Synthesis, analgesic and anti-inflammatory activities evaluation of some bi-, tri- and tetracyclic condensed pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2009.06.028

Novel series of bicyclic pyrrolo[1,2-c]pyrimidines 3a-g, 5, 6a, b, and 7a, b, tricyclic pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines 8a-c, 9a-g, 13a-c, 17, 18a, b, 19, 20a,b and 21 and tetracyclic condensed pyrimidines 14, 22 and 23 were synthesized through d

Full text of DOI:10.1016/j.ejmech.2009.06.028

European Journal of Medicinal Chemistry 44 (2009) 4572–4584
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, analgesic and anti-inflammatory activities evaluation
of some bi-, tri- and tetracyclic condensed pyrimidines
*
Kamilia M. Amin, Mona M. Hanna , Hanan E. Abo-Youssef, Riham F. George
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, ElKasr Eleini 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:
Novel series of bicyclic pyrrolo[1,2-c]pyrimidines 3a–g, 5, 6a, b, and 7a, b, tricyclic pyrimido[5,4-e]pyr-
rolo[1,2-c]pyrimidines 8a–c, 9a–g, 13a–c, 17, 18a, b, 19, 20a,b and 21 and tetracyclic condensed pyrimi-
dines 14, 22 and 23 were synthesized through different chemical reactions. Structures of all synthesized
pyrimidine derivatives were supported by spectral and elemental analyses. Analgesic activity evaluation
was carried out using acetic acid-induced writhing assay, and all compounds exerted comparable activity
to indomethacin. The anti-inflammatory activity evaluation was performed using carrageenan-induced
paw edema in rats, the potency of the bicyclic derivatives 3a–f and 7b revealed comparable activity to
indomethacin without gastric ulceration. The tricyclic derivatives 13a and 20a exerted good activity,
however, they induced gastric ulcers while 13b and 13c showed moderate activity without ulceration. In
case of tetracyclic derivatives, compound 14 exhibited the highest potency and safety profile.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 7 May 2009
Received in revised form
24 June 2009
Accepted 25 June 2009
Available online 1 July 2009
Keywords:
Condensed pyrimidines
Pyrrolo[1,2-c]pyrimidines
Tricyclic pyrimidines
Tetracyclic pyrimidines
Anti-inflammatory and analgesic
1. Introduction
and condensed pyrimidines are important classes of heterocyclic
compounds that exhibit a broad spectrum of biological activities
Inflammation is a complex defensive mechanism of the body to
any noxious stimulus; this process may vary from a localized to
a generalized response characterized by the accumulation of fluids
and leukocytes leading to edema and pain [1]. This inflammatory
response seems to be mediated by different physiological and
immunological mediators that play a role in acute and chronic
inflammation. The acute inflammation occurs as the initial response
to tissue injury, being mediated by the release of autacoids, for
example, histamine, bradykinin, prostaglandins and leukotrienes
[1,2]. On the other hand, the chronic inflammatory process involves
the release of diverse mediators, as interleukins, interferon and
such as anticancer [6–10], antiviral [11], antibacterial [12,13], anti-
oxidant [14,15], anxiolytic [16] and antidepressant activities [17].
Furthermore, they possess anti-inflammatory [18–24] and analgesic
activities that are well documented in the literature [25–27]. The
bicyclic pyrrolo[1,2-c]pyrimidine derivative I [28] and the tricyclic
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines IIa, b [29] and III [30],
prepared in our laboratory, were found to have significant analgesic
and anti-inflammatory activities, therefore, in continuation of these
efforts, we have been interested in the synthesis of further bi-, tri-,
and tetracyclic condensed pyrimidine derivatives retaining the
pyrrolo[1,2-c]pyrimidine as a basic nucleus and evaluating their
analgesic and anti-inflammatory activities.
tumor necrosis factor
a (TNF-a), a cytokine that plays a major role in
this kind of inflammatory process and whose production is associ-
ated with some inflammatory diseases such as rheumatoid arthritis
[1,2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are
commonly used for the treatment of pain and inflammation asso-
ciated with different diseases particularly rheumatoid arthritis [3],
however, their chronic use may cause GIT ulceration, bleeding and
renal injury [4,5]. Therefore, although there are a number of anti-
inflammatory analgesic drugs available in the market, there is
a need to develop novel drugs with better safety profile. Pyrimidine
Cl
H
N
CN
CN
O
N
R
NH
H2N
N
N
N
N
N
N
O
N
F
C3H7
O
IIa,b
I
S
III
IIa
IIb
R=CH
3
* Corresponding author.
E-mail address: mona_maurice@yahoo.com (M.M. Hanna).
R=CH Cl
2
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.028

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4573
CN
CN
NH
1
a
CN
CN
N
R`
NH
b
N
N
N
N
R
CN
O
H
N
X
NH2
2
3a-g
2a X=O, R=C6H5
2b X=S, R=C2H5
N
N
3a R`=CH3
3b R`=C2H5
O
3c R`=C2H4OH
3d R`=CH2CH=CH
3e R`=C4H9
3f R`=4-CNC6H4
3g R`=3-CF3C6H4
5
c
d
CN
H2N
N
N
X
NH2
R
NH2
e
N
N
N
X
O
4
6
4a X=O, R=C6H5
4b X=S, R=C2H5
6a X=OH
6b X=NH2
f
R`
N
OC2H5
HN
N
CN
N
R
g
N
N
N
N
C2H5
X
S
8
7
8a R`=CH3
7a X=O, R=C6H5
7b X=S, R=C2H5
8b R`=C2H5
8c R`=C3H7
Scheme 1. Reagents and solvents: a: RCNX, CH2Cl2; b: DMS, dry benzene for 3a, R0X, dry DMF for 3b–g; c: NaBH4, absolute ethanol; d: ClCH2CH2NH2.HCl, NaH, dry DMF;
e: NH2OH.HCl, NaHCO3,methanol for 6a, NH2NH2.H2O, absolute ethanol for 6b; f: CH(OC2H5)3, acetic anhydride; g: R0NH2, absolute ethanol.
2. Results and discussion
dimethyl formamide (DMF) in the presence of sodium hydride. 1H
NMR spectrum of 5 showed exchangeable bands at 6.21 and
8.42 ppm corresponding to NH, NH2, respectively.
2.1. Chemistry
Furthermore, the target carboxamidines 6a, b were obtained by
reaction of the intermediate 4a with hydroxylamine hydrochloride
and hydrazine hydrate, respectively. The 1H NMR spectra displayed
three signals at 4.94, 5.97 and 7.78 ppm corresponding to the two
NH2 and OH groups, respectively, in case of 6a and at 5.80, 6.10 and
8.38 ppm assigned to the three NH2 groups of compound 6b.
On the other hand, condensation of the amino derivatives 4a,
b with Triethyl orthoformate in the presence of catalytic amount of
acetic anhydride resulted in the formamidic acid ethyl esters 7a, b,
respectively. The 1H NMR spectra showed the presence of signals at
0.98, 3.85 ppm and at 1.32, 4.30 ppm due to OC2H5 group of 7a, b,
respectively.
The synthetic pathways adopted for the preparation of the
desired new compounds are illustrated in Schemes 1–3. The known
starting materials 2a, b, [30,31] and 4a, b [30,32] proved to be
versatile compounds by virtue of their vicinal imino or amino and
nitrile functions. Thus, compound 2a was subjected to react with
dimethyl sulfate (DMS) and with alkyl or aryl halides to give 3a and
a number of N-substituted imino derivatives 3b–g, respectively
(Scheme 1). The structures of 3a–g were confirmed on the basis of
their elemental and spectral data. The IR spectrum of 3c revealed
the disappearance of the NH band at 3316 cmꢀ1 and the presence of
the OH stretching band at 3296 cmꢀ1 and an additional nitrile
stretching at 2187 cmꢀ1 has been observed in case of 3f. The
N-substituted analog 5 was similarly prepared through reaction of
the intermediate 4a with 2-chloroethylamine hydrochloride in
Ring closure of 7b with a proper aliphatic primary amine at
room temperature afforded the fused pyrimido[5,4-e]pyrrolo[1,2-
c]pyrimidines 8a–c. The IR spectra of the condensed pyrimidines

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K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
CN
R`
N C
H
N
N
R
X
10
R`
H
N
H
N
O
N
O
N
CN
NH2
R
N
a
b
NH
N
N
N
N
R
X
4a,b
O
X
c
11
9a-g
9a X=O, R=C6H5, R`=H
9b X=O, R=C6H5, R`=4-Cl
9c X=O, R=C6H5, R`=4-OCH3
9d X=S, R=C2H5, R`=4-OCH3
Cl
N
N
9e X=S, R=C2H5, R`=4-OCH3, 3-OH
9f X=O, R=C6H5, R`=2-NO2
9g X=O, R=C6H5, R`=4-NO2
N
N
O
12
d
e
R
N
N
N
O
COOH
N
N
N
N
N
N
H
N
O
N
N
N
N
13
13a R=-NHC3H7
O
O
N
13b R=
13c R=
15
14
N
O
Scheme 2. Reagents and solvents: a: the appropriate aldehyde, gl.acetic acid; b: PhCOCl, dry benzene; c: POCl3; d: the appropriate amine, absolute ethanol for 13a, K2CO3, dry DMF
for 13b, c; e: glycine, K2CO3, dry DMF.
8a–c showed the NH band at 3370 cmꢀ1 in addition they lacked
corresponding to the propyl, pyrrolidino and the morpholino
moieties, respectively.
the absorption band assigned to the nitrile stretching vibration at
2196 cmꢀ1
.
However, treatment of the chloro derivative 12 with glycine in
dry DMF in the presence of anhydrous potassium carbonate
resulted in a tetracyclic derivative 14 instead of the expected
glycine derivative 15. The proposed structure of 14 was supported
by the 1H NMR that showed the characteristic signal of the imi-
dazolidinone CH2 at 3.03 ppm, and the mass spectrum presented
the molecular ion peak at 397.
On the other hand, the known intermediate 1-hydrazino-3,5-
diphenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyr-
imidin-6-one 16, [32] wasin turn, provedtobe a useful intermediate
for the synthesis of the designed condensed pyrimidines 17–23
(Scheme 3). Treatment of 16 with acetylacetone in dry DMF resulted
in the formation of 1-(3,5-dimethylpyrazol-1-yl) derivative 17
whose structure was confirmed by the presence of singlet peaks at
2.30, 2.53, and 6.07 ppm in the 1H NMR corresponding to the two
methyl groups and the pyrazolo proton, respectively. Furthermore,
Other target polycyclic azines 9a–g, 11–13 were obtained as
depicted in Scheme 2. Thus, condensation of the intermediates 4a,
b with different aromatic aldehydes in glacial acetic acid yielded
only the tricyclic derivatives 9a–g instead of the benzylidene
derivatives 10 (Scheme 2). The mechanism of this reaction was
outlined in Fig. 1 [33]. The proposed structures 9a–g were
confirmed by IR spectra which showed the absence of the nitrile
stretching band in the region of 2178 cmꢀ1 and the presence of NH
and an additional carbonyl stretching bands at 3392 and 1725 cmꢀ1
,
respectively. Moreover, 1H NMR data showed two exchangeable
singlet signals corresponding to the two NH groups.
Furthermore, the reported 1-chloropyrimido[5,4-e]pyrrolo[1,2-
c]pyrimidine derivative 12 [32], yielded 13a–c upon alkylation with
propylamine, pyrrolidine or morpholine. 1H NMR spectra of the
target compounds 13a–c revealed extra aliphatic bands

