Synthesis of [1,2,4]triazolo[1,5-a]pyridines of potential PGE2 inhibitory properties

Article abstract of DOI:10.1016/j.ejmech.2008.09.049

A variety of 5-amino-6,8-dicyano-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate containing compounds 3a-i, 5a-c were prepared via reaction of arylidenemalononitriles 1a-c, 4a and 4b with 2-[(substituted amino)thiocarbonyl]cyanoacetohydrazides 2a-d in r

Full text of DOI:10.1016/j.ejmech.2008.09.049

European Journal of Medicinal Chemistry 44 (2009) 1972–1977
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of [1,2,4]triazolo[1,5-a]pyridines of potential PGE2
inhibitory properties
,
b
Adel S. Girgis a , Flora F. Barsoum
*
a Pesticide Chemistry Department, National Research Centre, El-Behoos Street, Dokki, 12622 Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A
variety of 5-amino-6,8-dicyano-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate containing
Received 10 June 2008
Received in revised form
23 September 2008
Accepted 30 September 2008
Available online 11 October 2008
compounds 3a–i, 5a–c were prepared via reaction of arylidenemalononitriles 1a–c, 4a and 4b with 2-
[(substituted amino)thiocarbonyl]cyanoacetohydrazides 2a–d in refluxing ethanol in the presence of
triethylamine. Anti-inflammatory activity screening of the synthesized compounds (at a dose of 50 mg/kg
body weight) utilizing in vivo acute carrageenan-induced paw oedema standard method in rats exhibited
that the prepared heterocycles possess considerable pharmacological properties especially, 3f, 3h, 5b and
5c which reveal remarkable activities relative to indomethacin (which was used as a reference standard at
a dose of 10 mg/kg body weight). PGE2 inhibitory properties of the highly promising synthesized anti-
inflammatory active agents (3f, 3h, 5b and 5c) were determined by PGE2 assay kit technique, which reveal
remarkable activity coinciding greatly with the observed anti-inflammatory properties. Anti-tumor
activity screening of 3b and 3e, as representative examples of the synthesized compounds, at a dose of
Keywords:
5-Amino-6,8-dicyano-1H-
[1,2,4]triazolo[1,5-a]pyridines
Ylidenemalononitriles
2-[(Substituted
amino)thiocarbonyl]cyanoacetohydrazides
Anti-inflammatory
PGE2
10 mM utilizing 59 different human tumor cell lines, representing leukemia, melanoma and cancers of the
lung, colon, brain, ovary, breast, prostate and kidney exhibited that the tested compounds reflect mild or
no activity at all against most of the used human tumor cell lines. However, compound 3e reveals
considerable anti-tumor properties against leukemia CCRF-CEM and HL-60(TB) cell line.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Anti-tumor
1. Introduction
two isoforms of the COX enzyme [5,6]. One isoform (COX-1) is
constitutive and regularly expressed producing prostaglandins
Non-steroidal anti-inflammatory drugs (NSAIDs) are mainly
used in the treatment of pain and inflammation related to a large
variety of pathologies [1,2]. Their mechanism of action involves
inhibition of production of prostaglandins by the enzyme cyclo-
oxygenase (COX) [3]. Prostaglandins are among the most important
mediators of inflammation. They promote blood vessel dilation and
vascular permeability, causing the typical redness, heat and
swelling phenomena involved in inflammation. Moreover, they
promote pain transmission from nociceptors to the brain by
increasing the sensitivity of the nerve endings. However, prosta-
glandins also play a cytoprotective role in the gastrointestinal tract
and they are necessary for normal platelet aggregation and renal
function. Therefore, a reduction in the production of circulating
prostaglandins leads to numerous side effects, the most important
being gastrointestinal ulcers [4]. The double nature (i.e. inflam-
mation reduction vs. gastrolesivity) of the effects caused by early
NSAIDs acting as COX inhibitors was explained by the discovery of
involved in the cytoprotection of the gastrointestinal tract. The
other isoform (COX-2) is associated with inflammatory states and is
generally absent from all tissues, unless induced by inflammation
mediators. Therefore, the inducible isoform COX-2 constitutes the
real target for anti-inflammatory drugs. Several lines of evidence
suggest that selective COX-2 inhibitors may also provide an
opportunity for both cancer prevention and therapy [7]. Further-
more, promising in vitro data also indicate that treatment with
selective COX-2 inhibitors may reduce the risk of Alzheimer’s [8,9]
and Parkinson’s diseases [10,11] and may also be effective in the
treatment of asthma [12].
