Synthesis and biological evaluation of thieno [2′,3′:4,5] pyrimido[1,2-b][1,2,4]triazines and thieno[2,3-d][1,2,4]triazolo[1,5-a] pyrimidines as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2012.12.003

A new series of thieno[2′,3′:4,5]pyrimido[1,2-b][1,2,4] triazines and thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidines was synthesized. The newly synthesized compounds were evaluated for their anti-inflammatory and analgesic activity using diclofenac Na as a reference standard. Additionally, the ulcerogenic effects and acute toxicity (ALD₅₀) values of the active compounds were also determined. In general, the thieno[2,3-d][1,2,4] triazolo[1,5-a]pyrimidine derivatives exhibited better biological activities than the thieno[2′,3′:4,5]pyrimido[1,2-b][1,2,4]triazines. Collectively, the thienotriazolopyrimidine derivatives 9, 13 and 14a were proved to display distinctive anti-inflammatory activity at the acute and subacute models as well as good analgesic profile with a delayed onset of action. Moreover, they revealed good gastrointestinal safety profile and are well tolerated by experimental animals with high safety margin (ALD₅₀ > 0.3 g/kg).

Full text of DOI:10.1016/j.ejmech.2012.12.003

European Journal of Medicinal Chemistry 62 (2013) 341e351
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of thieno [20,30:4,5]pyrimido[1,2-b]
[1,2,4]triazines and thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidines as
anti-inflammatory and analgesic agentsq
,
a
a
b
Hayam M. Ashour a , Omaima G. Shaaban , Ola H. Rizk , Ibrahim M. El-Ashmawy
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt
b Department of Pharmacology, Faculty of Veterinary Medicine, University of Alexandria, Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
A new series of thieno[20,30:4,5]pyrimido[1,2-b][1,2,4]triazines and thieno[2,3-d][1,2,4]triazolo[1,5-a]
pyrimidines was synthesized. The newly synthesized compounds were evaluated for their anti-
inflammatory and analgesic activity using diclofenac Na as a reference standard. Additionally, the
ulcerogenic effects and acute toxicity (ALD50) values of the active compounds were also determined. In
general, the thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidine derivatives exhibited better biological activities
than the thieno[20,30:4,5]pyrimido[1,2-b][1,2,4]triazines. Collectively, the thienotriazolopyrimidine
derivatives 9, 13 and 14a were proved to display distinctive anti-inflammatory activity at the acute and
subacute models as well as good analgesic profile with a delayed onset of action. Moreover, they revealed
good gastrointestinal safety profile and are well tolerated by experimental animals with high safety
margin (ALD50 > 0.3 g/kg).
Article history:
Received 30 April 2012
Received in revised form
2 December 2012
Accepted 3 December 2012
Available online 28 December 2012
Keywords:
Thienopyrimidotriazines
Thienotriazolopyrimidines
Anti-inflammatory activity
Ulcerogenic effect
Ó 2012 Elsevier Masson SAS. All rights reserved.
Analgesic activity
1. Introduction
for the design and development of novel anti-inflammatory agents
as an alternative to NSAIDs.
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the
most widely used therapeutics, primarily for the treatment of pain,
rheumatic arthritis and various types of inflammatory conditions.
However, their use is mainly restricted by their well known and
serious adverse gastrointestinal side effects such as gastroduodenal
erosions, ulcerations and nephrotoxicity [1e6]. Attempts to
develop non-steroidal anti-inflammatory drugs that are devoid of
classical NSAID toxicity, especially gastrointestinal injury, follow
several strategies, one of which is selective cyclooxygenase-2 (COX-
2) inhibition [7,8]. Although agents that inhibit COX-2 while sparing
COX-1 represented a new attractive therapeutic development, they
also gave rise to some of the side effects seen with NSAIDs, namely,
effects on the kidney that might manifest as an increased incidence
of hypertension, edema and associated clinical states [9e11].
Therefore, there is an urgent need for new targets that are required
In the past few years, thienopyrimidines have attracted great
attention due to their impressive array of pharmacological activities.
They were found to display antiviral [12,13], antimicrobial [14,15],
anticancer [16], analgesic and anti-inflammatory activities [17,18].
Moreover, a literature survey revealed that novel tri- and tetracyclic
ring systems, containing the thienopyrimidine skeleton have been
endowed with potential anti-inflammatory activity [19e21]. On the
other hand, it had been reported that many compounds having
a 1,2,4-triazole or triazine skeleton possessed significant anti-
inflammatory activity [22e31]. Thus the introduction of a 1,2,4-
triazole or 1,2,4-triazine moiety to the thienopyrimidine system is
expected to influence the biological activity significantly [31].
However, little is known about these tricyclic ring systems and few
reports exist describing the features of such compounds as biolog-
ically active agents or pharmaceuticals.
In the course of a research program devoted to the development
of new lead structures [32e37] devoid of the undesirable side
effects associated with classical NSAIDs, we have been interested in
the synthesis and anti-inflammatory evaluation of thienopyr-
imidines and tricyclic ring systems comprising the thienopyr-
imidine skeleton fused with a triazole or triazine moieties [37]. In
this context, we have reported the interesting anti-inflammatory
q
This work was presented in the Medicinal chemistry conference, Lanzarote,
Spain, 2nde5th March 2012, Poster highlight presentation No. 2.
* Corresponding author. Tel.: þ20 3 4871317; fax: þ20 3 4873273.
E-mail address: hayamashour@ymail.com (H.M. Ashour).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.003

342
H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
[20,30:4,5]pyrimido[1,2-b] [1,2,4] triazines as anti-inflammatory and
O
O
H3C
H3C
analgesic agents. To ameliorate the pharmacological profile of the
target compounds, various substituents were introduced at position 2
of the thienotriazolopyrimidine and thienopyrimidotriazine ring
systems. In fact, these groups are endowed with different lipophilic
and electronic properties that would influence the targeted biological
activity. It should be pointed out that, in addition to the targeted anti-
inflammatory and analgesic activities, the ulcerogenic and acute
toxicity profiles of the most active compounds were also evaluated.
The results revealed that some derivatives showed promising
activities.
CH2C6H5
N
CH2C6H5
NHNH2
H2N
O
H2N
N
N
S
S
O
N
N
NH
A
X
B
X = O, S
O
N
H3C
CH2C6H5
N
H2N
O
N
S
N
2. Results and discussion
S
O
O
2.1. Chemistry
C
The synthetic strategies adopted for the synthesis of the inter-
mediate and final compounds are depicted in Schemes 1 and 2. In
Scheme 1, ethyl 2-amino-4,5-dimethyl thiophene-3-carboxylate 1
[38] was conveniently converted to ethyl 4,5-dimethyl-2-(1H-tet-
Fig. 1. Chemical structures of previously synthesized anti-inflammatory thienopyr-
imidines and fused thienopyrimidines.
activity of some lead compounds (AeC) (Fig. 1) namely 3-benzyl-2-
hydrazino-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d] pyrimidine-6-
carboxamide (A), 5-benzyl-3-methyl-4-oxo-8-substituted-4,5,7,8-
tetra hydrothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxa-
mide (B) and ethyl[(5-benzyl-2-carbamoyl-3-methyl-4-oxo-4,5-
dihydrothieno[3,2-e][1,2,4] triazolo[4,3-a] pyrimidin-8-yl)sulfanyl]
acetate (C) [37]. These compounds displayed pronounced anti-
inflammatory activity comparable to diclofenac sodium in the
acute and subacute inflammatory models in addition to their low
ulcerogenic potential and high safety margin.
razol-1-yl)thiophene-3-carboxylate
2 by reaction with triethyl
orthoformate and sodium azide in glacial acetic acid [39]. 1H
NMR spectrum of compound 2 showed a characteristic singlet at
9.89 ppm integrated for tetrazole C5-proton. Compound 2 was
readily cyclized to 2,3-diaminothieno[2,3-d]pyrimidine 3 via cyan-
amide intermediate through reflux in hydrazine hydrate [39]. All
trials to cyclize 3 into the corresponding thienopyrimidotriazepine 4
utilizing reaction conditions previously reported for the synthesis of
analogous compounds [40] was unsuccessful. Heating 3 with excess
acetyl acetone yielded the 1-methyl-3-oxobutylideneamino deriv-
ative 5. IR spectrum of 5 revealed an additional absorption band at
1650 cmꢀ1 corresponding to the ketonic C]O group, whereas, its 1H
NMR spectrum displayed five singlets due to CH3 and CH2 protons.
Encouraged by these results, we got interested in extending the
scope of this approach for the synthesis and biological evaluation of
some new thieno[2,3-d] [1,2,4] triazolo[1,5-a]pyrimidines, and thieno
H3C
H3C
COOEt
N
COOEt
NH2
H3C
H3C
i
N
N
S
2
S
N
1
ii
O
H3C
H3C
O
N
O
N
H3C
H3C
H3C
CH3
NH2
NH2
N
N
N
N
R1
N
iii
N
N
v
H3C
X
S
N
S
S
3
4
8a-d
CH3
iv
iii
iv
X
O
O
H3C
H3C
H3C
N
N
R
O
N
N
N
C
H
H3C
H3C
H3C
O
N
S
N
S
NH2
N
N
NH2
R
5
7a-f
S
N
H
6a-f
For 7:a, R = C6H5; b, R= 4-Cl-C6H4; c, R= 4-OCH3-C6H4; d, R= 3,4-(OCH3)2-C6H3;
e, R= 3,4,5-(OCH3)3-C6H2; f, R= 4-NO2-C6H4
8:a, R1=C6H5; b, R1= 4-ClC6H4; c, R1= 4-BrC6H4; d, R1= 4-CH3C6H4
Reagents and conditions: i) NaN3 / CH(OC2H5)3 /gl.CH3COOH/ reflux; ii ) N2H4 99 % / reflux;
iii) CH2(COCH3)2 / reflux; iv) RCHO / fusion at 180oC
v) 4-R1COCH2Br / 4-CH3C6H4-SO3H / absolute EtOH / reflux
Scheme 1. Synthetic pathways for the key intermediate 3 and compounds 5, 7aef and 8aed.

H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
343
O
H3C
H
N
N
H3C
S
S
N
H
N
10
O
O
H3C
H3C
H3C
N
N
N
N
H3C
CH2CN
CH3
ii
S
N
H
S
N
H
N
N
9
iii
i
11
O
H3C
NH2
NH2
N
H3C
S
N
3
iv
v
O
N
O
H3C
H3C
H3C
H3C
N
N
N
N
N
NHR2
CH2COOEt
CH2CONH
S
N
H
S
N
H
N
13
12a-c
vi
O
H3C
H3C
N
R3
S
N
H
N
14a-c
For 12: a, R2= C6H5; b, R2= 4-ClC6H4; c, R2= 4-CH3C6H4
14: a, R3= H; b, R3=Cl; c, R3= OCH3
Reagents and conditions: i) Ac2O / reflux ; ii) CS2 / NaOEt / ethanol / reflux ; iii ) CNCH2COOEt / reflux;
iv) R2NCS / fusion 180oC or R2 NCS / DMF / NaOH / r.t. ;
v) CH2(COOC2H5)2 / reflux; vi) R3C6H4NH2 /fusion at 180oC
Scheme 2. Synthetic pathways for the target compounds 9, 10, 11, 12aec, 13 and 14aec.
