Pyrrolizines: Design, synthesis, anticancer evaluation and investigation of the potential mechanism of action

Article abstract of DOI:10.1016/j.bmc.2017.08.039

A novel set of pyrrolizine-5-carboxamides has been synthesized and evaluated for their anticancer potential against human breast MCF-7, lung carcinoma A549 and hepatoma Hep3B cancer cell lines. Compound 10c was the most active against MCF-7 with IC₅₀

Full text of DOI:10.1016/j.bmc.2017.08.039

Accepted Manuscript
Pyrrolizines: design, synthesis, anticancer evaluation and investigation of the
potential mechanism of action
Ahmed M. Gouda, Ahmed H. Abdelazeem, Hany A. Omar, Ashraf N. Abdalla,
Mohammed A.S. Abourehab, Hamed I. Ali
PII:
S0968-0896(17)31494-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.039
BMC 13943
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
21 July 2017
18 August 2017
22 August 2017
Please cite this article as: Gouda, A.M., Abdelazeem, A.H., Omar, H.A., Abdalla, A.N., Abourehab, M.A.S., Ali,
H.I., Pyrrolizines: design, synthesis, anticancer evaluation and investigation of the potential mechanism of action,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.039
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Pyrrolizines: design, synthesis, anticancer evaluation and investigation of
the potential mechanism of action
Ahmed M. Gouda1,2,*, Ahmed H. Abdelazeem2, Hany A. Omar3,4, Ashraf N. Abdalla5,6,
Mohammed A.S. Abourehab7, Hamed I. Ali8,9, *
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Umm Al-Qura University, Makkah
21955, Saudi Arabia; 2Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef 62514, Egypt; 3Sharjah Institute for Medical Research and College of Pharmacy, University of
Sharjah, Sharjah 27272, United Arab Emirates; 4Department of Pharmacology and Toxicology, Faculty
of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt; 5Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia; 6Department
of Pharmacology, Medicinal and Aromatic Plants Research Institute, National Center for Research,
Khartoum 2404, Sudan; 7Department of Pharmaceutics, Faculty of Pharmacy, Umm Al-Qura University,
Makkah 21955, Saudi Arabia; 8Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan
University, Cairo, Egypt; 9Rangel College of Pharmacy, Health Science Center, Texas A&M University,
Kingsville, Texas 78363, United States.
* To whom correspondence should be addressed
Ahmed M. Gouda, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef 62514, Egypt.
Tel.: +2010-0287-7405
Fax: +2082-231-7958
E-mail address: ahmed.gouda@pharm.bsu.edu.eg, amsaid@uqu.edu.sa
Hamed I. Ali
Rangel College of Pharmacy, Health Science Center, Texas A&M University, Kingsville, Texas 78363,
United States.
Tel.: +1361-221-0758
Fax: +1361-221-0793
E-mail address: alyismail@pharmacy.tamhsc.edu
1

List of abbreviations
ALK1, activin receptor-like kinase; CDCl3, deuterated chloroform; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DSV, discovery studio visualizer;
DYRK3, dual specificity tyrosine phosphorylation-regulated kinase; CDK2, cyclin-dependent
kinase-2; COX, cyclooxygenase; DMSO, dimethyl sulfoxide, EGFR, epidermal growth factors
receptor; eV, electron volte; FITC, fluorescein isothiocyanate; GSK3, glycogen synthase kinase-
3; HEPES: 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid; IC50, half maximal inhibitory
concentration; 5-LOX, 5-lipoxygenase; MAPK, mitogen-activated protein kinase; ODC,
ornithine decarboxylase inhibitor; PBS, Phosphate-buffered saline; PC, pancreatic cancer; PDB,
protein data bank; PI, propidium iodide; PPARꢀ, peroxisome proliferator-activated receptor
gamma; PS, phosphatidylserine; SAR, structure activity relationship; SEM, standard error of the
mean; SI, selectivity index; VEGF, vascular endothelial growth factor.
Highlights
•
•
•
•
•
•
•
A novel series of twelve pyrrolizines (9-12a-c) were designed and synthesized
The anticancer activity was evaluated against MCF-7, A549, and Hep3B cells using MTT assay
Compound 12b showed IC50 value of 3.24 µM against A549 cell line
The new compounds inhibited COX-1 and COX-2 with the selectivity index of 8.38-113.1 range
Compound 12b revealed a weak to moderate inhibition of six different protein kinases
Compounds 12a and 12b exhibited the highest activation of caspase-3/7 in A549 cells
Cell cycle analysis and Annexin V PI/FITC assay revealed an accumulation of MCF-7 tumor
cells in the S-phase and an apoptotic induction by compound 12b.
Key words. Pyrrolizine, anticancer, COX-2, Kinase, Caspase-3/7, Cell cycle, Annexin V,
Apoptosis
2

Graphical Abstract
3

Abstract
A novel set of pyrrolizine-5-carboxamides has been synthesized and evaluated for their
anticancer potential against human breast MCF-7, lung carcinoma A549 and hepatoma Hep3B
cancer cell lines. Compound 10c was the most active against MCF-7 with IC50 value of 4.72 µM,
while compound 12b was the most active against A549 and Hep3B cell lines. Moreover,
kinases/COXs inhibition and apoptosis induction were suggested as potential molecular
mechanisms for the anticancer activity of the novel pyrrolizines based on their structural
features. The new compounds significantly inhibited COX-1 and COX-2 with IC50 values in the
ranges of 5.78-11.96 µM and 0.1-0.78 µM, respectively with high COX-2 selectivity over COX-
1. Interestingly, the most potent compound in MTT assay, compound 12b, exhibited high
inhibitory activity against COX-2 with selectivity index (COX-1/COX-2) > 100. Meanwhile,
compound 12b displayed weak to moderate inhibition of six kinases with inhibition% (7-20%)
compared to imatinib (inhibition% = 1-38%). The results of cell cycle analysis, annexin V
PI/FITC apoptosis assay and caspase-3/7 assay revealed that compound 12b has the ability to
induce apoptosis. The docking results of compound 12b into the active sites of COXs, ALK1
and Aurora A indicated that it fits nicely inside their active sites. Overall, the current study
highlighted the significant anticancer activity of the newly synthesized pyrrolizines with a
potential multi-targeted mechanism which could serve as a base for future studies and further
structural optimization into potential anticancer agents.
4

1. Introduction
The persistent need for the development of new anticancer agents was augmented by several
factors including the high rate of incidence and mortality of cancer, the poor bioavailability of
some anticancer agents, and finally the evolution of resistance against many of the currently
used anticancer agents [1-5].
As a multifactorial disease, treatment of cancer can be better achieved using multi-target therapy
to overcome the resistance problem. Multi-target therapy can be obtained by either drug
combination therapy or polypharmacology [6]. The combination of licofelone 1 (Fig. 1) with
gefitinib exhibited significant reduction in pancreatic cancer (PC) at a dose lower than doses of
the individual agents [7,8]. Moreover, combination of licofelone 1 with peroxisome proliferator-
activated receptor gamma (PPARꢀ) ligand and ornithine decarboxylase inhibitor (ODC) could
provide chemopreventive and therapeutic effects [9-11]. Combination therapy showed an
enhanced survival rate for the treatment of different types of cancers [12]. But, it still has some
limitations, especially those related to the increased risk of drug-drug interaction and toxicity.
On the other hand, anticancer agent with multi-targeted mechanism lack the problems of drug-
drug interaction and dosage/sequences optimization needed in combination therapy.
The design of multi-targeted anticancer agents could provide an alternative approach for drug
combination in complex diseases [13,14]. In anticancer research, several drugs act as multi-
kinase inhibitors have displayed high therapeutic efficacy. But, another class of anticancer
agents which can hit other targets together with or without kinase inhibition have been reported.
Of these class, the dual COX/5-LOX inhibitor (licofelone) 1 has displayed potent anticancer
activities against several types of hormonal dependent/independent cancer cells [15,16].
Exploring the mechanism of action of licofelone 1 revealed its ability to induce apoptosis [17].
Fig. 1. Pyrrolizine-based anticancer agents with their IC50 values against different cancer cells
5

