Design, synthesis and biological evaluation of some novel thienopyrimidines and fused thienopyrimidines as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.007

Some new substituted thienopyrimidine derivatives comprising thioxo, thioalkyl and pyrazolyl derivatives as well as fused thienotriazolopyrimidine and thienopyrimidinotriazine ring systems were prepared from 3-benzyl-2-hydrazino-5-methyl-4-oxo-3,4-dihydro

Full text of DOI:10.1016/j.ejmech.2012.07.007

European Journal of Medicinal Chemistry 55 (2012) 85e93
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of some novel thienopyrimidines
and fused thienopyrimidines as anti-inflammatory agentsq
,
b
Ola H. Rizk a, Omaima G. Shaaban a , Ibrahim M. El-Ashmawy
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, 1 Meedan Elkhartoom, Alexandria 21521, Egypt
b Department of Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new substituted thienopyrimidine derivatives comprising thioxo, thioalkyl and pyrazolyl deriva-
tives as well as fused thienotriazolopyrimidine and thienopyrimidinotriazine ring systems were prepared
from 3-benzyl-2-hydrazino-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide 4. The
designed compounds were evaluated for their anti-inflammatory activity. Compounds 4, 9, 10 and 13
showed the highest anti-inflammatory effect compared with the reference drug diclofenac sodium.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 25 September 2011
Received in revised form
1 July 2012
Accepted 4 July 2012
Available online 14 July 2012
Keywords:
Synthesis
Thienopyrimidines
Fused thienopyrimidines
Anti-inflammatory activity
1. Introduction
Based on the above mentioned findings, we decided to
synthesize some novel thienopyrimidines and fused thienopyr-
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most widely used therapeutic agents, primarily for the treatment of
pain and inflammation in arthritis. However, long-term clinical
usage of NSAIDs is associated with significant side effects including
gastrointestinal lesions, bleeding and nephrotoxicity [1e4]. NSAID-
induced gastropathy is estimated to affect up to half of chronic
NSAID users, with major world health implications [5]. Therefore
the discovery of new safer anti-inflammatory drugs represents
a challenging goal for such a research area.
In the last few years, thienopyrimidines and fused thienopyr-
imidines have gained much attention due to their diverse biological
activities [6e10]. Recently, a large number of thienopyrimidines
and fused thienopyrimidines (Fig. 1) were found to possess prom-
ising anti-inflammatory activities [11e13]. Moreover, triazoles,
pyrazoles and triazines moieties have been gaining prominence for
their wide spectrum of activities especially as anti-inflammatory
[14e18].
imidines aiming at developing more active anti-inflammatory
agents devoid of the undesirable side effects associated with clas-
sical NSAIDs. The newly synthesized compounds were designed so
as to comprise a thienopyrimidine moiety directly attached to
a pyrazole or fused with a triazole or triazine rings. Such structural
assembly was supposed to enhance the biological potential of this
class of compounds [13].
2. Results and discussion
2.1. Chemistry
The synthetic strategies adopted for the synthesis of the inter-
mediate and final compounds are depicted in Schemes 1e3.
In Scheme 1, the starting compound ethyl 2-amino-5-
carbamoyl-4-methylthiophene-3-carboxylate 1 [19] was prepared
by heating a mixture of acetoacetamide, ethyl cyanoacetate and
sulfur in the presence of morpholine as a basic catalyst. Refluxing 1
with benzyl isothiocyanate in acetonitrile using anhydrous potas-
sium carbonate afforded the sulfanyl thienopyrimidine derivative 2
following the method previously used for preparing such analogs
compounds [20]. The structure of 2 was confirmed by its elemental
analysis and spectral data. 1H NMR spectrum indicated the pres-
ence of signals assigned to the benzyl moiety and the sulfanyl
q
This work was partially presented at the 16th SCI/RSC Medicinal Chemistry
Symposium, Churchill College, Cambridge, UK, Sunday 11
September, 2011, as a Poster. P 2.
e Wednesday 14
* Corresponding author. Fax: þ20 3 4873273.
E-mail address: omimagaber@yahoo.com (O.G. Shaaban).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.007

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O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
anilino moiety. The thiol tautomer of compound 9 was S-alkylated
with ethyl bromoacetate in absolute ethanol to give the thioalkyl
derivative 10 in an excellent yield. 1H NMR spectrum of 10 showed
a triplet and a quartet signals assigned for the ethyl moiety, while its
13CNMRspectrumdisplayedsignals at23.27and70.83ppmduetothe
ethyl group.
In Scheme 3, the key intermediate 4 was cyclized with
aromatic carboxylic acids by refluxing with phosphorous oxy-
chloride to afford compounds 11a,b which were identified by IR
and 1H NMR spectra. Reacting compound 4 with formic acid gave
the corresponding 5-benzyl-3-methyl-4-oxo-4,5-dihydrothieno
[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide 12. Further-
more, heating compound 4 with ethyl chloroformate in dry
dioxane induced cyclization to the 5-benzyl-3-methyl-4,8-dioxo-
4,5,7,8-tetrahydrothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-
carboxamide 13. 1H NMR spectrum of compound 13 showed a D2O
exchangeable singlet assigned for the triazolo NH whereas; its 13C
NMR spectrum revealed two signals at 146.74 and 157.62 ppm
corresponding to triazolothienopyrimidine-C3a and C8 respec-
tively. Synthesis of the triazolo derivative 14 was accomplished by
heating a mixture of 4 and diethyl malonate in glacial acetic acid.
1H NMR spectrum of compound 14 showed a triplet and a quartet
characteristic for the ethyl moiety, while its MS spectrum revealed
a molecular ion peak at m/z 425 (12.51%) which match its
molecular weight. Additionally, compound 4 was cyclized with
the appropriate phenacyl bromides in boiling absolute ethanol
and anhydrous sodium acetate to give the thieno[30,20:5,6]pyr-
imidino[2,1-c][1,2,4]triazine derivatives 15aec. 1H NMR spectra
of compounds 15aec showed the presence of singlet at
5.13e5.20 ppm assigned to the triazino C-9 protons. On the other
hand, refluxing compound 4 with the appropriate phenacyl
cyanides in glacial acetic acid resulted in the formation of the
pyrazolyl derivatives 16a,b. Their 1H NMR spectrum showed
singlets at 10.33 and 10.31 ppm assigned to additional NH2 group
and singlets at 3.21 and 2.88 ppm corresponding to the pyrazolyl
C-4 protons. The pyrazolyl derivatives 17 and 18 were successfully
achieved by heating 4 with acetylacetone and ethyl acetoacetate
respectively in glacial acetic acid. 1H NMR spectra of compounds
17 and 18 were characterized by pyrazolyl C-4 protons and signals
for the methyl groups on the pyrazole ring. 13C NMR spectrum for
compound 17 revealed two signals for the two methyl groups of
the pyrazole ring at 23.86 and 49.80 ppm and a signal at
125.01 ppm due to pyrazole C-4. Moreover, the MS spectrum of 17
showed a molecular ion peak at m/z 393 (7.15%) which matched its
molecular weight.
Fig.
1. Examples
of
anti-inflammatory
thienopyrimidines
and
fused
thienopyrimidines.
group. Treatment of compound 2 with methyl iodide and anhy-
drous potassium carbonate in dry acetone yielded the methyl-
sulfanyl derivative 3 according to a literature procedure [21].
Analytical and spectral data of compound 3 are in agreement with
the proposed structure, confirmed by the appearance of a methyl
singlet at 2.71 ppm in the 1H NMR spectrum. Refluxing 3 with 99%
hydrazine hydrate in ethanol furnished the corresponding hydra-
zino derivative 4. 1H NMR showed the absence of the methyl singlet
and the appearance of the D2O exchangeable signals at 9.32 and
11.40 ppm assigned to NH2 and NH respectively.