In the present work, it is intended to investigate synthesis of
[1,2,4]triazolo[1,5-a]pyridine containing compounds adopting
facile synthetic approaches and utilizing easily accessible starting
chemicals. The anti-inflammatory properties of the synthesized
heterocycles will be screened. The prostaglandin (PGE2) inhibitory
activity for the most promising prepared anti-inflammatory active
agents will be determined. Additionally, anti-tumor properties of
representative examples of the prepared heterocycles will be
investigated against various human tumor cell lines. The interest for
construction of this heterocyclic nucleus is attributed to the
* Corresponding author. Tel.: þ20 2 22352405; fax: þ20 2 33370931.
E-mail address: girgisas10@yahoo.com (A.S. Girgis).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.049

A.S. Girgis, F.F. Barsoum / European Journal of Medicinal Chemistry 44 (2009) 1972–1977
1973
biological and pharmacological properties associated with its
structure. Where many publications reported their efficiency as
adenosine (A2a) receptor antagonist [13–18], ALK5 receptor modu-
lator activity [19] beside anti-implantation properties (pregnancy-
terminating activity) [20]. Other reports describe their ability to
inhibit secretion of gastric acid so, useful for treating gastrointes-
tinal disorders [21], in addition to their anti-tumor [22], antibac-
terial [23,24], antifungal [25] and herbicidal [26–28] properties.
The reaction was assumed to proceed via active methylene
nucleophilic attack of 2 at the -carbon of -unsaturated dinitrile
b
a,b
system of 1 followed by cyclization, dehydration with subsequent
dehydrogenation giving finally 3. Similarly, reaction of bis(ylide-
nemalononitrile) containing compounds 4a, 4b with 2a, 2b under
similar reaction conditions, proceeded smoothly affording the
corresponding bis([1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolates)
5a–c (Scheme 2).
2. Results and discussion
2.2. Anti-inflammatory activity
2.1. Chemistry
Anti-inflammatory activity of the synthesized compounds 3a–i,
5a–c (at a dose of 50 mg/kg body weight) was determined in vivo
by the acute carrageenan-induced paw oedema standard method in
rats [29–32]. The anti-inflammatory properties were recorded at
successive time intervals (1, 2, 3 and 4 h) and compared with that of
indomethacin (at a dose of 10 mg/kg body weight) which was used
as a reference standard. From the obtained results (Table 1), it has
been noticed that many of the tested compounds exhibit consid-
erable anti-inflammatory properties, especially 3f, 3h, 5b and 5c
which reveal remarkable activities with potency (% oedema inhi-
bition of the tested compounds relative to % oedema inhibition of
indomethacin) 99.4, 101.0, 110.9 and 114.0, respectively.
Reaction of arylidenemalononitriles 1a–c with 2-[(substituted
amino)thiocarbonyl]cyanoacetohydrazides 2a–d in refluxing
ethanol in the presence of triethylamine as a basic catalyst, afforded
directly
5-amino-6,8-dicyano-1,7-disubstituted-1H-[1,2,4]tri-
azolo[1,5-a]pyridin-4-ium-2-thiolates 3a–i. The structure of 3 was
established through spectroscopic (IR, 1H NMR) as well as
elemental analyses data. The IR spectra of 3a–i reveal the presence
of amino stretching vibration bands at
the nitrile stretching vibration band at
n
¼ 3350–3130 cmꢀ1 besides
n
¼ 2225–2220 cmꢀ1
.
1H
NMR spectra of 3a–i add a sharp evidence for the presumed
structure, exhibiting only one D2O exchangeable amino signal at
Structure–activity relationships based on the obtained results
indicated that, the type of the alicyclic-amino moiety attached to
the phenyl group oriented at the 7-position of heterocyclic nucleus
seems to be a controlling factor in developing the total pharma-
cological properties. Utilization of piperidinyl function seems more
favorable than the case of using morpholinyl residue for
d
¼ 9.00–9.44, excluding any other assumed pyridine intermediate
isolation. The alicyclic-amino moiety signals (1.59–1.61, 3.26–3.37
‘‘assignable for piperidinyl 3a, 3d, 3f, 3h’’; 3.21–3.29, 3.74–3.77
‘‘assignable for morpholinyl 3b, 3e, 3g, 3i’’ and 2.27, 2.53 ‘‘assign-
able for piperazinyl 3c’’) are also well recognized (Scheme 1).
