Novel substituted and fused pyrrolizine derivatives: Synthesis, anti-inflammatory and ulcerogenecity studies

Article abstract of DOI:10.1016/j.ejmech.2009.10.031

Synthesis of several substituted pyrrolizines 10a-f, 11a-f, 13a-c, pyrimidopyrrolizines 14a-c, 15a-c, and pyrrolizinopyrimidoisoindoles 12a-c was discussed. The starting compounds 6-amino-7-cyano-N-(4-(un)substitutedphenyl)-2,3-dihydro-1H-pyrrolizine-5 -c

Full text of DOI:10.1016/j.ejmech.2009.10.031

European Journal of Medicinal Chemistry 45 (2010) 482–491
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel substituted and fused pyrrolizine derivatives: Synthesis, anti-inflammatory
and ulcerogenecity studies
,
b
b
Safinaz E. Abbas a, Fadi M. Awadallah a , Nashwa A. Ibrahim , Ahmed M. Gouda
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Eini street 11562 Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Beni-Sweif University, 62111 Beni-Sweif, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of several substituted pyrrolizines 10a–f, 11a–f, 13a–c, pyrimidopyrrolizines 14a–c, 15a–c, and
pyrrolizinopyrimidoisoindoles 12a–c was discussed. The starting compounds 6-amino-7-cyano-N-(4-
(un)substitutedphenyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamides 9a–c were reacted with different
aldehydes, acid chlorides, and acid anhydrides to give the target compounds. The structures of the new
compounds were characterized by spectral and elemental analyses. All compounds were tested for their
anti-inflammatory activity using the carrageenan-induced rat paw oedema model and exhibited weak to
good activities compared to ketorolac as the reference drug. Also, analgesic activity of selected
compounds, which are the most active in the anti-inflammatory screening, was measured using the
acetic acid-induced writhing model; revealing activities comparable to or higher than ketorolac. Ulcer
indices for the most active compounds were calculated and some compounds showed no or minimal
ulcerogenic effect compared to ketorolac.
Received 6 August 2009
Received in revised form
15 October 2009
Accepted 20 October 2009
Available online 25 October 2009
Keywords:
Pyrrolizines
Pyrimidopyrrolizines
Anti-inflammatory activity
Analgesic activity
Ulcerogenicity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
a continuous attempt to produce anti-inflammatory drugs that are
devoid of classical NSAID toxicity, especially gastrointestinal injury.
Inflammation is a normal and essential response to any noxious
stimulus, which threatens the host and may vary from a localized
response to a more generalized one [1]. The inflammatory process
is designed to provide a rapid mechanism by which the host can
respond to the invasion of foreign materials and return to
homeostatic equilibrium. Acute inflammation is mediated by the
release of autacoids such as histamine, serotonin, bradykinin,
prostaglandins and leukotrienes [2]. On the other hand, the chronic
inflammatory process involves the release of diverse mediators, as
Pyrrolizine ring constitutes a skeleton for many natural and
synthetic compounds of diverse biological activities [5–7]. Licofe-
lone (ML3000) 1 is an anti-inflammatory drug with potent activity
in various animal experimental models for acute and chronic
inflammation. It is a balanced inhibitor of 5-lipoxygenase (5-LOX)
and cyclooxygenase-1 and -2 (COX-1 and COX-2). In addition, it has
antiplatelet, and anti-bronchoconstrictive activity with excellent
gastrointestinal safety [8,9]. Ketorolac 2 is another important
pyrrolizine derivative that is a nonselective inhibitor of COX
enzymes that may play an important role in the treatment of sever
neuropathic pain (Fig.1) [10]. Other effective pyrrolizine derivatives
3 and 4 lacking the free carboxylic group characteristic for most
NSAIDs are reported (Fig. 1) [11].
interleukins, interferon and tumor necrosis factor
a (TNF-a),
a cytokine that plays a major role in this kind of inflammatory
process and whose production is associated with some inflamma-
tory diseases such as rheumatoid arthritis, Crohn’s disease
and others [2].
Non-steroidal anti-inflammatory drugs (NSAIDs), which are
widely used for reducing pain and swelling associated with
inflammation, represent a research area of continuous and ever-
growing development [3]. NSAIDs cause several serious adverse
effects; the most important one is gastric injury that might later
cause gastric ulceration and renal injury [4]. Therefore, there is
Prompted by these findings, and aiming to discover novel and
potent anti-inflammatory agents that might be devoid of the
gastrointestinal side effects, the pyrrolizine ring, and specifically,
the
N-(4-(un)substitutedphenyl)-2,3-dihydro-1H-pyrrolizine-5-
carboxamide moiety was used as the core heterocyclic scaffold of
our target compounds (Fig. 2). The design concept of this scaffold
lies on its structural similarity to ketorolac 2, both having a pyrro-
lizine ring system bearing an out-of-plane phenyl ring at position 5.
Our target compounds differ in that the phenyl ring at position 5 is
attached to the pyrrolizine nucleus through an amide link and in
that they lack the carboxyl group at position1. Structure extension
* Corresponding author. Tel.: þ20 2 26070436.
E-mail address: fadi_mae@hotmail.com (F.M. Awadallah).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.031

S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
483
with the appropriate aldehydes in absolute ethanol in the presence
of glacial acetic acid gave the benzylidine compounds 10a–f, and
their reaction with the appropriate acid chlorides, usually prepared
in situ via the action of thionyl chloride on the corresponding acids,
furnished the amide derivatives 11a–f.
HOOC
N
According to Scheme 2, the reaction of 9a–c with phthalic
anhydride in glacial acetic acid yielded, unexpectedly, two products
as revealed by the TLC of the reaction mixture. Removal of the
reaction solvent under vacuum left out the residue containing the
two products which were successfully separated by fractional
crystallization from acetone whereby the pyrrolizinopyr-
imidoisoindoles 12a–c were obtained from the acetone-insoluble
fraction and the 6-isoindolyl derivatives 13a–c were isolated from
the acetone-soluble fraction, as later confirmed by the spectral and
elemental analyses. The major products are 13a–c. The main clear
difference between the two products was in their IR spectra which
revealed the presence of a cyano absorption band in the range
2226–2222 cmꢀ1 for compounds 13a–c and the absence of this
band in the IR spectra of compounds 12a–c.
The target compounds 14a–c and 15a–c were obtained from the
reaction of compounds 9a–c with maleic anhydride and succinic
anhydride, respectively. The synthesized compounds were charac-
terized by spectral and elemental analyses. Evidence for cyclization
and formation of the fused pyrimidinone ring was drawn from IR
spectra that showed OH carboxylic absorption band at about
2550 cmꢀ1 together with the disappearance of the characteristic
cyano band. Also, 1H NMR spectra showed 3 exchangeable protons
in the range of 9.60–11.70 ppm assigned for 2NH protons and one
COOH proton.
Cl
O
N
COOH
R2
2
1
R2
R1
N
N
Y
R1
3
4
R1= H, Cl, CH3, SO2CH3, SCH3
R2= H, Cl, CH3
Y= CH2, S, O
Fig. 1. Examples of pyrrolizine compounds with anti-inflammatory activity.
has been made through introduction of additional substitutions at
positions 6 and 7 of the pyrrolizine ring, which have been modified
to evolve our target compounds (Fig. 2). Accordingly, the designed
compounds fall into two main groups, the first includes the
The proposed mechanism for the formation of products 12–
15a–c is demonstrated in Figs. 3 and 4 [12,13].
substituted pyrrolizine derivatives having in the
6 position
different benzylidineamino 10a–f, phenylpropionylamino 11a–f
and isoindolyl groups 13a–c (general formula A). The amide
derivatives 11a–c and 11d–f can be viewed as hybrid molecules
between our pyrrolizine scaffold, and ibuprofen and ketoprofen
structures, respectively, hoping to have synergistic action. The
second group of compounds comprises the fused pyrrolizine
derivatives, (general formula B) exemplified by the pyrrolizino-
pyrimidoisoindole compounds 12a–c, which can be viewed as the
restrained analogues of compounds 13a–c, and the pyr-
imidopyrrolizines 14a–c and 15a–c.
2.2. Pharmacological screening
2.2.1. Anti-inflammatory activity
Evaluation of anti-inflammatory activity of the synthesized
compounds was performed using the carrageenan-induced rat paw
oedema model using ketorolac as the reference drug [14]. Mean
changes in paw oedema thickness of animals pretreated with the
tested compounds after 1, 2, 3, and 4 h. from induction of inflam-
mation was measured and shown in Table 1 together with the
inhibition percent of oedema by the tested compounds.