In Scheme 2, the new hydrazones 5a,b were prepared by conden-
sation of the key intermediate 4with 4-bromo or 4-nitrobenzaldehyde
utilizing a previously reported procedure [22]. Reaction of 5a,b with
bromine in glacial acetic acid and anhydrous sodium acetate yielded
the cyclized counterparts 6a,b. 1H NMR spectra of compounds 6a,b
lacked signals characteristic for N]CHand NH protons, while 13CNMR
spectrum for compound 6a provided further confirmation of the
structure. Moreover, heating the hydrazones 5a,b with acetic anhy-
dride yielded 7a,b in good yields. 1H NMR spectra of 7a,b showed
additional singlets at 2.82 or 2.88 ppm attributed to COCH3 group and
lacked signals assigned for N]CH and NH protons. The structure of
these compounds was further verified by 13C NMR spectral data.
Refluxing the hydrazino derivative 4 with phenyl isothiocyanate in dry
dimethylformamide produced the cyclized tetrahydrothieno[3,2-e]
[1,2,4]triazolo[4,3-a]pyrimidine derivative 9 instead of the thio-
semicarbazide 8. Compound 9 was also prepared through heating the
hydrazine derivative 4 with carbon disulfide in ethanolic potassium
hydroxide. Structure of 9 was substantiated by its 1H NMR spectral
data which revealed the disappearance of signals attributed to the
2.2. Anti-inflammatory (AI) activity
To assess the AI activity of the designed compounds, 16
compounds namely; 2, 3, 4, 5a, 6a, 7a, 9, 10, 11b, 12, 13, 14, 15c,
16b, 17 and 18 were evaluated by two screening protocols;
namely, the formalin-induced paw edema [23] and turpentine oil-
induced granuloma pouch [24] bioassays, using diclofenac Na
(10 mg/kg) as a reference standard anti-inflammatory agent. The
paw edema was employed as a model for acute and sub-acute
inflammation, while the turpentine oil-induced granuloma
pouch assay was utilized as another model for sub-acute inflam-
matory condition. The data obtained were presented in Tables 1e3
and expressed as means ꢀ SE. Statistical differences of control and
test groups were carried out using the Analysis of Variance
(ANOVA) followed by ‘StudenteNewmaneKeuls Multiple
Comparison Test. They were performed using computer package
of the Statistical Analysis System (SAS, 1987), SAS Incorporation
Institute. The difference in results was considered significant
when P < 0.05.
Scheme 1. Synthetic routes of compounds 2e4.

O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
87
Scheme 2. Synthetic routes of compounds 5e10.
2.2.1. Formalin-induced paw edema bioassay (acute inflammatory
model)
In this acute inflammatory model [23] each test compound was
2.2.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model)
For this sub-acute inflammatory model [23] inflammation was
induced by formalin injection in the first and third days, and test
compounds were administered orally (at 10 mg/kg daily) for 7 days.
Again, diclofenac Na was used as a reference anti-inflammatory
agent in this assay. 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, compounds 4, 9,
10, 13, 14 and 15c showed superior anti-inflammatory activities
(41e46%) than diclofenac Na (39%). On the other hand, compounds
5a, 12, 17 and 18 displayed anti-inflammatory activities (39%) equal
to diclofenac Na. At the 8th day, compound 4 was found to be
superior over the reference drug with anti-inflammatory activity
(43%), compounds 10, 13, 14 and 15c were nearly effective (36%,
37%) as diclofenac Na (36%).
dosed orally (10 mg/kg body weight) 1 h prior to induction of
inflammation by formalin injection. Diclofenac Na was utilized as
a reference anti-inflammatory drug at a dose of 10 mg/kg, po. The
anti-inflammatory activity was then calculated 1e4 h after induc-
tion 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, three compounds, 4 (69%), 5a
(69%) and 13 (75%) showed promising pharmacokinetic profiles as
revealed from their potent and rapid onset of action which was
higher than diclofenac Na (63%) at a dose of 10 mg/kg, po. Whereas,
the rest of the compounds showed less activities. After 2 h interval,
the data indicated that compounds 4, 9, 10, 13 and 16b displayed
better anti-inflammatory activity (52e61%) compared to diclofenac
Na (43%). Whereas, compounds 3, 11b, 12, 14, 15c and 18
were nearly as effective (43e48%) as diclofenac Na. The anti-
inflammatory activity after 4 h interval for compounds 10, 11b, 13
and 17 were found to be equally effective to diclofenac Na (49%).
Whereas, compounds 4, 9 and 12 proved to be more effective (51%,
51%, and 54%, respectively) than diclofenac Na (49%) at the same
time interval.
2.2.3. Turpentine oil-induced granuloma pouch bioassay (sub-acute
inflammatory model)
In this bioassay [24] each test compound was administered
orally (10 mg/kg) 1 h prior to turpentine oil injection and continued
for 7 days. At the 8th day, the exudates volume (mL) was measured
and the percentage of granuloma inhibition was calculated. Diclo-
fenac Na (10 mg/kg) was used as a reference drug. The results
depicted in Table 3 revealed that, while most of the test compounds
showed anti-inflammatory activity less than the reference drug,
compound 4 was proved to be more effective (44.79%) than diclo-
fenac Na (41.17%). Compounds 6a and 10 were equipotent (41.17%)
with diclofenac Na. While, compounds 7a, 12, 14, 16b, and 17
showed moderate anti-inflammatory activity in this bioassay with
Some of the most active compounds 4, 9, 10, 13, 16b and 18 were
further tested at 5, 10, 20, 40 and 50 mg/kg body weight in order to
determine their ED50 values. Most of these compounds were found
to be nearly equipotent (ED50 ¼ 8.36e11.53 mg/kg, Table 1).
Meanwhile, compounds 4, 9 and 10 were found to be the most
potent (ED50 ¼ 8.36e8.72 mg/kg) compared to the reference drug
diclofenac Na (ED50 ¼ 11.53 mg/kg of body weight).

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O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
Scheme 3. Synthetic routes of compounds 11a,be18.
Table 1
Anti-inflammatory activity (AI) of some selected compounds in formalin-induced rat paw edema bioassay (acute inflammatory model).
