Potential anti-inflammatory activity and ulcerogenicity study of some novel pyrimido[4′,5′:4,5]pyrimido[1,6-a]azepine derivatives

Article abstract of DOI:10.1007/s00044-010-9545-5

A series of novel tricyclic pyrimido[4′,5′: 4,5]pyrimido[1,6-a]azepine derivatives were synthesized using the starting compound 3-amino-1-oxo-2-phenyl-5-(pyrrolidin-1-yl)-1,2,4a,5,6,7,8,9- octahydropyrimido[1,6-a] azepine-4-carbonitrile 4. This series includes the 3-aryl derivatives 6a, b, the 3-cycloaminoalkyl derivatives 8a-f, the 3-mercaptomethyl derivatives 10 and 11a, b, the 2-cycloaminomethyl derivatives 13a-c, the 1-cycloamino derivatives 15a-c and the 1-amino derivative 16. The structures of the newly synthesized compounds were elucidated by IR, ¹H NMR, ¹³C NMR, mass spectroscopy and elemental analyses. The anti-inflammatory activity of all newly synthesized compounds was evaluated using the carrageenan-induced paw oedema test in rats using diclofenac sodium as the reference drug. Ulcer indices for the most active compounds were calculated. The 3-mercaptomethylacetic acid derivative 10 was the most active compound, showing activity comparable to diclofenac sodium with minimal ulcerogenic effect while the rest of the tested compound exhibited moderate anti-inflammatory activity. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9545-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:395–405
DOI 10.1007/s00044-010-9545-5
ORIGINAL RESEARCH
Potential anti-inflammatory activity and ulcerogenicity study
of some novel pyrimido[40,50:4,5]pyrimido[1,6-a]azepine
derivatives
•
Nehad A. El-Sayed Fadi M. Awadallah
•
•
Nashwa A. Ibrahim Mohamed T. El-Saadi
Received: 6 November 2010 / Accepted: 15 December 2010 / Published online: 7 January 2011
Ó Springer Science+Business Media, LLC 2010
Abstract A series of novel tricyclic pyrimido[40,50:
4,5]pyrimido[1,6-a]azepine derivatives were synthesized
using the starting compound 3-amino-1-oxo-2-phenyl-5-
(pyrrolidin-1-yl)-1,2,4a,5,6,7,8,9-octahydropyrimido[1,6-a]
azepine-4-carbonitrile 4. This series includes the 3-aryl
derivatives 6a, b, the 3-cycloaminoalkyl derivatives 8a–f,
the 3-mercaptomethyl derivatives 10 and 11a, b, the
2-cycloaminomethyl derivatives 13a–c, the 1-cycloamino
derivatives 15a–c and the 1-amino derivative 16. The
structures of the newly synthesized compounds were elu-
cidated by IR, 1H NMR, 13C NMR, mass spectroscopy and
elemental analyses. The anti-inflammatory activity of all
newly synthesized compounds was evaluated using the
carrageenan-induced paw oedema test in rats using dic-
lofenac sodium as the reference drug. Ulcer indices for the
most active compounds were calculated. The 3-mercap-
tomethylacetic acid derivative 10 was the most active
compound, showing activity comparable to diclofenac
sodium with minimal ulcerogenic effect while the rest of
the tested compound exhibited moderate anti-inflammatory
activity.
Introduction
Inflammation is the complex biological response of vascu-
lar tissues to harmful stimuli, such as pathogens, damaged
cells or irritants (Ferrero-Miliani et al., 2007). Acute
inflammation is the initial response of the body to harmful
stimuli and is achieved by the increased movement of
plasma and leukocytes from the blood into the injured tis-
sues. A cascade of biochemical events propagates and
matures the inflammatory response, involving the local
vascular system, the immune system, and various cells
within the injured tissue. On the other hand, chronic
inflammation leads to a progressive shift in the type of cells
which are present at the site of inflammation and is char-
acterized by simultaneous destruction and healing of the
tissue from the inflammatory process (Lacerda et al., 2009).
Non-steroidal anti-inflammatory drugs (NSAIDs) are
commonly prescribed for the treatment of acute and
chronic inflammation, pain and fever. However, long term
usage of NSAIDs is associated with significant side effects
of gastrointestinal lesions, bleeding and nephrotoxicity
(Alagarsamy et al., 2007).
Keywords Pyrimidopyrimidoazepine ꢀ
Anti-inflammatory activity ꢀ Ulcerogenicity
Therefore, there is an unrelenting effort to discover new
and safe NSAIDs. In this field, several studies were
reported on the nitrogen bridgehead condensed pyrimidine
derivatives which unveil new potent anti-inflammatory
agents belonging to the pyrimido[40,50:4,5]pyrimido[1,6-
a]azepines I–III (Fig. 1) (Ebeid et al., 1991; El-Sayed
et al., 1993, 2003). A later study revealed that the intro-
duction of a morpholino group at position 12 of the tricy-
clic system afforded derivatives that were found markedly
more potent than unsubstituted ones, as exemplified by
compound IV and at the same time they are of minimal
ulcerogenic potential (El-Sayed et al., 2006). In continua-
tion to our previous reports and for further exploration of
N. A. El-Sayed ꢀ F. M. Awadallah (&)
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Cairo University, Kasr El-Eini Street, 11562 Cairo, Egypt
e-mail: fadi_mae@hotmail.com
N. A. Ibrahim ꢀ M. T. El-Saadi
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Beni-Sweif University, 62111 Beni-Suef, Egypt
123

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Med Chem Res (2012) 21:395–405
H
N
H
N
CN
N
O
(CH2)4
O
N
O
N
CH2SH
R1
N
R2
N
N
N
N
N
CH2CH3
N
C2H5
R3
S
O
S
III
II
I
R1 = H3CCONH-, H5C6CONH-
OH
R2 =
,
OCH3
OH
R3 = -C2H5, -C6H5.
O
N
H
N
CH2SCH2COOH
O
N
N
N
O
IV
Fig. 1 Examples of pyrimido[40,50:4,5]pyrimido[1,6-a]azepine compounds with anti-inflammatory activity
this fused tricyclic ring system, it deemed of interest to
prepare a set of new tricyclic pyrimido[40,50:4,5]pyrimi-
do[1,6-a]azepine derivatives substituted with the more
lipophilic pyrrolidine moiety at position 12 instead of the
morpholino group to study the influence of this replace-
ment on the anti-inflammatory activity. This approach of
increasing lipophilicity by introducing the more lipophilic
pyrrolidine ring has been recently explored by the authors
and proved beneficial in improving the anti-inflammatory
activity which urged us to continue in this direction
(El-Sayed et al., 2010). In addition, various substituents
were introduced at positions 1, 2 or 3 of the pyrimido[4,5-
d]pyrimido[1,6-a]azepine nucleus, which have been
modified to evolve our target compounds (Fig. 2).
R2
N
Fig. 2 General structure of the
target compounds
R1
R3
N
N
N
N
O
prepared as previously reported by the authors (El-Sayed
et al., 2010).
Refluxing of compounds 5a, b in ethanolic hydrochloric
acid gave the tricyclic compounds 6a, b, respectively. IR
spectra of the target compounds supported the postulated
structure due to the disappearance of the nitrile absorption
band.
Synthesis of compounds 7a, b was achieved by refluxing
the enaminonitrile derivative 4 with the appropriate chlo-
roacyl chloride in dry benzene, using triethylamine as a
catalyst. The data drawn from IR spectra of 7a supported
Results and discussion
Chemistry
1
the postulated structure. Moreover, H-NMR spectrum of
The synthetic pathways adopted for the preparation of
the target compounds are illustrated in Scheme 1. The
known chloropyrimidoazepine intermediate 2 was prepared
according to the previously reported method from capro-
lactam 1 in several steps (Ebeid and Bitter, 1978; Ebeid
et al., 1995; El-Sayed and Hussein, 2003; El-Sayed et al.,
2006).