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4575
N
Cl
CH3
N
O
O
H
N
H3C
S
N
N
N
N
N
N
N
N
N
H
N
N
O
12
N
H3C
N
a
O
O
b
19
17
d
NH2
HN
N
N
O
R
NH2
H
N
N
N
X
N
H
c
N
N
N
N
N
e
N
N
N
O
16
N
N
O
20a X=CH
20b X=N
O
f
i
18a R=CN
18b R=COOC2H5
h
O
O
H
N
N
N
O
HN
N
H
N
N
H3C
O
N
H
N
N
N
N
N
N
N
N
g
N
O
N
21
O
O
23
22
Scheme 3. Reagents and solvents: a: NH2NH2.H2O, absolute ethanol; b: acetylacetone, dry DMF; c: ethoxymethylene malononitrile for 18a, ethyl ethoxymethylenecyanoacetate,
dry DMF for 18b; d: p-toluenesulphonyl chloride, Et3N, dry benzene; e: the acid chloride, pyridine; f: ethyl chloroformate, Et3N, dry benzene; g: dry DMF; h: ethylchloroformate,
NaOCH3,CH3OH; i: chloroacetyl chloride, Et3N, dry benzene.
the 5-aminopyrazolyl derivatives 18a, b were obtained directly by
condensation reaction of the hydrazino derivative 16 with ethoxy-
methylene malononitrile or ethyl ethoxymethylene cyanoacetate,
respectively. The mechanism of this reaction is outlined in Fig. 2
[34,35], it is noteworthy that the NH of the hydrazine moietyand not
the pyrimidino nitrogen was involved in the cyclization, therefore,
resulting in the formation of 1-substituted-5-aminopyrazole-4-
carbonitrile or carboxylic acid ethyl ester 18a, b rather than the
1-substituted-3-aminopyrazole-4-carbonitrile or carboxylic acid
ethyl ester 18c nor the triazepine derivative 18d. The 1H NMR
showed the presence of the amino group signal at 7.14 ppm in case of
18a and signals at 1.31, 4.26, 6.75 ppm corresponding to ethox-
ycarbonyl and amino groups, respectively, of 18b. Additionally, mass
spectrum of 18b revealed molecular ion peak at 495; moreover, the
structure of the product 18a was identified on the basis of X-ray
crystal structure analysis (Fig. 3) which confirmed that the
substituted nitrogen of the pyrazole ring is adjacent to the carbon
atom carrying the amino group.
performed in methanol in the presence of sodium methoxide the
tetracyclic derivative 22 was obtained. It was also possible to obtain
compound 22 by refluxing 21 in dry DMF. The IR spectrum of 21
showed stretching bands at 3358, 3328, 1748 cmꢀ1 corresponding
to the two NHs and carbonyl group of the ester moiety, respectively,
and these bands disappeared in the IR spectrum of 22. In addition,
the 1H NMR of 21 revealed the presence of the triplet and quartet
peaks of the ethyl group at 1.31 and 4.25 ppm respectively which
disappeared in the 1H NMR spectrum of 22.
Finally, the reaction of the hydrazino derivative 16 with chlor-
oacetyl chloride in dry benzene in the presence of equimolar
amount of triethylamine afforded the tetracyclic triazine derivative
23 rather than the open acylated tricyclic derivative. The 1H NMR
confirmed the obtained structure by the presence of an
exchangeable signal of NH moiety at 9.60 ppm and a singlet peak of
CH2 group at 4.04 ppm.
2.2. Pharmacological screening
N-acylation of the hydrazino derivative 16 with different acid
chlorides as p-toluenesulphonyl chloride, benzoyl chloride or nic-
otinoyl chloride under different conditions resulted in the expected
derivatives 19, 20a, b, respectively (Scheme 3). The 1H NMR spectra
of the derivatives 19, 20a, b confirmed the obtained structures. The
hydrazino derivative 16 was also reacted with ethyl chloroformate
in dry benzene and in the presence of triethylamine to yield the
ethoxyhydrazide derivative 21, however, when the reaction was
2.2.1. Analgesic activity
The analgesic activity of the synthesized compounds 3a–g, 5, 6a,
b, 7a, b, 9a–g, 13a–c, 17, 18a, b, 19, 20a, b, 21, 14, 22, and 23 was
studied by the acetic acid-induced writhing test in mice [36–39]
using indomethacin as a reference standard. The tested compounds
presented a significant analgesic profile as it was noticed that all
compounds revealed analgesic activity comparable to or higher

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K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
R`
R`
N
N
HO
N
CN
X
O
N
HN
N
R`
NH2
R
NH
R
CHO
NH
R
+
N
N
X
X
4a,b
4-imino-1,3-oxazine
derivative
HZ
R`
H
N
O
R`
HO
NH
N
-HZ
+
NH
R
NH
N
N
N
X
R
X
9a-g
Fig. 1. Mechanism of formation of 9a–g.
than that of indomethacin at the same dose level and test condi-
tions (Table 1). Regarding the bicyclic nucleus, the 3-substituted
derivatives 3a–f, 5 and 7b exhibited higher analgesic activity
compared to indomethacin. It is noted that elongation of the
aliphatic side chain of the N-alkylated derivatives 3a–e increased
the analgesic activity and the N-ethylamino derivative 5 exhibited
the highest % inhibition compared to the other bicyclic analogs.
However, a decrease in potency was only observed in m-aryl
substituted derivative 3g compared with its p-substituted analog
3f. Moreover, replacement of the 1-oxo in the 3-formamidic acid
derivative 7a by 1-thioxo group resulted in 7b with a higher
activity, this may be due to the increase in lipophilicity. It was also
noted that the N-hydroxy carboxamidine derivative 6a had a better
analgesic effect than the N-amino analog 6b. Concerning the
tricyclic nucleus, N-methyl derivative 8a presented higher activity
than both the N-ethyl 8b and N-propyl derivative 8c, and substi-
tution on 3-aryl group by an electron withdrawing moiety as in 9b,
9f and 9g or electron donating group as in 9c–e resulted in higher
potency than the unsubstituted compound 9a. However, substitu-
tion at position 1 with aliphatic amine 13a, cycloalkylamine 13b, c
or pyrazole ring 17, 18a, b did not affect the activity except for the
pyrrolidin-1-yl substituted compound 13b that exhibited higher
significant activity than indomethacin. In addition, the hydrazino
derivatives 19, 20a, b, 21 showed comparable activity to indo-
methacin while the ethoxyhydrazide derivative 21 had higher
potency. All the tetracyclic derivatives exerted comparable activity
H
R
OC2H5
NH2
HN
NC
R
N
H
N
NH
HN
N
N
R
N
N
N
N
N
N
HN
N
N
N
N
N
O
O
O
16
N
R
N
N
H2N
R
N
H2N
NH2
N
N
N
N
N
R
N
N
N
N
O
N
18a,b
R= CN, COOC2H5
N
N
N
N
O
18c
O
18d
Fig. 2. Mechanism of formation of 18a and 18b.

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4577
Table 2
IC 50 values of the analgesic activity for compounds 5, 9e and 23.
Compound Response (% inhibition) at
0.4 mg/kg 0.8 mg/kg 1 mg/kg 2 mg/kg 4 mg/kg 8 mg/kg
IC 50
(mg/kg)
5
9e
23
49.36
53.65
43.13
81.33
75.97
63.31
90.56
86.91
76.61
94.43
92.00
82.40
95.97
95.27
86.06
96.36
97.00
88.83
0.402
0.359
0.446
2.2.2. Anti-inflammatory activity
The tested compounds for analgesic activity were further eval-
uated for their anti-inflammatory activity using the carrageenan-
induced paw edema method in rats [40–43]. The percentage
inhibition of edema was calculated and the data were expressed as
the mean ꢁ SEM. The anti-inflammatory activity of the test
compounds relative to that of indomethacin, as a reference drug,
was also calculated. The results are recorded in Tables 3 and 4. It has
been noticed that the bicyclic derivatives 3a–c showed comparable
activity to indomethacin, the N-butyl derivative 3e had higher
potency than the N-allyl derivative 3d, and the N-ethylamino
derivative 5 showed moderate activity. The p-substituted aromatic
derivative 3f was more potent than the m-substituted one 3g. In
case of the formamidic acid ethyl ester derivative 7a, the activity
decreased lower than that of the thioxo analog 7b and this may be
due to the lipophilicity of 7b (Table 3). Furthermore, the N-hydroxy
carboxamidine 6a was equipotent to indomethacin at the forth
hour while the N-amino analog 6b had the maximum effect at the
second hour. On the other hand, the tested tricyclic derivatives
displayed moderate to weak anti-inflammatory activity except 13a
and 20a that exhibited higher activity compared to the reference
drug (Table 4). The tetracyclic imidazolo derivative 14 revealed
higher potency than the triazolo derivative 22 and its homolog 23
(Table 4). It should be noted that the ethoxyhydrazide derivative 21
had the same activity profile as its cyclized triazole derivative 22,
they may be interconvertable in the body.
Fig. 3. ORTEP projection of single crystal X-ray diffraction of 18a.
to indomethacin, however, the triazino derivative 23 displayed the
highest activity compared to its 5-membered imidazo and triazolo
analogs 14 and 22, respectively.
Compounds 5, 9e and 23 were further tested for their analgesic
activity at 0.4, 0.8, 1, 2, 4 and 8 mg/kg in order to determine their IC
50 values. The data revealed that the tricyclic derivative 9e is more
potent than both the bicyclic and the tetracyclic derivatives 5 and
23, respectively (Table 2).
Table 1
Effects of the compounds on acetic acid-induced abdominal writhing in mice.
Compound
Dose (mg/kg)
Writhing reflex ꢁ SEM
Inhibition %
Control
–
10
76.83 ꢁ 1.08
–
Indomethacin
3a
21.83 ꢁ 0.91a
20.00 ꢁ 0.58a
19.67 ꢁ 0.49a
9.33 ꢁ 0.88a,b
14.50 ꢁ 0.76a,b
8.67 ꢁ 0.88a,b
13.17 ꢁ 0.79a,b
27.83 ꢁ 0.95a,b
2.83 ꢁ 0.83a,b
10.83 ꢁ 1.05a,b
16.17 ꢁ 0.79a,b
23.17 ꢁ 1.05a
9.17 ꢁ 0.70a,b
1.83 ꢁ 0.48a,b
18.50 ꢁ 0.76a,b
17.50 ꢁ 0.85a,b
17.17 ꢁ 0.87a,b
3.00 ꢁ 0.77a,b
12.00 ꢁ 0.86a,b
11.50 ꢁ 0.76a,b
1.00 ꢁ 0.26a,b
10.17 ꢁ 0.48a,b
4.17 ꢁ 0.87a,b
71.58
73.97
74.40
87.85
81.13
88.72
82.86
63.77
96.31
85.90
78.96
69.85
88.07
97.61
75.92
77.22
77.66
96.10
84.38
85.03
98.70
86.77
94.58
69.63
95.01
67.02
78.52
76.57
76.78
74.84
76.57
71.80
77.87
91.76
75.92
91.76
7.69
8.12
8.59
8.47
Moreover, the IC 50 values for the most active compounds 3a,
13a and 14 were calculated using the doses of 10, 15, 20, 25 mg/kg
(Table 5). It was found that the bicyclic compound 3a is nearly
equipotent to the tetracyclic one 14 whereas 13a, the tricyclic
derivative, is more potent (IC 50 ¼ 16.50 mg/kg).
3b
3c
3d
3e
3f
3g
5
6a
6b
7a
7b
8a
8b
8c
9a
9b
8.94
10.24
11.49
8.68
In conclusion, all the tested compounds showed remarkable
analgesic activity comparable to that of indomethacin in systemic
in vivo bioassay at the same dose level. Regarding, the anti-
inflammatory activity, generally the bicyclic derivatives exhibited
higher activity than the tri- and tetracyclic compounds. Finally, it
was concluded that the tested compounds exerted their analgesic
and anti-inflammatory activities via different mechanisms.
8.33
8.36
9.00
8.12
7.63
8.04
8.45
10.45
11.45
11.32
10.39
10.86
11.75
11.75
11.66
12.04
12.40
11.52
12.65
13.00
14.37
15.26
13.81
13.84
12.87
11.55
11.96
2.2.3. Ulcerogenic activity
9c
9d
9e
9f
The prepared compounds were tested for their ulcerogenic effect
after testing for their anti-inflammatory activity. It was found that
the bicyclic N-substituted imino derivatives 3a–f and the for-
mamidic ethyl esters 7a, b had gastric safety profile. Furthermore,
all the tricyclic derivatives were ulcerogenic except 8a, 8c, 13b, 13c
and 18b, on the other hand, all the tetracyclic derivatives displayed
super gastric safety pattern when compared to the ulcerogenic
effect of indomethacin.
9g
a
13a
13b
13c
14
23.33 ꢁ 0.88
a,b
3.83 ꢁ 0.87
a,b
25.33 ꢁ 0.84
16.50 ꢁ 0.76a,b
18.00 ꢁ 0.58a,b
17.83 ꢁ 0.70a,b
17
18a
18b
19
20a
20b
21
a,b
19.33 ꢁ 0.76
18.00 ꢁ 0.97a,b
3. Experimental
a
21.67 ꢁ 0.88
17.00 ꢁ 0.73a,b
6.33 ꢁ 0.84a,b
18.50 ꢁ 1.03a,b
6.33 ꢁ 0.42a,b
3.1. Chemistry
22
23
Melting points were determined on Electrothermal Stuart 5MP3
digital melting point apparatus and were uncorrected. Elemental
microanalyses were performed at the microanalytical center,
a
Statistically significant from control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b