X
N
S
H
N
EtOH, Et3N
X
N
CH=C(CN)2
+
NC
N
H
NHR
O
NC
CN
O
(2)
(1)
H2N
N
NHCSNHR
-H2O
-2H
1a, X = CH2
1b, X = O
3a, R = CH3, X = CH2
3b, R = CH3, X = O
X
N
1c, X = NCH3
3c, R = CH3, X = NCH3
3d, R = C2H5, X = CH2
3e, R = C2H5, X = O
2a, R = CH3
2b, R = C2H5
3f, R = CH2Ph, X = CH2
3g, R = CH2Ph, X = O
NC
CN
2c, R = CH2Ph
2d, R = CH2CH=CH2
R
+
H2N
N
N
N
3h, R = CH2CH=CH2, X = CH2
3i, R = CH2CH=CH2, X = O
S
(3)
Scheme 1.

1974
A.S. Girgis, F.F. Barsoum / European Journal of Medicinal Chemistry 44 (2009) 1972–1977
O(CH2)nO
CH=C(CN)2
CH=C(CN)2
O(CH2)nO
(2)
NC
CN
NC
CN
N
EtOH, Et3N
R
+
R
+
H2N
N
N
N
H2N
N
(4)
N
S
S
(5)
4a, n = 2
4b, n = 3
5a, R = CH3, n = 2
5b, R = CH3, n = 3
5c, R = C2H5, n = 2
Scheme 2.
constructing an anti-inflammatory active agent as exhibited in
pairs 3a, 3b (potency, 75.4, 67.0, respectively), 3d, 3e (potency, 90.6,
81.1, respectively), 3f, 3g (potency, 99.4, 58.0, respectively) and 3h,
3i (potency, 101.0, 89.4, respectively). Alternatively, adoption of
piperazinyl function as an alicyclic-amino residue is associated
with decrease in the gained anti-inflammatory properties as
exhibited in compounds 3a, 3b and 3c (potency, 75.4, 67.0, 54.0,
respectively).
3e, 3i (potency, 67.0, 81.1, 89.4, respectively) and 5a, 5c (potency,
86.4, 114.0, respectively). It is also noticed that, construction of
compounds possessing two pharmacologically active moieties (i.e.
two triazolopyridinyl systems) is considered an effective tool for
developing an enhanced pharmacological active agent as exhibited
in compounds 5a–c.
2.3. PGE2 inhibitory properties
It is also obvious that, the length of carbon chain attached to the
1-position of heterocyclic nucleus is an effective factor governing
the observed pharmacological properties. The allyl (3 carbon chain
length) function seems more enhancing element for developing an
anti-inflammatory active agent than ethyl (2 carbon chain length)
and methyl (one carbon chain length) residues as exhibited in
compounds 3a, 3d, 3h (potency, 75.4, 90.6, 101.0, respectively), 3b,
PGE2 inhibitory properties of the most promising prepared anti-
inflammatory active agents (3f, 3h, 5b and 5c) were determined
using the previously described 6-day air pouch standard method in
rats [33] via a commercial PGE2 assay kit (R&D Systems, Inc., Min-
neapolis, USA). From the obtained results (Table 2), it has been
noticed that all the tested compounds effectively reduce PGE2 level.
Table 1
Anti-inflammatory activity of the tested compounds using acute carrageenan-induced paw oedema in rats.