7a revealed a singlet signal resonating at 4.35 ppm attrib-
uted to the two protons of the chloromethyl moiety. Also,
1H-NMR of 7b showed two triplets at 2.12 and 4.23 ppm
assigned to the four protons of the chloroethyl moiety.
Condensation of the chloro derivatives 7a, b with dif-
ferent cyclic secondary amines in absolute ethanol in the
presence of anhydrous potassium carbonate resulted in the
formation of compounds 8a–f. Physical, elemental and
The 5-pyrrolidino derivative 3, the key intermediate
aminonitrile 4 and the amide derivatives 5a, b were
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Med Chem Res (2012) 21:395–405
397
Scheme 1 Reagents and
solvents: a 1 (CH3)2SO4, 2
CH2(CN)2, 3 SO2Cl2, 4 PhNCO;
b pyrrolidine in abs. ethanol;
c NaBH4 in abs. ethanol;
N
CN
Cl
CN
NH
O
NH
b
a
N
N
N
N
NH
O
O
d RCOCl, dry benzene,
3
1
2
triethylamine; e ethanolic HCl;
f ClCO(CH2)nCl, dry benzene,
triethylamine; g appropriate
amine, methylene chloride, anh.
potassium carbonate; h thiourea,
ethanol, 10% NaOH;
c
O
N
CN
H2N
N
CN
R
N
N
NH
NH2
N
i ClCH2COOH, ethanol, KOH;
j RCOCl, dry benzene,
d
o
N
N
N
N
N
O
triethylamine; k 85% formic
acid; l appropriate amine,
HCHO, acetonitrile, acetic acid;
m POCl3; n appropriate amine,
abs. ethanol, anh. potassium
carbonate; o HCONH2
N
O
5a,b
4
O
k
e
16
f
H
H
N
O
O
Cl
R
N
N
N
N
N
N
H
m
O
N
N
N
N
N
Cl
n
N
N
N
N
N
N
O
N
N
O
O
l
12
O
14
6a,b
7a,b
n
R'
g
O
N
N
h
N
R'
N
N
N
H
H
O
N
N
N
O
N
N
CH2SH
n
R'
N
N
N
N
O
N
13a-c
N
O
N
N
N
O
15a-c
O
9
8a-f
j
i
O
Cl
H
O
HO
N
H
N
S
R
HN
O
N
R= a
b
N
Cl
S
N
COOH
N
N
N
N
N
N
N
O
R'= a
b
O
11a,b
10
N
c
O
7a, 8a-c n= 1
7b, 8d-f n= 2
spectral data were in accordance with the structure of the
titled compounds.
The 3-mercaptomethylacetic acid derivative 10 was
obtained through the reaction of the mercapto derivative 9
with chloroacetic acid in absolute ethanol in the presence
of potassium hydroxide. The 1H-NMR spectrum of 10
revealed a singlet signal at 4.21 ppm correlated to the
methylene protons of the acetic acid moiety, beside another
singlet resonating at 6.82 ppm corresponding to the proton
of the carboxylic function group.
The reaction of thiourea with the 3-chloromethyl
derivative 7a yielded the corresponding 3-mercaptomethyl
derivative 9. The IR spectral analysis of compound 9
revealed a stretching band of the SH group, weakly
1
absorbing around at 2455 cm-1. In addition, its H-NMR
spectrum showed a singlet signal resonating at 10.62 ppm,
assigned to the proton of the SH group, in addition to
another singlet signal at 2.82 ppm related to the methylene
protons of the mercaptomethyl group.
On the other hand, reaction of 9 with the appropriate
acid chloride in dry benzene in the presence of triethyl-
amine afforded the thioesters 11a, b.
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Med Chem Res (2012) 21:395–405
Reaction of the key enaminonitrile 4 with formic acid
The results of the preliminary anti-inflammatory activity
revealed that the intermediate bicyclic derivatives 5a, b,
which could be considered as hybrid molecules between
the pyrimidoazepine nucleus and salicylic acid or diclofe-
nac moieties, respectively, possessed higher activity than
their cyclized tricyclic derivatives 6a, b. However, when
these moieties were separated from the pyrimidine ring by
a carbonyl thiomethyl spacer as in compounds 11a, b, the
activity slightly increased. Introduction of a saturated five-
or six-membered aza heterocyclic ring separated by a
methyl spacer from the pyrimidine ring 8a–c were slightly
more active than those with an ethyl spacer 8d–f. However,
movement of the azaheterocyclic methyl moiety to position
2 as in compounds 13a–c or to position 1 as in compounds
15a–c led to a decrease in activity.
yielded the corresponding tricyclic derivative 12. IR
spectrum of compound 12 exhibited the disappearance of
the characteristic stretching vibration band of the cyano
group, in addition to the presence of vibrations bands
absorbing at 3265, 1656 cm-1 related to the NH and two
carbonyl groups. 1H-NMR spectrum of 12 showed a singlet
signal at 8.15 ppm attributed to the proton at C3.
In a successful method to obtain the Mannich bases
13a–c, compound 12 was reacted with the appropriate
amine, formaldehyde and acetic acid, in acetonitrile. The
suggested structure of the target compounds was referred
from the IR spectra which revealed the disappearance of
1
the characteristic NH stretching band. H-NMR spectra of
13a–c were in consistency with their structure.
The preparation of the chloro derivative 14 was
achieved via heating compound 12 with excess phospho-
rous oxychloride under dry conditions. The synthesized
compound was characterized by its physical, analytical and
spectral data. IR spectrum demonstrated only one band at
1675 cm-1 attributed to one carbonyl group, in addition to
the vanishing of the NH band. 1H-NMR spectrum was
complying with the postulated structure of our titled
compound.
No significant difference was perceived in the activity of
the primary amino derivative 16 compared to its tertiary
amino analogues 15a–c. Finally the 3-mercaptomethyl
acetic acid derivative 10 retains good potency comparable
to that of diclofenac sodium (relative potency = 97.71%).
The high activity of this compound could suggest that this
compound is acting mechanistically as the classical NSA-
IDs. Compound IV, the morpholino analogue of 10, was
previously reported to have 84.5% relative potency which
evidenced the positive impact of lipophilicity on the
activity of these compounds and which supported our
rationale of improving activity by increasing lipophilicity.
The IC 50 of the most active compound 10 was calcu-
lated using doses of 12, 18 and 24 lmol/kg after 3 h from
induction of inflammation (Table 2).
Nucleophilic substitution of the chloro group in 14 with
the appropriate secondary amine was performed to afford
the target compounds 15a–c. 1H-NMR spectra of 15a–
c were complying with their expected structure as that of
15c showed two triplets at 2.75 and 4.15 ppm related to the
8 protons of the morpholino group.
Alternatively, fusion of the key compound 4 with
formamide gave the target 1-amino derivative 16. Reaction
was confirmed by the IR spectrum which revealed the
disappearance of the characteristic stretching vibration of
the cyano group; and the 1H-NMR spectrum which showed
a singlet signal at 8.46 ppm related to the proton C3.
Acute ulcerogenicity study
The most active compounds, 5a, 5b, 8c, 8f, 10 and 11a, b,
were evaluated for gastric ulcerogenic potential in rats
(Table 3) (Robert et al., 1968; Meshali et al., 1983;
El-Sayed et al., 2010). In comparison with diclofenac
sodium all tested compounds showed better gastric toler-
ance. The highest ulcer index was exhibited by the acetic
acid derivative 10 but it is still much lower than that of
diclofenac sodium.
Pharmacological screening
Anti-inflammatory activity
Evaluation of anti-inflammatory activity of the synthesized
compounds was performed using the carrageenan-induced
rat paw oedema model using diclofenac sodium as the
reference drug (Winter et al., 1962; Kasahara et al., 1985;
Tozkoparan et al., 2007; Abdel-Megeed et al., 2009;
El-sayed et al., 2010). Mean changes in paw oedema
thickness of animals pretreated with the tested compounds
after 1, 2 and 3 h from induction of inflammation was
measured and the inhibition percent of oedema by the
tested compounds was calculated. The relative potencies %
of the tested compounds compared to diclofenac sodium at
the third hour was also calculated (Table 1).