The 6-amino-7-cyano-N-(un)substitutedphenyl-5-carboxamides
9a–f showed moderate activities compared to ketorolac.
Regarding the effect of the electronic nature of the para-sub-
stituent on the activity, results revealed that compounds with
electron withdrawing groups (Cl, NO2, COOH) 9c, 9d and 9f
exhibited higher activities than the unsubstituted compound 9a
which in turn possessed higher activity than compounds with
electron donating groups (CH3, OH) 9b and 9e. This relation
between the electronic nature of the substituent and activity
holds true in most of the other series.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, compounds 9a–f were prepared from the
reaction of 2-pyrrolidin-2-ylidinemalononitrile 8 with the corre-
sponding a-chloroacetanilides 6a–f. Compounds 9a–c served as the
starting materials for other target compounds, where condensation
The benzylidene amino derivatives 10a–f exhibited weak to
moderate activities relative to ketorolac. Within this series, it could
be observed that the nature and position of the substituent on the
benzylidene ring had minor effect on activity.
As for the amide derivatives 11a–f, which can be viewed as
hybrid molecules bearing the ibuprofen and ketoprofen moieties, it
is noteworthy that they showed activities slightly lower than that of
ketorolac but having the advantage of being less ulcerogenic as will
be shown later.
CN
R
N
N
O
O
N
H
N
H
Interestingly, activities of the 6-isoindolyl derivatives 13a–c
were much lower than that of ketorolac while their restrained
analogues 12a–c were much higher. Another important
A
B
Fig. 2. General structures of the target compounds.

484
S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
R1
NH2
O
N
H
7
5a-f
CN
a
b
NH2
N
H
N
c
CN
CN
R1
R1
+
N
H
O
O
N
H
8
6a-f
Cl
9a-f a R1=H
a R1=H
b R1=CH3
b R1=CH3
e
c R1=Cl
c R1=Cl
d R1= NO2
d R1= NO2
d
e R1= OH
e R1= OH
f R1= COOH
f R1= COOH
R2
CN
O
CH3
CN
R3
N
H
N
N
N
R1
R1
O
N
H
11a-f
O
N
H
R2
10a-f
a R1=H
b R1= CH3 R2=H
c R1= Cl R2=H
d R1= H R2=OCH3 R3= OH
e R1=CH3 R2=OCH3 R3= OH
f R1= Cl R2=OCH3 R3= OH
R2=H
R3= OCH3
R3= OCH3
R3= OCH3
a R1=H
R2= CH2-CH(CH3)2
b R1=CH3 R2= CH2-CH(CH3)2
c R1=Cl
d R1=H
e R1=CH3 R2=COC6H5
f R1=Cl R2=COC6H5
R2= CH2-CH(CH3)2
R2=COC6H5
Scheme 1. Reagents and solvents: a: ClCH2COCl, glacial acetic acid, sodium acetate; b: dimethylsulfate in benzene then malononitrile; c: dry acetone, anhydrous K2CO3; d: ArCHO,
ethanol, glacial acetic acid; e: ArCOCl, dry benzene.
O
CN
CN
O
N
N
a
NH2
N
N
N
+
N
R
R
O
O
R
O
O
O
N
H
N
H
N
H
12a-c
a R=H
b R=CH3
c R=Cl
13a-c
9a-c
a R=H
b R=CH3
c R=Cl
b
O
N
N
H
N
HOOC
c
R
O
N
H
14a-c
a R=H
b R=CH3
c R=Cl
O
N
COOH
N
H
N
R
O
N
H
a R=H
b R=CH3
15a-c
c R=Cl
Scheme 2. Reagents and solvents: a: phthalic anhydride, glacial acetic acid; b: maleic anhydride glacial acetic acid; c: succinic anhydride, glacial acetic acid.

S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
CN
485
O
CN
O
CN
O
NH2
O
N
N
H
-H2O
R1
N
N
R1
O
N
O
HOOC
R1
N
H
O
O
N
H
O
9a-c
N
H
13a-c
CN
OH
HN
O
N
N
N
N
HOOC
R1
HOOC
O
R1
N
H
O
N
H
rearrangement
O
HN
O
-
N
-
O
NH
N
H
+
N
+
N
N
N
N
COOH
COOH
O
COOH
R1
R1
R1
O
O
N
H
N
N
H
H
-H2O
O
N
N
N
R1
O
O
N
H
12a-c
Fig. 3. The proposed reaction mechanism of the formation of compounds 12a–c and 13a–c.
O
CN
CN
O
CN HO
N
O
O
NH2
N
H
N
N
N
R1
HOOC
HOOC
O
R1
R1
O
N
H
O
N
H
N
H
9a-c
rearrangement
O
N
N
H
N
HOOC
R1
O
N
H
14a-c, 15a-c
Fig. 4. The proposed reaction mechanism of the formation of compounds 14a–c and 15a–c.

486
S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
Table 1
Oedema thickness and inhibition percent of control, ketorolac and tested compounds.
Comp.
Oedema thickness(ꢁ10ꢀ2 mm) ꢂ SEM (Inhibition%)
1h
2h
3h
4h
Control
Ketorolac
9a
9b
9c
9d
9e
9f
140.8 ꢂ 1.39
155.6 ꢂ 2.80
167.4 ꢂ 2.09
195.6 ꢂ 3.08
41.6 ꢂ 2.02 (70.46)
112.6 ꢂ 1.03 (20.03)
121.2 ꢂ 2.27 (13.92)
103.6 ꢂ 2.25 (26.42)
108.4 ꢂ 2.68 (23.01)
121.8 ꢂ 1.02 (13.49)
100.2 ꢂ 1.86 (28.84)
117.6 ꢂ 1.72 (16.48)
109.2 ꢂ 2.65 (22.44)
96.2 ꢂ 2.08 (31.68)
99.2 ꢂ 1.56 (29.55)
112.6 ꢂ 2.93 (20.03)
91.6 ꢂ 1.91 (34.94)
98.8 ꢂ 3.07 (29.83)
89.8 ꢂ 2.22 (36.22)
85.6 ꢂ 2.18 (39.20)
114.6 ꢂ 1.63 (18.61)
103.2 ꢂ 2.78 (26.71)
108.6 ꢂ 3.61 (22.87)
57.6 ꢂ 1.91 (59.09)
64.8 ꢂ 1.66 (53.98)
54.4 ꢂ 1.69 (61.36)
119.6 ꢂ 1.33 (15.06)
129.2 ꢂ 2.75 (8.24)
118.2 ꢂ 2.54 (16.05)
64.6 ꢂ 1.96 (54.12)
68.6 ꢂ 2.2 (51.28)
61.2 ꢂ 1.59 (56.53)
76.2 ꢂ 1.07 (45.88)
82.2 ꢂ 2.08 (41.62)
71.6 ꢂ 2.14 (49.15)
40.2 ꢂ 1.62 (74.17)
116.2 ꢂ 0.86 (25.32)
123.6 ꢂ 1.6 (20.57)
101.0 ꢂ 2.17 (35.09)
113.8 ꢂ 2.58 (26.86)
128.4 ꢂ 1.72 (17.48)
102.6 ꢂ 2.32 (34.06)
112.4 ꢂ 2.44 (27.76)
108.4 ꢂ 3.16 (30.33)
94.4 ꢂ 2.27 (39.33)
102.8 ꢂ 2.3 (34.32)
116.2 ꢂ 2.06 (25.32)
90.6 ꢂ 2.62 (41.77)
99.6 ꢂ 3.75 (35.99)
88.2 ꢂ 3.65 (43.32)
87.8 ꢂ 2.63 (43.57)
108.4 ꢂ 3.39 (30.33)
104.4 ꢂ 3.92 (32.91)
103.4 ꢂ 2.77 (33.56)
70.6 ꢂ 2.29 (54.63)
73.6 ꢂ 2.11 (52.70)
62.4 ꢂ 2.23 (59.90)
130.4 ꢂ 2.36 (16.20)
139.4 ꢂ 2.71 (10.41)
127.8 ꢂ 2.92 (17.87)
68.4 ꢂ 2.06 (56.04)
71.6 ꢂ 2.42 (53.98)
63.6 ꢂ 2.32 (59.13)
76.4 ꢂ 3.04 (50.90)
78.8 ꢂ 2.63 (49.36)
66.4 ꢂ 2.64 (57.33)
41.2 ꢂ 2.06 (75.39)
112.8 ꢂ 1.59 (32.62)
124.4 ꢂ 1.36 (25.69)
101.4 ꢂ 2.86 (39.43)
107.2 ꢂ 3.22 (31.11)
135.8 ꢂ 2.04 (18.88)
93.4 ꢂ 2.16 (44.21)
113.2 ꢂ 2.85 (32.38)
109.4 ꢂ 3.14 (34.65)
95.2 ꢂ 2.42 (43.13)
106.4 ꢂ 1.86 (36.44)
117.6 ꢂ 2.4 (29.75)
90.2 ꢂ 2.42 (46.12)
75.2 ꢂ 2.01 (55.08)
72.6 ꢂ 2.73 (56.63)
66.2 ꢂ 1.77 (60.45)