Compounda
Volume of edema (mL)b
0
1 h
2 h
4 h
ED50 (mg/kg)
Control
2
3
4
5a
6a
7a
9
10
11b
12
13
15c
16b
17
18
0.27 ꢀ 0.01
0.26 ꢀ 0.01
0.29 ꢀ 0.02
0.30 ꢀ 0.01
0.28 ꢀ 0.03
0.26 ꢀ 0.01
0.25 ꢀ 0.01
0.29 ꢀ 0.02
0.30 ꢀ 0.01
0.24 ꢀ 0.03
0.27 ꢀ 0.01
0.29 ꢀ 0.02
0.28 ꢀ 0.01
0.28 ꢀ 0.03
0.28 ꢀ 0.02
0.28 ꢀ 0.01
0.28 ꢀ 0.02
0.43 ꢀ 0.01
0.50 ꢀ 0.01
0.66 ꢀ 0.02
0.38 ꢀ 0.01* (25)c
0.38 ꢀ 0.01* (44)
0.35 ꢀ 0.02* (69)
0.33 ꢀ 0.01* (69)
0.37 ꢀ 0.02* (31)
0.36 ꢀ 0.01* (31)
0.37 ꢀ 0.01* (50)
0.37 ꢀ 0.01* (56)
0.34 ꢀ 0.03* (37)
0.34 ꢀ 0.01* (56)
0.33 ꢀ 0.01* (75)
0.37 ꢀ 0.02* (44)
0.35 ꢀ 0.01* (56)
0.37 ꢀ 0.02* (44)
0.38 ꢀ 0.01* (37)
0.34 ꢀ 0.01* (63)
0.49 ꢀ 0.01 (0)
0.63 ꢀ 0.02 (5)
0.42 ꢀ 0.01* (43)
0.39 ꢀ 0.01* (61)
0.42 ꢀ 0.02* (39)
0.40 ꢀ 0.01* (39)
0.41 ꢀ 0.02* (30)
0.39 ꢀ 0.02* (56)
0.39 ꢀ 0.01* (61)
0.37 ꢀ 0.01* (43)
0.40 ꢀ 0.02* (43)
0.38 ꢀ 0.01* (61)
0.41 ꢀ 0.02* (43)
0.39 ꢀ 0.02* (52)
0.42 ꢀ 0.01* (39)
0.40 ꢀ 0.02* (48)
0.41 ꢀ 0.03* (43)
0.53 ꢀ 0.01* (38)
0.49 ꢀ 0.01* (51)
0.50 ꢀ 0.01* (44)
0.49 ꢀ 0.02* (41)
0.50 ꢀ 0.01* (36)
0.48 ꢀ 0.01* (51)
0.50 ꢀ 0.02* (49)
0.44 ꢀ 0.03* (49)
0.45 ꢀ 0.02* (54)
0.49 ꢀ 0.03* (49)
0.49 ꢀ 0.02* (46)
0.49 ꢀ 0.01* (46)
0.48 ꢀ 0.01* (49)
0.49 ꢀ 0.01* (46)
0.48 ꢀ 0.01* (49)
8.36
8.72
8.36
11.53
9.65
10.5
11.53
Diclofenac Na
*Significantly different compared to corresponding control. P ꢁ 0.05.
a
Dose levels, po: test compounds (10 mg/kg body weight), diclofenac Na (10 mg/kg body weight).
Values are expressed as mean ꢀ S.E. (Number of animals N ¼ 5 rats).
b
c
Between parentheses (Percentage anti-inflammatory activity, AI %).

O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
89
Table 2
turpentine oil-induced granuloma pouch screens (sub-acute
inflammatory models). This would indicate that these compounds
are effective in acute inflammation and less active in chronic
inflammatory conditions.
Anti-inflammatory activity (AI) of some selected compounds in formalin-induced
rat paw edema bioassay (sub-acute inflammatory model).
Compounda
Volume of edema (mL)b
0
1st day
8th day
2.2.4. Ulcerogenic activity
Control
2
3
4
5a
6a
7a
9
10
11b
12
13
14
15c
16b
17
0.27 ꢀ 0.01
0.26 ꢀ 0.01
0.29 ꢀ 0.02
0.30 ꢀ 0.01
0.28 ꢀ 0.03
0.26 ꢀ 0.01
0.25 ꢀ 0.01
0.29 ꢀ 0.02
0.30 ꢀ 0.01
0.24 ꢀ 0.03
0.27 ꢀ 0.01
0.29 ꢀ 0.02
0.28 ꢀ 0.01
0.28 ꢀ 0.01
0.28 ꢀ 0.03
0.28 ꢀ 0.01
0.28 ꢀ 0.02
0.28 ꢀ 0.02
0.73 ꢀ 0.01
0.83 ꢀ 0.01
0.70 ꢀ 0.02 (4)c
0.67 ꢀ 0.02* (17)
0.56 ꢀ 0.01* (43)
0.56 ꢀ 0.01* (39)
0.55 ꢀ 0.01* (37)
0.56 ꢀ 0.02* (33)
0.56 ꢀ 0.01* (41)
0.57 ꢀ 0.01* (41)
0.55 ꢀ 0.01* (33)
0.55 ꢀ 0.01* (39)
0.55 ꢀ 0.02* (43)
0.53 ꢀ 0.01* (46)
0.53 ꢀ 0.02* (46)
0.58 ꢀ 0.02* (35)
0.56 ꢀ 0.01* (39)
0.56 ꢀ 0.02* (39)
0.56 ꢀ 0.01* (39)
0.79 ꢀ 0.02 (5)
Six tested compounds namely 4, 9, 10, 13, 16b and 18 that
exhibited variable anti-inflammatory profiles in the pre-mentioned
animal models were further evaluated for their ulcerogenic
potential in rats [25]. Gross observation of the isolated rat stomachs
showed a normal stomach texture for compounds 13, 16b and 18
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 4, 9 and 10 showed weak ulcer-
ation effect (10, 10 and 20% respectively) compared to the reference
drug diclofenac Na (no ulceration). It is worth-mentioning that,
indomethacin; the reference standard anti-inflammatory drug; was
found to cause 100% ulceration under the same experimental
conditions.
0.73 ꢀ 0.02* (21)
0.62 ꢀ 0.01* (43)
0.65 ꢀ 0.01* (34)
0.65 ꢀ 0.01* (30)
0.64 ꢀ 0.01* (30)
0.66 ꢀ 0.01* (34)
0.66 ꢀ 0.01* (36)
0.63 ꢀ 0.01* (30)
0.65 ꢀ 0.01 (32)
0.64 ꢀ 0.02* (37)
0.63 ꢀ 0.02* (37)
0.63 ꢀ 0.01* (37)
0.67 ꢀ 0.02* (30)
0.66 ꢀ 0.01* (32)
0.65 ꢀ 0.02* (34)
0.64 ꢀ 0.02* (36)
18
Diclofenac Na
*Significantly different compared to corresponding control. P ꢁ 0.05.
a
2.2.5. Acute toxicity
Dose levels, po: test compounds (10 mg/kg body weight), diclofenac Na (10 mg/
Six of the selected compounds namely 4, 9, 10, 13, 16b and 18
were further evaluated for their approximate acute lethal dose
ALD50 in male rats using a literature method [26]. The results
indicated that all of the tested compounds were proved to be non-
toxic and are well tolerated by the experimental animals. The
compounds showed a high safety margin when screened at graded
doses (0.1e0.3 g/kg, po), where their ALD50 values were found to be
>0.3 g/kg.
kg body weight).
b
Values are expressed as mean ꢀ S.E. (Number of animals N ¼ 5 rats).
c
Between parentheses (Percentage anti-inflammatory activity, AI %).
percentage of granuloma inhibition of 39.82%, 39.36%, 39.82%,
40.27% and 40.27%, respectively.
A collective interpretation of the anti-inflammatory activity of
the test compounds in pre-mentioned screens (Tables 1e3)
revealed that the hydrazinothienopyrimidine 4 and the thieno-
triazolopyrimidine 10 showed pronounced activity in the formalin-
induced paw edema screen (acute inflammatory model), in
formalin-induced paw edema and turpentine oil-induced granu-
loma pouch screens (sub-acute inflammatory models). These facts
would suggest that such compounds might be effective in
managing acute inflammation and chronic inflammatory condi-
tions. Whereas, the thienotriazolopyrimidines 9 and 13 proved to
be highly active in the formalin paw edema screen (acute inflam-
matory model) and less active in formalin paw edema and
2.3. Conclusion
In conclusion, the aim of the present investigation was to
synthesize novel thienopyrimidines and fused tricyclic thienopyr-
imidines as possible anti-inflammatory agents. Among the tested
analogs, compounds 4, 9, 10 and 13 showed pronounced anti-
inflammatory activity comparable with diclofenac Na in the acute
inflammatory model and the sub-acute inflammatory models,
suggesting that they might be effective in managing acute and
chronic inflammatory conditions. On the other hand, compounds
14 and 15c were nearly effective as diclofenac Na in the sub-acute
inflammatory model and compound 6a was equipotent with
diclofenac Na in the turpentine oil-induced granuloma pouch
screen. Additionally, all of the tested compounds revealed super GI
safety profile and are well tolerated by the experimental animals
with high safety margin (ALD50 > 3.0 g/kg). Consequently, such
type of compounds would represent a fruitful matrix for further
development of more potent and selective anti-inflammatory
agents that deserve further investigation and derivatization in
order to explore the scope and limitation of its biological activities.