4578
K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
Table 3
Anti-inflammatory effect of the bicyclic derivatives against carrageenan-induced paw edema in rats and ratio of ulceration (n ¼ 6).
Compound
Ratio of ulceration
Edema thickness (mm) ꢁ SEM (% inhibition)
Potencyc
1 h
2 h
3 h
4 h
Control
3a
3b
3c
3d
3e
3f
3g
5
6a
6b
7a
7b
0/6
0/6
0/6
0/6
0/6
0/6
0/6
2/6
3/6
4/6
2/6
0/6
0/6
5/6
1.483 ꢁ 0.070b
2.017 ꢁ 0.070b
2.317 ꢁ 0.095b
2.683 ꢁ 0.095b
–
1.200 ꢁ 0.052a,b (19.08)
1.367 ꢁ 0.099b (7.82)
1.400 ꢁ 0.052b (5.60)
1.45 ꢁ 0.085b (2.23)
1.317 ꢁ 0.105b (11.19)
1.233 ꢁ 0.092a,b (16.86)
1.35 ꢁ 0.072b (8.97)
1.250 ꢁ 0.096b (15.71)
1.417 ꢁ 0.040b (4.45)
1.35 ꢁ 0.081b (8.97)
1.15 ꢁ 0.112a,b,c (22.45)
1.300 ꢁ 0.082b,c (12.34)
0.783 ꢁ 0.117a (47.20)
1.483 ꢁ 0.054a (26.47)
1.550 ꢁ 0.120a (23.15)
1.567 ꢁ 0.067a (22.31)
1.633 ꢁ 0.080a,b (19.04)
1.533 ꢁ 0.096a (24.00)
1.450 ꢁ 0.067a (28.11)
1.867 ꢁ 0.080b (7.44)
1.467 ꢁ 0.092a (27.27)
1.717 ꢁ 0.091a,b (14.87)
1.467 ꢁ 0.062a (27.27)
1.833 ꢁ 0.088b,c (9.12)
1.633 ꢁ 0.056a,b,c (19.04)
1.300 ꢁ 0.179a (35.55)
1.617 ꢁ 0.048a (30.21)
1.750 ꢁ 0.152a (24.47)
1.717 ꢁ 0.065a (25.90)
1.783 ꢁ 0.048a (23.05)
1.867 ꢁ 0.080a (19.42)
1.667 ꢁ 0.148a (28.05)
2.117 ꢁ 0.070b (8.63)
1.717 ꢁ 0.087a (25.90)
1.817 ꢁ 0.087a (21.58)
1.717 ꢁ 0.079a (25.90)
2.183 ꢁ 0.091b,c (5.78)
1.833 ꢁ 0.092a (20.89)
1.600 ꢁ 0.210a (30.95)
1.750 ꢁ 0.067a (34.77)
1.867 ꢁ 0.152a (30.41)
1.950 ꢁ 0.103a (27.32)
2.300 ꢁ 0.089a,b (14.28)
2.083 ꢁ 0.079a (22.36)
2.050 ꢁ 0.206a (23.59)
2.433 ꢁ 0.102b (9.31)
2.230 ꢁ 0.092a (16.88)
1.950 ꢁ 0.062a (27.32)
2.067 ꢁ 0.156a (22.96)
2.550 ꢁ 0.099b,c (4.96)
2.133 ꢁ 0.120a,c (20.50)
1.950 ꢁ 0.239a (27.32)
1.27
1.11
1.00
0.52
0.81
0.86
0.34
0.61
1.00
0.84
0.18
0.75
1.00
Indo.
a
Statistically significant from control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
Potency was expressed as % inhibition of the tested compounds relative to % inhibition of indomethacin at 4 h effect.
b
c
Faculty of Science, Cairo University. UV spectra were recorded on
a Shimadzu UV–Visible spectrophotometer, UV-1650 PC in chlo-
roform. The IR spectra were recorded on a Bruker FT-IR spectro-
photometer as potassium bromide discs in case of solids and as thin
films in case of oils. The 1H NMR spectra were recorded in CDCl3
ethoxymethylene cyanoacetate [46] and nicotinyl chloride hydro-
chloride [47] were prepared according to the reported procedures.
3.1.1. (E/Z) 3-Methylimino-1-oxo-2-phenyl-1,2,3,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3a
and DMSO-d6 on
a
Varian Mercury spectrometer (200 and
To a suspension of 2a (0.5 g, 2 mmol) in dry benzene (5 ml)
dimethyl sulfate (2.2 mmol) was added, and the reaction mixture
was refluxed for 3 h. The solvent was removed under vacuum, the
residue was dissolved in water, and the pH of the solution was
adjusted at 10 using 5 N sodium hydroxide. The obtained precipitate
was filtered, washed with water and crystallized from absolute
ethanol to give a white solid, 0.47 g, 90% yield, m.p. 186–188 ꢂC. IR:
ymax./cmꢀ13053 (CH aromatic), 2937–2866 (CH aliphatic), 2204 (CN),
300 MHz) and 13C NMR spectra were recorded at 75.45 MHz. Mass
spectra were performed on HP MODEL: MS_5988 mass spectrom-
eter and on Shimadzu QP-2010 Plus (EI, 70 eV). Silica gel used for
column chromatography was obtained from Fluka, 70–230 mesh.
Thin layer chromatography was carried out on silica gel TLC plates
with fluorescence indicator (F254). Compounds 1 [44], 2a [31], 2b
[30], 4a [32], 4b [30], 11 [32,45], 12, 16 [32] and the reagents ethyl
Table 4
Anti-inflammatory effect of the tri- and tetracyclic derivatives against carrageenan-induced paw edema in rats and ratio of ulceration (n ¼ 6).
Compound
Ratio of ulceration
Edema thickness (mm) ꢁ SEM (% inhibition)
Potencyc
1 h
2 h
3 h
4 h
b
Control
8a
8b
8c
9a
9b
9c
9d
9e
9f
9g
13a
13b
13c
14
0/6
0/6
1/6
0/6
2/6
3/6
2/6
2/6
4/6
3/6
1/6
3/6
0/6
0/6
0/6
2/6
1/6
0/6
2/6
3/6
2/6
1/6
0/6
0/6
5/6
1.483 ꢁ 0.070b
2.017 ꢁ 0.070b
2.317 ꢁ 0.095
2.683 ꢁ 0.095 b
–
1.317 ꢁ 0.048b (11.19)
1.367 ꢁ 0.049b (11.63)
1.283 ꢁ 0.105b (13.46)
1.417 ꢁ 0.048b (4.47)
0.850 ꢁ 0.118a (42.68)
1.383 ꢁ 0.098b (6.72)
1.333 ꢁ 0.115b (10.09)
1.150 ꢁ 0.177a,b (22.45)
1.300 ꢁ 0.097b (12.34)
1.467 ꢁ 0.084b (1.10)
1.283 ꢁ 0.105b (13.49)
1.367 ꢁ 0.012b (7.82)
1.317 ꢁ 0.098b (11.19)
1.267 ꢁ 0.067 b (14.57)
1.217 ꢁ 0.070a,b (17.94)
1.300 ꢁ 0.052b (12.34)
1.383 ꢁ 0.133b (6.74)
1.150 ꢁ 0.085a,b (22.45)
0.95 ꢁ 0.085a (35.94)
1.000 ꢁ 0.058a (32.57)
1.200 ꢁ 0.093a,b (19.08)
1.330 ꢁ 0.088b (10.11)
1.450 ꢁ 0.056 b (2.23)
0.783 ꢁ 0.117a (47.20)
1.733 ꢁ 0.096a,b (14.06)
1.783 ꢁ 0.065b (11.59)
1.700 ꢁ 0.097a,b (15.72)
1.767 ꢁ 0.084b (12.41)
1.567 ꢁ 0.152a (22.31)
1.833 ꢁ 0.122b (6.64)
1.833 ꢁ 0.088b (9.12)
1.833 ꢁ 0.096b (9.12)
1.733 ꢁ 0.096a,b (14.06)
1.733 ꢁ 0.096a,b (14.06)
1.500 ꢁ 0.097a (25.63)
1.483 ꢁ 0.125a (26.47)
1.633 ꢁ 0.126a,b (19.04)
1.567 ꢁ 0.056a (22.31)
1.600 ꢁ 0.058a,b (20.67)
1.817 ꢁ 0.087b (9.92)
1.65 ꢁ 0.106a,b (18.20)
1.683 ꢁ 0.019a,b (16.56)
1.433 ꢁ 0.152a (28.95)
1.783 ꢁ 0.095b (11.60)
1.667 ꢁ 0.072a,b (17.37)
1.767 ꢁ 0.080b (12.39)
1.867 ꢁ 0.062b (7.44)
1.300 ꢁ 0.179a (35.55)
2.050 ꢁ 0.062b (11.52)
2.067 ꢁ 0.033b (10.80)
2.100 ꢁ 0.107b (9.37)
2.050 ꢁ 0.089b (11.52)
2.117 ꢁ 0.172b (8.63)
2.000 ꢁ 0.134a,b (13.68)
2.067 ꢁ 0.076b (10.79)
2.250 ꢁ 0.106b (2.89)
2.117 ꢁ 0.040b (8.65)
1.983 ꢁ 0.098a,b (14.40)
1.600 ꢁ 0.103a (30.94)
1.733 ꢁ 0.115a (25.21)
1.883 ꢁ 0.145a (18.73)
1.683 ꢁ 0.048a (27.36)
1.833 ꢁ 0.056a (20.89)
2.100 ꢁ 0.037b (9.37)
1.767 ꢁ 0.109a (23.74)
1.783 ꢁ 0.196a (23.05)
2.417 ꢁ 0.054b (9.93)
2.417 ꢁ 0.095b (9.91)
2.500 ꢁ 0.151b (6.82)
2.400 ꢁ 0.139b (10.55)
2.417 ꢁ 0.185b (9.91)
2.183 ꢁ 0.087a (18.64)
2.283 ꢁ 0.087a,b (14.90)
2.600 ꢁ 0.113b (3.09)
2.583 ꢁ 0.060b (3.73)
2.167 ꢁ 0.076a (19.23)
1.700 ꢁ 0.103a (36.64)
2.267 ꢁ 0.076a (15.51)
2.383 ꢁ 0.164b (11.18)
1.900 ꢁ 0.113a (29.18)
2.217 ꢁ 0.114a (17.37)
2.500 ꢁ 0.068b (6.82)
2.383 ꢁ 0.075b (11.18)
2.100 ꢁ 0.195a (21.73)
2.000 ꢁ 0.144a (25.46)
2.533 ꢁ 0.115b (5.59)
2.250 ꢁ 0.067a (16.14)
2.267 ꢁ 0.080a (15.51)
2.117 ꢁ 0.054a (21.10)
1.950 ꢁ 0.239a (27.32)
0.36
0.36
0.24
0.38
0.36
0.68
0.54
0.11
0.13
0.70
1.34
0.56
0.40
1.06
0.63
0.24
0.40
0.79
0.93
0.20
0.59
0.56
0.77
1.00
17
18a
18b
19
20a
20b
21
22
23
Indo.
a
1.683 ꢁ 0.142 (27.36)
2.117 ꢁ 0.031b (8.63)
1.900 ꢁ 0.037a,b (17.99)
1.917 ꢁ 0.048a,b (17.26)
1.950 ꢁ 0.067a,b (15.84)
1.600 ꢁ 0.210a (30.95)
a
Statistically significant from control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b
c
Potency was expressed as % inhibition of the tested compounds relative to % inhibition of indomethacin at 4 h effect.