100.6 ꢂ 1.47 (39.91)
92.4 ꢂ 2.84 (44.80)
93.4 ꢂ 2.25 (44.21)
85.2 ꢂ 2.06 (49.10)
83.6 ꢂ 2.11 (50.06)
80.4 ꢂ 1.69 (51.7)
131.8 ꢂ 2.13 (21.27)
135.8 ꢂ 1.56 (18.88)
130.4 ꢂ 1.96 (22.10)
70.4 ꢂ 2.14 (57.95)
76.2 ꢂ 2.62 (54.48)
61.8 ꢂ 1.66 (63.08)
76.8 ꢂ 2.31 (54.12)
80.4 ꢂ 2.04 (51.97)
66.8 ꢂ 2.18 (60.10)
40.4 ꢂ 1.54 (79.35)
121.2 ꢂ 1.66 (38.04)
130.2 ꢂ 1.16 (33.44)
111.0 ꢂ 1.76 (43.25)
116.6 ꢂ 3.08 (40.39)
149.2 ꢂ 2.44 (23.72)
100.6 ꢂ 3.06 (48.57)
126.6 ꢂ 2.23 (35.28)
110.6 ꢂ 2.25 (43.46)
97.8 ꢂ 3.01 (50.00)
122.4 ꢂ 1.44 (37.42)
128.0 ꢂ 2.97 (34.56)
96.6 ꢂ 4.37 (50.61)
80.8 ꢂ 1.66 (58.69)
75.4 ꢂ 1.96 (61.45)
71.2 ꢂ 1.77 (63.60)
92.8 ꢂ 2.42 (52.56)
86.4 ꢂ 2.2 (55.83)
92.4 ꢂ 2.4 (52.76)
106 ꢂ 2.88 (45.81)
109.8 ꢂ 2.18 (43.87)
101.4 ꢂ 1.63 (48.16)
152.2 ꢂ 3.02 (22.19)
159.8 ꢂ 3.22 (18.30)
146.8 ꢂ 2.08 (24.95)
65.2 ꢂ 2.62 (66.67)
73.6 ꢂ 3.01 (62.37)
59.4 ꢂ 2.62 (69.63)
75.4 ꢂ 2.38 (61.45)
86.2 ꢂ 2.44 (55.93)
73.6 ꢂ 2.48 (62.37)
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
12a
12b
12c
13a
13b
13c
14a
14b
14c
15a
15b
15c
Data analyzed by one way ANOVA, (n ¼ 5), all compounds were significantly different from control and from ketorolac using Student’s t-test, P < 0.05.
observation is the unexpected decline in activity of compounds
12a–c with time deviating from all other screened compounds.
On the other hand, the pyrimidopyrrolizine compounds 14a–c
and 15a–c exhibited anti-inflammatory activities comparable to
that of ketorolac. The double bond in the side chain of compounds
14a–c, which may confer some restriction in the free rotation of the
carboxyl group, had no effect on the activities of compounds 14a–c
compared to their saturated derivatives 15a–c.
using the acetic acid-induced writhing test in mice [15–18] using
ketorolac as a reference drug. All tested compounds, except 15c,
possessed good analgesic activity comparable to or higher than
ketorolac. The most active compound was 15a. (Table 2)
2.2.3. Acute ulcerogenicity study
Ulcerogenic effect of derivatives 11a–f, 14a–c and 15a–c with
best overall profile in animal efficacy model was evaluated for
gastric ulcerogenic potential in rats (Table 3) [19,20]. When
compared to ketorolac, compounds 11a, 11c and 11d did not cause
any gastric ulceration at the same dose level while compounds 11b,
11e and 11f exhibited only little gastric ulceration; about 30% that
of ketorolac. On the other hand, compounds 14a–c and 15a–c
showed higher gastric ulceration than ketorolac. These results
In conclusion, it could be seen that the most active compounds
were 11a–f, 14a–c and 15a–c possessing, in average, 75% the
activity of ketorolac.
2.2.2. Analgesic activity
The most potent drugs in the anti-inflammatory screening 11a–
c,e, 14a–c and 15a,c were further tested for their analgesic activity
Table 2
Table 3
Effects of the tested compounds on acetic acid-induced abdominal writhing in mice.
Ulcer index of the most active compounds.
Writhing reflex ꢂ SEM
Inhibition%
%Incidence/10 Average no of ulcer Average severity Ulcer index
Control
Ketorolac
11a
11b
11c
11e
14a
14b
14c
76.83 ꢂ 1.08a
4.60 ꢂ 0.50
3.00 ꢂ 0.95
3.40 ꢂ 0.51
2.20 ꢂ 0.86a
6.60 ꢂ 0.98
3.80 ꢂ 0.58
5.00 ꢂ 0.71
1.80 ꢂ 0.37a
0.40 ꢂ 0.24a
10.00 ꢂ 0.71a
–
Control
Ketorolac
11a
11b
11c
11d
11e
11f
14a
14b
14c
15a
15b
0
8
0
2
0
0
2
2
10
10
10
8
10
8
0
1.6
0
0.2
0
0
0.6
0.6
5.8
6.8
5.2
7
0
2
0
1
0
0
1
1
1.66
1.5
1.89
1.66
1.71
1.86
0
11.6
0
3.2
0
0
3.6
3.6
17.46
18.3
17.09
16.66
17.91
15.66
94.01
96.09
95.57
97.13
91.40
95.05
93.49
97.65
99.47
86.98
15a
15c
Data analyzed by one way ANOVA, (n ¼ 5), all compounds were significantly
6.2
5.8
different from control.
15c
a
Statistically significant from ketorolac using Student’s t-test, P < 0.05.

S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
487
revealed the advantageously high gastric tolerance to compounds
11a–f compared to ketorolac.
4.1.1.2. 6-Amino-7-cyano-N-(4-hydroxyphenyl)-2,3-dihydro-1H-pyr-
rolizine-5-carboxamide (9e). White crystals, m.p. 187–188 ꢃC, yield
51%. IR ymax/cmꢀ1 3391 (OH), 3266 (NHs), 3071 (CH aromatic), 2925
(CH aliphatic), 2218 (CN), 1671 (COs), 1606 (NH), 1545–1512
3. Conclusions
(C]C).1H NMR (DMSO-d6-400 MHz):
d 2.51 (m, 2H, CH2-2), 2.89 (t,
2H, J ¼ 7.5 Hz, CH2-1), 4.22 (t, 2H, J ¼ 7.2 Hz, CH2-3), 4.62 (s, 2H,
NH2), 5.47 (s, 1H, OH), 6.85–7.58 (m, 4H, aromatic protons), 10.01 (s,
1H, NH). MS: m/z(%): 282 (Mþ, 37), 250 (10), 174 (100), 146 (57), 147
(63), 119 (55), 92 (27), 66 (62). Anal. Calcd. for C15H14N4O2 (282.30):
C, 63.82; H, 5.00; N, 19.85. Found: C, 64.10; H, 4.87; N, 20.16.
Various substituted pyrrolizines and pyrimido-fused pyrroli-
zines were synthesized and screened for anti-inflammatory and
ulcerogenic potential. Hybrid compounds 11a–f, which bear the
ibuprofen and ketoprofen moieties, and the pyrimido-fused
pyrrolzine derivatives 14a–c and 15a–c, exhibited the highest anti-
inflammatory activity. Despite their slightly lower activity,
compared to ketorolac, compounds 11a–f achieved the advantage
of being much less ulcerogenic as revealed from the results of acute
ulcerogenicity studies. Furthermore, analgesic activity performed
on selected compounds with the higher anti-inflammatory profile
showed promising analgesic activity. Combining the analgesic
activity with the ulcerogenic potential of the tested compounds, it
could be revealed that the most active and, at the same time, safest
compound was 11c.