Tavolari et al. have presented a new evidence that the induction of apoptosis is mediated
through mitochondrial pathway rather than COX inhibition and arachidonic acid pathway [18].
licofelone 1 displayed also indirect blockade of EGFR kinase, mitogen-activated protein kinase
(MAPK) and AKT cascades. The inhibition of these kinases results from the changes in cell
membrane fluidity and contribute at least in part to the induction of apoptosis in HCA-7 colon
cancer cells [19].
Moreover, the tripentone (MR22388) 2 (Fig. 1) exhibited strong anticancer activity against
leukemia L1210 cells with IC50 of 15 nM [20]. MR22388 induced apoptosis through the MAP
kinase pathway and displayed strong inhibition of several other kinases, in addition to inhibition
of tubulin polymerization [21-22].
We have previously reported compounds 3 among series of caspase-3/7 inducers [23].
Compound 3 displayed potent anticancer activity with IC50 values in the range of 7.9 and 8.54
µM against MCF-7 and PC-3 cell lines respectively, Fig. 1.
Although the anti-inflammatory/anticancer potential of licofelone was extensively studied in
several reports [24,25], but little data are available about its heteroaryl analogs. In the current
work, we aimed to manipulate the chemical structure of the lead compound 3. Based on the
structure-activity relationship of licofelone [24]. We have designed the new analogs lacking the
non-essential 3,3-dimethyl and carboxylic group, with 2 atoms spacer in between the pyrrolizine
nucleus and phenyl rings, Fig. 2.
Fig. 2. Rational design and structural modification of compound 3
6

The new analogs were obtained through two modifications of the chemical structure of
compound 3, Fig. 2. The first was the replacement of the 4-nitrophenyl moiety by five-
membered heterocyclic ring (furanyl or thiophenyl ring), six-membered (pyridinyl) ring or bulky
(naphthalenyl) ring. The second modification was conducted through variation of the type of
substituents on the second phenyl ring, using the 4-methyl (+σ) and 4-chloro (-σ) substituents in
addition to the unsubstituted analogs.
2. Results and discussion
2.1. Chemistry
Synthesis of compounds 5, 7a-c and 8a-c (Scheme 1) was performed following the reported
protocols [26-28].
Scheme 1. Reagents and reaction conditions: (a) (CH3)2SO4, benzene, CH2(CN)2; (b)
ClCH2COCl, AcOH, CH3COONa; (c) K2CO3, acetone, reflux, 24 h.
The Schiff’s base derivatives 9-12 were prepared by refluxing the pyrrolizines 8a-c with
different aryl aldehydes including furan-2-carbaldehyde, thiophene-2-carbaldehyde, pyridine-3-
carbaldehyde, or 2-naphthaldehyde in absolute ethanol in the presence of a catalytic amount of
glacial acetic acid as depicted in Scheme 2.
The structural characterization of all the synthesized derivatives was accomplished using IR, 1H-
NMR, 13C NMR, and mass spectroscopy, in addition to the elemental analyses. The IR spectra
of compounds 9-12 revealed their characteristic absorption bands in the ranges of 3211-3421,
2204-2215, and 1656-1679 cm-1 corresponding to the amide, cyano, and carbonyl group,
7

1
respectively. The H-NMR spectra of compounds 9-12 showed singlet signal owing to Ph-CH3
protons assigned for compound 9b, 10b, 11b and 12b in a range of 2.34-2.36 ppm, a multiplet
signal due to the protons of CH2-2 at range of 2.48-2.62 ppm, triplet at the range of 2.90-3.12
ppm attributed to the CH2-1 protons, another triplet at the range of 4.46-4.60 ppm corresponding
to the CH2-3. The aromatic protons in compounds 9-12 appeared as a multiplet at the range of
6.62-9.17 ppm. The methylene protons (N=CH) in compounds 9-12 appeared as a singlet at the
range of 88.8-9.97 ppm, while the amide protons (CONH) appeared as singlet at the range of
10.38-11.25 ppm.
Scheme 2. Reagent and reaction conditions: (a) pyridine-3-carbaldehyde, AcOH, EtOH, reflux, 4 h; (b)
furan-2-carbaldehyde, AcOH, EtOH, reflux, 4 h; (c) thiophene-2-carbaldehyde, AcOH, EtOH, reflux, 4 h; (d)
2-naphthaldehyde, AcOH, EtOH, reflux, 4 h.
The 1H-NMR spectra of compounds 9a-c revealed the aromatic protons of the 2-furanyl ring as a
quartet signal attributed to the furanyl CH-4ꢁ at the range of 6.62-6.64 ppm, the furanyl CH-3ꢁ
appeared as a doublet at the range of 6.99-7.03 ppm, while the furanyl CH-5ꢁ appeared as a
1
singlet at the range of 7.71-7.73 ppm. The H-NMR spectra of compounds 10a-c revealed the
aromatic protons of the 2-thiophenyl ring as a triplet signal attributed to the furanyl CH-4ꢁ at
7.17 ppm for compounds 10a,b. The thiophene CH protons at positions 3ꢁ and 5ꢁ were detected
1
as two doublets at the range of 7.49-6.61 ppm for compounds 10a,b. The H-NMR spectra of
8

compounds 11a-c revealed the aromatic protons of the 3-pyridinyl ring as a triplet signal
attributed to the pyridinyl CH-5ꢁ at 7.13 and 7.53 ppm for compounds 11a and 11b,
respectively. The pyridinyl CH-4ꢁ and CH-6ꢁ appeared as two doublets at a range of 8.18-8.79
ppm, while the pyridinyl CH-2ꢁ appeared as singlet at a range of 9.15-9.17 ppm. The 13C-NMR
spectra of compounds 9-12 revealed signal at 20.86-24.57 ppm, corresponding to the
aliphatic carbon of the methyl group in compounds 9b, 10b, 11b and 12b. The carbon
signals of the three methylene groups of the pyrrolizine ring appeared in the range 24.38-
50.27 ppm. The two signals at the range of 151.55-158.82 ppm are corresponding to the
N=CH and C=O carbons. On the other hand, the mass spectra for compounds 9a-c-12a-c
revealed the molecular ion peaks at 344 (9a), 358 (9b), 378 (9c), 360 (10a), 374 (10b), 394
(10c), 355 (11a), 369 (11b), 389 (11c), 404 (12a), 418 (12b), and 438 (12c), respectively. The
fragmentation patterns of these compounds were in concordance with their chemical structures.
2.2. Pharmacology
2.2.1. Anticancer activity
2.2.1.1. MTT assay results
The growth inhibitory effect of the new compounds 9-12 against three cancer cell lines was
performed to evaluate their anticancer activities. The target compounds were examined against
MCF-7 (human breast cancer), A549 (lung carcinoma) and Hep3B (hepatoma) cell lines using
MTT assay [29]. The results were expressed in IC50 and doxorubicin was used as a reference
drug, Table 1. All the tested compounds showed inhibitory activities with IC50 values in the
range of 3.24-34.75 µM against the three cancer cell lines. Compound 10c was the most active
against MCF-7 with IC50 value of 4.72 µM, while compound 12b was the most active against
both A549 and Hep3B cell lines.
2.2.1.2. Structure activity relationship (SAR)
Based on the MTT assay results, the structure activity relationship could be explained according
to two main structural features; the steric bulkiness of the aryl/heteroaryl moiety in the side
chain at C6 and the electronic effects of the various substituents on the phenyl ring. Correlating
the results of the MTT assay (Table 1) with aryl/heteroaryl moiety bulkiness, it could be
observed that the bulkiness of the aryl/heteroaryl ring has an important impact on the activity of
the unsubstituted phenyl derivatives 9a, 10a, 11a and 12a where the electronic effect of
methyl/chloro substituents was absent. Accordingly, the naphthalenyl derivatives 12a was the
most active compared to their corresponding thiophenyl and furanyl analogs. Conversely, the
9

