New pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines: Synthesis, 2D-QSAR, anti-inflammatory, analgesic and ulcerogenicity studies

Article abstract of DOI:10.1016/j.ejmech.2012.06.048

New pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines were synthesized. A series of ylidene carbohydrazides 14a-i, and hydrazonate 15, were obtained from the prepared 3-carbohydrazide derivative 13. Pyrazole derivatives 12, 16a,b, 18, 19, 20, were also prepared through different reactions. The anti-inflammatory and analgesic activities of all new compounds were evaluated and most of them exerted comparable activity to indomethacin and celecoxib. Ulcer indexes for the most active compounds were calculated and most of them showed less ulcerogenic effect than the reference drugs. The most potent anti-inflammatory compound 12 showed an IC₅₀ of 6.00 μmol/kg and low ulcer index. COX-1/COX-2 activity ratio of compounds 12 and 16b showed almost equal inhibitory effect on both isoenzymes. 2D-QSAR studies revealed good predictive and statistically significant QSAR models.

Full text of DOI:10.1016/j.ejmech.2012.06.048

European Journal of Medicinal Chemistry 55 (2012) 12e22
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines: Synthesis, 2D-QSAR,
anti-inflammatory, analgesic and ulcerogenicity studies
Mona M. Hanna
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, 11562 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
New pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines were synthesized. A series of ylidene carbohydrazides
14aei, and hydrazonate 15, were obtained from the prepared 3-carbohydrazide derivative 13. Pyrazole
derivatives 12, 16a,b, 18, 19, 20, were also prepared through different reactions. The anti-inflammatory
and analgesic activities of all new compounds were evaluated and most of them exerted comparable
activity to indomethacin and celecoxib. Ulcer indexes for the most active compounds were calculated
and most of them showed less ulcerogenic effect than the reference drugs. The most potent anti-
Received 26 February 2012
Received in revised form
19 June 2012
Accepted 25 June 2012
Available online 4 July 2012
inflammatory compound 12 showed an IC50 of 6.00
mmol/kg and low ulcer index. COX-1/COX-2
Keywords:
activity ratio of compounds 12 and 16b showed almost equal inhibitory effect on both isoenzymes.
2D-QSAR studies revealed good predictive and statistically significant QSAR models.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine
Anti-inflammatory
Analgesic
Ulcer index
2D-QSAR
COX-1/COX-2
1. Introduction
Pyrimidines and condensed pyrimidines constitute an impor-
tant skeleton for compounds with diverse biological activities
Inflammation is part of the non-specific immune response that
occurs as a reaction to any exogenous or endogenous insult which
[7e9] including anti-inflammatory [10e12] and analgesic activities
[13e16]. In previous studies, pyrimido[5,4-e]pyrrolo[1,2-c]pyrimi-
dine derivatives prepared in our lab were found to possess signif-
icant anti-inflammatory and analgesic activities, whereby, some of
them were superior to the reference drugs and with low ulcero-
genic potential [17]. Compounds I and II (Fig. 1) are shown as
examples [18]. As a continuation of this work and aiming to further
explore this ring system in the field of non-ulcerogenic anti-
inflammatory agents, it was of interest to investigate a hybrid
pharmacophoric approach by combining the pyrimido[5,4-e]pyr-
rolo[1,2-c]pyrimidine a privileged lead molecule with several other
important pharmacophoric moieties and to evaluate their analgesic
and anti-inflammatory activities. Therefore, active pharmacophoric
groups were introduced at position 3 of the pyrimidopyrrolopyr-
imidine core including carboxylic and acetic acid moieties and their
esters, as possible prodrugs with low ulcerogenic activity.
Furthermore, different substituted ylidene groups were also
incorporated as it was reported that hydrazone moiety present in
some compounds was suggested to be anti-inflammatory phar-
macophoric element [19]. Additionally, pyrazole derivatives,
including antipyrine molecule, that are well known to be important
integral moieties in many analgesic and anti-inflammatory drugs
[20,21] were also used and hybridized to the tricyclic core.
threatens the host. It is
a complex phenomenon involving
biochemical as well as immunological factors [1]. Non-steroidal
anti-inflammatory drugs (NSAIDs) have a wide clinical use for the
treatment of inflammatory and painful conditions including rheu-
matoid arthritis, soft tissue lesions, fever and respiratory tract
infections [2]. However, long term administration of NSAIDs gives
rise to unacceptable side effects mainly gastric lesions [3]. The
effectiveness of NSAIDs in reducing pain and swelling lies in their
ability to inhibit cyclooxygenases COX-1 and COX-2 that are the key
enzymes involved in the prostaglandin biosynthesis [4]. COX-1
inhibition is thought to be a major mechanism of gastric damage
by NSAIDs [5]. Selective COX-2 inhibitors were developed to
decrease the incidence of gastrointestinal toxicity. However, recent
concerns regarding these drugs and their association with signifi-
cant cardiovascular side effects led to reconsideration of their
appropriate use [6]. Therefore, there is always a constant need for
discovery of novel and safer anti-inflammatory drugs.
E-mail address: mmauricebta@gmail.com.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.048

M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
13
The reaction of the 3-chlorocarbonyl intermediate 9 with
4-aminoantipyrine, in dry dimethylformamide, generated the
target compound 3-N-pyrazolylcarboxamide derivative 12. The 1H
NMR spectrum of 12 showed 2 singlet peaks at 0.94 and 3.16 ppm
corresponding to the 2 methyl protons (CeCH3 and NeCH3),
respectively.
The synthesis of the 3-carbohydrazide derivative 13 was carried
out by treating the carboxylate ester 10 with hydrazine hydrate in
absolute ethanol. The presence of absorption bands at 3398,
3317and 3244 cmꢀ1 in the IR spectrum were attributed to the NH
and NH2 groups, furthermore, the 3 C]O bands appeared in the
range of 1700e1681 cmꢀ1 (Scheme 2).
O
H
N
R
N
N
N
N
R
N
H
N
N
N
N
O
O
I
CH3
II
N
R = -NHC3H7,
N
C H
R= -OC2H5 ,
6
5
Condensation of the obtained acid hydrazide derivative 13 with
different aldehydes, ketones and triethylorthoformate in the pres-
ence of absolute ethanol and catalytic amount of glacial acetic acid
furnished the ylidene 14aei and the formamidic acid ethyl ester 15.
The IR spectra of 14aei and 15 revealed the disappearance of NH2
bands. 1H NMR of 14c showed 2 singlets at 3.87 and 8.61 ppm
attributed to the 3 protons of the OCH3 group and the CH]N
proton, respectively, in addition to multiplets of 9 aromatic protons
in the range of 6.91e7.80 ppm denoting the presence of the 2
phenyl groups. The 1H NMR of 15 displayed the triplet and quartet
signals corresponding to the ethyl group at 1.32 and 4.09 ppm,
respectively (Scheme 3).
H3C
Fig. 1. Examples of tricyclic pyrimidopyrrolopyrimidine derivatives with anti-
inflammatory and analgesic activities.
Ulcerogenic studies were carried out for compounds with the best
activity profile. 2D-QSAR studies were also performed to correlate
between the structures of the synthesized compounds and their
pharmacological activities.
2. Results and discussion
The 3-carbohydrazide 13 was also reacted with different
reagents in order to obtain the desired substituted pyrazolyl
derivatives 16a,b, 18, 19 and 20. Therefore, 1,3-diketones, namely
acetylacetone and benzoylacetone were allowed to react with 13 in
dry DMF to give the 3,5-dimethylpyrazole-1-carbonyl derivative
16a and its 5-methyl-3-phenyl analogue 16b. The IR spectrum of
16b revealed the disappearance of the amino stretching bands. 1H
NMR of 16b showed the presence of 2 singlet peaks at 2.21 and
6.18 ppm corresponding to the methyl group and the pyrazolyl
proton respectively. The 3-methyl-5-hydroxypyrazolyl derivative
18 was obtained from the reaction of 13 with ethyl acetoacetate,
whereby, the uncyclized ester derivative 17 was first separated.
Subsequently, ring closure to the corresponding pyrazole derivative
18 was successfully performed by refluxing 17 with 2 M KOH. The IR
spectrum of 17 showed the presence of the ester carbonyl
stretching band at 1720 cmꢀ1 and the disappearance of the amino
band. The 1H NMR of 17 revealed the presence of triplet and quartet
signals at 1.18 and 4.09 ppm attributed to ethyl group confirming
that condensation occurred with the acetyl group. 1H NMR of 18
showed the disappearance of the characteristic pattern of the ethyl
group in addition to the presence of 2 singlet peaks of the CH3 and
CH pyrazole protons at 2.49 and 8.00 ppm, respectively. Further-
more, the 3-hydroxypyrazolone derivative 19 was synthesized by
treating the carbohydrazide 13 with diethyl malonate. The struc-
ture of 19 was confirmed by the presence of the OH absorption
band at 3294 cmꢀ1 in the IR spectrum and the 1H NMR that
revealed the signals of the CH2 pyrazole and OH protons at 1.90 and
12.85 ppm respectively (Scheme 3).