The resonance of the N]CeCH3 protons and the methyl carbon
upfield confirms that the configuration at C1 is E [41,42]. Fusion of 3
with the appropriate aldehyde in an oil bath at 180 ꢁC following
previously published reaction conditions [43] didn’t yield the ex-
pected thienotriazolopyrimidines 6aef and Schiff bases 7aef were
isolated. IR spectra of the latter compounds showed strong bands at
3491e3262 and 1684e1671 cmꢀ1 indicating presence of NH2 and
C]O groups respectively. Inspection of the 1H NMR spectra of
compounds 7aee revealed a singlet attributed to N]CH proton in
the range 8.72e9.03 ppm indicating existence of these compounds
as E isomers [44] while, the 1H NMR spectrum of 7f revealed two
singlets corresponding to N]CH proton at 9.28 and 9.47 ppm
confirming presence of this compound as a mixture of Z and E
isomers. Refluxing 3 with phenacyl or 4-substituted phenacyl
bromides in ethanol using p-toluenesulfonic acid afforded the target
thieno[30,20: 4,5]pyrimido[1,2-b][1,2,4] triazin-6-ones 8aed. IR
spectra of 8aed revealed absence of absorption bands for the NH2
group while their 1H NMR spectra showed a singlet for the thie-
nopyrimidotriazine C3 proton at 9.47e9.50 ppm. The MS spectrum
of 8c showed a molecular ion peak at m/z 386 (16.2%) and Mþ2 at m/
z 388 (19.1) which matches its molecular formula C16H11BrN4OS.
Additionally, 13C NMR spectra of compounds 8a and 8b revealed
signals due to thienopyrimidotriazine C2 and C3 at their expected
chemical shifts.
ethoxide resulted in the 2-thioxo analog 10. The structure of 10 was
confirmed from its mass spectrum that showed a molecular ion peak
at m/z 252 (26.26%) in addition to 1H NMR, 13C NMR and micro-
analysis. The 3-cyanomethyl derivative 11 was obtained in a good
yield by reaction of 3 with ethyl cyanoacetate in an oil bath at 180 ꢁC.
The IR spectrumof compound 11 revealed strongabsorptionbands at
3357, 2265, and 1677 cmꢀ1 due to NH, CN and C]O groups respec-
tively, while its 1H NMR spectrum displayed a singlet at 4.32 ppm
attributed to CH2 protons, whereas, its 13C NMR showed signals at
28.20, 124.72, 152.18 characteristic for CH2, CN and thieno-
triazolopyrimidine C2 respectively. 2-Arylaminothienotriazolopy-
rimidines12aec were obtained through fusion of 3 with aryl iso-
thiocyanates in an oil bath at 180 ꢁC (Method A) or by stirring 3 with
aryl isothiocyanates in dimethyl formamide containing NaOH
(Method B). IR spectra of these compounds exhibited strong
absorption bands at 3309e3149 and 1679e1674 due to NH and C]O
groups, while their 1H NMR and 13C NMR spectra matched their
proposed structures. Heating 3 with diethylmalonate resulted in the
ethyl acetate ester 13. The IR spectrum of 13 revealed strong
absorption bands due to NH, ester and amidic C]O, while the 1H
NMR spectrum displayed the triplet and quartet of the ester group at
their expected chemical shifts and a singlet at 3.98 ppm integrated
for the CH2CO protons. Further confirmation of the structure was
derived from its mass spectrum that showed a molecular ion peak at
m/z 306 (75.7%) which is in accordance with its molecular formula.
Finally, synthesis of the N-substituted acetamides 14aec was ach-
ieved by fusion of the ester 13 with aniline or 4-substituted anilines.
The chemical structure of compounds 14aec was confirmed by their
IR, 1H NMR, 13C NMR and MS spectral data. The IR spectra revealed
absorption bands due to NH and C]O groups, while their 1H NMR
Referring to Scheme 2, heating 3 in acetic anhydride for a long
time yielded the cyclized thienotriazolopyrimidine 9 in a good yield.
Literature survey revealed that compound 9 was previously prepared
in a lower yield via different reaction conditions [45]. The chemical
structure of compound 9 was verified by analytical and spectral data.
Heating 3 with carbon disulphide in ethanol containing sodium

344
H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
Table 2
spectra showed D2O exchangeable singlets attributed to NH protons
in addition to singlets corresponding to CH2CO protons. Additionally,
13C NMR spectrum for 14b showed signals at 44.72, 157.77, 161.69,
174.53 due to CH2, thienotriazolopyrimidine C2, and the two C]O
respectively providing a substantial proof for the assigned structure
of these compounds.
Anti-inflammatory activities (AI) of the fused thienopyrimidines in formalin-
induced rat paw edema bioassay (sub-acute inflammatory model).
Compounda
Volume of edema (ml)b
0
1st day
8th day
Control
8a
8b
8c
8d
9
10
11
12a
12b
12c
13
14a
14b
14c
0.32 ꢂ 0.01
0.30 ꢂ 0.02
0.33 ꢂ 0.01
0.29 ꢂ 0.01
0.32 ꢂ 0.02
0.31 ꢂ 0.01
0.34 ꢂ 0.01
0.31 ꢂ 0.01
0.29 ꢂ 0.02
0.30 ꢂ 0.01
0.31 ꢂ 0.01
0.35 ꢂ 0.02
0.33 ꢂ 0.02
0.30 ꢂ 0.01
0.32 ꢂ 0.01
0.32 ꢂ 0.02
0.68 ꢂ 0.01
0.86 ꢂ 0.03
0.52 ꢂ 0.01c (38)d
0.56 ꢂ 0.02c (36)
0.53 ꢂ 0.01c (33)
0.59 ꢂ 0.02c (25)
0.51 ꢂ 0.03c (44)
0.53 ꢂ 0.02c (47)
0.50 ꢂ 0.02c (47)
0.52 ꢂ 0.01c (36)
0.53 ꢂ 0.02c (36)
0.55 ꢂ 0.01c (33)
0.54 ꢂ 0.01c (47)
0.51 ꢂ 0.01c (50)
0.53 ꢂ 0.02c (36)
0.53 ꢂ 0.01c (41)
0.52 ꢂ 0.02c (44)
0.63 ꢂ 0.01c (38)
0.77 ꢂ 0.02c (18)
0.69 ꢂ 0.02c (25)
0.74 ꢂ 0.01c (22)
0.68 ꢂ 0.02c (31)
0.66 ꢂ 0.01c (40)
0.64 ꢂ 0.03c (38)
0.72 ꢂ 0.03c (20)
0.71 ꢂ 0.04c (24)
0.72 ꢂ 0.02c (24)
0.67 ꢂ 0.02c (40)
0.67 ꢂ 0.02c (37)
0.63 ꢂ 0.03c (38)
0.65 ꢂ 0.01c (38)
0.64 ꢂ 0.02c (40)
2.2. Biological evaluation
2.2.1. Anti-inflammatory (AI) activity
The AI activity of the designed tricyclic compounds was evalu-
ated by utilizing the formalin-induced paw edema bioassay [46]
using diclofenac Na (20 mg/kg) as a reference standard anti-
inflammatory agent. The paw edema was employed as a model
for acute and sub-acute inflammation. The data obtained were
presented in Tables 1 and 2 and expressed as means ꢂ SE. Statistical
differences of control and test groups were carried out using the
Analysis of Variance (ANOVA) followed by ‘StudenteNewmane
Keuls Multiple Comparison Test’. They were performed using
computer package of the Statistical Analysis System (SAS, 1987),
SAS Incorporation Institute. The difference in results was consid-
ered significant when P < 0.05.
Diclofenac Na
a
Dose levels, po: test compounds (20 mg/kg b. wt.), diclofenac Na (20 mg/kg b.
wt.).
b
c
Values are expressed as mean ꢂ S.E. (Number of animals N ¼ 5 rats).
Significantly different compared to corresponding control P ꢃ 0.05.
Between parentheses (Percentage anti-inflammatory activity, AI %).
d
2.2.1.1. Formalin-induced paw edema bioassay (acute inflammatory
model). In this acute inflammatory model [46] each test compound
was dosed orally (20 mg/kg body weight) 1 h prior to induction of
inflammation by formalin injection. Diclofenac Na was utilized as
a reference drug at a dose of 20 mg/kg, po. The anti-inflammatory
activity was then calculated 1e4 h after induction and presented in
Table 1 as the mean paw volume (ml) in addition to the percentage
anti-inflammatory activity (AI%).
A comparative study of the anti-inflammatory activity of test
compounds relative to the reference drug at different time intervals
indicated the following: after 1 h, six compounds showed distinctive
pharmacokinetic profiles as revealed from their potent and rapid
onset of action which was equal to or higher than diclofenac Na at
a dose of 20 mg/kg, po. A remarkable AI% was recorded for the
thienotriazolopyrimidines 9 (38%), 10 (42%), 11 (42%), 13 (47%), 14a
(42%) and 14c (47%), when compared with diclofenac Na (38%),
whereas, the thienotriazolopyrimidine 14b showed AI% (33%) lower
than diclofenac Na. The other compounds showed moderate to weak
activity. After 2 h interval, the data indicated that the thieno-
triazolopyrimidine ester 13 displayed anti-inflammatory activity
(53%), superior to diclofenac Na (43%) whereas, the 2-methyl-
thienotriazolopyrimidine 9, the 2-thioxo thienotriazolopyrimidine
10, and the 2-thienotriazolopyrimidinyl acetamide 14c were nearly
effective in inhibiting the paw edema with percentage activity of
(43e40%) compared to the reference drug (43%). Taking the anti-
inflammatory activity after 4 h time interval as a criterion for
comparison, it can be concluded that the thienotriazolopyrimidine
ester 13 and the 2-methylthienotriazolopyrimidine 9 showed anti-
inflammatory activity (56 and 52% respectively) similar to diclofe-
nac Na (52%), whereas, the 2-thienotriazolopyrimidinyl acetamides
14a, 14c displayed lower anti-inflammatory activity (40, 43%
respectively). The other compounds showed weak activity.
The most active compounds (9, 10, 11, 13, 14a, 14c) were further
tested at 5, 10, 20, 40 and 50 mg/kg body weight in order to
Table 1
Anti-inflammatory activities (AI) of the fused thienopyrimidines in formalin-induced rat paw edema bioassay (acute inflammatory model).