three pyridinyl derivatives 11a-c showed the least activity among all the new compounds against
MCF-7 and Hep3G cell lines. Moreover, the thiophenyl bearing derivatives 10a-c were nearly
more active than their corresponding furanyl derivatives 9a-c against the three cell lines.
Table 1. IC50 values of compounds 9-12 against MCF-7, A549 and Hep3B cancer cell lines
IC50 (µM)
Comp. No.
R1
R2
MCF-7
A549
Hep3B
H
4-CH3
4-Cl
H
9a
9b
8.35 1.1
9.75 1.2
12.80 0.4
16.15 1.5 29.44 2.4 24.41 2.2
15.68 1.4 28.38 3.4 27.52 3.5
9c
10a
10b
10c
11a
6.97 0.8 12.53 2.6 18.62 0.6
11.39 1.6
4-CH3
4-Cl
H
5.35 1.1
13.75 1.5
4.72 0.4 22.80 1.3 11.62 1.7
11.65 0.9 17.54 3.2 25.82 2.4
18.26 1.2 20.53 1.9 34.75 2.7
22.52 1.9 23.03 1.3 31.17 2.6
4-CH3
4-Cl
H
11b
11c
12a
5.77 1.1
7.76 0.8
7.11 0.7
3.24 0.3
10.17 0.9
8.69 0.4
4-CH3
4-Cl
12b
12c
9.82 0.7 11.19 0.8 12.49 1.1
7.9 1.1a 16.25 1.5 14.04 1.9
3,5-dichloro
3
-
-
-
-
Licofelone
Doxorubicin
5.5 0.6 b
-
-
1.7 1.1
2.3 1.8
2.4 1.3
Cells were treated with the new compound or vehicle for 72 h. and the results were presented as
mean S.E, a quoted from previous publication [23], b quoted from previous publication [16]
10

Concerning the impact of electronic effect of the 4-methyl/chloro substituents on activity of the
new compounds, it was clear that, (1) The unsubstituted derivative 9a was the most active in the
three furanyl derivatives against both MCF-7 and A549 cell lines, followed by the chloro analog 9c
and finally the methyl analog 9b. (2) The three thiophenyl derivatives 10a-c displayed variable
activity against the three cell lines where compound 10c with the electron withdrawing chloro
group was the most active against MCF-7 and Hep3B cells, while 10b displayed the highest
activity against A549 cell line. (3) Substitution with both electron donating methyl and electron
withdrawing chloro groups reduced the activity of the pyridinyl derivatives where the
unsubstituted analog 11a displayed the highest activity against the three cell lines. (4) The 4-
methyl substituent enhanced the anticancer activity of the naphthalenyl derivatives 12a-c while
the 4-chloro substitution decreased activity against both A549 and Hep3B cells.
2.2.2. In vitro COX-1/2 inhibitory activity
The discovery of several COX-2 selective inhibitors with potent anticancer activity represents an
evidence for the important role of COX-2 receptor in regulation of cellular growth and death in
human cancer [30-32]. In this work, COX-1/2 inhibition by the new compounds 9-12 was
evaluated using COX colorimetric inhibitor screening assay kit (Catalog No. 560131, Cayman
Chemicals INC., Ann Arbor, MI, USA). The assay was done following the manufacturer’s
instructions and as previously reported [33]. The results were expressed in IC50 values, Table 2.
The new compounds displayed inhibition of COX-1 and COX-2 with IC50 values in the ranges
of 5.78-11.96 µM and 0.1-0.78 µM respectively. Compound 11c was the most active COX-1
inhibitor, while compound 11a was the most active COX-2 inhibitor. The new compounds 9-12
displayed selective inhibition of COX-2 over COX-1 with selectivity index (SI) in the range of
8.38-113.1. Compound 11a was the most selective for COX-2 (SI = 113.1) followed by
compound 12b (SI = 108.73), Table 2. A weak relationship was observed between the
selectivity of the new compounds to COX-2 with their anticancer activity. Compound 12b, the
second selective COX-2 inhibitor in the new derivatives displayed the highest anticancer activity
with IC50 values in the range of 11.6-19.1 µM against the three cell lines. The third selective
COX-2 inhibitor (10b) displayed IC50 values in the range of 16.9-28.9 µM, being less active
than 12b. Moreover, lack of the anticancer activity of compound 11c (IC50 >60 µM) was
11

associated with its very weak selectivity for COX-2 selectivity (SI = 8.5). Similarly, compounds
9c and 10c with selectivity index of 18.69 and 21.33 displayed IC50 >60 µM against MCF-7
cells.
Table 2: In vitro COX-1/2 enzymes inhibition results of compound 9-12.
COX-1
(IC50 µM)
6.54
COX-2
(IC50 µM)
0.78
b
SI
Comp. No
a
a
9a
8.38
9.23
0.19
48.58
18.69
8.86
9b
9c
8.41
0.45
4.96
0.56
10a
10.41
7.68
0.11
94.64
21.33
113.10
59.63
8.50
10b
0.36
10c
11.31
9.54
0.10
11a
0.16
11b
5.78
0.68
11c
12a
7.98
0.52
15.35
108.73
62.53
0.0796
296.0
0.045
11.96
10.63
0.039
14.8
0.11
12b
0.17
12c
0.49
Indomethacin
Celecoxib
0.05
b
b
a
0.21
4.7
Licofelone
a
IC50 was calculated using three determinations for COX-1 (ovine) and COX-2
(human recombinant) screening assay kit (Cat. No 560131, Cayman
b
Chemicals Inc., Ann Arbor, MI, USA); In vitro COX-2 selectivity index (SI) =
b
IC50 of COX-1/ IC50 of COX-2. IC50 values quoted from previous
publication [24]
Taken together, a remarkable relationship between COX-2 selectivity of the new compounds
and their anticancer activities could be observed. Compounds 10a-c displayed higher selectivity
against COX-2 enzyme than their furanyl analogs 9a-c which was in agreement with the results
of the MTT assay. Compound 12b with the highest anticancer displayed high selectivity index
of 108.73, Table 2. Compound 11a with the highest selectivity for COX-2 showed potent
activity against MCF-7 and moderate activity against A549 and Hep3G cell lines.
However, compounds 9a, 10a and 11c which displayed different anticancer activities in MTT
assay have nearly the same selectivity for COX-2. Moreover, compounds 10b and 12c which
12

displayed nearly the same anticancer activity against A549 cells displayed different selectivity
for COX-2 enzyme. These findings suggested that COX-2 enzyme may not be the only target for
the new compounds.
2.2.3. Kinase profiling radiometric assay
In attempt to explore other targets that may contribute to the mechanism of action of the new
compounds as anticancer agents, the most active compound 12b was selected for the kinase
profiling test. The profiling test was done in KINEXUS Corporation, Vancouver, BC, Canada,
using the radiolabeled ATP determination method. The test was done following the previous
report [34]. The inhibitory activity of compound 12b was evaluated against 20 kinases. The 20
kinases were selected from different groups, families and types including protein-tyrosine kinase
and protein-serine/threonine kinase.
The results revealed that compound 12b inhibits 6 kinases with inhibition% in the range of 7-
20%, Table 3. Although this result showed weak to moderate inhibitory activity of compound
12b, but it was higher than imatinib (inhibition% = 1-38). The highest inhibition caused by
compound 12b was against DYRK3 kinase (20%), followed by 18% inhibition in the enzyme
activity of both ALK1 and Aurora A. Moreover, compound 12b exhibited 11% inhibition in
CDK2/Cyclin A1 kinase compared to only 2% for imatinib.
In conclusion, compound 12b showed low to moderate inhibitory activity of 6 different kinases
in comparison with imatinib. This finding suggests that kinase inhibition may participate
partially to the overall anticancer activity of compound 12b.
13