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4579
Table 5
3.67–3.79 (m, 2H, pyrrolidine), 4.56 (br.s, 2H, CH2CH]CH2), 5.15
(d, 2H, CH2CH]CH2), 5.82–5.86 (m, 1H, CH2CH]CH2), 6.99–7.51
(m, 5H, aromatic protons). Anal. Calcd. for C17H16N4O (292.34): C,
69.85; H, 5.52; N, 19.16. Found: C, 69.71; H, 5.34; N, 18.99.
IC 50 values of the anti-inflammatory activity for compounds 3a, 13a and 14.
Compound
Response (% inhibition) at
IC 50
(mg/kg)
10 mg/kg
15 mg/kg
20 mg/kg
25 mg/kg
3a
13a
14
36.00
27.56
17.00
43.04
41.63
40.00
50.77
63.29
61.31
62.73
69.05
68.40
17.77
16.50
17.62
3.1.2.4. (E/Z) 3-Butylimino-1-oxo-2-phenyl-1,2,3,5,6,7-hexahy-
dropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3e. Compound 3e was
prepared from 2a and butyl bromide for 8 h. Brown oil, 0.17 g, 28%
yield. IR: ymax./cmꢀ1 3058 (CH aromatic), 2956–2858 (CH aliphatic),
2195 (CN), 1704 (C]O), 1624 (C]N), 1593–1492 (C ¼ C).1H NMR
1706 (C]O), 1636 (C]N), 1590 (C]C). 1H NMR (CDCl3, 300 MHz):
2.22–2.32 (m, 2H, pyrrolidine), 3.24 (t, J ¼ 7.8 Hz, 2H, pyrrolidine),
d
(CDCl3, 300 MHz):
d
0.92 (t, J ¼ 6.9 Hz, 3H, NCH2CH2CH2CH3), 1.26–
3.38 (s, 3H, CH3), 4.04 (t, J ¼ 7.2 Hz, 2H, pyrrolidine), 7.13–7.51 (m, 5H,
aromatic protons). 13C NMR (CDCl3, 75.45 MHz): 20.38, 32.13, 49.75
(pyrrolidine carbons), 36.43 (N–CH3), 116.50 (CN), 126.02, 128.14,
128.70, 128.97, 129.26 (aromatic protons), 136.90 (C]C–CN), 145.26
(C]C–CN), 148.71 (C]O), 163.63 (C]N). MS: m/z (%): 266
(Mþ, 18.82), 265 (100), 251 (17.34). Anal. Calcd. for C15H14N4O
(266.31): C, 67.65; H, 5.30; N, 21.04. Found: C, 67.81; H, 4.98; N, 21.03.
1.45 (m, 4H, NCH2CH2CH2CH3), 1.45–1.75 (m, 2H, pyrrolidine), 3.10–
3.15 (m, 2H, pyrrolidine), 4.17–4.20 (m, 4H, NCH2CH2CH2CH3 and
2H pyrrolidine), 7.20–7.58 (m, 5H, aromatic protons). Anal. Calcd.
for C18H20N4O (308.39): C, 70.11; H, 6.54; N, 18.17. Found: C, 70.20;
H, 6.31; N, 18.44.
3.1.2.5. (E/Z) 3-(4-Cyanophenyl)imino-1-oxo-2-phenyl-1,2,3,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3f. Compound 3f
was prepared from 2a and 4-fluorobenzonitrile for 6 h. Reddish
brown crystals, 0.36 g, 51% yield, m.p. 185–187 ꢂC. IR: ymax./cmꢀ1
3063 (CH aromatic), 2960–2887 (CH aliphatic), 2212 (CN), 2187(CN),
1683 (C]O), 1602 (C]N), 1542–1494 (C]C). 1H NMR (CDCl3,
3.1.2. General procedure for the preparation of 3b–g
To a solution of 2a (0.5 g, 2 mmol) in dry DMF (5 ml), sodium
hydride (0.1 g, 60% in mineral oil, washed with hexane, 2.5 mmol)
was added, and the reaction mixture was heated at 80 ꢂC for 1 h.
Then the appropriate alkyl or aryl halide was added (2.5 mmol),
and the mixture was further heated for a specified time, cooled to
R.T. and poured on ice water (20 ml). The obtained solid was filtered
and recrystallized from ethanol/water in case of 3b–d, or purified
by column chromatography using silica gel (230 mesh size) as
stationary phase and chloroform:ethanol (9.5:0.5) as elution
system in case of 3e–g.
300 MHz):
d
2.14–2.24 (m, 2H, pyrrolidine), 2.93 (t, J ¼ 7.8 Hz, 2H,
pyrrolidine), 3.69 (t, J ¼ 7.2 Hz, 2H, pyrrolidine), 7.09 (d, J ¼ 8.1 Hz,
2H, aromatic protons), 7.22–7.44 (m, 5H, aromatic protons), 7.54
(d, J ¼ 8.1 Hz, 2H, aromatic protons). MS: m/z (%): 353 (Mþ, 10.85),
215 (100). Anal. Calcd. for C21H15N5O.2H2O (389.42): C, 64.77; H,
4.92; N, 17.98. Found: C, 64.47; H, 4.63; N, 17.86.
3.1.2.6. (E/Z) 1-Oxo-2-phenyl-3-(3-trifluoromethylphenyl)imino-
1,2,3,5,6,7-hexahydropyrrolo[1,2-c]pyrimidine-4-carbonitrile
3g. Compound 3g was prepared from 2a and 3-fluorobenzotri-
fluoride for 15 h. Brown crystals, 0.25 g, 32% yield, m.p. 209–211 ꢂC.
IR: ymax./cmꢀ1 3062 (CH aromatic), 2955–2853 (CH aliphatic),
2209 (CN), 1704 (C]O), 1629(C]N), 1596–1494 (C]C). 1H NMR
3.1.2.1. (E/Z) 3-Ethylimino-1-oxo-2-phenyl-1,2,3,5,6,7-hexahy-
dropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3b. Compound 3b was
prepared from 2a and ethyl iodide for 3 h. Brown crystals, 0.20 g,
36% yield, m.p. 264–265 ꢂC (decomposition). UV (CHCl3), Conc.
7.13 ꢃ10ꢀ5 M, nm: 227.5 (log
3: 3.65), 241 (log 3: 4.01), 287 (log 3:
3.91). IR: ymax./cmꢀ1 3061 (CH aromatic), 2923–2852 (CH aliphatic),
(CDCl3, 300 MHz): d 2.06–2.13 (m, 2H, pyrrolidine), 2.91 (t,
2203 (CN), 1705 (C]O), 1633 (C]N), 1593–1492 (C]C). 1H NMR
J ¼ 7.8 Hz, 2H, pyrrolidine), 3.69 (t, J ¼ 7.05 Hz, 2H, pyrrolidine),
7.14–7.59 (m, 9H, aromatic protons). Anal. Calcd. for C21H15F3N4O
(396.38): C, 63.64; H, 3.81; N,14.13. Found: C, 64.18; H, 3.63; N,14.35.
(DMSO-d6, 200 MHz):
d
1.10 (t, J ¼ 6.8 Hz, 3H, CH2CH3), 1.94–2.09
(m, 2H, pyrrolidine), 2.90–2.95 (m, 2H, pyrrolidine), 3.63–3.73 (m,
2H, CH2CH3), 3.90–4.00 (m, 2H, pyrrolidine), 7.00–7.55 (m, 5H,
aromatic protons). MS: m/z (%): 280 (Mþ, 11.87), 57 (100). Anal.
Calcd. for C16H16N4O (280.33): C, 68.55; H, 5.75; N, 19.99. Found: C,
68.20; H, 5.75; N, 19.91.
3.1.3. 3-(2-Aminoethylamino)-1-oxo-2-phenyl-1,2,4a,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine-4-carbonitrile 5
Compound 5 was prepared according to the general procedure of
preparation of 3b–g by heating 4a (0.5 g, 2 mmol), sodium hydride
(0.20 g, 60% in mineral oil washed with hexane, 5 mmol) and 2-
chloroethylamine hydrochloride (0.28 g, 2.5 mmol) at 80 ꢂC for 10 h.
Buff solid, 0.29 g, 50% yield, m.p.190–192 ꢂC dec. IR: ymax./cmꢀ13348
(NH, NH2 stretching), 3050 (CH aromatic), 2925–2855 (CH
aliphatic), 2189 (CN), 1655 (C]O), 1598–1495 (C]C). 1H NMR
3.1.2.2. (E/Z) 3-(2-Hydroxyethyl)imino-1-oxo-2-phenyl-1,2,3,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3c. Compound 3c
was prepared from 2a and 2-chloroethanol for 10 h. Brown crystals,
0.22 g, 38% yield, m.p. >330 ꢂC (decomposition). IR: ymax./cmꢀ1 3296
(OH), 3058 (CH aromatic), 2923–2853 (CH aliphatic), 2206 (CN),
1705 (C]O), 1634 (C]N), 1596–1492 (C]C). 1H NMR (DMSO-d6,
(DMSO-d6, 200 MHz): d 1.40–1.80 (m, 4H, pyrrolidine), 2.19 (br.s,1H,
D2O, 200 MHz):
d
1.95–2.00 (m, 2H, pyrrolidine), 2.91–2.96 (m, 2H,
pyrrolidine), 2.51–2.72 (m, 2H, NHCH2CH2NH2), 2.91–2.93 (m, 2H,
NHCH2CH2NH2), 3.12 (br.s, 1H, pyrrolidine), 4.00 (br.s, 1H, pyrroli-
dine), 6.21 (br.s, 1H, NH exchanged by D2O), 6.88–7.35 (m, 5H,
aromatic protons), 8.42 (br.s, 2H, NH2 exchanged by D2O). Anal.
Calcd. for C16H19N5O (297.36): C, 64.63; H, 6.44; N, 23.55. Found: C,
64.58; H, 6.62; N, 23.89.
pyrrolidine), 3.80–3.83 (m, 2H, NCH2CH2OH), 3.96–4.09 (m, 2H,
pyrrolidine), 4.32–4.43 (m, 2H, NCH2CH2OH), 6.99–7.51 (m, 5H,
aromatic protons), 9.19 (s, 1H, OH exchanged by D2O). Anal. Calcd.
for C16H16N4O2 (296.33): C, 64.85; H, 5.44; N, 18.91. Found: C, 64.63;
H, 5.45; N, 18.55.
3.1.2.3. (E/Z) 3-Allylimino-1-oxo-2-phenyl-1,2,3,5,6,7-hexahy-
dropyrrolo[1,2-c]pyrimidine-4-carbonitrile 3d. Compound 3d was
prepared from 2a and allyl bromide for 6 h. Black crystals, 0.15 g,
26% yield, m.p. 177–178 ꢂC (decomposition). IR: ymax./cmꢀ1 3074
(CH aromatic), 2923–2853 (CH aliphatic), 2212 (CN), 1702 (C]O),
1627 (C]N), 1590–1491 (C]C). 1H NMR (DMSO-d6, 200 MHz):
3.1.4. 3-Amino-N-hydroxy-1-oxo-2-phenyl-1,2,4a,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine-4-carboxamidine 6a
To a suspension of 4a (0.5 g, 2 mmol) in methanol (5 ml), sodium
bicarbonate (0.17 g, 2.1 mmol) and hydroxylamine hydrochloride
(0.14 g, 2.1 mmol) were added, and the mixture was refluxed for
24 h. The inorganic precipitate was filtered and the filtrate was
concentrated under vacuum, the obtained white crystals were
d
2.00–2.10 (m, 2H, pyrrolidine), 2.40–2.59 (m, 2H, pyrrolidine),