Compounda
Volume of edema (ml)b
0
ED50 (mg/Kg)
1 h
2 h
4 h
Control
8a
8b
8c
8d
9
10
11
12a
12b
12c
13
14a
14b
14c
0.32 ꢂ 0.01
0.30 ꢂ 0.02
0.33 ꢂ 0.01
0.29 ꢂ 0.01
0.32 ꢂ 0.02
0.31 ꢂ 0.01
0.34 ꢂ 0.01
0.31 ꢂ 0.01
0.29 ꢂ 0.02
0.30 ꢂ 0.01
0.31 ꢂ 0.01
0.35 ꢂ 0.02
0.33 ꢂ 0.02
0.30 ꢂ 0.01
0.32 ꢂ 0.01
0.32 ꢂ 0.02
0.55 ꢂ 0.01
0.64 ꢂ 0.02
0.76 ꢂ 0.01
0.48 ꢂ 0.03 (14)d
0.51 ꢂ 0.02 (14)
0.49 ꢂ 0.02 (13)
0.50 ꢂ 0.01 (21)
0.44 ꢂ 0.02c (38)
0.46 ꢂ 0.02c (42)
0.43 ꢂ 0.02c (42)
0.48 ꢂ 0.01 (9)
0.49 ꢂ 0.01 (9)
0.49 ꢂ 0.02 (21)
0.46 ꢂ 0.01c (47)
0.46 ꢂ 0.02c (42)
0.44 ꢂ 0.01c (33)
0.43 ꢂ 0.01c (47)
0.45 ꢂ 0.01c (38)
0.53 ꢂ 0.02c (28)
0.61 ꢂ 0.01 (12)
0.56 ꢂ 0.01 (5)
0.55 ꢂ 0.01c (28)
0.49 ꢂ 0.01c (43)
0.52 ꢂ 0.02c (43)
0.47 ꢂ 0.02c (25)
0.57 ꢂ 0.02 (12)
0.60 ꢂ 0.02 (6)
0.58 ꢂ 0.01 (5)
0.50 ꢂ 0.01c (53)
0.53 ꢂ 0.01c (37)
0.53 ꢂ 0.02c (28)
0.51 ꢂ 0.02c (40)
0.50 ꢂ 0.02c (43)
0.59 ꢂ 0.01c (34)
0.67 ꢂ 0.02 (20)
0.63 ꢂ 0.01 (27)
0.64 ꢂ 0.02c (27)
0.52 ꢂ 0.02c (52)
0.56 ꢂ 0.02c (27)
0.59 ꢂ 0.01c (36)
0.62 ꢂ 0.01c (25)
0.69 ꢂ 0.02 (11)
0.67 ꢂ 0.02 (18)
0.54 ꢂ 0.02c (56)
0.59 ꢂ 0.02c (40)
0.61 ꢂ 0.01c (29)
0.57 ꢂ 0.01c (43)
0.53 ꢂ 0.02c (52)
23.45e
27.50
29.00
28.15
26.12
18.45
25.13
Diclofenac Na
a
Dose levels, po: test compounds (20 mg/kg b. wt.), diclofenac Na (20 mg/kg b. wt.).
Values are expressed as mean ꢂ S.E. (Number of animals N ¼ 5 rats).
Significantly different compared to corresponding control. P ꢃ 0.05.
Between parentheses (Percentage anti-inflammatory activity, AI %).
ED50 is the effective dose calculated after 2 h.
b
c
d
e

H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
345
determine their ED50 values after 2 h interval. Compound 14c was
found to be more potent (ED50 ¼ 18.45, Table 1) than the reference
drug diclofenac Na (ED50 ¼ 25.13 mg/kg), whereas, the rest of the
compounds were found to be nearly equipotent (ED50 ¼ 23.45e
29.00 mg/kg, Table 1) compared to the reference drug.
isolated rat stomachs showed a normal stomach texture for
compounds 9 and 14c with no observable hyperemia indicating
a superior GI safety profile (no ulceration) in the population of the
test animals at an oral dose of 300 mg/kg, when administered twice
at 2 h interval in fasted rats. Whereas, compounds 10, 11, 13, 14a
showed weak ulceration effect (20e30%). It is worth-mentioning
that, diclofenac Na was found to cause 20% ulceration under the
same experimental conditions.
2.2.1.2. Formalin-induced paw edema bioassay (sub-acute inflam-
matory model). For this sub-acute inflammatory model [46],
inflammation was induced by formalin injection in the first and
third days, and test compounds were administered once orally
(20 mg/kg daily) for 7 days. Again, diclofenac Na was used as
a reference agent. The anti-inflammatory activity was calculated at
1st and 8th day after induction and presented in Table 2 as the mean
paw volume and the percentage anti-inflammatory activity (AI%).
The obtained data revealed that, at the 1st day, the thieno-
triazolopyrimidine 14a showed higher anti-inflammatory activity
(50%) than the reference drug diclofenac Na (44%), while the thie-
notriazolopyrimidines 9, 10, 11, 13, 14c displayed anti-inflammatory
activity (41e47%) nearly equivalent to the reference drug
(44%). Meanwhile, the thienopyrimidotriazines 8a, 8b and the
thienotriazolopyrimidines 12a, 12b, 14b displayed weaker anti-
inflammatory activity (36e38%). At the 8th day, the thienopyr-
imidotriazine 8a and thienotriazolopyrimidines 10, 11, 13, 14a, 14b,
14c were found to be nearly as effective (37e40%) as diclofenac Na.
The other compounds displayed moderate to weak activity.
A further interpretation of the anti-inflammatory activity of the
test compounds in pre-mentioned screens (Tables 1 and 2) revealed
that the thienotriazolopyrimidines 9, 13, 14a and 14c showed
equivalent to moderate anti-inflammatory activity in both models
compared to diclofenac Na. On the other hand, the thienopyr-
imidotriazine 8a and the thienotriazolopyrimidines 10, 11, 14b were
nearly equipotent or slightly less potent compared to diclofenac Na
in the subacute model at all time intervals nevertheless they proved
to be less active in the acute inflammatory model. This fact would
suggest that such type of compounds might be effective in
managing chronic inflammation, while they would be less effective
in controlling acute inflammatory conditions.
2.2.1.4. Acute toxicity. The same selected compounds; 9, 10, 11, 13,
14a, 14c were further evaluated for their approximate acute lethal
dose ALD50 in male rats using a literature method [48]. The results
indicated that all of the tested compounds proved to be non-toxic
and are well tolerated by experimental animals. The compounds
showed a high safety margin when screened at graded doses (0.1e
0.3 g/kg, po), where their ALD50 values were found to be >0.3 g/kg.
2.2.2. Analgesic activity
The analgesic activity of the synthesized tricyclic compounds
was evaluated using the rat tail withdrawal technique in response
to immersion in water at 55 ꢁC [49] using diclofenac Na as a refer-
ence drug (20 mg/kg, po). The analgesic activity was measured at
1e4 h time intervals after pain induction. The results were recorded
as the average values of five administrations and the percentage
increase of the reaction time in comparison with the basal values.
The results were presented in Table 3 and expressed as
means ꢂ S.E. Statistical differences of control and test groups were
carried out as described under Section 2.2.1.
A comparative study of the analgesic activity of the test
compounds relative to the reference drug at different time intervals
revealed the following: after 1 h, the cyanomethylthienotriazo-
lopyrimidine 11 exhibited potent analgesic activity (41%) compared
to diclofenac Na (30%), while the thienotriazolopyrimidines 9, 10,
12b, 14b showed analgesic activity (percentage increase of reaction
time 26e30%) nearly equivalent to diclofenac Na. After 2 h, the
results showed that the thienotriazolopyrimidines 11,13 were found
to be nearly equipotent (53 and 47% respectively) to the reference
drug (49%), whereas, the thienotriazolopyrimidines 9, 12b showed
lower activity (40%). On the other hand, after 4 h, thieno-
triazolopyrimidines 11 and 13 showed higher analgesic activity (64%
and 62%) compared to the reference drug (56%), whereas the thie-
nopyrimidotriazine 8a and thienotriazolopyrimidines 9, 12b, 14a
2.2.1.3. Ulcerogenic activity. The tested compounds namely; 9, 10,
11, 13, 14a, 14c that exhibited promising anti-inflammatory profiles
in the pre-mentioned animal models were further evaluated for
their ulcerogenic potential in rats [47]. Gross observation of the
Table 3
Analgesic activities of the fused thienopyrimidines using the rat tail withdrawal technique.
Compounda
Reaction time (sec)b
0
1 h
2 h
4 h
Control
8a
8b
8c
8d
9
10
11
12a
12b
12c
13
14a
14b
14c
2.69 ꢂ 0.20
2.75 ꢂ 0.30
2.95 ꢂ 0.20
2.74 ꢂ 0.01
2.82 ꢂ 0.01
3.00 ꢂ 0.20
3.11 ꢂ 0.20
2.62 ꢂ 0. 10
2.76 ꢂ 0.10
3.00 ꢂ 0.20
3.11 ꢂ 0.02
2.89 ꢂ 0.20
2.60 ꢂ 0.10
3.20 ꢂ 0.20
2.80 ꢂ 0.20
2.77 ꢂ 0.20
2.75 ꢂ 0.10
2.82 ꢂ 0.30
2.88 ꢂ 0.20
3.24 ꢂ 0.20 (17)d
3.15 ꢂ 0.20 (6)
3.11 ꢂ 0.02 (13)
3.20 ꢂ 0.03 (13)
3.91 ꢂ 0.10c (30)
3.95 ꢂ 0.20c (27)
3.71 ꢂ 0.10c (41)
2.91 ꢂ 0.10 (5)
3.80 ꢂ 0.30c (26)
3.85 ꢂ 0.02 (23)
3.45 ꢂ 0.20c (19)
3.15 ꢂ 0.20 (21)
4.10 ꢂ 0.30c (28)
3.20 ꢂ 0.20 (14)
3.62 ꢂ 0.20c (30)
3.69 ꢂ 0.20c (34)
3.30 ꢂ 0.30 (11)
3.40 ꢂ 0.01 (24)
3.53 ꢂ 0.01 (25)
4.20 ꢂ 0.20c (40)
4.20 ꢂ 0.30c (35)
4.02 ꢂ 0.20c (53)
3.25 ꢂ 0.20 (17)
4.22 ꢂ 0.30c (40)
4.10 ꢂ 0.02 (31)
4.25 ꢂ 0.30c (47)
3.54 ꢂ 0.10c (36)
4.25 ꢂ 0.40c (32)
3.80 ꢂ 0.20c (35)
4.15 ꢂ 0.40c (49)
4.15 ꢂ 0.40c (50)
3.65 ꢂ 0.20c (23)
3.53 ꢂ 0.02c (28)
3.64 ꢂ 0.01c (29)
4.63 ꢂ 0.10c (54)
4.35 ꢂ 0.20c (39)
4.32 ꢂ 0.10c (64)
3.49 ꢂ 0.30c (26)
4.62 ꢂ 0.30c (54)
4.50 ꢂ 0.03c (44)
4.69 ꢂ 0.20c (62)
3.95 ꢂ 0.30c (51)
4.49 ꢂ 0.40c (40)
3.98 ꢂ 0.20c (42)
4.33 ꢂ 0.20c (56)
Diclofenac Na
a
Dose levels, po: test compounds (20 mg/kg b. wt.), diclofenac Na (20 mg/kg b. wt.).
Values are expressed as mean ꢂ S.E. (Number of animals N ¼ 5 rats).
Significantly different compared to corresponding control. P ꢃ 0.05.
Between parentheses (Percentage analgesic activity).
b
c
d

346
H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
were found to be nearly equipotent (50e54%) to the reference drug.
The rest of the tested compounds showed variable degree of anal-
gesic activities ranging between weak to moderate (23e44%).