Table 3: The activation and inhibition of compound 12b and imatinib against 20 different
kinases at 10 µM concentration.
% Inhibition
Kinase
12b
Imatinib
ALK1
AMPK (A1/B1/G1)
ASK1
Aurora A
BLK
BRAF
CDK2/Cyclin A1
CK1 Alpha 1
DYRK3
EGFR
EPHA1
FLT1
GRK1
GSK3 alpha
MSK1
NEK1
p38 Alpha
PDK1
14%
18%
5%
-38%
-3%
13%
-2%
0%
-10%
13%
22%
-10%
2%
-1%
49%
-2%
17%
34%
4%
-18%
11%
19%
-18%
16%
56%
-11%
3%
-20%
47%
37%
10%
-7%
5%
43%
-8%
28%
27%
4%
PRKG1
SGK1
16%
3%
Negative values indicate inhibition and positive value indicate
activation
2.2.4. Caspase-3/7 activation assay
The role of caspase-3 and caspase-7 in as terminal enzymes in apoptosis was previously
discussed [35,36]. Here in this study, it was of interest to evaluate the effect of the new
compounds on these caspases using caspase 3/7 activation assay. Compounds 9a and 12a-c, the
most active against lung carcinoma A549 cells were selected to evaluate their effect on caspase
enzymes. Caspase 3/7 activity in A549 cells treated with compounds 9a and 12a-c was
measured using caspase-Glo 3/7 luminescence assay kit (Promega, Madison, WI) following the
manufacturer's instructions and as mentioned before [37]. Compounds 12a and 12b were the
most potent in activating caspase enzymes in A549 cells at concentrations of 5, 10 and 20 µM.
In general, the results of caspase-3/3 activation assay were in concordance with the results of
MTT assay in A549 cells, Fig. 3.
14

7000
6000
5000
4000
3000
2000
1000
0
**
**
**
**
*
0
5
10
20
Concentration (µM)
Fig. 3. Caspase-Glo 3/7 assay of lung carcinoma A549 cells treated with compounds 9a (ꢂ), 12a ((ꢂ), 12b
(ꢂ), and 12c (ꢂ) (24 h, x axis); luminesence (RLU, y axis). Experiment was repeated 3x. controls received
equivalent amount of DMSO (vehicle). Columns, mean; bars, S.D. (n = 5). * p < 0.05, ** p < 0.01 as
compared to vehicle (DMSO)-treated cells using one-way ANOVA followed by Dunnett’s multiple
caparison test
2.2.5. Determination of cell cycle perturbations
The cell division was analysed by flow cytometry using Propidium iodide-stained cell
populations. The cell cycle analysis (BC, FC500) was conducted to test the effect of compound
12b in MCF-7 cells (24 h) according to the reported protocol [38]. MCF-7 cells were treated by
compound 12b (1.0 ꢃM), which demonstrated more than two-folds increase in S phase
compared to control; this accumulation in S phase was maintained in the higher doses (5 and 10
µM); all at the expense of other stages, Fig. 4. Cells in G2/M phase slightly increased at 10 ꢃM,
compared to control. The results indicate that compound 12b caused considerable S phase arrest.

Fig. 4. Cell cycle phases of MCF-7 treated with compound 12b for 24h (concentration, x axis); cell count
(percent, y axis). Controls received equivalent amount of DMSO (vehicle). Columns, mean; bars, S.D. (n =
3). * p < 0.05, ** p < 0.01 as compared to vehicle (DMSO)-treated cells using one-way ANOVA followed
by Dunnett’s multiple caparison test
2.2.6. Annexin V PI/FITC apoptosis assay
Induction of apoptosis was reported to mediate the anticancer activity of licofelone [17].
Accordingly, compound 12b the most active in MTT assay was selected for Annexin V
apoptosis assay. MCF-7 cells were treated in this study with compound 12b (24 h), then cells
were double stained with Annexin PI/V FITC and analyzed by flow cytometry for detection of
apoptosis according to the previous report [39]. The obtained results revealed MCF-7 cells in
different phases according to their staining status. The results showed a slight increase in the
apoptosis events (C2 and C4) at 1 ꢃM compared to control, while in the higher doses
cytotoxicity slightly increase, Fig. 5. At 1 µM, compound 12b blocked MCF-7 in the S phase,
which was consistent without increase in the higher doses; and increased apoptosis, suggesting
that it is the therapeutic window.
16

Fig. 5. Annexin V phases of MCF-7 cancer cells treated for 24 h with DMSO control (a), compound 12b
at 1 µM (b), 5 µM (c), 10 µM (d); Data shown in the mean % SD (n = 3). Experiment was repeated 3x.
2.3. Molecular docking study
The role of COX-2 receptor in cancers was supported by several evidences which described the
anticancer potential of several COX-2 selective inhibitors [30-32]. Accordingly, the molecular
docking study was conducted by comparative docking studies into COX-1/COX-2 and into the
most targeted kinases namely: ALK1 and Aurora A kinases.
2.3.1. Comparative Docking study into COX-1 and COX-2 enzymes
GOLD 5.2.2. docking program was used for a differential docking study of compounds 9a-c,
10a-c, 11a-c, and 12a-c into COX-1 and COX-2 enzymes. The Gold score fitness, Gold score
external vdw, and Gold score external hydrogen bonding (HB) are obtained as presented in
17

Table 1 for the best top five docked compounds. Our task for this docking study, is to compare
the binding score fitness of each pose and to determine the best-fit docking style.
The Gold docking was initially validated by docking of the co-crystallized IBP ligand
(ibuprofen) and RCX (viox) into COX-1 (PDB: 1eqg) and COX-2 (PDB: 5kir), respectively.
Both of the docked ligands were almost superimposed on the co-crystallized one within a root
mean square deviation (RMSD) of 0.64 and 0.81Å, respectively as depicted in Fig. 6.
Fig. 6. a) Validation of the GOLD program by docking of ibuprofen into COX-1 (PDB: 1eqg); b)
Validation of the GOLD program by docking of rofecoxib (Vioxx) into COX 2 (PDB: 5kir). The docked
ligands (ball and stick) were identically superimposed on the co-crystallized IBP and RCX ligands
(yellow sticks) within RMSD of 0.64 and 0.81 Å, respectively revealing a successful docking protocol.
Compounds 9a-c, 10a-c, 11a-c, and 12a-c were docked into COX-1 and COX-2 for a
comparative binding study to correlate their differential selectivity with the experimental results.
Remarkably, the docked compounds exhibited higher binding affinities into COX-2 of
Goldscore fitness of the range of 73.73 -93.18. Whereas these compounds demonstrated inferior
Goldscore fitness into COX-1 was with range of 28.78-62.23 as indicated in Table 4.
In addition, the assigned compounds were identically superimposed on the co-crystallized ligand
(RCX601) in COX-2 enzyme of RMSD of 0.36-1.82Å in a closer proximity than that of COX-1
(RMSD of 3.00-3.23Å). These results distinctly demonstrated the higher selectivity of our
compounds into COX-2 over COX-1.
18

