Synthesis and antitumor evaluation of some novel pyrrolizine derivatives

Article abstract of DOI:10.1007/s00044-011-9761-7

Novel series of pyrrolizines (7, 9a-d, 10a-d, 11a, b, 14a-d, 16, 19, 20a, b, 24, 25a, b), pyrimido[5,4-a]pyrrolizines (12a, b, 13, 15a, b, 18, 21a, b, 22, 23a-d) and pyrido[3,2-a]pyrrolizines (17, 26a, b) were synthesized through different reactions. The chemical structures of all the synthesized pyrrolizine derivatives were determined by spectral and elemental analyses. Antitumor activity evaluation of all the prepared compounds was carried out using NR assay method against breast cancer cell line (MCF-7). The novel pyrrolizine scaffold 7 and all its prepared derivatives showed high antitumor activity comparable to that of doxorubicin. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9761-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2349–2362
DOI 10.1007/s00044-011-9761-7
ORIGINAL RESEARCH
Synthesis and antitumor evaluation of some novel pyrrolizine
derivatives
•
Mona M. Hanna Nagwa M. Abdelgawad
•
•
Nashwa A. Ibrahim Amany B. Mohammed
Received: 3 February 2011 / Accepted: 19 July 2011 / Published online: 3 August 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Novel series of pyrrolizines (7, 9a–d, 10a–d,
11a, b, 14a–d, 16, 19, 20a, b, 24, 25a, b), pyrimido[5,4-
a]pyrrolizines (12a, b, 13, 15a, b, 18, 21a, b, 22, 23a–d)
and pyrido[3,2-a]pyrrolizines (17, 26a, b) were synthesized
through different reactions. The chemical structures of all
the synthesized pyrrolizine derivatives were determined by
spectral and elemental analyses. Antitumor activity evalu-
ation of all the prepared compounds was carried out using
NR assay method against breast cancer cell line (MCF-7).
The novel pyrrolizine scaffold 7 and all its prepared
derivatives showed high antitumor activity comparable to
that of doxorubicin.
heterogeneous chemical compounds that treat cancer pri-
marily by being toxic to the rapidly growing and dividing
cells. Despite the long history of their use and the devel-
opment of several new multiple drug regimens to improve
clinical success, treatment failure is still frequently
encountered even in patients with malignancies that are
highly sensitive to chemotherapeutic agents (e.g., breast
cancer).
In comparison to other drug classes, cytotoxic antican-
cer drugs present unique problems that come primarily
from the relative lack of specificity of their systemic bio-
distribution, their susceptibility to induce drug resistance
and the administration of high drug doses due to their
´
unfavorable pharmacokinetics (Wong et al., 2007; Duran
et al., 2008).
Keywords Pyrrolizines ꢀ Pyrimidopyrrolizines ꢀ
Pyridopyrrolizines ꢀ Antitumor
The pyrrolizine ring constitutes an important skeleton
for many natural and synthetic compounds of diverse
biological activities including analgesic, anti-inflammatory
(Ulbrich et al., 2002; Abbas, et al., 2010), antimalarial
(Sparatore et al., 2008), antiarrhythmic (Hattori et al.,
1988), nootropic (Oka et al., 2000), anticonvulsant (Diafi
et al., 1991), antibacterial (Barsoum and Nawar, 2003), and
antiviral activities (Argyropoulos et al., 2008). Further-
more, it represents a key nucleus for the synthesis of many
antitumor drugs, the ring itself being a good alkylator for
DNA such as Mitomycin C 1. This drug is known to be
reductively activated intracellularly to the bifunctional
pyrrolizine intermediate 2 that binds to the minor groove
and selectively forms interstrand cross-linking with DNA
(Rajaraman and Jimenez, 2002). Other pyrrolizine deriva-
tives 3, 4 have been reported to be cytotoxic against breast
cancer cells (Sonnet et al., 2000; El-Moghazy et al., 2009).
In addition, the antitumor activity has been observed in
several pyrimidopyrrolizines 5, 6 (Mezentseva et al., 1991;
El-Moghazy et al., 2009).
Introduction
Cancer has posed a great challenge to the fields of medicine
and immunology. Finding novel and efficient compounds
has been a major point of concern for research in phar-
maceutical sciences. Apart from surgery, radiation,
immunotherapy, and hormonal therapy, the therapeutic
tactics presently used in the clinical practice for cancer
treatment, are focused on cytotoxic drugs as the main form
of chemotherapy for cancer. Cytotoxic drugs are very
M. M. Hanna ꢀ N. M. Abdelgawad
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo, Egypt
N. A. Ibrahim (&) ꢀ A. B. Mohammed
Medicinal Chemistry Department, Faculty of Pharmacy,
Beni-Suef University, Beni-Suef, Egypt
e-mail: nashwaahmed_im@yahoo.com
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Med Chem Res (2012) 21:2349–2362
Based on these findings and for further exploration of
Results and discussion
pyrrolizines and pyrrolizine–heterocyclic hybrids, a new
derivative 7 was designed and synthesized to be used as a
heterocyclic scaffold for the targeted compounds. Both
morpholine and hydroxyl group moieties were incorporated
in the scaffold 7 as hydrogen bond donors (Nuss et al.,
2009). Preliminary antitumor screening of 7 was promising
as it revealed IC50 of 0.035 lmol/ml that was found to be
less than that of doxorubicin.
Chemistry
The synthetic pathways used in the synthesis of the target
compounds are illustrated in Schemes 1 and 2. 2-Pyrroli-
din-2-ylidene-malononitrile 8 was prepared according to
the previously reported procedure (Etienne and Correia,
1969; Ebeid and Bitter, 1978). The target compound 7 was
obtained by stirring pyrrolidin-2-ylidenemalononitrile 8,
morpholine, and formaldehyde at room temperature. The
IR spectrum of compound 7 showed sharp band at
3477 cm-1 corresponding to hydroxyl group, two stretch-
ing absorption bands at 3410 and 3289 cm-1 characteristic
for amino group and an absorption band at 2197 cm-1
The obtained results focused our interest to perform
several modifications on this novel pyrrolizine system 7
that were targeted toward two groups of compounds. The
first group included the pyrrolizine derivatives with dif-
ferent substituents at position 6 (general formula A), and
the second group of compounds involved new pyrrolizine-
heterocyclic hybrids formed by the fusion of 7 with other
heterocyclic moieties exemplified by pyrimido and pyri-
dopyrrolizines (general formula B) (Figs. 1, 2).
1
attributed to cyano group. The H-NMR of compound 7
revealed three signals at 2.09, 2.90, and 3.64 ppm corre-
sponding to the pyrrolizine protons CH2-2, CH2-1, CH2-3,
Fig. 1 Examples of pyrrolizine
compounds with antitumor
activity
O
CH2OCONH2
OCH3
O(C=O)NH2
H2N
H3C
OH
H2N
H3C
N
NH
N
NH
O
OH
1
2
Mitomycin C
O
CN
R
R
N
N
NH
O
R1
R=NHCONHC2H5,NHCOOC2H5
R= 3-, 4- pyridinyl, R1= H, OCF3
4
3
H5C2
O
H
N
N
O
H2N
NH
N
N
N
CONHC6H5
COC6H5
5
6
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Med Chem Res (2012) 21:2349–2362
2351
Fig. 2 General structures of the
target compounds
R
X
NH2
NC
R
NC
Y
O
R1
O
O
N
N
N
N
OH
N
OH
N
OH
7
A
B
1
respectively, a doublet signal at 4.10 ppm corresponding to
CH-6, that appeared as singlet upon deuteration, two
multiplets at 2.53 and 3.64 ppm confirming the presence of
the morpholino protons and two singlets at 6.14 and
9.51 ppm, exchangeable with D2O that corresponding to
the OH and NH2 groups, respectively. Furthermore, the
H–H cosy spectrum also showed the coupling between the
CH-6 and the OH groups. The structure was confirmed by
13C-NMR spectrum and HSQC that showed the direct
relation between each carbon atom and the directly
attached proton, the presence of three carbons C-5, C-7,
C-8, and the CN group free from protons.
derivative 7 with ethyl acetoacetate. The H-NMR spec-
trum of compound 16 revealed a singlet signal at 2.67 ppm
corresponding to the methyl group and another singlet at
3.78 ppm representing COCH2CO protons. The pyrimido-
pyrrolizine derivative 18 was prepared by refluxing com-
pound 7 with formic acid. The IR spectrum of 18 showed
the disappearance of the absorption bands corresponding to
both cyano and amino groups confirming intramolecular
cyclization and the appearance of an absorption band at
1626 cm-1 corresponding to the carbonyl group. Form-
amidic acid ethyl ester derivative 19 was obtained by
refluxing 7 with triethyl orthoformate while the amidine
derivative 24 was prepared by reacting 7 with excess
formamide. The reaction of 19 with isonicotinic acid
hydrazide or benzoic acid hydrazide yielded 20a, b,
respectively. These compounds were cyclized to the
pyrimidopyrrolizine derivatives 21a, b by refluxing with
ethanolic HCl. The IR spectrum of 21a revealed the dis-
appearance of the stretching absorption band of the cyano
group and the appearance of an absorption band at
1661 cm-1 corresponding to two CO groups. The 1H-NMR
of both compounds 21a, b revealed the presence of the
aromatic signals at 7.54–7.69 and 7.06–7.42 ppm charac-
terizing the pyridine and phenyl ring, respectively.