Conclusions
Various 1, 2 or 3 substituted pyrimido[40,50:4,5]pyrimi-
do[1,6-a]azepine derivatives were prepared and screened
for their anti-inflammatory activity and ulcerogenic
potential. The 3-mercaptomethylacetic acid derivative 10
showed activity comparable to diclofenac sodium while the
rest of the tested compound exhibited moderate anti-
inflammatory activity. Taking the results of the ulceroge-
nicity study into consideration it could be claimed that
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Med Chem Res (2012) 21:395–405
399
Table 1 Oedema thickness, inhibition percent in oedema thickness and relative potency % of control, diclofenac sodium and tested compounds
Comp.
Oedema thickness (9 10-2 mm) SEM (% inhibition)
Relative potency
% at 3 h
1 h
2 h
3 h
Control
137.80 1.067
156.60 1.967
171.40 1.435
Diclofenac-sodium
43.20 1.562 (68.6)
76.21 1.392 (44.7)
93.81 2.596 (31.9)
112.82 1.319 (21.9)
101.43 1.939 (29.7)
90.83 1.241 (36.2)
99.22 1.414 (28.01)
71.61 1.363 (51.9)
107.61 2.267 (20.5)
105.80 2.059 (26.8)
84.23 2.477 (38.8)
46.41 1.860 (66.4)
98.81 1.772 (29.8)
92.23 1.827 (33.09)
122.60 1.949 (11.03)
124.40 1.886 (16.2)
150.20 1.319 (11.9)
127.61 1.772 (13.06)
145.60 1.631 (9.5)
137.81 1.714 (10.3)
127.81 2.477 (17.7)
128.81 2.395 (17.6)
25.80 1.392 (83.5)
71.22 1.71 (53.8)
80.65 1.326 (48.5)
111.83 2.083 (27.9)
96.81 2.457 (35.2)
89.01 1.288 (42.2)
98.62 1.536 (37.2)
66.43 1.536 (57.5)
105.62 2.358 (32.5)
101.26 1.913 (35.7)
74.82 1.854 (52.2)
41.20 2.064 (73.2)
96.72 1.655 (36.9)
84.83 2.498 (45.8)
120.40 1.029 (26.8)
121.63 1.720 (22.3)
130.82 2.011 (16.4)
131.22 1.593 (18.5)
133.45 2.785 (14.8)
129.20 1.462 (17.4)
123.41 1,568 (21.2)
120.61 1.854 (22.8)
21.09 1.428 (87.6)
59.32 1.870 (65.4)
56.22 2.267 (67.2)
107.64 2.976 (37.19)
93.62 2.227 (45.3)
87.80 1.933 (48.7)
97.96 1.772 (42.4)
64.33 1.984 (62.4)
95.41 2.315 (44.3)
100.21 2.014 (41.4)
64.22 1.773 (52.5)
20.43 1.661 (85.6)
86.21 1.714 (49.7)
80.43 1.568 (53.07)
114.60 1.990 (33.13)
115.41 1.923 (32.6)
121.23 1.861 (29.27)
119.81 2.973 (30.09
124.61 2.420 (27.29)
123.51 2.863 (27.94)
113.42 1.861 (33.82)
113.52 2.024 (33.7)
5a
74.65
76.71
42.45
51.71
53.59
48.40
71.23
50.57
47.26
59.93
97.71
56.37
61.30
37.81
37.21
33.41
34.34
31.15
31.89
38.60
38.47
5b
6a
6b
8a
8b
8c
8d
8e
8f
10
11a
11b
12
13a
13b
13c
15a
15b
15c
16
Data analyzed by one way ANOVA, (n = 5), all compounds were significantly different from control and from diclofenac sodium using
Student’s t-test, P \ 0.05
compound 10 outweigh diclofenac sodium as a safe anti-
Table 2 IC 50 values of compound 10 and Diclofenac sodium
inflammatory agent of comparable potency.
Comp
% Inhibition in oedema thickness at
IC 50
(lmol/kg)
12 lmol/kg 18 lmol/kg 24 lmol/kg
Diclofenac- 59.5
sodium
79.3
69.3
88.2
89.6
7.26
Experimental protocols
10
51.2
11.73
Chemistry
Table 3 Ulcer indices of compounds 5a, 5b, 8c, 8f, 10, 11a and 11b
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 wave number (cm-1) using potassium bro-
Compound
%
Average
Incidence/10 no. of ulcer severity index
Average Ulcer
Control
–
6
2
2
2
1
2
1
1
–
–
Nil
9
Diclofenac-sodium
1.2
0.4
0.6
0.8
1.2
2.2
1.2
0.2
1.8
1
5a
3.4
4.1
4.3
3.4
2.7
3.4
2.6
5b
8c
1.5
1.5
1.2
1.5
1.2
1.4
1
mide disc. H-NMR spectra were recorded on a Varian
Mercury VX-300 NMR spectrometer at 300 MHz in the
specified solvent. Vulnerable protons (NH, OH, SH) were
deuterium exchanged. Chemical shifts were reported on the
d scale and were related to that of the solvent and J values
are given in Hz. 13C-NMR spectra were obtained on a
8f
10
11a
11b
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Med Chem Res (2012) 21:395–405
3-Chloroalkyl-5-phenyl-12-(pyrrolidin-1-yl)-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:
4,5]pyrimido[1,6-a]azepine-1,6-dione (7a, b)
Varian Mercury VX-300 NMR spectrometer at 100 MHz
in the specified solvent. Mass spectra were recorded on
Fennigan MAT, SSQ 7000, Mass spectrometer, at 70 eV
(EI). IUPAC chemical nomenclature was assigned using
CS Chemdraw ultra version 5.0. Reaction time was mon-
itored using thin layer chromatography; performed using
Macherey–Nagel Alugram Sil G/UV254 silica gel plates
and chloroform-ethanol (9:1) as the eluting system.
A mixture of the aminonitrile 4 (3.5 g, 10 mmol), the
appropriate chloroacyl chloride (15 mmol) and triethyl-
amine (2.5 ml) in dry benzene (10 ml) was refluxed at a
temperature not exceeding 70°C for 2 h and evaporated
under vacuum to dryness. The residue was triturated with
petroleum ether; the separated solid was filtered and crys-
tallized from aqueous ethanol.
3-(2-Hydroxyphenyl)-5-phenyl-12-(pyrrolidin-1-yl)-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido
[40,50:4,5]pyrimido[1,6-a]azepine-1,6-dione (6a)
and 3-[2-(2,6-dichlorophenylamino) benzyl]-
5-phenyl-12-(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,12, 12a-
decahydropyrimido[40,50:4,5]pyrimido[1,6-a]
azepine-1,6-dione (6b)
3-Chloromethyl-5-phenyl-12-(pyrrolidin-1-yl)-1,2,5,6,8,
9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrimido[1,6-
a]azepine-1,6-dione (7a): m.p. 184–186°C, yield 72.8%. IR
(KBr, cm-1): 3265 (NH); 3055 (CH aromatic); 2933–2857
1
(CH aliphatic); 1675 (2CO). H-NMR (CDCl3: d, ppm):
1.28–1.41(m, CH2-10, 2H); 1.61–2.28 (m, CH2-9, CH2-11,
–CH2CH2– pyrrolidine, 8H); 2.75 (t, J = 3.6 Hz, 2CH2N–
pyrrolidine, 4H); 3.33 (t, J = 4.3 Hz, CH2-8, 2H); 3.71-
3.78 (m, CH-12, 1H); 4.35 (s, –CH2Cl, 2H); 4.72
(d, J = 5.4 Hz, CH-12a, 1H); 7.21–7.75 (m, aromatic
protons, 5H); 8.53 (s, NH, 1H, disappeared on deuteration).
MS: m/z (%) M? 427 (23), 191 (100). Anal. Calcd. for
C22H26ClN5O2 (427.93): C, 61.75; H, 6.12; N, 16.37.
Found: C, 61.45; H, 6.66; N, 16.85.