Table 4. The docking results for the top five ranks of the compounds docked into COX-1
(1EQG) [40] and COX-2 (5KIR) [41] in comparison to the native bound inhibitors involving
GOLD5.2.2
Hydrogen bonds between
compounds and COX enzymes
Gold
Score
Fitness
S
S
RMSDc
(Å)
COX Ligand
9b
(hb-ext)a (vdw-ext)b
Atom of compd.
Amino acid
61.84
61.39
0.07
0.15
55.83
53.06
d
2.14
3.00
1HN of Arg120
1HN of Arg120
2HN of Arg120
NHC=O
Furan-CH=N
NHC=O
9c
1HN of Arg120
NHC=O
Thiophen-CH=N
NHC=O
62.23
61.51
63.22
0.86
1.40
0.48
52.90
52.30
54.62
1HN of Arg120 3.23
2HN of Arg120
10a
10b
10c
1HN of Arg120
NHC=O
1HN of Arg120
3.02
Thiophen-CH=N
NHC=O
2HN of Arg120
1HN of Arg120
NHC=O
Thiophen-CH=N
NHC=O
1HN of Arg120 3.06
2HN of Arg120
51.13
85.11
84.91
93.18
9.58
1.73
3.72
1.56
71.08
56.35
65.97
71.10
p-C(CH3)C=O
1HN of Arg120 0.64
1HN of Arg120 1.82
0.76
IBP701f
9b
Furan-O
d
9c
Ph-NHC=O
HO of Tyr355
0.39
12a
Ph-NHC=O
7-CN
HO of Tyr355
HN of Phe518
88.15
88.21
1.65
1.82
2.89
68.13
69.44
51.82
0.58
12b
12c
Ph-NHC=O
7-CN
HO of Tyr355
HN of Phe518
0.36
0.81
p-S=O
p-S=O
HN of Ile517
HN of Phe518
RCX601h 72.23
a S(hb_ext) Gold score protein-ligand hydrogen bonding
b S(vdw_ext): Gold score external Vdw (the van der waals interactions between protein and ligand)
c RMSD: Root mean square deviation.
d No hydrogen bond detected.
eOvine COX-1 complexed with ibuprofen
fIbuprofen: 2-(4-isobutylphenyl)propionic acid
gHuman COX-2 complexed with vioxx
hRofecoxib (Vioxx)
19

The docked compounds into COX-1 were bound into the binding site through up to four
hydrogen bonds mainly with NH moiety of Arg120 amino acid. In addition, these compounds
interacted hydrophobically revealing Gold score (external vdw) within a range of 34.91-55.83.
In regard to COX-2, the designed compounds bound hydrophilically into the binding domain by
up to two hydrogen bonds substantially with Tyr355 (OH), Phe518 (NH), and Arg120 (NH) and
they docked deeply and robustly interacted hydrophobically by external vdw interactions within
a range of 55.83-71.22. In this regard, compound 12b exhibited remarkable Goldscore fitness of
88.15 into the binding domain COX-2 within 0.58Å from the native ligand (RCX) as shown in
Fig. 7. It bound tightly into COX-2 by two hydrogen bonds with Tyr355 and Phe518 amino
acids. Predominantly, compound 12b interacts hydrophobically (vdw-ext: 69.44) into the
binding site of COX-2 as shown in Fig. 8. Its naphthyl ring interacted by π-alkyl and π-π
hydrophobic interactions with Leu352 and Trp387, respectively. Likewise, its N-(4-tolyl) moiety
revealed π-alkyl interaction with Val349 and Ala527. And its 1H-pyrrolizine interacted by π-
cation electrostatic interaction with Arg513 and hydrophobic-alkyl interaction with Ala516. The
aforementioned results demonstrated that our designed and synthesized compounds namely 9b,
9c, and 12a-c revealed a significant selectivity towards COX-2 over COX-1. Also, the
calculated molecular docking results are correlated to a high extent to the experimental ones.
Fig. 7. a) Comparative docking mode of compound 12b (yellow ball and stick) into COX1 (PDB:
1eqg), it exhibited a Goldscore fitness of 35.92, RMSD of 2.66Å, and one hydrogen bond with
20

Arg120; b) docking mode of compound 12b into COX 2 (PDB: 5kir), it revealed a Goldscore fitness
of 88.15, RMSD of 0.58Å, and two hydrogen bonds with Tyr355 and Phe518. (--) Hydrogen bond;
(--) π-alkyl and π-π hydrophobic interaction; and (--) π-cation-electrostatic interaction.
2.3.2. Docking into ALK1 and Aurora A kinases
Targeting oncogenic such as ALK1, Aurora A, CDK2/cyclin A and DYRK3 represents
important strategy in treatment of cancer and other diseases [42-46]. The docking results into
ALK1 kinase (pdb code: 3MY0) [47] and Aurora A kinases (pdb code: 2W1C) [48] are presented
in Table 5 for compound 12b in comparison to the co-crystallized ligands.
Compounds 12b, the most active in MTT assay was selected to evaluate its inhibitory activity
against 20 kinases. The results revealed that compound 12b inhibited DYRK3A, ALK1, Aurora
A and CDK2/cyclin A kinases with inhibition percent in the range of 11-20%. Although the
inhibition of these kinases was apparently weak to moderate, but it was higher than that of
imatinib. Docking studies were performed to investigate the binding modes and binding
affinities of compound 12b into the active site of ALK1 and Aurora A kinases. AutoDock 4.2
was used in these studies. The crystal structure of ALK1 (pdb code: 3MY0) [47] and Aurora A
(pdb code: 2W1C) [48] were obtained from protein data bank (http://www.rcsb.org/pdb). The
protein structure was prepared by delating water molecules and the native ligands. Validation of
the docking studies were done by re-docking the native ligands with their corresponding kinases.
The binding modes and interactions of the native ligands with the key amino acids in the active
site were identified and compared with the reported data. The re-docked native ligands
superimposed onto the position of the native ligands in the original pdb files.
Table 5. Results of the docking of compounds 12b into ALK1 (pdb code: 3MY0) and Aurora A (pdb
code: 2W1C) incomparison to the native ligands
d
a
Atoms involved in H-bonding
Ligand atom Kinase moiety
Length
Target
Kinase
∆Gb
b
Ligand
Ki
(Å)
(kcal/mol)
N of CN
NH of HIS280
OH of SER284
OH of ASP287
2.09
2.64
2.10
-10.58
17.48nM O of CO
O of CO
12b
NAT
26.98nM
NH of HIS280
O of ARG291
2.37
2.24
LDN
-10.33
-9.2
HAU1
O of CONH NH of ALA213
1.97
2.62
181.90nM
12b
N of CN
NH of THR217
21

H34
N15
H14
N24
O of ALA213
NH of ALA213
O of GLU211
O of PRO214
d
1.99
2.06
2.20
1.92e
-8.94
281.04nM
L0C
a
e
b
c
Binding free energy; Inhibition constant; Hydrogen bonds; length in angstrom (Å),
electrostatic attraction (ionic bond).
The docking studies of compound 12b into the active sites of ALK1 and Aurora A revealed a
binding free energy (∆Gb) of -10.58, -9.2, -10.83 and -10.54 kcal/mol, respectively. The binding
modes of compound 12b and the types of interactions with the amino acid in the active site of the
three kinases were represented in Fig. 8 and Table 5. Compound 12b formed a network of
hydrogen bonds within the active sites of ALK1 with HIS280, SER284 and ASP287 amino acids
with bond length of 2.09, 2.64 and 2.10 Å, respectively.
Fig. 8. a) 3D Docking mode of compound 12b into ALK1 (pdb code: 3MY0); b) 2D Docking mode of
compound 12b into ALK1; c) 3D Docking mode of compound 12b into Aurora A (pdb code: 2WIC); d)
22