As shown in Scheme 1, the p-substituted benzylidene
derivatives 9a–d were obtained by the reaction of the
pyrrolizine derivative 7 with different p-substituted benz-
1
aldehydes in absolute ethanol. The H-NMR spectrum of
9a showed the appearance of a singlet signal at 9.53 ppm,
with an integration of one proton corresponding to the
benzylidene proton. Subsequent treatment of 9a–d with
thioglycolic acid in dry benzene yielded the thiazolidinone
derivatives 10a–d. The 1H-NMR spectrum of 10a exhibited
a singlet signal at 5.65 ppm corresponding to the methy-
lene protons of the thiazolidinone ring. The thiourea and
the urea derivatives 11a, b were synthesized by refluxing
compound 7 with ethyl isothiocyanate or phenyl isocya-
nate, respectively. Stirring 11a, b with 1% sodium ethoxide
at room temperature gave 12a, b. The disappearance of the
cyano group in their IR spectra confirmed the intramolec-
ular cyclization. However, the pyrimidopyrrolizine deriv-
ative 13 was obtained by reacting the pyrrolizine derivative
7 with p-chlorophenyl isocyanate at room temperature, its
structure was confirmed by 13C-NMR spectrum that
showed signals for the aromatic carbons besides the car-
bonyl carbon at 168.44 ppm. Different acylating agents
such as acetyl chloride, benzoyl chloride, chloroacetyl
chloride, and ethyl chloroformate reacted with compound 7
in dioxane at room temperature yielding the acylated
pyrrolizine derivatives 14a–d. The pyrimidopyrrolizine
derivatives 15a, b were obtained by cyclization of 14a, b
with 1% ethanolic sodium ethoxide.
The pyrimidopyrrolizine derivative 22 was synthesized
by two pathways; either by treating 19 with methanolic
solution saturated with ammonia or by cyclization of the
amidine derivative 24 using 1% sodium ethoxide. The
disappearance of the cyano stretching band in the IR
spectrum of 22 indicated the intramolecular cyclization.
In addition, it revealed the presence of broad stretching
bands at 3437 and 3289 cm-1, corresponding to the OH
1
and NH2 groups, respectively. The H-NMR showed two
singlet signals at 4.77 and 9.53 ppm representing the NH2
and N=CH protons, respectively. The reaction of the
pyrrolizine derivative 7 with acetyl acetone or benzoyl
acetone in dioxane afforded 25a, b, respectively. The IR
spectrum of 25a revealed an absorption band at
1705 cm-1 corresponding to the carbonyl group. The
pyridopyrrolizine derivatives 17, 26a, b were obtained by
treating 16 and 25a, b with 1% sodium ethoxide. Reac-
tion of the pyrimidopyrrolizine 22 with different
As described in Scheme 2, the butanamide derivative 16
was synthesized by the reaction of the pyrrolizine
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Med Chem Res (2012) 21:2349–2362
NC
CN
NH
8
a
NH
2
NC
O
O
g
N
R
NH
NC
N
OH
O
R
7
N
OH
b
N
f
d
14a-d
N
NC
a: R=CH3
O
b: R= C6H5
c: R= CH2Cl
d: R= OC2H5
N
N
OH
X
Cl
R
9a-d
NH
a: R=Cl
O
NH
NC
b: R= NO2
N
O
c: R= OCH3
N
NH
HN
e
d: R= N(CH3)2
N
OH
O
N
11a, b
a: R= C2H5, X= S
N
OH
R
H
c
b: R= C6H5, X= O
13
N
N
e
O
S
O
R
N
X
N
OH
N
R
O
NC
N
O
NH
15a, b
N
HN
a: R= CH3
N
OH
O
N
b: R= C6H5
10a-d
N
OH
a: R=Cl
b: R= NO2
12a, b
a: R= C2H5, X= S
c: R= OCH3
b: R= C6H5, X= O
d: R= N(CH3)2
Scheme 1 Reagents and solvents: a morpholine, HCHO; b Ar–CHO,
ethanol; c thioglycolic acid, dry benzene; d ethyl isocyanate or phenyl
isothiocyanate, dichloromethane, triethylamine; e 1% NaOC2H5; f
p-chlorophenyl isocyanate, dichloromethane, triethylamine; g acetyl
chloride, benzoyl chloride, chloroacetyl chloride or ethyl chlorofor-
mate, dioxane
Antitumor activity
acylating agents as acetyl chloride, benzoyl chloride,
chloroacetyl chloride, and ethyl chloroformate in dioxane
1
yielded 23a–d. The H-NMR of compound 23d showed
All compounds were tested for their antitumor activity
against breast cancer cell line (MCF-7) using neutral red
assay method (Doyle and Griffiths, 1998). The novel
the characteristic signals of the ethyl group at 1.72 and
4.66 ppm.
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Med Chem Res (2012) 21:2349–2362
2353
CH
3
O
CH
3
NH
2
NC
O
O
O
N
N
R
NC
a
j
O
N
OH
N
NH
NC
7
N
OH
O
25a, b
i
N
a: R= CH3
b: R= C6H5
NH
c
2
N
OH
d
16
N
NC
O
N
b
NH
OC H
2
5
N
b
N
OH
N
O
O
NC
24
O
N
O
CH
N
3
N
OH
O
CH
3
N
OH
O
18
R
NH
N
b
19
H N
2
O
H N
2
O
e
N
g
N
O
N
OH
N
OH
R
17
N
N
NH NH
26a, b
H N
2
O
N
a: R= CH3
NC
N
O
b: R= C6H5
N
OH
N
22
N
OH
20 a,b
a: R= 4-C5H4N
b: R= C6H5
h
f
O
R
N
N
NH
NH
NH
O
NH
N
N
R
O
N
OH
O
O
N
23a-d
N
OH
a: R= CH3
b: R= C6H5
21a,b
a: R= 4-C5H4N
b: R= C6H5
c: R= CH2Cl
d: R= OC2H5
Scheme 2 Reagents and solvents: a CH3COCH2COOC2H5, dioxane;
b 1%NaOC2H5; c HCOOH; d CH(OC2H5)3; e INH or benzoic acid
hydrazide, dioxane; f ethanolic HCl; g methanolic solution of
ammonia; h acetyl chloride, benzoyl chloride, chloroacetyl chloride
or ethyl chloroformate, dioxane; i HCONH2; j CH3COCH2COCH3 or
C6H5COCH2COCH3, dioxane
pyrrolizine system 7 and all its prepared derivatives
revealed significant antitumor profile which was better
than that of doxorubicin. The p-nitro 9b and p-methoxy
9c benzylidine derivatives showed better activity than the
starting compound 7, while the p-chloro 9a and
p-dimethylamino derivative 9d were less active. How-
ever, the impact of cyclization of benzylidine derivatives
9a–d into thiazolidinone derivatives 10a–d on activity is
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Med Chem Res (2012) 21:2349–2362
not absolute as p-chloro 10a and p-dimethylamino sub-
stituents 10d revealed an increase in activity when
compared to 10b and 10c. All the 6-substituted amide
derivatives 14a–d and 16 showed an increase in activity
compared with the pyrrolizine system 7. Substitution on
the acetamido group of 14a with a lipophilic chlorine
atom 14c or an acetyl group 16 enhanced the activity;
however, aryl substitution 14b decreased the activity.
The thiourea and urea derivatives 11a, b, respectively,
exhibited a decrease in activity in comparison with
compound 7. Most of the 6-substituted imines (–N=C–)
19, 20a, b, 24, 25a, b revealed higher activity than
compound 7. Benzoic acid hydrazide derivative 20b was
found to be highly cytotoxic; however, its bioisosteric
analog isonicotinic acid hydrazide 20a exhibited a
decrease in activity, but it was still higher than that of
compound 7. The amidine 24 revealed good antitumor
activities, on the other hand, the ethoxyimino derivative
19 was less active. Distribution on the imino group with
methyl and phenacyl moities 25b enhanced the activity,
however, by substituting phenacyl with propionyl group
25a, a decrease in activity was observed compared with
compound 7. All the pyrimidopyrrolizines revealed better
activity than the lead compound 7 except compounds 13,
15a, 26a, that showed a slight deviation in their activity.
The highest cytotoxic activity was attributed to the
2-phenyl pyrimidinone derivative 15b, however, replace-
ment of the 2-phenyl with a methyl group 15a decreased
the activity to reach nearly that of compound 7. The
4-iminopyrimidinone derivative 12b having a phenyl
group at position 3 revealed a remarkable activity, on the
other hand, replacement of phenyl with p-chlorophenyl
group 13 or alkyl 12a decreased the activity. The 4-amino
pyrimidopyrrolizine derivative 22 was equipotent with 7,
however, its 4-oxo analog 18 and the 4-substituted amino
derivatives 23a–d exhibited an increase in activity that
reached the highest value in the benzamide derivative 23b.
The benzoic acid hydrazide derivative 21b and its isoni-
cotinic acid bioisostere 21a were nearly equal in activity
but were more active than the bicyclic system 7. Bioisos-
teric replacement of the pyrimido with pyrido nucleus
revealed good activity except for compound 26a.
Table 1 IC50 and S.E. of the tested compounds
Comp. no.