2.1. Chemistry
The synthetic pathways for the synthesis of the targeted
compounds are illustrated in Schemes 2 and 3. The known starting
compound pyrrolo[1,2-c]pyrimidine-4-carbonitrile derivative 5,
was synthesized according to previously reported procedures.
2-Pyrrolidin-2-ylidine malononitrile 3 was prepared by methyla-
tion of 2-pyrrolidinone 1 into the ether intermediate 2 followed by
condensation with malononitrile. The reaction of 3 with phenyl
isocyanate followed by reduction of the obtained enaminonitrile 4
with sodium borohydride, via 1,4-addition reaction, afforded the
key starting compound 5 (Scheme 1) [22e24].
Cyclization and formation of the 3-chloroacetyl and
3-chlorocarbonyl pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine inter-
mediates 6 and 9, respectively, was achieved by acylation of 5 with
either malonyl or oxalyl chloride in dry benzene at room temper-
ature (Scheme 2).
The ester derivatives 7 and 10 were prepared by refluxing the
non-isolated intermediates 6 and 9, with absolute ethanol. The IR
spectrum of 7 revealed the presence of an additional C]O
stretching band at 1743 cmꢀ1, furthermore, the disappearance of
the nitrile band confirmed intramolecular cyclization. 1H NMR of 7
showed a singlet signal at 3.51 attributed to CH2 protons of the
acetate group and the characteristic triplet and quartet signals of
the ethyl protons at 1.08 and 3.93 ppm, respectively. Alkaline
hydrolysis of 7 and 10 with 10% NaOH solution afforded the acetic
acid and carboxylic acid analogues 8 and 11, respectively. The IR
spectrum of 8 revealed the presence of the OH and C]O absorption
bands at 2785e2600 cmꢀ1 and 1743 cmꢀ1 respectively. 1H NMR of 8
showed an additional exchangeable singlet at 12.28 ppm assigned
to the COOH proton (Scheme 2).
Finally, the reaction of ethyl cyanoacetate with 13 yielded the
5-amino-3-hydroxypyrazole-1-carbonyl derivative 20. The IR
spectrum of 20 showed the absorption bands of OH and NH2 group
in the range of 3425e3367 cmꢀ1 (Scheme 3).
CN
CN
CN
OCH3
O
NH
NH2
CN
a
d
b
c
N
NH
N
N
N
N
NH
2
4
1
3
5
O
O
Scheme 1. Reagents and solvents. a: Dimethyl sulphate, dry benzene; b: malonnitrile; c: phenyl isocyanate, methylene chloride; d: sodium borohydride, absolute ethanol.

14
M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
H
H
N
H
N
N
O
CH2COOC2H5
O
CH2COOH
O
CH2COCl
N
N
N
c
d
N
N
N
N
N
N
O
O
O
8
7
a
b
6
5
H
H
N
H
N
O
O
COOC2H5
COOH
N
O
COCl
N
N
d
N
c
N
N
N
N
N
N
O
O
9
O
10
11
f
e
CH3
H3C
O
N
N
H
N
H
N
O
O
CONHNH2
C6H5
N
H
N
N
O
N
N
N
N
O
O
13
12
Scheme 2. Reagents and solvents. a: Malonyl chloride, dry benzene; b: oxalyl chloride, dry benzene; c: absolute ethanol; d: 10% NaOH; e: 4-aminoantipyrine, DMF, K2CO3;
f: hydrazine hydrate, absolute ethanol.
2.2. Pharmacological screening
activity. Regarding the effect of the electronic nature of the
substituent on the activity, it was observed that substitution on
the phenyl group of benzylidene derivative 14a with an electron
withdrawing group led to the less active 4-chlorobenzylidene
derivative 14b. On the other hand, a non-significant change in
activity was recorded for electron donating substituents as shown
in compounds 14c, 14d, and 14e that were equipotent having %
inhibition values approaching compound 14a and indomethacin.
The ethyl formohydrazonate derivative 15 revealed activity higher
than the standard drugs.
As expected, all pyrazolyl derivatives, exhibited remarkable
anti-inflammatory profile and were more active than indomethacin
and celecoxib as shown in compounds 12, 16a, 16b, 18 and 19 or
equipotent as compound 20. The pyrazolone derivative 12 proved
to exert excellent activity higher than the other pyrazole deriva-
tives and the 2 reference drugs. The 3,5-dimethyl pyrazolyl deriv-
ative 16a showed activity higher than its 3-phenyl analogue 16b.
Replacing one methyl group in 16a with a hydroxyl group in 18 or
replacing both methyl groups by OH and C]O in compound 19, led
to slight decrease in activity; however, it was still more potent than
indomethacin. On the other hand, the presence of an additional
hydrophilic amino group as shown in compound 20 further
decreased the activity approaching that of indomethacin. The
uncyclized ethyl hydrazono butanoate derivative 17 showed anti-
inflammatory activity equal to that of indomethacin but less than
its cyclized pyrazolyl bioisostere 18.
2.2.1. Anti-inflammatory activity
Evaluation of anti-inflammatory activity of the newly synthe-
sized compounds was determined by the carrageenan-induced rat
paw oedema model using indomethacin and celecoxib as reference
drugs [25e27]. Mean changes in paw oedema thickness after 1, 2, 3,
and 4 h, from induction of inflammation and inhibition% of oedema
by the tested compounds were recorded in Table 1.
The newly synthesized compounds presented significant anti-
inflammatory profile and most of the tested compounds revealed
anti-inflammatory activity comparable to or higher than that of the
reference drugs at the same dose level. The ethyl acetate ester
derivative 7 and its acetic acid analogue 8 exerted an excellent
activity that was the highest among the rest of tested compounds
and the 2 reference drugs. Reduction of the chain length in 7 and 8
to the ethyl carboxylate derivative 10 and its 3-carboxylic acid
analogue 11, led to some decrease in activity, suggesting the
importance of the acetic acid moiety for optimum activity; never-
theless, they were still more potent than indomethacin, celecoxib
and most of the tested compounds.
The 3-carbohydrazide derivatives 13 and 14aei displayed good
to moderate activity when compared with the reference drugs.
The disubstituted methylene derivatives 14gei were noticed to be
more potent than the monosubstituted benzylidene analogues
14aee and the reference drugs. Unexpectedly, the dimethyl-
methylene derivative 14f showed a marked decrease in activity
being the least active in the series, suggesting that the presence of
an aromatic phenyl group might be important for optimum
IC50 values of the most active compounds 7, 8, 12, 16b, were
calculated and the results were recorded in Table 2. The pyrazolone
derivatives 12 exhibited the highest activity.

M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
15
Scheme 3. Reagents and solvents. a: Aldehydes or ketones, absolute ethanol, glacial acetic acid; b: triethylorthoformate; c: acetylacetone or benzoylacetone, DMF; d: ethyl ace-
toacetate; e: 2 M KOH; f: diethyl malonate; g: ethyl cyanoacetate, DMF.
2.2.2. Analgesic activity
compound was still more active than indomethacin. On the other
hand, a further decrease in activity was noted when the 2 phenyl
were replaced by methyl groups 14f. Introduction of electron
withdrawing group on the phenyl ring 14h also decreased the
activity.
All pyrazole derivatives displayed good activity; compounds 12,
16a,18 and 19 were nearly equipotent. However, substitution of one
methyl group in 16a by a phenyl group 16b, led to a decrease in
activity, on the other hand, substitution with hydrophilic NH2 and
OH groups 20 led to increased in activity that exceeded that of the
other pyrazole derivatives and indomethacin.
The newly synthesized compounds were evaluated for their
analgesic activity by applying the acetic acid-induced writhing test
in mice using indomethacin as a reference standard (Table 3)
[28,29]. The tested compounds exhibited significant analgesic
activity, whereby, some of them 7, 14c, 14d, 14g, 14i, 20, showed
excellent activity exceeding that of indomethacin at the same dose
level. Furthermore, compounds 8, 11, 12, 14a, 14f, 14h, 16a, 18 also
exhibited good analgesic activity with
% inhibition values
approaching or equal to that of the standard drug. The 3-ethyl
acetate derivative 7 revealed the highest activity compared with
the carboxylate ester 10 and the acid derivatives 8 and 11. The
unsubstituted benzylidene derivative 14a exerted good activity,
furthermore, substitution on the phenyl ring with electron
donating groups, either OCH3 group 14c or OH group 14d, increased
the activity to exceed that of indomethacin. On the other hand, the
presence of these 2 groups together on the phenyl ring, as shown in
compound 14e, or the presence of an electron withdrawing
substituent 14b led to decrease in activity. The diphenyl methylene
derivative 14i showed the best analgesic profile compared with all
tested compounds and indomethacin. Replacement of one phenyl
group with methyl group 14g decreased the activity; however, the
2.2.3. Ulcerogenicity study
The newly synthesized compounds were tested for their
ulcerogenic effect. The ratio of ulceration was recorded in Table 1. It
was observed that the ester derivatives 7 and 10 had gastric safety
profile better than their acid analogues 8 and 11. The carbohy-
drazide derivatives 14b, 14c, 14e, 14f and 15 were found safe
however, the unsubstituted carbohydrazide 13 and compounds
14a, 14d, 14g and 14h revealed certain ulcerogenic effect. On the
other hand, pyrazoles 12, 16a, 16b, 18, 19, 20, showed good gastric
tolerance.