ꢂ
2D Docking mode of compound 12b into Aurora A. Hydrogen bonds were represented as (ꢂ ꢂ),
ꢂ
ꢂ
ꢂ
hydrophobic interactions including pi-sigma as (ꢂ ꢂ), alkyl as (ꢂ ꢂ) and pi-alkyl as (ꢂ ꢂ) and
unfavorable interaction as (ꢂ ꢂ ꢂ).
Moreover, five hydrophobic interactions of the pi-sigma, alkyl and pi-alkyl types were observed.
Also, one unfavorable interaction between carbonyl oxygen in compound 12b and VAL208 was
formed. On the other hand, compound 12b formed two hydrogen bonds with ALA213 and
THR217 amino acids within the Aurora active site. In addition, seven hydrophobic interactions
including pi-sigma, alkyl and pi-alkyl was observed.
3. Conclusion
In this study, a novel series of pyrrolizine-5-carboxamide has been synthesized and evaluated for
their anticancer potential against a panel of human cancer cell lines. All the newly synthesized
compounds showed IC50 values in the range of 3.24-34.75 µM against the tested cell lines.
Compound 10c was the most active against MCF-7 with IC50 value of 4.72 µM, while compound
12b was the most active against A549 and Hep3B cell lines. Additionally, the new compounds
displayed inhibition of COX-1 and COX-2 with IC50 values in the ranges of 5.78-11.96 µM and
0.1-0.78 µM, respectively, being more selective for COX-2 over COX-1 with selectivity index
(SI) in the range of 8.38-113.1. Compound 12b, the most active compound in the MTT assay,
was more than 100 time more selective for COX-2. The results of the kinase profiling revealed
the ability of compound 12b to inhibit various kinases with weak to moderate inhibition% in the
range of 7-20% which is considered an appreciable activity in comparison with imatinib
(inhibition% = 1-38%). The results of caspase activation assay revealed the ability of the new
pyrrolizines to activate caspase-3/7 in A549 cells. Compounds 12a and 12b were the most
potent in activating caspase enzymes in lung carcinoma A549 cells at 5, 10 and 20 µM. Cell
cycle analysis and Annexin V PI/FITC apoptosis assay revealed that compound 12b
accumulated MCF-7 cells in the S phase and induced apoptosis. Docking studies of some
selected compounds into the active site of COX-1/2, ALK1 and Aurora A were performed to
study the binding pattern and binding free energy compared to the native ligands of these
proteins. Totally, the activation of caspase-3/7, inhibitions of COX-2 and some kinases could
contribute in part to the overall mechanism of action of the anticancer activity of the new
compounds.
23

4. Experimental
4.1. Chemistry
All the chemical reagents and solvents used were purchased from Sigma-Aldrich. Solvents were
dried according to the literature when necessary. The purity of the new compounds was checked
with TLC using benzene-ethanol mixture (9:1). Melting points (m.p.) were determined by IA
9100MK-Digital melting point apparatus. Elemental analysis was performed at the
microanalytical center, Cairo University. Infrared spectra (IR) were done using BRUKER
TENSOR 37 spectrophotometer and expressed in wave number (cm-1) using KBr disc (faculty of
pharmacy, Umm Al-Qura University, KSA). The proton magnetic spectra were recorded on
BRUKER APX400 spectrometer at 400 (faculty of pharmacy, Beni-Suef University, Egypt)
and BRUKER AVANCE III at 500 MHz (faculty of pharmacy, Umm Al-Qura University, KSA)
in chloroform and j constant are given in Hz. The 13C-NMR spectra of the new compounds in
chloroform were done at 100 and 125 MHz. Mass spectra were recorded using GCMS on
Shimadzu Qp-2010 Plus mass spectrometer at 70 eV (EI) in the microanalytical center,
Cairo University. Elemental analyses were done in the microanalytical center, Cairo
University. Compounds 5, 7a-c and 8a-c were prepared according to the previous reports [26-
28].
4.1.1. General procedure for the preparation of compounds (9-12).
A mixture of pyrrolizine-5-carboxamides 8a-c (2 mmol) and the aldehyde (2.2 mmol) in
absolute ethanol (30 mL) in the presence of glacial acetic acid (0.5 ml) was refluxed for 4 hours.
The reaction mixture was then concentrated, set aside to cool, whereby yellow crystals were
formed, collected and recrystallized from chloroform-acetone (1:1).
4.1.1.1.
(EZ)-7-Cyano-6-((furan-2-ylmethylene)amino)-N-phenyl-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (9a)
24

Compound 8a was refluxed with furan-2-carbaldehyde to afford compound 9a as yellow crystals,
m.p. 227-30 °C, yield 75%. IRꢄmax/cm-1 3117 (NHs), 3049 (Ar C-H), 2215 (CN), 1673
(C=O). 1H-NMR (CDCl3-400 MHz) δ (ppm): 2.50 (m, 2H, pyrrolizine CH2-2), 2.93 (t, 2H, J =
7.6 Hz, pyrrolizine CH2-1), 4.49 (t, 2H, J = 7.3 Hz, pyrrolizine CH2-3), 6.62 (q, 1H, J = 3.2
Hz, furanyl CH-4ꢁ), 6.99 (d, 1H, J = 3.4 Hz, furanyl CH-3ꢁ), 7.09 (t, 1H, J = 7.4 Hz, phenyl
CH-4′), 7.35 (t, 2H, J = 7.8 Hz, phenyl CH-3′+CH-5′), 7.72 (s, 1H, furanyl CH-5ꢁ), 7.76 (d,
2H, J = 8.2 Hz, phenyl CH-2′+CH-6′), 8.88 (s, 1H, methylene amino (N=CH)), 11.13 (s, 1H,
CONH). 13C-NMR (CDCl3-100 MHz) δ (ppm): 24.38, 25.35, 50.01, 112.87, 116.32, 118.31,
118.60, 118.65, 119.35, 123.57, 128.97, 138.30, 138.81, 144.57, 146.62, 148.31, 151.55,
158.41. MS (EI): m/z (%) 346 (M++2, 2), 345 (M++1, 17), 344 (M+, 71), 343 (M+-1, 8),
327 (5), 316 (6), 299 (3), 252 (100), 224 (19), 196 (17), 184 (7), 169 (9) 156 (6), 141 (4),
106 (4), 95 (6), 80 (18). Anal. Calcd. for C20H16N4O2 (344.37): C, 69.76; H, 4.68; N, 16.27.
Found: C, 69.95; H, 4.89; N, 16.72.
4.1.1.2.
(EZ)-7-Cyano-6-((furan-2-ylmethylene)amino)-N-(4-tolyl)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (9b)
Compound 8b was refluxed with furan-2-carbaldehyde to afford compound 9b as yellow crystals,
m.p. 236-8°C, yield 78%. IRꢄmax/cm-1 3211 (NHs), 3050 (Ar C-H), 2214 (CN), 1673 (C=O),
1612 (C=C). 1H-NMR (CDCl3-400 MHz) δ (ppm): 2.34 (s, 3H, CH3), 2.52 (m, 2H, pyrrolizine
CH2-2), 2.98 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-1), 4.51 (t, 2H, J = 7.3 Hz, pyrrolizine CH2-
3), 6.62 (q, 1H, J = 2 Hz, furanyl CH-4ꢁ), 7.01 (d, 1H, J = 3.4 Hz, furanyl CH-3ꢁ), 7.16 (d,
2H, J = 8.1 Hz, phenyl CH-3′+CH-5′), 7.65 (d, 2H, J = 8.2 Hz, phenyl CH-2′+CH-6′), 7.71 (s,
1H, furanyl CH-5ꢁ), 8.91 (s, 1H, methylene amino (N=CH)), 11.06 (s, 1H, CONH). 13C-NMR
(CDCl3-100 MHz) δ (ppm): 20.91 (CH3), 24.44, 25.41, 50.02, 112.86, 116.37, 118.46, 118.52,
119.38, 119.41, 129.47, 133.14, 136.23, 138.21, 144.63, 146.55, 148.15, 151.69, 158.32. MS
(EI): m/z (%) 360 (M++2, 2), 359 (M++1, 16), 358 (M+, 63), 357 (M+-1, 7), 341 (3), 330
(3), 291 (2), 252 (100), 224 (16), 196 (14), 184 (7), 169 (7), 156 (5), 141 (4), 129 (3), 106
(6), 95 (4), 80 (33). Anal. Calcd. for C21H18N4O2 (358.39): C, 70.38; H, 5.06; N, 15.63. Found:
C, 70.78; H, 5.50; N, 15.45.
4.1.1.3. (EZ)-N-(4-Chlorophenyl)-7-cyano-6-((furan-2-ylmethylene)amino)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (9c)
Compound 8c was refluxed with furan-2-carbaldehyde to afford compound 9c as yellow crystals,
m.p. 260-2°C, yield 68%. IRꢄmax/cm-1 3272, 3223 (NHs), 2983 (Ar C-H), 2211 (CN), 1679
25