IC50 lmol/ml SE
7
0.035 0.002
0.045 0.001
0.019 0.001
0.016 0.001
0.037 0.002
0.018 0.004
0.055 0.001
0.035 0.001
0.019 0.011
0.037 0.001
0.044 0.001
0.034 0.001
0.018 0.003
0.037 0.001
0.029 0.001
0.034 0.001
0.023 0.002
0.027 0.001
0.036 0.001
0.063 0.008
0.016 0.001
0.022 0.001
0.029 0.002
0.030 0.004
0.041 0.001
0.029 0.001
0.018 0.004
0.020 0.001
0.019 0.002
0.035 0.001
0.029 0.003
0.021 0.001
0.023 0.001
0.025 0.002
0.026 0.001
0.042 0.001
0.028 0.001
0.038 0.001
9a
9b
9c
9d
10a
10b
10c
10d
11a
11b
12a
12b
13
14a
14b
14c
14d
15a
Doxorubicin
15b
16
17
18
19
20a
20b
21a
21b
22
23a
23b
23c
23d
24
25a
25b
26a
In conclusion, it could be presumed that all the tested
pyrrolizine derivatives adopted in this study, exerted high
antitumor activity against breast cancer cells compared to
that of doxorubicin. From the previous results of antitumor
screening, it was found that 25 out of the 38 prepared
compounds showed remarkable activity compared with the
scaffold system 7. It was noteworthy that, the bicyclic
p-methoxy benzylidene derivative 9c and the tricy-
clic phenyl substituted pyrimidine derivative 15b were
found to be the most active and equipotent compounds
(Table 1).
Experimental
Chemistry
Melting points were uncorrected and were carried out by
open capillary tube method using IA 9100MK-Digital
Melting Point Apparatus. Microanalyses were carried out
at the microanalytical Center, Faculty of Science, Cairo
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Med Chem Res (2012) 21:2349–2362
2355
University. Infrared spectra were made on Bruker Vector
22 (Japan) infrared spectrophotometers and were expressed
in wave number (cm-1) using potassium bromide disc. The
proton magnetic resonance 1H-NMR spectra were recorded
on a Varian Mercury VX-300 NMR spectrometer at
300 MHz in the specified solvent. 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 Bruker APX400 at 100 MHz. Mass
spectra were recorded on Fennigan MAT, SSQ 7000, Mass
spectrometer, at 70 eV (EI). Compound 8 was prepared
according to the reported procedures (Etienne and Correia,
1969; Ebeid and Bitter, 1978). All the newly synthesized
compounds were obtained as racemic mixtures.
(CN), 1624, 1576 (N=C, C=C), 1514, 1474 (C–N, C–O),
1
996 (C–Cl). H-NMR (300 MHz, CDCl3: d, ppm): 2.06–
2.16 (m, 2H, CH2-2), 2.54–2.57 (m, 4H, N(CH2)2), 2.91
(t, J = 6.3 Hz, 2H, CH2-1), 3.65–3.70 (m, 4H, O(CH2)2),
4.11 (t, J = 6.6 Hz, 2H, CH2-3), 4.75 (d, J = 6.9 Hz, 1H,
CH-6), 7.23–7.42 (m, 4H, aromatic protons), 9.35 (s, 1H,
OH exchangeable with D2O), 9.53 (s, 1H, N=CH). Anal.
Calcd. For C19H21ClN4O2 (372.85): C, 61.21, H, 5.68, N,
15.03. Found: C, 61.50, H, 6.18, N, 15.16.
6-[(4-Nitrobenzylidene)amino]-5-hydroxy-5-morpholin-4-
yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-carbonitrile 9b
White crystals, m.p. 172–4°C, yield 65%. IR: tmax/cm-1
3324 (OH), 3050 (CH aromatic), 2959, 2924 (CH ali-
phatic), 2191 (CN), 1628, 1580, 1519 (NO2, N=C, C=C),
6-Amino-5-hydroxy-5-morpholin-4-yl-2,3,5,6-tetrahydro-
1H-pyrrolizine-7-carbonitrile 7
1
1423, 1298, 1246 (C–N, C–O, NO2). H-NMR (300 MHz,
CDCl3: d, ppm): 2.06–2. 16 (m, 2H, CH2-2), 2.54–2.57 (m,
4H, N(CH2)2), 2.91 (t, J = 6.3 Hz, 2H, CH2-1), 3.65–3.72
(m, 4H, O(CH2)2), 4.11 (t, J = 6.6 Hz, 2H, CH2-3), 4.75
(d, J = 6.9 Hz, 1H, CH-6), 7.27–7.37 (m, 4H, aromatic
protons), 9.53 (s, 2H, N=CH, OH exchangeable with D2O).
Anal. Calcd. For C19H21N5O4 (383.40): C, 59.52, H, 5.52,
N, 18.27. Found: C, 59.17, H, 5.27, N, 18.70.
Compound 8 (1 g, 7.5 mmol) was dissolved in morpholine
(5 ml), then formaldehyde solution (34%) (0.75 ml,
7.5 mmol) was added portion wise. The reaction mixture
was stirred at room temperature for 24 h, poured over
cooled water and left overnight in a refrigerator. The
formed crystals were filtered, washed with water and
crystallized from ethanol. m.p. 150–152°C, yield 75%. IR:
t
max/cm-1 3477 (OH), 3410, 3289 (NH2), 2956, 2845 (CH
aliphatic), 2197 (CN), 1632, 1586 (NH, C=C), 1482, 1312
1
6-[(4-Methoxybenzylidene)amino]-5-hydroxy-5-
morpholin-4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-
carbonitrile 9c
(C–N, C–O). H-NMR (300 MHz, CDCl3: d, ppm): 2.09–
2.15 (m, 2H, CH2-2), 2.53–2.56 (m, 4H, N(CH2)2), 2.90 (t,
J = 7.8 Hz, 2H, CH2-1), 3.64–3.71 (m, 6H, O(CH2)2,
CH2-3), 4.10 (d, J = 6.3 Hz, 1H, CH-6), 6.14 (s, 2H, NH2
exchangeable with D2O), 9.51 (s, 1H, OH) exchangeable
with D2O. 13C NMR (DMSO-d6): d 20.22 (C-2), 33.42 (C-
1), 49.03 (C-3), 49.97 (N(CH2)), 51.55 (C-7), 60.06 (C-6),
66.12 (O(CH2)),120.07 (CN), 167.02 (C-5), 171.55 (C-7a).
MS: m/z (%) 250 (M?, 5), 100 (100). Anal. Calcd. for
C12H18N4O2 (250.30): C, 57.58, H, 7.25, N, 22.38. Found:
C, 57.81, H, 6.87, N, 21.97.
White crystals, m.p. 195–7°C, yield 73%. IR: tmax/cm-1
3475 (OH), 3100 (CH aromatic), 2955, 2925 (CH ali-
phatic), 2196 (CN), 1631, 1585 (N=C, C=C), 1482, 1312
(C–N, C–O). 1H-NMR (300 MHz, DMSO-d6: d, ppm):
1.04–1.08 (m, 2H, CH2-2), 1.88–1.98 (m, 6H, N(CH2)2,
CH2-1), 2.74–2.82 (m, 4H, O(CH2)2), 3.54 (t, J = 6.9 Hz,
2H, CH2-3), 4.10 (s, 3H, OCH3), 4.53 (d, J = 6.9 Hz, 1H,
CH-6), 7.52–7.98 (m, 4H, aromatic protons), 9.62 (s, 1H,
N=CH), 9.66 (s, 1H, OH exchangeable with D2O). Anal.
Calcd. For C20H24N4O3 (368.43): C, 65.20, H, 6.57, N,
15.21. Found: C, 65.36, H, 6.76, N, 15. 65.
General method for the preparation of 9a–d
A mixture of compound 7 (0.78 g, 3.12 mmol) and the
appropriate p-substituted benzaldehyde (3.12 mmol) was
refluxed in absolute ethanol (20 ml) in presence of glacial
acetic acid (0.5 ml) for 24 h. The solvent was evaporated
under vacuum. The separated solid were collected, dried,
and recrystallized from acetone.
6-[(4-Dimethylaminobenzylidene)amino]-5-hydroxy-5-
morpholin-4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-
carbonitrile 9d
White crystals, m.p. 210–3°C, yield 68%. IR: tmax/cm-1
3300 (OH), 2989 (CH aromatic), 2930 (CH aliphatic), 2184
(CN), 1631, 1601 (N=C, C=C), 1513, 1386 (C–N, C–O).
1H-NMR (300 MHz, DMSO-d6: d, ppm): 1.91–1.96
(m, 2H, CH2-2), 2.39–2.49 (m, 4H, N(CH2)2), 2.79
6-[(4-Chlorobenzylidene)amino]-5-hydroxy-5-morpholin-
4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-carbonitrile 9a
White crystals, m.p. 183–5°C, yield 74%. IR: tmax/cm-1
3375 (OH), 3171 (CH aromatic), 2955 (CH aliphatic), 2186
123

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6-[2-(4-Methoxyphenyl)-4-oxo-thiazolidine-3-yl]-5-
hydroxy-5-morpholin-4-yl-2,3,5,6-tetrahydro-1H-
pyrrolizine-7-carbonitrile 10c
(t, J = 8.1 Hz, 2H, CH2-1), 3.44–3.54 (m, 6H, O(CH2)2,
CH2-3), 3.70 (s, 6H, N(CH3)2), 3.96 (d, J = 6 Hz, 1H, CH-6),
7.28–7.58 (m, 4H, aromatic protons), 9.62 (s, 2H, N=CH,
OH exchangeable with D2O). Anal. Calcd. For
C21H27N5O2 (381.47): C, 66.12, H, 7.13, N, 18.36. Found:
C, 65.95, H, 7.53, N, 18.06.