16
M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
Table 1
Anti-inflammatory activity of the synthesized compounds against carrageenan-induced paw oedema in rats and ratio of ulceration (n ¼ 6).
Compd No
Ratio of ulceration
Oedema thickness (mm) ꢁ SEM (% inhibition)
1 h
2 h
3 h
4 h
Control
7
8
10
11
0/6
1/6
3/6
1/6
2/6
0/6
2/6
2/6
0/6
0/6
2/6
0/6
0/6
2/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
5/6
5/6
3.573 ꢁ 0.231b,c
3.808 ꢁ 0.052b,c
4.060 ꢁ 0.152b,c
4.162 ꢁ 0.233b,c
0.660 ꢁ 0.191a,b,c (81)
0.805 ꢁ 0.189a,b,c (77)
1.020 ꢁ 0.277a (71)
1.377 ꢁ 0.216a (68)
0.780 ꢁ 0.280a,b,c (78)
1.580 ꢁ 0.308a (56)
1.140 ꢁ 0.264a (68)
1.640 ꢁ 0.127a (54)
1.748 ꢁ 0.279a (51)
1.692 ꢁ 0.206a (53)
2.933 ꢁ 0.206b,c (18)
2.653 ꢁ 0.235a,b,c (26)
1.402 ꢁ 0.102a (61)
2.042 ꢁ 0.254a (43)
2.013 ꢁ 0.223a (44)
1.888 ꢁ 0.425a (47)
1.493 ꢁ 0.182a (58)
0.776 ꢁ 0.034a (78)
1.961 ꢁ 0.231a (45)
1.545 ꢁ 0.166a (57)
1.138 ꢁ 0.246a (68)
1.780 ꢁ 0.168a (50)
1.540 ꢁ 0.300a (57)
1.650 ꢁ 0.170 (54)
0.763 ꢁ 0.143a,b,c (80)
1.143 ꢁ 0.199a,b,c (70)
1.293 ꢁ 0.537a,b,c (67)
1.661 ꢁ 0.912a,c (56)
1.190 ꢁ 0.252a,b,c (68)
2.092 ꢁ 0.282a (45)
2.243 ꢁ 0.241a (41)
2.603 ꢁ 0.274a (32)
3.000 ꢁ 0.203a,b (21)
2.520 ꢁ 0288a (34)
3.242 ꢁ 0.099a,b,c (15)
2.937 ꢁ 0.205a (23)
2.115 ꢁ 0.261a (44)
1.898 ꢁ 0.209a (50)
1.853 ꢁ 0.233a (51)
1.690 ꢁ 0.312a,c (55)
1.631 ꢁ 0.163a,b,c (57)
1.311 ꢁ 0.171a,b,c (65)
2.223 ꢁ 0.311a (41)
2.498 ꢁ 0.226a (34)
2.131 ꢁ 0.179a (44)
3.303 ꢁ 0.090a,b (20)
2.300 ꢁ 0.133a (39)
2.51 ꢁ 0.172a (34)
0.580 ꢁ 0.173a,b,c (90)
1.126 ꢁ 0.116a,b,c (72)
1.292 ꢁ 0.518a,b,c (68)
1.375 ꢁ 0.147a,b,c (66)
0.863 ꢁ 0.192a,b,c (78)
2.022 ꢁ 0.298a (50)
1.900 ꢁ 0.240a (53)
2.270 ꢁ 0.150a (44)
2.271 ꢁ 0.208a (44)
2.197 ꢁ 0.168a (46)
2.177 ꢁ 0.154a (46)
2.623 ꢁ 0.199a (35)
1.913 ꢁ 0.141a (53)
1.997 ꢁ 0.258a (51)
1.997 ꢁ 0.160a (51)
1.456 ꢁ 0.208a,b,c (64)
1.426 ꢁ 0.217a,b,c (65)
1.636 ꢁ 0.241a (59)
2.120 ꢁ 0.169a (47)
1.765 ꢁ 0.188a (57)
2.090 ꢁ 0.215a (49)
2.228 ꢁ 0.057a (45)
2.160 ꢁ 0.161a (47)
2.16 ꢁ 0.186a (47)
0.452 ꢁ 0.163a,b,c (89)
0.168 ꢁ 0.800a,b,c (80)
1.398 ꢁ 0.466a,b,c (66)
1.133 ꢁ 0.923a,b,c (72)
0.803 ꢁ 0.208a,b,c (83)
1.831 ꢁ 0.274a (56)
2.072 ꢁ 0.245a (50)
2.572 ꢁ 0.160a (38)
2.223 ꢁ 0.208a (47)
2.208 ꢁ 0.185a (47)
2.177 ꢁ 0.154a (46)
2.652 ꢁ 0.170a,b (36)
1.608 ꢁ 0.130a (61)
1.790 ꢁ 0.0.125a (57)
1.960 ꢁ 0.217a (53)
1.863 ꢁ 0.247a (55)
1.341 ꢁ 0.253a,b,c (67)
1.555 ꢁ 0.201a,c (62)
1.993 ꢁ 0.144a (52)
1.582 ꢁ 0.192a (62)
1.456 ꢁ 0.190a,c (65)
2.073 ꢁ 0.146a (50)
2.020 ꢁ 0.161a (51)
2.15 ꢁ 0.175a (48)
12
13
14a
14b
14c
14d
14e
14f
14g
14h
14i
15
16a
16b
17
18
19
20
Indo.
Celec.
a
Statistically significant from control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
Statistically significant from celecoxib at p < 0.05.
b
c
Acute ulcerogenicity study was performed for compounds 7, 8,12
activity are shown in Table 6; and those derived for the analgesic
activity are shown in Table 7. The relation between the experi-
mental and predicted values for anti-inflammatory and analgesic
activities is illustrated in Figs. 2 and 3, respectively. These models
were validated with the leave-one-out (LOO) method.
As per the anti-inflammatory activity, the best derived QSAR
model for the 21 pyrimidopyrrolopyrimidine derivatives was pre-
sented by the following estimated penta-parametric equation with
and 16b with the best overall anti-inflammatory profile. The
ulcerogenic effect was evaluated for gastric ulcerogenic potential in
rats and all compounds were found less ulcerogenic than the stan-
dard drugs (Table 4) [30,31]. The ester derivative 7 exhibited gastric
ulceration lower than that produced by its acid analogue 8 and the
reference drugs indomethacin and celecoxib at the same dose level.
2.2.4. COX-1/COX-2 selectivity assay
Compounds 12 and 16b were evaluated for their selectivity to
inhibit COX-1 and COX-2 isoenzymes using Cayman’s COX activity
assay kit according to the manufacturer’s instructions [32]. The ratio
of % inhibition of COX-1/COX-2 would suggest the non-selectivity of
the tested compounds. The COX-1/COX-2 ratio showed that
compounds 12 and 16b have almost equal inhibitoryactivity on both
isoenzymes compared with indomethacin (Table 5).
Table 3
Effects of the compounds on acetic acid induced abdominal writhing in mice.
Compound No
Dose
(mg/kg)
Writhing
reflex ꢁ SEM
Inhibition
%
Potency
%
Control
Indomethacin
7
8
10
11
12
13
14a
14b
14c
14d
14e
14f
14g
14h
14i
15
16a
16b
17
18
19
e
70.83 ꢁ 1.45b
24.00 ꢁ 0.93a
20.66 ꢁ 0.88a,b
30.00 ꢁ 1.15a,b
50.00 ꢁ 2.22a,b
25.00 ꢁ 0.96a
26.00 ꢁ 0.96a
48.00 ꢁ 0.85a,b
30.00 ꢁ 0.96a,b
46.16 ꢁ 1.16a,b
20.00 ꢁ 1.06a,b
22.00 ꢁ 0.96a
39.83 ꢁ 0.90a,b
29.16 ꢁ 1.13a,b
23.00 ꢁ 0.96a
26.83 ꢁ 0.60a
12.83 ꢁ 0.60a,b
53.16 ꢁ 0.07a,b
26.83 ꢁ 0.94a,b
42.76 ꢁ 0.83a,b
73.00 ꢁ 0.96a,b
27.71 ꢁ 0.60a
27.00 ꢁ 0.66a
21.00 ꢁ 0.96a,b
e
e
10
10.68
9.86
10.26
9.46
15.29
9.86
12.42
13.42
13.29
12.88
13.75
11.03
12.83
13.83
14.63
11.49
11.72
13.51
13.12
11.78
11.84
11.81
66.12
70.83
57.65
29.41
64.70
63.29
32.23
57.65
34.83
71.76
68.94
43.77
58.83
67.53
62.12
81.89
24.95
62.12
39.63
1.17
100
107.12
87.19
44.48
97.85
95.72
48.74
87.19
52.68
108.53
104.26
66.20
88.97
102.13
93.95
123.85
37.73
93.95
59.79
1.77
2.3. QSAR studies
In an attempt to correlate the anti-inflammatory and analgesic
activities of the new compounds with their physicochemical
properties, QSAR study was undertaken. Descriptors of the
molecular modelling software, Molecular Operating Environment
(MOE version 2008.10.2), 57 were used. The anti-inflammatory and
analgesic activity data were expressed as % inhibition. The most
relevant descriptors derived for modelling the anti-inflammatory
Table 2
IC50 of the most active compounds 7, 8, 12, 16b.