1
(C=O), 1618 (C=C). H-NMR (CDCl3-400 MHz) δ (ppm): 2.55 (m, 2H, pyrrolizine CH2-2),
3.02 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-1), 4.50 (t, 2H, J = 8.0 Hz, pyrrolizine CH2-3), 6.64
(q, 1H, J = 3.5 Hz, furanyl CH-4ꢁ), 7.03 (d, 1H, J = 3.5 Hz, furanyl CH-3ꢁ), 7.31 (d, 2H, J =
8.8 Hz, phenyl CH-3′+CH-5′), 7.72 (m, 3H, J = 8.2 Hz, phenyl CH-2′+CH-6′ + furanyl CH-
5ꢁ), 8.92 (s, 1H, methylene amino (N=CH)), 11.25 (s, 1H, CONH). 13C-NMR (CDCl3-100
MHz) δ (ppm): 24.50, 25.42, 50.03, 113.01, 116.22, 118.15, 119.11, 120.48, 120.54, 128.38,
128.97, 137.42, 138.47, 144.69, 146.72, 148.41, 151.48, 158.44. MS (EI): m/z (%) 381
(M++3, 3), 380 (M++2, 13), 379 (M++1, 10), 378 (M+, 39), 377 (M+-1, 3), 350 (2), 326 (2),
310 (3), 283 (1), 252 (100), 224 (15), 196 (13), 184 (5), 169 (7), 156 (5), 141 (4), 129 (4),
106 (3), 95 (4), 80 (25). Anal. Calcd. for C20H15ClN4O2 (378.81): C, 63.41; H, 3.99; N, 14.79.
Found: C, 63.83; H, 4.29; N, 14.43.
4.1.1.4.
(EZ)-7-Cyano-N-phenyl-6-((thiophen-2-ylmethylene)amino)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (10a)
Compound 8a was refluxed with thiophene-2-carbaldehyde to afford compound 10a as yellow
crystals, m.p. 230-2 °C, yield 73%. IRꢄmax/cm-13231 (NHs), 3086 (Ar C-H), 2212 (CN), 1667
1
(C=O). H-NMR (CDCl3-500 MHz) δ (ppm): 2.49 (m, 2H, pyrrolizine CH2-2), 2.91 (t, 2H, J =
7.5 Hz, pyrrolizine CH2-1), 4.47 (t, 2H, J = 7.1 Hz, pyrrolizine CH2-3), 7.13 (t, 1H, J = 7.3 Hz
phenyl CH-4′), 7.17 (t, 1H, J = 4.0 Hz, thiophenyl CH-4ꢁ), 7.38 (t, 2H, J = 7.6 Hz, phenyl CH-
3′+CH-5′), 7.50 (d, 1H, J = 3.0 Hz, thiophenyl CH-3ꢁ), 7.61 (d, 1H, J = 4.6 Hz, thiophenyl
CH-5ꢁ), 7.78 (d, 2H, J = 8.0 Hz, phenyl CH-2′+CH-6′), 9.23 (s, 1H, methylene amino
(N=CH)), 10.48 (s, 1H, CONH). 13C-NMR (CDCl3-125 MHz) δ (ppm): 24.40, 25.27, 50.20,
116.40, 117.76, 119.51, 119.59, 123.78, 128.61, 129.00, 131.48, 134.29, 138.42, 138.51,
141.92, 148.46, 151.80, 158.34. MS (EI): m/z (%) 363 (M++3, 1), 362 (M++2, 5), 361
(M++1, 16), 360 (M+, 62), 359 (M+-1, 7), 343 (3), 331 (4), 270 (6), 268 (100), 249 (4), 240
(13), 212 (9), 199 (3), 185 (7), 156 (6), 146 (2), 129 (3), 111 (9), 96 (6), 80 (25). Anal.
Calcd. for C20H16N4OS (360.43): C, 66.65; H, 4.47; N, 15.54. Found: C, 66.48; H, 4.57; N,
15.11.
4.1.1.5.
(EZ)-7-Cyano-6-((thiophen-2-ylmethylene)amino)-N-(4-tolyl)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (10b)
Compound 8b was refluxed with thiophene-2-carbaldehyde to afford compound 10b as yellow
crystals, m.p. 228-30 °C, yield 76%. IRꢄmax/cm-13317, 3280 (NHs), 3076 (Ar C-H), 2212
1
(CN), 1656 (C=O), 1603 (C=C). H-NMR (CDCl3-500 MHz) δ (ppm): 2.36 (s, 3H, CH3),
26

2.48 (m, 2H, pyrrolizine CH2-2), 2.90 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-1), 4.46 (t, 2H, J =
7.2 Hz, pyrrolizine CH2-3), 7.17 (t, 3H, phenyl CH-3′+CH-5′+ thiophenyl CH-4ꢁ), 7.49 (d,
1H, J = 3.3 Hz, thiophenyl CH-3ꢁ), 7.60 (d, 1H, J = 4.8 Hz, thiophenyl CH-5ꢁ), 7.66 (d, 2H, J
= 8.2 Hz, phenyl CH-2′+CH-6′), 9.22 (s, 1H, methylene amino (N=CH)), 10.41 (s, 1H,
CONH). 13C-NMR (CDCl3-125 MHz) δ (ppm): 20.94 (CH3), 24.39, 25.28, 50.18, 116.44,
117.88, 119.46, 119.55, 128.58, 129.48, 131.42, 133.29, 134.18, 135.86, 138.30, 141.98,
148.34, 151.67, 158.20. MS (EI): m/z (%) 376 (M++2, 2), 375 (M++1, 6), 374 (M+, 22), 373
(M+-1, 2), 280 (48), 268 (37), 251 (3), 238 (8), 215 (9), 200 (2), 185 (2), 174 (9), 156 (2),
147 (70), 131 (4), 119 (100), 106 (52), 91 (33), 80 (36). Anal. Calcd. for C21H18N4OS
(374.46):C, 67.36; H, 4.85; N, 14.96. Found: C, 67.38, H, 4.89; N, 15.35.
4.1.1.6. (EZ)-N-(4-Chlorophenyl)-7-cyano-6-((thiophen-2-ylmethylene)amino)-2,3-dihydro-
1H-pyrrolizine-5-carboxamide (10c)
Compound 8c was refluxed with thiophene-2-carbaldehyde to afford compound 10c as Yellow
crystals, m.p. 250-2°C, yield 74%. IRꢄmax/cm-1 3271, 3232 (NHs), 3003 (Ar C-H), 2204
1
(CN), 1666 (C=O). H-NMR (CDCl3-500 MHz) δ (ppm): 2.59 (m, 2H, pyrrolizine CH2-2),
3.08 (t, 2H, J = 7.5 Hz, pyrrolizine CH2-1), 4.55 (t, 2H, J = 7.1 Hz, pyrrolizine CH2-3), 7.24
(broad s, 1H, thiophenyl CH-4ꢁ), 7.35 (d, 2H, J = 8.4 Hz, phenyl CH-3′+CH-5′), 7.65 (m, 2H,
thiophenyl CH-3ꢁ+CH-5ꢁ), 7.77 (d, 2H, J = 8.4 Hz, phenyl CH-2′+CH-6′), 9.34 (s, 1H,
methylene amino (N=CH)), 10.59 (s, 1H, CONH). 13C-NMR (CDCl3-125 MHz) δ (ppm):
24.57, 25.42, 50.20, 116.20, 117.64, 120.94, 128.72, 128.76, 129.02, 131.47, 134.47, 137.02,
138.93, 141.97, 148.39, 152.24, 156.95, 158.47. MS (EI): m/z (%) 398 (M++4, 2), 397
(M++3, 6), 396 (M++2, 20), 395 (M++1, 17), 394 (M+, 50), 393 (M+-1, 10), 377 (1), 311
(1), 300 (2), 283 (2), 268 (100), 240 (11), 212 (6), 199 (2), 184 (6), 174 (6), 156 (5), 146
(3), 129 (4), 127 (5), 111 (6), 96 (3), 80 (36). Anal. Calcd. for C20H15ClN4OS (394.88): C,
60.83; H, 3.83; N, 14.19. Found: C, 60.46; H, 4.24; N, 14.16.
4.1.1.7.
(EZ)-7-Cyano-N-phenyl-6-((pyridin-3-ylmethylene)amino)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (11a)
Compound 8a was refluxed with pyridine-3-carbaldehyde to afford compound 11a as yellow
crystalline product, m.p. 233-4°C, yield 83%. IRꢄmax/cm-1 3272, 3236 (NHs), 3012, 2922 (Ar
1
C-H), 2215 (CN), 1667 (CO). H-NMR (CDCl3-400 MHz) δ (ppm): 2.58 (m, 2H, pyrrolizine
CH2-2), 3.07 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-1), 4.56 (t, 2H, J = 7.3 Hz, pyrrolizine CH2-
3), 7.13 (t, 1H, J = 7.4 Hz, pyridinyl CH-5ꢁ), 7.37 (t, 2H, J = 7.8 Hz, phenyl CH-3′+CH-5′),
27