4.1.1.3. 4-[(6-Amino-7-cyano-2,3-dihydro-1H-pyrrolizine-5-carbon-
yl)amino]benzoic acid (9f). White crystals, m.p. 245–248 ꢃC, yield
59%. IR ymax/cmꢀ1 3347 (OH), 3285 (NHs), 3018 (CH aromatic), 2968
(CH aliphatic), 2848–2561 (broad band OH) 2224 (CN), 1685 (CO),
1604 (NH), 1535 (C]C). 1H NMR (DMSO-d6-400 MHz):
d 2.40 (m,
2H, CH2-2), 2.89 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.22 (t, 2H, J ¼ 7.2 Hz,
CH2-3), 4.30 (s, 2H, NH2), 7.71–7.92 (m, 4H, aromatic protons), 9.50
and 10.65 (2 s, 2H, NH and COOH). MS: m/z(%): 310 (Mþ, 32), 281
(4), 252 (4), 214 (7.2), 196 (2.5), 174 (100), 146 (44), 119 (32), 92 (33),
65 (59). Anal. Calcd. for C16H14N4O3 (310.31): C, 61.93; H, 4.55; N,
18.06. Found: C, 61.70; H, 4.49; N, 18.08.
4. Experimental protocols
4.1.2. General method for preparation of compounds (10a–f)
A mixture of the carboxamide 9a–c (3.75 mmol), the appro-
priate aldehyde (3.75 mmol) and glacial acetic acid (0.5 ml) in
absolute ethanol (20 ml) was refluxed for 4 h. The reaction mixture
was concentrated, set aside to cool, whereby crystals were formed,
collected and recrystallized from ethanol.
4.1. Chemistry
Melting points were uncorrected and were carried out by open
capillary tube method using IA 9100MK-Digital Melting Point
Apparatus. Microanalyses were carried out at the microanalytical
Center, Faculty of Science, Cairo University. Infrared spectra were
made on Bruker Vector 22 (Japan) infrared spectrophotometers and
were expressed in wavenumber (cmꢀ1) using potassium bromide
disc. The proton magnetic resonance 1H NMR spectra were recor-
ded on a Varian Mercury VX-300 NMR spectrometer at 300 MHz
and Bruker APX400 spectrometer at 400 MHz in the specified
4.1.2.1. (EZ) 7-Cyano-6-[(4-methoxybenzylidene)amino]-N-phenyl-
2,3-dihydro-1H-pyrrolizine-5-carboxamide (10a). Yellow crystals,
m.p. 203–205 ꢃC, yield 84%. IR ymax/cmꢀ1 3229 (NH), 3056, 3030
(CH aromatic) 2979–2947 (CH aliphatic), 2206 (CN),1674 (CO),1598
(NH), 1543 (C]C). 1H NMR (CDCl3-300 MHz):
d 2.56 (m, 2H, CH2-2),
solvent. Chemical shifts were reported on the
d
scale and were
3.04 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.92 (s, 3H, OCH3), 4.54 (t, 2H,
J ¼ 7.2 Hz, CH2-3), 7.03–7.39 (m, 5H, 5 aromatic protons), 7.66 and
7.90 (2 d, 4H, J ¼ 8.7 Hz, para-substituted aromatic H), 9.10 (s, 1H,
N]CH) and 10.70 (s, 1H, NH, which disappeared on deuteration).
MS: m/z(%): 384 (Mþ, 48), 355 (6), 293 (24), 292 (100), 277 (14), 264
(7), 249 (11), 135 (24). C23H20N4O2 (384.43): C, 71.86; H, 5.24; N,
14.57. Found: C, 71.80; H, 4.83; N, 14.40.
related to that of the solvent and J values are given in Hz. 13C NMR
spectra were obtained on a Bruker APX400 at 100 MHz. Mass
spectra were recorded on Fennigan MAT, SSQ 7000, Mass spec-
trometer, at 70 eV (EI) and Waters Micromass Q-Tof Micro mass
spectrometer (ESI). All mass spectra were recorded in EI mode
unless otherwise stated. Compounds 6a [21], 6b, 6c [22], 6d [23,24],
6e [25], 6f [26], 8 [27] and 9a–c [28] were prepared according to the
reported procedures.
4.1.2.2. (EZ) 7-Cyano-6-[(4-methoxybenzylidene)amino]-N-(4-tolyl)-
2,3-dihydro-1H-pyrrolizine-5-carboxamide (10b). Yellow crystals,
m.p. 209–211 ꢃC, yield 87%. IR ymax/cmꢀ1 3219 (NH), 3015 (CH
aromatic), 2958–2918 (CH aliphatic), 2213 (CN), 1663 (CO), 1595
4.1.1. General method for preparation of compounds (9d–f)
A
mixture of 2-pyrrolidin-2-ylidenemalononitrile 8 (1 g,
7.5 mmol), the appropriate acetanilide 6d–f (7.5 mmol) and anhy-
drous potassium carbonate (1.04 g, 7.5 mmol) in dry acetone
(50 ml) was stirred under reflux for 24 h. The mixture was filtered,
concentrated and left to cool, whereby crystals were formed,
collected, dried and recrystallized from ethanol–acetone.
(NH), 1542 (C]C). 1H NMR (CDCl3-300 MHz):
d 2.34 (s, 3H, CH3),
2.55 (m, 2H, CH2-2), 3.04 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.92 (s, 3H,
OCH3), 4.52 (t, 2H, J ¼ 7.2 Hz, CH2-3), 7.04, 7.95 (2d, 4H, J ¼ 8.7 Hz,
para-substituted aromatic H), 7.17 and 7.57 (2d, 4H, J ¼ 8.4 Hz, para-
substituted aromatic H), 9.03 (s, 1H, N]CH) and 10.69 (s, 1H, NH,
which disappeared on deuteration). Anal. Calcd. for C24H22N4O2
(398.46): C, 72.34; H, 5.57; N, 14.06. Found: C, 72.42; H, 5.26; N,
14.16.
4.1.1.1. 6-Amino-7-cyano-N-(4-nitrophenyl)-2,3-dihydro-1H-pyrroli-
zine-5-carboxamide(9d). Yellow crystals, m.p. 235–237 ꢃC, yield
55%. IR ymax/cmꢀ1 3360, 3290 (NHs), 3074, 3049 (CH aromatic)
2993–2847 (CH aliphatic), 2220 (CN), 1673 (CO), 1602 (NH), 1559
4.1.2.3. (EZ) N-(4-Chlorophenyl)-7-cyano-6-[(4-methoxybenzylidene
amino)]-2,3-dihydro-1H-pyrrolizine-5-carboxamide (10c). Yellow
crystals, m.p. 213–216 ꢃC, yield 81%. IR ymax/cmꢀ1 3224 (NH), 3060
(CH aromatic) 2981–2939 (CH aliphatic), 2211 (CN),1665 (CO),1597
(C]C). 1H NMR (DMSO-d6-400 MHz):
d
2.50 (m, 2H, CH2-2), 2.99 (t,
2H, J ¼ 7.5 Hz, CH2-1), 4.30 (t, 2H, J ¼ 7.2 Hz, CH2-3), 4.78 (s, 2H,
NH2), 7.88–8.37 (m, 4H, aromatic protons), and 9.80 (s, 1H, NH). 13
NMR (DMSO-d6): 19.69 (C-1), 36.39 (C-2), 50.62 (C-3), 78.28 (C-7),
C
d
(NH), 1542 (C]C). 1H NMR (CDCl3-300 MHz):
d 2.54 (m, 2H, CH2-2),
116.28 (CN), 118.06 (C-6), 119.77 (C-20, -60), 124.07 (C-5), 129.28
(C-30, -50), 139.06 (C-7a), 146.28 (C-10), 164.55 (C-40), 172.53 (CO).
Anal. Calcd. for C15H13N5O3 (311.30): C, 57.87; H, 4.21; N, 22.50.
Found: C, 57.63; H, 4.01; N, 22.99.
3.01 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.91 (s, 3H, OCH3), 4.48 (t, 2H,
J ¼ 7.2 Hz, CH2-3), 7.02, 7.87 (2d, 4H, J ¼ 7.5, aromatic H), 7.29 (d, 2H,
J ¼ 6.9 Hz, aromatic H), 7.59 (d, 2H, J ¼ 6.9 Hz, aromatic H), 9.02 (s,
1H, N]CH) and 10.75 (s, 1H, NH, which disappears on deuteration).

488
S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
C23H19ClN4O2 (418.88): C, 63.52; H, 4.40; N, 12.88. Found: C, 63.81;
H, 4.29; N, 12.69.
9.33 (s, 1H, NHCOCH, which disappeared on deuteration) and 10.22
(s, 1H, CONH–Ph, which disappeared on deuteration). MS (ESI): m/
z: 454 (Mþ), 453 (M-1), 389, 274, 227, 174. Anal. Calcd. for
C28H30N4O2 (454.56): C, 73.98; H, 6.65; N, 12.33. Found: C, 73.71; H,
6.27; N, 12.67.
