all acetic acid
analogues of 5-acylamino-1,2,4-triazole-3-acetates 4aee
with small differences in contacts with the hydrophobic
pocket.
4.1.2. Synthesis of ethyl 5-amino-1H-1,2,4-triazole-3-
acetate (2) [14]
The conformational superposition of indomethacin (from
the X-ray crystal structure of indomethacin-COX-2 complex)
and the acetic acid analogue of compound 4b (from the dock-
ing simulation) are shown in Fig. 6.
A suspension of compound 1 (4.0 g, 0.028 mol) in ethanol
(50 mL) saturated with hydrogen chloride gas was refluxed for
4 h. The reaction mixture was cooled to room temperature and
adjusted to pH 7e8 with potassium hydroxide solution (20%
w/v). The solvent was evaporated under reduced pressure
and the residue was crystallized from hot water. Yield 64%,
mp 160e162
3. Conclusion
ꢀC. IR (KBr, cmꢁ1): 3425e3255 (NH2, NH),
We reported here the synthesis of different acylated 1,2,4-
triazole-3-acetates. The synthesized compounds were tested
for their anti-inflammatory activity. Results showed that the
most tested compounds exhibited significant anti-inflamma-
tory activity in the carageenan-induced rat paw edema test
with low gastric ulcerogenicity compared with indomethacin.
Furthermore, the molecular modeling studies suggested that
the 1-acyl-5-amino-1,2,4-triazole derivatives 3aee exhibited
higher affinity for the COX-2 active site and hence increased
anti-inflammatory activity than the corresponding 5-acyla-
mino-1,2,4-triazole derivatives 4aee.
1
1719 (C]O), 1654, 1611 (C]N), and 1199 (CeOeC). H
NMR (60 MHz, DMSO-d6, d ppm): 1.1 (t, 3H, CH2CH3);
3.6 (s, 2H, CH2CO); 4.2 (q, 2H, CH2CH3); 6.2 (s, 2H, NH2 ex-
changeable); 12.2 (br s, 1H, N1eH exchangeable).
4.1.3. General procedure for the synthesis of ethyl 5-amino-1-
(un)substituted benzoyl-1H-1,2,4-triazole-3-acetate (3aee)
A solution of the (un)substituted benzoyl chloride
(0.01 mol) in dioxane (5 mL) was added dropwise to a mixture
of compound 2 (1.7 g, 0.01 mol), pyridine (1.2 mL, 0.015 mol)

122
A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
and dioxane (10 mL) with constant stirring at 0e5 ꢀC. The re-
action mixture was stirred for 30 min at this temperature, then
for 4 h at room temperature. The mixture was then poured onto
water (50 mL) and the formed precipitate was filtered, washed
with water and crystallized from methanol.
(br s, 1H, NHCO exchangeable); 14.3 (br s, 1H, N1eH ex-
changeable). Anal. (C13H14N4O3) C, H, N.
4.1.4.2. Ethyl 5-(4-chlorobenzamido)-1H-1,2,4-triazole-3-ace-
tate (4b). Yield 96%; mp 273e275 ꢀC. IR (KBr, cmꢁ1):
3290, 3150 (NH), 1732, 1677 (C]O ester, C]O amide).
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.1 (t, 3H,
4.1.3.1. Ethyl 1-benzoyl-5-amino-1H-1,2,4-triazole-3-acetate
(3a). Yield 69%; mp 126e128 ꢀC. IR (KBr, cmꢁ1): 3415
(NH2), 1734, 1691 (C]O ester, C]O amide). 1H NMR
(60 MHz, DMSO-d6, d ppm): 1.1 (t, 3H, CH2CH3); 3.6 (s,
2H, CH2CO); 4.2 (q, 2H, CH2CH3); 7.9 (s, 2H, NH2 exchange-
able); 8.3 (m, 5H, Ar-H). Anal. (C13H14N4O3) C, H, N.
CH2CH3); 3.8 (s, 2H, CH2CO); 4.3 (q, 2H, CH2CH3); 7.9
(d, 2H, Ar-H); 8.4 (d, 2H, Ar-H); 12.5 (br s, 1H, NHCO ex-
changeable); 13.7 (br s, 1H, N1eH exchangeable). MS (EI):
m/z 308 [3%, Mþ], m/z 310 [1.1%, M þ 2]. Anal.