Compd. No.
Response (% inhibition) at doses of
IC50 mg/kg
mol/kg)
(
m
1 mg/kg
2 mg/kg
4 mg/kg
6 mg/kg
61.00
61.88
70.35
92.25
93.58
106.40
7
8
12
16b
12
20
30
20
32
40
48
45
60
64
78
65
64
74
77
60
4.07 (11.00)
3.56 (10.00)
3.16 (6.00)
3.90 (8.00)
20
a
Statistically significant from control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b

M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
17
Table 4
Ulcer index of the most active compounds 7, 8, 12, 16b.
3. Experimental
3.1. Chemistry
Cpd
% incidence
Average
Severity
Ulcer index
Indo.
Celec.
7
8
12
10
6.66
5
8.33
5
6.66
3.33
3.16
1
1.16
1
1.40
1.31
1
1
1.16
1.28
14.73
11.13
7
10.49
7.16
10.94
Melting points were uncorrected and were determined on
Electrothermal Stuart 5MP3 digital melting point apparatus.
Elemental microanalyses were performed at the microanalytical
center, Faculty of Science, Cairo University and the Regional Center
for Mycology and Biotechnology, Al-Azhar University. The infrared
spectra were carried out on a Bruker FT-IR spectrophotometer and
Shimadzu FT-IR 8400S infrared spectrophotometer and were
expressed in wave number (cmꢀ1) using potassium bromide disc.
The 1H NMR spectra were recorded on a Varian Mercury VX-300
NMR spectrometer at 300 MHz and on NMR JOEL (Eclipse) 400 at
400 MHz in the specified solvent (exchanged with D2O). 13C NMR
spectra were recorded on NMR JOEL (Eclipse) 400 at 100 MHz in
DMSO-d6. Mass spectra were performed on HP MODEL: MS_5988
mass spectrometer and on Shimadzu QP-2010 Plus (EI, 70 eV).
Compounds 2e5 were prepared according to the reported
procedures [22e24].
16b
3
correlation coefficient (R2) ¼ 0.7528 and root mean square error
(RMSE) ¼ 0.06641.
Anti-inflammatory (% inhibition) ¼ ꢀ5.43095e1.54154 a_nO e
10.18241 log P þ 464.65895 vsurf_CP þ 2.64391 mr þ 9.17934
dipole.
From the equation positive correlation of vsurf_CP (i3D critical
packing parameter), mr (2D molar refractivity) and dipole (i3D
dipole moment) with the anti-inflammatory activity was observed.
On the other hand, the activity was negatively correlated with a_nO
(2D number of oxygen atoms) and log P (2D log octanol/water
partition coefficient). The high coefficient value of vsurf_CP re-
flected its high impact on the anti-inflammatory activity i.e. it was
the most influencing parameter. Critical packing parameter defines
a ratio between the hydrophilic and lipophilic part of a molecule. In
contrast to the hydrophilicelipophilic balance, critical packing
refers just to molecular shape. It is defined as: volume (lipophilic
part)/[(surface)(hydrophilic part)(length of lipophilic part)]. Lipo-
philic and hydrophilic calculations are performed at ꢀ0.6
and ꢀ3.0 kcal/mol, respectively. Critical packing is a good param-
eter to predict molecular packing such as in micelle formation, and
may be relevant in solubility studies in which the melting point
plays an important role [33,34]. The influence of CP was in agree-
ment with the experimental data, where the most active
compound 7 (inhibition % ¼ 89) had vsurf_CP ¼ 0.1562; whereas,
the compounds of low inhibition% (around 50%) possessed
vsurf_CP values of 0.0628e0.0981.
3.1.1. General procedure for the preparation of 7, 10
To a suspension of 5 (1 mmol) in dry benzene (10 ml) the
appropriate acid chloride malonyl or oxalyl chloride (1.1 mmol) was
added, and the reaction mixture was stirred for 3 h at room
temperature (R.T.). The solvent was removed under vacuum, the
residue washed twice with dry benzene (5 ml) and the solvent was
removed by distillation at each time. To the obtained 3-chloroacetyl
or 3-chlorocarbonyl intermediate 6 or 9, absolute ethanol (30 ml)
was added and the reaction mixture was refluxed for 2 h. The
separated precipitate was filtered, washed with water and crys-
tallized from absolute ethanol.
3.1.1.1. Ethyl 2-(1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydropyr-
imido[5,4-e]pyrrolo[1,2-c]pyrimidin-3-yl)acetate 7. Yellow solid,
76% yield, m.p. 239e240 ꢂC. IR: ymax./cmꢀ1 3515 (NH stretching),
3035 (CH aromatic), 2962e2831 (CH aliphatic), 1743, 1685, 1674
Regarding the analgesic activity, the best derived QSAR model
was presented by the following estimated penta-parametric
equation with correlation coefficient (R2) ¼ 0.6547 and root mean
square error (RMSE) ¼ 0.12196.
(3C]O). 1H NMR (DMSO-d6, 400 MHz):
d 1.08 (t, 3H, CH2CH3,
J ¼ 8 Hz), 1.60e1.95 (m, 3H, pyrrolidine), 2.62e2.75 (m, 1H, pyr-
rolidine), 3.43 (m, 1H, pyrrolidine), 3.51 (s, 2H, CH2COOC2H5),
3.60e3.67 (m, 1H, pyrrolidine), 3.93 (q, 2H, CH2CH3, J ¼ 8 Hz),
4.42e4.47 (m,1H, pyrrolidine), 7.12e7.37 (m, 5H, aromatic protons),
12.46 (s, 1H, NH). 13C NMR: 14.3, 32.9, 45.4, 55.5, 61.3, 62.9, 98.8,
128.8, 130.6, 137.7, 142.0, 151.0, 186.5, 195.4. Anal. Calcd. for
C19H20N4O4 (368.39): C, 61.95; H, 5.47; N, 15.21. Found: C, 62.18; H,
5.52; N, 15.43.
Analgesic activity (% inhibition)
dipole 14.84819 Slog
TPSA þ 0.40038 zagreb.
¼
67.43728
0.9148 vsurf_D1
þ
24.50080
0.22627
þ
P
ꢀ
þ
From the equation positive correlation of dipole (i3D dipole
moment), Slog P (2D log octanol/water partition coefficient), TPSA
(2D topological polar surface area) and Zagreb (2D Zagreb index)
with the analgesic activity was observed. The activity was nega-
tively correlated with vsurf_D1 (i3D hydrophobic volume). The
most influencing parameter was the dipole moment as revealed
from its high coefficient value.
3.1.1.2. Ethyl 1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydropyrimido
[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carboxylate 10. Light brown solid,
80% yield, m.p. 270e272 ꢂC. IR: ymax./cmꢀ1 3140 (NH stretching),
3050 (CH aromatic), 2982e2860 (CH aliphatic), 1759, 1688, 1645
Comparing the QSAR models for both anti-inflammatory and
analgesic activities, it could be revealed that the parameters that
affected one activity were different from those which affected the
other activity. This could explain the experimental biological
activities in which the highly active compounds in anti-
inflammatory screening (e.g. 8, 12) were not as potent in the
analgesic activity and vice versa.
(3C]O). 1H NMR (DMSO-d6, 300 MHz):
d 1.17 (t, 3H, CH2CH3,
J ¼ 10 Hz), 1.60e1.98 (m, 3H, pyrrolidine), 2.62e2.78 (m, 1H, pyrro-
lidine), 3.23e3.27 (m, 1H, pyrrolidine), 3.60e3.75 (m, 1H, pyrroli-
dine), 4.14 (q, 2H, CH2CH3, J ¼ 10 Hz), 4.5e4.6 (m, 1H, pyrrolidine),
7.10e7.50 (m, 5H, aromatic protons), 12.90 (s, 1H, NH). MS: m/z (%):
355.30 (Mþ þ 1, 1.03), 354.35 (Mþ, 2.28), 119.15 (100). Anal. Calcd. for
C18H18N4O4 (354.13): C, 61.01; H, 5.12; N, 15.81. Found: C, 61.27; H,
5.33; N, 16.22.
Table 5
COX-1/COX-2 ratio of compounds 12, 16b and indomethacin.
3.1.2. General procedure for the preparation of 8, 11
Compound No
COX-1/COX-2
The ester derivative 7 or 10 (1 mmol) was refluxed with 10%
NaOH (20 ml) for 2 h, the solution was cooled, diluted with water
(10 ml) and acidified with 1 N HCl. The precipitated solid was
filtered, washed with water and crystallized from ethanol.
12
16b
Indomethacin
1.30
1.02
3.98

18
M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
Table 6
The molecular descriptor values, experimental and predicted activity values of the anti-inflammatory activities of the studied compounds.