Compound
Mean swelling volume ‘‘ml’’ (% inhibition of oedema)
Potencyc
1 h
2 h
3 h
4 h
Control
0.207 ꢁ 0.101b
0.332 ꢁ 0.146b
0.520 ꢁ 0.134b
0.705 ꢁ 0.134b
–
(00.0)
(00.0)
(00.0)
(00.0)
Indomethacin
0.090 ꢁ 0.081a
(56.5)
0.158 ꢁ 0.102a
(52.4)
0.180 ꢁ 0.132a
(65.4)
0.227 ꢁ 0.170a
(67.8)
100.0
75.4
67.0
54.0
90.6
81.1
99.4
58.0
101.0
89.4
86.4
110.9
114.0
3a
3b
3c
3d
3e
3f
0.202 ꢁ 0.047b
(2.4)
0.298 ꢁ 0.133
0.353 ꢁ 0.067b
(32.1)
0.345 ꢁ 0.127a
(51.1)
(10.2)
0.203 ꢁ 0.123b
0.275 ꢁ 0.109
(17.2)
0.303 ꢁ 0.086a
(41.7)
0.385 ꢁ 0.143a
(45.4)
(1.9)
0.172 ꢁ 0.063
(16.9)
0.287 ꢁ 0.170
0.322 ꢁ 0.168a
(38.1)
0.447 ꢁ 0.175a,b
(36.6)
(13.6)
0.142 ꢁ 0.123
0.110 ꢁ 0.104a
(66.9)
0.230 ꢁ 0.110a
(55.8)
0.272 ꢁ 0.121a
(61.4)
(31.4)
0.118 ꢁ 0.100
(43.0)
0.232 ꢁ 0.158
0.322 ꢁ 0.169a
(38.1)
0.317 ꢁ 0.125a
(55.0)
(30.1)
0.192 ꢁ 0.116
0.325 ꢁ 0.112b
(2.1)
0.313 ꢁ 0.144a
(39.8)
0.230 ꢁ 0.124a
(67.4)
(7.2)
3g
3h
3i
0.207 ꢁ 0.122b
(00.0)
0.328 ꢁ 0.180b
0.485 ꢁ 0.196b
(6.7)
0.428 ꢁ 0.157a,b
(39.3)
(1.2)
0.160 ꢁ 0.062
0.307 ꢁ 0.105
(7.5)
0.338 ꢁ 0.128a
(35.0)
0.222 ꢁ 0.129a
(68.5)
(22.7)
0.188 ꢁ 0.063
(9.2)
0.268 ꢁ 0.131
0.387 ꢁ 0.242b
(25.6)
0.278 ꢁ 0.190a
(60.6)
(19.3)
5a
5b
5c
0.097 ꢁ 0.096a
0.293 ꢁ 0.120
(11.7)
0.370 ꢁ 0.167b
(28.8)
0.292 ꢁ 0.250a
(58.6)
(53.1)
0.190 ꢁ 0.063
(8.2)
0.257 ꢁ 0.114
0.307 ꢁ 0.102a
(41.0)
0.175 ꢁ 0.105a
(75.2)
(22.6)
0.188 ꢁ 0.067
0.232 ꢁ 0.084
(30.1)
0.312 ꢁ 0.108a
(40.0)
0.160 ꢁ 0.066a
(77.3)
(9.2)
a
Statistically significant from the control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
Potency was expressed as % oedema inhibition of the tested compounds relative to % oedema inhibition of indomethacin ‘‘reference standard’’ at 4 h effect.
b
c

A.S. Girgis, F.F. Barsoum / European Journal of Medicinal Chemistry 44 (2009) 1972–1977
1975
It has also been noticed that the PGE2 inhibitory properties of the
tested compounds coincide greatly with our described anti-
inflammatory properties. In other words, compound 5c, which
exhibits the highest promising anti-inflammatory properties also
reveals the highest inhibitory level of PGE2 (87.67 pg/ml) compa-
rable with indomethacin (98.33 pg/ml) which was used as a refer-
ence standard in this study. Similarly, compounds 3f, 3h and 5b
reveal considerable PGE2 inhibitory activities (102.33, 94.50,
92.17 pg/ml, respectively).
while refluxing was collected, washed with ethanol and crystal-
lized from a suitable solvent affording 3a–i as yellow crystals.
3.1.1. 5-Amino-6,8-dicyano-1-methyl-7-[4-(1-piperidinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3a)
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
methanol mixture as 1:1 v/v, mp 318–320 ꢂC, yield 69%. IR: nmax
/
cmꢀ1 3330, 3138 (NH2), 2222 (C^N), 1648, 1595 (C]N, C]C). 1H
NMR: 1.61 (br. s, 6H, piperidinyl 3 CH2), 3.33 (br. s, 4H, piperidinyl
d
2 NCH2), 3.90 (s, 3H, CH3), 7.08 (d, 2H, arom. H, J ¼ 9.0 Hz), 7.41 (d,
2H, arom. H, J ¼ 9.0 Hz), 9.32 (br s, 2H, D2O exchangeable NH2).
Anal. Calcd. for C20H19N7S (389.471): C, 61.67; H, 4.92; N, 25.18.
Found: C, 61.87; H, 5.03; N, 25.27.
2.4. Anti-tumor activity
Anti-tumor activity screening of 3b and 3e, as representative
examples of the synthesized compounds adopted by develop-
mental therapeutics program of National Cancer Institute (Bethe-
3.1.2. 5-Amino-6,8-dicyano-1-methyl-7-[4-(4-morpholinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3b)
sida, Maryland, USA), at a dose of 10 mM utilizing 59 different
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
human tumor cell lines, representing leukemia, melanoma and
cancers of the lung, colon, brain, ovary, breast, prostate and kidney
was carried out according to the previously reported standard
procedure [34–37]. The obtained results represent percentage
growth of the tumor cell lines treated with compounds under
investigation relative to control cell experiments.