4.1.2.4. (EZ) 7-Cyano-6-[(4-hydroxy-3-methoxybenzylidene)amino]-
N-phenyl-2,3-dihydro-1H-pyrrolizine-5-carboxamide (10d). Yellow
crystals, m.p. 233–236 ꢃC, yield 84%. IR ymax/cmꢀ1 3444 (OH), 3234
(NH), 3069, 3040 (CH aromatic) 2995–2843 (CH aliphatic), 2211
(CN), 1672 (CO), 1595 (NH), 1512 (C]C). 1H NMR (CDCl3-300 MHz):
4.1.3.2. (RS) 7-Cyano-6-[2-(4-isobutylphenyl)propionylamino]-N-(4-
tolyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(11b). White
d
2.55 (m, 2H, CH2-2), 3.03 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.98 (s, 3H,
crystals, m.p. 195–197 ꢃC, yield 57%. IR ymax/cmꢀ1 3281, 3203 (NHs),
3074, 3017 (CH aromatic), 2954–2866 (CH aliphatic), 2214 (CN),
1703, 1663 (COs), 1593 (NH), 1540–1514 (C]C). 1H NMR (DMSO-d6-
OCH3), 4.52 (t, 2H, J ¼ 7.2 Hz, CH2-3), 6.16 (s, 1H, OH, which dis-
appeared on deuteration), 7.03–7.67 (m, 8H, aromatic H), 9.05 (s,
1H, N]CH) and 10.71 (s, 1H, NH, which disappeared on deutera-
400 MHz):
d
0.82 (d, 6H, J ¼ 6.3 Hz, (CH3)2CH), 1.44 (d, 3H,
tion). 13C NMR (DMSO-d6):
d
24.19 (C-1), 25.16 (C-2), 50.18 (C-3),
J ¼ 7.2 Hz, CH3CHCO), 1.76 (m, 1H, (CH3)2CH), 2.25 (s, 3H, CH3 tolyl),
2.36–2.51 (m, 4H, CH2-2, CH2CH), 2.97 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.92
(q,1H, J ¼ 7.2 Hz, CHCO), 4.25 (t, 2H, J ¼ 7.2 Hz, CH2-3), 7.01–7.71 (m,
8H, aromatic proton), 9.25 (s, H, NHCOCH, which disappeared on
deuteration) and 10.17 (s, H, CONH–Ph, which disappeared on
55.88 (CH3), 76.72 (C-7), 100.00 (CN), 109.71 (C-6), 116.00 (C-200),
116.31 (C-500), 119..21 (C-20, -60), 123.88 (C-600), 125.63 (C-40), 126.80
(C-100), 129.35 (C-30, -50), 138.64 (C-5), 140.03 (C-10), 148.75 (C-7a),
148.87 (C-400) 152.06 (C-300), 157.92 (CH]N), 160.00 (CO). MS m/
z(%): 401 (Mþ þ 1, 51), 400 (Mþ, 62), 308 (100), 293 (9), 277 (23),
250 (46), 184 (6), 157 (7). Anal. Calcd. for C23H20N4O3 (400.43):
C,68.99; H, 5.03; N, 13.99. Found: C, 68.95; H, 4.89; N, 13.80.
deuteration). 13C-NMR (DMSO-d6):
d 18.45 (CH3CHCO), 20.38 (C-1),
22.10 ((CH3)2CH), 24.27 (CH3–Ph), 29.47 (C-2), 35.90 (CH(CH3)2),
44.39(CHCO), 49.21(C-3), 58.99 (CH2CH), 84.14 (C-7), 114.33 (CN),
119.32 (C-20, -60), 120.42 (C-5), 126.97 (C-2", -6"), 128.89 (C-3", -5"),
129.11 (C-30, -50), 132.53 (C-1"), 135.54 (C-40), 138.02 (C-7a), 145.75
(C-10), 156.97 (C-6), 163.74 (C-4"), 171.98 (CONHPh), 174.71 (COCH).
Anal. Calcd. for C29H32N4O2 (468.59): C, 74.33; H, 6.88; N, 11.96.
Found: C, 74.33; H, 7.06; N, 11.99.
4.1.2.5. (EZ) 7-Cyano-6-[(4-hydroxy-3-methoxybenzylidene)amino]-
N-(4-tolyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(10e). Yellow crystals, m.p. 245–246 ꢃC, yield 89%. IR ymax/cmꢀ1
3324 (OH), 3224 (NH), 3050 (CH aromatic), 3000–2854 (CH
aliphatic), 2217 (CN), 1661 (CO), 1599 (NH), 1545–1516 (C]C). 1H
NMR (CDCl3-300 MHz):
d
2.34 (s, 3H, CH3), 2.56 (m, 2H, CH2-2),
4.1.3.3. (RS)
N-(4-Chlorophenyl)-7-cyano-6-[2-(4-isobutylphenyl)
3.05 (t, 2H, J ¼ 7.5 Hz, CH2-7), 4.01 (s, 3H, OCH3), 4.53 (t, 2H,
J ¼ 7.2 Hz, CH2-3), 6.17 (s, H, OH, which disappeared on deutera-
tion), 7.04–7.61 (m, 7H, aromatic protons), 9.00 (s, H, N]CH) and
10.69 (s, H, NH, which disappeared on deuteration). Anal. Calcd. for
C24H22N4O3 (414.46): C, 69.55; H, 5.35; N, 13.52. Found: C, 69.63; H,
5.31; N, 13.68.
propionylamino]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(11c). White crystals, m.p. 205–207 ꢃC, yield 55%. IR ymax/cmꢀ1
3404, 3260 (NHs), 3096, 3049, 3011 (CH aromatic), 2953–2869 (CH
aliphatic), 2222 (CN), 1663 (COs), 1597 (NH), 1539–1519 (C]C). 1H
NMR (DMSO-d6-300 MHz):
d
0.81 (d, 6H, J ¼ 6.3, (CH3)2CH), 1.44 (d,
3H, J ¼ 7.2, CH3CHCO), 1.74 (m, 1H, (CH3)2CH), 2.34 (d, 2H, J ¼ 6.9,
CHCH2), 2.50 (m, 2H, CH2-2), 2.98 (t, 2H, J ¼ 7.5, CH2-1), 3.87 (q, 1H,
J ¼ 7.2 Hz, CHCO), 4.24 (t, 2H, J ¼ 7.2, CH2-3), 6.98–7.39 (m, 8H,
aromatic protons), 9.45 (s, 1H, NHCOCH, which disappeared on
deuteration) and 10.13 (s, 1H, CONH–Ph, which disappeared on
deuteration) which disappeared on deuteration. Anal. Calcd. for
C28H29ClN4O2 (489.01): C, 68.77; H, 5.98; N, 11.46. Found: C, 68.93;
H, 5.72; N, 11.22.
4.1.2.6. (EZ) N-(4-Chlorophenyl)-7-cyano-6-[(4-hydroxy-3-methox-
ybenzylidene)amino]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(10f). Yellow crystals, m.p. 255–258 ꢃC, yield 84%. IR ymax/cmꢀ1
3318 (OH), 3232 (NH), 3067 (CH aromatic) 2943–2846 (CH
aliphatic), 2210 (CN), 1669 (CO), 1613 (NH), 1583–1546 (C]C). 1H
NMR (CDCl3-300 MHz):
d
2.58 (m, 2H, CH2-2), 3.07 (t, 2H, J ¼ 7.5 Hz,
CH2-1), 4.00 (s, 3H, OCH3), 4.53 (t, 2H, J ¼ 7.2 Hz, CH2-3), 6.09 (s, H,
OH, which disappeared on deuteration), 7.06–7.64 (m, 7H, aromatic
protons), 9.06 (s, H, N]CH) and 10.76 (s, H, NH, disappeared on
deuteration). Anal. Calcd. for C23H19ClN4O3 (434.87): C, 63.52; H,
4.40; N, 12.88. Found: C, 63.81; H, 4.29; N, 12.69.
4.1.3.4. (RS)
6-[2-(3-Benzoylphenyl)propionylamino]-7-cyano-N-
phenyl-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(11d). White
crystals, m.p. 192–194 ꢃC, yield 64%. IR ymax/cmꢀ1 3207, 3135
(NHs), 3031 (CH aromatic) 2998–2878 (CH aliphatic), 2220 (CN),
1662 (COs), 1602 (NH), 1560 (C]C). 1H NMR (DMSO-d6-
4.1.3. General method for preparation of compounds (11a–f)
The corresponding acid (3.75 mmol) and thionyl chloride (1 ml)
were heated on water bath for one hour, the excess thionyl chloride
was distilled under vacuum and the residue was dissolved in 20 ml
dry benzene. The appropriate compound 9a–c (3.75 mmol) was
added and the reaction mixture was stirred for one hour then left to
stand for 48 h at room temperature. The precipitate formed was
filtered, washed with water and hot ethanol, and recrystallized
from ethanol-acetone.