(C13H13ClN4O3) C, H, N.
4.1.4.3. Ethyl 5-(4-bromobenzamido)-1H-1,2,4-triazole-3-ace-
tate (4c). Yield 96%; mp 270e272 ꢀC. IR (KBr, cmꢁ1):
3270, 3075 (NH), 1728, 1669 (C]O ester, C]O amide).
4.1.3.2. Ethyl 1-(4-chlorobenzoyl)-5-amino-1H-1,2,4-triazole-
3-acetate (3b). Yield 75%; mp 164e165 ꢀC. IR (KBr,
cmꢁ1): 3415 (NH2), 1731, 1684 (C]O ester, C]O amide).
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.2 (t, 3H,
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.2 (t, 3H,
CH2CH3); 3.8 (s, 2H, CH2CO); 4.3 (q, 2H, CH2CH3); 8.1
(d, 2H, Ar-H); 8.5 (d, 2H, Ar-H); 12.4 (br s, 1H, NHCO ex-
changeable); 13.2 (br s, 1H, N1eH exchangeable). Anal.
(C13H13BrN4O3) C, H, N.
CH2CH3); 3.6 (s, 2H, CH2CO); 4.1 (q, 2H, CH2CH3); 7.6
(d, 2H, Ar-H); 7.8 (s, 2H, NH2 exchangeable); 8.0 (d, 2H,
Ar-H). MS (EI): m/z 308 [11%, Mþ], m/z 310 [3.7%,
M þ 2]. Anal. (C13H13ClN4O3) C, H, N.
4.1.4.4. Ethyl 5-(4-methoxybenzamido)-1H-1,2,4-triazole-3-ac-
etate (4d). Yield 95%; mp 256e258 ꢀC. IR (KBr, cmꢁ1):
3275, 3120 (NH), 1733, 1672 (C]O ester, C]O amide).
4.1.3.3. Ethyl 1-(4-bromobenzoyl)-5-amino-1H-1,2,4-triazole-
3-acetate (3c). Yield 71%; mp 165e167 ꢀC. IR (KBr,
cmꢁ1): 3400 (NH2), 1724, 1680 (C]O ester, C]O amide).
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.1 (t, 3H,
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.1 (t, 3H,
CH2CH3); 3.8 (s, 2H, CH2CO); 4.0 (s, 3H, OCH3); 4.2 (q,
2H, CH2CH3); 7.3 (d, 2H, Ar-H); 8.2 (d, 2H, Ar-H); 12.6
(br s, 1H, NHCO exchangeable); 14.3 (br s, 1H, N1eH ex-
changeable). Anal. (C14H16N4O4) C, H, N.
CH2CH3); 3.6 (s, 2H, CH2CO); 4.2 (q, 2H, CH2CH3); 8.1
(d, 2H, Ar-H); 8.2 (s, 2H, NH2 exchangeable); 8.5 (d, 2H,
Ar-H). Anal. (C13H13BrN4O3) C, H, N.
4.1.3.4. Ethyl 1-(4-methoxybenzoyl)-5-amino-1H-1,2,4-tria-
zole-3-acetate (3d). Yield 66%; mp 137e139 ꢀC. IR (KBr,
cmꢁ1): 3465 (NH2), 1733, 1684 (C]O ester, C]O amide).
4.1.4.5. Ethyl 5-(4-nitrobenzamido)-1H-1,2,4-triazole-3-ace-
tate (4e). Yield 92%; mp > 300 ꢀC. IR (KBr, cmꢁ1): 3190,
3090 (NH), 1728, 1670 (C]O ester, C]O amide). 1H
NMR (60 MHz, DMSO-d6, d ppm): 1.2 (t, 3H, CH2CH3);
3.7 (s, 2H, CH2CO); 4.3 (q, 2H, CH2CH3); 8.8 (m, 4H, Ar-
H); 12.4 (br s, 1H, NHCO exchangeable); 13.2 (br s, 1H,
N1eH exchangeable). Anal. (C13H13N5O5) C, H, N.