Compound
No
a_nO
logP
vsurf_CP
Mr
Dipole
Experimental
activity
(% inhibition)
Predicted
activity
(% inhibition)
Residual
values
(RES)
7
8
10
11
12
13
14a
14b
14c
14d
14e
14g
14h
14i
15
16a
16b
17
18
19
4.000
4.000
4.000
4.000
4.000
3.000
3.000
3.000
4.000
4.000
5.000
3.000
3.000
3.000
4.000
3.000
3.000
5.000
4.000
4.000
5.000
1.5260
0.1562
0.1073
0.1574
0.1258
0.1236
0.0889
0.0981
0.0956
0.0893
0.0904
0.0698
0.1215
0.1362
0.1256
0.0992
0.1091
0.1195
0.1023
0.0809
0.0807
0.0628
9.7686
8.7860
9.2891
8.3080
14.0352
8.7324
1.0673
1.3034
0.9900
1.3834
2.1066
0.4813
1.8238
1.3925
1.7770
1.9134
2.1237
1.5722
1.4587
1.5560
1.2188
1.1649
1.1115
0.6455
0.8494
1.5504
1.1028
89
80
66
72
83
56
50
38
47
47
46
61
57
53
55
67
62
52
62
65
50
81.0541
65.8236
74.6393
74.8131
80.9965
63.7136
49.9208
45.1661
51.6870
54.1963
47.2239
60.4411
66.5770
45.5488
58.2716
65.0872
52.8851
58.4882
53.1575
60.6814
47.6276
7.9459
14.1764
ꢀ8.6393
ꢀ2.8131
2.0035
0.7490
2.0200
0.6610
2.0880
ꢀ0.4870
3.3520
3.3630
2.7570
2.4610
2.4150
3.2780
3.2890
5.5210
1.4410
1.4600
3.7030
1.2790
0.7720
0.6930
0.5170
ꢀ7.7136
12.0228
12.1989
12.6920
11.8350
12.4840
12.4706
12.6571
14.8254
10.3103
10.8234
13.1991
11.7958
10.2970
10.4401
10.1106
0.0792
ꢀ7.1661
ꢀ4.6870
ꢀ7.1963
ꢀ1.2239
0.5589
ꢀ9.5770
7.4512
ꢀ3.2716
1.9128
9.1149
ꢀ6.4882
8.8425
4.3187
2.3724
20
3.1.2.1. 2-(1,6-Dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydropyrimido
[5,4-e]pyrrolo[1,2-c]pyrimidin-3-yl)acetic acid 8. Pale yellow solid,
68% yield, m.p. >340 ꢂC (decomposition). IR: ymax./cmꢀ1 3100 (NH
stretching), 3035 (CH aromatic), 2962e2831 (CH aliphatic),
2785e2600 (br, OH), 1743, 1674, 1635 (3C]O). 1H NMR (DMSO-d6,
3H, pyrrolidine), 2.70e2.75 (m, 1H, pyrrolidine), 3.24e3.32 (m, 1H,
pyrrolidine), 3.60e3.69 (m, 1H, pyrrolidine), 4.45e4.54 (m, 1H,
pyrrolidine), 7.16e7.42 (m, 5H, aromatic protons), 7.90 (s, 1H, NH),
12.60 (br, s, 1H, OH). MS: m/z (%): 327.20 (Mþ þ 1, 0.09), 326.20
(0.05), 281 (100). Anal. Calcd. for C16H14N4O4 (326.31): C, 58.89; H,
4.32; N, 17.17. Found: C, 59.13; H, 4.41; N, 17.36.
300 MHz): d 1.62e1.88 (m, 3H, pyrrolidine), 2.05 (s, 2H, CH2COOH),
2.68e2.72 (m, 1H, pyrrolidine), 3.21e3.29 (m, 1H, pyrrolidine),
3.58e3.64 (m, 1H, pyrrolidine), 4.39e4.44 (m, 1H, pyrrolidine),
7.13e7.41 (m, 6H, aromatic protons þ NH), 12.28 (s, 1H, OH). Anal.
Calcd. for C17H16N4O4 (340.33): C, 59.99; H, 4.74; N, 16.46. Found: C,
60.17; H, 4.92; N, 16.78.
3.1.3. N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a-octahydropyrimido[5,4-e]
pyrrolo[1,2-c]pyrimidine-3-carboxamide 12
To a solution of the acid chloride intermediate 9 (1 mmol) in DMF
(10 ml), K2CO3 (1 mmol) and 4-aminoantipyrine (1 mmol) were
added. The reaction mixture was heated at 100 ꢂC for 8 h and then
cooled to R.T. The reaction mixture was poured on cold water; the
separated solid was filtered, washed, dried and crystallized from
ethanol to give yellowish white crystals, 60% yield, m.p. 228e229 ꢂC.
IR: ymax./cmꢀ1 3205, 3132 (2NH stretching), 3070 (CH aromatic),
3.1.2.2. 1,6-Dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-3-carboxylic acid 11. White solid, 71%
yield, m.p. 282e284 ꢂC. IR: ymax./cmꢀ1 3394 (NH stretching), 3050
(CH aromatic), 2974e2877 (CH aliphatic), 2792e2600 (br, OH),1759,
1666, 1635 (3C]O). 1H NMR (DMSO-d6, 300 MHz):
d 1.70e1.90 (m,
Table 7
The molecular descriptor values, experimental and predicted activity values of the analgesic activities of the studied compounds.
Compound
No
Dipole
Slog P
vsurf_D1
TPSA
Zagreb
Experimental
activity
(% inhibition)
Predicted
activity
% inhibition
Residual
values
(RES)
8
10
11
12
1.3034
0.9900
1.3834
2.1066
0.4813
1.8238
1.3925
1.7770
1.9134
2.1237
1.8301
1.5722
1.4587
1.5560
1.2188
1.1649
1.1115
0.6455
1.5504
1.1028
0.8494
1.3056
713.6250
847.8750
737.8750
1077.8750
653.6250
1013.6250
1038.3750
882.6250
957.2500
967.0000
834.6250
908.0000
1027.2500
1080.2500
747.8750
870.3750
1044.8750
981.2500
808.0000
738.6250
796.1250
102.3100
91.3100
138.0000
142.0000
134.0000
206.0000
138.0000
166.0000
180.0000
174.0000
180.0000
190.0000
152.0000
172.0000
186.0000
192.0000
154.0000
170.0000
190.0000
174.0000
170.0000
170.0000
170.0000
57.6500
29.4100
64.7000
63.2900
32.2300
57.6500
34.8300
71.7600
68.9400
43.7700
58.8300
67.5300
62.1200
81.8900
24.9500
62.1200
39.6300
1.1700
60.5151
32.7158
50.4388
58.1084
33.8463
51.6960
38.0054
75.3796
56.8046
66.3104
57.3502
73.9510
49.9520
77.5407
55.6965
49.4555
49.6817
14.0198
59.0755
58.4816
46.6750
ꢀ2.8651
ꢀ3.3058
14.2612
5.1816
1.7416
0.9155
2.4479
ꢀ0.1792
2.9033
2.6346
2.5298
1.6868
1.6954
1.3429
3.2934
3.0247
5.2439
0.9269
1.9846
3.7990
1.2762
0.9657
0.4613
0.6556
102.3100
117.6600
120.1300
106.4700
106.4700
115.7000
126.7000
135.9300
106.4700
106.4700
106.4700
106.4700
115.7000
99.9000
13
ꢀ1.6163
14a
14b
14c
14d
14e
14f
14g
14h
14i
15
16a
16b
17
18
19
5.9540
ꢀ3.1754
ꢀ3.6196
12.1354
ꢀ22.5404
1.4798
ꢀ6.4210
12.1680
4.3493
ꢀ30.7465
12.6645
ꢀ10.0517
ꢀ12.8498
1.9245
99.9000
132.7700
114.7500
134.9800
146.1500
61.0000
61.8800
70.3500
3.3984
23.6750
20

M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
19
under vacuum; the separated solid was filtered, dried and crystal-
lized from the appropriate solvent.
3.1.5.1. N0-Benzylidene-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahy-
dropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydrazide 14a.
Light brown solid, crystallized from ethanol, 69% yield, m.p.
264e265 ꢂC. IR: ymax./cmꢀ1 3425, 3264 (2NH stretching), 3063 (CH
aromatic), 2974 (CH aliphatic), 1724e1650 (br, 3C]O). 1H NMR
(CHCl3, 300 MHz):
d 1.80e2.10 (m, 3H, pyrrolidine), 2.85e3.20 (m,
1H, pyrrolidine), 3.42e3.57 (m, 1H, pyrrolidine), 3.80e3.98 (m, 1H,
pyrrolidine), 4.59e4.69 (m, 1H, pyrrolidine), 7.27e7.74 (m, 10H,
aromatic protons), 7.77, (s, 1H, CH]N), 9.61, (s, 1H, NH), 10.25 (s, 1H,
NH). Anal. Calcd. for C23H20N6O3 (428.44): C, 64.48; H, 4.71; N,19.62.
Found: C, 65.08; H, 4.91; N, 19.24.
Fig. 2. Experimental vs predicted anti-inflammatory activity represented as
inhibition%.