Collectively, the thienopyrimidotriazine analog 8a and the
thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidine derivatives 9, 11, 12b,
13, 14a were proved to be the most active analgesic agents with
a delayed onset of action (percentage increase of reaction time 50e
64% after 4 h) compared to diclofenac Na (percentage increase of
reaction time 56% after 4 h).
analgesic profile. Moreover, the thieno[2,3-d] [1,2,4] triazolo [1,5-a]
pyrimidine derivatives exhibited better anti-inflammatory and
analgesic activities than the thieno[20,30:4,5] pyrimido[1,2-b] [1,2,4]
triazine variants. Collectively, the thieno[2,3-d] [1,2,4]triazolo[1,5-a]
pyrimidines 9, 10, 11, 13, 14a and 14c were proved to display
distinctive anti-inflammatory activity at both models, while the
thieno[20,30:4,5]pyrimido[1,2-b] [1,2,4]triazine analog 8a and the
thieno[2,3-d] [1,2,4]triazolo[1,5-a] pyrimidine derivatives 9, 11, 12b,
13, 14a were proved to be the most active analgesic agents with
a delayed onset of action compared to diclofenac Na. Additionally,
the active compounds revealed good GI safety profile and are well
tolerated by experimental animals with high safety margin
(ALD50 > 0.3 g/kg). Finally, these compounds represent new struc-
ture scaffolds that could be further optimized for future develop-
ment of more potent anti-inflammatory and analgesic agents.
2.2.3. Structure activity relationship
A deep insight in the structures of the synthesized compounds
revealed that they represent two different classes of compounds,
namely the thieno[20,30:4,5] pyrimido[1,2-b][1,2,4]triazines (Scheme
1) and the thieno[2,3-d][1,2,4]triazolo[1,5-a] pyrimidines (Scheme
2). Within the thieno[20,30:4,5] pyrimido[1,2-b] [1,2,4] triazines 8ae
d, activity was confined to the phenyl derivative 8a which dis-
played weak anti-inflammatory activity in the acute model, never-
theless, it showed activity (38%) equipotent to diclofenac Na in the
subacute model at the 8th day in addition to analgesic activity (50%)
slightly less potent than diclofenac Na (56%) after 4 h. Regarding the
thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidines (Scheme 2), the anti-
inflammatory and analgesic activities appear to be closely related
to the nature of substituent at position 2 of the triazole ring. In this
respect, the 2-methylthienotriazolopyrimidine 9 showed anti-
inflammatory activity in the acute model (38e52%) equipotent to
diclofenac Na at all time intervals, however, in the subacute model
the activity (44%) was equivalent to diclofenac sodium on the 1st day
but it subsided on the 8th day. Meanwhile it displayed a delayed
analgesic activity (54%) equipotent to the reference drug after 4 h.
3. Experimental
3.1. Chemistry
All reagents and solvents were purchased from commercial
suppliers and were dried and purified when necessary by standard
techniques.
Melting points were determined in open glass capillaries using
Stuart capillary melting point apparatus (Stuart Scientific Stone,
Staffordshire, UK) and are uncorrected. Infrared (IR) spectra were
recorded on PerkineElmer 1430 infrared spectrophotometer (Per-
kin Elmer, Beaconsfield, UK) and measured by Ú cmꢀ1 scale using
KBr cell. 1H NMR spectra were scanned on Varian Mercury VX-300,
using tetramethylsilane (TMS) as internal standard and DMSO-d6 as
Replacing the 2-methyl group with
a
2-thioxo function
the solvent (chemical shifts are given in d ppm). Splitting patterns
(compound10) maintained the activity in the acute model at 1 and
2 h intervals but it decreased after 4 h, while in the subacute model,
the activity (47% & 40%) was equipotent to the reference drug at both
time intervals, however, the analgesic activity was markedly reduced
at all time intervals. Conversion of the 2-methyl group to a 2-
cyanomethyl moiety (compound 11) decreased the activity in the
acute model after 2& 4 h while, the activity at the subacute model
(47% & 38%) was equipotent to diclofenac Na at both time intervals,
meanwhile, the analgesic activity was markedly improved (41%e
64%) to exceed that of diclofenac Na (30%e56%) at all time inter-
vals. Substitution of the 2-methyl with 2-substituted amino group
(compounds 12aec) greatly decreased the anti-inflammatory
activity in both models, while the analgesic activity was main-
tained only for compound 12b that showed equipotent activity (54%)
to diclofenac Na after 4 h. Replacement of the 2-methyl group by
ethyl acetate one (compound 13) resulted in an obvious improve-
ment in the anti-inflammatory activity in both models in addition to
better analgesic activity. In this view, it exhibited anti-inflammatory
activity (47%e56%) higher than diclofenac Na (38%e52%) in the acute
model and equipotent to it (47% & 40%) in the subacute one, mean-
while, it exhibited analgesic activity (47%) equivalent to the reference
drug after 2 h and higher than it (62%) after 4 h. Finally, derivatisation
of the ester 13 to the corresponding substituted amides 14aec
reduced the anti-inflammatory activity in the acute model as well
as the analgesic activity, while the activity was not noticeably
affected in the subacute model.
were designated as follows: s: singlet; d: doublet; dd: doublet of
doublet; t: triplet; m: multiplet. 13C NMR proton decoupled spectra
were recorded on a Varian Mercury VX-300 spectrometer in DMSO-
d6 and measured in
d scale. Mass spectra were run on a Finnigan
mass spectrometer model SSQ/7000 (70 eV) or on a gas chro-
matograph/mass spectrometer Schimadzu GCMS-QP 2010 Plus
(70 eV). Microanalyses were performed at the Microanalytical Unit,
Faculty of Science, Cairo University, Egypt and the found values
were within ꢂ0.4% of the theoretical values. Follow up of the
reactions and checking the purity of the compounds was made by
thin layer chromatography (TLC) on silica gel-precoated aluminum
sheets (Type 60 GF254; Merck; Germany) and the spots were
detected by exposure to UV lamp at
l 254 nm for few seconds.
3.1.1. Ethyl 4,5-dimethyl-2-(1H-tetrazol-1-yl)thiophene-3-carboxyl-
ate (2)
A suspension of 1 (9.95 g, 50 mmol), triethyl orthoformate
(37.9 ml, 230 mmol), and sodium azide (3.9 g, 60 mmol) in glacial
acetic acid (40 ml) was stirred and heated under reflux for 2 h. The
reaction mixture was cooled to room temperature and 7 ml of conc.
HCl was added. The separated solid was filtered and the filtrate was
evaporated under reduced pressure. The remaining residue was
crystallized from ethanol. Yield: 65%; Mp: 114e116 ꢁC. IR (KBr,
cmꢀ1): 2981, 2930 (CH3), 1718 (C]O), 1613 (C]N), 1565, 1504, 1452
(C]C). 1H NMR (300 MHz, DMSO-d6)
d
0.95 (t, J ¼ 6.9 Hz, 3H, ester
CH3), 2.29 (s, 3H, thiophene C4eCH3), 2.43 (s, 3H, thiophene C5e
CH3), 4.03 (q, J ¼ 6.9 Hz, 2H, ester CH2), 9.89 (s, 1H, tetrazole C5e
H). Anal. Calcd for C10H12N4O2S (252.29): C, 47.61; H, 4.79; N,
22.21. Found: C, 47.98; H, 4.86; N, 22.39.
2.3. Conclusion
The present study reports the synthesis and biological evaluation
of some fused tricyclic thienopyrimidines as anti-inflammatory and
analgesic agents. The obtained results clearly revealed that most of
the tested compounds showed better activity at the subacute
inflammatory model than the acute one in addition to good
3.1.2. 2,3-Diamino-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (3)
A suspension of 2 (10 mmol) in hydrazine hydrate (15 ml) was
heated under reflux for 7 h, then cooled and diluted with 50 ml
water. The precipitated solid was filtered, washed with diethyl

H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
347
ether, dried and crystallized from ethanol. Yield: 80% (reported
85%); Mp: 285 ꢁC (as reported). IR (KBr, cmꢀ1): 3455, 3296, 3209,
3152 (NH), 2954, 2915 (CH3), 1677 (C]O), 1616 (C]N), 1510, 1440
3.1.4.3. 2-Amino-3-[(4-methoxyphenylmethylidene)amino]-5,6-
dimethylthieno[2,3-d]pyrimidin-4(3H)-one (7c). Yield: 65%; Mp:
222 ꢁC (ethanol). IR (KBr, cmꢀ1): 3474, 3266 (NH), 3067, 2982 (CH),
1676 (C]O), 1629, 1606 (C]N), 1568, 1506, 1468 (C]C), 1253, 1019
(C]C). 1H NMR (300 MHz, DMSO-d6)
d 2.18 (s, 3H, thienopyr-
imidine C5eCH3), 2.25 (s, 3H, thienopyrimidine C6eCH3), 5.32 (s,
2H, NeNH2, D2O exchangeable), 7.02 (s, 2H, CeNH2, D2O
exchangeable). Anal. Calcd for C8H10N4OS (210.26): C, 45.70; H,
4.79; N, 26.65. Found: C, 45.68; H, 4.96; N, 26.95.
(CeOeC). 1H NMR (300 MHz, DMSO-d6)
d 2.20 (s, 3H, thienopyr-
imidine C5eCH3), 2.24 (s, 3H, thienopyrimidine C6eCH3), 3.79 (s,
3H, OCH3), 6.96 (s, 2H, NH2, D2O exchangeable), 7.07, 7.90 (2ꢄ d,
J ¼ 8.7 Hz, each 2H, methoxyphenyl C3,5eH and C2,6eH), 8.72 (s, 1H,
N]CH). 13C NMR (300 MHz, DMSO-d6)
d 12.25 (thienopyrimidine
3.1.3. 2-Amino-5,6-dimethyl-3-{[(1E)-1-methyl-3-oxobutylidene]
amino}thieno[2,3-d] pyrimidin-4(3H)-one (5)
C5eCH3), 12.79 (thienopyrimidine C6eCH3), 55.45 (OCH3), 113.98
(methoxyphenyl C3,5), 120.69 (thienopyrimidine C4a), 121.69
(methoxyphenyl C1), 125.08 (thienopyrimidine C6), 128.08 (thie-
nopyrimidine C5), 131.03 (methoxyphenyl C2,6), 150.82 (N]CH),
154.98 (thienopyrimidine C7a), 162.65 (thienopyrimidine C4),
163.12 (methoxyphenyl C4), 168.34 (thienopyrimidine C2). Anal.
Calcd for C16H16N4O2S (328.39): C, 58.52; H, 4.91; N,17.06. Found: C,
58.80; H, 4.62; N, 16.92.