From the observed data it has been noticed that, the tested
compounds reflect mild or no activity at all against most of the used
human tumor cell lines. However, compound 3e exhibits consid-
erable anti-tumor properties against leukemia CCRF-CEM and HL-
60(TB) cell lines (percentage growth of the tumor cell lines are
43.55 and 42.85, respectively), considering cell line growth inhi-
methanol mixture as 1:2 v/v, mp 320–322 ꢂC, yield 69%. IR: nmax
/
cmꢀ1 3350, 3276, 3241, 3179 (NH2), 2225 (C^N), 1641, 1591 (C]N,
C]C). 1H NMR:
d
3.29 (t, 4H, morpholinyl 2 NCH2, J ¼ 4.8 Hz), 3.76
(t, 4H, morpholinyl 2 OCH2, J ¼ 4.8 Hz), 3.90 (s, 3H, CH3), 7.12 (d, 2H,
arom. H, J ¼ 9.0 Hz), 7.44 (d, 2H, arom. H, J ¼ 8.7 Hz), 9.34 (br s, 2H,
D2O exchangeable NH2). Anal. Calcd. for C19H17N7OS (391.451): C,
58.29; H, 4.38; N, 25.05. Found: C, 58.43; H, 4.46; N, 25.21.
3.1.3. 5-Amino-6,8-dicyano-1-methyl-7-[4-(4-
methylpiperazinyl)phenyl]-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-
2-thiolate (3c)
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 308–310 ꢂC, yield 67%. IR: nmax/cmꢀ1
3332, 3177 (NH2), 2220 (C^N), 1644, 1597 (C]N, C]C). 1H NMR:
bition with >50% at a concentration of 10 mM usually seems
a noticeable activity.
Finally, it could be concluded that the constructed ring system,
[1,2,4]triazolo[1,5-a]pyridine, could be a hint for developing good
promising pharmacological agent, especially as anti-inflammatory
properties. However, the variety of attached functional residues
must be considered during designing of these hint targets.
d
2.27 (s, 3H, piperazinyl NCH3), 2.53 (br s, 4H, piperazinyl 2
H3CNCH2), 3.31 (br s, 4H, piperazinyl 2 ArNCH2), 3.89 (s, 3H, CH3), 7.11
(d, 2H, arom. H, J ¼ 8.7 Hz), 7.42 (d, 2H, arom. H, J ¼ 8.7 Hz), 9.00 (br s,
2H, D2O exchangeable NH2). Anal. Calcd. for C20H20N8S (404.494): C,
59.38; H, 4.98; N, 27.71. Found: C, 59.57; H, 5.15; N, 27.91.
3. Experimental
3.1.4. 5-Amino-6,8-dicyano-1-ethyl-7-[4-(1-piperidinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3d)
Reaction time 5 h, crystallizes from n-butanol, mp 292–294 ꢂC,
yield 62%. IR: nmax/cmꢀ1 3264 (NH2), 2220 (C^N),1634,1588 (C]N,
Melting points are uncorrected and recorded on an Electro-
thermal 9100 digital melting point apparatus. IR spectra (KBr) were
recorded on Bruker Vector 22 and Jasco FT/IR plus 460 spectro-
photometers. 1H NMR spectra were recorded on a Varian MERCURY
300 (300 MHz) in DMSO-d6. The starting compounds 1a–c [38,39],
2a–d [40,41] and 4a, 4b [42] were prepared according to the
previously reported procedures.
C]C). 1H NMR:
d
1.33 (t, 3H, CH3, J ¼ 6.9 Hz), 1.61 (br s, 6H, piper-
idinyl 3 CH2), 3.37 (m, 4H, piperidinyl 2 NCH2), 4.49 (q, 2H, CH3CH2,
J ¼ 6.9 Hz), 7.08 (d, 2H, arom. H, J ¼ 9.0 Hz), 7.42 (d, 2H, arom. H,
J ¼ 8.7 Hz), 9.33 (br s, 2H, D2O exchangeable NH2). Anal. Calcd. for
C21H21N7S (403.501): C, 62.51; H, 5.25; N, 24.30. Found: C, 62.45; H,
5.18; N, 24.49.