Compound 5a/5b(10 mmol) was refluxed in ethanolic
hydrochloric acid solution (15 ml) for about 4 h and left to
cool. The solution was diluted with water and neutralized
with sodium carbonate and the separated solid was filtered
and crystallized from aqueous ethanol.
3-(2-Hydroxyphenyl)-5-phenyl-12-(pyrrolidin-1-yl)-1,2,
5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrimido
[1,6-a]azepine-1,6-dione (6a): m.p. 175–177°C, yield
62.2%. IR (KBr, cm-1): 3445 (OH); 3310 (NH); 3017 (CH
aromatic); 2912–2866 (CH aliphatic); 1702, 1645 (2CO).
1H-NMR (DMSO-d6: d, ppm): 1.41–1.53 (m, CH2-10, 2H);
1.73–2.24 (m, CH2-9, CH2-11, –CH2CH2– pyrrolidine,
8H); 2.64 (t, J = 3.2 Hz, 2CH2N– pyrrolidine, 4H); 3.12
(t, J = 4.2 Hz, CH2-8, 2H); 3.48–3.52 (m, CH-12, 1H);
4.34 (d, J = 4.7 Hz, CH-12a, 1H); 6.82–7.52 (m, aromatic
protons, 9H); 9.21 (s, NH, 1H, disappeared on deuteration);
9.73 (s, OH, 1H, disappeared on deuteration). Anal. Calcd.
for C27H29N5O3 (471.55): C, 68.77; H, 6.20; N, 14.85.
Found: C, 68.45; H, 6.31; N, 14.61.
3-(2-Chloroethyl)-5-phenyl-12-(pyrrolidin-1-yl)-1,2,5,6,
8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrimido[1,
6-a]azepine-1,6-dione (7b): m.p. 183–185°C, yield 54.5%.
IR (KBr, cm-1): 3235 (NH); 3094 (CH aromatic);
1
2963–2875 (CH aliphatic); 1681, 1641 (2CO). H-NMR
(CDCl3: d, ppm): 1.08–1.24 (m, CH2-10, 2H); 1.54–1.93
(m, CH2-9, CH2-11, –CH2CH2– pyrrolidine, 8H); 2.12
(t, J = 2.9 Hz, –CH2CH2Cl), 2H); 2.54 (t, J = 3.5 Hz,
2CH2N– pyrrolidine, 4H); 3.21 (t, J = 4.8 Hz, CH2-8, 2H);
3.51–3.58 (m, CH-12, 1H); 4.23 (t, J = 2.9 Hz,
–CH2CH2Cl, 2H); 4.81 (d, J = 5.2 Hz, CH-12a, 1H);
7.15–7.82 (m, aromatic protons, 5H); 8.76 (s, NH, 1H,
disappeared on deuteration). Anal. Calcd. for C23H28
ClN5O2 (441.95): C, 62.51; H, 6.39; N, 15.85. Found: C,
62.88; H, 6.12; N, 15.63.
3-[2-(2,6-Dichlorophenylamino)-benzyl]-5-phenyl-12-
(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,12,12a-decahydropy-
rimido[40,50:4,5]pyrimido[1,6-a]azepine-1,6-dione (6b):
m.p. 173–175°C, yield 58.5%. IR (KBr, cm-1): 3180, 3218
(2NH); 3043 (CH aromatic); 2933–2857 (CH aliphatic);
1675, 1630 (2C=O). 1H-NMR (DMSO-d6: d, ppm):
1.38–1.42 (m, CH2-10, 2H); 1.82–2.31 (m, CH2-9, CH2-11,
–CH2CH2– pyrrolidine, 8H); 2.62 (t, J = 4.1 Hz, 2CH2N–
pyrrolidine, 4H); 2. 81 (s, –CH2–C6H4, 2H); 3.21 (t,
J = 4.5 Hz, CH2-8, 2H); 3.58–3.64 (m, CH-12, 1H); 4.61
(d, J = 4.9 Hz, CH-12a, 1H); 6.42–7.31(m, aromatic pro-
tons, 12H); 9.12 (s, NH, 1H, disappeared on deuteration);
10.34 (s, NH, 1H, disappeared on deuteration). Anal.
Calcd. for C34H34Cl2N6O2 (629.58): C, 64.86; H, 5.44; N,
13.35. Found: C, 64.41; H, 6.02; N, 12.96.
5-Phenyl-12-(pyrrolidin-1-yl)-3-
[(cycloalkylamino)methyl]-1,2,5,6,8,9,10,11,12,12a-
decahydropyrimido[40,50: 4,5]pyrimido[1,6-a]azepine-
1,6-dione (8a–f)
A
mixture of the 3-chloroalkyl derivative 7a/7b
(10 mmol), the appropriate amine (10 mmol) and anhy-
drous potassium carbonate (1.9 g, 20 mmol), in methylene
chloride (15 ml) was refluxed for about 7 h and filtered
while hot. The filtrate was distilled under vacuum and the
remaining residue was crystallized from ethanol.
123

Med Chem Res (2012) 21:395–405
401
5-Phenyl-12-(pyrrolidin-1-yl)-3-[(pyrrolidin-1-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (8a): m.p. 205–207°C, yield
73%. IR (KBr, cm-1): 3229 (NH); 3054 (CH aromatic);
2912–2853 (CH aliphatic); 1655 (2CO). 1H-NMR (DMSO-
d6: d, ppm): 1.23–1.29 (m, CH2-10, 2H); 1.72–2.14 (m, CH2-
9, CH2-11, –CH2CH2– of 2 pyrrolidine rings, 12H); 2.55
(t, J = 4.6 Hz, 2CH2-N of 2 pyrrolidine rings, 8H); 2.84
(s, N=C(NH)–CH2–N, 2H); 3.41 (t, J = 4.1 Hz, CH2-8, 2H);
3.73–3.79 (m, CH-12, 1H); 4.51 (d, J = 5.7 Hz, CH-12a,
1H); 7.17–7.64 (m, aromatic protons, 5H); 9.14 (s, NH, 1H,
disappeared on deuteration). MS: m/z (%) M? 462 (15), 191
(100). Anal. Calcd. for C26H34N6O2 (462.59): C, 67.51; H,
7.41; N, 18.17. Found: C, 67.22; H, 7.31; N, 17.74.
(t, J = 2.8 Hz, CH2CH2–N, 2H); 3.23 (t, J = 4.2 Hz, CH2-
8, 2H); 3.58–3.62 (m, CH-12, 1H); 4.52 (d, J = 5.6 Hz,
CH-12a, 1H); 7.15–7.91 (m, aromatic protons, 5H); 8.47
(s, NH, 1H, disappeared on deuteration). Anal. Calcd. for
C27H36N6O2 (476.62): C, 68.04; H, 7.61; N, 17.63. Found:
C, 68.54; H, 7.45; N, 17, 41.
5-Phenyl-12-(pyrrolidin-1-yl)-3-[2-(piperidin-1-yl)ethyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (8e): m.p. 172–174°C, yield
33%. IR (KBr, cm-1): 3313 (NH); 3045 (CH aromatic);
2916–2865 (CH aliphatic); 1685, 1641 (2CO).1H-NMR
(DMSO-d6: d, ppm): 1.01–1.23 (m, CH2-10, 2H); 1.51 (t,
J = 3.2 Hz, –CH2CH2–N, 2H); 1.82–2.36 (m, CH2-9, CH2-
11, –CH2CH2– pyrrolidine, –CH2CH2CH2– piperidine,
14H); 2.62 (t, J = 4.5 Hz, 2CH2–N piperidine, 2CH2–N
pyrrolidine, 8H); 2.93 (t, J = 3.2 Hz, –CH2CH2–N, 2H);
3.37 (t, J = 4.3 Hz, CH2-8, 2H); 3.68-3.74 (m, CH-12, 1H);
4.62 (d, J = 5.9 Hz, CH-12a, 1H); 7.21–7.76 (m, aromatic
protons, 5H); 9.15 (s, NH, 1H, disappeared on deuteration).
Anal. Calcd. for C28H38N6O2 (490.31): C, 68.54; H, 7.81;
N, 17.13. Found: C, 68.72; H, 7.35; N, 17.26.