A mixture of 3 (0.42 g, 2.0 mmol) in acetyl acetone (2.0 g,
2.0 ml, 20 mmol) was heated under reflux for 45 min and left to
cool. The precipitated product was filtered, washed with diethyl
ether, dried and crystallized from ethanol. Yield: 60%; Mp: 222e
224 ꢁC. IR (KBr, cmꢀ1): 3446, 3273 (NH), 3066, 2954, 2915 (CH),
1679, 1650 (C]O), 1611 (C]N), 1569, 1479, 1436 (C]C). 1H NMR
(300 MHz, DMSO-d6) d 1.65 (s, 3H, N]CeCH3), 2.03 (s, 3H, COCH3),
2.22 (s, 3H, thienopyrimidine C5eCH3), 2.23 (s, 3H, thienopyr-
imidine C6eCH3), 2.28 (s, 2H, CH2CO), 7.22 (s, 2H, NH2, D2O
3.1.4.4. 2-Amino-3-[(3,4-dimethoxyphenylmethylidene)amino]-5,6-
dimethylthieno[2,3-d]pyrimidin-4(3H)-one (7d). Yield: 68%; Mp:
174 ꢁC (ethanol). IR (KBr, cmꢀ1): 3477, 3280 (NH), 3052, 2976, 2916
(CH), 1682 (C]O), 1632, 1601 (C]N), 1509, 1465 (C]C), 1275, 1021
exchangeable). 13C NMR (300 MHz, DMSO-d6)
d 12.18 (thienopyr-
imidine C5eCH3), 12.66 (thienopyrimidine C6eCH3), 17.01 (oxobu-
tylidene C1eCH3), 29.32 (oxobutylidene C4), 31.46 (oxobutylidene
C2), 120.63 (thienopyrimidine C4a), 122.19 (thienopyrimidine C6),
127.89 (thienopyrimidine C5), 153.15 (oxobutylidene C1), 156.80
(thienopyrimidine C7a), 161.52 (thienopyrimidine C4), 164.16
(thienopyrimidine C2), 196.41 (oxobutylidene C3). Anal. Calcd for
C13H16N4O2S (292.36): C, 53.41; H, 5.52; N, 19.16. Found: C, 53.12;
H, 5.22; N, 18.84.
(CeOeC). 1H NMR (300 MHz, DMSO-d6)
d 2.23 (s, 3H, thienopyr-
imidine C5eCH3), 2.24 (s, 3H, thienopyrimidine C6eCH3), 3.87 (s,
6H, 2 OCH3), 7.01 (s, 2H, NH2, D2O exchangeable), 7.10 (d, J ¼ 8.8 Hz,
1H, dimethoxyphenyl C5eH), 7.46 (dd, J ¼ 8.8, 2.0 Hz, 1H, dime-
thoxyphenyl C6eH), 7.67 (d, J ¼ 2.0 Hz, 1H, dimethoxyphenyl C2e
H), 8.77 (s, 1H, N]CH). Anal. Calcd for C17H18N4O3S (358.41): C,
56.97; H, 5.06; N, 15.63. Found: C, 56.85; H, 4.76; N, 15.30.
3.1.4. 2-Amino-5,6-dimethyl-3-[(substituted phenylmethylidene)
amino]thieno[2,3-d] pyrimidin-4(3H)-ones (7aef)
3.1.4.5. 2-Amino-5,6-dimethyl-3-[(3,4,5-trimethoxyphenylmethylid-
ene)amino]thieno[2,3-d]pyrimidin-4(3H)-one (7e). Yield: 60%; Mp:
202e203 ꢁC (dimethyl formamide/water) (2: 1). IR (KBr, cmꢀ1):
3468, 3309 (NH), 3057, 2973, 2921 (CH), 1684 (C]O), 1628, 1605
(C]N), 1512, 1470 (C]C), 1271, 1024 (CeOeC). 1H NMR (300 MHz,
A mixture of 3 (0.42 g, 2.0 mmol) and the appropriate aromatic
aldehyde (4.0 mmol) was heated in an oil bath at 180 ꢁC for 30 min.
The reaction mixture was left to cool and the solidified mass was
triturated with ethyl alcohol, filtered, dried and crystallized from
the proper solvent.
DMSO-d6)
d 2.31(s, 3H, thienopyrimidine C5eCH3), 2.38 (s, 3H,
thienopyrimidine C6eCH3), 3.65 (s, 3H, OCH3), 3.89 (s, 6H, 2 OCH3),
6.59 (s, 2H, trimethoxy phenyl C2,6eH), 7.01 (s, 2H, NH2, D2O
exchangeable), 8.79 (s, 1H, N]CH). Anal. Calcd for C18H20N4O4S
(388.44): C, 55.66; H, 5.19; N, 14.42. Found: C, 55.42; H, 5.50; N,
14.17.
3.1.4.1. 2-Amino-5,6-dimethyl-3-[(phenylmethylidene)amino]thieno
[2,3-d]pyrimidin-4(3H)-one (7a). Yield: 55%; Mp: 220 ꢁC (ethanol).
IR (KBr, cmꢀ1): 3468, 3262 (NH), 3043, 2978, 2909 (CH), 1675 (C]
O), 1627 (C]N), 1571, 1505, 1443 (C]C). 1H NMR (300 MHz,
DMSO-d6)
d
2.24 (s, 3H, thienopyrimidine C5eCH3), 2.28 (s, 3H,
3.1.4.6. 2-Amino-5,6-dimethyl-3-[(4-nitrophenylmethylidene)amino]
thieno[2,3-d] pyrimidin-4(3H)-one (7f). Yield: 70%; Mp: 242 ꢁC
(Dimethyl formamide/ethanol) (2: 1). IR (KBr, cmꢀ1): 3491, 3322
(NH), 3103, 2914, 2850 (CH), 1673 (C]O), 1588 (C]N), 1520, 1437
thienopyrimidine C6eCH3), 7.07 (s, 2H, NH2, D2O exchangeable),
7.52e7.63 (m, 3H, phenyl C3,4,5eH), 8.0 (d, J ¼ 7.6 Hz, 2H, phenyl
C
2,6eH), 8.97 (s, 1H, N]CH). 13C NMR (300 MHz, DMSO-d6)
d 21.79
(thienopyrimidine C5eCH3), 22.34 (thienopyrimidine C6eCH3),
123.34 (thienopyrimidine C4a), 131.27 (thienopyrimidine C6),
137.57 (thienopyrimidine C5), 138.22 (phenyl C3,5), 138.59 (phenyl
C2,6), 141.89 (phenyl C4), 142.01 (phenyl C1), 160.24 (N]CH),
164.32 (thienopyrimidine C7a), 172.75 (thienopyrimidine C4),
178.36 (thienopyrimidine C2). Anal. Calcd for C15H14N4OS
(298.36): C, 60.38; H, 4.73; N, 18.78. Found: C, 60.51; H, 4.85; N,
18.66.
(C]C), 1343 (NO2). 1H NMR (300 MHz, DMSO-d6)
d 2.19 (s, 3H,
thienopyrimidine C5eCH3), 2.23 (s, 3H, thienopyrimidine C6eCH3),
7.19 (s, 2H, NH2, D2O exchangeable), 8.18, 8.29, 8.36, 8.39 (4ꢄ d,
J ¼ 8.6 Hz, each 2H, nitro phenyl C2,6eH & C3,5eH, Z and E isomers),
9.28, 9.47 (2ꢄ s, each 1H, 2 N]CH, Z and E isomers). Anal. Calcd for
C15H13N5O3S (343.36): C, 52.47; H, 3.82; N, 20.40. Found: C, 52.22;
H, 3.50; N, 20.17.
3.1.5. 7,8-Dimethyl-2-(phenyl or 4-substituted phenyl)-9H-thieno
[20,30:4,5] pyrimido [1,2-b][1,2,4]triazin-9-ones (8aed)
3.1.4.2. 2-Amino-3-[(4-chlorophenylmethylidene)amino]-5,6-dimeth-
yl-thieno[2,3-d] pyrimidin-4(3H)-one (7b). Yield: 62%; Mp >300 ꢁC
(dimethylformamide/water) (2:1). IR (KBr, cmꢀ1): 3429, 3304 (NH),
3029, 2975, 2920 (CH), 1671 (C]O), 1598 (C]N), 1444 (C]C), 1089
A mixture of 3 (0.42 g, 2.0 mmol), phenacyl bromide or 4-
substituted phenacyl bromide (2.0 mmol) and p-toluenesulfonic
acid (0.52 g, 3.0 mmol) in ethanol (20 ml), was heated under reflux
for 8 h. The precipitated solid was filtered, washed with ethanol,
dried and crystallized from dimethyl formamide.
(CeCl). 1H NMR (300 MHz, DMSO-d6)
d 2.32 (s, 3H, thienopyr-
imidine C5eCH3), 2.41 (s, 3H, thienopyrimidine C6eCH3), 7.07 (s, 2H,
NH2, D2O exchangeable), 7.63, 8.07 (2ꢄ d, J ¼ 8.6 Hz, each 2H,
chlorophenyl C3,5eH & C2,6eH), 9.03 (s, 1H, N]CH). Anal. Calcd for
C15H13ClN4OS. 1/2H2O (341.81): C, 52.66; H, 4.10; N, 16.38. Found: C,
52.31; H, 3.72; N, 16.72.
3.1.5.1. 7,8-Dimethyl-2-phenyl-9H-thieno[20,30:4,5]pyrimido[1,2-b]
[1,2,4]triazin-9-one (8a). Yield: 68%; Mp >300 ꢁC. IR (KBr, cmꢀ1):
3063, 2980, 2915 (CH), 1687 (C]O), 1631, 1600 (C]N), 1510, 1466

348
H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
(C]C). 1H NMR (300 MHz, DMSO-d6)
d
2.44 (s, 6H, 2 CH3), 7.60e
(thienotriazolopyrimidine C7eCH3), 21.96 (thienotriazolopyrimidine
C6eCH3), 22.59 (thienotriazolopyrimidine C2eCH3), 124.47 (thieno-
triazolopyrimidine C7a),133.22 (thienotriazolopyrimidine C6),137.27
(thienotriazolopyrimidine C7),150.29 (thienotriazolopyrimidine C2),
157.49 (thienotriazolopyrimidine C4a), 161.70 (thienotriazolopyr-
imidine C8), 169.54 (thienotriazolopyrimidine C3a). MS (m/z, %): 234
(Mþ, 51.1), 192 (11.4), 164 (13.6), 138 (25.0), 110 (20.5), 85 (20.50), 59
(100.0). Anal. Calcd for C10H10N4OS (234.28): C, 51.27; H, 4.30; N,
23.91. Found: C, 50.94; H, 4.55; N, 23.64.
7.80 (m, 3H, phenyl C3,4,5eH), 8.41 (d, J ¼ 7.6 Hz, 2H, phenyl C2,6e
H), 9.49 (s, 1H, thienopyrimidotriazine C3eH). 13C NMR (300 MHz,
DMSO-d6)
d 22.63 (thienopyrimidotriazine C8eCH3), 23.18 (thie-
nopyrimidotriazine C7eCH3), 124.43 (thienopyrimidotriazine C8a),
132.12 (thienopyrimidotriazine C7), 138.31 (thienopyrimidotriazine
C8), 139.24 (phenyl C3,5), 140.18 (phenyl C2,6), 141.21 (phenyl C4),
143.16 (phenyl C1), 148.54 (thienopyrimidotriazine C4a), 156.29
(thienopyrimidotriazine C2), 157.64 (thienopyrimidotriazine C3),
163.84 (thienopyrimidotriazine C5a), 169.68 (thienopyrimido-
triazine C9). Anal. Calcd for C16H12N4OS (308.36): C, 62.32; H, 3.92;
N, 18.17. Found: C, 62.34; H, 3.57; N, 18.24.