3.1. Synthesis of 5-amino-6,8-dicyano-1,7-disubstituted-1H-
[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolates 3a–i (general
procedure)
3.1.5. 5-Amino-6,8-dicyano-1-ethyl-7-[4-(4-morpholinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3e)
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 305–307 ꢂC, yield 62%. IR: nmax/cmꢀ1
3332, 3133 (NH2), 2220 (C^N), 1648, 1594 (C]N, C]C). 1H NMR:
A mixture of equimolar amounts of 1a–c and 2a–d (10 mmol) in
absolute ethanol (25 ml) containing triethylamine (11 mmol) was
boiled under reflux for the appropriate time. The separated solid
d
1.33 (t, 3H, CH3, J ¼ 6.9 Hz), 3.24 (t, 4H, morpholinyl 2 NCH2,
J ¼ 4.8 Hz), 3.77 (t, 4H, morpholinyl 2 OCH2, J ¼ 4.8 Hz), 4.49 (q, 2H,
CH3CH2, J ¼ 6.9 Hz), 7.12 (d, 2H, arom. H, J ¼ 9.3 Hz), 7.45 (d, 2H,
arom. H, J ¼ 8.7 Hz), 9.36 (s, 2H, D2O exchangeable NH2). Anal.
Calcd. for C20H19N7OS (405.471): C, 59.24; H, 4.72; N, 24.18. Found:
C, 59.35; H, 4.82; N, 24.40.
Table 2
PGE2 inhibitory activity of the tested compounds.
Compound
Concentration of PGE2 (pg/ml)
Control
Indomethacin
3f
3h
5b
5c
533.33 ꢁ 19.26b
98.33 ꢁ 3.33a
102.33 ꢁ 4.74a
94.50 ꢁ 3.63a
92.17 ꢁ 2.86a
87.67 ꢁ 5.55a
3.1.6. 5-Amino-1-benzyl-6,8-dicyano-7-[4-(1-piperidinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3f)
Reaction time 6 h, crystallizes from N,N-dimethylformamide–
water mixture as 4:1 v/v, mp 297–299 ꢂC, yield 54%. IR: nmax/cmꢀ1
Results are means of 6 experiments ꢁ standard error.
3274 (NH2), 2220 (C^N), 1637, 1592 (C]N, C]C). 1H NMR:
d
1.59
a
Statistically significant from the control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b
(br s, 6H, piperidinyl 3 CH2), 3.26 (br s, 4H, piperidinyl 2 NCH2), 5.74

1976
A.S. Girgis, F.F. Barsoum / European Journal of Medicinal Chemistry 44 (2009) 1972–1977
(s, 2H, NCH2Ph), 7.02–7.35 (m, 9H, arom. H), 9.40 (br s, 2H, D2O
exchangeable NH2). Anal. Calcd. for C26H23N7S (465.561): C, 67.07;
H, 4.98; N, 21.06. Found: C, 66.90; H, 4.86; N, 21.24.
d
2.09 (quintet, 2H, OCH2CH2, J ¼ 6.0 Hz), 3.82 (s, 6H, 2 NCH3), 4.17
(br s, 4H, 2 OCH2), 7.15–7.55 (m, 8H, arom. H), 9.43 (s, 4H, D2O
exchangeable 2 NH2). Anal. Calcd. for C33H24N12O2S2 (684.74): C,
57.88; H, 3.53; N, 24.55. Found: C, 57.99; H, 3.57; N, 24.78.
3.1.7. 5-Amino-1-benzyl-6,8-dicyano-7-[4-(4-morpholinyl)phenyl]-
1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3g)
Reaction time 6 h, crystallizes from N,N-dimethylformamide–
water mixture as 4:1 v/v, mp 283–285 ꢂC, yield 54%. IR: nmax/cmꢀ1
3301, 3252 (NH2), 2221 (C^N), 1642, 1595 (C]N, C]C). 1H NMR:
3.2.3. 7,70-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis(5-amino-6,8-
dicyano-1-ethyl-1H-[1,2,4]triazolo[1,5-a]pyridin-
4-ium-2-thiolate) (5c)
Reaction time 24 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 308–310 ꢂC, yield 49%. IR: nmax/cmꢀ1
3270, 3177 (NH2), 2226 (C^N), 1634, 1600 (C]N, C]C). 1H NMR:
d
3.21 (t, 4H, morpholinyl 2 NCH2, J ¼ 5.1 Hz), 3.74 (t, 4H, morpholinyl
2 OCH2, J ¼ 5.1 Hz), 5.74 (s, 2H, NCH2Ph), 7.07–7.38 (m, 9H, arom. H),
9.44 (br s, 2H, D2O exchangeable NH2). Anal. Calcd. for C25H21N7OS
(467.541): C, 64.22;H, 4.53;N, 20.97. Found:C, 63.99; H, 4.39;N, 21.17.