Table 3
Anti-inflammatory activity of some selected compounds in turpentine oil induced
granuloma pouch in rats.
Compounda
Volume of exudates (mL)b
Percentage of inhibition
Control
2
3
4
5a
6a
7a
9
10
11b
12
13
14
15c
16b
17
2.21 ꢀ 0.09
2.12 ꢀ 0.03
1.96 ꢀ 0.04
1.22 ꢀ 0.05*
1.45 ꢀ 0.17*
1.30 ꢀ 0.06*
1.33 ꢀ 0.08*
1.70 ꢀ 0.15*
1.30 ꢀ 0.08*
1.42 ꢀ 0.09*
1.34 ꢀ 0.06*
1.36 ꢀ 0.08*
1.33 ꢀ 0.07*
1.36 ꢀ 0.12*
1.32 ꢀ 0.09*
1.32 ꢀ 0.07*
1.35 ꢀ 0.05*
1.30 ꢀ 0.07*
e
4.07
11.31
44.79
34.38
41.17
39.82
23.07
41.17
35.74
39.36
38.46
39.82
38.46
40.27
40.27
38.91
41.17
3. Experimental
3.1. Chemistry
Melting points were determined in open-glass capillaries using
a Gallen-Kamp melting point apparatus and are uncorrected. The IR
spectra (KBr) were recorded using a PerkineElmer 1430 spectro-
photometer. The 1H NMR and 13C NMR spectra were determined on
a Varian spectrometer (300 MHz), Faculty of Science, Cairo
University using tetramethylsilane (TMS) as internal standard and
18
Diclofenac Na
*Significantly different compared to corresponding control. P ꢁ 0.05.
DMSO-d6 as the solvent (chemical shifts are given in
d ppm).
a
Dose levels, po: test compounds (10 mg/kg body weight), diclofenac Na (10 mg/
Splitting patterns were designated as follows: s: singlet; d: doublet;
t: triplet; m: multiplet. Mass spectra were carried out using an
kg body weight).
b
Values are expressed as mean ꢀ S.E. (Number of animals N ¼ 5 rats).

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O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
Shimadzu GCMS-QP-1000EX mass spectrometer at 70 eV, Faculty
of Science, Cairo University. Microanalyses were performed at the
Microanalytical Unit, Faculty of Science, Cairo University. The
results of the microanalyses were within ꢀ0.4% of the calculated
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)
mp: 290e92 ꢂC. IR (KBr, cmꢃ1): 3495, 3381 (NH), 1674, 1639 (C]
O), 1613 (C]N), 1253, 1068 (CeSeC). 1H NMR (
ppm): 2.67 (s, 3H,
d
CH3), 5.26 (s, 2H, CH2), 7.22e7.34 (m, 5H, AreH), 7.39 (s, 2H, NH2,
D2O exchangeable), 7.61 (d, J ¼ 8.4 Hz, 2H, bromophenyleC2,6eH),
7.81 (d, J ¼ 8.4 Hz, 2H, bromophenyleC3,5eH), 8.29 (s, 1H, N]CH),
11.94 (s, 1H, NH, D2O exchangeable). Anal. Calcd. for
C22H18BrN5O2S (496.39): C 53.23, H 3.66, N 14.11. Found: C 53.01, H
3.32, N 14.33.
and the spots were detected by exposure to UV lamp at l 254 nm for
few seconds. Compound 1 was synthesized as described in Ref. [19].
3.1.4.2. 3-Benzyl-2-[4-nitrobenzylidenehydrazino]-5-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (5b). Yield: 75%,
mp: 268e70 ꢂC. IR (KBr, cmꢃ1): 3382, 3332 (NH), 1686, 1644 (C]O),
3.1.1. 3-Benzyl-5-methyl-4-oxo-2-sulfanyl-3,4-dihydrothieno[2,3-d]
pyrimidine-6-carboxamide (2)
A mixture of 1 (2.28 g, 10 mmol), benzyl isothiocyanate (1.49 g,
1.3 mL, 10 mmol) and anhydrous potassium carbonate (1.4 g,
10 mmol) in acetonitrile (30 mL) was heated under reflux for 15 h.
The reaction mixture was left to cool and filtered. The obtained
potassium salt was dissolved in water, neutralized with acetic acid
then the obtained crude product was filtered, washed with water,
dried and crystallized from ethanol.
1615 (C]N), 1252, 1062 (CeSeC). 1H NMR (
d
ppm): 2.66 (s, 3H,
CH3), 5.26 (s, 2H, CH2), 7.23e7.35 (m, 5H, phenyleH), 7.47 (s, 2H,
NH2, D2O exchangeable), 8.24 (d, 8.2 Hz, 2H,
nitrophenyleC2,6eH), 8.32 (d, 8.2 Hz, 2H,
J
¼
¼
J
nitrophenyleC3,5eH), 8.46 (s, 1H, N]CH), 12.05 (s, 1H, NH, D2O
exchangeable). Anal. Calcd. for C22H18N6O4S (462.49): C 57.14, H
3.92, N 18.17. Found: C 56.89, H 3.76, N 18.14.
Yield: 78%, mp: 292e4 ꢂC. IR (KBr, cmꢃ1): 3476, 3171 (NH), 1686
(C]O), 1638 (C]N), 1289, 1079 (CeSeC). 1H NMR (
d
ppm): 2.62
3.1.5. 5-Benzyl-3-methyl-4-oxo-8-(4-substituted phenyl)-4,5-dihy-
drothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide
(6a,b)
To a stirred mixture of the appropriate hydrazone 5a or
b (1 mmol) and anhydrous sodium acetate (0.25 g, 3 mmol) in gl.
acetic acid (5 mL), bromine (0.32 g, 0.1 mL, 2 mmol) was added
dropwise. The mixture was stirred at room temp. for 12 h then
poured on 0.5 N cold NaOH. The obtained precipitate was filtered,
washed with water, dried and crystallized from DMF/H2O (5:1).
(s, 3H, CH3), 5.59 (s, 2H, CH2), 7.22e7.31 (m, 5H, AreH), 7.56 (s, 2H,
NH2, D2O exchangeable), 11.24 (s, 1H, SH, D2O exchangeable). Anal.
Calcd. for C15H13N3O2S2 (331.42): C 54.36, H 3.95, N 12.68. Found: C
54.12, H 3.86, N 12.92.
3.1.2. 3-Benzyl-5-methyl-2-(methylsulfanyl)-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (3)
A stirred mixture of 2 (0.33 g, 1 mmol), methyl iodide (2 mmol)
and anhydrous potassium carbonate (0.276 g, 2 mmol) in dry
acetone (10 mL) was heated under reflux for 4 h. The reaction
mixture was cooled, poured into ice-cold water; the precipitate
formed was dried and crystallized from DMF.
