Design, synthesis and pharmacological evaluation of novel pyrrolizine derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2014.01.001

A new series of novel pyrrolizine derivatives has been synthesized and biologically evaluated as potential anticancer agents. The starting compounds, 6-amino-7-cyano-N-(3,5-disubstitutedphenyl)-2,3-dihydro-1H-pyrrolizine-5- carboxamides 11a-b, were reacte

Full text of DOI:10.1016/j.bioorg.2014.01.001

Bioorganic Chemistry 53 (2014) 1–7
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and pharmacological evaluation of novel pyrrolizine
derivatives as potential anticancer agents
Ahmed M. Gouda a,b, Ahmed H. Abdelazeem a,c, El-Shaimaa A. Arafa d, Khaled R.A. Abdellatif e,
⇑
a Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
b Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al-Qura University, Mekkah 21955, Saudi Arabia
c Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif 21974, Saudi Arabia
d Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
e Department of Pharmaceutical Organic Chemistry, Beni-Suef University, Beni-Suef 62514, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 September 2013
Available online 4 January 2014
A new series of novel pyrrolizine derivatives has been synthesized and biologically evaluated as potential
anticancer agents. The starting compounds, 6-amino-7-cyano-N-(3,5-disubstitutedphenyl)-2,3-dihydro-
1H-pyrrolizine-5-carboxamides 11a–b, were reacted with different acid chlorides, aldehydes and
isocyanates to give the target compounds 12–14. Structural characterizations of the new compounds
were performed using spectral and elemental analysis. All compounds were tested for their anticancer
activity against human breast cancer and prostate cancer cell lines, MCF-7 and PC-3 respectively. With
exception of compounds 11a and 13a, results revealed that all the tested compounds showed half max-
Keywords:
Pyrrolizine
Apoptosis
Caspase activation
Breast cancer
Prostate cancer
imal inhibitory concentration (IC50) values less than 40
l
M. Compound 12b and the three urea deriva-
M. The
tives 14b–d showed the most potent anticancer activity with IC50 values less than 2.73
l
anticancer activity of these compounds was mediated, at least in part, via the induction of apoptosis
as indicated by its ability to activate caspase-3/7. In light of the high potency of our novel compounds
in targeting both breast and prostate cancers, these compounds warrant continued preclinical develop-
ment as potential anticancer agents.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
of these promising agents [6]. The activation of caspase-3, the main
executioner in apoptosis, was useful in the discovery of many
Although many anticancer chemotherapeutic agents have been
proven to be successful in the clinical trials, the high rate of mor-
tality presents an urgent need for more safe and more efficient
agents [1,2]. The clinical use of many chemotherapeutic agents still
associated with many severe adverse reactions and rapid develop-
ment of resistance despite of the great advances in targeted anti-
cancer therapy [3,4]. The design of anticancer agents targeting
cancer cell survival pathways improved the clinical promise of
these agents as a component of anticancer therapy [5]. The selec-
tive induction of apoptosis in cancer cells is one of the main aims
potential anticancer agents [7,8].
The pyrrolizine scaffold constitutes the basic skeleton in many
compounds with diverse pharmacological activities [9–11]. Many
pyrrolizine derivatives exhibit antitumor activities due to their
ability to cross link DNA [12,13]. Recently, several pyrrolizine
derivatives were developed lacking the alkylating functions and
displayed potent antitumor activities. One of the diphenyl substi-
tuted pyrrolizine derivatives that showed a good activity against
breast cancer is the selective COX-2 inhibitor, Licofelone
1
[14–16]. The high selectively for COX-2 could play the major role
in its anticancer effects [14–16]. In addition, licofelone was found
to enhance apoptosis in prostate and colon cancer cells through
the mitochondrial pathway [17]. The replacement of the acetic acid
moiety of licofelone by additional phenyl acyl side chain signifi-
cantly increased the anticancer activity, compound 2 (Fig. 1) [17].
On the other hand, many nitrile and aminonitrile derivatives
showed anticancer activity through induction of apoptosis and
activation of caspase-3 compounds [18]. EpiCept Corporation USA
has developed the Anticancer Screening Apoptosis Program (ASAP)
that helped in the discovery of the 1-benzoyl-3-cyanopyrrolo
Abbreviations: ASAP, Anticancer Screening Apoptosis Program; CDCl3, deuter-
ated chloroform; DMEM/F-12, Dulbecco’s Modified Eagle Medium: Nutrient Mix-
ture F-12; DMSO, dimethyl sulfoxide; eV, electron volt; FBS, fetal bovine serum;
COX-2, cyclooxygenase-2; IC50, half maximal inhibitory concentration; IR, Infrared
spectroscopy; m.p., melting point; m/z, mass-to-charge ratio; MTT, 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; SAR, structural activity
relationship.
⇑
Corresponding author. Address: Department of Pharmaceutical Organic Chem-
istry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. Fax: +20
(002) 082 2317958.
E-mail address: khaled.ahmed@pharm.bsu.edu.eg (K.R.A. Abdellatif).
0045-2068/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2014.01.001

2
A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
Fig. 1. Structures of some reported apoptosis inducers and caspase activators.