4580
K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
filtered and recrystallized from ethanol. White solid, 0.28 g, 50%
yield, m.p. 166–169 ꢂC. IR: ymax./cmꢀ1 3403 (OH stretching), 3334–
3144 (NH2 stretching), 2969–2882 (CH aliphatic), 1648 (C]O),
159.26 (C]C), 175.24 (C]S). Anal. Calcd. for C13H18N4OS (278.38):
C, 56.09; H, 6.52; N, 20.13. Found: C, 56.36; H, 6.32; N, 20.16.
1595–1534 (C ¼ C). 1H NMR (DMSO-d6, 300 MHz):
d
1.77–2.07 (m,
3.1.7. General procedure for the preparation of 8a–c
3H, pyrrolidine), 3.10–3.15 (m, 1H, pyrrolidine), 3.51–3.65 (m, 2H,
pyrrolidine), 4.70 (t, J ¼ 6.6 Hz, 1H, pyrrolidine), 4.94 (s, 2H, NH2
exchanged with D2O), 5.97 (s, 2H, NH2 of amidine exchanged with
D2O), 6.88–7.43 (m, 5H, aromatic protons), 7.78 (s,1H, OH exchanged
with D2O). 13C NMR (DMSO-d6, 75.45 MHz): 24.50, 31.27, 46.99,
50.47 (pyrrolidine carbons), 82.66 (C]C), 117.58, 118.85, 121.56,
128.32, 128.54 (aromatic carbons), 140.20 (C]O), 154.27 (C]C),
162.90, 164.81 (C]N). MS: m/z (%): 287.15 (Mþ, 2.87), 93.05 (100).
Anal. Calcd. for C14H17N5O2 (287.32): C, 58.52; H, 5.96; N, 24.37.
Found: C, 58.70; H, 5.80; N, 24.87.
To a solution of 7b (0.25 g, 0.89 mmol) in absolute ethanol (5 ml),
the appropriate alkyl amine (0.98 mmol) was added, and the
mixture was stirred at R.T. for 3 h. The solvent was evaporated under
vacuum and the residue was purified by column chromatography
using silica gel as stationary phase and chloroform:ethanol (5.5:4.5)
as elution system.
3.1.7.1. 5-Ethyl-1-imino-2-methyl-6-thioxo-1,2,8,9,10,10a-hexahy-
dro-5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine 8a. Reddish brown
solid, 0.08 g, 32% yield, m.p. 114–115 ꢂC. IR: ymax./cmꢀ1 3370 (NH
stretching), 2923–2853 (CH aliphatic), 1638 (C]N), 1565–1461
3.1.5. 3-Amino-N-amino-1-oxo-2-phenyl-1,2,4a,5,6,7-
hexahydropyrrolo[1,2-c]pyrimidine -4-carboxamidine 6b
(C]C), 1250 (C]S). 1H NMR (CDCl3, 300 MHz):
d
1.28 (t, J ¼ 6.9 Hz,
3H, NCH2CH3), 1.91–2.03 (m, 3H, pyrrolidine), 2.90–2.96 (m, 1H,
pyrrolidine), 3.21 (br.s, 1H, NH exchanged with D2O), 3.52 (s, 3H,
NCH3), 3.73–3.80 (m,1H, pyrrolidine), 4.20–4.28 (m,1H, pyrrolidine),
4.45–4.56 (m, 3H, 1H pyrrolidine þ NCH2CH3), 7.81 (s, 1H, N]CH).
MS: m/z (%): 263 (Mþ, 90.07), 234 (100). Anal. Calcd. for C12H17N5S
(263.36): C, 54.73; H, 6.51; N, 26.59. Found: C, 55.18; H, 7.03; N, 26.39.
A mixture of 4a (0.5 g, 2 mmol) and hydrazine hydrate (98%,
5 mmol) in absolute ethanol (5 ml) was refluxed for 10 h, the
solvent was removed under vacuum and the residue was crystal-
lized from ethanol/water. Buff solid, 0.30 g, 56% yield, m.p. 163–
165 ꢂC. IR: ymax./cmꢀ1 3304–3133 (NH2 stretching), 3057 (CH
aromatic), 2934–2864 (CH aliphatic), 1636 (C]O), 1599 (NH2
bending), 1562–1496 (C]C). 1H NMR (DMSO-d6, 300 MHz):
d
1.52–
3.1.7.2. 2,5-Diethyl-1-imino-6-thioxo-1,2,8,9,10,10a-hexahydro-5H-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine 8b. Reddish brown solid,
0.09 g, 36% yield, m.p. 125–127 ꢂC. IR: ymax./cmꢀ1 3370 (NH
stretching), 2923–2853 (CH aliphatic), 1638 (C]N), 1575–1463
1.60 (m, 3H, pyrrolidine), 2.04–2.09 (m, 2H, pyrrolidine), 3.04–
3.11(m, 2H, pyrrolidine), 5.80–5.90 (m, 2H, NH2–C]N exchanged
with D2O), 6.10–6.18 (m, 2H]N–NH2 exchanged with D2O), 6.85–
7.35 (m, 5H, aromatic protons), 8.38 (s, 2H, NH2 exchanged with
D2O). MS: m/z (%): 285 (M ꢀ 1þ, 15.38), 55 (100). Anal. Calcd. for
C14H18N6O. 1.5 H2O (313.36): C, 53.67; H, 6.75; N, 26.82. Found:
C, 53.75; H, 6.09; N, 26.83.
(C]C), 1251 (C]S). 1H NMR (CDCl3, 300 MHz):
d
1.29 (t, J ¼ 6.5 Hz,
3H, NCH2CH3), 1.45 (t, J ¼ 7.2 Hz, 3H, NCH2CH3), 1.86–2.03 (m, 3H,
pyrrolidine), 2.93–3.00 (m, 1H, pyrrolidine), 3.70–3.80 (m, 1H, pyr-
rolidine), 4.05–4.26 (m, 4H, 1H pyrrolidine þ NCH2CH3 þ NH,
exchanged with D2O), 4.47–4.57(m, 3H,1H pyrrolidine þ NCH2CH3),
7.84 (s, 1H, N]CH). Anal. Calcd. for C13H19N5S (277.39): C, 56.29;
H, 6.90; N, 25.25. Found: C, 56.18; H, 6.89; N, 25.13.
3.1.6. General procedure for the preparation of 7a, b
A mixture of 4a or 4b (2 mmol) and catalytic amount of acetic
anhydride in Triethyl orthoformate (3 ml) is heated for 3 h, the
mixture was cooled to R.T and poured on ice water (30 ml). The
separated solid was filtered, washed with water and crystallized
from ethanol.
3.1.7.3. 5-Ethyl-1-imino-2-propyl-6-thioxo-1,2,8,9,10,10a-hexahydro-
5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine 8c. Brown solid, 0.10 g,
40% yield, m.p. 137–139 ꢂC. IR: ymax./cmꢀ1 3361 (NH stretching),
2963–2873 (CH aliphatic), 1637 (C]N), 1569–1472 (C]C), 1254
3.1.6.1. N-(4-Cyano-1-oxo-2-phenyl-1,2,4a,5,6,7-hexahydropyrrolo-
[1,2-c]pyrimidin-3-yl) formimidic acid ethyl ester 7a. White solid,
0.54 g, 88% yield, m.p. 103–104 ꢂC. IR: ymax./cmꢀ1 3053 (CH
aromatic), 2983–2894 (CH aliphatic), 2202 (CN), 1697 (C]O), 1628
(C]S). 1H NMR (CDCl3, 300 MHz):
d
0.98 (t, J ¼ 7.5 Hz, 3H,
NCH2CH2CH3), 1.25 (t, J ¼ 6.6 Hz, 3H, NCH2CH3), 1.76–1.97 (m, 5H,
3H pyrrolidine þ NCH2CH2CH3), 2.90–2.92 (m, 1H, pyrrolidine),
3.65–3.95 (m, 3H,1H pyrrolidine þ NCH2CH2CH3), 4.16–4.20 (m, 1H,
pyrrolidine), 4.42–4.50 (m, 3H, 1H pyrrolidine þ NCH2CH3), 5.40
(br.s,1H, NH, exchanged with D2O), 7.74 (s, 1H, N]CH). Anal. Calcd.
for C14H21N5S (291.42): C, 57.70; H, 7.26; N, 24.03. Found: C, 58.16;
H, 6.70; N, 23.65.
(C]N), 1613 (C]C). 1H NMR (CDCl3, 200 MHz):
d
0.98 (t, J ¼ 7 Hz,
3H, OCH2CH3, 1.87–2.01(m, 3H, pyrrolidine), 2.35–2.43 (m, 1H,
pyrrolidine), 3.47–3.57 (m, 1H, pyrrolidine), 3.64–3.74 (m, 1H,
pyrrolidine), 3.85 (q, J ¼ 7 Hz, 2H, OCH2CH3), 4.35–4.42 (m, 1H,
pyrrolidine), 7.08–7.34 (m, 5H, aromatic protons), 7.72 (s, 1H,
N]CH). MS: m/z (%): 310 (Mþ, 18.72), 56 (100%). Anal. Calcd. for
C17H18N4O2 (310.36): C, 65.79; H, 5.85; N, 18.05. Found: C, 65.90; H,
5.92; N, 18.41.
3.1.8. General procedure for the preparation of 9a–g
A mixture of 4a or 4b (2 mmol) and an appropriate aldehyde
(2.5 mmol) in glacial acetic acid (5 ml) was heated for 5 h, the
solvent was removed under vacuum and the residue was crystal-
lized from ethanol to produce the pure compounds 9 a–g.
3.1.6.2. N-(4-Cyano-2-ethyl-1-thioxo-1,2,4a,5,6,7-hexahydropyrrolo
[1,2-c]pyrimidin -3-yl) formimidic acid ethyl ester 7b. Grey solid,
0.48 g, 88% yield, m.p. 87–88 ꢂC. IR: ymax./cmꢀ1 2977–2829 (CH
aliphatic), 2196 (CN), 1634 (C]N), 1594 (C]C), 1248–1219 (C]S).
3.1.8.1. 3,5-Diphenyl-3,4,8,9,10,10a-hexahydro-2H,5H-pyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-1,6-dione 9a. White solid, 0.38 g, 54%
yield, m.p. 236–237 ꢂC. IR: ymax./cmꢀ1 3392 (NH stretching), 3061
(CH aromatic), 2977–2882 (CH aliphatic), 1725 (C]O), 1680 (C]O),
1H NMR (CDCl3, 300 MHz):
d
1.13 (t, J ¼ 6.9 Hz, 3H, NCH2CH3), 1.32
(t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.80–2.00 (m, 3H, pyrrolidine), 2.35–
2.40 (m, 1H, pyrrolidine), 3.78–3.83 (m, 2H, pyrrolidine), 3.98–4.03
(m, 1H, pyrrolidine), 4.11–4.18 (m, 2H, NCH2CH3), 4.30 (q, J ¼ 7.2 Hz,
2H, OCH2CH3), 7.89 (s, 1H, N]CH). 13C NMR (CDCl3, 75.45 MHz):
13.69 (NCH2CH3), 14.33 (OCH2CH3), 21.72, 32.57 (pyrrolidine
carbons), 42.95 (NCH2CH3), 52.53, 54.68 (pyrrolidine carbons),
64.28 (OCH2CH3), 69.96 (C]C), 117.41 (CN), 155 (N]CH),
1613–1492(C]C).1HNMR(DMSO-d6, 200 MHz):
d 1.88–2.64 (m, 4H,
pyrrolidine), 3.37–3.78 (m, 2H, pyrrolidine), 4.27–4.36 (m, 1H, pyr-
rolidine), 4.62–4.69 (m, 1H, NHCHNH), 4.79 (s, 1H, NH exchanged
with D2O), 4.85 (s, 1H, NH exchanged with D2O), 7.19–7.74 (m, 10H,
aromatic protons). Anal. Calcd. for C21H20N4O2 (360.42): C, 69.98;
H, 5.59; N, 15.54. Found: C, 69.69; H, 5.70; N, 15.44.