d
1.21 (t, 3H, NCH2CH3, J ¼ 7.5 Hz), 1.26 (t, 3H, NCH2CH3, J ¼ 7.2 Hz),
4.29-4.45 (m, 8H, 2 NCH2 þ 2 OCH2), 7.11–7.49 (m, 8H, arom. H),
9.42 (br s, 4H, D2O exchangeable NH2). Anal. Calcd. for
2
3.1.8. 5-Amino-6,8-dicyano-7-[4-(1-piperidinyl)phenyl]-1-(prop-2-
en-1-yl)-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3h)
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 276–277 ꢂC, yield 58%. IR: nmax/cmꢀ1
3333, 3271, 3130 (NH2), 2223 (C^N), 1643, 1595 (C]N, C]C). 1H
C34H26N12O2S2 (698.77): C, 58.44; H, 3.75; N, 24.06. Found: C,
58.64; H, 3.94; N, 24.22.
3.3. Anti-inflammatory activity screening
NMR: d 1.61 (br s, 6H, piperidinyl 3 CH2), 3.28 (br s, 4H, piperidinyl 2
Anti-inflammatory activity screening for the prepared
compounds was determined in vivo by the acute carrageenan-
induced paw oedema standard method in rats [29–32]. Wister
albino rats of either sex (pregnant female animals were excluded)
weighing 160–190 g were divided into 14 groups of 6 animals each.
Administration of indomethacin (reference standard at a dose of
10 mg/kg body weight) and the tested compounds (3a–i and 5a–c)
dissolved in DMSO, at a dose of 50 mg/kg (body weight) was given
intraperitoneally 1 h before induction of inflammation. The control
group was given DMSO only. Carrageenan paw oedema was
induced by subcutaneous injection of 1% solution of carrageenan in
saline (0.1 ml/rat) into the right hind paw of rats. Paw volumes were
measured volumetrically after successive time intervals (1, 2, 3 and
4 h) with plethysmometer 7140 (UGO BASILE, Italy) and compared
with the initial hind paw volume of each rat for determining the
oedema volume. Data were collected, checked, revised and
analyzed. Quantitative variables from normal distribution were
expressed as means ꢁ SE ‘‘standard error’’. The significant differ-
ence between groups was tested by using one-way ANOVA fol-
lowed by post hoc test and the chosen level of significance was
p < 0.05.
NCH2), 5.04-5.24 (m, 4H, CH2]CHCH2 þ NCH2CH]CH2), 5.91–6.03
(m, 1H, NCH2CH]CH2), 7.07 (d, 2H, arom. H, J ¼ 9.0 Hz), 7.38 (d, 2H,
arom. H, J ¼ 8.7 Hz), 9.35 (br s, 2H, D2O exchangeable NH2). Anal.
Calcd. for C22H21N7S (415.511): C, 63.59; H, 5.09; N, 23.60. Found: C,
63.50; H, 4.98; N, 23.43.
3.1.9. 5-Amino-6,8-dicyano-7-[4-(4-morpholinyl)phenyl]-1-(prop-
2-en-1-yl)-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-thiolate (3i)
Reaction time 5 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 282–283 ꢂC, yield 53%. IR: nmax/cmꢀ1
3330, 3271, 3131 (NH2), 2223 (C^N), 1645, 1597 (C]N, C]C). 1H
NMR:
d
3.29 (t, 4H, morpholinyl 2 NCH2, J ¼ 4.8 Hz), 3.76 (t, 4H,
morpholinyl 2 OCH2, J ¼ 4.8 Hz), 5.04–5.24 (m, 4H, CH2]CHCH2 þ
NCH2CH]CH2), 5.91–6.04 (m, 1H, NCH2CH]CH2), 7.11 (d, 2H, arom.
H, J ¼ 9.0 Hz), 7.42 (d, 2H, arom. H, J ¼ 8.7 Hz), 9.38 (s, 2H, D2O
exchangeable NH2). Anal. Calcd. for C21H19N7OS (417.481): C, 60.41;
H, 4.59; N, 23.49. Found: C, 60.29; H, 4.53; N, 23.25.