[1,2-a]quinoline
3
as
a
potent apoptosis inducer [19]. The
that their cumulative effects would be stronger and the impact of
these effects on activity would be higher than the monosubstituted
pyrrolizine derivatives. Concomitantly, the distance between the
additional phenyl ring and pyrrolizine nucleus was varied (two
or three atoms), and the substitution pattern of this phenyl ring
could affect the target cancer cells and the overall activity.
structural activity relationship (SAR) study of compound 3 re-
vealed that replacing the 3-cyano group by an ester or ketone
group led to inactive compounds [18]. Other compounds 4–6 shar-
ing certain structural similarity were reported as apoptosis induc-
ers [20–24]. These compounds have a substituted phenyl moiety
attached by two or three atoms spacer that contains double bond
to another aromatic or heteroaromatic ring. The spacer either con-
sists of sp2 carbons as in compound 4 or may include one or two
nitrogen atoms as in compounds 5 and 6 (Fig. 1). Generally, it
was observed that the activities of compounds 1–6 vary according
to the electronic effect and the position of the monosubstituents
where the chloro, methoxy and trifluoromethy substituted deriva-
tives possessed the highest activity [20–24]. In addition, com-
pounds 1–2, 4 and 5 have two phenyl rings attached to the basic
nucleus either directly or through three or four atoms spacer.
Promoted by the aforementioned findings and aiming to devel-
op novel and potent anticancer agents, two general scaffolds (A
and B, Fig. 2) incorporating the heterocyclic core, 7-cyano-N-
(3,5-disubstitutedphenyl)-2,3-dihydro-1H-pyrrolizin-5-carboxam-
ide, were developed. The design concept is based mainly on the
structural similarity with compounds 1–6 with maximizing the
electronic effect of the substituents to investigate their influence
on the anticancer activity of the newly designed pyrrolizine
derivatives. In order to maximize this electronic effect, we used a
disubstituted phenyl ring with two electron withdrawing (di-
chloro) or electron donating (dimethyl) groups. It was expected
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, preparation of the intermediates 8a, 8b
and 10 was done according to previously reported procedures
[25,26]. Compounds 11a and 11b were prepared from the reaction
of 2-pyrrolidin-2-ylidine malononitrile 10 with the corresponding
a-chloroacetanilide 8a and 8b in dry acetone according to previ-
ously reported procedures [27]. The reaction proceeded directly
via the un-isolated intermediate, N-substituted pyrrolidin-2-
ylidine malononitrile which cyclized spontaneously by addition
of the a-methylene group to one of the two nitrile groups to give
compounds 11a–b. Characterization of the newly synthesized
compounds was carried out using spectral and elemental analysis.
The IR spectrum showed the presence of one sharp absorption
band at 2217 and 2220 cmÀ1 assigned for the cyano group in the
compounds 11a and 11b respectively. On the other hand, the mass
spectrum revealed the molecular ions of the compounds 11a and
11b at 294 and 335 respectively, and the fragmentation patterns
were in concordance with the chemical structure. 1H NMR spectra
showed the presence of a singlet proton at 9.09 and 9.29 ppm
assigned for the amide protons in the compounds 11a and 11b
respectively. Further characterization of compound 11a was done
using 13C NMR spectrum as well.
Compounds 11a and 11b were used as starting materials for
preparing the other target compounds 12–14, as illustrated in
Scheme 2. The reaction of compounds 11a and 11b with the appro-
priate acid chloride in dry benzene gave compounds 12a–d. The
synthesized compounds were characterized by the spectral and
elemental analysis. 1H NMR spectra revealed the presence of two
protons at the range of 9.29–10.93 ppm assigned for the amide
protons in compounds 12a–d.
Moreover, the condensation of the compounds 11a and 11b
with the appropriate aldehyde in absolute ethanol in the presence
Fig. 2. General scaffolds for the newly designed ligands.

A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
3
Scheme 1. Reagents and conditions: (a) ClCH2COCl, gl. acetic acid, CH3COONa; (b) (CH3)2SO4, benzene, malononitrile; and (c) K2CO3, acetone, reflux, 24 h.
Scheme 2. Reagents and conditions: (a) ArCOCl, dry benzene, 48 h; (b) ArCHO, EtOH, gl. acetic acid, reflux, 4 h; and (c) ArNCO, CH2Cl2, N(Et)3, reflux 12 h.
of glacial acetic yielded the compounds 13a–d which were
characterized by spectral and elemental analysis. The 1H NMR
spectra showed the appearance of one new proton at the range
of 10.37–11.47 ppm assigned for the benzylidene proton CH.
In addition, the target compounds 14a–d were obtained from
the reaction of compounds 11a and 11b with the appropriate iso-
cyanate in methylene chloride in the presence of triethylamine.
The reaction proceeded without intramolecular rearrangement of
the cyano group yielding ureido derivatives. Characterization of
compound 14a–d was done using spectral and elemental analysis.
The 1H NMR spectra revealed the presence of two protons in the
range of 8.86–10.74 ppm assigned for the two NH protons, while
the 13C NMR spectrum of compound 14a showed two signals at
158.1 and 178.3 ppm assigned for the two carbonyl groups.
inhibitory effect on both breast (MCF-7) and prostate cancer
(PC-3) cell lines. The effect of the test compounds on cancer cell
viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay in six replicates as
previously reported [28]. All tested compound showed an IC50 in
the micromolar range, going parallel against the two cell lines
(Table 1). With exception of compound 13a, all compounds signif-
icantly inhibited the cell viability of cancer cells. The parent com-
pound 11a showed the lowest anticancer activity while its dichloro
analog 11b showed improved activity on both MCF-7 and PC-3
cells with IC50 of 4.17 and 6.12 respectively.