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4581
3.1.8.2. 3-(4-Chlorophenyl)-5-phenyl-3,4,8,9,10,10a-hexahydro-2H,-
5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6-dione 9b. White
solid, 0.30 g, 37% yield, m.p. 255–256 ꢂC. IR: ymax./cmꢀ1 3389 (NH
stretching), 3061 (CH aromatic), 2978–2880 (CH aliphatic), 1725
(C]O), 1680 (C]O), 1593–1493 (C]C). 1H NMR (DMSO-d6,
149.68 (N–(CO)–N), 163.30 (C–CO–NH). Anal. Calcd. for C21H19N5O4
(405.42): C, 62.22; H, 4.72; N,17.27. Found: C, 62.00; H, 4.78; N,17.29.
3.1.8.7. 3-(4-Nitrophenyl)-5-phenyl-3,4,8,9,10,10a-hexahydro-2H,5H-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6-dione 9g. Orange red
solid, 0.62 g, 78% yield, m.p. 245–246 ꢂC. IR: ymax./cmꢀ1 3392 (NH
stretching), 3063 (CH aromatic), 2978–2881 (CH aliphatic), 1725
(C]O), 1681 (C]O) 1596–1493 (C]C). 1H NMR (DMSO-d6,
200 MHz):
d 1.88–2.37 (m, 4H, pyrrolidine), 3.47–3.51 (m, 2H,
pyrrolidine), 4.32–4.34 (m, 2H, 1H pyrrolidine þ NHCHNH), 4.81
(s, 1H, NH exchanged with D2O), 4.88 (s, 1H, NH exchanged with
D2O), 7.20–7.65 (m, 9H, aromatic protons). Anal. Calcd. for
C21H19ClN4O2.H2O (412.88): C, 61.09; H, 5.13; N, 13.57. Found: C,
60.86; H, 5.05; N, 13.66.
300 MHz):
d 1.86–2.49 (m, 4H, pyrrolidine), 3.43–3.52 (m, 2H, pyr-
rolidine), 4.31–4.42 (m, 2H, 1H pyrrolidine þ NHCHNH), 4.79 (s, 1H,
NH exchanged with D2O), 4.83 (s, 1H, NH exchanged with D2O),
7.15–7.48 (m, 9H, aromatic protons). MS: m/z (%): 403 (M ꢀ 2þ, 4.41),
401 (100), 283 (12.36). Anal. Calcd. for C21H19N5O4 (405.42):
C, 62.22; H, 4.72; N, 17.27. Found: C, 62.19; H, 4.74; N, 17.28.
3.1.8.3. 3-(4-Methoxyphenyl)-5-phenyl-3,4,8,9,10,10a-hexahydro-
2H,5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6-dione 9c. White
solid, 0.27 g, 35% yield, m.p. 255–256 ꢂC. IR: ymax./cmꢀ1 3389 (NH
stretching), 3061(CH aromatic), 2978–2880 (CH aliphatic), 1725
(C]O), 1680 (C]O), 1593–1493 (C]C). 1H NMR (DMSO-d6,
3.1.9. 3,5-Diphenyl-1-propyl-8,9,10,10a-tetrahydro-5H-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-one 13a
300 MHz):
d
1.88–2.38 (m, 4H, pyrrolidine), 3.40–3.60 (m, 5H, 2H
A mixture of 12 (0.5 g, 1.32 mmol) and an ethanolic solution of
propylamine (5 ml) was refluxed for 5 h, ethanol was removed under
vacuum and the residue was crystallized from ethanol/water to give
dark brown solid, 0.33 g, 63% yield, mp 102 ꢂC. IR: ymax./cmꢀ1 3421
(NH stretching), 3061 (CH aromatic), 2957–2852 (CH aliphatic),1676
(CO stretching), 1657 (NH bending), 1591–1545 (C]C). 1H NMR
pyrrolidine þ OCH3), 4.23–4.30 (m, 2H, 1H pyrrolidine þ NHCHNH),
4.78 (s, 1H, NH exchanged with D2O), 4.82 (s, 1H, NH exchanged
with D2O), 7.18–7.46 (m, 9H, aromatic protons). Anal. Calcd. for
C22H22N4O3 (390.45): C, 67.68; H, 5.68; N, 14.35. Found: C, 67.79; H,
5.33; N, 14.32.
(CDCl3, 300 MHz):
d
1.05 (t, J ¼ 7.4 Hz, 3H, NCH2CH2CH3), 1.65–2.15
3.1.8.4. 5-Ethyl-3-(4-methoxyphenyl)-6-thioxo-3,4,8,9,10,10a-hexahydro-
2H,5H-pyrimido[5,4-e]pyrrolo[1,2-c] pyrimidin-1-one 9d. Yellow
solid, 0.25 g, 35% yield, m.p. 123–125 ꢂC. IR: ymax./cmꢀ1 3402 (NH
stretching), 3069 (CH aromatic), 2972–2843 (CH aliphatic), 1712
(C]O), 1500–1447 (C]C), 1255 (C]S). 1H NMR (CDCl3, 200 MHz):
(m, 6H, 3H pyrrolidine þ NCH2CH2CH3 þ NH exchanged with D2O),
2.55–2.60 (m, 1H, pyrrolidine), 3.40–3.50 (m, 1H, pyrrolidine), 3.55–
3.75 (m, 2H, NCH2CH2CH3), 4.00–4.10 (m,1H, pyrrolidine), 4.54–4.60
(m, 1H, pyrrolidine), 7.27–8.08 (m, 10H, aromatic protons). Anal.
Calcd. for C24H25N5O (399.50): C, 72.16; H, 6.31; N, 17.53. Found: C,
71.61; H, 6.34; N, 17.36.
d
1.21 (t, J ¼ 6.8 Hz, 3H, NCH2CH3), 1.93–2.21 (m, 3H, pyrrolidine),
2.55–2.63 (m, 1H, pyrrolidine), 3.63 (s, 1H, NH exchanged with
D2O), 3.70 (s, 1H, NH exchanged with D2O), 3.82–4.36 (m, 9H, 3H
pyrrolidine þ NHCHNH þ OCH3 þ NCH2CH3), 6.97–7.46 (m, 2H,
aromatic protons), 8.04–8.20 (m, 2H, aromatic protons). MS: m/z
(%): 357 (M ꢀ 1þ, 9.92), 355 (100). Anal. Calcd. for C18H22N4O2S
(358.47): C, 60.31; H, 6.19; N, 15.63. Found: C, 60.36; H, 6.21; N,
15.24.
3.1.10. General procedure for the preparation of 13b, c and 14
To the mixture of 12 (0.5 g, 1.32 mmol) and anhydrous potas-
sium carbonate (0.23 g, 1.66 mmol) in dry DMF (5 ml) equimolar
amount (1.32 mmol) of the corresponding amine was added. The
reaction mixture was heated at 100 ꢂC for 7 h, cooled to RT, and
poured on ice/water (30 ml). The separated solid was filtered,
washed with water and recrystallized from ethanol.
3.1.8.5. 5-Ethyl-3-(3-hydroxy-4-methoxyphenyl)- 6-thioxo-3,4,8,9,-
10,10a-hexahydro-2H, 5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
3.1.10.1. 3,5-Diphenyl-1-pyrrolidin-1-yl-8,9,10,10a-tetrahydro-5H-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-one 13b. Buff solid, 0.38 g,
70% yield, m.p. 205–206 ꢂC (decomposition). IR: ymax./cmꢀ13054 (CH
aromatic), 2948–2847 (CH aliphatic), 1678 (CO stretching), 1587–
1-one 9e. Yellow solid, 0.21 g, 28% yield, m.p. 126–128 ꢂC. IR: ymax.
/
cmꢀ1 3504 (OH stretching), 3374 (NH stretching), 3050 (CH
aromatic), 2972–2882 (CH aliphatic), 1699 (C]O), 1563–1492
(C]C), 1254 (C]S). 1H NMR (CDCl3, 300 MHz):
d
1.24 (t, J ¼ 6.9 Hz,
1539 (C]C). 1H NMR (CDCl3, 200 MHz):
d 1.60–2.15 (m, 7H, 3H
3H, NCH2CH3), 1.91–2.64 (m, 4H, pyrrolidine), 3.62 (s, 1H, NH
exchanged with D2O), 3.67 (s, 1H, NH exchanged with D2O), 3.80–
4.11 (m, 5H, 2H pyrrolidine þ OCH3), 4.21–4.41 (m, 4H, 1H pyrroli-
dine þ NHCHNH þ NCH2CH3), 6.05 (s, 1H, OH exchanged with D2O),
7.00–7.20 (m, 2H, aromatic protons), 8.20 (s, 1H, aromatic proton).
Anal. Calcd. for C18H22N4O3S (374.47): C, 57.74; H, 5.92; N, 14.96.
Found: C, 57.82; H, 5.78; N, 14.68.
pyrrolidine þ 4H pyrrolidinyl), 2.40–2.50 (m, 1H, pyrrolidine), 3.40–
3.50 (m, 3H, 1H pyrrolidine þ 2H pyrrolidinyl), 3.75–3.85 (m, 2H
pyrrolidinyl), 4.05–4.20 (m, 1H, pyrrolidine), 4.65–4.80 (m, 1H, pyr-
rolidine), 7.26–8.08 (m, 10H, aromatic protons). MS: m/z, (%): 411
(Mþ, 100), 383 (53.06), 368 (74.46), 355 (27.07). Anal. Calcd. for
C25H25N5O (411.51): C, 72.97; H, 6.12; N, 17.02. Found: C, 72.55; H,
6.02; N, 17.17.
3.1.8.6. 3-(2-Nitrophenyl)-5-phenyl-3,4,8,9,10,10a-hexahydro-
3.1.10.2. 1-Morpholin-4-yl-3,5-diphenyl-8,9,10,10a-tetrahydro-5H-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-one 13c. Greyish brown
solid, 0.35 g, 63% yield, m.p. 179–180 ꢂC (decomposition). UV
2H,5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6-dione
9f. Buff
solid, 0.35 g, 45% yield, m.p. 242–244 ꢂC. IR: ymax./cmꢀ1 3388 (NH
stretching), 3062 (CH aromatic), 2978–2881(CH aliphatic), 1724
(C]O), 1681 (C]O), 1596–1493 (C]C). 1H NMR (DMSO-d6,
(CHCl3), Conc. 3.50 ꢃ 10ꢀ5 M, nm: 225 (log
3: 3.71), 258 (log 3: 4.43).
IR: ymax./cmꢀ1 3061 (CH aromatic), 2954–2851 (CH aliphatic), 1686
300 MHz):
d 1.86–2.34 (m, 4H, pyrrolidine), 3.50–3.52 (m, 2H, pyr-
(CO stretching), 1563–1545 (C]C). 1H NMR (CDCl3, 200 MHz):
rolidine), 4.30–4.38 (m, 2H, 1H pyrrolidine þ NHCHNH), 4.79 (s, 1H,
NH exchanged with D2O), 4.84 (s, 1H, NH exchanged with D2O),
7.19–7.47 (m, 9H, aromatic protons). 13C NMR (DMSO-d6,
75.45 MHz): 22.27, 31.15, 40.71 (pyrrolidine carbons), 46.36
(NHCHNH), 53.20 (pyrrolidine), 114.86 (O]C–C]C), 128.05, 128.10,
128.61, 128.78, 128.95 (aromatic carbons), 135.61 (O]C–C]C–NH),
d 1.80–2.00 (m, 3H, pyrrolidine), 2.60–2.70 (m, 2H, pyrrolidine),
3.16–3.20 (m, 2H, morpholino NCH2), 3.42–3.50 (m, 3H, 1H pyrro-
lidine þ morpholino NCH2), 3.75–3.90 (m, 4H, morpholino OCH2),
4.60–4.70 (m, 1H, pyrrolidine), 7.18–7.97 (m, 10H, aromatic
protons). Anal. Calcd. for C25H25N5O2 (427.51): C, 70.24; H, 5.89; N,
16.38. Found: C, 69.71; H, 5.62; N, 16.52.