3.2. Synthesis of [alkanediylbis(oxy-2,1-phenylene)]bis(5-amino-
6,8-dicyano-1-substituted-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-
2-thiolates) 5a–c (general procedure)
The anti-inflammatory activity was expressed as percentage
inhibition of oedema volume in treated animals in comparison with
the control group (Table 1).
A mixture of 4a, 4b (5 mmol) and the corresponding 2a, 2b
(10 mmol) in absolute ethanol (25 ml) containing triethylamine
(11 mmol) was boiled under reflux for the appropriate time. The
separated solid while refluxing was collected, washed with ethanol
and crystallized from a suitable solvent affording 5a–c as yellow
crystals.
Vc ꢀ Vt
%Inhibition of oedema ¼
ꢃ 100
Vc
where Vc and Vt are the volumes of oedema for the control and
drug-treated animal groups, respectively.
Potency of the tested compounds was calculated relative to
indomethacin ‘‘reference standard’’ treated group according to the
following equation.
3.2.1. 7,70-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis(5-amino-6,8-
dicyano-1-methyl-1H-[1,2,4]triazolo[1,5-a]pyridin-4-ium-2-
thiolate) (5a)
Reaction time 16 h, crystallizes from N,N-dimethylformamide–
water mixture as 2:1 v/v, mp 341–343 ꢂC, yield 54%. IR: nmax/cmꢀ1
3291, 3186 (NH2), 2226 (C^N), 1642, 1603 (C]N, C]C). 1H NMR:
Potency ¼ (% Oedema inhibition of tested compound treated
group)/(% Oedema inhibition of indomethacin treated group).
d
3.79 (s, 3H, NCH3), 3.81 (s, 3H, NCH3), 4.39 (br. s, 4H, 2 OCH2),
3.4. Measurement of PGE2 level
7.11–7.48 (m, 8H, arom. H), 9.40 (s, 4H, D2O exchangeable 2 NH2).
Anal. Calcd. for C32H22N12O2S2 (670.72): C, 57.30; H, 3.31; N, 25.06.
Found: C, 57.11; H, 3.11; N, 25.20.
Measurement of PGE2 level was determined by the previously
described 6-day air pouch standard method in rats [33]. Male
albino rats weighing 200–250 g were divided into 6 groups of 6
animals each. The air pouch was induced as follows, on the first day
of the experiment; 20 ml of air was injected subcutaneously at the
back of each rat. Two days later, another 10 ml of air was injected at
the same site. On the fifth day after the first injection, a further
10 ml of air was injected into the pouch. Then, 24 h later and before
injecting the pouch with carrageenan (2 ml of 1% solution in saline),
3.2.2. 7,70-[1,3-Propanediylbis(oxy-2,1-phenylene)]bis(5-amino-6,8-
dicyano-1-methyl-1H-[1,2,4]triazolo[1,5-a]pyridin-
4-ium-2-thiolate) (5b)
Reaction time 10 h, crystallizes from N,N-dimethylformamide–
water mixture as 1:1 v/v, mp 302–304 ꢂC, yield 53%. IR: nmax/cmꢀ1
3291, 3187 (NH2), 2228 (C^N), 1638, 1601 (C]N, C]C). 1H NMR:

A.S. Girgis, F.F. Barsoum / European Journal of Medicinal Chemistry 44 (2009) 1972–1977
1977
four groups of animals were treated orally with the tested
compounds (3f, 3h, 5b and 5c) ‘‘at a dose of 50 mg/kg body weight’’,
one group with indomethacin (reference standard) ‘‘at a dose of
10 mg/kg body weight’’ suspended in saline solution by the aid of
few drops of Tween 80 and the last group with sterile saline
(control group). All injections were given under light ether anaes-
thesia. Six hours after the carrageenan injection, animals were
lightly anaesthetised with ether and the contents of the pouch were
aspirated using a Pasteur pipette and transferred into graduated
plastic tube kept in ice. The bulk of the exudates was frozen and
stored at ꢀ20 ꢂC until required for PGE2 assay. PGE2 was measured
by an ELISA (Beckman BiomekÔ 1000 automated laboratory
workstation apparatus) technique using PGE2 assay kit supplied by
R&D Systems Inc., Minneapolis, USA, according to the manu-
facturer’s specifications.
also to Prof. M.T. Khayyal (Faculty of Pharmacy, Cairo University)
and Dr. Ebtehal El-Demerdash (Faculty of Pharmacy, Ain-Shams
University) for facilities allowed and valuable discussion.
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Acknowledgment
This work is sponsored by the U.S.–Egypt Science and Tech-
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