3.1.5.1. 5-Benzyl-8-(4-bromophenyl)-3-methyl-4-oxo-4,5-dihydrothi-
eno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide (6a). Yield:
68%, mp: 272e4 ꢂC. IR (KBr, cmꢃ1): 3456, 3351 (NH), 1685 (C]O),
Yield: 72%, mp: 252e4 ꢂC. IR (KBr, cmꢃ1): 3363, 3173 (NH), 1690,
1622 (C]N), 1238,1073 (CeSeC). 1H NMR (
d ppm): 2.71 (s, 3H, CH3),
1644 (C]O), 1605 (C]N), 1286, 1077 (CeSeC). 1H NMR (
d
ppm):
5.38 (s, 2H, CH2), 7.28e7.36 (m, 3H, phenyleC3,4,5eH), 7.48 (d,
J ¼ 7.2 Hz, 2H, phenyleC2,6eH), 7.7 (s, 2H, NH2, D2O exchangeable),
7.72 (d, J ¼ 8.2 Hz, 2H, BrephenyleC2,6eH), 7.86 (d, J ¼ 8.2 Hz, 2H,
2.56 (s, 3H, CH3eC), 2.71 (s, 3H, CH3eS), 5.3 (s, 2H, CH2), 7.22e7.36
(m, 5H, AreH), 7.59 (s, 2H, NH2, D2O exchangeable). Anal. Calcd. for
C16H15N3O2S2 (345.44): C 55.63, H 4.38, N 12.16. Found: C 55.41, H
4.27, N 12.08.
BrephenyleC3,5eH). 13C NMR
(d ppm): 19.55 (CH3), 50.40
(CH2ebenzyl), 123.59 (bromophenyleC4), 129.53 (benzyleC4),
130.46 (bromophenyleC1), 132.64 (benzyleC3 and C5), 133.15
(benzyleC2 and C6), 133.48 (thienotriazolopyrimidineeC2), 137.00
(bromophenyleC2 and C6), 137.22 (bromophenyleC3 and C5), 137.48
(benzyleC1), 141.70 (thienotriazolopyrimidineeC3), 141.88 (thienot-
riazolopyrimidineeC3e), 147.07 (thienotriazolopyrimidineeC3a),
151.14 (thienotriazolopyrimidineeC8), 154.05 (thienotriazolo-
3.1.3. 3-Benzyl-2-hydrazino-5-methyl-4-oxo-3,4-dihydrothieno
[2,3-d]pyrimidine-6-carboxamide (4)
To a suspension of 3 (3.45 g, 10 mmol) in ethanol (50 mL),
hydrazine hydrate 99% (4 mL, 80 mmol) was added. The reaction
mixture was refluxed for 3 h, during which a precipitate was
formed. After cooling, the product was filtered, washed with water,
dried and crystallized from DMF/EtOH (5:1).
pyrimidineeC2e), 161.12
(thienotriazolopyrimidineeC4eC]O),
167.88 (CONH2). Anal. Calcd. for C22H16BrN5O2S$1/2H2O (503.38): C
52.49, H 3.40, N 13.91. Found: C 52.26, H 3.01, N 13.87.
Yield: 62%, mp: 314e6 ꢂC. IR (KBr, cmꢃ1): 3364, 3230 (NH), 1673
(C]O), 1614 (C]N), 1266, 1061 (CeSeC). 1H NMR (
d ppm): 2.73 (s,
3H, CH3), 5.33 (s, 2H, CH2), 7.26e7.34 (m, 3H, phenyleC3,4,5eH), 7.42
(d, J ¼ 7.2 Hz, 2H, phenyleC2,6eH), 7.76 (s, 2H, CONH2, D2O
exchangeable), 9.32 (s, 2H, NH2, D2O exchangeable), 11.40 (s, 1H,
NH, D2O exchangeable). Anal. Calcd. for C15H15N5O2S (329.38): C
54.70, H 4.59, N 21.26. Found: C 54.63, H 4.45, N 20.98.
3.1.5.2. 5-Benzyl-3-methyl-8-(4-nitrophenyl)-4-oxo-4,5-dihydrothi-
eno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide
Yield: 63%, mp: 224e6 ꢂC. IR (KBr, cmꢃ1): 3464, 3350 (NH), 1683
(C]O), 1622 (C]N), 1234, 1045 (CeSeC). 1H NMR (
ppm): 2.73
(6b).
d
(s, 3H, CH3), 5.41 (s, 2H, CH2), 7.29e7.38 (m, 3H, phenyleC3,4,5eH),
7.49 (d, J ¼ 7.5 Hz, 2H, phenyleC2,6eH), 7.67 (s, 2H, NH2, D2O
exchangeable), 8.09 (d, J ¼ 8.7 Hz, 2H, nitrophenyleC2,6eH), 8.46
3.1.4. 3-Benzyl-2-[4-substituted benzylidenehydrazino]-5-methyl-
4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (5a,b)
The title compounds were prepared by refluxing a mixture of
the hydrazine 4 (0.329 g, 1 mmol) and the appropriate aryl alde-
hyde (1 mmol) in absolute ethanol (20 mL) containing 2 drops of
acetic acid for 2 h. The reaction mixture was allowed to cool and the
obtained product was filtered, dried and crystallized from DMF.
(d,
J
¼
8.7 Hz, 2H, nitrophenyleC3,5eH). Anal. Calcd. for
C22H16N6O4S (460.47): C 57.39, H 3.50, N 18.25. Found: C 57.18,
H 3.21, N 18.29.
3.1.6. 7-Acetyl-5-benzyl-3-methyl-4-oxo-8-(4-substituted phenyl)-
4,5,7,8-tetrahydrothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-
carboxamide (7a,b)
3.1.4.1. 3-Benzyl-2-[4-bromobenzylidenehydrazino]-5-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (5a). Yield: 78%,
The appropriate hydrazone 5a or b (1 mmol) was heated under
reflux in Ac2O (5 mL) for 8 h then the reaction mixture was

O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
91
concentrated to small volume, poured onto ice cold water, stirred
for 2 h and refrigerated for an over night. The separated products
were filtered, washed with water, dried and crystallized from DMF.
Yield: 82%, mp: 238e40 ꢂC. IR (KBr, cmꢃ1): 3334, 3171 (NH), 1729,
1678 (C]O),1596 (C]N),1250,1040 (CeOeC),1225,1062 (CeSeC).1H
NMR (
d
ppm): 1.1 (t, J ¼ 6.9 Hz, 3H, CH2CH3), 2.75 (s, 3H, CH3), 3.29 (s,
2H, CH2eS), 4.04(q, J¼ 6.9 Hz, 2H, CH2CH3), 5.33 (s, 2H, CH2), 7.26e7.34
3.1.6.1. 7-Acetyl-5-benzyl-8-(4-bromophenyl)-3-methyl-4-oxo-4,5,7,
8-tetrahydrothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxa-
mide (7a). Yield:60%, mp:290e2 ꢂC. IR(KBr, cmꢃ1):3441, 3261 (NH),
(m, 3H, phenyleC3,4,5eH), 7.42 (d, J ¼ 7.8 Hz, 2H, phenyleC2,6eH), 7.78
(s, 2H, NH2, D2O exchangeable). 13C NMR (
d ppm): 23.27 (CH2CH3),
23.79 (CH3), 49.81 (CH2ebenzyl), 54.58 (SeCH2), 70.83 (CH2CH3),
127.84 (benzyleC4), 136.99 (thienotriazolopyrimidineeC2), 137.37
(benzyleC3 and C5), 137.82 (benzyleC2 and C6), 137.78 (benzyleC1),
145.28 (thienotriazolopyrimidineeC3), 146.11 (thienotriazolopyrim-
idineeC3e),149.94 (thienotriazolopyrimidineeC3a),150.99 (thienotria-
zolopyrimidineeC8), 158.85 (thienotriazolopyrimidineeC2e), 165.45
(thienotriazolopyrimidineeC4eC]O), 172.45 (CONH2), 177.34
(COOC2H5). Anal. Calcd. for C20H19N5O4S2 (457.53): C 52.50, H 4.19, N
15.31. Found: C 52.36, H 3.99, N 15.18.
1692 (C]O),1639 (C]N),1227,1071 (CeSeC). 1H NMR (
d ppm): 2.66
(s, 3H, CH3), 2.82 (s, 3H, COCH3), 5.39 (s, 2H, CH2), 7.28e7.37 (m, 3H,
phenyleC3,4,5eH), 7.48 (d, J ¼ 6.9 Hz, 2H, phenyleC2,6eH), 7.51 (s, 2H,
NH2, D2O exchangeable), 7.72 (d,
J
¼
8.1 Hz, 2H,
bromophenyleC2,6eH), 7.87e7.91 (m, 3H, bromophenyleC3,5eH and
triazoloeC8eH). Anal. Calcd. for C24H20BrN5O3S (538.43): C 53.54, H
3.74, N 13.01. Found: C 53.26, H 3.45, N 12.96.