3.1.5.2. N0-(4-Chlorobenzylidene)-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,
10a,-octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohy-
drazide 14b. Light brown solid, crystallized from ethanol, 64%
yield, m.p. 261e262 ꢂC. IR: ymax./cmꢀ1 3439e3320 (br, 2NH), 3044
(CH aromatic), 2992e2938 (CH aliphatic), 1691e1620 (3C]O). 1H
2927e2885 (CH aliphatic), 1766, 1743, 1675, 1651 (4C]O). 1H NMR
(DMSO-d6, 400 MHz): d 0.94 (s, 3H, CH3eC), 1.72e1.90 (m, 3H, pyr-
NMR (DMSO-d6, 300 MHz):
d 1.72e1.93 (m, 3H, pyrrolidine),
rolidine), 2.35e2.42 (m, 1H, pyrrolidine), 3.16 (s, 3H, NeCH3)
3.32e3.45 (m, 1H, pyrrolidine), 3.96e4.04 (m, 1H, pyrrolidine),
5.32e5.40 (m, 1H, pyrrolidine), 7.05e7.65 (m, 11H, aromatic
protons þ NH), 11.63 (s, 1H, NH). 13C NMR; 11.3, 35.3, 53.1, 116.2,
126.3, 128.1, 129.6, 134.8, 151.8, 153.3, 156.9, 160.2. MS: m/z (%):
526.20 (Mþ ꢀ 1, 1.87), 80.00 (100). Anal. Calcd. for C27H25N7O4
(527.57): C, 61.41; H, 4.73; N,18.57. Found: C, 61.58; H, 4.85; N,18.79.
2.62e2.79 (m, 1H, pyrrolidine), 3.23e3.40 (m, 1H, pyrrolidine),
3.62e3.79 (m, 1H, pyrrolidine), 4.5e4.6 (m, 1H, pyrrolidine),
7.15e7.74 (m, 9H, aromatic protons), 8.08, (s, 1H, CH]N), 11.19, (s,
1H, NH), 13.00 (s, 1H, NH). Anal. Calcd. for C23H19ClN6O3 (462.89):
C, 59.68; H, 4.14; N, 18.16. Found: C, 60.10; H, 4.17; N, 18.38.
3.1.5.3. N0-(4-Methoxybenzylidene)-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,
10a,-octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydr-
azide 14c. Orange solid, crystallized from aqueous ethanol, 72%
yield, m.p. 252e254 ꢂC. IR: ymax./cmꢀ1 3474, 3284 (2NH), 3050
(CH aromatic), 2930e2839 (CH aliphatic), 1720, 1646, 1602 (3C]
3.1.4. 1,6-Dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydropyrimido
[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydrazide 13
To a mixture of 10 (0.35 g, 1 mmol) in absolute ethanol (20 ml),
hydrazine hydrate (99%) (10 mmol) was added. The mixture was
stirred for 5 h at R.T. The separated white precipitate was filtered
and crystallized from absolute ethanol to give white crystals, 70%
yield, m.p. >340 ꢂC (decomposition). IR: ymax./cmꢀ1 3398, 3317,
3244 (NH2, 2NH stretching), 3063 (CH aromatic), 2884 (CH
aliphatic), 1700e1681 (br, 3C]O). 1H NMR (CHCl3, 300 MHz):
O). 1H NMR (CHCl3, 300 MHz):
d 1.80e2.10 (m, 3H, pyrrolidine),
2.90e3.20 (m, 1H, pyrrolidine), 3.42e3.55 (m, 1H, pyrrolidine),
3.70e3.79 (m, 1H, pyrrolidine), 3.87 (s, 3H, OeCH3) 4.60e4.69 (m,
1H, pyrrolidine), 6.91e7.80 (m, 9H, aromatic protons), 8.61, (s, 1H,
CH]N), 9.53 (s, 1H, NH), 10.1 (s, 1H, NH). 13C NMR: 19.3, 45.7,
56.4, 115.0, 127.2, 128.1, 129.6, 130.8, 137.9, 138.2, 157.0, 162.1,
162.5. Anal. Calcd. forC24H22N6O4 (458.47): C, 62.87; H, 4.84; N,
18.33. Found: C, 63.02; H, 4.70; N, 18.71.
d
1.84e2.04 (m, 3H, pyrrolidine), 2.97e2.99 (m, 1H, pyrrolidine),
3.43e3.50 (m,1H, pyrrolidine), 3.80e4.10 (m, 3H, pyrrolidine þ NH2),
4.58e4.63 (m, 1H, pyrrolidine), 7.20e7.48 (m, 6H, aromatic
protons þ NH), 7.90 (s, 1H, NH). Anal. Calcd. for C16H16N6O3 (340.34):
C, 56.47; H, 4.74; N, 24.69. Found: C, 56.72; H, 4.80; N, 25.06.
3.1.5.4. N0-(2-Hydroxybenzylidene)-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,
10a,-octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydr-
azide 14d. Light brown solid, crystallized from acetic acid, 72% yield,
m.p. 217e218 ꢂC. IR: ymax./cmꢀ1 3444 (OH), 3290, 3271 (2NH), 3043
(CH aromatic), 2939e2854 (CH aliphatic),1700e1610 (br, 3C]O).1H
3.1.5. General procedure for the preparation of 14aei
To a solution of the hydrazide 13 (0.34 g, 1 mmol) in ethanol
(20 ml), few drops of acetic acid and the appropriate aldehyde or
ketone (1 mmol) were added. The reaction mixture was heated
under reflux for 5 h and then cooled. The solvent was removed
NMR (DMSO-d6, 300 MHz):
d 1.70e2.00 (m, 3H, pyrrolidine),
2.65e2.80 (m, 1H, pyrrolidine), 3.29e3.40 (m, 1H, pyrrolidine),
3.70e3.79 (m, 1H, pyrrolidine), 4.50e4.60 (m, 1H, pyrrolidine),
6.89e7.70 (m, 9H, aromatic protons), 9.00 (s,1H, CH]N),10.70 (s,1H,
NH), 11.13 (s, 1H, OH), 11.30 (s, 1H, NH). Anal. Calcd. for C23H20N6O4
(444.44): C, 62.16; H, 4.54; N,18.91. Found: C, 62.34; H, 4.80; N,19.37.
3.1.5.5. N0-(3-Hydroxy-4-methoxybenzylidene)-1,6-dioxo-5-phenyl-1,2,
5,6,8,9,10,10a,-octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-car-
bohydrazide 14e. Light brown solid, crystallized from ethanol, 77%
yield, m.p. 278e280 ꢂC. IR: ymax./cmꢀ1 3474 (OH), 3255, 3217 (2NH),
3059 (CH aromatic), 2931e2893 (CH aliphatic),1681,1639, 1630 (3C]
O). 1H NMR (DMSO-d6, 300 MHz):
d 1.71e2.00 (m, 3H, pyrrolidine),
2.63e2.80 (m, 1H, pyrrolidine), 3.29e3.40 (m, 1H, pyrrolidine),
3.65e3.72 (m, 1H, pyrrolidine), 3.80 (s, 3H, OeCH3), 4.52e4.57 (m,1H,
pyrrolidine), 6.81e7.48 (m, 8H, aromatic protons), 7.92 (s, 1H, CH]N),
9.68 (s, 1H, NH), 10.87 (s, 1H, OH), 12.8 (s, 1H, NH). Anal. Calcd. for
C24H22N6O5 (474.47): C, 60.75; H, 4.67; N, 17.71. Found: C, 60.71; H,
4.80; N, 17.93.
Fig. 3. Experimental vs predicted analgesic activity represented as inhibition%.

20
M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
3.1.5.6. 1,6-Dioxo–5-phenyl-N0-(propan-2-ylidene)-1,2,5,6,8,9,10,10a,-
octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydrazide
14f. Light brown solid, crystallized from absolute ethanol, 70% yield,
m.p. 268e270 ꢂC. IR: ymax./cmꢀ1 3325 (br, 2NH), 3020 (CH aromatic),
2950e2850 (CH aliphatic), 1690, 1674 (br) (3C]O). 1H NMR (DMSO-
J ¼ 7.2 Hz), 4.61e4.65 (m, 1H, pyrrolidine), 6.67 (s, 1H, N]CH),
7.25e7.49 (m, 5H, aromatic protons), 9.84 (s, 1H, NH), 10.32 (s, 1H,
NH). Anal. Calcd. for C19H20N6O4 (396.4): C, 57.57; H, 5.09; N, 21.20.
Found: C, 57.99; H, 4.79; N, 21.53.
d6, 300 MHz):
d
1.39, (s, 3H, CH3),1.70e1.87 (m, 3H, pyrrolidine),1.92
3.1.7. General procedure for the preparation of 16a,b
To a solution of 13 (0.34 g, 1 mmol) in dry DMF (5 ml) acetyla-
cetone or benzoylacetone (2 mmol), was added. The solution was
heated at 100 ꢂC for 8 h, cooled to R.T. and poured on ice water
(20 ml). The separated solid was filtered, and crystallized from
absolute ethanol.