Yellow crystals, m.p. 222–5°C, yield 65%. IR: tmax/cm-1
3323 (OH), 3132, 3036 (CH aromatic), 2919, 2851 (CH
aliphatic), 2191 (CN), 1643 (CO), 1595 (C=C), 1487, 1305,
1229 (C–N, C–O). 1H-NMR (300 MHz, DMSO-d6: d,
ppm): 1.90–1.98 (m, 2H, CH2-2), 2.74–2.81 (m, 6H,
N(CH2)2, CH2-1), 3.52–3.58 (m, 6H, O(CH2)2, CH2-3),
3.64 (s, 3H, OCH3), 4.34 (d, J = 6 Hz, 1H, CH-6), 6.55
(s, 2H, COCH2S), 7.76–7.80 (m, 4H, aromatic protons),
9.63 (s, 1H, NCHS), 10.62 (s, 1H, OH exchangeable with
D2O). Anal. Calcd. For C22H26N4O4S (442.53): C, 59.71,
H, 5.92, N, 12.66. Found: C, 59.96, H, 5.91, N, 12.42.
General method for the preparation of 10a–d
A mixture of the appropriate compound 9a–d (3.12 mmol)
and thioglycolic acid (0.29 g, 3.12 mmol) was refluxed in
dry benzene (20 ml) for 24 h. The excess solvent was
evaporated under vacuum. The formed solid was collected,
dried and crystallized from ethanol.
6-[2-(4-Dimethylaminophenyl)-4-oxo-thiazolidine-3-yl]-5-
hydroxy-5-morpholin-4-yl-2,3,5,6-tetrahydro-1H-
pyrrolizine-7-carbonitrile 10d
6-[2-(4-Chlorophenyl)-4-oxo-thiazolidine-3-yl]-5-hydroxy-
5-morpholin-4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-
carbonitrile 10a
Yellow crystals, m.p. 232–4°C, yield 68%. IR: tmax/cm-1
3426 (OH), 3089 (CH aromatic), 2919 (CH aliphatic), 2178
(CN), 1767 (CO), 1601, 1508, 1376 (C–N, C–O). 1H-NMR
(300 MHz, DMSO-d6: d, ppm): 2.06–2.16 (m, 2H, CH2-2),
2.89–2.95 (m, 6H, N(CH2)2, CH2-1), 3.64–3.72 (m, 6H,
CH2-3, O(CH2)2), 4.23 (s, 6H, N(CH3)2), 4.59 (d, 1H,
J = 9 Hz, CH-6), 5.65 (s, 2H, COCH2S), 6.79 (s, 1H,
NCHS), 7.56,7.60 (2d, 4H, J = 6 Hz, aromatic protons),
9.51 (s, 1H, OH exchangeable with D2O). Anal. Calcd. For
C23H29N5O3S (455.57): C, 60.64, H, 6.42, N, 15.37.
Found: C, 60.46, H, 6.80, N, 15.84.
Yellow crystals, m.p. 214–6°C, yield 79%. IR: tmax/cm-1
3414 (OH), 3050 (CH aromatic), 2930 (CH aliphatic), 2189
(CN), 1697 (CO), 1587, 1387 (C–N, C–O), 775 (C–Cl). 1H-
NMR (300 MHz, CDCl3: d, ppm): 2.06–2.16 (m, 2H, CH2-
2), 2.89–2.95 (m, 6H, N(CH2)2, CH2-1), 3.64–3.72 (m, 6H,
CH2-3, O(CH2)2), 4.58 (d, J = 9 Hz, 1H, CH-6), 5.65 (s,
2H, COCCH2S), 6.79 (s, 1H, NCHS), 7.55–7.61 (m, 4H,
aromatic protons), 9.51 (s, 1H, OH exchangeable with
D2O). 13C-NMR (DMSO-d6, 75.45 MHz): 20.19 (C-2),
20.24 (C-1), 32.92 (CH2–S), 33.28 (C-3), 33.46 (N(CH2)2),
48.94 (N–C–S), 49.14 (C-6), 66.08 (O(CH2)2), 66.33 (C-7),
119.80 (C–OH), 120.40 (CN), 120.60, 166.30, 168.44,
171.58 (aromatic carbons), 171.62 (C=C-7), 171.71 (C=O).
Anal. Calcd. For C21H23ClN4O3S (446.03): C, 56.43, H,
5.19, N, 12.54. Found: C, 56.83, H, 5.60, N, 12.71.
General method for the preparation of 11a, b
A mixture of compound 7 (0.78 g, 3.12 mmol), and equi-
molar amount of ethyl isothiocyanate or phenyl isocyanate
(3.12 mmol) in dichloromethane was refluxed for 10 h in
presence of triethylamine (5 drops). The reaction mixture
was evaporated under vacuum; the formed solid was col-
lected, dried, and crystallized from ethanol.
6-[2-(4-Nitrophenyl)-4-oxo-thiazolidine-3-yl]-5-hydroxy-
5-morpholin-4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-
carbonitrile 10b
1-(7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1h-pyrrolizin-6-yl)-3-ethylthiourea 11a
Yellow crystals, m.p. 205–7°C, yield 76%. IR: tmax/cm-1
3431 (OH), 3055 (CH aromatic), 2958, 2924 (CH ali-
phatic), 2183 (CN), 1640 (br. CO), 1579, 1449 (NO2,
C=C), 1368, 1299, 1165 (NO2, C–N, C–O). 1H-NMR
(300 MHz, DMSO-d6: d, ppm): 1.88–1.98 (m, 2H, CH2-2),
2.74–2.85 (m, 6H, N(CH2)2, CH2-1), 3.52–3.57 (m, 6H,
O(CH2)2, CH2-3), 3.96 (d, J = 6.3 Hz, 1H, CH-6), 6.56 (s,
2H, COCH2S), 7.28–7.58 (m, 4H, aromatic protons), 9.62
(s, 1H, NCHS), 11.68 (s, 1H, OH exchangeable with D2O).
Anal. Calcd. For C21H23N5O5S (457.50): C, 55.13, H, 5.07,
N, 15.31. Found: C, 55.09, H, 5.33, N, 15.64.
White crystals, m.p. 175–7°C, yield 79%. IR: tmax/cm-1
3396 (OH), 3359, 3297 (NHs), 2988, 2919 (CH aliphatic),
2184 (CN), 1630 (C=S), 1510, 1388 (NH, C=C), 1292,
1246 (C–N, C–O). 1H-NMR (300 MHz, DMSO-d6:
d, ppm): 1.23 (t, 3H, J = 4.8 Hz, CH2CH3), 1.91–1.96 (m,
2H, CH2-2), 2.39–2.49 (m, 4H, N(CH2)2), 2.79
(t, J = 6 Hz, 2H, CH2-1), 3.44–3.80 (m, 8H, O(CH2)2,
CH2CH3, CH2-3), 3.96 (d, 1H, J = 6 Hz, CH-6), 6.55,
7.27, 9.62 (3s, 3H, 2NHs, OH exchangeable with D2O).
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Med Chem Res (2012) 21:2349–2362
2357
Anal. Calcd. For C15H23N5O2S (337.44): C, 53.39, H, 6.87,
N, 20.75. Found: C, 53.00, H, 7.27, N, 20.35.
(m, 6H, O(CH2)2, CH2-7), 5.36 (d, J = 7.8 Hz, 1H, CH-
9a), 6.55, 9.62 (2s, 3H, 2NHs, OH exchangeable with
D2O), 7.06–7.42 (m, 5H, aromatic protons). Anal. Calcd.
for C19H23N5O3 (369.42): C, 61.77, H, 6.28, N, 18.96.
Found: C, 61.45, H, 5.98, N, 19.14.
1-(7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro -1H-pyrrolizin-6-yl)-3-phenylurea 11b
White crystals, m.p. 183–5°C, yield 83%. IR: tmax/cm-1
3435 (OH), 3336, 3283 (NHs), 3100 (CH aromatic), 2966,
2947 (CH aliphatic), 2184 (CN), 1643 (CO), 1585,
3-(4-Chlorophenyl)-9-hydroxy-4-imino-9-morpholin-4-yl-
3,4,6,7,9,9a-hexahydro-1H-pyrimido[5,4-a]pyrrolizine-
2(5H)-one 13
1
1483,1454 (NH, C=C), 1369, 1337 (C–N, C–O). H-NMR
(300 MHz, CDCl3: d, ppm): 2.06–2.16 (m, 2H, CH2-2),
2.62 (s, 4H, N(CH2)2), 2.88 (t, 2H, J = 9.9 Hz, CH2-1),
3.65–3.75 (m, 6H, O(CH2)2, CH2-3), 4.17 (d, 1H,
J = 6.3 Hz, CH-6), 5.06, 6.26, 9.51 (3s, 3H, 2NHs, OH,
exchangeable with D2O), 7.06–7.42 (m, 5H, aromatic
protons). Anal. Calcd. For C19H23N5O3 (369.42): C, 61.77,
H, 6.28, N, 18.96. Found: C, 61.57, H, 6.18, N, 19.13.