1H NMR (60 MHz, DMSO-d6,
d ppm): 1.1 (t, 3H,
CH2CH3); 3.7 (s, 2H, CH2CO); 4.0 (s, 3H, OCH3); 4.2 (q,
2H, CH2CH3); 7.4 (d, 2H, Ar-H); 8.2 (s, 2H, NH2 exchange-
able); 8.7 (d, 2H, Ar-H). Anal. (C14H16N4O4) C, H, N.
4.1.3.5. Ethyl 1-(4-nitrobenzoyl)-5-amino-1H-1,2,4-triazole-3-
acetate (3e). Yield 63%; mp 263e265 ꢀC. IR (KBr, cmꢁ1):
3400 (NH2), 1729, 1690 (C]O ester, C]O amide). 1H
NMR (60 MHz, DMSO-d6, d ppm): 1.2 (t, 3H, CH2CH3);
3.7 (s, 2H, CH2CO); 4.3 (q, 2H, CH2CH3); 8.3 (s, 2H, NH2 ex-
changeable); 8.8 (m, 4H, Ar-H). Anal. (C13H13N5O5) C, H, N.
4.2. Anti-inflammatory activity
Adult male albino rats of average weight (100 g ꢂ 10%)
were divided into groups, each of six rats. Each group was
treated with a suspension of the tested compound or the refer-
ence drug orally by gastric tubes at dose level of 5 mg/kg. The
control animal group, on the other hand, was treated with the
vehicle, CMC. After 30 min, 0.1 mL of freshly prepared carra-
geenan solution (1% in normal saline) was injected into the
subplanar region of the right hind paw of each rat. The thick-
ness of rat paw was measured at different time intervals (60,
120, 180 min) after administration of the test samples. The dif-
ference between the thicknesses of two paws (right and left)
was taken as a measure of edema.
4.1.4. General procedure for the synthesis of ethyl 5-
(un)substituted benzamido-1H-1,2,4-triazole-3-acetate (4aee)
The appropriate ethyl 1-acyl-5-amino-1,2,4-triazole-3-ace-
tate 3aee (0.01 mol) was heated above the melting points (at
220e270 ꢀC) for 10 min without solvent. After cooling, the
product obtained was collected and crystallized from dioxane.
4.1.4.1. Ethyl 5-benzamido-1H-1,2,4-triazole-3-acetate (4a).
Yield 95%; mp 256e258 ꢀC. IR (KBr, cmꢁ1): 3235, 3110
(NH), 1735, 1673 (C]O ester, C]O amide). 1H NMR
(60 MHz, DMSO-d6, d ppm): 1.1 (t, 3H, CH2CH3); 3.8 (s,
2H, CH2CO); 4.2 (q, 2H, CH2CH3); 7.3 (m, 5H, Ar-H); 12.8
4.3. Gastric ulcerogenic effect
Adult male albino rats (120e200 g) were divided into
groups; each of six animals. Animals were starved for 24 h

A.M. Abdel-Megeed et al. / European Journal of Medicinal Chemistry 44 (2009) 117e123
123
before the experiment but had free access to water. The an-
imals were then treated orally by means of a stomach tube
with suspensions of the tested compounds and the reference
drug in aqueous solution of carboxymethyl cellulose, CMC,
(0.5% w/v) at dose levels of 10, 50, 100 mg/kg. Control
animals were treated with an equal volume of 0.5%
carboxymethyl cellulose. After 6 h, the rats were sacrificed
and the stomachs were removed, dissected along the greater
curvature and washed with water. The lesions in gastric
mucosa were determined by using stereoscopic microscope.
Ulcer was defined as at least one lesion that was 0.5 mm
or more in length. Lesion size (mm) was measured along
its greatest length and in the case of patches; five such
lesions were considered the equivalent of a 1 mm ulcer.
The sum of the lesions’ lengths in each group of animals
was divided by their number and expressed as the gastric
ulcer index.
alpha spheres. (iv) The obtained model was then used in pre-
dicting the ligandeenzyme interactions at the active site.
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A
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