300 MHz):
d
1.53 (d, 3H, J ¼ 6.6 Hz, CH3CHCO), 2.50 (m, 2H, CH2-
2), 3.03 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.13 (q, 1H, J ¼ 6.6 Hz, CHCO),
4.31 (t, 2H, J ¼ 6.6 Hz, CH2-3), 7.08–7.84 (m, 14H, aromatic
protons), 9.26 (s, 1H, NHCOCH, which disappeared on deutera-
tion) and 10.24 (s, 1H, CONH–Ph, which disappeared on deuter-
ation). MS (ESI): m/z: 501 (Mþ ꢀ 1), 412, 267, 240, 159. Anal.
Calcd. for C31H26N4O3 (502.56): C, 74.09; H, 5.21; N, 11.15. Found:
C, 74.07; H, 5.03; N, 11.00.
4.1.3.5. (RS) 6-[2-(3-Benzoylphenyl)propionylamino]-7-cyano-N-(4-
tolyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (11e). White crys-
tals, m.p. 197–199 ꢃC, yield 61%. IR ymax/cmꢀ1 3405, 3266 (NHs),
3058, 3027(CH aromatic) 2975–2875 (CH aliphatic), 2222 (CN),
1659 (COs), 1599 (NH), 1562–1517 (C]C). 1H NMR (CDCl3-
4.1.3.1. (RS)
7-Cyano-6-[2-(4-isobutylphenyl)propionylamino]-N-
phenyl-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(11a). White
crystals, m.p. 189–191 ꢃC, yield 61%. IR ymax/cmꢀ1 3284, 3209 (NHs),
3097, 3044 (CH aromatic), 2991–2866 (CH aliphatic), 2211 (CN),
1701 (COs), 1592 (NH and C]C). 1H NMR (DMSO-d6-400 MHz):
300 MHz):
d
1.67 (d, 3H, J ¼ 6.6 Hz, CH3CHCO), 2.26 (s, 3H, tolyl
d
0.82 (d, 6H, J ¼ 6.3 Hz, (CH3)2CH), 1.44 (d, 3H, J ¼ 7.2 Hz,
CH3), 2.50 (m, 2H, CH2-2), 2.95 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.96 (q, 1H,
J ¼ 6.6 Hz, CHCO), 4.33 (t, 2H, J ¼ 6.6 Hz, CH2-3), 7.02–7.85 (m, 13H,
aromatic protons), 8.03 (s, 1H, NHCOCH, which disappeared on
deuteration) and 9.13 (s, 1H, CONH–Ph, which disappeared on
CH3CHCO), 1.76 (m, 1H, (CH3)2CH), 2.37–2.51 (m, 4H, CH2-2,
CH2CH), 2.98 (t, 2H, J ¼ 7.5 Hz, CH2-1), 3.91 (q, 1H, J ¼ 7.2 Hz, CHCO),
4.26 (t, 2H, J ¼ 7.2 Hz, CH2-3), 7.00–7.58 (m, 9H, aromatic protons),

S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
489
deuteration). Anal. Calcd. for C32H28N4O3, (516.59): C, 74.40; H,
5.46; N, 10.85. Found: C, 74.29; H, 5.19; N, 10.62.
aromatic), 2994–2962(CH aliphatic), 2222 (CN), 1782, 1715, 1675
(COs),1598 (NH),1534 (C]C). 1H NMR (DMSO-300 MHz):
2.51 (m,
d
2H, CH2-2), 3.09 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.37 (t, 2H, J ¼ 7.2 Hz, CH2-
3), 7.03–7.44 (m, 5H, aromatic protons), 7.91–8.01 (m, 4H, aromatic
protons) and 9.93 (s,1H, NH, which disappeared on deuteration).13C
NMR (DMSO-d6): 24.95 (C-1), 25.92 (C-2), 49.84 (C-3), 84.71 (C-7),
114.57 (CN), 120.06 (C-6), 120.40 (C-40), 121.22 (C-20, -60), 124.28 (C-
400, -700), 124.44 (C-5), 129.00 (C-30, -50), 131.78 (C-500, -600), 135.56 (C-
3a00, -7a00), 138.70 (C-10), 146.71 (C-7a), 157.81 (CONH), 166.18 (2 CO,
C-100, -300). MS: m/z(%): 396 (Mþ, 14), 304 (100), 276 (6), 248 (6), 221
(3), 192 (3), 130 (8), 65 (16). Anal. Calcd. for C23H16N4O3 (396.40): C,
69.69; H, 4.07; N, 14.13. Found: C, 69.23; H, 3.75; N, 14.43.
4.1.3.6. (RS)
6-[2-(3-Benzoylphenyl)propionylamino]-N-(4-chlor-
ophenyl)-7-cyano-2,37-dihydro-1H-pyrrolizine-5-carboxamide
(11f). White crystals, m.p. 234–6 ꢃC, yield 54%. IR ymax/cmꢀ1 3287,
3210 (NHs), 3099, 3045 (CH aromatic) 2991–2805 (CH aliphatic),
2211 (CN), 1702 (COs), 1591 (NH), 1559 (C]C). 1H NMR (DMSO-d6-
300 MHz):
d
1.54 (d, 3H, J ¼ 6.6 Hz, CH3CHCO), 2.51 (m, 2H, CH2-2),
3.02 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.10 (q, 1H, J ¼ 6.6 Hz, CHCO), 4.32 (t,
2H, J ¼ 6.6 Hz, CH2-3), 7.05–7.81 (m, 13H, aromatic protons), 9.33 (s,
1H, NHCOCH, which disappeared on deuteration) and 10.26 (s, 1H,
CONH–Ph, which disappeared on deuteration). Anal. Calcd. for
C31H25ClN4O3 (537.01): C, 69.33; H, 4.69; N, 10.43. Found: C, 69.19;
H, 4.58; N, 10.60.
4.1.4.5. 7-Cyano-6-(1,3-dioxoisoindoinl-2-yl)-N-(4-tolyl)-2,3-dihy-
dro-1H-pyrrolizine-5-carboxamide (13b). White crystals, m.p.
271–273 ꢃC, yield 81%. IR ymax/cmꢀ1 3360 (NH), 3030 (CH
aromatic), 2922–2855 (CH aliphatic), 2226 (CN), 1781, 1713, 1665
(COs), 1593 (NH), 1568–1519 (C]C).1H NMR (DMSO-d6-300 MHz):
4.1.4. General method for preparation of compounds (12a–c and
13a–c)
A mixture of the carboxamide derivatives 9a–c (3.75 mmol) and
phythalic anhydride (0.56 g, 3.75 mmol) in glacial acetic acid
(20 ml) was refluxed for 3 h, the solution was evaporated under
reduced pressure, and the product was washed with water and
dried. The crude powder was dissolved in acetone, the insoluble
product was filtered and washed with hot acetone, the residue was
recrystallized from ethanol to give compounds 12a–c. The acetone
filtrate and washings were evaporated till dryness to leave out
compounds 13a–c which were recrystallized from acetone–water.
d
2.22 (s, 3H, CH3), 2.51 (m, 2H, CH2-2), 3.09 (t, 2H, J ¼ 7.5 Hz, CH2-
1), 4.36 (t, 2H, J ¼ 7.2 Hz, CH2-3), 7.05–7.29 (m, 4H, aromatic H),
7.91–8.01 (m, 4H, aromatic protons of isoindolyl ring) and 9.83 (s,
1H, NH, which disappeared on deuteration). Anal. Calcd. for
C24H18N4O3 (410.42): C, 70.23; H, 4.42; N, 13.65. Found: C, 70.22; H,
4.01; N, 13.35.