A mixture of compound 7 (0.78 g, 3.12 mmol), and
p-chlorophenyl isocyanate (4.8 g, 3.12 mmol) in dichlo-
romethane (20 ml) and triethylamine (5 drops) was stirred
for 24 h at room temperature, the formed white precipitate
was filtered, dried and crystallized from acetone. m.p. 176–
8°C, yield 89%. IR: tmax/cm-1 3427 (OH), 3329, 3274
(NHs), 3100 (CH aromatic), 2920 (CH aliphatic), 1643
(CO), 1577, 1452 (NH, C=C), 1364, 1299 (C–N, C–O), 778
General method for the preparation of 12a, b
1
(C–Cl). H-NMR (300 MHz, CDCl3: d, ppm): 2.06–2.19
(m, 2H, CH2-6), 2.26–2.65 (m, 4H, N(CH2)2), 2.67 (t, 2H,
CH2-5), 3.52–3.78 (m, 6H, O(CH2)2, CH2-7), 4.21 (d,
J = 6.6 Hz, 1H, CH-9a), 6.38, 6.98, 9.51 (3s, 3H, 2NH,
OH exchangeable with D2O), 7.23–7.32 (m, 4H, aromatic
protons). 13C-NMR (DMSO-d6-75.45 MHz): 20.19 (C-6),
20.24 (C-5), 41.83 (C-7), 49.14 (N(CH2)2), 66.33 (C-9a),
70.05 (O(CH2)2), 70.19 (C=C), 107.05 (C–OH), 119.82,
120.40, 130.40, 130.82 (aromatic carbons), 156.01
(C=NH), 166.30 (C=C), 168.44 (C=O). Anal. Calcd. For
C19H22ClN5O3 (403.86): C, 56.51, H, 5.49, N, 17.34.
Found: C, 56.90, H, 5.62, N, 17.04.
A solution of the appropriate compound 11a, b in 1%
sodium ethoxide (30 ml) was stirred for 24 h at room
temperature. The obtained solid was filtered, dissolved in
the least amount of water and acidified with diluted
hydrochloric acid. The separated precipitate was filtered,
dried, and crystallized from ethanol.
3-Ethyl-9-hydroxy-4-imino-9-morpholin-4-yl-3,4,6,7,9,9a-
hexahydro-1H-pyrimido[5,4-a]pyrrolizine-2 (5H)-thione
12a
Yellow crystals, m.p. 205–7°C, yield 82%. IR: tmax/cm-1
3384 (OH, NHs), 2923, 2854 (CH aliphatic), 1448 (C–N,
C–C). 1H-NMR (300 MHz, DMSO-d6: d, ppm): 1.23
(t, 3H, J = 6 Hz, CH2CH3), 1.91–1.96 (m, 2H, CH2-6),
2.39–2.49 (m, 4H, N(CH2)2), 2.79 (t, J = 7.8 Hz, 2H,
CH2-5), 3.44–3.54 (m, 6H, CH2-7, O(CH2)2), 3.65 (q,
J = 5.7 Hz, 2H, CH2CH3), 4.60 (d, J = 6.3 Hz, 1H, CH-
9a), 5.37, 6.55, 9.62 (3s, 3H, 2NHs, OH, exchangeable with
D2O). MS: m/z (%) 337 (M?, 3), 57 (100). Anal. Calcd. for
C15H23N5O2S (337.44): C, 53.39, H, 6.87, N, 20.75.
Found: C, 53.39, H, 6.82, N, 20.27.
General method for the preparation of 14a–d
A mixture of compound 7 (1 g, 3.99 mmol) and the
appropriate acid chloride or ethyl chloroformate (4 mmol)
in dioxane (20 ml) was stirred for 24 h at room tempera-
ture. The formed precipitate was filtered, dried and crys-
tallized from methanol.
N-(7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro)-1H-pyrrolizin-6-yl)acetamide 14a
White crystals, m.p. 225–7°C, yield 75%. IR: tmax/cm-1
3357 (OH), 3299 (NH), 2983, 2922 (CH aliphatic), 2185
(CN), 1631 (CO), 1511, 1429 (NH, C=C), 1386, 1301,
9-Hydroxy-4-imino-9-morpholin-3-phenyl-4-yl-
3,4,6,7,9,9a-hexahydro-1H-pyrimido[5,4-a]pyrrolizine-
2(5H)-one 12b
1
1248 (C–N, C–O). H-NMR (300 MHz, CDCl3: d, ppm):
Yellow crystals, m.p. 227–9°C, yield 85%. IR: tmax/cm-1
3324, 3194 (OH, NHs), 3057 (CH aromatic), 2921 (CH
aliphatic), 1647 (CO), 1597, 1554, 1492 (NH, C=N, C=C),
1.62 (s, 3H, CH3), 2.05–2.15 (m, 2H, CH2-2), 2.87–2.92
(m, 6H, N(CH2)2, CH2-1), 3.66–3.71 (m, 6H, O(CH2)2),
CH2-3), 4.73 (t, 1H, J = 6.2 Hz, CH-6), 6.57, 9.55 (2s, 2H,
NH, OH exchangeable with D2O). Anal. Calcd. for
C14H20N4O3 (292.33): C, 57.52, H, 6.90, N, 19.17. Found:
C, 57.32, H, 7.20, N, 19.30.
1
1310, 1232 (C–N, C–O). H-NMR (300 MHz, DMSO-d6:
d, ppm): 1.91–1.96 (m, 2H, CH2-6), 2.39–2.49 (m, 4H,
N(CH2)2), 2.79 (t, J = 7.8 Hz, 2H, CH2-5), 3.52–3.54
123

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Med Chem Res (2012) 21:2349–2362
N-(7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro)-1H-pyrrolizin-6-yl)benzamide 14b
9-Hydroxy-2-methyl-9-morpholin-4-yl-3,4,6,7,9,9a-
hexahydro-3H-pyrimido[5,4-a]pyrrolizin-4(5H)-one 15a
White crystals, m.p. 232–4°C, yield 82%. IR: tmax/cm-1
3363 (OH), 3314 (NH), 3008 (CH aromatic), 2960 (CH
aliphatic), 2191 (CN), 1649 (CO), 1589, 1522, 1455 (NH,
C=C), 1435, 1403, 1309 (C–N, C–O). 1H-NMR (300 MHz,
CDCl3: d, ppm): 2.05–2.15 (m, 2H, CH2-2), 2.87–2.92 (m,
6H, N(CH2)2, CH2-1), 3.66–3.71 (m, 6H, O(CH2)2, CH2-3),
4.73 (t, 1H, J = 6.7 Hz, CH-6), 6.57, 9.55 (2s, 2H, NH,
OH exchangeable with D2O), 7.42–7.52 (m, 5H, aromatic
protons). Anal. Calcd. for C19H22N4O3 (354.40): C, 64.39,
H, 6.26, N, 15.81. Found: C, 64.79, H, 5.96, N, 15.74.
Yellow crystals, m.p. 247–9°C, yield 68%. IR: tmax/cm-1
3419 (broad band OH, NH), 2967 (CH aliphatic), 1635
(CO), 1449 (C–O, C–N). 1H-NMR (300 MHz, CDCl3:
d, ppm): 1.26 (s, 3H, CH3), 1.52–1.55 (m, 2H, CH2-6),
2.01–2.15 (m, 6H, N(CH2)2, CH2-5), 2.87–2.92 (m, 4H,
O(CH2)2), 3.69 (t, J = 7.2 Hz, 2H, CH2-7), 4.72 (s, 1H,
CH-9a), 6.92, 7.61 (2s, 2H, NH, OH exchangeable with
D2O). Ms: m/z (%) 292 (M?, 3), 57 (100). Anal. Calcd. for
C14H20N4O3 (292.33): C, 57.52, H, 6.90, N, 19.17. Found:
C, 57.32, H, 7.20, N, 19.30.
9-Hydroxy-9-morpholin-2-phenyl–3,4,6,7,9,9a-hexahydro-
5H-pyrimido[5,4-a]pyrrolizin-4(5H)-one 15b
2-Chloro-N-(7-cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro)-1H-pyrrolizin-6-yl)acetamide 14c
Yellow crystals, m.p. 283–5°C, yield 73%. IR: tmax/cm-1
3437 (br., OH, NH), 2967 (CH aliphatic), 1632 (CO), 1498,
1443 (N–H, C–O, C–N). 1H-NMR (300 MHz, CDCl3:
d, ppm): 2.06–2.16 (m, 4H, CH2-5,6), 2.89–2.95 (m, 4H,
N(CH2)2), 3.64–3.72 (m, 6H, O(CH2)2, CH2-7), 4.58
(d, J = 6.3 Hz, 1H, CH-9a), 5.65, 9.51 (2s, 2H, NH, OH
exchangeable with D2O), 7.56–7.90 (m, 5H, aromatic
protons). Anal. Calcd. for C19H22N4O3 (354.40): C, 64.39,
H, 6.26, N, 15.81. Found: C, 63.92, H, 6.56, N, 15.87.
White crystals, m.p. 217–9°C, yield 85%. IR: tmax/cm-1
3363 (OH), 3313 (NH), 2960, 2871 (CH aliphatic), 2191
(CN), 1650 (CO), 1589, 1521, 1456 (NH, C=C), 1404,
1310, 1272 (C–N, C–O), 944,775 (C–Cl). 1H-NMR
(300 MHz, CDCl3: d, ppm): 2.05–2.15 (m, 2H, CH2-2),
2.87–2.92 (m, 6H, N(CH2)2, CH2-1), 3.56–3.71 (m, 6H,
O(CH2)2, CH2-3), 4.73 (t, 1H, J = 6.7 Hz, CH-6), 5.26
(s, 2H, COCH2Cl), 6.57, 9.55 (2s, 2H, NH, OH
exchangeable with D2O). Anal. Calcd. for C14H19ClN4O3
(326.78): C, 51.46, H, 5.86, N, 17.15. Found: C, 51.85, H,
6.16, N, 17.26.