The second substituted phenyl group at position
6 in
compounds 12–14 increased the activity compared to the parent
compound 11a. Also, the activity of these compounds varied with
the length of the spacer between the second phenyl and the
pyrrolizine nucleus, where compounds with three atoms spacer
(14b–d) showed higher activity than those with two atoms spacer
13a–d.
2.2. Pharmacological screening
2.2.1. Anticancer activity
Evaluation of the anticancer activity for the synthesized com-
pounds 11–14 were performed by determination of the growth
The three urea derivatives 14b–d in addition to 6-benzoylamino-
7-cyano-N-[3,5-dichlorophenyl]-1H-pyrrolizine-5-carboxamide 12b

4
A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
Table 1
IC50 values for the 7-cyano-2,3-dihydro-1H-pyrrolizine-5-carboxamides derivatives
on MCF-7 and PC-3 cell lines.
Comp.
R1
R2
R3
IC50
(lM)
MCF-7
PC-3
11a
11b
12a
CH3
Cl
CH3
CH3
Cl
CH3
NH2
NH2
59.66 4.2
4.17 0.9
10 1.2
67.02 3.1
6.12 1.4
9.61 1.7
12b
12c
12d
13a
13b
Cl
Cl
2.34 0.8
8.81 0.9
8.58 2.1
>100
2.73 0.3
7.91 0.7
8.35 1.8
>100
Fig. 3. The caspase activation assay Columns, mean; bars, S.D. (n = 5).
CH3
Cl
CH3
Cl
3. Conclusions
Various substituted pyrrolizines were synthesized and screened
for anticancer activity. The pyrrolizine scaffold 11b possesses good
anticancer activity in the cell viability assay and activity increase
by substitution at position 6 with substituted phenyl. The most
active derivatives were those bearing two phenyl rings substituted
with electron withdrawing group (13c, 13d, 14c and14d). Com-
pounds with three atoms spacer between the pyrrolizine nucleus
and the additional phenyl (12c, 12d and 12a–d) showed higher
activity than those with two atoms spacer (12a, 12b and 13a–d)
and compound 12c was the most active. Based on the overall
results, it was conceptualized that the anticancer activity of the
newly synthesized compounds were affected by the length of the
spacer, the presence or absence of two phenyl rings attached to
the pyrrolizine nucleus and finally the type of the substituents.
Combining the results of the cell viability assay and caspase activa-
tion assay, we can suggest that, at least in the breast cell line MCF-
7, the caspase activation may contribute to the mechanism of
anticancer activity of these compounds.
CH3
Cl
CH3
Cl
17.7 1.9
14.3 2.2
13c
13d
CH3
Cl
CH3
Cl
8.76 1.5
7.9 1.1
6.43 0.9
8.54 1.4
14a
14b
CH3
Cl
CH3
Cl
38.4 2.3
2.7 0.3
32.6 2.3
2.09 0.2
14c
14d
CH3
Cl
CH3
Cl
1.12 0.1
2.3 0.3
0.98 0.1
2.0 0.2
Cell were treated with the test compounds or vehicle for 48 h. Data were reported
as mean S.D. (n = 6).
showed the highest anticancer activity, where their IC50 were less
than 3 lM. When the additional phenyl were substituted with elec-
4. Experimental protocol
tron withdrawing group as in compounds 13c–d and 14c–d, the ob-
served IC50 values were lower than the IC50 values of those
substituted with electron donating groups (13a–b). Taken
together, it was found that there are two requirements for the anti-
cancer activity of the pyrrolizine derivatives. First, it is imperative to
incorporate two phenyl rings substituted with electron withdrawing
groups into the pyrrolizine nucleus. Secondly, the distance between
these two phenyl rings and the pyrrolizine nucleus is crucial for
activity where the anticancer activity is enhanced with the increase
in the length of the spacer. It should be mentioned that the three
atoms spacer was the optimal for activity.
4.1. Chemistry
Chemical reagents and solvents were obtained from commercial
sources. Solvents are dried by standard methods when necessary.
Melting points (m.p.) were uncorrected and were carried out by
open capillary tube method using IA 9100MK-Digital Melting Point
Apparatus. Microanalyses were carried out at the microanalytical
Center, Faculty of Science, Cairo University. Infrared spectra were
made on BRUKER Vector 22 (Japan), infrared spectrophotometers
and were expressed in wavenumber (cmÀ1) using potassium bro-
mide disc. The proton magnetic resonance 1H NMR spectra were re-
corded on a Varian Mercury VX-300 NMR spectrometer at 400 MHz
and BRUKER APX400 spectrometer at 400 MHz in the specified sol-
vent, 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 at the faculty of
pharmacy, Beni-Suef University. Mass spectra were recorded on
Fennigan MAT, SSQ 7000, Mass spectrometer, at 70 eV (EI) at the
microanalytical Center, Faculty of Science, Cairo University. All mass
spectra were recorded in the EI mode. Thin layer chromatography,
was done using Macherey–Nagel Alugram Sil G/UV254 silica gel
plates and benzene–ethanol (9.5:0.5) as the eluting system.
2.2.2. Caspase activation assay
Caspase activity was determined for the most active com-
pounds in Cell Viability Analysis. The caspase-3/7 in MCF-7 cells
treated with test agents were measured using Caspase-Glo 3/7
luminescence assay kit (Promega, Madison, WI) according to the
manufacturer’s instructions and as mentioned before [29] (Fig. 3).