(s, 3H, CH3), 2.65e2.80 (m, 1H, pyrrolidine), 3.29e3.40 (m, 1H,
pyrrolidine), 3.63e3.78 (m, 1H, pyrrolidine), 4.48e4.59 (m, 1H,
pyrrolidine), 7.26e7.46 (m, 5H, aromatic protons), 9.61 (s, 1H, NH),
12.85 (s, 1H, NH). Anal. Calcd. for C19H20N6O3 (380.40): C, 59.99; H,
5.30; N, 22.09. Found: C, 60.40; H, 5.90; N, 22.34.
3.1.5.7. 1,6-Dioxo-5-phenyl-N0-(1-phenylethylidene)-1,2,5,6,8,9,10,10a,-
octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydrazide
14g. Brown solid, crystallized from ethanol, 73% yield, m.p.
310e311 ꢂC. IR: ymax./cmꢀ1 3309, 3151 (2NH stretching), 3059 (CH
aromatic), 2958e2877 (CH aliphatic), 1720, 1689, 1647 (3C]O). 1H
3.1.7.1. 3-(3,5-Dimethyl-1H-pyrazole-1-carbonyl)-5-phenyl-8,9,10,10a,-
tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6(2H,5H)-dione
16a. Yellow solid, crystallized from ethanol, 70% yield, m.p.
209e210 ꢂC. IR: ymax./cm-1 3250 (NH), 3050 (CH aromatic),
2939e2827 (CH aliphatic), 1700e1651 (br, 3C]O). 1H NMR (CDCl3,
NMR (DMSO-d6, 300 MHz):
d
1.79, (s, 3H, CH3), 1.92e2.00 (m, 3H,
300 MHz): d 1.85e2.24 (m, 3H, pyrrolidine), 2.44 (s, 3H, CH3), 2.52
pyrrolidine), 2.65e2.80 (m, 1H, pyrrolidine), 3.29e3.40 (m, 1H,
pyrrolidine), 3.64e3.71 (m, 1H, pyrrolidine), 4.50e4.59 (m, 1H,
pyrrolidine), 7.30e7.78 (m, 10H, aromatic protons), 9.99 (s, 1H, NH),
13.04 (s, 1H, NH). MS: m/z (%): 442.45 (Mþ, 29.69), 77.10 (100). Anal.
Calcd. for C24H22N6O3 (442.47): C, 65.15; H, 5.01; N, 18.99. Found: C,
65.24; H, 4.98; N, 18.99.
(s, 3H, CH3), 2.95e3.00 (m, 1H, pyrrolidine), 3.40e3.52 (m, 1H,
pyrrolidine), 3.78e3.92 (m, 1H, pyrrolidine), 4.51e4.71 (m, 1H,
pyrrolidine), 6.20 (s, 1H, pyrazole proton), 7.24e7.47 (m, 5H,
aromatic protons), 9.61 (s, 1H, NH). MS: m/z (%): 404.30 (Mþ, 2.08),
96.15 (100). Anal. Calcd. for C21H20N6O3 (404.42): C, 62.37; H, 4.98;
N, 20.78. Found: C, 62.49; H, 4.87; N, 21.14.
3.1.5.8. N0-[1-(4-Chlorophenyl)ethylidene]-1,6-dioxo-5-phenyl-
1,2,5,6,8,9,10,10a,-octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimi-
dine-3-carbohydrazide 14h. Brown solid, crystallized from
ethanol, 76% yield, m.p. 157e158 ꢂC. IR: ymax./cmꢀ1 3367, 3305
(2NH), 3039 (CH aromatic), 2958e2881 (CH aliphatic), 1720,
3.1.7.2. 3-(5-Methyl-3-phenyl-1H-pyrazole-1-carbonyl)-5-phenyl-8,9,
10,10a,-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6(2H,5H)-
dione 16b. Orange solid, crystallized from ethanol, 80% yield, m.p.
260e262 ꢂC. IR: ymax./cmꢀ1 3329 (NH), 3062 (CH aromatic),
2970e2850 (CH aliphatic), 1720, 1685, 1647 (3C]O). 1H NMR
1697, 1643 (3C]O). 1H NMR (DMSO-d6, 400 MHz):
d
1.72e2.00
(CDCl3, 300 MHz): d 1.86e2.10 (m, 3H, pyrrolidine), 2.21 (s, 3H,
(m, 3H, pyrrolidine), 1.78 (s, 3H, CH3), 2.65e2.78 (m, 1H, pyrro-
lidine), 3.35e3.40 (m, 1H, pyrrolidine), 3.65e3.72 (m, 1H, pyr-
rolidine), 4.53e4.63 (m, 1H, pyrrolidine), 7.31e7.80 (m, 9H,
aromatic protons), 10.1 (s, 1H, NH), 12.81 (s, 1H, NH). Anal. Calcd.
for C29H21ClN6O3 (476.91): C, 60.44; H, 4.44; N, 17.62. Found: C,
60.72; H, 4.81; N, 18.09.
CH3), 2.95e3.00 (m, 1H, pyrrolidine), 3.40e3.52 (m, 1H, pyrroli-
dine), 3.78e3.93 (m, 1H, pyrrolidine), 4.50e4.71 (m, 1H, pyrroli-
dine), 6.18 (s, 1H, pyrazole proton), 7.26e7.95 (m, 10H, aromatic
protons), 9.68 (s, 1H, NH). Anal. Calcd. for C26H22N6O3 (466.18): C,
66.94; H, 4.75; N, 18.02. Found: C, 66,69; H, 5.00; N, 18.37.
3.1.8. Ethyl 3-[2-(1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-
octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbonyl)
hydrazono]butanoate 17
3.1.5.9. N0-(Diphenylmethylene)-1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-
octahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbohydrazide
14i. Brown solid, crystallized from ethanol, 68% yield, m.p.
287e289 ꢂC. IR: ymax./cmꢀ1 3306, 3150 (2NH stretching), 3043 (CH
aromatic), 2974e2860 (CH aliphatic), 1710, 1697, 1647 (3C]O). 1H
A mixture of the hydrazide 13 (0.34 g, 1 mmol) and ethyl ace-
toacetate (10 ml) were heated under reflux for 5 h. The reaction
mixture was cooled to R.T. and the separated solid was filtered
washed with water and crystallized from ethanol to give orange
solid, 85% yield, m.p. 239e240 ꢂC. IR: ymax./cmꢀ1 3367, 3232 (2NH),
3062 (CH aromatic), 2989e2893 (CH aliphatic), 1720e1685,
NMR (DMSO-d6, 400 MHz):
d 1.65e1.92 (m, 3H, pyrrolidine),
2.65e2.75 (m, 1H, pyrrolidine), 3.24e3.35 (m, 1H, pyrrolidine),
3.55e3.65 (m, 1H, pyrrolidine), 4.45e4.55 (m, 1H, pyrrolidine),
6.95e7.70 (m, 15H, aromatic protons), 9.65 (s, 1H, NH), 12.80 (s, 1H,
NH). 13C NMR: 21.3, 45.0, 45.3, 92.9, 127.3, 128.2, 128.5, 128.8, 130.1,
130.2, 136.1, 137.0, 150.0, 150.1, 150.2, 150.3. Anal. Calcd. for
C29H24N6O3 (504.54): C, 69.04; H, 4.79; N, 16.66. Found: C, 69.43; H,
4.88; N, 16.97.
1670e1647 (br, 4C]O). 1H NMR (DMSO-d6, 400 MHz):
d 1.18 (t,
3H, CH2CH3, J ¼ 8 Hz), 1.45 (s, 3H, CH3eC), 1.67e1.95 (m, 3H, pyr-
rolidine), 2.68e2.78 (m, 1H, pyrrolidine), 3.25e3.30 (m, 1H, pyr-
rolidine), 3.31 ( s, 2H, CH2), 3.64e3.73 (m, 1H, pyrrolidine), 4.09 (q,
2H, CH2CH3, J ¼ 8 Hz), 4.50e4.59 (m,1H, pyrrolidine), 7.28e7.45 (m,
5H, aromatic protons), 9.75(s, 1H, NH), 10.30 (s, 1H, NH). 13C NMR:
14.6,15.6, 21.5, 44.3, 45.3, 60.9,115.1,129.2,130.5,137.4,150.5,165.8,
169.9, 195.0. MS: m/z (%): 454.30 (Mþ þ 2, 36.56), 96.15 (100). Anal.
Calcd. For C22H24N6O5 (452.46): C, 58.46; H, 5.35; N,18.57. Found: C,
58.91; H, 5.49; N, 18.74.
3.1.6. Ethyl N0-(1,6-dioxo-5-phenyl-1,2,5,6,8,9,10,10a,-octahydro-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-3-carbonyl)
formohydrazonate 15
A mixture of 13 (0.34 g, 1 mmol) and a catalytic amount of acetic
anhydride in triethylorthoformate (5 ml) was heated for 3 h, the
mixture was cooled to R.T. and poured on ice water (20 ml). The
separated solid was filtered, washed with water and crystallized
from ethanol to give orange solid, 72% yield, m.p. 234e235 ꢂC. IR:
ymax./cmꢀ1 3450, 3331, 3150 (2NH stretching), 3059 (CH aromatic),
2929e2884 (CH aliphatic),1720e1639, (3C]O). 1H NMR (DMSO-d6,
3.1.9. 3-(3-Methyl-5-hydroxy-1H-pyrazole-1-carbonyl)-5-phenyl-
8,9,10,10a,-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
1,6(2H,5H)-dione 18
Compound 17 (0.45 g, 1 mmol) and 2 M KOH (20 ml) were
refluxed for 5 h, the reaction mixture was then cooled and acidified
with HCL. The resulting solid was filtered, washed with water, dried
and crystallized from acetic acid to give yellow solid, 65% yield, m.p.