N-(7-cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro)-1H-pyrrolizin-6-yl)-3-oxobutanamide 16
To a mixture of compound 7 (0.26 g, 2 mmol) and trieth-
ylamine (0.5 ml) in dioxane (20 ml), ethyl acetoacetate
(2 mmol) was added, the reaction mixture was refluxed for
10 h then concentrated by distillation under vacuum. The
separated solid was filtered, dried, and crystallized from
acetone, m.p. 195–7°C, yield 82%. IR: tmax/cm-1 3422
(OH), 3325 (NH), 2959 (CH aliphatic), 2193 (CN), 1679
(br., 2CO), 1628, 1581, 1520 (NH, C=C, C=O), 1294, 1245
Ethyl-(7-cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizin-6-yl) carbamate 14d
White crystals, m.p. 242–4°C, yield 78%. IR: tmax/cm-1
3358 (OH), 3297 (NH), 2919 (CH aliphatic), 2185 (CN),
1631 (CO), 1600, 1513 (NH, C=C), 1458, 1388, 1332 (C–N,
1
C–O). H-NMR (300 MHz, CDCl3: d, ppm): 1.21 (t, 3H,
1
J = 6.9 Hz, CH2CH3), 2.06–2.16 (m, 2H, CH2-2,), 2.54–
2.57 (m, 4H, N(CH2)2), 2.91 (t, 2H, J = 6 Hz, CH2-1),
3.65–3.72 (m, 8H, O(CH2)2, CH2-3, CH2CH3), 4.12 (d, 1H,
J = 6.3 Hz), 6.12, 9.53 (2s, 2H, NH, OH exchangeable with
D2O). Anal. Calcd. for C15H22N4O4 (322.36): C, 55.89, H,
6.88, N, 17.38. Found: C, 55.72, H, 6.81, N, 17.78.
(C–O, C–N). H-NMR (300 MHz, CDCl3: d, ppm): 2.00–
2.17 (m, 2H, CH2-2), 2.67 (s, 3H, CH3), 2.89–2.97 (m, 6H,
N(CH2)2), CH2-1), 3.60–3.73 (m, 6H, O(CH2)2, CH2-3),
3.78 (s, 2H, COCH2), 4.21 (d, 1H, J = 5.7 Hz, CH-6),
6.34, 9.51 (2s, 2H, NH, OH, exchangeable with D2O).
Anal. Calcd. for C16H22N4O4 (334.37): C, 57.47, H, 6.63,
N, 16.76. Found: C, 57.17, H, 6.23, N, 16.62.
General method for the preparation of 15a, b
3-Acetyl-4-amino-9-hydroxy-9-morpholin-4-yl-6,7,9,9a-
tetrahydro-1H-pyrido[3,2-a]pyrrolizin-2(5H)-one 17
A solution of the appropriate compound 14a, b (0.36 mmol)
in 1% sodium ethoxide in ethanol (20 ml) was treated in the
same method used for the preparation of compounds 12a, b.
The formed precipitate was filtered, dried, and crystallized
from acetone.
Applying the same procedure used in the preparation of
compound 15a, b, compound 17 was prepared from com-
pound 16 (0.66 g, 2 mmol). The separated solid
after acidification was filtered, dried, crystallized from
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Med Chem Res (2012) 21:2349–2362
2359
ethanol–acetone mixture. White crystals, m.p. 222–3°C,
yield 67%. IR: tmax/cm-1 3431,3332, 3280 (br., OH, NH2,
NH), 2958, 2924 (CH aliphatic), 1640 (2CO), 1579, 1449
(NH, C=C), 1299, 1109 (C–O, C–N). 1H-NMR (300 MHz,
CDCl3: d, ppm): 2.06–2.16 (m, 2H, CH2-6), 2.54–2.57
(m, 4H, N(CH2)2), 2.91 (t, J = 6.3 Hz, 2H, CH2-5), 3.21
(s, 3H, CH3), 3.65–3.72 (m, 6H, O(CH2)2), CH2-7), 4.12
(d, J = 6.6 Hz, 1H, CH-9a), 6.13 (s, 2H, NH2, exchangeable
with D2O), 6.34, 9.53 (2s, 2H, NH, OH exchangeable with
D2O). Anal. Calcd. for C16H22N4O4 (334.37): C, 57.47, H,
6.63, N, 16.76. Found: C, 57.46, H, 6.59, N, 16.33.
2 mmol) in dioxane (20 ml) was refluxed for 20 h. The
reaction mixture was concentrated by distillation under
vacuum and cooled. The obtained solid was filtered, dried,
and crystallized from ethanol.
N0-((7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizin-6-yl)imino)methyl)iso
nicotinohydrazide 20a
White crystals, m.p. 228–30°C, yield 75%. IR: tmax/cm-1
3435 (OH), 3334, 3282 (NHs), 2985 (CH aliphatic), 2185
(CN), 1643 (CO), 1618, 1585, 1487 (NH, C=C, C=N),
1
9-Hydroxy-9-morpholin-4-yl-6,7,9,9a-tetrahydro-3H-
pyrimido[5,4-a] pyrrolizin-4(5H)-one 18
1427, 1369 (C–O, C–N). H-NMR (300 MHz, DMSO-d6:
d, ppm):1.91–1.95 (m, 2H, CH2-2), 2.74–2.84 (m, 6H,
N(CH2)2, CH2-1), 3.52–3.56 (m, 6H, O(CH2)2, CH2-3),
3.96 (d, 1H, J = 5.7, Hz, CH-6), 6.55, 6.76, 7.38 (3s, 3H,
2NHs, OH exchangeable with D2O), 7.71–7.76 (m, 2H,
CH-a), 8.68–8.75 (m, 2H, CH-b), 9.62 (s, 1H, N=CH). MS:
m/z (%) (397 (M?, 5), 134 (100). Anal. Calcd. for
C19H23N7O3 (397.43): C, 57.42, H, 5.83, N, 24.67. Found:
C, 57.17, H, 5.77, N, 24.59.
Compound 7 (0.78 g, 3.12 mmol) was refluxed in excess
formic acid (10 ml) for 6 h. The reaction mixture was left
aside to cool. The formed product was filtered, dried and
crystallized from methanol, Yellow crystals, m.p. 243–5°C,
yield 73%. IR: tmax/cm-1 3425 (OH), 3330, 3286 (NHs),
2925 (CH aliphatic), 1626 (CO), 1581, 1518, 1435 (C=N,
1
NH, C=C), 1300, 1237 (C–N, C–O). H-NMR (300 MHz,
DMSO-d6: d, ppm): 1.88–2.05 (m, 4H, CH2-5,6), 2.74–
2.84 (m, 4H, N(CH2)2), 3.25–3.31 (m, 4H, O(CH2)2), 3.68
(t, 2H, J = 7.6 Hz, CH2-7), 4.60 (d, J = 5.4 Hz, 1H,
CH-9a), 6.55, 7.19, 9.62 (2s, 2H, NH, OH exchangeable
with D2O), 7.82 (s, 1H, N=CH). Anal. Calcd. for
C13H18N4O3 (278.31) C, 56.01, H, 6.52, N, 20.13. Found:
C, 56.05, H, 6.62, N, 20.17.
N0-((7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizin-6-yl)imino)methyl)benzo
hydrazide 20b
White crystals, m.p. 218–20°C, yield 83%. IR: tmax/cm-1
3435 (OH), 3335, 3283 (NHs), 3008 (CH aromatic), 2903
(CH aliphatic), 2184 (CN), 1644 (CO), 1584, 1483, 1427
(NH, C=C, C=N), 1369, 1304 (C–O, C–N). 1H-NMR
(300 MHz, DMSO-d6: d, ppm): 1.88–1.98 (m, 2H, CH2-2),
2.74–2.85 (m, 6H, N(CH2)2, CH2-1), 3.43–3.54 (m, 6H,
O(CH2)2, CH2-3), 3.96 (d, J = 6.3 Hz, 1H, CH-6), 6.56,
11.68 (2s, 3H, NHs, OH exchangeable with D2O), 7.28–
7.82 (m, 5H, aromatic protons), 9.62 (s, 1H, N=CH). Anal.
Calcd. for C20H24N6O3 (396.44): C, 60.59, H, 6.10, N,
21.20. Found: C, 60.64, H, 6.15, N, 21.32.
Ethyl (7-cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizin-6-yl)imidoformate 19
A mixture of compound 7 (1 g, 3.99 mmol) and triethyl
orthoformate (10 ml) was refluxed for 5 h. The excess
solvent was removed under reduced pressure and the sep-
arated solid was filtered, dried and crystallized from ace-
tone. White crystals, m.p. 232–4°C, yield 73%. IR: tmax
/
cm-1 3356 (OH), 2961 (CH aliphatic), 2186 (CN), 1599,
1514, 1458 (C=C, C=N), 1421, 1388, 1332 (C–O, C–N).
1H-NMR (300 MHz, DMSO-d6: d, ppm): 1.60 (s, 3H,
CH2CH3), 2.06–2.16 (m, 2H, CH2-2), 2.54–2.57 (m, 4H,
N(CH2)2), 2.91 (t, J = 7.8 Hz, 2H, CH2-1), 3.65–3.72 (m,
8H, O(CH2)2, CH2CH3, CH2-3), 4.12 (d, J = 6.3 Hz, 1H,
CH-6), 6.12 (s, 1H, OH exchangeable with D2O), 9.53
(s, 1H, N=CH). Anal. Calcd. for C15H22N4O3 (306.36):
C, 58.81, H, 7.24, N, 18.29. Found: C, 59.07, H, 7.39, N,
18.67.
General method for the preparation of 21a, b
A mixture of compound 20a or 20b (2 mmol) in ethanolic
HCl (30 ml) was refluxed for 2 h. The formed precipitate
was filtered, dried, and crystallized from methanol/chlo-
roform mixture.