5-Phenyl-12-(pyrrolidin-1-yl)-3-[(piperidin-1-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]py-
rimido[1,6-a]azepine-1,6-dione (8b): m.p. 163–166°C,
yield 56%. IR (KBr, cm-1): 3312(NH); 3098 (CH aro-
1
matic); 2921–2866 (CH aliphatic); 1640, 1623 (2CO). H-
NMR (CDCl3: d, ppm): 1.01–1.23 (m, CH2-10, 2H);
1.53–1.96 (m, CH2-9, CH2-11, –CH2CH2– pyrrolidine,
–CH2CH2CH2– of piperidine, 14H); 2.22 (t, J =
3.9 Hz, 2CH2–N of piperidine, 2CH2–N of pyrrolidine,
8H); 2.71 (s, N=C(NH)–CH2N–, 2H); 3.26 (t, J = 4.5 Hz,
CH2-8, 2H); 3.58–3.61 (m, CH-12, 1H); 4.58 (d, J =
5.4 Hz, CH-12a, 1H); 6.96–7.32 (m, aromatic protons, 5H);
8.77 (s, NH, 1H, disappeared on deuteration). Anal. Calcd.
for C27H36N6O2 (476.61): C, 68.04; H, 7.61; N, 17.63.
Found: C, 68.31; H, 7.32; N, 17.23.
5-Phenyl-12-(pyrrolidin-1-yl)-3-[2-(morpholin-4-yl)ethyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (8f): m.p. 191–193°C, yield
54%. IR (KBr, cm-1): 3281 (NH); 3062 (CH aromatic);
1
2945–2862 (CH aliphatic); 1675, 1623 (2CO). H-NMR
(DMSO-d6: d, ppm): 1.11–1.34 (m, CH2-10, 2H); 1.56 (t,
J = 2.6 Hz, CH2CH2–N, 2H); 1.74–2.31 (m, CH2-9, CH2-
11, –CH2CH2– pyrrolidine, 8H); 2.61 (t, J = 4.3 Hz,
2CH2–N pyrrolidine, 4H); 2.93 (m, CH2CH2–N–, 2CH2–N
morpholine, 6H); 3.34 (t, J = 4.7 Hz, CH2-8, 2H);
3.63–3.72 (m, CH-12, 1H); 4.15 (t, J = 3.4 Hz, 2CH2–O
morpholine, 4H); 4.61 (d, J = 5.5 Hz, CH-12a, 1H);
7.11–7.61 (m, aromatic protons, 5H); 8.82 (s, NH, 1H,
disappeared on deuteration). Anal. Calcd. for C27H36N6O3
(492.28): C, 65.83; H, 7.37; N, 17.06. Found: C, 65.61; H,
6.85; N, 16.67.
5-Phenyl-12-(pyrrolidin-1-yl)-3-[(morpholin-4-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (8c): m.p. 185–186°C, yield
51%. IR (KBr, cm-1): 3215 (NH); 3094 (CH aromatic);
1
2944–2862 (CH aliphatic); 1661, 1641 (2CO). H-NMR
(CDCl3: d, ppm): 1.32–1.37 (m, CH2-10, 2H); 1.53–1.96
(m, CH2-9, CH2-11, –CH2CH2– pyrrolidine, 8H); 2.22 (t,
J = 4.1 Hz, 2CH2–N pyrrolidine, 4H); 2.51 (t, J = 4.7 Hz,
N=C(NH)–CH2N–, 2CH2–N morpholine, 6H); 3.18 (t,
J = 5.5 Hz, CH2-8, 2H); 3.50–3.56 (m, CH-12, 1H); 4.23
(t, J = 4.5 Hz, 2CH2–O morpholine, 4H); 4.61 (d,
J = 5.2 Hz, CH-12a, 1H); 7.14–7.73 (m, aromatic protons,
5H); 8.24 (s, NH, 1H, disappeared on deuteration). Anal.
Calcd. for C26H34N6O3 (478.59): C, 65.25; H, 7.16; N,
17.56. Found: C, 65.72; H, 6.94; N, 17.82.
5-Phenyl-12-(pyrrolidin-1-yl)-3-sulfanylmethyl-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]
pyrimido[1,6-a]azepine-1,6-dione (9)
A mixture of compound 7a (4.27 g, 10 mmol) and thiourea
(0.76 g, 10 mmol) in ethanol (10 ml) was heated under
reflux for about 3 h then left to cool. The separated crystals
were filtered, dissolved in ice cooled solution of 10%
sodium hydroxide and filtered; the alkaline filtrate was
acidified with dilute hydrochloric acid to pH 4. The formed
precipitate was filtered, washed with water and crystallized
from aqueous ethanol to give 2.76 g (64.6%) of 9, m.p.
205–209°C. IR (KBr, cm-1): 3218 (NH); 3044 (CH aro-
matic); 2913–2843 (CH aliphatic); 2455 (SH), 1670, 1625
5-Phenyl-12-(pyrrolidin-1-yl)-3-[2-(pyrrolidin-1-yl)ethyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (8d): m.p. 166–169°C, yield
63%. IR (KBr, cm-1): 3294 (NH); 3084 (CH aromatic);
2915–2867 (CH aliphatic); 1656 (2CO). 1H-NMR (DMSO-
d6: d, ppm): 1.14–1.21 (m, CH2-10, 2H); 1.45 (t, J =
2.8 Hz,–CH2CH2–N, 2H); 1.83–2.32 (m, CH2-9, CH2-11,
–CH2CH2– of 2 pyrrolidine rings, 12H); 2.64 (t, J =
4.6 Hz, 2CH2–N of 2 pyrrolidine rings, 8H); 2.96
1
(2CO). H-NMR (CDCl3: d, ppm): 1.23–1.31 (m, CH2-10,
123

402
Med Chem Res (2012) 21:395–405
2H); 1.98-2.29 (m, CH2-9, CH2-11, –CH2CH2– pyrrolidine,
8H); 2.55 (t, J = 4.1 Hz, 2CH2–N pyrrolidine, 4H); 2.82
(s, –CH2SH, 2H); 3.43 (t, J = 4.4 Hz, CH2–8, 2H);
4.10–4.13 (m, CH-12, 1H); 4,84 (d, J = 5.2 Hz, CH-12a,
1H); 6.94–7.58 (m, aromatic protons, 5H); 8.64 (s, NH, 1H,
disappeared on deuteration); 10.62 (s, SH, 1H, disappeared
on deuteration). MS: m/z (%) M? 425.19 (14), 160 (100).
Anal. Calcd. for C22H27N5O2S (425.19): C, 62.09; H, 6.40;
N, 16.46. Found: C, 62.21; H, 6.34; N, 15.97.
triturated with ether; the separated solid was filtered and
crystallized from aqueous ethanol.
3-[(2-Hydroxybenzoyl)sulfanylmethyl]-5-phenyl-12
(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,12,12a-decahydropyrim-
ido[40,50:4,5]pyrimido[1,6-a]azepine-1,6-dione (11a): m.p.
190–193°C, yield 44%. IR (KBr, cm-1): 3415 (OH); 3315
(NH); 3052 (CH aromatic); 2934-2882 (CH aliphatic);
1
1710, 1664, 1633 (3CO). H-NMR (DMSO-d6: d, ppm):
1.28–1.41 (m, CH2-10, 2H); 1.52–1.91 (m, CH2-9, CH2-11,
–CH2CH2– pyrrolidine, 8H); 2.38 (t, J = 3.5 Hz, 2CH2–N
pyrrolidine, 4H); 2.93 (s, –CH2S–CO, 2H); 3.48 (t,
J = 4.2 Hz, CH2-8, 2H); 3.78–3.82 (m, CH-12, 1H); 4.65
(d, J = 5.1 Hz, CH-12a, 1H); 6.94–7.63 (m, aromatic
protons, 9H); 8.56 (s, NH, 1H, disappeared on deuteration);
10.13 (s, OH, 1H, disappeared on deuteration). Anal.