3.1.7. 6,7-Dimethyl-2-thioxo-2,3-dihydrothieno[2,3-d][1,2,4]triazolo
[1,5-a] pyrimidin-8(1H)-one (10)
Carbon disulphide (1.2 ml, 20 mmol) was added dropwise over
a period of 15 min to a mixture of 3 (0.42 g, 2.0 mmol) and sodium
ethoxide (4.0 mmol) in absolute ethanol (15 ml). After complete
addition, the reaction mixture was stirred at room temperature for
1 h followed by heating under reflux for 24 h. The solvent was
evaporated in vacuo. Water was added to the residue and the
alkaline solution was filtered. The clear filtrate was acidified with
dilute hydrochloric acid and the separated solid was collected and
crystallized from dimethyl formamide. Yield: 66%; Mp >300 ꢁC. IR
(KBr, cmꢀ1): 3214 (NH), 2920, 2855 (CH), 1669 (C]O), 1611 (C]N),
1520, 1468 (C]C), 1300, 1271, 1168, 978 (NeC]S). 1H NMR
3.1.5.2. 2-(4-Chlorophenyl)-7,8-dimethyl-9H-thieno[20,30:4,5]pyr-
imido[1,2-b] [1,2,4]triazin-9-one (8b). Yield: 65%; Mp >300 ꢁC. IR
(KBr, cmꢀ1): 3063, 2923 (CH), 1701 (C]O), 1600 (C]N), 1578, 1519,
1497 (C]C). 1H NMR (300 MHz, DMSO-d6)
d 2.25 (s, 3H, thieno-
pyrimidotriazine C8eCH3), 2.42 (s, 3H, thienopyrimidotriazine C7e
CH3), 7.08, 7.44 (2ꢄ d, J ¼ 8.8 Hz, each 2H, chlorophenyl C3,5eH &
C
2,6eH), 9.48 (s, 1H, thienopyrimidotriazine C3eH). 13C NMR
(300 MHz, DMSO-d6)
d 22.46 (thienopyrimidotriazine C8eCH3),
23.19 (thienopyrimidotriazine C7eCH3), 124.12 (thienopyrimido-
triazine C8a), 132.36 (thienopyrimidotriazine C7), 138.26 (thieno-
pyrimidotriazine C8), 140.09 (chlorophenyl C3,5), 141.18 ( chloro-
phenyl C2,6), 141.47 (chlorophenyl C1), 146.50 (chlorophenyl C4),
148.89 (thienopyrimidotriazine C4a), 156.41 (thienopyrimido-
triazine C2), 157.93 (thienopyrimidotriazine C3), 163.68 (thieno-
pyrimidotriazine C5a), 169.71 (thienopyrimidotriazine C9). Anal.
Calcd for C16H11ClN4OS (342.80): C, 56.06; H, 3.23; N, 16.34. Found:
C, 55.93; H, 3.36; N, 16.30.
(300 MHz, DMSO-d6)
d 2.28 (s, 3H, thienotriazolopyrimidine C7e
CH3), 2.37 (s, 3H, thieno triazolopyrimidine C6eCH3), 7.95, 12.09
(2ꢄ s, each 1H, 2 NH, D2O exchangeable). 13C NMR (300 MHz,
DMSO-d6)
d 21.42 (thienotriazolopyrimidine C7eCH3), 22.13 (thie-
notriazolopyrimidine C6eCH3), 123.82 (thienotriazolopyrimidine
C7a), 133.26 (thienotriazolopyrimidine C6), 137.53 (thienotriazolo-
pyrimidine C7), 156.46 (thienotriazolopyrimidine C4a), 162.18
(thienotriazolopyrimidine C8), 169.51 (thienotriazolopyrimidine
C3a), 176.47 (thienotriazolopyrimidine C2). MS (m/z, %): 252.30 (Mþ,
26.26), 239 (51.54), 212 (46.33), 190 (41.18), 177 (41.82), 156 (43.45),
151.15 (36.97), 129 (37.26), 105 (43.30), 91 (62.97), 71 (100.0), 67
(51.59), 55 (49.98). Anal. Calcd for C9H8N4OS2 (252.32): C, 42.84; H,
3.20; N, 22.21. Found: C, 42.54; H, 3.27; N, 21.98.
3.1.5.3. 2-(4-Bromophenyl)-7,8-dimethyl-9H-thieno[20,30:4,5]pyr-
imido[1,2-b] [1,2,4]triazin-9-one (8c). Yield: 70%; >300 ꢁC. IR (KBr,
cmꢀ1): 3053, 2917, 2849 (CH), 1696 (C]O), 1584 (C]N), 1515, 1459
(C]C), 1070 (CeBr). 1H NMR (300 MHz, DMSO-d6)
d 2.34 (s, 3H,
thienopyrimidotriazine C8eCH3), 2.38 (s, 3H, thienopyrimido-
triazine C7eCH3), 7.90, 8.34 (2ꢄ d, J ¼ 8.8 Hz, each 2H, bromophenyl
C
3,5eH & C2,6eH), 9.50 (s, 1H, thienopyrimidotriazine C3eH). MS
3.1.8. 6,7-Dimethyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]triazolo
[1,5-a]pyrimidin-2-ylacetonitrile (11)
(m/z, %): 388 (Mþþ2, 19.1), 386 (Mþ, 16.2), 179 (33.8), 178 (52.9),153
(17.6), 129 (10.3), 101 (25.0), 76 (26.5), 59 (100.0). Anal. Calcd for
C16H11BrN4OS (387.25): C, 49.62; H, 2.86; N, 14.47. Found: C, 49.98;
H, 3.17; N, 14.24.
A mixture of 3 (0.42 g, 2.0 mmol) and ethyl cyanoacetate (2.26 g,
20.0 mmol, 2.13 ml) was heated in an oil bath at 180 ꢁC for 30 min.
The reaction mixture was left to cool and the solidified mass was
triturated with ethyl alcohol, filtered, dried and crystallized from
dimethyl formamide/water (2:1). Yield: 60%; Mp >300 ꢁC. IR (KBr,
cmꢀ1): 3357 (NH), 2965, 2919 (CH), 2265 (CN), 1677 (C]O), 1625
(C]N), 1589, 1524, 1464 (C]C). 1H NMR (300 MHz, DMSO-d6)
3.1.5.4. 7,8-Dimethyl-2-(4-methylphenyl)-9H-thieno[20,30:4,5]pyr-
imido[1,2-b] [1,2,4]triazin-9-one (8d). Yield: 62%; Mp >300 ꢁC. IR
(KBr, cmꢀ1): 3058, 2973, 2917 (CH), 1685 (C]O), 1600 (C]N), 1578,
1518, 1469 (C]C). 1H NMR (300 MHz, DMSO-d6)
d
2.44 (s, 3H,
d
2.31 (s, 3H, thienotriazolopyrimidine C7eCH3), 2.39 (s, 3H, thie-
notriazolopyrimidine C6eCH3), 4.32 (s, 2H, CH2), 12.36 (s, 1H, NH,
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6)
21.56 (thie-
thienopyrimidotriazine C8eCH3), 2.45 (s, 3H, thienopyrimido-
triazine C7eCH3), 2.49 (s, 3H, phenyl CH3), 7.49, 8.32 (2ꢄ d, J ¼ 8.1 Hz,
each 2H, methylphenyl C3,5eH & C2,6eH), 9.47 (s, 1H, thienopyr-
imidotriazine C3eH). Anal. Calcd for C17H14N4OS (322.38): C, 63.33;
H, 4.38; N, 17.38. Found: C, 63.12; H, 4.67; N, 17.74.
d
notriazolopyrimidine C7eCH3), 21.94 (thienotriazolopyrimidine
C6eCH3), 28.20 (CH2), 121.70 (thienotriazolopyrimidine C7a), 124.72
(CN), 133.14 (thienotriazolopyrimidine C6), 137.17 (thieno-
triazolopyrimidine C7),152.18 (thienotriazolopyrimidine C2),154.63
(thienotriazolopyrimidine C4a), 161.92 (thienotriazolopyrimidine
C8), 169.59 (thienotriazolopyrimidine C3a), MS (m/z, %): 259 (Mþ,
2.3), 233 (100.0), 219 (28.8), 193 (13.8), 178 (18.8), 165 (40.0), 164
(33.8), 138 (15.0), 110 (21.3), 94 (32.5), 59 (80.0). Anal. Calcd for
C11H9N5OS (259.29): C, 50.95; H, 3.50; N, 27.01. Found: C, 50.62; H,
3.30; N, 26.76.
3.1.6. 2,6,7-Trimethylthieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-
8(3H)-one (9)
A mixture of 3 (0.42 g, 2.0 mmol) in acetic anhydride (5 ml) was
heated under reflux for 36 h. The reaction mixture was left to cool
and the precipitated solid was filtered, washed with ethanol, dried
and crystallized from dimethyl formamide/ethanol (1:2). Yield: 72%
(reported 49%); Mp: 293e294 ꢁC (reported 290e292 ꢁC). IR (KBr,
cmꢀ1): 3329 (NH), 2985 (CH), 1672 (C]O), 1639 (C]N), 1544, 1501,
3.1.9. 6,7-Dimethyl-2-(phenyl or 4-substituted phenyl)aminothieno
[2,3-d][1,2,4] triazolo[1,5-a]pyrimidin-8(3H)-ones (12aec)
1453 (C]C), 1H NMR (300 MHz, DMSO-d6)
d 2.3 (s, 3H, thieno-
triazolopyrimidine C7eCH3), 2.36 (s, 3H, thienotriazolopyrimidine
C6eCH3), 2.41 (s, 3H, thieno triazolo pyrimidine C2eCH3), 7.96 (s,1H,
Method A: A mixture of 3 (0.42 g, 2.0 mmol) and the appropriate
aryl isothiocyanate (6.0 mmol) was heated in an oil bath at 180 ꢁC
for 30 min. The reaction mixture was left to cool and the solidified
NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6)
d 21.48

H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
349
mass was triturated with ethanol, filtered, dried and crystallized
from dimethyl formamide/ethanol (2:1).
J ¼ 6.9 Hz, 2H, CH2CH3), 10.90 (s, 1H, NH, D2O exchangeable). 13
C
NMR (300 MHz, DMSO-d6)
d
12.31 (thienotriazolopyrimidine C7e
Method B: A mixture of 3 (0.42 g, 2.0 mmol), finely powdered
potassium hydroxide (0.11 g, 2.0 mmol) and the appropriate aryl
isothiocyanate (2.0 mmol) in dry dimethyl formamide (5 ml) was
stirred at room temperature for 6 h. The reaction mixture was then
poured into ice-cold water and the obtained precipitate was
filtered, washed with water, dried and crystallized from dimethyl
formamide/ethanol (2:1).
CH3), 12.39 (thienotriazolopyrimidine C6eCH3), 13.93 (ester CH3),
33.15 (CH2), 61.13 (OCH2), 114.59 (thienotriazolopyrimidine C7a),
120.67 (thienotriazolopyrimidine C6), 120.69 (thienotriazolopyr-
imidine C7), 123.88 (thieno triazolopyrimidine C2), 127.91 (thie-
notriazolopyrimidine C4a), 148.43 (thienotriazolopyrimidine C8),
152.13 (thienotriazolopyrimidine C3a), 167.56 (ester C]O). MS (m/
z, %): 306 (Mþ, 75.7), 259 (17.0), 234 (55.1), 232 (100.0), 204 (21.1),
164 (59.9), 110 (18.6), 59 (72.9). Anal. Calcd for C13H14N4O3S
(306.34): C, 50.97; H, 4.61; N, 18.29. Found: C, 51.22; H, 4.96; N,
18.65.