N0-((9-Hydroxy-9-morpholin-4-yl-4-oxo-3,4,6,7,9,9a-
hexahydro-5H-pyrimido[5,4-a]pyrrolizin-2-yl)
isonicotinohydrazide 21a
General method for the preparation of 20a, b
Yellow crystals, m.p. 238–40°C, yield 78%. IR: tmax/cm-1
3432 (OH), 3292, 3262 (NHs), 2925, 2880 (CH aliphatic),
1661 (2CO), 1571, 1524, 1427 (NH, C=C, C=N), 1304,
A mixture of compound 19 (0.6 g, 2 mmol) and isonazide
(0.274 g, 2 mmol) or benzoic acid hydrazide (0.27 g,
1
1255 (C–O, C–N). H-NMR (300 MHz, CDCl3: d, ppm):
123

2360
Med Chem Res (2012) 21:2349–2362
General method for the preparation of 23a–d
2.06–2.16 (m, 2H, CH2-6), 2.89–2.95 (m, 6H, N(CH2)2,
CH2-5), 3.64–3.72 (m, 6H, O(CH2)2, CH2-7), 4.58 (d,
J = 6.3 Hz, 1H, CH-9a), 5.65, 6.79, 9.51, 10.51 (4s, 4H,
3NHs, OH exchangeable with D2O), 7.54–7.69 (m, 4H,
a,b). Anal. Calcd. for C19H23N7O4 (413.43): C, 55.20, H,
5.61, N, 23.72. Found: C, 55.17, H, 5.57, N, 23.29.
Compounds 23a–d were prepared by treating compound 22
(0.6 g, 2 mmol) with the appropriate acid chloride or ethyl
chloroformate (2 mmol) by the same method used for the
preparation of compound 15a, b. The separated solid was
filtered, dried, and crystallized from acetone/ethanol
mixture.
N0-((9-Hydroxy-9-morpholin-4-yl-4-oxo-3,4,6,7,9,9a-
hexahydro-5H-pyrimido[5,4-a]pyrrolizin-2-yl)
benzohydrazide 21b
N-(9-Hydroxy-9-morpholin-4-yl-,6,7,9,9a-tetrahydro-5H-
pyrimido[5,4-a]pyrrolizin-4-yl)acetamide 23a
Yellow crystals, m.p. 225–7°C, yield 73%. IR: tmax/cm-1
3431 (OH), 3332, 3280 (NHs), 3055 (CH aromatic), 2958,
2924 (CH aliphatic), 1640 (br., 2CO), 1579, 1449, 1368
(NH, C=C, C=N), 1299, 1165 (C–O, C–N). 1H-NMR
(300 MHz, DMSO-d6: d, ppm): 1.91–1.96 (m, 2H, CH2-6),
2.39–2.49 (m, 4H, N(CH2)2), 2.79 (t, J = 7.8 Hz, 2H,
CH2-5), 3.44–3.54 (m, 6H, O(CH2)2), CH2-7), 5.32 (d,
J = 6.3 Hz, 1H, CH-9a), 6.55, 9.62, 10.62, 11.40 (4s, 4H,
3NHs, OH exchangeable with D2O), 7.06–7.42 (m, 5H,
aromatic protons). Anal. Calcd. For C20H24N6O4 (412.44):
C, 58.24, H, 5.87, N, 20.38. Found: C, 51.50, H, 5.86, N,
20.16.
White crystals, m.p. 258–60°C, yield 82%. IR: tmax/cm-1
3426 (OH), 3328 (NH), 2919 (CH aliphatic), 1632 (CO),
1578, 1508, 1433 (NH, C=C, C=N), 1376, 1295 (C–O, C–N).
1H-NMR (300 MHz, CDCl3: d, ppm): 1.65 (s, 3H, CH3),
2.09–2.16 (m, 2H, CH2-6), 2.89–2.95 (m, 6H, N(CH2)2,
CH2-5), 3.64–3.72 (m, 6H, O(CH2)2, CH2-7), 4.58 (d,
J = 6.3 Hz, 1H, CH-9a), 5.65, 9.51 (2s, 2H, NH, OH
exchangeable with D2O), 9.55 (1s, 1H, N=CH). Anal.
Calcd. for C15H21N5O3 (319.36): C, 56.41, H, 6.63, N,
21.93. Found: C, 56.28, H, 6.56, N, 21.97.
N-(9-hydroxy-9-morpholin-4-yl-,6,7,9,9a-tetrahydro-5H-
pyrimido[5,4-a]pyrrolizin-4-yl)benzamide 23b
4-Amino-9-morpholin-4-yl-,6,7,9,9a-tetrahydro-5H-
pyrimido[5,4-a]pyrrolizin-9-ol 22
White crystals, m.p. 272–3°C, yield 73%. IR: tmax/cm-1
3323, 3282 (OH, NH), 3036 (CH aromatic), 2919 (CH
aliphatic), 1643 (CO), 1595, 1551, 1487 (NH, C=C, C=N),
A solution of compound 19 (0.6 g, 2 mmol) in methanol
(20 ml) was stirred at 0°C for one hour; during this hour
methanol saturated with ammonia (20 ml) was added
portionwise. The reaction mixture was stirred for another
6 h at room temperature. The precipitated solid was fil-
tered, dried, and crystallized from acetone.
1
1305, 1229 (C–O, C–N). H-NMR (300 MHz, CDCl3: d,
ppm): 2.06–2.16 (m, 2H, CH2-6), 2.54–2.57 (m, 4H,
N(CH2)2), 2.91 (t, 2H, J = 7.8 Hz, CH2-5), 3.65–3.72 (m,
6H, O(CH2)2, CH2-7), 4.12 (d, J = 6.3 Hz, 1H, CH-9a),
4.77, 6.11 (2s, 2H, NH, OH exchangeable with D2O), 7.75–
7.90 (m, 5H, aromatic protons), 9.53 (1s, 1H, N=CH).
Anal. Calcd. for C20H23N5O3 (381.43): C, 62.98, H, 6.08,
N, 18.36. Found: C, 63.65, H, 6.36, N, 18.56.
Compound 22 was also synthesized by stirring a solu-
tion of compound 24 (1 g, 4.1 mmol) in 1% sodium eth-
oxide in ethanol (20 ml) for 24 h at room temperature. The
obtained product was filtered, dried, and crystallized from
acetone.
2-Chloro-N-(9-hydroxy-9-morpholin-4-yl-,6,7,9,9a-
tetrahydro-5H-pyrimido[5,4-a]pyrrolizin-4-yl) acetamide
23c
White crystals, m.p. 256–8°C, yield 75%. IR: tmax/cm-1
3437–3285 (br., OH, NH2), 2918 (CH aliphatic), 1567,
1514 (C=C, C=N), 1379, 1304 (C–O, C–N). 1H-NMR
(300 MHz, DMSO-d6: d, ppm): 2.06–2.16 (m, 2H, CH-6),
2.45–2.57 (m, 4H, N(CH2)2), 2.91 (t, J = 7.8 Hz, 2H,
CH2-5), 3.65–3.72 (m, 6H, O(CH2)2, CH2-7), 4.12 (d, 1H,
J = 6.3 Hz, CH-9a), 4.77 (s, 2H, NH2 exchangeable with
D2O), 6.11 (s, 1H, OH exchangeable with D2O), 9.53 (s,
1H, N=CH). 13C-NMR (DMSO-d6-75.45 MHz): 20.24 (C-
5), 33.28 (C-6), 33.46 (C-7), 41.83 (N(CH2)2), 48.94
(O(CH2)2), 49.14 (C-9a), 97.08 (C=C), 97.33 (C–OH),
156.05 (C=C), 156.08 (C-NH2), 166.30 (C=N). Anal.
Calcd. for C13H19N5O2 (277.32): C, 56.30, H, 6.91, N,
25.25. Found: C, 56.51, H, 6.91, N, 24.95.
White crystals, m.p. 263–5°C, yield 86%. IR: tmax/cm-1
3453 (OH), 3274 (NH), 2920 (CH aliphatic), 1771 (CO),
1628, 1574, 1522, 1453 (NH, C=C, C=N), 1301, 1254 (C–
1
O, C–N). H-NMR (300 MHz, CDCl3: d, ppm): 2.06–2.16
(m, 2H, CH2-6), 2.54–2.57 (m, 4H, N(CH2)2), 2.91 (t,
J = 7.8 Hz, 2H, CH2-5), 3.65–3.72 (m, 6H, O(CH2)2,
CH2-7), 4.12 (d, 1H, J = 6.3 Hz, CH-9a), 4.77 (s, 2H,
CH2Cl), 6.11 (s, 2H, NH,OH exchangeable with D2O),
9.53 (s, 1H, N=CH). Anal. Calcd. for C15H20ClN5O3
(353.80): C, 50.92, H, 5.70, N, 19.79. Found: C, 50.62, H,
5.67, N, 19.92.
123

Med Chem Res (2012) 21:2349–2362
2361
Ethyl-(9-hydroxy-9-morpholin-4-yl-,6,7,9,9a-tetrahydro-
5H-pyrimido[5,4-a]pyrrolizin-4-yl)carbamate 23d
J = 6.3 Hz, CH-6), 9.53 (s, 1H, OH exchangeable with
D2O). Anal. Calcd. for C17H24N4O3 (332.40): C, 61.43, H,
7.28, N, 16.86. Found: C, 61.23, H, 7.52, N, 17.15.