Calcd. for C29H31N5O4S (545.21): C, 63.83; H, 5.73; N,
12.83. Found: C, 63.41; H, 5.32; N, 12.52.
(1,6-Dioxo-5-phenyl-12-pyrrolidino-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]
pyrimido[1,6-a]azepin-3-ylmethylsulfanyl)acetic acid (10)
A mixture of compound 9 (4.2 g, 10 mmol), chloroacetic
acid (0.94 g, 10 mmol) and potassium hydroxide (0.72 g,
20 mmol), in absolute ethanol (20 ml) was refluxed for
about 8 h then filtered while hot. The filtrate was evapo-
rated to dryness; the residue was diluted with water and
neutralized with dilute hydrochloric acid. The separated
crystals were filtered, washed with water and recrystallized
from aqueous ethanol to give 2.46 g (50.9%) of 10, m.p.
212–215°C. IR (KBr, cm-1): 3235 (OH), 3145 (NH), 3072
(CH aromatic); 2915, 2863 (CH aliphatic); 1660, 1618
3-{[2-(2,6-Dichlorophenylamino)phenyl]acetyl]sulfa-
nylmethyl}-5-phenyl-12-(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,
12,12a-decahydroprimido[40,50:4,5]pyrimido[1,6-a]azepine-
1,6-dione (11b): m.p. 206–209°C, yield 56%. IR (KBr,
cm-1): 3371 (NH); 3081 (CH aromatic); 2913–2833 (CH
1
aliphatic); 1680, 1655, 1630 (3CO). H-NMR (DMSO-d6:
d, ppm): 1.22–1.34 (m, CH2-10, 2H); 1.42–1.84 (m, CH2-9,
CH2-11, –CH2CH2– pyrrolidine, 8H); 2.45 (t, J = 4.7 Hz,
2CH2–N pyrrolidine, 4H); 2.88 (s, CH2S-CO, 2H); 3.28 (t,
J = 4.3 Hz, CH2-8, 2H); 3.61–3.72 (m, CH-12, 1H); 4.12
(s, –S–CO–CH2–, 2H); 4.55 (d, J = 4.9 Hz, CH-12a, 1H);
6.92–7.55 (m, aromatic protons, 12H); 8.66 (s, NH, 1H);
9.22 (s, NHCO, 1H). Anal. Calcd. for C36H36Cl2N6O3S
(702.19): C, 61.45; H, 5.16; N, 11.94. Found: C, 61.12; H,
4.78; N, 11.61.
1
(3CO). H-NMR (CDCl3: d, ppm): 1.28–1.34 (m, CH2-10,
2H); 1.58–1.99 (m, CH2-9, CH2-11, –CH2CH2– pyrroli-
dine, 8H); 2.45 (t, J = 4.7 Hz, 2CH2–N pyrrolidine, 4H);
2.91(s, –CH2SCH2COOH, 2H); 3.23 (t, J = 5.3 Hz,
CH2-8, 2H); 3.61–3.65 (m, CH-12, 1H); 4.21 (s, –CH2
SCH2COOH, 2H); 4.51 (d, J = 5.6 Hz, CH-12a, 1H); 6.82
(s, –COOH, 1H, disappeared on deuteration); 7.25–7.73
(m, aromatic protons, 5H); 8.71 (s, NH, 1H, disappeared on
deuteration). 13C-NMR(CDCl3: d): 22.4 (C-10), 24.1
(–CH2CH2– pyrrolidine), 29.1 (C-9, C-11), 35.2 (–CH2S–
CH2), 37.8 (CH2S–CH2), 45.3 (C-8), 48.3 (C-12a), 50.2
(2CH2–N pyrrolidine), 52.3 (C-12), 106.2 (C-12b), 119.1
(C-2\, C-6\), 123.8 (C-4\), 129.2 (C-3\, C-5\), 140.2 (C-1\),
152.3 (C-4a), 154.6 (C-1), 165.1 (C-3), 170.4 (C=O), 177.2
(–COOH). MS: m/z (%) M? 483.19 (20), 162 (100). Anal.
Calcd. for C24H29N5O4S (483.19): C, 59.61; H, 6.04; N,
14.48. Found: C, 59.15; H, 6.41; N, 14.97.
5-Phenyl-12-(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,12,12a-
decahydropyrimido[40,50:4,5]pyrimido[1,6-a]azepine-1,
6-dione (12)
A mixture of the aminonitrile 4 (3.48 g, 10 mmol) and
formic acid (85%, 30 ml) was refluxed for 10 h, cooled and
then poured onto ice-water. The precipitate was filtered,
washed several times by water, dried and then crystallized
from ethanol to afford 1.58 g (41.6%) of 12, m.p.
152–155°C. IR (KBr, cm-1): 3265 (NH); 3083 (CH aro-
1
matic); 2913-2873 (CH aliphatic); 1656 (2CO). H-NMR
3-[(Substituted benzoyl/substituted acetyl)sulfanylmethyl]-
5-phenyl-12(pyrrolidin-1-yl)-1,2,5,6,8,9,10,11,12,12a-
decahydropyrimido[40,50:4,5]pyrimido[1,6-a]azepine-1,6-
dione (11a, b)
(CDCl3: d, ppm): 1.28–1.41 (m, CH2-10, 2H); 1.63–2.28
(m, CH2-9, CH2-11, –CH2CH2– pyrrolidine, 8H); 2.55 (t,
J = 4.5 Hz, 2CH2–N pyrrolidine, 4H); 3.25 (t, J = 4.8 Hz,
CH2-8, 2H); 3.58–3.64 (m, CH-12, 1H); 4,67 (d,
J = 5.7 Hz, CH-12a, 1H); 6.94–7.65 (m, aromatic protons,
5H); 8.15 (s, NH-CH=N, 1H); 9.26 (s, NH, 1H). MS: m/
z (%) M? 379.2 (16), 196 (100). Anal. Calcd. for
C21H25N5O2 (379.2) : C, 66.47; H, 6.64; N, 18.46 Found:
C, 65.82; H, 6.25; N, 18.24.
A mixture of compound 9 (4.2 g, 10 mmol), the appro-
priate acid chloride (11 mmol) and triethylamine (0.5 ml)
in dry benzene (10 ml) was refluxed for 4 h. The solvent
was removed under vacuum and the obtained residue was
123

Med Chem Res (2012) 21:395–405
403
5-Phenyl-12-(pyrrolidin-1-yl)-2-
CH-12a, 1H); 6.92–7.54 (m, aromatic protons, 5H); 8.31
(s, N=CH–N, 1H). Anal. Calcd. for C26H34N6O3, (478.59):
C, 65.25; H, 7.16; N, 17.56. Found: C, 65.73; H, 7.52; N,
17.34.
[(cycloalkylamino)methyl]-1,2,5,6,8,9,10,11,12,12a-
decahydropyrimido[40,50:4,5]pyrimido[1,6-a]azepine-1,
6-dione (13a–c)
To a stirred solution of compound 12 (3.8 g, 10 mmol) in
acetonitrile (20 ml), a mixture of the appropriate amine
(10 mmol), formaldehyde (10 mmol) and acetic acid
(5 ml) was added drop wise. The mixture was stirred at
room temperature for 10 h then treated with a solution of
20% sodium hydroxide and extracted with ethyl acetate.
The organic extracts were collected, evaporated under
vacuum and the residue was crystallized from aqueous
ethanol.