The results revealed that compound 12b, 14b–d stimulate cas-
pase 3/7, where compound 14c was the most active, and this re-
sults were in concordance with the results obtained in the cell
viability assay.

A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
5
4.1.1. General method for preparation of compounds (11)
mixture of 2-pyrrolidin-2-ylidenemalononitrile 10 (1 g,
NMR (DMSO-d6-400 MHz): d 2.40 (m, 2H, CH2-2), 3.02 (t, 2H,
J = 7.5 Hz, CH2-1), 4.28 (t, 2H, J = 7.2 Hz, CH2-3), 6.52 (m, H, aromatic
proton), 7.24 (s, H, aromatic proton), 7.52 and 760 (two t, 2H,
J = 7.2 Hz, aromatic protons), 7.85 (s, 2H, aromatic proton), 8.14
and 8.15 (2 d, 2H, J = 7.2 Hz, aromatic protons), and 9.88, 10.93
(two s, 2H, NH). 13C NMR (DMSO-d6): d 24.9, 25.7, 49.6, 85.8,
115.1, 118.3, 118.5, 123.1, 128.4, 128.6, 128.9, 132.6, 133.6, 134.4,
141.7, 146.8, 158.4, 166.3. MS (EI): m/z (%): 440 (M++2, 3), 439
(M++1, 2), 438 (M+, 4), 278 (27), 174 (6), 145 (4), 105 (100), 92
(4), 77 (52). Anal. Calcd. for C22H16Cl2N4O2 (439.29): C, 60.15; H,
3.67; N, 12.75. Found: C, 60.31; H, 4.11; N, 12.67.
A
7.5 mmol), the appropriate acetanilide 8a and 8b (7.5 mmol) and
anhydrous potassium carbonate (1.04 g, 7.5 mmol) in dry acetone
(50 ml) was stirred under reflux for 24 h. The mixture was filtered,
concentrated and left to cool, whereby a white crystal were
formed, collected, dried and recrystallized from ethanol–acetone.
4.1.1.1. 6-Amino-7-cyano-N-(3,5-dimethylphenyl)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (11a). White crystals, m.p. 217–9 °C,
yield 71%, IRt
max/cmÀ1 3338, 3254 (NHs), 2961, 2914 (CAH ali-
phatic), 2217 (CN), 1664 (CO), 1613 1567, 1542 (C@C, NH), 1436,
1330 (CAN, CAO). 1H NMR (DMSO-d6-400 MHz): d 2.36 (s, 6H,
two PhCH3), 2.44 (m, 2H, CH2-2), 2.89 (t, 2H, J = 7.5 Hz, CH2-1),
4.21 (t, 2H, J = 7.2 Hz, CH2-3), 5.43 (s, 2H, NH2), 6.71 (s, H, aromatic
4.1.2.3.
2,3-dihydro-1H-pyrrolizine-5-carboxamide (12c). White crystals,
m.p. 253–5 °C, yield 72%. IR
max/cmÀ1 3408, 3235 (NHs), 3094,
6-(2-Benzylacetylamino)-7-cyano-N-[3,5-dimethylphenyl]-
t
protons), 7.21 (s, 2H, aromatic protons), and 9.09 (s, H, CONH)). 13
C
3057 (CAH aromatic), 2961 (CAH aliphatic), 2222 (CN), 1667
(broad band due to C@Os), 1594, 1566, 1525 (C@C, NH), 1490,
1425, 1379 (CAN, CAO). 1H NMR (DMSO-d6-400 MHz): d 2.24 (s,
6H, two PhCH3), 2.42 (m, 2H, CH2-2), 2.90 (t, 2H, J = 7.5 Hz,
CH2-1), 3.32 (s, 2H, COCH2), 4.21 (t, 2H, J = 7.2 Hz, CH2-3), 6.68–
7.62 (m, 8 H, aromatic protons), 9.31, 9.67 (two s, 2H, two CONH).
13C NMR (DMSO-d6): d 21.2, 24.8, 25.3, 29.1, 49.7, 79.3, 109.0,
117.1, 118.1, 124.9, 125.3, 138.0, 138.5, 138.7, 139.4, 144.9,
146.6, 147.2, 159.7. MS (EI): m/z (%): 414 (M++2, 58), 413 (M++1,
56), 412 (M+, 65), 365 (50) 340 (58%), 326 (49), 309 (58), 242
(46), 220 (51), 185 (44), 179 (65), 137 (78), 121 (59), 92 (9), 78
(56), 69 (100). Anal. Calcd. for C25H24N4O2 (412.48): C, 72.79; H,
5.86; N, 13.58. Found: C, 73.22; H, 5.49; N, 13.86.
NMR (DMSO-d6): d 21.7 (two PhCH3), 24.8, 25.3, 49.7, 77.9, 108.6,
115.9, 118.1 (two aromatic carbons), 124.9, 138 (two aromatic car-
bons), 138.6, 144.9, 146.6, 159.7 (CO). MS (EI): m/z (%): 295 (M++1,
4), 294 (M+, 23), 174 (100), 146 (12), 121 (14), 92 (11), 65 (12).
Anal. Calcd. for C17H18N4O (294.35): C, 69.37; H, 6.16; N, 19.03.
Found: C, 68.93; H, 5.72; N, 18.80.