3.1.6.2. 7-Acetyl-5-benzyl-3-methyl-8-(4-nitrophenyl)-4-oxo-4,5,7,8-
tetrahydrothieno [3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide
(7b). Yield: 61%, mp: 252e4 ꢂC. IR (KBr, cmꢃ1): 3440, 3243 (NH), 1677
3.1.9. 5-Benzyl-3-methyl-4-oxo-8-(2-substituted phenyl)-4,5-dihydr-
othieno [3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide(11a,b)
A mixture of 4 (0.329 g, 1 mmol) and the appropriate carboxylic
acid (2 mmol) in phosphorous oxychloride (4 mL) was refluxed for
2 h and allowed to cool. The products were poured onto crushed
ice while stirring, filtered, washed with sodium bicarbonate
solution then with water, left to dry and crystallized from EtOH/
H2O (5:1).
(C]O), 1620 (C]N), 1229, 1047 (CeSeC). 1H NMR (
d ppm): 2.66, 2.71
(2s, 3H, CH3), 2.83, 2.88 (2s, 3H, COCH3), 5.41 (s, 2H, CH2), 7.29e7.38 (m,
3H, phenyleC3,4,5eH), 7.49 (d, J ¼ 7.2 Hz, 2H, phenyleC2,6eH), 7.52 (s,
2H, NH2, D2O exchangeable), 7.95 (s, 1H, triazoloeC8eH), 8.09 (d,
J ¼ 8.1 Hz, 2H, nitrophenyleC2,6eH), 8.49 (d, J ¼ 8.1 Hz, 2H,
nitrophenyleC3,5eH). 13C NMR (
d ppm): 23.59 (CH3), 24.99 (COCH3),
54.98 (CH2ebenzyl), 122.17 (nitrophenyleC3 and C5), 126.75
(benzyleC4),133.53 (nitrophenyleC2 and C6),133.81 (nitrophenyleC1),
137.55 (benzyleC3 and C5), 137.59 (thienotriazolopyrimidineeC2),
137.86 (benzyleC2 and C6), 141.26 (benzyleC1), 145.07 (nitro-
phenyleC4), 154.46 (thienotriazolopyrimidineeC3), 155.02 (thie-
notriazolopyrimidineeC3e), 158.19 (triazolothienopyrimidineeC3a),
158.85 (thienotriazolopyrimidineeC8), 164.66 (thienotriazolo-
pyrimidineeC2e), 165.28 (thienotriazolopyrimidineeC4eC]O), 171.71
(CONH2), 172.22 (COCH3). Anal. Calcd. for C24H20N6O5S (504.53): C
57.14, H 4.0, N 16.66. Found: C 56.95, H 3.87, N 16.96.
3.1.9.1. 5-Benzyl-3-methyl-4-oxo-8-phenyl-4,5-dihydrothieno [3,2-e]
[1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide (11a). Yield: 72%,
mp: 232e4 ꢂC. IR (KBr, cmꢃ1): 3438, 3243 (NH), 1687 (C]O), 1624
(C]N),1235,1075 (CeSeC). 1H NMR (
d ppm): 2.65 (s, 3H, CH3), 5.39
(s, 2H, CH2), 7.28e7.37 (m, 3H, phenyleC3,4,5eH), 7.5 (d, J ¼ 7.2 Hz,
2H, phenyleC2,6eH), 7.65 (d, J ¼ 7.2 Hz, 2H, 8-phenyl-C2,6eH),
7.69 (s, 2H, NH2, D2O exchangeable), 7.72e7.77 (m, 3H,
8-phenyleC3,4,5eH). Anal. Calcd. for C22H17N5O2S$1/2H2O (424.48):
C 62.25, H 4.27, N 16.50. Found: C 62.05, H 3.99, N 16.38.
3.1.7. 5-Benzyl-3-methyl-4-oxo-8-thioxo-4,5,7,8-tetrahydrothieno
[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide (9)
3.1.9.2. 5-Benzyl-8-(2-chlorophenyl)-3-methyl-4-oxo-4,5-dihydroth-
ieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide
Yield: 74%, mp: 114e6 ꢂC. IR (KBr, cmꢃ1): 3439, 3238 (NH), 1685
(C]O), 1620 (C]N), 1237, 1075 (CeSeC). 1H NMR (
ppm): 2.65 (s,
3H, CH3), 5.4 (s, 2H, CH2), 7.3e7.39 (m, 3H, phenyleC3,4,5eH), 7.52
(d, 7.2 Hz, 2H, phenyleC2,6eH), 7.6e7.81 (m, 4H,
(11b).
3.1.7.1. Method A. To a solution of 4 (0.329 g, 1 mmol) in dry DMF
(5 mL), phenyl isothiocyanate (1 mmol) was added dropwise, the
reaction mixture was then refluxed for 6 h and cooled. The
precipitated product was filtered, washed with ethanol and crys-
tallized from DMF/EtOH (5:1). Yield: 68%, mp: >300 ꢂC.
d
J
¼
chlorophenyleC3,4,5,6eH), 7.83 (s, 2H, NH2, D2O exchangeable).
Anal. Calcd. for C22H16ClN5O2S$1/2H2O (458.92): C 57.58, H 3.73, N
15.26. Found: C 57.31, H 3.34, N 15.15.
3.1.7.2. Method B. A mixture of 4 (0.658 g, 2 mmol), potassium
hydroxide (0.11 g, 2 mmol) and carbon disulfide (2 mL) in ethanol
(20 mL) was heated under reflux for 12 h. The reaction mixture was
concentrated, cooled, diluted with water and acidified with dil HCl.
The separated solid was filtered, washed with ethanol, dried and
crystallized from DMF/EtOH (5:1).
3.1.10. 5-Benzyl-3-methyl-4-oxo-4,5-dihydrothieno[3,2-e][1,2,4]
triazolo[4,3-a]pyrimidine-2-carboxamide (12)
A solution of 4 (0.329 g, 1 mmol) in formic acid (5 mL) was
heated under reflux for 2 h, then concentrated to small volume and
diluted with ice-cold H2O. The obtained precipitate was filtered,
washed with H2O, dried and crystallized from dioxane.
Yield: 69%, mp: 312e4 ꢂC. IR (KBr, cmꢃ1): 3363, 3192 (NH), 1673
Yield: 72%. IR (KBr, cmꢃ1): 3384, 3225 (NH), 1680, 1649 (C]O),
1624 (C]N), 1266, 1069 (CeSeC). 1H NMR (
d ppm): 2.72 (s, 3H,
CH3), 5.16 (s, 2H, CH2), 7.26e7.34 (m, 3H, phenyleC3,4,5eH),
7.38e7.42 (m, 3H, phenyleC2,6eH & triazolo NH), 7.69 (s, 2H, NH2,
D2O exchangeable). Anal. Calcd. for C16H13N5O2S2 (371.44): C 51.74,
H 3.53, N 18.85. Found: C 51.45, H 3.33, N 18.69.