3.1.9.1. 6,7-Dimethyl-2-phenylaminothieno[2,3-d][1,2,4]triazolo[1,5-
a]pyrimidin-8(3H)-one (12a). Yield: 56%, method A; 68%, method
B, Mp >300 ꢁC. IR (KBr, cmꢀ1): 3298, 3159 (NH), 3048, 2919 (CH),
1674 (C]O), 1614 (C]N), 1589, 1472 (C]C). 1H NMR (300 MHz,
3.1.11. 2-(6,7-Dimethyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]triazolo
[1,5-a] pyrimidin-2-yl)-N-phenyl or 4-substituted phenyl acetamides
(14aec)
A mixture of 13 (0.31 g, 1.0 mmol) and the proper amine
(3.0 mmol) was heated in an oil bath at 180 ꢁC for 1h. The reaction
mixture was left to cool and the solidified mass was triturated with
ethyl alcohol, filtered, dried and crystallized from dimethyl form-
amide/ethanol (1:2).
DMSO-d6)
d 2.25 (s, 3H, thienotriazolopyrimidine C7eCH3), 2.34
(s, 3H, thienotriazolopyrimidine C6eCH3), 6.91 (t, J ¼ 7.6 Hz, 1H,
phenyl C4eH), 7.30 (t, J ¼ 7.6 Hz, 2H, phenyl C3,5eH), 7.66 (d,
J ¼ 7.6 Hz, 2H, phenyl C2,6eH), 9.55, 12.30 (2ꢄ s, each 1H, 2 NH,
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6)
d 21.68 (thie-
notriazolopyrimidine C7eCH3), 22.45 (thienotriazolopyrimidine
C6eCH3), 123.31 (thienotriazolopyrimidine C7a), 126.32 (phenyl
C2,6), 129.92 (phenyl C4), 132.46 (thienotriazolopyrimidine C6),
137.81 (thienotriazolopyrimidine C7), 138.16 (phenyl C3,5), 150.07
(phenyl C1), 158.18 (thienotriazolopyrimidine C2), 161.64 (thieno-
triazolopyrimidine C4a), 163.90 (thienotriazolopyrimidine C8),
168.30 (thienotriazolopyrimidine C3a). MS (m/z, %): 312 (Mþ þ 1,
27.4), 311 (Mþ, 100.0), 282 (24.2), 179 (22.6), 153 (67.7), 118 (14.5),
104 (21.0), 66 (43.5), 59 (74.2). Anal. Calcd for C15H13N5OS
(311.36): C, 57.86; H, 4.21; N, 22.49. Found: C, 57.49; H, 4.50; N,
22.18.
3.1.11.1. 2-(6,7-Dimethyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]tri-
azolo[1,5-a] pyrimidin-2-yl)-N-phenyl acetamide (14a). Yield: 82%;
Mp >300 ꢁC. IR (KBr, cmꢀ1): 3274, 3164 (NH), 2975, 2912 (CH), 1674
(C]O), 1622 (C]N), 1592, 1537, 1471, 1443 (C]C). 1H NMR
(300 MHz, DMSO-d6)
d 2.29 (s, 3H, thienotriazolopyrimidine C7e
CH3), 2.35 (s, 3H, thienotriazolopyrimidine C6eCH3), 3.96 (s, 2H,
CH2), 7.08 (t, J ¼ 7.7 Hz, 1H, phenyl C4eH), 7.33(t, J ¼ 7.7 Hz, 2H,
phenyl C3,5eH), 7.60 (d, J ¼ 7.7 Hz, phenyl C2,6eH), 10.34, 12.62 (2ꢄ
s, each 1H, 2 NH, D2O exchangeable). Anal. Calcd for C17H15N5O2S
(353.40): C, 57.78; H, 4.28; N, 19.82. Found: C, 57.52; H, 4.05; N,
20.06.
3.1.9.2. 2-[(4-Chlorophenyl)amino]- 6,7-dimethylthieno[2,3-d][1,2,4]
triazolo[1,5-a]pyrimidin-8(3H)-one (12b). Yield: 60%, method A;
69% method B, Mp >300 ꢁC. IR (KBr, cmꢀ1): 3309, 3175 (NH), 3045,
2975, 2852 (CH), 1679 (C]O), 1615 (C]N), 1572, 1476 (C]C), 1094
3.1.11.2. N-(4-Chlorophenyl)-2-(6,7-dimethyl-8-oxo-3,8-dihydrothi-
eno[2,3-d][1,2,4] triazolo[1,5-a] pyrimidin-2-yl)acetamide (14b).
Yield: 79%; Mp >300 ꢁC. IR (KBr, cmꢀ1): 3263, 3163 (NH), 2979,
2911 (CH), 1674 (C]O), 1624 (C]N), 1585, 1531, 1477, 1447 (C]C),
(CeCl). 1H NMR (300 MHz, DMSO-d6)
d 2.30 (s, 3H, thieno-
triazolopyrimidine C7eCH3), 2.39 (s, 3H, thienotriazolopyrimidine
C6eCH3), 7.34, 7.68 (2ꢄ d, J ¼ 8.7 Hz, each 2H, chlorophenyl C3,5eH
& C2,6eH), 9.78, 12.76 (2ꢄ s, each 1H, 2 NH, D2O exchangeable).
Anal. Calcd for C15H12ClN5OS (345.81): C, 52.10; H, 3.50; N, 20.25.
Found: C, 52.42; H, 3.85; N, 20.57.
1085 (CeCl). 1H NMR (300 MHz, DMSO-d6)
d 2.31(s, 3H, thieno-
triazolopyrimidine C7eCH3), 2.37 (s, 3H, thienotriazolopyrimidine
C6eCH3), 3.97 (s, 2H, CH2), 7.36, 7.62 (2ꢄ d, J ¼ 8.6 Hz, each 2H,
chlorophenyl C3,5eH & C2,6eH), 10.39, 12.64 (2ꢄ s, each 1H, 2 NH,
3.1.9.3. 6,7-Dimethyl-2-[(4-methylphenyl)amino]thieno[2,3-d][1,2,4]
triazolo[1,5-a]pyrimidin-8(3H)-one (12c). Yield: 54%, method A;
67%, method B, Mp >300 ꢁC. IR (KBr, cmꢀ1): 3300, 3149 (NH), 3029,
2918 (CH), 1674 (C]O), 1615 (C]N), 1571, 1514, 1473 (C]C). 1H
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6)
d 21.91 (thie-
notriazolopyrimidine C7eCH3), 22.56 (thienotriazolopyrimidine
C6eCH3), 44.72 (CH2), 124.0 (thienotriazolopyrimidine C7a), 130.18
(chlorophenyl C2,6), 133.39 (thienotriazolopyrimidine C6), 136.64
(chlorophenyl C4), 137.35 (thienotriazolopyrimidine C7), 138.19
(chlorophenyl C3,5), 147.09 (chlorophenyl C1), 157.77 (thieno-
triazolopyrimidine C2), 160.19 (thienotriazolopyrimidine C4a),
161.69 (thienotriazolopyrimidine C8), 169.98 (thienotriazolopyr-
imidine C3a), 174.53 (acetamido C]O). Anal. Calcd for
C17H14ClN5O2S (387.84): C, 52.65; H, 3.64; N, 18.06. Found: C, 52.95;
H, 3.86; N, 18.37.
NMR (300 MHz, DMSO-d6)
d 2.23 (s, 3H, thienotriazolopyrimidine
C7eCH3), 2.24 (s, 3H, thienotriazolopyrimidine C6eCH3), 2.46 (s,
3H, phenyl CH3), 7.30, 8.06 (2ꢄ d, J ¼ 8.6 Hz, each 2H, methylphenyl
C
3,5eH and C2,6eH), 9.55, 12.30 (2ꢄ s, each 1H, 2 NH, D2O
exchangeable). Anal. Calcd for C16H15N5OS (325.39): C, 59.06; H,
4.65; N, 21.52. Found: C, 59.42; H, 4.95; N, 21.87.
3.1.10. Ethyl (6,7-dimethyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]
triazolo[1,5-a]pyrimidin-2-yl)acetate (13)
3.1.11.3. 2-(6,7-Dimethyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]tri-
azolo[1,5-a] pyrimidin-2-yl)-N-(4-methoxyphenyl)acetamide (14c).
Yield: 84%; Mp >300 ꢁC. IR (KBr, cmꢀ1): 3274, 3148 (NH), 2971, 2917
(CH), 1672 (C]O), 1620 (C]N), 1575, 1538 (C]C), 1242, 1032 (Ce
A suspension of 3 (0.42 g, 2.0 mmol) in diethylmalonate
(3.27 g, 20 mmol, 3.1 ml) was heated under reflux for 4 h. The
reaction mixture was left to cool and the precipitated solid was
filtered, washed with cold ethanol, dried and crystallized from
dimethyl formamide. Yield: 78%; Mp: 245 ꢁC. IR (KBr, cmꢀ1): 3157
(NH), 2981, 2925 (CH), 1742 (ester C]O), 1687 (amide C]O),
1616 (C]N), 1572, 1475, 1447 (C]C), 1248, 1026 (CeOeC). 1H
OeC). 1H NMR (300 MHz, DMSO-d6)
d 2.31(s, 3H, thieno-
triazolopyrimidine C7eCH3), 2.41 (s, 3H, thienotriazolopyrimidine
C6eCH3), 3.72 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 6.90, 7.50 (2ꢄ d,
J ¼ 9.0 Hz, each 2H, methoxyphenyl C3,5eH & C2,6eH), 10.21, 12.62
(2ꢄ s, each 1H, 2NH, D2O exchangeable). Anal. Calcd for
C18H17N5O3S (383.42): C, 56.38; H, 4.47; N, 18.27. Found: C, 56.06;
H, 4.58; N, 18.56.
NMR (300 MHz, DMSO-d6)
3H, thienotriazolopyrimidine C7eCH3), 2.40 (s, 3H, thieno-
triazolopyrimidine C6eCH3), 3.98 (s, 2H, CH2CO), 4.15 (q,
d
1.21 (t, J ¼ 6.9 Hz, CH2CH3), 2.31 (s,

350
H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
3.2. Anti-inflammatory (AI) activity
3.2.6. Statistical analysis of data
The data obtained are presented as means ꢂ SE of the mean. The
concentration-dependent effects of various drugs in vitro were
evaluated statistically by the randomized block design analysis of
Variance (ANOVA) followed by StudenteNewmaneKeuls Multiple
Comparison Test. The difference in results was considered signifi-
cant when P < 0.05.
3.2.1. Formalin-induced paw edema bioassay (acute inflammatory
model)
Male albino rats weighing 180e200 g were used throughout the
assay. They were kept in the animal house under standard condi-
tion of light and temperature with free access to food and water.
The animals were randomly divided into groups each of five rats.