4.1.4.6. N-(4-Chlorophenyl)-7-cyano-6-(1,3-dioxoisoindolin-2-yl)-
2,3-dihydro-1H- pyrrolizine-5-carboxamide (13c). White crystals,
m.p. 260–263 ꢃC, yield 74%. IR ymax/cmꢀ1 3245 (NH), 3067, 3032
(CH aromatic), 2952 (CH aliphatic), 2226 (CN), 1787, 1727, 1669
(COs), 1640 (NH), 1598–1521 (C]C). 1H NMR (CDCl3-300 MHz):
4.1.4.1. 7,13-Dioxo-N-phenyl-2,3,7,13-tetrahydro-1H-pyrrolizi-
no[10,20:5,6]pyrimido[2,1-a] isoindole-5-carboxamide (12a). Yellow
powder, m.p. 340–343 ꢃC, yield 9%. IR ymax/cmꢀ1 3303 (NH), 3058,
3027 (CH aromatic), 2955–2915 (CH aliphatic), 1772, 1703, 1667
d
2.59 (m, 2H, CH2-2), 3.09 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.41 (t, 2H,
J ¼ 7.2 Hz, CH2-3), 7.24–7.42 (m, 4H, aromatic H), 7.84–8.02 (m, 4H,
aromatic protons) and 8.65 (s, 1H, NH, which disappeared on
deuteration). Anal. Calcd. for C23H15ClN4O3 (430.84): C, 64.12; H,
3.51; N, 13.00. Found: C, 63.96; H, 3.74; N, 13.15.
(COs), 1610 (NH), 1548 (C]C). 1H NMR (DMSO-d6-300 MHz):
d 2.56
(m, 2H, CH2-2), 3.11 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.45 (t, 2H, J ¼ 7.2 Hz,
CH2-3), 7.14–8.19 (m, 9H, aromatic H), 10.00 (s, 1H, NH, which dis-
appeared on deuteration). MS: m/z (%): 397 (Mþ þ 1, 10), 396 (Mþ,
29), 304 (70), 209 (8), 130 (58), 102 (61), 91 (26), 65 (100). Anal.
Calcd. for C23H16N4O3 (396.40): C, 69.69; H, 4.07; N, 14.13. Found: C,
69.89; H, 4.02; N, 13.86.
4.1.5. General method for preparation of compounds (14a–c)
A mixture of compounds 9a–c (3.75 mmol) and maleic anhy-
dride (0.37 g, 3.75 mmol) in 20 ml glacial acetic anhydride was
refluxed for 3 h, the solution was evaporated under reduced pres-
sure, and the product was washed with water and recrystallized
from ethanol/acetone.
4.1.4.2. 7,13-Dioxo-N-(4-tolyl)-2,3,7,13-tetrahydro-1H-pyrrolizi-
no[10,20:5,6]pyrimido[2,1-a]iso- indole-5-carboxamide (12b). Yellow
powder, m.p. 339–342 ꢃC, yield 11%. IR ymax/cmꢀ1 3311 (NH), 3063
(CH aromatic), 2989–2920 (CH aliphatic), 1774, 1703, 1666 (COs),
4.1.5.1. (EZ) 3-(4-Oxo-9-(phenylcarbamoyl)-4,5,6,7-tetrahydro-1H-
pyrimido[5,4-a]pyrrolizin-2-yl) acrylic acid (14a). Yellow crystals,
m.p. 300–302 ꢃC, yield 64%. IR ymax/cmꢀ1 3446, 3294 (NH), 3081,
3017 (CH aromatic) 2955–2918 (CH aliphatic), 2855 (broad band,
OH), 1757, 1701, 1670 (COs), 1601 (NH), 1548 (C]C). 1H NMR
1609 (NH), 1545–1507 (C]C). 1H NMR (DMSO-d6-300 MHz):
d 2.30
(s, 3H, CH3-tolyl), 2.50 (m, 2H, CH2-2), 2.93 (t, 2H, J ¼ 7.5 Hz, CH2-1),
4.27 (t, 2H, J ¼ 7.2 Hz, CH2-3), 7.19–7.93 (m, 8H, aromatic protons),
9.71 (s, 1H, CONH, which disappeared on deuteration). Anal. Calcd.
for C24H18N4O3 (410.42): C, 70.23; H, 4.42; N, 13.65. Found: C, 70.72;
H, 4.03; N, 13.96.
(DMSO-d6-300 MHz):
d
2.51 (m, 2H, CH2-6), 3.06 (t, 2H, J ¼ 7.5 Hz,
CH2-5), 4.43 (t, 2H, J ¼ 7.2 Hz, CH2-7), 6.83–7.74 (m, 7H, 5 aromatic
protons þ 2 vinylic protons CH]CHCOOH), 9.92, 10.16 and 11.96
(3s, 3H, 2 NHs and COOH, which disappeared on deuteration). MS
(ESI): m/z: 363 (Mþ ꢀ 1), 305, 280, 218, 203, 175, 162, 150, 115. Anal.
Calcd. for C19H16N4O4 (364.35): C, 62.63; H, 4.43; N, 15.38. Found: C,
62.50; H, 4.30; N, 15.45.
4.1.4.3. N-(4-Chlorophenyl)-7,13-dioxod2,3,7,13-tetrahydro-1H-pyr-
rolizino[10,20:5,6]pyrimido [2,1-a]isoindole-5-carboxamide (12c).
Yellow powder, m.p. 365–369 ꢃC, yield 7%. IR ymax/cmꢀ1 3313 (NH),
3069 (CH aromatic), 2926–2857 (CH aliphatic), 1776, 1709, 1670
(COs), 1616 (NH), 1545 (C]C). 1H NMR (DMSO-d6-300 MHz):
d 2.50
(m, 2H, CH2-2), 3.05 (t, 2H, J ¼ 7.5 Hz, CH2-1), 4.37 (t, 2H, J ¼ 7.2 Hz,
CH2-3), 7.40–8.13 (m, 8H, aromatic protons), 9.96 (s, 1H, CONH,
which disappeared on deuteration). Anal. Calcd. for C23H15ClN4O3
(430.84): C, 64.12; H, 3.51; N, 13.00. Found: C, 64.01; H, 3.42;
N, 13.54.
4.1.5.2. (EZ) 3-(4-Oxo-9-(4-tolylcarbamoyl)-4,5,6,7-tetrahydro-1H-
pyrimido[5,4-a]pyrrolizin-2-yl) acrylic acid (14b). Yellow crystals,
m.p. 334–336 ꢃC, yield 67%. IR ymax/cmꢀ1 3308, 3135 (NH), 3080
(CH aromatic), 2921 (CH aliphatic), 2560 (OH), 1700, 1657 (COs),
1603, (NH) 1545–1514 (C]C). 1H NMR (DMSO-d6-300 MHz):
d 2.28
(s, 3H, CH3), 2.50 (m, 2H, CH2-6), 3.00 (t, 2H, J ¼ 7.5 Hz, CH2-5), 4.27
(t, 2H, J ¼ 7.2 Hz, CH2-7), 7.10–7.61 (m, 6H, 4 aromatic protons þ 2
vinylic protons CH]CHCOOH), 9.68, 10.14 and 11.67 (3s, 3H, 2 NHs
and COOH, which disappeared on deuteration). Anal. Calcd. for
4.1.4.4. 7-Cyano-
6-(1,3-dioxoisoindolin-2-yl)-N-phenyl-2,3-dihy-
dro-1H-pyrrolizine-5-carboxamide (13a). White crystals, m.p.
243–246 ꢃC, yield 76%. IR ymax/cmꢀ1 3316 (NH), 3065, 3020 (CH

490
S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
C20H18N4O4 (378.38): C, 63.48; H, 4.79; N, 14.81. Found: C, 63.37; H,
5.20; N, 15.35.
C19H17ClN4O4 (400.82): C, 56.93; H, 4.28; N, 13.98. Found: C, 57.14;
H, 4.25; N, 14.40.
4.1.5.3. (EZ) 3-(9-(4-Chlorophenylcarbamoyl)-4-oxo-4,5,6,7-tetrahy-
dro-1H-pyrimido[5,4-a] pyrrolizin-2-yl)acrylic acid (14c). Yellow
crystals, m.p. 332–334 ꢃC, yield 65%. IR ymax/cmꢀ1 3440, 3300, 3258
(NHs), 3082 (CH aromatic) 2964–2916 (CH aliphatic), 2550 (OH),
1765–1667 (COs), 1601 (NH), 1546 (C]C). 1H NMR (DMSO-d6-
4.2. Pharmacological screening
4.2.1. Anti-inflammatory activity [14]
Adult albino rats of both sexes weighing between 120–150 g
were used. Rats were uniformly hydrated by giving 3 ml water/rat
through gastric inoculation to reduce variability to oedema
response. Animals were divided into groups each of five animals.
The control group was given saline solution containing few drops
of Tween 80. Ketorolac tromethamine (39.23 mmol/kg) was
administrated as standard drug for comparison and compounds
under examination (39.23 mmol/kg) were suspended in distilled
water by the aid of few drops of Tween 80 and were given
intraperitoneally one hour before induction of inflammation.