4.1.1.2.
pyrrolizine-5-carboxamide (11b). White crystals, m.p. 244–7 °C,
yield 62%. IR
max/cmÀ1 3351, 3278 (NHs), 3075 (CAH aromatic),
6-Amino-7-cyano-N-(3,5-dichlorophenyl)-2,3-dihydro-1H-
t
2957 (CAH aliphatic), 2220 (CN), 1670 (CO), 1587, 1536 (C@C,
NH), 1438, 1311 (CAN, CAO), 834 (CACl). 1H NMR (DMSO-d6-
400 MHz): d 2.40 (m, 2H, CH2-2), 2.98 (t, 2H, J = 7.5 Hz, CH2-1),
4.32 (t, 2H, J = 7.2 Hz, CH2-3), 5.70 (s, 2H, NH2), 7.06 (s, H, aromatic
protons), 7.65 (s, 2H, aromatic protons), and 9.29 (s, H, NH). 13C
NMR (DMSO-d6): d 19.7, 36.4, 43.6, 50.7, 59.6, 115.7, 116.3, 118,
121.7, 127.7, 132.2, 138.5, 164.8. MS (EI): m/z (%): 336 (M++2, 7),
335 (M++1, 3), 334 (M+, 10), 161 (10), 174 (100), 145 (11), 146
(21), 147 (10), 92 (16), 77 (3). Anal. Calcd. for C15H12Cl2N4O
(335.19): C, 53.75; H, 3.61; N, 16.72. Found: C, 54.23; H, 4.07; N,
16.20.
4.1.2.4.
2,3-dihydro-1H-pyrrolizine-5-carboxamide (12d). White crystals,
m.p. 271–4 °C, yield 66%. IR
max/cmÀ1 3294, 3250 (NHs), 3077
6-(2-Benzylacetylamino)-7-cyano-N-[3,5-dichlorophenyl]-
t
(CAH aromatic), 2955 (CAH aliphatic), 2220 (CN), 1680, 1650
(COs), 1588, 1537 (C@C, NH), 1436, 1373 (CAN, CAO), 838 (CACl).
1H NMR (DMSO-d6-400 MHz): d 2.40 (m, 2H, CH2-2), 2.99 (t, 2H,
J = 7.5 Hz, CH2-1), 3.45 (s, 2H, COCH2), 4.33 (t, 2H, J = 7.2 Hz, CH2-
3), 6.62–7.65 (m, 8H, aromatic protons), and 9.29, 9.54 (two s,
2H, two CONH). 13C NMR (DMSO-d6): d 24.3, 25.2, 28.9, 47.5,
81.4, 116.2, 117.3, 119.0, 122.7, 127.6, 128.3, 128.9, 131.5, 134.1,
136.8, 140.2, 147.5, 156.6, 164.1. MS (EI): m/z (%): 453 (M++1, 3),
372 (48), 200 (73), 173 (96), 145 (53), 117 (100), 92 (14), 77
(16). Anal. Calcd. for C23H18Cl2N4O2 (453.32): C, 60.94; H, 4.00; N,
12.36. Found: C, 61.43; H, 4.34; N, 11.99.
4.1.2. General method for preparation of compounds (12a–d)
A mixture of 6-Amino-7-cyano-N-[3,5-disubstituted-phenyl]-
2,3-dihydro-1H-pyrrolizine-5-carboxamide 11 (5.3 mmol), appro-
priate acyl chloride (10.6 mmol) in dry benzene (50 ml) is stirred
for one hour and left to stand for 48 h at room temperature. Filter
the separated product, wash with water, dry and recrystallize from
ethanol/acetone.
4.1.3. General procedure for the preparation of compounds (13a–d)
A mixture of the carboxamide derivatives 11 (3.75 mmol) and
the appropriate aldehyde (3.75 mmol) was refluxed in absolute
ethanol (20 ml) in the presence of glacial acetic acid (0.5 ml) for
4 h. The reaction mixture was concentrated, set aside to cool, the
formed crystals was collected and recrystallized from ethanol.
4.1.2.1. 6-Benzoylamino-7-cyano-N-[3,5-dimethylphenyl]-2,3-dihydro-
1H-pyrrolizine-5-carboxamide (12a). White crystals, m.p. 263–5 °C,
yield 81%. IRt
max/cmÀ1 3338, 3276, 3254 (NHs), 2997, 2914, 2853
(CAH aliphatic), 2217 (CN), 1664 (broad band due to COs), 1613,
1567, 1542 (C@C, NH), 1436, 1377 (CAN, CAO). 1H NMR (DMSO-
d6-400 MHz): d 2.38 (s, 6H, two PhCH3), 2.42 (m, 2H, CH2-2), 2.91
(t, 2H, J = 7.5 Hz, CH2-1), 4.23 (t, 2H, J = 7.2 Hz, CH2-3), 6.68–7.81
(m, 8H, aromatic protons), and 9.51, 10.23 (two s, 2H, two CONH).