4582
K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
3.1.10.3. 5,7-Diphenyl-2,7,10,11,12,12a-hexahydropyrrolo[1,2-c]imi-
dazo[1‘,2‘:1,6]pyrimido[5,4-e]pyrimidine-3,8-dione 14. Light brown
solid, 0.29 g, 53% yield, m.p. 230 ꢂC (decomposition). IR: ymax./cmꢀ1
3063 (CH aromatic), 2921–2851 (CH aliphatic), 1655 (2 CO), 1595–
467 (96.82), 421 (45.96), 354 (75.71), 329 (58.48). Anal. Calcd.
for C27H25N7O3 (495.55): C, 65.44; H, 5.09; N, 19.79. Found: C,
65.45; H, 5.56; N, 19.11.
1507 (C]C). 1H NMR (CDCl3, 300 MHz):
d
1.70–2.15 (m, 3H, pyr-
3.1.12. General method for the preparation of 19, 21, and 23
A mixture of 16 (0.37 g, 1 mmol), p-toluenesulphonyl chloride,
ethyl chloroformate or chloroacetyl chloride (1.25 mmol) and
triethylamine (1 mmol) in dry benzene (5 ml) was refluxed for 6 h;
the solvent was removed under vacuum. The residue was triturated
with water (15 ml) and filtered, washed with water and crystallized
from ethanol.
rolidine), 2.65–2.75 (m, 1H, pyrrolidine), 3.03 (s, 2H, CH2)
3.35–3.45 (m, 1H, pyrrolidine), 3.90–3.95 (m, 1H, pyrrolidine),
4.75–4.80 (m, 1H, pyrrolidine), 7.27–8.09 (m, 10H, aromatic
protons). MS: m/z (%) ¼ 397.24 (Mþ, 1.95), 357.10 (100%). Anal.
Calcd. for C23H19N5O2 (397.43): C, 69.51; H, 4.82; N, 17.62. Found: C,
69.32; H, 5.31; N, 17.92.
3.1.12.1. 3,5-Diphenyl-1-p-tolylsulphonylhydrazino-8,9,10,10a-tetra-
hydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-one 19. Pink solid,
0.37 g, 72% yield, m.p. 145–147 ꢂC (decomposition). IR: ymax./cmꢀ1
3333 (NH stretching), 3063 (CH aromatic), 2923–2852 (CH
aliphatic), 1664 (C]O), 1589–1567 (C]C), 1321, 1152 (SO2 stretch-
3.1.11. General procedure for the preparation of 17 and 18a, b
To a solution of 16 (0.37 g, 1 mmol) in dry DMF (5 ml) was
added acetylacetone (10 mmol), ethoxymethylene malononitrile
(0.12 g, 1 mmol) or ethyl ethoxymethylene cyanoacetate (0.16 g,
1 mmol). 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,
washed with ethanol to produce 17, 18a or 18b, respectively, in
a pure form.
ing). 1H NMR (CDCl3, 300 MHz):
d 1.70–2.05 (m, 3H, pyrrolidine),
2.16 (s, 3H, CH3), 2.70–2.80 (m, 1H, pyrrolidine), 3.45–3.50 (m, 1H,
pyrrolidine), 3.97–4.05 (m, 1H, pyrrolidine), 4.52–4.57 (m, 1H, pyr-
rolidine), 6.80 (s, NH exchanged with D2O), 7.01 (d, J ¼ 8.1 Hz, 2H, o-
CH3), 7.20–7.48 (m, 8H, aromatic protons), 7.73–7.80 (m, 4H,
aromatic protons), 8.05 (s, NH exchanged with D2O). MS: m/z (%):
526 (Mþ, 1.64), 341 (100). Anal. Calcd. for C28H26N6O3S (526.55): C,
63.87; H, 4.98; N, 15.96. Found: C, 64.12; H, 4.67; N, 16.14.
3.1.11.1. 1-(3,5-Dimethyl-pyrazol-1-yl)–3,5–diphenyl-8,9,10,10a-tet-
rahydro-5H-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-one 17. Dark
brown solid, 0.34 g, 79% yield, m.p. 201–203 ꢂC. UV (CHCl3), Conc.
2.29 ꢃ 10ꢀ5 M, nm: 228.5 (log
3: 3.89), 260 (log 3: 4.61), 303 (log 3:
4.14). IR: ymax./cmꢀ1 3062 (CH aromatic), 2923–2853 (CH
3.1.12.2. N0-(3,5-Diphenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]
aliphatic), 1686 (CO stretching), 1583–1554 (C]C). 1H NMR (CDCl3,
pyrrolo[1,2-c]pyrimidin-6-one-1-yl)-ethoxyhydrazide
21. Yellow
200 MHz):
d 1.46–1.87 (m, 4H, pyrrolidine), 2.30 (s, 3H, CH3), 2.53
solid, 0.39 g, 89% yield, m.p. 226–228 ꢂC. IR: ymax./cmꢀ1 3358, 3328
(NH stretching), 3059 (CH aromatic), 2922–2852 (CH aliphatic),
1748 (C]O ester), 1645 (C]O), 1591–1562 (C]C). 1H NMR (CDCl3,
(s, 3H, CH3), 3.42–3.55 (m, 1H, pyrrolidine), 3.80–3.90 (m, 1H,
pyrrolidine), 5.15–5.22 (m, 1H, pyrrolidine), 6.07 (s, 1H, pyrazole),
7.26–8.05 (m, 10H, aromatic protons). MS: m/z, (%): 436 (Mþ,
26.70), 421 (9.42), 394 (100), 329 (21.80). Anal. Calcd. for
C26H24N6O.H2O (454.53): C, 68.71; H, 5.77; N, 18.49. Found: C,
68.81; H, 5.79; N, 18.72.
300 MHz):
d
1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 2.00–2.20 (m, 3H,
pyrrolidine), 2.60–2.70 (m, 1H, pyrrolidine), 3.40–3.50 (m, 1H,
pyrrolidine), 3.95–4.05 (m, 1H, pyrrolidine), 4.25 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 4.65–4.75 (m, 1H, pyrrolidine), 6.50 (s, 1H, NH
exchanged with D2O), 6.85 (s, 1H, NH exchanged with D2O), 7.27–
8.02 (m, 10H, aromatic protons). MS: m/z (%): 444 (Mþ, 28.67), 342
(100). Anal. Calcd. for C24H24N6O3 (444.49): C, 64.85; H, 5.44; N,
18.91. Found: C, 64.81; H, 5.54; N, 18.68.
3.1.11.2. 5-Amino-1-(6-oxo-3,5-diphenyl-5,6,8,9,10,10a-hexahy-
dropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-1-yl)-1H-pyrazole-4-car-
bonitrile 18a. Brown solid, 0.31 g, 70% yield, m.p. 328–329 ꢂC. IR:
ymax./cmꢀ1 3430, 3259 (NH2 stretching), 3064 (CH aromatic), 2922–
2852 (CH aliphatic), 2215 (CN), 1664 (CO stretching), 1607 (NH
bending), 1550–1512 (C ¼ C). 1H NMR (DMSO-d6, 300 MHz):
3.1.12.3. 6,8-Diphenyl-11,12,13,13a-tetrahydro-2H-pyrrolo[1,2-c]
[1,2,4]triazino[40,30:1,6]pyrimido[5,4-e]pyrimidine-3,8-dione
23. Grey solid, 0.33 g, 80% yield, m.p. 230 ꢂC (decomposition). IR:
ymax./cmꢀ1 3214 (NH stretching), 3063 (CH aromatic), 2924–2853
(CH aliphatic), 1674 (2 C]O), 1577–1548 (C]C). 1H NMR (CDCl3,
d
1.60–1.95 (m, 4H, pyrrolidine), 3.30–3.40 (m, 1H, pyrrolidine),
3.65–3.70 (m, 1H, pyrrolidine), 4.95–5.05 (m, 1H, pyrrolidine), 7.14
(s, 2H, NH2 exchanged with D2O), 7.34–7.93 (m, 11H, 10 aromatic
protons þ 1H pyrazole). Anal. Calcd. for C25H20N8O (448.49): C,
66.95; H, 4.49; N, 24.98. Found: C, 66.77; H, 4.95; N, 24.90.
300 MHz):
d 1.95–2.30 (m, 3H, pyrrolidine), 2.50–2.60 (m, 1H, pyr-
rolidine), 3.50–3.60 (m, 1H, pyrrolidine), 3.75–3.85 (m, 1H, pyrroli-
dine), 4.04 (s, 2H, CH2), 4.79–4.83 (m, 1H, pyrrolidine), 7.27–7.98 (m,
10H, aromatic protons), 9.60 (s, 1H, NH exchanged with D2O). 13C
NMR (CDCl3, 75.45 MHz): 21.93, 29.65, 41.12 (pyrrolidine carbons),
45.22 (CH2), 55.26 (pyrrolidine carbon), 120.94 (C]C–N), 125.89,
126.18, 127.92, 128.08, 128.40, 128.89, 129.57, 131.26 (aromatic
carbons), 136.59 (C]C–N and N–C]N), 150.84 (N–CO–N), 159.78
(NHCOCH2), 172.06 (NH–N]C–N). Anal. Calcd. for C23H20N6O2
(412.44): C, 66.98; H, 4.89; N, 20.38. Found: C, 66.52; H, 5.22; N, 20.51.
3.1.11.3. 5-Amino-1-(6-oxo-3,5-diphenyl-5,6,8,9,10,10a-hexahy-
dropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-1-yl)-1H-pyrazole-4-
carboxylic acid ethyl ester 18b. Buff solid, 0.35 g, 70% yield, m.p.
238–239 ꢂC. IR: ymax./cmꢀ1 3451, 3343 (NH2 stretching), 3061
(CH aromatic), 2920–2850 (CH aliphatic), 1690 (CO stretching),
1608 (NH bending), 1581–1542(C]C). 1H NMR (CDCl3,
300 MHz):
d
1.31 (t, J ¼ 7.2 Hz, 3H, COOCH2CH3), 1.60–2.25 (m,
4H, pyrrolidine), 3.35–3.45 (m, 1H, pyrrolidine), 3.85–4.00 (m,
1H, pyrrolidine), 4.26 (q, J ¼ 7.2 Hz, 2H, COOCH2CH3), 5.20–5.30
(m, 1H, pyrrolidine), 6.75 (s, 2H, NH2 exchanged with D2O),
7.18–7.85 (m, 11H, 10 aromatic protons þ 1H pyrazole). 13C NMR
(CDCl3, 75.45 MHz): 14.29 (CH2CH3), 20.83, 29.39, 45.15, 55.30
(pyrrolidine carbons), 59.58 (CH2CH3), 95.58 (C]C–NH2), 104.06
(C]C), 125.76, 127.66, 128.30, 128.63, 129.36, 131.05, 135.75,
136.53 (aromatic carbons), 141.38 (pyrazole carbon), 150.33 (N–
(CO)–N), 152.25 (C–NH2), 154.65 (C-phenyl), 159.48 (C]O ester),
161.47 (C]C), 163.79 (C-pyrazole). MS: m/z, (%): 495 (Mþ, 100),
3.1.13. General procedure for the preparation of 20a, b
A mixture of 16 (0.50 g, 1.34 mmol) and the corresponding acid
chloride (1.34 mmol) in dry pyridine (5 ml) was stirred at RT for 6 h.
The reaction mixture was poured on ice/water (40 ml). The sepa-
rated solid was filtered, washed with water and crystallized from
ethanol.
3.1.13.1. N0-(3,5-Diphenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyr-
rolo[1,2-c]pyrimidin-6-one-1-yl)benzoic acid hydrazide 20a. Buff