300 MHz):
d
2.51 (m, 2H, CH2-6), 2.99 (t, 2H, J ¼ 7.5 Hz, CH2-5), 4.44
(t, 2H, J ¼ 7.2 Hz, CH2-7), 6.93–7.75 (m, 6H, 4 aromatic protons and 2
vinylic protons CH]CHCOOH), 9.97, 10.18 and 11.85 (3s, 3H, 2 NHs
and COOH, which disappeared on deuteration). Anal. Calcd. for
C19H15ClN4O4 (398.80): C, 57.22; H, 3.79; N, 14.05. Found: C, 57.54;
H, 3.84; N, 13.80.
4.1.6. General method for preparation of compounds (15a–c)
The target compounds 15a–c were prepared from compounds
9a–c using the same method adopted for the preparation of
compound 14a–c using succinic anhydride (0.38 g, 3.75 mmol).
Induction of inflammation was performed by S.C. injection of 50 ml
of 1% carrageenan-sodium gel (Sigma–Aldrich, USA), into the sub-
plantar region of the right hind paw. The dorso-ventral diameter
(thickness) of the right and left hind paws of each rat was
measured using a pair of dial thickness gauge calipers accurate to
0.0001 cm 1 h, 2 h, 3 h and 4 h after induction of inflammation.
The left hind paw diameter served as a control for the degree of
inflammation in the right hind paw. The percentage of anti-
inflammatory activity (% inhibition of inflammation) was calcu-
lated according to the following equation
4.1.6.1. 3-(4-Oxo-9-(phenylcarbamoyl)-3,4,5,6-tetrahydro-1H-pyr-
imido[5,4-a]pyrrolizin-2-yl) propanoic acid (15a). Buff crystals, m.p.
271–274 ꢃC, yield 69%. IR ymax/cmꢀ1 3435, 3291, 3198 (NHs), 3090
(CH aromatic), 2973–2865 (CH aliphatic), 2593 (OH), 1723–1649
(COs), 1618 (NH), 1560 (C]C). 1H NMR (DMSO-d6-300 MHz):
d 2.51
(m, 2H, CH2-6), 2.76 (t, 2H, J ¼ 6 Hz, CH2CH2COOH) þ 2.87 (t, 2H,
J ¼ 6 Hz, CH2COOH), 3.08 (t, 2H, J ¼ 7.5 Hz, CH2-5), 4.44 (t, 2H,
J ¼ 7.2 Hz, CH2-7), 7.06–7.72 (m, 5H, aromatic protons), 9.78, 10.22,
11.69 (3s, 3H, two NHs and COOH, which disappeared on deutera-
% inhibition [ ð1 LLt=LcÞ 3 100
Lt is the mean increase in paw thickness in rats treated with the
tested compounds.
Lc is the mean increase in paw thickness in control group.
Data were analyzed by SPSS statistical package version 10.
Results are presented in Table 1.
tion). 13C NMR (DMSO-d6):
d
24.89 (C-50), 25.89 (C-60), 29.01 (C-2),
29.92 (C-3), 49.32 (C-7), 101.71 (C-4a0), 110.96 (C-9a0), 119.32 (C-200,
-600), 123.36 (C-400), 129.32 (C-300, -500), 139.38 (C-90), 139.64 (C-100),
142.31 (C-4b0), 157.02 (C-20þ), 158.52 (CONH), 158.94 (COOH), 173.94
(C-40). MS: m/z(%): 366 (M , 7), 349 (6), 256 (45), 228 (68), 201 (5),
120 (11), 92 (34), 65 (100). Anal. Calcd. for C19H18N4O4 (366.37): C,
62.29; H, 4.95; N, 15.29. Found: C, 62.79; H, 4.55; N, 15.62.
4.2.2. Analgesic activity [15–18]
Analgesic activity was measured using the acetic acid-induced
writhing test in mice. Five albino mice of either sex (18–22 g) were
used in each group. One hour after intraperitoneal administration
of a suspension of the tested compound or ketorolac (39.23 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 mascular contractions in each mouse was counted for
a period of 30 min. A significant reduction in the number of
writhings by any test compound as compared to control animals
was considered as a positive analgesic response. Percentage inhi-
bition was calculated using the following formula, where n is the
average number of writhings in control group and n0 is the average
number of writhings in treated group.
4.1.6.2. 3-(4-Oxo-9-(4-tolylcarbamoyl)-3,4,5,6-tetrahydro-1H-pyr-
imido[5,4-a]pyrrolizine-2-yl) propanoic acid (15b). Buff crystals,
m.p. 280–283 ꢃC, yield 73%. IR ymax/cmꢀ1 3448, 3292, 3205 (NHs),
3082 (CH aromatic), 2963–2862 (CH aliphatic), 2594 broad band
(OH), 1790–1648 (COs), 1616 (NH), 1553–1516 (C]C). 1H NMR
(DMSO-d6-300 MHz):
d 2.27 (s, 3H, CH3), 2.49 (m, 2H, CH2-6), 2.78
(t, 2H, J ¼ 6 Hz, CH2CH2COOH), 2.85 (t, 2H, J ¼ 6 Hz, CH2COOH), 3.03
(t, 2H, J ¼ 7.5 Hz, CH2-5), 4.39 (t, 2H, J ¼ 7.2 Hz, CH2-7), 7.12–7.60 (m,
4H, aromatic protons), 10.12, 11.69 and 12.25 (3 br s, 3H, two NHs
and COOH, which disappeared on deuteration). 13C NMR (DMSO-
d6):
d
20.42 (CH3), 24.35(C-50), 25.39 (C-60), 28.49(C-2, C-3),
49.28(C-70), 83.75(C-4a0), 113.88(C-9a0), 119.54 (C-400), 120.49 (C-
90),120.72 (C-200, -600), 128.89(C-300, -500), 132.98 (C-100), 135.70(C-
4b0), 146.04(C-20), 156.99(CONH),, 175.51(COOH, C-40). MS: m/z(%):
380 (Mþ, 13), 362 (39), 274 (25), 256 (100). Anal. Calcd. for
C20H20N4O4 (380.40): C, 63.15; H, 5.30; N, 14.73. Found: C, 63.14; H,
5.07; N, 15.11.
% Inhibition [ ½ðn L n0Þ=nꢄ 3 100
Results are tabulated in Table 2.
4.2.3. Acute ulcerogenicity studies [19,20]
Adult albino rats of both sexes weighing between 120–150 g
were used. Animals were divided into groups each of five animals.
Rats were fasted 20 h before drug administration. The tested
4.1.6.3. 3-(9-(4-Chlorophenylcarbamoyl)-4-oxo-3,4,5,6-tetrahydro-
1H-pyrimido[5,4-a] pyrrolizin-2-yl)propanoic acid (15c). Buff crys-
tals, m.p. 290–293 ꢃC, yield 72%. IR ymax/cmꢀ1 3400, 3284, 3247
(NHs), 3062 (CH aromatic), 2922 (CH aliphatic), 1787, 1671 (COs),
1616, 1598 (NH, C]C), 1487, 1440 (C–N, C–O), 827, 783 (C–Cl). 1H
compounds and ketorolac were given orally in
a dose of
39.23 mmol/kg suspended in 1% Tween while one group received
vehicle (1% Tween). Rats were fasted for two hours, allowed to feed
for 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, opened along with the greater
curvature and rinsed with 0.9% saline. The number of mucosal
damage (red spots) was counted and their severity (ulcerogenic
NMR (DMSO-d6-300 MHz):
d 2.50 (m, 2H, CH2-6), 2.77 (t, 2H,
J ¼ 6 Hz, CH2CH2COOH), 2.89 (t, 2H, J ¼ 6 Hz, CH2COOH), 3.05 (t, 2H,
J ¼ 7.5 Hz, CH2-5), 4.40 (t, 2H, J ¼ 7.2 Hz, CH2-7), 7.34–7.73 (m, 4H,
aromatic protons), 10.22, 10.35 and 11.62 (3 br s, 3H, two NHs and
COOH, which disappeared on deuteration). Anal. Calcd. for.

S.E. Abbas et al. / European Journal of Medicinal Chemistry 45 (2010) 482–491
491
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Score
Score
¨
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Normal (No injury)
Latent small red spot
Wide red spot
0
1
2
Slight injury
Severe injury
3
4
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divided by total number of rats in the group ꢁ 100]/10.
- Average number of ulcers: number of ulcers in the group/total
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- Average severity:
S [each ulcer multiplied by its score of
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Ulcer index ¼ the sum of the 3 figures
Results are tabulated in Table 3.
Acknowledgements
The authors are grateful to Dr. Gamal Ahmed El-Sherbeny
Mostafa, lecturer of Pharmacology, Faculty of Pharmacy, Beni-Suef
University for his kind help in performing the pharmacological
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