1-Chloro-5-phenyl-12-pyrrolidino-5,6,8,9,10,11,12,12a-
octahydropyrimido[40,50:4,5]pyrimido[1,6-a]azepin-
6-one (14)
A mixture of compound 12 (0.38 gm, 1 mmol) and phos-
phorus oxychloride (10 ml) was heated at 110°C for about
6 h. The mixture was evaporated under vacuum and tritu-
rated with sodium carbonate solution (5%). The precipitate
was filtered, dried and crystallized from aqueous ethanol to
yield 0.18 g (47%) of 14, m.p. 150–152°C. IR (KBr,
cm-1): 3072 (CH aromatic); 2954–2831 (CH aliphatic);
5-Phenyl-12-(pyrrolidin-1-yl)-2-[(pyrrolidin-1-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (13a): m.p. 175–179°C, yield
56%. IR (KBr, cm-1): 3081 (CH aromatic); 2932–2817
(CH aliphatic); 1680, 1650 (2CO). 1H-NMR (DMSO-d6: d,
ppm): 1.21-1.29 (m, CH2-10, 2H); 1.85–2.21 (m, CH2-9,
CH2-11, –CH2CH2– 2 pyrrolidines, 12H); 2.63 (t, J =
3.8 Hz, 2CH2–N 2 pyrrolidines, 8H); 3.14 (t, J = 4.8 Hz,
CH2-8, 2H); 3.49–3.54 (m, CH-12, 1H); 4.25 (s, N–CH2–
N, 2H); 4,67 (d, J = 5.3 Hz, CH-12a, 1H); 6.98–7.72
(m, aromatic protons, 5H); 8.27 (s, N–CH=N, 1H). MS:
m/z (%) M? 462.27 (32), 195 (100). Anal. Calcd. for
C26H34N6O2 (462.59): C, 67.51; H, 7.41; N, 18.17. Found:
C, 67.11; H, 7.25; N, 18.36.
1
1675 (CO). H-NMR (DMSO-d6: d, ppm): 1.19–1.32 (m,
CH2-10, 2H); 1.72–2.15 (m, CH2-9, CH2-11, CH2CH2–
pyrrolidine, 8H); 2.62 (t, J = 4.5 Hz, 2CH2–N pyrrolidine,
4H); 3.31 (t, J = 4.7 Hz, CH2-8, 2H); 3.60–3.68 (m, CH-
12, 1H); 4,51 (d, J = 5.3 Hz, CH-12a, 1H); 7.14–7.68
(m, aromatic protons, 5H); 8.31 (s, N–CH=N, 1H). MS:
m/z (%) M? 397.17 (21), 161 (100). Anal. Calcd. for
C21H24ClN5O (397.17): C, 63.39; H, 6.08; N, 17.60.
Found: C, 62.97 H, 6.34; N, 18.01.
5-Phenyl-1-(cycloalkylamino)-12-(pyrrolidin-1-yl)-
5,6,8,9,10,11,12,12a-octahydropyrimido[40,50:4,5]
pyrimido[1,6-a]azepin-6-one (15a–c)
5-Phenyl-12-(pyrrolidin-1-yl)-2-[(piperidin-1-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (13b): m.p. 164–168°C, yield
58%. IR (KBr, cm-1): 3075 (CH aromatic); 2923–2877
(CH aliphatic); 1670, 1635 (2CO). 1H-NMR (DMSO-d6: d,
ppm): 1.22–1.34 (m, CH2-10, 2H); 1.63–1.96 (m, CH2-9,
CH2-11, CH2CH2– pyrrolidine, –CH2CH2CH2– piperidine,
14H); 2.36 (t, J = 4.2 Hz, 2CH2–N piperidine, 2CH2–N
pyrrolidine, 8H); 3.18 (t, J = 5.2 Hz, CH2-8, 2H);
3.49–3.58 (m, CH-12, 1H); 4.22(s, N–CH2–N, 2H); 4.63
(d, J = 5.8 Hz, CH-12a, 1H); 6.92–7.32 (m, aromatic
protons, 5H); 8.77 (s, N=CH–N, 1H). Anal. Calcd. for
C27H36N6O2 (476.61): C, 68.04; H, 7.61; N, 17.63. Found:
C, 67.74; H, 7.35; N, 17.33.
A mixture of compound 14 (3.97 g, 10 mmol), the appro-
priate amine (10 mmol) and anhydrous potassium carbon-
ate (1.9 g, 20 mmol), in absolute ethanol (15 ml) was
refluxed for about 8 h and filtered while hot. The filtrate
was distilled under vacuum; the residue was triturated with
ethanol and the obtained solid was filtered, dried and
crystallized from aqueous ethanol.
5-Phenyl-1,12-di(pyrrolidin-1-yl)-5,6,8,9,10,11,12,12a-
octahydropyrimido[40,50:4,5]pyrimido[1,6-a]azepin-6-one
(15a): m.p. 165–168°C, yield 75%. IR (KBr, cm-1): 3073
(CH aromatic); 2913–2865 (CH aliphatic); 1645 (CO). 1H-
NMR (DMSO-d6: d, ppm): 1.29–1.36 (m, CH2-10, 2H);
1.72–2.13 (m, CH2-9, CH2-11, –CH2CH2– of 2 pyrrolidine
rings, 12H); 2.33 (t, J = 4.6 Hz, 2CH2–N of 12-pyrrolid-
inyl, 4H); 2.72 (t, J = 3.8 Hz, 2CH2–N of 1-pyrrolidinyl,
4H); 3.25 (t, J = 5.7 Hz, CH2-8, 2H); 3.31–3.48 (m, CH-
12, 1H); 4,67 (d, J = 5.3 Hz, CH-12a, 1H); 7.23–7.81
(m, aromatic protons, 5H); 8.42 (s, N–CH=N, 1H). 13C-
NMR(DMSO-d6: d): 22.6 (C-10), 23.5 (2C-12-pyrrolidi-
nyl), 24.1 (2C-1-pyrrolidinyl), 29.3 (C-9, C-11), 43.6 (C-
12a), 47.2 (C-8), 50.3 (2C-N-1-pyrrolidinyl), 53.8 (2C-N-
12-pyrrolidinyl), 60.2 (C-12), 104.3 (C-12b), 118.8 (C-2\,
C-6\), 125.1 (C-4\), 129.6 (C-3\, C-5\), 139.1 (C-1|), 155.2
5-Phenyl-12-(pyrrolidin-1-yl)-2-[(morphlin-4-yl)methyl]-
1,2,5,6,8,9,10,11,12,12a-decahydropyrimido[40,50:4,5]pyrim-
ido[1,6-a]azepine-1,6-dione (13c): m.p. 171–174°C, yield
41%. IR (KBr, cm-1): 3072 (CH aromatic); 2935–2861
(CH aliphatic); 1685, 1645 (2CO). 1H-NMR (DMSO-d6: d,
ppm): 1.21–1.36 (m, CH2-10, 2H); 1.62–1.99 (m, CH2-9,
CH2-11, –CH2CH2– pyrrolidine, 8H); 2.29 (t, J = 4.3 Hz,
2CH2–N of pyrrolidine, 4H); 2.64 (t, J = 3.9 Hz, 2CH2–N
morpholine, 4H); 3.25 (t, J = 4.6 Hz, CH2-8, 2H); 3.68-
3.76 (m, CH-12, 1H); 4.23 (t, J = 3.9 Hz, 2CH2–O mor-
pholine, 4H); 4.42 (s, N–CH2–N, 2H); 4.81 (d, J = 5.5 Hz,
123

404
Med Chem Res (2012) 21:395–405
(C-1), 157.2 (C-3), 165.4 (C-4a), 172.2 (C=O). MS:
m/z (%) M? 432.26 (25), 196 (100). Anal. Calcd. for
C25H32N6O (432.26): C, 69.42; H, 7.46; N, 19.43. Found:
C, 69.12; H, 7.54; N, 18.96.
C, 66.29; H, 7.42; N, 22.09. Found: C, 65.83; H, 6.83; N,
21.73.
Pharmacological screening
5-Phenyl-1-(piperidin-1-yl)-12-(pyrrolidin-1-yl)-5,6,8,9,
10,11,12,12a-octahydropyrimido[40,50:4,5]pyrimido[1,6-a]-
azepin-6-one (15b): m.p. 174–176°C, yield 65%. IR (KBr,
cm-1): 3083 (CH aromatic); 2933–2857 (CH aliphatic);
Anti-inflammatory activity
Adult albino rats of both sexes weighing between 120 and
150 g were used. Rats were uniformly hydrated by giving
3 ml water/rat through gastric inoculation to reduce vari-
ability to oedema response. Animals were divided into 23
groups each of five animals. The control group was given
saline solution containing few drops of 1% Tween 80.