K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
4583
solid, 0.50 g, 78% yield, m.p. 230–232 ꢂC. IR: ymax./cmꢀ1 3276
measured 4428 of which 1747 reflections with threshold expression
(NH stretching), 3063 (CH aromatic), 2923–2852 (CH aliphatic),
I > 3s(I) were used in the structural analysis. Convergence for 307
1669 (2C]O), 1591–1567 (C]C). 1H NMR (CDCl3, 300 MHz):
d
1.59
variable parameters by least-squares refinement on F2 with w ¼ 1/
(s, 1H, NH exchanged with D2O), 1.97–2.17 (m, 3H, pyrrolidine),
2.80–2.84 (m, 1H, pyrrolidine), 3.48–3.54 (m, 1H, pyrrolidine),
3.97–4.06 (m, 1H, pyrrolidine), 4.71–4.74 (m, 1H, pyrrolidine), 7.23–
7.99 (m, 15H, aromatic protons), 9.26 (s, 1H, NH exchanged with
D2O). 13CNMR (CDCl3, 75.45 MHz): 21.72, 29.67, 45.05, 54.06 (pyr-
rolidine carbons), 93.11 (C]C), 125.99, 127.67, 128.15, 128.65,
128.93,129.76,130.61,132.32,136.90 (aromatic carbons),151.40 (N–
(CO)–N), 156.50 (C-phenyl), 157.59 (C]O benzoyl), 161.99 (C]C),
164.89 (C–NH). MS: m/z (%): 476.25 (Mþ, 3.75), 356.20 (100). Anal.
Calcd. for C28H24N6O2 (476.54): C, 70.57; H, 5.08; N, 17.64. Found: C,
70.22; H, 5.58; N, 17.42.
[
s
2(Fo2) þ 0.10000 Fo2]. The final agreement factors were R ¼ 0.045 and
wR ¼ 0.087 with a goodness-of-fit of 1.422.
3.3. Pharmacological studies
3.3.1. Animals
The albino rats and mice were obtained from the animal house
of National Research Center, Dokki, Cairo. The animals were accli-
matized to the lab conditions for two days with free access to the
food and water. On the day before the experiment, the food was
withdrawn, but the animals were allowed free access to water.
3.1.13.2. N0-(3,5-diphenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyr-
rolo[1,2-c]pyrimidin-6-one-1-yl)
nicotinic acid hydrazide
3.3.2. Preparation of test samples
20b. Reddish brown solid, 0.48 g, 75% yield, m.p. 245–246 ꢂC. UV
All compounds in 0.029 mmol/kg concentration were sus-
pended in saline solution with the aid of few drops of tween 80, and
these test samples were given orally to the animals by the oral
stomach tube. The control group animals received the same
experimental handling as those of the test groups except for the
drug treatment. Indomethacin (10 mg/kg, 0.029 mmol) was used as
a reference drug.
(CHCl3), Conc. 2.09 ꢃ 10ꢀ5 M, nm: 227.5 (log
3: 4.02), 257 (log 3:
4.40), 308 (log 3
: 3.93). IR: ymax./cmꢀ1 3415–3271 (NH stretching),
3060 (CH aromatic), 2923–2853(CH aliphatic), 1660 (2C]O), 1590–
1565 (C]C). 1H NMR (CDCl3–300 MHz):
d
1.90–2.20 (m, 4H, 3H
pyrrolidine þ NH exchanged with D2O), 2.55 (br.s., 1H, pyrrolidine),
3.48–3.52 (m, 1H, pyrrolidine), 3.75–3.85 (m, 1H, pyrrolidine), 4.80
(br.s., 1H, pyrrolidine), 7.20–7.50 (m, 10H, aromatic protons), 7.85
(br.s., 1H, C50), 8.12 (br.s., 1H, C40), 8.74 (br.s., 1H, C60), 9.04 (br.s., 1H,
C20), 10.50 (br.s., 1H, NH amide exchanged with D2O). Anal. Calcd.
for C27H23N7O2 (477.53): C, 67.91; H, 4.85; N, 20.53. Found: C, 68.14;
H, 4.96; N, 20.77.
3.3.3. Analgesic activity
Analgesic activity was measured by the acetic acid-induced
writhing test in mice [36–39]. Six albino mice of either sex (18–
22 g) were used in each group. One hour after oral administration of
a suspension of 0.029 mmol/kg of the test compounds or indo-
methacin, 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 writhings by any test compound as compared to control
3.1.14. 5,7-Diphenyl-10,11,12,12a-tetrahydro-2H-pyrrolo[1,2-c]
[1,2,4]triazolo[40,30:1,6]pyrimido[5,4-e]pyrimidine-3,8-dione 22
To a mixture of 16 (0.37 g, 1 mmol) and sodium methoxide
(0.05 g, 1 mmol) in methanol (5 ml) was added ethyl chloroformate
(1.25 mmol), the reaction was refluxed for 12 h, and the solvent was
removed under vacuum. The residue was triturated with water
(15 ml) and filtered, washed with water and purified by column
chromatography using silica gel as stationary phase and chloroform
as mobile phase to give brown solid, 0.32 g, 80% yield, m.p. 182–
183 ꢂC. This compound was also obtained by heating a solution of
21 in dry DMF for 12 h then poured on ice water. The obtained solid
was filtered, washed with water and crystallized from ethanol to
give reddish brown solid, m.p. 180–182 ꢂC. IR: ymax./cmꢀ1 3420 (NH
stretching), 3053–3018 (CH aromatic), 2926–2852 (CH aliphatic),
animals was considered as
a positive analgesic response.
Percentage protection was calculated using the following formula,
where n is the average number of writhings in control group and n0
is average number of writhings in treated group.
% Protection ¼ ½ðn ꢀ n0Þꢄ ꢃ 100
3.3.4. Anti-inflammatory activity
Anti-inflammatory activity screening was carried out using
carrageenan-induced paw edema in albino rats [40–43]. Adult
albino rats of either sex weighing 120–150 g were divided into 38
groups of 6 animals each. One hour after the oral administration of
the test compounds or the reference drug (indomethacin), each rat
was injected with 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 successive time
intervals (1, 2, 3 and 4 h) after induction of the inflammation and
compared with the initial hind paw thickness of each rat for
determining the edema thickness. The anti-inflammatory activity
was expressed as percentage inhibition of edema and was calcu-
lated by the following equation:
1692 (2C]O), 1586–1556 (C]C). 1H NMR (CDCl3, 300 MHz):
d 1.90
(br.s., 1H, NH exchanged with D2O), 2.07–2.12 (m, 3H, pyrrolidine),
2.65–2.75 (m, 1H, pyrrolidine), 3.60–3.67 (m, 1H, pyrrolidine),
3.70–3.77 (m, 1H, pyrrolidine), 4.70–4.80 (m, 1H, pyrrolidine), 7.26–
8.11 (m, 10H, aromatic protons). MS: m/z (%): 398 (Mþ, 11.1), 341
(17.8), 77 (100). Anal. Calcd. for C22H18N6O2 (398.42): C, 66.32; H,
4.55; N, 21.09. Found: C, 66.52; H, 4.52; N, 21.26.
3.2. Single crystal X-ray crystallographic data of 18a
Compound 18a was recrystallized as brown cubic crystals from
ethanol. The crystallographic data were collected at T ¼ 298 K on
a Kappa CCD Enraf Nonius FR 590 diffractometer using a graphite
monochromator with Mo-Ka radiation (
l
¼ 0.71073 Å). The crystal
% Inhibition ¼ 100 ꢃ ½Vc ꢀ Vt=Vcꢄ
structure was determined by SIR92 [48] and refined by maXus [49]
(Bruker Nonius, Delft and MacScience, Japan). Chemical formula
C25H20N8O, Mr ¼ 448.490, monoclinic, crystallizes in space group P21/
C; cell lengths ‘‘a ¼ 10.2720 (4), b ¼ 20.8375 (12), c ¼ 12.3140 (9) Å’’;
where Vc is the mean of paw thickness of rats after administration
of carrageenan in the positive control group, Vt is the mean of paw
thickness of rats after administration of the tested compounds or
the reference drug. Mean values of treated groups were compared
with mean values of a control group and analyzed using statistical
methods.
cell angles ‘‘
a
¼ 90.00,
b
¼ 13.(18) ꢃ 1010
,
g
¼ 90.00ꢂ, V ¼ 2169.8 (2) Å3,
Z ¼ 4, Dc ¼ 1.373 mg/m3,
q
values 2.910–21.726ꢂ, absorption coefficient
m
(Mo-Ka) ¼ 0.09 mmꢀ1
,
F(000) ¼ 720. The unique reflections

4584
K.M. Amin et al. / European Journal of Medicinal Chemistry 44 (2009) 4572–4584
R. Kumar, R.A. Mook Jr., G. Moorthy, S. Patnaik, Bioorg. Med. Chem. Lett. 19
Potency of the tested compounds was calculated relative to
indomethacin (reference standard) treated group according to the
following equation:
(2009) 360–364.
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3.3.5. Gastric–ulcerogenic effect
Five hours after the oral treatment of rats with the tested
compounds for anti-inflammatory activity tests, they were killed
under deep ether anaesthesia and their stomachs were removed.
Then the stomach of each rat was opened through great curvature
and examined for lesions or bleedings.
3.3.6. Statistical analysis of data
Data obtained from animal experiments were expressed as
mean ꢁ standard error (ꢁSEM). Statistical differences between the
treatments and the control were tested by one-way analysis of
variance (ANOVA) followed by post hoc test using SPSS 11.0 soft-
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