13C NMR (DMSO-d6): d 21.2, 24.8, 25.3, 50.2, 76.7, 109.7, 116.0,
119.2, 123.9, 125.6, 126.8, 129.4, 138.6, 140.1, 148.8, 148.9, 152.1,
157.9 and 160.0. MS (EI): (%) 397 ((M+À1, 10), 396 (M+À2, 29), 304
(70), 209 (8), 130 (58), 102 (61), 91 (26), 65 (100). Anal. Calcd. for
4.1.3.1.
dimethylphenyl]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(13a). Yellow crystals, m.p. 278–81 °C, yield 83%. IR
max/cmÀ1
7-Cyano-6-[(4-dimethylaminobenzylidene)-amino]-N-[3,5-
t
3244 (NHs), 3095 (CAH aromatic), 2965, 2920, 2852 (CAH ali-
phatic), 2215 (CN), 1661 (CO), 1617, 1566, 1518 (C@C, NH), 1343
(CAN, CAO). 1H NMR (CDCl3-400 MHz): d 2.19 (s, 6H, two PhCH3),
2.56 (m, 2H, CH2-2), 2.93 (t, 2H, J = 7.5 Hz, CH2-1), 3.29 (s, 6H,
N(CH3)2), 4.53 (t, 2H, J = 7.2 Hz, CH2-3), 6.77–7.87 (m, 7H, aromatic
protons), and 9.02 (s, 1H, CONH). 10.92 (s, H, N@CH). 13C NMR
(CDCl3): d 21.5, 24.6, 25.4, 40.2, 50.0, 77.4, 111.6, 116.8, 117.4,
123.3, 124.3, 130.8, 138.5, 138.6, 140.9, 147.7, 153.2, 158.9,
159.7. MS (EI): m/z (%): 427 (M++2, 16), 426 (M++1, 33), 425 (M+,
60), 395 (13), 362 (100), 334 (33), 305 (24), 272 (42), 255 (22),
C24H22N4O2 (398.46): C, 72.34; H, 5.57; N, 14.06. Found: C, 72.42; H,
5.26; N, 14.40.
4.1.2.2. 6-Benzoylamino-7-cyano-N-[3,5-dichlorophenyl]-2,3-dihydro-
1H-pyrrolizine-5-carboxamide
(12b). White
crystals,
m.p.
279–81 °C, yield 69%. IR
t
max/cmÀ1 3446, 3274 (NHs), 3070 (CAH
aromatic), 2971, 2907 (CAH aliphatic), 2212 (CN), 1668 (CO),
1592, 1537 (C@C, NH), 1434, 1364 (CAN, CAO), 812 (CACl). 1H
174 (16), 148 (76), 105 (127). Anal. Calcd. for
C26H27N5O

6
A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
(425.53): C, 73.39; H, 6.40; N, 16.46. Found: C, 73.71; H, 6.27; N,
16.58.
7.48 (m, 7H, aromatic protons), and 8.86, 9.23 (two s, 2H, two
CONH). 13C NMR (DMSO-d6): d 21.6, 24.6, 25.9, 50.1, 79.3, 112.4,
115.8, 117.0, 118.6, 122.2, 125.5, 129.3, 138.6, 140.3, 141.4,
147.3, 153.1, 158.1, 178.3. MS (EI): m/z (%): 413 (M+, 38), 370
(49), 341 (43), 319 (42), 292 (42), 278 (44), 258 (31), 248 (45),
225 (31), 214 (100), 187 (60), 173 (63), 162 (44), 151 (35), 138
(63), 121 (35), 92 (25), 77 (52). Anal. Calcd. for C24H23N5O2
(413.47): C, 69.72; H, 5.61; N, 16.94. Found: C, 69.63; H, 5.31; N,
17.29.
4.1.3.2.
dichlorophenyl]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
(13b). Yellow crystals, m.p. 296–9 °C, yield 72%. IR
max/cmÀ1 3408,
7-Cyano-6-[(4-dimethylaminobenzylidene)-amino]-N-[3,5-
t
3313, 3282, 3226(NHs), 3097, 3032 (CAH aromatic), 2972, 2910
(CAH aliphatic), 2209 (CN), 1678, 1657 (COs), 1592, 1540 (C@C,
NH), 1438, 1433, 1316 (CAN, CAO), 835 (CACl). 1H NMR (CDCl3-
400 MHz): d 2.58 (m, 2H, CH2-2), 3.07 (t, 2H, J = 7.5 Hz, CH2-1),
3.15 (s, 6H, N(CH3)2), 4.51 (t, 2H, J = 7.2 Hz, CH2-3), 6.81–7.87 (m,
7H, aromatic protons), and 8.96 (s, 1H, CONH). 11.24 (s, H, N@CH).
13C NMR (CDCl3): d 25.1, 25.2, 26.0, 41.6, 48.4, 50.2, 114.3, 118.9,
124.1, 127.9, 129.4, 134.9, 136.1, 140.3, 147.5, 150.1, 154.2,
157.6, 162.3. MS (EI): m/z (%): 467 (M++2, 25), 466 (M++1, 17),
465 (M+, 35), 305 (55), 265 (13), 148 (100), 77 (18). Anal. Calcd.
for C24H21Cl2N5O (466.36): C, 61.81; H, 4.54; N, 15.02. Found: C,
61.70; H, 4.49; N, 14.89.
4.1.4.2.
dihydro-1H-pyrrolizine-5-carboxamide (14b). White crystals, m.p.