Diclofenac sodium (20 lmol/kg) was taken as standard
drug for comparison and compounds under examination
(20 lmol/kg) were suspended in distilled water by the aid
of few drops of Tween 80 and were given intraperitoneally
1 h before induction of inflammation. Induction of
inflammation was performed by S.C. injection of 50 ll 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 paw
of each rat was measured using a pair of dial thickness
gauge calipers accurate to 0.001 cm 1 h, 2 h and 3 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 calculated
according to the following equation:
1
1680 (CO). H-NMR (DMSO-d6: d, ppm): 1.19–1.29 (m,
CH2-10, 2H); 1.62–1.98 (m, CH2-9, CH2-11, CH2CH2–
pyrrolidine, –CH2CH2CH2– of piperidine, 14H); 2.36
(t, J = 4.6 Hz, 2CH2–N of pyrrolidine, 4H); 2.82 (t, J =
3.9 Hz, 2CH2–N of piperidine, 4H); 3.29 (t, J = 4.4 Hz,
CH2-8, 2H); 3.59–3.64 (m, CH-12, 1H); 4.82 (d, J =
5.4 Hz, CH-12a, 1H); 7.26–7.75 (m, aromatic protons, 5H);
8.25 (s, N=CH–N, 1H). Anal. Calcd. for C26H34N6O
(446.28): C, 69.93; H, 7.67; N, 18.82. Found: C, 69.74; H,
8.11; N, 19.21.
5-Phenyl-1-(morpholin-4-yl)-12-(pyrrolidin-1-yl)-5,6,8,
9,10,11,12,12a-octahydropyrimido[40,50:4,5]pyrimido[1,6-a]
azepin-6-one (15c): m.p. 181–183°C, yield 53%. IR (KBr,
cm-1): 3055 (CH aromatic); 2925–2857 (CH aliphatic);
1
1665 (CO). H-NMR (DMSO-d6: d, ppm): 1.13–1.24 (m,
CH2-10, 2H); 1.65–1.99 (m, CH2-9, CH2-11, CH2CH2–
pyrrolidine, 8H); 2.34 (t, J = 4.2 Hz, 2CH2–N of pyrroli-
dine, 4H); 2.75 (t, J = 4.5 Hz, 2CH2–N morpholine, 4H);
3.16 (t, J = 5.2 Hz, CH2-8, 2H); 3.58–3.64 (m, CH-12,
1H); 4.15 (t, J = 4.5 Hz, 2CH2–O morpholine, 4H); 4.86
(d, J = 5.6 Hz, CH-12a, 1H); 6.98–7.67 (m, aromatic
protons, 5H); 8.24 (s, N=CH–N, 1H). Anal. Calcd. for
C25H32N6O2 (448.25): C, 66.94; H, 7.19; N, 18.74. Found:
C, 66.61; H, 7, 53; N, 18.34.
% Inhibition ¼ ð1 ꢁ Lt= LcÞ ꢂ 100
where Lt is the mean increase in paw thickness in rats
treated with the tested compounds and 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.
1-Amino-5-phenyl-12-(pyrrolidin-1-yl)-
5,6,8,9,10,11,12,12a-octahydropyrimido[40,50:4,5]
pyrimido[1,6-a]azepin-6-one (16)
Acute ulcerogenicity study
A mixture of the aminonitrile 4 (1.75 g, 5 mmol) and
formamide (20 ml) was refluxed for 2 h; cooled and the
reaction mixture was poured into cold water. The solid
precipitate was filtered, washed with water, dried and
crystallized from aqueous ethanol to yield 1.03 g (54%) of
12, m.p. 163–165°C. IR (KBr, cm-1): 3215 (NH2); 3064
(CH aromatic); 2940–2865 (CH aliphatic); 1635 (CO). 1H-
NMR (CDCl3: d, ppm): 1.29–1.36 (m, CH2-10, 2H);
1.83–2.35 (m, CH2-9, CH2-11, –CH2CH2– pyrrolidine,
8H); 2.64 (t, J = 4.5 Hz, 2CH2–N pyrrolidine, 4H); 3.27 (t,
J = 4.6 Hz, CH2-8, 2H); 3.69–3.74 (m, CH-12, 1H); 4.25
(d, J = 5.7 Hz, CH-12a, 1H); 7.12–7.73 (m, aromatic
protons, 5H); 8.46 (s, N–CH=N, 1H); 8.89 (s, NH2, 2H,
disappeared on deuteration). MS: m/z (%) M? 380.23
(22%), 166 (100%). Anal. Calcd. for C21H26N6O (380.23):
Adult albino rats of both sexes weighing between 120 and
150 g were used. Animals were divided into groups each of
five animals. Rats were fasted 20 h before drug adminis-
tration. The tested compounds and diclofenac sodium were
given orally in a dose of 20 lmol/kg suspended in 1%
Tween while one group received vehicle (1% Tween). Rats
were fasted for 2 h; allowed to feed for 2 h then fasted for
another 20 h. Rats were given another two doses in the
second and third day. In the fourth day, rats were killed, 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 severity) was graded from 0 to 4 according to
the following score assignment:
123

Med Chem Res (2012) 21:395–405
Score
405
Ebeid MY, Hassanein HH, Obidan N (1995) Heterocyclic synthesis
from o-aminonitriles condensed pyrimidines of potential phar-
macological activity. Bull Fac Pharm Cairo Univ 33:35–40
El-Sayed NM, Hussein MM (2003) Synthesis and evaluation of some
bicyclic and tricyclic azepine derivatives as antimicrobial agents.
Egypt J Pharm Sci 44:319–325
Score
Normal (no injury)
Latent small red spot
Wide red spot
0
1
2
Slight injury
Severe injury
3
4
El-Sayed NM, Hanna MM, Abdel Gawad NM, Hassanein HH, Ebeid
MY (1993) Condensed pyrimidines XV. Bull Fac Pharm Cairo
Univ 31:171–176
The following figures were calculated:
El-Sayed NM, Eissa AA, Arafa RK, Ebeid MY, Soliman GA (2003)
Synthesis of some novel pyrido[20,30:4,5]pyrimido[1,6-a]azepine
derivatives of anti-inflammatory, analgesic and antipyretic
activities. Egypt J Pharm Sci 44:207–225
–
% Incidence/10 = [number of rats showing ulcer of
any grade divided by total number of rats in the
group 9 100]/10.
El-Sayed NA, Farag AE, El-Saadi MT, El-Moghazy SM, Abd Ellatif
HA (2006) Synthesis and anti-inflammatory activity of some
fused pyrimidines. Bull Pharm Sci Assiut Univ 29:520–546
El-Sayed NA, Awadallah FM, Ibrahim NA, El-Saadi MT (2010)
Synthesis, anti-inflammatory and ulcerogenicity studies of some
substituted pyrimido[1,6-a]azepine derivatives. Eur J Med Chem
45:3147–3154
–
–
Average number of ulcers: number of ulcers in the
group/total number of rats in the group.
P
Ulcer index = the sum of the three figures
Average severity: [each ulcer multiplied by its score
of severity]/number of ulcers in the group.
Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE (2007)
Chronic inflammation: importance of NOD2 and NALP3 in
interleukin-1b generation. Clin Exp Immunol 147:227–235
Kasahara Y, Hikino H, Tsurufuji S, Watanabe M, Ohuchi K (1985)
Anti-inflammatory actions of ephedrines in acute inflammations.
Planta Med 51:325–331
Results are tabulated in Table 3.
Acknowledgments The authors are grateful to Prof. Dr. Hekma
Abdl-Tawab, professor of Pharmacology for her kind help in per-
forming the pharmacological screening.
Lacerda RB, de Lima CKF, da Silva LL, Romeiro NC, Miranda ALP,
Barreiro EJ, Fraga CAM (2009) Discovery of novel analgesic
and anti-inflammatory 3-arylamine-imidazo[1,2-a]pyridine sym-
biotic prototypes. Bioorg Med Chem 17:74–84
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