284e285 ꢂC. IR: ymax./cmꢀ1 3441 (OH), 3267 (NH), 3012 (CH
300 MHz):
d
1.32 (t, 3H, CH2CH3, J ¼ 7.2 Hz), 1.80e2.10 (m, 3H,
pyrrolidine), 2.90e3.10 (m, 1H, pyrrolidine), 3.42e3.57 (m, 1H,
pyrrolidine), 3.80e3.88 (m, 1H, pyrrolidine), 4.09 (q, 2H, CH2CH3,

M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
21
aromatic), 2978e2850 (CH aliphatic), 1675e1600 (3C]O). 1H NMR
(DMSO-d6, 300 MHz): 1.80e1.85 (m, 3H, pyrrolidine), 2.49 (s, 3H,
inhibition of oedema and was calculated by the following
equation:
d
CH3), 3.13e3.18 (m, 2H, pyrrolidine), 3.86e3.91 (m, 2H, pyrroli-
dine), 7.07e7.51 (m, 5H, aromatic protons), 8.00 (s, 1H, pyrazole
proton), 11.90 (s, 1H, NH), 13.86 (s, 1H, OH). Anal. Calcd. for
C20H18N6O4 (406.39): C, 59.11; H, 4.46; N, 20.68. Found: C, 59.29; H,
4.38; N, 21.04.
%Inhibition ¼ 100 ꢃ ½1 ꢀ Vt=Vcꢄ
Where Vt is the mean increase of paw thickness of rats after
administration of the tested compounds or the reference drug. Vc
is the mean increase in paw thickness in rats after administration
of carrageenan in the positive control group. Data were analyzed
by SPSS statistical package version 10. Results are presented in
Table 1.
3.1.10. 3-(3-Hydroxy-5-oxo-4,5-dihydro-1H-pyrazole-1-carbonyl)-
5-phenyl-8,9,10,10a,-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]
pyrimidine-1,6(2H,5H)-dione 19
A mixture of the hydrazide 13 (0.34 g, 1 mmol) and diethyl
malonate (10 ml) was heated under reflux for 5 h. The resulting
solid was filtered, washed with water, dried and crystallized from
ethyl alcohol to give orange solid, 60% yield, m.p. >350 ꢂC. IR:
ymax./cmꢀ1 3294 (OH), 3192 (NH), 3059 (CH aromatic),
2981e2850 (CH aliphatic), 1720e1700, 1680e1665(br, 4C]O). 1H
IC50 values was calculated by the same method using the doses
of 1, 2, 4, 6 mg/kg (Table 2).
3.2.2. Analgesic activity
Analgesic activity was measured using the acetic acid induced
writhing test in mice [28,29]. Six Swiss albino mice of either sex
(18e22 g) were used in each group. One hour after oral admin-
istration of a suspension of the tested compounds or indometh-
acin, (0.029 mmol/kg), each mouse was injected with 0.3 ml of 1%
acetic acid solution intraperitoneally. Starting 5 min after the
acetic acid injection, the number of muscular contractions in
each mouse was counted for a period of 30 min. A significant
reduction in the number of writhing by any test compound as
compared to control animals was considered as a positive anal-
gesic response. Percentage protection was calculated using the
following formula, where n is average number of writhing in
control group and n0 is average number of writhing in treated
groups (Table 3).
NMR (DMSO-d6, 300 MHz):
d 1.68e1.88 (m, 3H, pyrrolidine), 1.90
(s, 2H, pyrazole proton), 2.62e2.78 (m, 1H, pyrolidine), 3.22e3.29
(m, 1H, pyrrolidine), 3.60e3.74 (m, 1H, pyrrolidine), 4.49e4.60
(m, 1H, pyrrolidine), 7.20e7.41 (m, 5H, aromatic protons), 9.89
(s, 1H, NH), 12.85 (s, 1H, OH). Anal. Calcd. for C19H16N6O5
(408.37): C, 55.88; H, 3.95; N, 20.58. Found: C, 55.96; H, 4.08; N,
21.04.
3.1.11. 3-(5-Amino-3-hydroxy-1H-pyrazole-1-carbonyl)-5-phenyl-
8,9,10,10a,-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
1,6(2H,5H)-dione 20
A mixture of the hydrazide 13 (0.34 g, 1 mmol) and ethyl
cyanoacetate (1 mmol) in dioxane (20 ml) was heated under
reflux for 5 h. The solution was cooled and poured on ice cold
water, the resulting solid was filtered, washed with water, dried
and crystallized from ethyl alcohol to give brown solid, 60% yield,
m.p. >350 ꢂC (decp). IR: ymax./cmꢀ1 3425e3367 (OH, NH2), 3228
(NH stretching), 3070 (CH aromatic), 2981e2850 (CH aliphatic),
1720e1680 (br, 3C]O). 1H NMR (DMSO-d6, 300 MHz):
% Protection ¼ ½ðn ꢀ n0Þ=nꢄ ꢃ 100
Potency of the tested compounds was calculated according to
the following equation:
Potency% ¼ % inhibition of the tested compd:=
% inhibition of Indomethacin ꢃ 100
d
1.68e1.84 (m, 3H, pyrrolidine), 2.62e2.79 (m, 1H, pyrrolidine),
3.20e3.29 (m, 1H, pyrrolidine), 3.58e3.79 (m, 1H, pyrrolidine),
4.05e4.20 (m, 1H, pyrrolidine), 6.30 (s, 2H, NH2), 7.20e7.52 (m,
6H, aromatic protons þ pyrazole proton), 9.89 (s, 1H, NH), 11.25
(s, 1H, OH). Anal. Calcd. for C20H17N7O4 (407.38): C, 56.02; H,
4.21; N, 24.07. Found: C, 56.15; H, 4.32; N, 24.38.
3.2.3. Gastric-ulcerogenic effect
Five hours after the oral treatment of rats with the tested
compounds and standard drugs, they were killed under deep ether
anaesthesia and their stomachs were removed. The stomach of
each rat was opened through great curvature and examined for
lesions or bleedings (Table 1).
3.2. Pharmacological screening
3.2.4. Acute ulcerogenicity studies [30,31]
3.2.1. Anti-inflammatory activity
Adult Wistar albino rats of both sexes weighing between 120
and 150 g were used. Animals were divided into groups each of 6
animals. Rats were fasted for 20 h before drug administration. The
tested compounds and the reference drugs indomethacin and cel-
ecoxib were given orally in a dose of 0.029 mmol/kg suspended in
1% Tween while one group received vehicle (1% Tween). Rats were
fasted for 2 h, allowed to feed 2 h then fasted for another 20 h. Rats
were given another two doses in the second and third days. In the
fourth day, rats were sacrificed, the stomach removed, open along
with the greater curvature and rinsed with 0.9% saline. The number
of mucosal damage (red spots) was counted and their severity
(ulcerogenic severity) was graded from 0 to 4 according to the
following score assignment:
Anti-inflammatory activity screening was carried out using
carrageenan induced paw oedema in albino rats [25e27]. Adult
Wistar albino rats of both sexes weighing 120e150 g were divided
into groups of 6 animals each. The animals were acclimatized to
the lab conditions for two days with free access to food and water.
On the day before the experiment, the food was withdrawn, but
the animals were allowed free access to water. The control group
was given saline solution containing few drops of Tween 80. The
synthesized compounds (0.029 mmol/kg) and the reference drugs
indomethacin and celecoxib (0.029 mmol) were suspended in
saline solution with few drops of tween 80 and were given orally,
using the oral stomach tube, 1 h before induction of inflammation.
Induction of inflammation was performed by S.C. injection of
freshly prepared suspension of carrageenan (0.1 ml of 1%, Fluka) in
saline into subplantar tissue of the right hind paw. The thickness
of the paw was measured at 1, 2, 3 and 4 h intervals after
induction of the inflammation and compared with the initial hind
paw thickness of each rat for the determination of oedema
thickness. The anti-inflammatory activity was expressed as %
Score
Score
Normal (no injury)
Latent small red spot
Wide red spot
0
1
2
Slight injury
Severe injury
3
4

22
M.M. Hanna / European Journal of Medicinal Chemistry 55 (2012) 12e22
The following figures were calculated:
Acknowledgements
- % Incidence/10 ¼ number of rats showing ulcer of any grade
divided by total number of rats in the group ꢃ 100/10.
The author would like to thank the Pharmacology Department,
Faculty of Pharmacy, Cairo University for performing the pharma-
cological screening of the newly prepared compounds.
- Average number of ulcers: number of ulcers in the group/total
number of rats in the group.
P
- Average severity:
severity]/number of ulcers in the group.
[each ulcer multiplied by its score of
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