281–4 °C, yield 73%. IR
max/cmÀ13330, 3282 (NHs), 3077 (CAH
7-Cyano-6-(3-phenylureido)-N-(3,5-dichlorophenyl)-2,3-
t
aromatic), 2221 (CN), 1681, 1650 (COs), 1593, 1538 (C@C, NH),
1498, 1439, 1313 (CAN, CAO). 1H NMR (DMSO-400 MHz): d 2.45
(m, 2H, CH2-2), 2.97 (t, 2H, J = 7.5 Hz, CH2-1), 4.31 (t, 2H,
J = 7.2 Hz, CH2-3), 5.72 (bs, 1H, NH), 6.94–7.65 (m, 7H, aromatic
protons), and 9.12, 10.52 (two s, 2H, two CONH). 13C NMR
(DMSO-d6): d 24.1, 25.2, 44.6, 77.2, 115.7, 116.3, 118.1, 122.7,
124.6, 126.3, 129.5, 132.2, 127.7, 138.5, 140.4, 153.1, 162.1,
174.6. MS (EI): m/z (%): 456 (M++3, 20), 455 ((M++2, 10), 454
((M++1, 11), 416 (15), 394 (12), 377 (13), 313 (17), 274 (15), 247
(13), 225 (16), 196 (14), 174 (10), 149 (17), 120 (20), 93 (55), 69
(100). Anal. Calcd. for C22H17Cl2N5O2 (454.31): C, 58.16; H, 3.77;
N, 15.42. Found: C, 57.83; H, 4.01; N, 15.11.
4.1.3.3.
7-Cyano-6-[(4-nitrobenzylidene)amino]-N-[3,5-dimethyl-
(13c). Yellow
phenyl]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
crystals, m.p. 261–4 °C, yield 86%. IRˆmax/cmÀ13321, 3277 (NHs),
3057 (CAH aromatic), 2929 (CAH aliphatic), 2216 (CN), 1732,
1661 (C@Os), 1591, 1530 (C@C, NH), 1458, 1439, 1303 (CAN,
CAO). 1H NMR (CDCl3-400 MHz): d 2.23 (s, 6H, two PhCH3), 2.61
(m, 2H, CH2-2), 3.01 (t, 2H, J = 7.5 Hz, CH2-1), 4.58 (t, 2H,
J = 7.2 Hz, CH2-3), 6.77–8.38 (m, 7H, aromatic protons), and 9.28
(s, 1H, CONH). 10.37 (s, H, N@CH). 13C NMR (CDCl3): d 21.5, 24.5,
25.5, 50.3, 116.0, 117.3, 119.3, 124.3, 126.1, 129.2, 130.5, 137.7,
137.8, 139.0, 140.9, 148.7, 149.7, 156.2, 157.9. MS (EI): m/z (%):
428 (M++1, 31), 427 (M+, 96), 307 (100), 261 (76), 174 (44), 147
(27), 120 (47), 77 (62). Anal. Calcd. for C24H21N5O3 (427.46): C,
67.44; H, 4.95; N, 16.38. Found: C, 67.38; H, 4.89; N, 16.80.
4.1.4.3.
phenyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamide
crystals, m.p. 282–5 °C, yield 85%. IR
max/cmÀ1 3295 (broad band,
7-Cyano-6-[3-(4-chlorophenylureido)]-N-(3,5-dimethyl-
(14c). White
t
NHs), 3076 (CAH aromatic), 2978, 2914 (CAH aliphatic), 2213
(CN), 1634 (broad band, COs), 1591, 1561(C@C, NH), 1491, 1435,
1397 (CAN, CAO), 823 (CACl). 1H NMR (DMSO-400 MHz): d 2.36
(s, 6H, two PhCH3), 2.50 (m, 2H, CH2-2), 3.33 (t, 2H, J = 7.5 Hz,
CH2-1), 4.33 (t, 2H, J = 7.2 Hz, CH2-3), 5.30 (bs, 1H, NH),
6.71–7.48 (m, 7H, aromatic protons), and 8.96, 9.63 (two s, 2H,
two CONH). 13C NMR (DMSO-d6): d 21.6, 24.7, 25.9, 50.1, 78.3,
112.4, 116.8, 117.3, 119.6, 123.3, 125.9, 130.9, 138.2, 141.3,
143.8, 149.4, 154.8, 165.1, 178.3. MS (EI): m/z (%): 446 (M+À1, 1),
428 (21), 256 (100), 228 (36), 160 (20). Anal. Calcd. for C24H22ClN5-
O2 (447.92): C, 64.36; H, 4.95; N, 15.64. Found: C, 64.10; H, 4.87; N,
15.35.
4.1.3.4.
phenyl]-2,3-dihydro-1H-pyrrolizine-5-carboxamide
crystals, m.p. 292–5 °C, yield 73%. IR
max/cmÀ1 3351, 3274, 3229
7-Cyano-6-[(4-nitrobenzylidene)amino]-N-[3,5-dichloro-
(13d). Yellow
t
(NHs), 3069 (CAH aromatic), 2217 (CN), 1666 (CO), 1588, 1540
(C@C, NH), 1434, 1345 (CAN, CAO). 1H NMR (CDCl3-400 MHz): d
2.59 (m, 2H, CH2-2), 3.17 (t, 2H, J = 7.5 Hz, CH2-1), 4.58 (t, 2H,
J = 7.2 Hz, CH2-3), 6.83–7.87 (m, 7H, aromatic protons), and 9.33
(s, 1H, CONH). 11.47 (s, H, N@CH). 13C NMR (CDCl3): d 24.6, 25.4,
50.3, 117.7, 119.0, 124.1, 124.5, 124.6, 129.2, 129.4, 135.5, 139.8,
140.6, 149.2, 150.0, 157.1, 158.1, 163.4. MS (EI): m/z (%): 469
(M++2, 10), 468 (M++1, 6), 467 (M+, 21), 466 (M+À1, 14), 345
(15), 307 (100), 261 (33), 120 (36), 77 (13). Anal. Calcd. for C22H15-
Cl2N5O3 (468.29): C, 56.43; H, 3.23; N, 14.96. Found: C, 56.52; H,
3.34; N, 15.35.