(C]O), 1600 (C]N), 1267, 1061 (CeSeC). 1H NMR (
d ppm): 2.73 (s,
3H, CH3), 5.33 (s, 2H, CH2), 7.26e7.41 (m, 3H, phenyleC3,4,5eH), 7.42
(d, J ¼ 7.5 Hz, 2H, phenyleC2,6eH), 7.75 (s, 2H, NH2, D2O
exchangeable), 9.31 (s, 1H, triazoloeC8eH). Anal. Calcd. for
C16H13N5O2S (339.38): C 56.63, H 3.86, N 20.64. Found: C 56.43, H
3.59, N 20.39.
3.1.8. 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 (10)
To a suspension of 9 (0.329 g, 1 mmol) in absolute ethanol
(10 mL), ethyl bromoacetate (0.18 g, 0.11 mL, 1 mmol) was added.
The reaction mixture was heated under reflux for 9 h. Anhydrous
sodium acetate (0.08 g, 1 mmol) was added and the reaction
mixture was heated for further 30 min, cooled and poured into ice-
cold water. The formed precipitate was filtered, washed with water
dried and crystallized from DMF/EtOH (5:1).
3.1.11. 5-Benzyl-3-methyl-4,8-dioxo-4,5,7,8-tetrahydrothieno[3,2-e]
[1,2,4]triazolo[4,3-a]pyrimidine-2-carboxamide (13)
A suspension of 4 (0.329 g, 1 mmol), and ethyl chloroformate
(0.165 g, 0.145 mL, 1.5 mmol) in dry dioxane (5 mL) was heated
under reflux for 4 h, then cooled. The obtained precipitate was
filtered, washed with ethanol, dried and crystallized from DMF.

92
O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
Yield: 71%, mp: >300 ꢂC. IR (KBr, cmꢃ1): 3362, 3231 (NH), 1674
(C]O),1600 (C]N),1267,1061 (CeSeC).1H NMR (
ppm): 2.73 (s, 3H,
1692, 1664 (C]O), 1612 (C]N), 1266, 1033 (CeSeC). 1H NMR
(d ppm): 2.66 (s, 3H, CH3), 5.20 (s, 2H, triazinoeC9eH), 5.39 (s, 2H,
d
CH3), 5.33 (s, 2H, CH2), 7.26e7.34 (m, 3H, phenyleC3,4,5eH), 7.42 (d,
J ¼ 8.1 Hz, 2H, phenyleC2,6eH), 7.76 (s, 2H, NH2, D2O exchangeable),
CH2), 7.14e7.3 (m, 5H, phenyleH), 7.49 (d, J ¼ 8.7 Hz, 2H,
ClephenyleC2,6eH), 7.87 (s, 2H, NH2, D2O exchangeable), 8.3 (d,
J ¼ 8.7 Hz, 2H, ClephenyleC3,5eH). Anal. Calcd. for C23H18ClN5O2S
(463.95): C 59.54, H 3.91, N 15.10. Found: C 59.24, H 3.65, N 14.97.
9.32 (s, 1H, triazolo NH, D2O exchangeable). 13C NMR (
d ppm): 24.02
(CH3), 54.94 (CH2ebenzyl), 127.53 (benzyleC4), 136.89 (thienotri-
azolopyrimidineeC2), 137.30 (benzyleC3 and C5), 137.79 (benzyleC2
and C6), 137.88 (benzyleC1), 145.46 (thienotriazolopyrimidineeC3),
145.75 (thienotriazolopyrimidineeC3e), 146.74 (thienotriazolo-
pyrimidineeC3a), 151.27 (thienotriazolopyrimidineeC2e), 157.62 (thi-
enotriazolopyrimidineeC8eC]O), 165.61 (thienotriazolopyrimid-
ineeC4eC]O), 172.36 (CONH2). Anal. Calcd. for C16H13N5O3S
(355.38): C 54.08, H 3.69, N 19.71. Found: C 53.94, H 3.66, N 19.43.
3.1.14. General procedure for the synthesis of compounds 16e18
A
mixture of the hydrazino 4 (0.329 g, 1 mmol) and
4-substituted phenacylcyanide, acetylacetone or ethyl acetoacetate
(1 mmol) in gl. acetic acid (5 mL) was refluxed for 12, 6 and 10 h
respectively, and then cooled. The obtained precipitates were
filtered, washed with ethanol and crystallized from DMF to give
compounds 16a,b, 17 or 18 respectively.
3.1.12. Ethyl (5-benzyl-2-carbamoyl-3-methyl-4-oxo-4,5-dihydroth-
ieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-8-yl)acetate (14)
A mixture of the hydrazino 4 (0.329 g, 1 mmol) and diethyl
malonate (0.32 g, 0.226 mL, 2 mmol) in gl. acetic acid (3 mL) was
heated under reflux for 4 h, and then cooled. The obtained
precipitate was filtered, washed with ethanol and crystallized from
DMF.
4.1.14.1. 3-Benzyl-2-[5-amino-3-(4-bromophenyl)-1H-pyrazol-1-yl]-
5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxa-mide
(16a). Yield: 68%, mp: 256e8 ꢂC. IR (KBr, cmꢃ1): 3332, 3165 (NH),
1678 (C]O),1594 (C]N),1260,1033 (CeSeC). 1H NMR (
d ppm): 2.67
(s, 3H, CH3), 3.21 (s,1H, pyrazolyleC4eH), 5.35 (s, 2H, CH2), 7.26e7.38
(m, 5H, phenyleH), 7.43 (d, J ¼ 8.7 Hz,2H, bromophenyleC2,6eH), 7.78
(d, J ¼ 8.7 Hz, 2H, bromophenyleC3,5eH), 7.96 (s, 2H, NH2, D2O
exchangeable), 10.33 (s, 2H, pyrazolyleC5eNH2, D2O exchangeable).
Anal. Calcd. for C24H19BrN6O2S (535.43): C 53.84, H 3.58, N 15.70.
Found: C 53.64, H 3.39, N 15.35.
Yield: 78%, mp: 314e6 ꢂC. IR (KBr, cmꢃ1): 3344, 3138 (NH), 1742,
1682 (C]O), 1619 (C]N), 1250, 1050 (CeOeC), 1237, 1066 (CeSeC).
1H NMR (
d
ppm): 2.6 (t, J ¼ 6.9 Hz, 3H, CH2CH3), 2.73 (s, 3H, CH3),
2.89 (s, 2H, CH2eCO), 3.55 (q, J ¼ 6.9 Hz, 2H, CH2CH3), 5.3 (s, 2H,
CH2), 7.25e7.39 (m, 3H, phenyleC3,4,5eH), 7.41 (d, J ¼ 7.8 Hz, 2H,
phenyleC2,6eH), 7.76 (s, 2H, NH2, D2O exchangeable). MS, m/z
(relative abundance %): 425 [Mþ, (12.51)], 352 (42.92), 248 (12.54),
91 (100), 65 (17.17). Anal. Calcd. for C20H19N5O4S (425.47): C 56.46,
H 4.50, N 16.46. Found: C 56.32, H 4.22, N 16.25.