One group of five rats was kept as a control and another group
received the standard drug diclofenac Na (at a dose of 20 mg/kg
body weight po). A solution of formalin (2%, 0.1 ml) was injected
into the subplanter region of the left hind paw under light ether
anesthesia 1 h after oral administration (po) of the test compound
(at a dose level of 20 mg/kg body weight). The paw volume (ml)
was measured by means of water plethysmometer and re-
measured again 1, 2 and 4 h after administration of formalin. The
edema was expressed as an increase in the volume of paw, and the
percentage of edema inhibition for each rat and each group was
obtained as follows:
3.3. Analgesic activity
Analgesic activity was determined using tail withdrawal
response to immersion of rat tail in water at 55 ꢁC according to the
procedure described by Janssen et al. [49]. Male albino rats
weighing 120e150 g were used throughout this assay. They were
kept in the animal house under standard condition of light and
temperature with free access to food and water. The animals were
randomly divided into groups each of five rats. One group of five
rats was kept as a control and another group received the stan-
dard drug diclofenac Na (at a dose of 20 mg/kg body weight po).
The tested compounds were administrated orally at a dose of
20 mg/kg and diclofenac Na was used as a reference drug (20 mg/
kg). The recorded values were the average of five
administrations ꢂ SE and the percentage increase of the reaction
time (after 1e4 h time intervals) was calculated in comparison
with the basal values.
% Inhibition ¼ (Vt ꢀ Vo) control ꢀ (Vt ꢀ Vo) tested compound/(Vt e
Vo) control ꢄ 100
Where Vt ¼ volume of edema at specific time interval and
Vo ¼ volume of edema at zero time interval.
3.2.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model)
References
Rats in the first experiment were given the same test
compounds at a dose level of 20 mg/kg body weight daily for 7
consecutive days. A solution of formalin (2%, 0.1 ml) was injected
into the subplanter region of the left hind paw under light ether
anesthesia 1 h after oral administration (po) of the test compound.
A second injection of formalin (2%, 0.1 ml) was given on the
third day. The changes in the volume of paw were measured
plethysmographically at the first and eighth days.
[1] J.M. Scheiman, Clin. Update 12 (2005) 1e4.
[2] S. Fiorucci, L. Santucci, E. Distrutti, Dig. Liver Dis. 39 (2007) 1043e1051.
[3] D.C. Kosegarten, E.F. LaSala, S.F. Long, in: L. Shargel, A.H. Mutnick, P.F. Souney,
L.N. Swanson, L.H. Block (Eds.), Comprehensive Pharmacy Review, Lippincott
Williams and Wilkins, Philadelphia, 2001, pp. 289e291.
[4] M.R. Griffin, A. Yared, W.A. Ray, Am. J. Epidemiol. 151 (2000) 488e496.
[5] F.K. Chan, Nat. Clin. Pract. 3 (10) (2006) 563e573.
[6] B.J. Whittle, Eur. J. Pharmacol. 500 (1e3) (2004) 427e439.
[7] G. Dannhardt, W. Kiefer, Eur. J. Med. Chem. 36 (2001) 109e126.
[8] W. Kiefer, G. Dannhardt, Expert Rev. Clin. Immunol. 1 (2005) 431e442.
[9] M. Burnier, Expert Opin. Drug Saf. 4 (2005) 491e499.
[10] D. Wong, M. Wang, Y. Cheng, G.A. FitzGerald, Curr. Opin. Pharmacol. 5 (2005)
204e210.
3.2.3. Ulcerogenic activity
Male albino rats (180e200 g) were divided into groups each
of five animals and were fasted for 12 h prior to the adminis-
tration of the test compounds. Water was given ad libitum.
Control group received 1% gum acacia orally. Other groups
received diclofenac Na or the test compounds orally in two equal
doses at 0 and 12 h for three successive days at a dose of 300 mg/
kg per day. Animals were sacrificed by diethyl ether 6 h after the
last dose and their stomachs were removed. An opening at the
greater curvature was made and the stomach was cleaned by
washing with cold saline and inspected with a 3ꢄ magnifying
lens for any evidence of hyperemia, hemorrhage, definite
hemorrhagic erosion or ulcer.
[11] R.D. Rudic, D. Brinster, Y. Cheng, S. Fries, W.L. Song, S. Austin, T.M. Coffman,
G.A. Fitz Gerald, Circ. Res. 96 (2005) 1240e1247.
[12] A.E. Rashad, A.H. Shamroukh, R.E. Abdel-Megeid, A. Mostafa, R. El-Shesheny,
A. Kandeil, M.A. Ali, K. Banert, Eur. J. Med. Chem. 45 (2010) 5251e5257.
[13] A.E. Rashad, M.A. Ali, Nucleosides Nucleotides 25 (2006) 17e28.
[14] M.H. Bhuiyan, K.M. Rahman, K.H. Abdur Rahim, M.I. Hossain, M. Abu Naser,
Acta Pharm. 56 (2006) 441e450.
[15] M.I. Hegab, N.A. Hassan, A.E. Rashad, A.A. Fahmy, F.M.E. Abdel-Megeid,
Phosphorus, Sulfur and Silicon 182 (2007) 1535e1556.
[16] S.I. Alqasoumi, F.A. Ragab, A.M. Alafeefy, M. Galal, M.M. Ghorab, Phosphorus,
Sulfur and Silicon 184 (2009) 3241e3257.
[17] V. Alagarsamy, S. Vijayakumar, V. Raja Solomon, Biomed. Pharmacother. 61
(2007) 285e291.
[18] A.K. El-Ansary, A.H. Omar, Bull. Fac. Pharm. Cairo Univ. 39 (2001) 17e25.
[19] V. Alagarsamy, S. Meena, K.V. Ramseshu, V.R. Solomon, K. Thirumurugan,
K. Dhanabal, M. Murugan, Eur. J. Med. Chem. 41 (2006) 1293e1300.
[20] B.V. Ashalatha, B. Narayana, K.K. Vijaya Raj, N. Suchetha Kumari, Eur. J. Med.
Chem. 42 (2007) 719e728.
[21] M. Modica, M. Santagati, A. Santagati, V. Cutuli, N. Mangano, A. Caruso,
Pharmazie 55 (2000) 500e502.
[22] M.D. Mullican, M.W. Wilson, D.T. Connor, C.R. Kostlan, D.J. Schrier, R.D. Dyer,
J. Med. Chem. 36 (1993) 1090e1099.
[23] D.H. Boschelli, D.T. Connor, D.A. Bornemeier, R.D. Dyer, J.A. Kennedy,
P.J. Kuipers, G.C. Okonkwo, D.J. Schrier, C.D. Wright, J. Med. Chem. 36 (1993)
1802e1810.
[24] L. Czollner, G. Szilágyi, J. Langó, J. Janáky, Arch. Pharm. (Weinheim) 323 (1990)
225e227.
[25] E. Palaska, G. S¸ ahin, P. Kelicen, N.T. Durlu, G. Altinok, Il Farmaco 57 (2002)
101e107.
[26] G. Roma, M. Di Braccio, G. Grossi, F. Mattioli, M. Ghia, Eur. J. Med. Chem. 35
(2000) 1021e1035.
3.2.4. Acute toxicity
Twelve groups of rats (180e200 g) each consists of five animals,
were used in this test. The animals were fasted for 24 h prior to
administration of the test compounds. The compounds were given
orally in graded doses of 0.1e0.3 g/kg body weight, po. The
compounds were screened at graded doses for their acute lethal
doses (ALD50) and the mortalities were recorded at each dose level
after 24 h.
3.2.5. Determination of effective dose 50 (ED50
)
The selected compounds were further tested at 5, 10, 20, 40,
and 50 mg/kg body weight and the ED50 was determined by
measuring the inhibition of the edema volume 2 h after formalin
injection.
[27] G. Grossi, M. Di Braccio, G. Roma, V. Ballabeni, M. Tognolini, E. Barocelli, Eur. J.
Med. Chem. 40 (2005) 155e165.
[28] G. Roma, G. Grossi, M. Di Braccio, D. Piras, V. Ballabeni, M. Tognolini, S. Bertoni,
E. Barocelli, Eur. J. Med. Chem. 43 (2008) 1665e1680.

H.M. Ashour et al. / European Journal of Medicinal Chemistry 62 (2013) 341e351
351
[29] G. Roma, M. Di Braccio, G. Grossi, D. Piras, V. Ballabeni, M. Tognolini, S. Bertoni,
E. Barocelli, Eur. J. Med. Chem. 45 (2010) 352e366.
[30] P. Mullick, S.A. Khan, T. Begum, S. Verma, D. Kaushik, O. Alam, Acta Pol. Pharm.
Drug Res. 66 (2009) 379e385.
[37] O.H. Rizk, O.G. Shaaban, I.M. El-Ashmawy, Eur. J. Med. Chem. 55 (2012) 85e93.
[38] K. Gewald, E. Schinke, H. Bottcher, Chem. Ber. 99 (1966) 94e100.
[39] N.T. Pokhodylo, V.S. Matiychuk, M.D. Obushak, Tetrahedron 64 (2008)
1430e1434.
[31] M.M. Kandeel, A.H. Omar, Bull. Fac. Pharm. Cairo Univ. 41 (2003) 43e50.
[32] A.A. Bekhit, H.M.A. Ashour, A.A. Guemei, Arch. Pharm. Chem. Life Sci. 338
(2005) 167e174.
[33] A.A. Bekhit, H.M.A. Ashour, Y.S. Abdel Ghany, A.E.A. Bekhit, A. Baraka, Eur. J.
Med. Chem. 43 (2008) 456e463.
[34] A.A. Bekhit, H.M.A. Ashour, A.-E.A. Bekhit, H.M. Abdel Rahman, S.A. Bekhit,
J. Enz. Inhib. Med. Chem. 24 (2009) 296e309.
[35] A.A. Bekhit, H.M.A. Ashour, A.-E.A. Bekhit, S.A. Bekhit, Med. Chem. 5 (2009)
103e117.
[40] D. Lloyd, R.H. McDougall, D.R. Marshall, J. Chem. Soc. (1965) 3785e3792.
[41] G.J. Karabatsos, R.A. Taller, J. Am. Chem. Soc. 85 (1963) 3624e3629.
[42] J. Quin, G.K. Friestad, Tetrahedron 59 (2003) 6393e6402.
[43] H. Thakuria, G. Das, Arkivoc xv (2008) 321e328.
[44] G.J. Karabatsos, J.D. Graham, F.M. Vane, J. Am. Chem. Soc. 84 (1962) 753e755.
[45] M. Modica, M. Santagati, A. Santagati, J. Heterocycl. Chem. 38 (2001) 973e978.
[46] H. Hosseinzadeh, H. Younesi, BMC Pharmacol. 2 (2002) 1e2.
[47] G. Daidone, B. Maggio, D. Raffa, Eur. J. Med. Chem. 29 (1994) 707e711.
[48] M. Verma, M. Tripathi, A.K. Saxena, K. Shanker, Eur. J. Med. Chem. 29 (1994)
941e946.
[36] S.A.F. Rostom, I.M. El-Ashmawy, H.A. Abd El Razik, M.H. Badr, H.M.A. Ashour,
Bioorg. Med. Chem. 17 (2009) 882e895.
[49] P.A.J. Janssen, D.N. Kellett, J.E. Davis, Arzneim. Forsch. 13 (1963) 633e639.