4.1.4.4.
phenyl)-2,3-dihydro-1H-pyrrolizine-5-carboxamide
crystals, m.p. 287 °C, yield 76%. IR
max/cmÀ1 3296 (NHs), 3077
7-Cyano-6-[3-(4-chlorophenylureido)]-N-(3,5-dimethyl-
(14d). White
t
(CAH aromatic), 2955 (CAH aliphatic), 2221 (CN), 1681, 1647
(broad band, COs), 1588, 1536 (C@C, NH), 1491, 1436, 1373
(CAN, CAO), 868. 1H NMR (DMSO-400 MHz): d 2.46 (m, 2H, CH2-
2), 3.32 (t, 2H, J = 7.5 Hz, CH2-1), 4.35 (t, 2H, J = 7.2 Hz, CH2-3),
5.35 (bs, 1H, NH), 7.26–7.64 (m, 7H, aromatic protons), and 9.29,
10.74 (two s, 2H, two CONH). 13C NMR (DMSO-d6): d 24.9, 25.6,
44.8, 79.1, 115.8, 117.6, 119.3, 122.7, 124.8, 125.2, 129.9, 133.5,
129.7, 139.1, 141.2, 155.5, 168.3, 177.1. MS (EI): m/z (%): 489
(M++2, 15), 488 (M++1, 19), 464 (12), 442 (15), 418 (17), 392
(15), 376 (12), 357 (20), 290 (16), 272 (17), 236 (19), 223 (18),
178 (14), 149 (15), 137 (13), 121 (21), 93 (19), 69 (100). Anal.
Calcd. for C22H16Cl3N5O2 (488.75): C, 54.06; H, 3.30; N, 14.33.
Found: C, 54.43 H, 3.57; N, 14.40.
4.1.4. General procedure for the preparation of compounds (14a–d)
A mixture of 6-amino-7-cyano-N-[3,5-disubstituted-phenyl]-
2,3-dihydro-1H-pyrrolizine-5-carboxamide 11 (3.8 mmol) in
methylene chloride (25 ml), appropriate isocyanate (4 mmol),
and three drops of triethylamine is refluxed for 12 h, evaporate
the solvent under reduced pressure. The residue is dissolved in
acetone, concentrated; set aside where the solid obtained is col-
lected, dried and recrystallized from ethanol–acetone.
4.1.4.1.
dihydro-1H-pyrrolizine-5-carboxamide (14a). White crystals, m.p.
261–4 °C, yield 79%. IR
max/cmÀ13284 (broad band due to NHs),
7-Cyano-6-(3-phenylureido)-N-(3,5-dimethylphenyl)-2,3-
4.2. Pharmacological screening
t
3067 (CAH aromatic), 2957 (CAH aliphatic), 2212 (CN), 1671,
1650 (broad band due to COs), 1597, 1560 (C@C, NH), 1498,
1437, 1365 (CAN, CAO). 1H NMR (DMSO-400 MHz): d 2.39 (s,
6H, two PhCH3), 2.46 (m, 2H, CH2-2), 2.97 (t, 2H, J = 7.5 Hz,
CH2-1), 4.33 (t, 2H, J = 7.2 Hz, CH2-3), 5.81 (bs, 1H, NH), 6.81–
4.2.1. Inhibition of growth
4.2.1.1. Cell culture. MCF-7, human breast cancer cells and PC-3,
human prostate cancer cell lines were obtained from the American
Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s
modified Eagle’s medium /F12 medium (DMEM/F-12, Gibco, Grand

A.M. Gouda et al. / Bioorganic Chemistry 53 (2014) 1–7
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4.2.1.2. Cell viability analysis. The effect of test compounds on
cancer cell viability was assessed using the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in
six replicates as previously reported [28]. Briefly, Cells were seeded
in 96-well flat-bottomed plates for 24 h, and treated with test
agents in 5% FBS-supplemented DMEM/F-12 or DMEM for the indi-
cated time intervals. Controls received DMSO vehicle at a concen-
tration equal to that in drug-treated cells. After treatment, cells
were incubated in the same medium containing 0.5 mg/ml MTT
at 37 °C for 2 h. Reduced MTT was solubilized in DMSO (200 lL/
well) for determination of absorbance at 570 nm using a micro-
plate reader. Results are presented in Table 1.
4.2.2. Caspase activation assay
Caspase-3/7 and caspase-8 activities in MCF-7 cells treated with
test agents were measured using Caspase-Glo 3/7 luminescence
assay kit (Promega, Madison, WI) according to the manufacturer’s
instructions and as mentioned before [29]. Briefly Cells were plated
at 1 Â 104 (100
ll/well) into clear bottom, opaque wall 96-well tis-
sue culture plates and incubated for 24 h. The medium was
removed and the cells were then treated with test compounds
for 48 h. Caspase-3/7 activity were then assayed according to the
instructions included in the kit. The luminescence of plates was
read using a luminometer. Results are represented in Fig. 3.
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