White crystals, m.p. 248–50°C, yield 75%. IR: tmax/cm-1
3329 (OH), 3274 (NH), 2920 (CH aliphatic), 1643 (CO),
1577, 1452, 1364 (NH, C=C, C=N), 1299, 1222 (C–O,
1
5-Hydroxy-6-[(1-methyl-3-oxo-3-
phenylpropylidene)amino]-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizine-7-carbonitrile 25b
C–N). H-NMR (300 MHz, CDCl3: d, ppm): 1.72 (s, 3H,
CH2CH3), 2.05–2.15 (m, 2H, CH2-6), 2.87–2.95 (m, 6H,
N(CH2)2), CH2-5), 3.66–3.74 (m, 6H, O(CH2)2, CH2-7),
4.66–4.75 (m, 3H, CH-9a, CH2CH3), 7.42, 9.45 (2s, 2H,
NH, OH exchangeable with D2O), 9.55 (s, 1H, N=CH).
Anal. Calcd. for C16H23N5O4 (349.39): C, 55.00, H, 6.64,
N, 20.04. Found: C, 55.45, H, 6.56, N, 19.62.
White crystals, m.p. 242–4°C, yield 75%. IR: tmax/cm-1
3323 (br., OH), 3132, 3036 (CH aromatic), 2919 (CH ali-
phatic), 2191 (CN), 1643 (CO), 1595, 1438 (C=N, C=C),
1
1305, 1229 (C–N, C–O). H-NMR (300 MHz, DMSO-d6:
d, ppm): 1.03 (s, 3H, CH3), 1.93–2.01 (m, 2H, CH2-2), 2.40
(t, J = 4.5 Hz, 2H, CH2-1), 2.74–2.85 (m, 6H, N(CH2)2,
COCH2), 3.52–3.56 (m, 6H, O(CH2)2, CH2-3), 3.96
(d, J = 6 Hz, 1H, CH-6), 7.28–7.58 (m, 5H, aromatic
protons), 9.62 (s, 1H, OH exchangeable with D2O). Anal.
Calcd. for C22H26N4O3 (394.47): C, 66.99, H, 6.64, N,
14.20. Found: C, 67.46, H, 6.80, N, 14.09.
N-(7-Cyano-5-hydroxy-5-morpholin-4-yl-2,3,5,6-
tetrahydro-1H-pyrrolizin-6-yl)imidoformamide 24
A mixture of compound 7 (0.78 g, 3.12 mmol) and form-
amide (80%) (20 ml) was refluxed for 5 h. The reaction
mixture was poured onto water (50 ml) and cooled. The
obtained precipitate was filtered, dried, and crystallized
from acetone. White crystals, m.p. 245–7°C, yield 68%.
IR: tmax/cm-1 3466 (OH), 3336, 3282 (NH2), 2955, 2922
(CH aliphatic), 2193 (CN), 1591, 1528 (NH, C=C), 1441,
1303 (C–N, C–O).1H-NMR (300 MHz, DMSO-d6: d,
ppm): 2.06–2.16 (m, 2H, CH2-2), 2.60–2.64 (m, 4H,
N(CH2)2), 2.91 (t, J = 7.8 Hz, 2H, CH2-1), 3.65–3.75 (m,
6H, O(CH2)2), CH2-3), 4.17 (d, J = 6.3 Hz, 1H, CH-6),
6.26 (s, 3H, N=CH, NH2), 9.51 (s, 1H, OH exchangeable
with D2O). MS: m/z (%) 277 (M?, 0.8), 132 (100). Anal.
Calcd. for C13H19N5O2 (277.32): C, 56.30, H, 6.91, N,
25.25. Found: C, 56.60, H, 7.29, N, 25.71.
General method for the preparation of 26a, b
Compounds 26a, b were prepared from compounds 25a, b
(2 mmol), respectively, by the same method used for the
preparation of compounds 12a, b and were crystallized
from methanol.
3-Acetyl-4-amino-9-hydroxy-2-methyl-9-morpholin-4-yl-
6,7,9,9a-tetrahydro-5H-pyrido[3,2-a]pyrrolizine 26a
White crystals, m.p. 225–7°C, yield 68%. IR: tmax/cm-1
3435–3316 (br., OH, NH2), 2922 (CH aliphatic), 1694
(CO), 1630, 1524, 1458 (NH, C=C, C=N), 1302, 1259 (C–O,
General method for the preparation of 25a, b
1
C–N). H-NMR (300 MHz, CDCl3: d, ppm): 1.23 (s, 3H,
To a solution of compound 7 (0.5 g, 2 mmol) in dioxane
(25 ml), a catalytic amount of triethylamine (2 drops) and
acetyl acetone or benzoyl acetone (2 mmol) was added.
The reaction mixture was heated under reflux for 6 h then
concentrated and left aside to cool. The separated solid was
filtered, dried, and crystallized from ethanol.
N=C–CH3), 1.62 (s, 3H, CH3C=O), 2.05–2.15 (m, 2H,
CH2-6), 2.87–2.92 (m, 4H, N(CH2)2), 3.66–3.71 (m, 6H,
O(CH2)2, CH2-5), 4.12 (d, J = 6.3 Hz, 1H, CH-9a), 4.73
(t, 2H, J = 6.5 Hz, CH2-7), 6.57, 9.55 (2s, 3H, NH2, OH
exchangeable with D2O). Anal. Calcd. for C17H24N4O3
(332.40): C, 61.43, H, 7.28, N, 16.86. Found: C, 61.23, H,
7.58, N, 16.85.
5-Hydroxy-6-[(1-methyl-3-oxobutylidene)amino]-5-
morpholin-4-yl-2,3,5,6-tetrahydro-1H-pyrrolizine-7-
carbonitrile 25a
4-Amino-3-benzoyl-9-hydroxy-2-methyl-9-morpholin-4-yl-
6,7,9,9a-tetrahydro-5H-pyrido[3,2-a] pyrrolizine 26b
White crystals, m.p. 235–7°C, yield 72%. IR: tmax/cm-1
3434 (OH), 2960 (CH aliphatic), 2192 (CN), 1705 (CO),
1585, 1455 (C=C, C=N), 1304, 1250 (C–N, C–O). 1H-
NMR (300 MHz, CDCl3: d, ppm): 1.23 (s, 3H, N=C–CH3),
1.60 (s, 3H, CH3C=O), 2.06–2.16 (m, 2H, CH2-2), 2.54–
2.57 (m, 4H, N(CH2)2), 2.91 (t, 2H, J = 7.8 Hz, CH2-1),
3.65–3.72 (m, 8H, O(CH2)2,CH2-3, COCH2), 4.12 (d, 1H,
White crystals, m.p. 235–7°C, yield 73%. IR: tmax/cm-1
3427 (OH), 3329, 3274 (NH2), 3050 (CH aromatic), 2920
(CH aliphatic), 1798 (CO), 1577, 1452, 1364 (NH, C=C,
C=N), 1299, 1125 (C–O, C–N). 1H-NMR (300 MHz,
CDCl3: d, ppm): 1.20 (s, 3H, CH3), 2.05–2.15 (m, 2H,
CH2-6), 2.87–2.92 (m, 4H, N(CH2)2), 3.66–3.71 (m, 6H,
123

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Med Chem Res (2012) 21:2349–2362
using L-erythrose glycosylhydroxylamine as a masked acyclic
chiral nitrone. Tetrahedron 64:8752–8758
Barsoum FF, Nawar NN (2003) Synthesis of Novel 1Hpyrrolizine-3-
crboxamide and their antimicrobial properties. Boll Chim Farm
142:160–166
Diafi L, Rubat C, Coudert P, Bastide P, Margoum N, Tronche P (1991)
Synthesis and influence of the stereochemistry on anticonvulsant
activity of 1-aza[3.3.0]bicyclooctanes. Eur J Med Chem 26:
231–235
O(CH2)2, CH2-5), 4.12 (d, J = 6.3 Hz, 1H, CH-9a), 4.73
(t, J = 6.3 Hz, 2H, CH2-7), 6.57 (s, 2H, NH2 exchangeable
with D2O), 7.56–7.90 (m, 5H, aromatic protons), 9.55
(s, 1H, OH exchangeable with D2O). Anal. Calcd. for
C22H26N4O3 (394.47): C, 66.99, H, 6.64, N, 14.20. Found:
C, 66.96, H, 6.76, N, 14.06.
Pharmacological screening
Doyle A, Griffiths JB (1998) Cell and tissue culture: laboratory
procedures in biotechnology. John Wiley & Sons Ltd, New York
´
Duran JDG, Arias JL, Gallardo V, Delgado AV (2008) Magnetic
The breast cancer cells were plated in 96-multiwell plate
(4 9 104 cells/well) at 37°C for 24 h before treatment with
the compounds to allow attachment of cells to the wall of
plate. Different concentrations of the compounds under test
(0, 1, 2.5, 5 and 10 lg/ml DMSO) were added to the cell
monolayer. Monolayer cells were incubated with the
compounds for 48 h at 37°C and in an atmosphere of 5%
CO2. After 48 h cells were fixed and incubated with fresh
medium containing 40 lg/ml NR dye. Incubation was
continued for 3 h to allow incorporation of vital dye into
surviving cells. Excess stain was washed with 1% acetic
acid/50% ethanol, and color intensity was measured in
ELISA-type spectrophotometer equipped with a 540-nm
filter. The relation between the surviving fraction and drug
concentration was plotted to get the survival curve of the
tumor cell line after the specified compound. Probit
regression analysis procedure was introduced to select the
best model that describes the relationship between the drug
concentration and the probit (percentage of protection) as a
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