3.1.14.2. 3-Benzyl-2-[5-amino-3-(4-chlorophenyl)-1H-pyrazol-1-yl]-
5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide
(16b). Yield: 65%, mp: 270e2 ꢂC. IR (KBr, cmꢃ1): 3342, 3150 (NH),
1681 (C]O),1596 (C]N),1257,1031 (CeSeC).1H NMR (
d ppm): 2.68
(s, 3H, CH3), 2.88(s,1H, pyrazolyleC4eH), 5.25(s, 2H, CH2), 7.24e7.34
(m, 5H, phenyleH), 7.41 (d, J ¼ 8.7 Hz, 2H, chlorophenyleC2,6eH),
7.68 (d, J ¼ 8.7 Hz, 2H, chlorophenyleC3,5eH), 7.77 (s, 2H, NH2, D2O
exchangeable), 10.31 (s, 2H, pyrazolyleC5eNH2, D2O exchangeable).
Anal. Calcd. for C24H19ClN6O2S (490.98): C 58.71, H 3.90, N 17.12.
Found: C 58.49, H 3.74, N 16.97.
3.1.13. 5-Benzyl-3-methyl-4-oxo-8-(substituted phenyl)-4,5-dihydro-
9H-thieno[30,20:5,6]pyrimidino[2,1-c][1,2,4]triazine-2-carboxamide
(15aec)
To a suspension of 4 (0.329 g, 1 mmol) in absolute ethanol
(10 mL), 4-substituted phenacyl bromide (1 mmol) was added. The
reaction mixture was heated under reflux for 8 h, then anhydrous
sodium acetate (0.08 g, 1 mmol) was added and the reaction
mixture was heated for further 30 min, cooled and poured into ice-
cold water. The formed precipitate was filtered, washed with water
and crystallized from CHCl3/EtOH (5:1).
3.1.14.3. 3-Benzyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (17). Yield: 77%,
mp: >300ꢂC. IR(KBr, cmꢃ1): 3342, 3137 (NH),1682 (C]O),1617 (C]N),
1237, 1067 (CeSeC). 1H NMR (
d ppm): 2.69 (s, 3H, CH3), 2.76 (s, 6H,
pyrazolyleCH3), 5.3 (s, 2H, CH2), 7.25e7.33 (m, 3H, phenyleC3,4,5eH),
7.39e7.42 (m, 3H, phenyleC2,6eH & pyrazolyleC4eH), 7.76 (s, 2H,
3.1.13.1. 5-Benzyl-3-methyl-4-oxo-8-phenyl-4,5-dihydro-9H-thieno
[30,20:5,6] pyrimidino[2,1-c][1,2,4]triazine-2-carboxamide (15a).
Yield: 69%, mp: 232e4 ꢂC. IR (KBr, cmꢃ1): 3429, 3290 (NH), 1690,
NH2, D2O exchangeable). 13C NMR (
d
ppm): 21.06 (CH3), 23.86
(pyrazoleeC5eCH3), 49.80 (pyrazoleeC3eCH3), 54.51 (CH2ebenzyl),
125.01 (pyrazoleeC4), 127.06 (benzyleC4), 133.11
1668 (C]O), 1616 (C]N), 1270, 1045 (CeSeC). 1H NMR (
d ppm):
2.65 (s, 3H, CH3), 5.16 (s, 2H, triazinoeC9eH), 5.34 (s, 2H, CH2),
7.16e7.29 (m, 5H, phenyleH), 7.47 (s, 2H, NH2, D2O exchangeable),
7.56e7.79 (m, 5H, 8-phenyleH). Anal. Calcd. for C23H19N5O2S
(429.5): C 64.32, H 4.46, N 16.31. Found: C 64.21, H 4.27, N 16.10.
(thienopyrimidineeC6), 136.88 (benzyleC3 and C5), 137.28 (benzyleC2
and C6), 137.53 (benzyleC1), 137.77 (pyrazoleeC5), 145.48
(thienopyrimidineeC5), 146.75 (thienopyrimidineeC3d), 151.78
(pyrazoleeC3),
154.08
(thienopyrimidineeC2),
157.74
(thienopyrimidineeC2d), 165.50 (thienopyrimidineeC4eC]O), 172.29
(CONH2). MS, m/z (relative abundance %): 393 [Mþ, (7.15)], 353 (96.39),
345 (24.03), 315 (100), 299 (20.21),182 (20.64), 147 (24.12),130 (15.11),
60 (20.64), 57 (33.37). Anal. Calcd. for C20H19N5O2S (393.47): C 61.05, H
4.87, N 17.80. Found: C 60.94, H 4.56, N 17.60.
3.1.13.2. 5-Benzyl-8-(4-bromophenyl)-3-methyl-4-oxo-4,5-dihydro-
9H-thieno[30,20:5,6]pyrimidino[2,1-c][1,2,4]triazine-2-carboxamide
(15b). Yield: 75%, mp: 223e5 ꢂC. IR (KBr, cmꢃ1): 3350, 3210 (NH),
1680, 1670 (C]O), 1618 (C]N), 1275, 1035 (CeSeC). 1H NMR
(d ppm): 2.67 (s, 3H, CH3), 5.13 (s, 2H, triazinoeC9eH), 5.42 (s, 2H,
CH2), 7.18e7.36 (m, 5H, phenyleH), 7.48 (d, J ¼ 8.4 Hz, 2H,
3.1.14.4. 3-Benzyl-5-methyl-2-(3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide
(18). Yield: 72%, mp: 292e4 ꢂC. IR (KBr, cmꢃ1): 3343, 3141 (NH),
BrephenyleC2,6eH), 7.89 (s, 2H, NH2, D2O exchangeable), 8.36 (d,
J
¼
8.7 Hz, 2H, BrephenyleC3,5eH). Anal. Calcd. for
C23H18BrN5O2S (508.4): C 54.34, H 3.57, N 13.78. Found: C 54.01, H
3.44, N 13.38.
1681 (C]O), 1597 (C]N), 1236, 1067 (CeSeC). 1H NMR (
d ppm):
2.63 (s, 2H, pyrazolyleC4eH), 2.68 (s, 3H, CH3), 2.75 (s, 3H,
pyrazolyleCH3), 5.3 (s, 2H, CH2), 7.23e7.33 (m, 3H,
phenyleC3,4,5eH), 7.39e7.42 (d, J ¼ 6.6 Hz, 2H, phenyleC2,6eH),
7.76 (s, 2H, NH2, D2O exchangeable). Anal. Calcd. for C19H17N5O3S
(395.44): C 57.71, H 4.33, N 17.71. Found: C 57.35, H 4.01, N 17.41.
3.1.13.3. 5-Benzyl-8-(4-chlorophenyl)-3-methyl-4-oxo-4,5-dihydro-
9H-thieno[30,20:5,6]pyrimidino[2,1-c][1,2,4]triazine-2-carboxamide
(15c). Yield: 73%, mp: 244e6 ꢂC. IR (KBr, cmꢃ1): 3434, 3310 (NH),

O.H. Rizk et al. / European Journal of Medicinal Chemistry 55 (2012) 85e93
93
3.2. Anti-inflammatory (AI) activity
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 3X
magnifying lens for any evidence of hyperemia, hemorrhage, defi-
nite hemorrhagic erosion or ulcer.
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 10 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 10 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:
3.2.5. 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.
3.2.6. 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.
% Inhibition ¼ (Vt ꢃ V0) control ꢃ (Vt ꢃ V0) tested compound/
(Vt ꢃ V0) control ꢄ 100
where Vt ¼ volume of edema at specific time interval and
V0 ¼ volume of edema at zero time interval.
3.2.7. Statistical analysis
The data obtained are presented as means ꢀ 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.
3.2.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model)
Rats in the first experiment were given the same test
compounds at a dose level of 10 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 plethy-
mographically at the first and eighth days.
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Male albino rats (180e200 g) were divided into groups each of
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the test compounds. Water was given ad libitum. Control group
received 1% gum acacia orally.