7.49 (dd, 1H, J = 7.7, 4.8 Hz, phenyl CH-4′), 7.64 (d, 2H, J = 7.8 Hz, phenyl CH-2′+CH-6′),
8.22 (d, 1H, J = 7.8 Hz, pyridinyl CH-4ꢁ), 8.78 (d, 1H, J = 7.4 Hz, pyridinyl CH-6ꢁ), 9.17 (s,
1H, pyridinyl CH-2ꢁ), 9.23 (s, 1H, methylene amino (N=CH)), 10.46 (s, 1H, CONH). 13C-
NMR (CDCl3-100 MHz) δ (ppm): 24.56, 25.46, 50.27, 116.11, 118.46, 119.70, 122.12,
124.05, 124.22, 129.24, 131.13, 135.14, 138.03, 138.46, 148.49, 150.17, 152.74, 156.84,
158.23. MS (EI): m/z (%) 356 (M++1, 2), 355 (M+, 7), 277 (12), 263 (16), 236 (4), 207 (2),
194 (1), 180 (2), 153 (1), 127 (1), 117 (2), 106 (2), 91 (3), 80 (100). Anal. Calcd. for
C21H17N5O (355.39): C, 70.97; H, 4.82; N, 19.71. Found: C, 71.17; H, 5.29; N, 20.16.
4.1.1.8.
(EZ)-7-Cyano-6-((pyridin-3-ylmethylene)amino)-N-(4-tolyl)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (11b)
Compound 8b was refluxed with pyridine-3-carbaldehyde to afford compound 11b as yellow
crystalline product, m.p. 242-5°C, yield 87%. IRꢄmax/cm-13279, 3178 (NHs), 3031, 2992 (Ar
1
C-H), 2212 (CN), 1658 (C=O), 1597, 1568 (C=C). H-NMR (CDCl3-500 MHz) δ (ppm):
2.35 (s, 3H, CH3), 2.58 (m, 2H, pyrrolizine CH2-2), 3.06 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-
1), 4.56 (t, 2H, J = 7.3 Hz, pyrrolizine CH2-3), 7.19 (d, 2H, J = 8.1 Hz, phenyl CH-3′+CH-5′),
7.53 (t, 3H, phenyl CH-2′+CH-6′+pyridinyl CH-5ꢁ), 8.24 (d, 1H, J = 7.9, pyridinyl CH-4ꢁ),
8.79 (d, 1H, J = 4.4, pyridinyl CH-6ꢁ), 9.15 (s, 1H, pyridinyl CH-2ꢁ), 9.23 (s, 1H, methylene
amino (N=CH)), 10.38 (s, 1H, CONH). 13C-NMR (CDCl3-125 MHz) δ (ppm): 20.93 (CH3),
24.53, 25.45, 50.27, 116.17, 118.63, 119.54, 119.64, 124.14, 129.74, 131.28, 133.86, 135.32,
135.44, 138.19, 148.46, 149.88, 152.32, 156.40, 158.06. MS (EI): m/z (%) 371 (M++2, 2),
370 (M++1, 12), 369 (M+, 43), 368 (M+-1, 4), 291 (62), 263 (100), 236 (23), 207 (10), 194
(2), 180 (7), 153 (3), 127 (2), 117 (5), 106 (14), 91 (9), 80 (35). Anal. Calcd. for C22H19N5O
(369.42):C, 71.53; H, 5.18; N, 18.96. Found: C, 71.80; H, 4.83; N, 18.55.
4.1.1.9. (EZ)-N-(4-Chlorophenyl)-7-cyano-6-((pyridin-3-ylmethylene)amino)-2,3-dihydro-
1H-pyrrolizine-5-carboxamide (11c)
Compound 8c was refluxed with pyridine-3-carbaldehyde to afford compound 11c as yellow
crystalline product, m.p. 278-80 °C, yield 76%. IRꢄmax/cm-1 3230, 3174 (NHs), 3053 (Ar C-
1
H), 2213 (CN), 1660 (C=O). H-NMR (CDCl3-400 MHz) δ (ppm): 2.59 (m, 2H, pyrrolizine
CH2-2), 3.08 (t, 2H, J = 7.6 Hz, pyrrolizine CH2-1), 4.55 (t, 2H, J = 7.2 Hz, pyrrolizine CH2-
3), 7.33 (d, 2H, J = 8.7 Hz, phenyl CH-3′+CH-5′), 7.49 (dd, 1H, J = 7.9, 4.9 Hz, pyridinyl CH-
5ꢁ), 7.59 (d, 2H, J = 8.7 Hz, phenyl CH-2′+CH-6′), 8.18 (d, 1H, J = 8.1 Hz, pyridinyl CH-4ꢁ),
8.79 (d, 1H, J = 4.3 Hz, pyridinyl CH-6ꢁ), 9.16 (s, 1H, pyridinyl CH-2ꢁ), 9.23 (s, 1H,
28

methylene amino (N=CH)), 10.49 (s, 1H, CONH). 13C-NMR (CDCl3-100 MHz) δ (ppm):
24.58, 25.45, 50.27, 115.98, 118.21, 120.82, 124.05, 129.08, 129.24, 131.05, 135.20, 136.64,
138.61, 148.06, 148.65, 150.05, 152.83, 157.05, 158.06. MS (EI): m/z (%) 392 (M++3, 2),
391 (M++2, 10), 390 (M++1, 7), 389 (M+, 28), 388 (M+-1, 2), 311 (33), 263 (100), 236
(18), 207 (9), 194 (6), 180 (9), 153 (4), 127 (4), 117 (5), 106 (8), 91 (6), 80 (21). Anal.
Calcd. for C21H16ClN5O (389.84): C, 64.70; H, 4.14; N, 17.96. Found: C, 65.10; H, 4.18; N,
18.08.
4.1.1.10.
(EZ)-7-Cyano-6-((naphthalen-2-ylmethylene)amino)-N-phenyl-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (12a)
Compound 8a was refluxed with 2-naphthaldehyde to afford compound 12a as yellow crystals,
m.p. 245-8°C, yield 79%. IRꢄmax/cm-1 3281, 3181 (NHs), 3063 (Ar C-H), 2207 (CN), 1670
(C=O). 1H-NMR (CDCl3-400 MHz) δ (ppm): 2.57 (m, 2H, pyrrolizine CH2-2), 3.06 (t, 2H, J =
7.6 Hz, pyrrolizine CH2-1), 4.56 (t, 2H, J = 7.2 Hz, pyrrolizine CH2-3), 7.09 (t, 1H, J = 7.4 Hz,
aromatic proton), 7.31 (t, 2H, J = 7.8 Hz, phenyl CH-3′+CH5′), 7.62 (m, 5H, aromatic
protons), 7.95 (d, 1H, J = 8.1 Hz, naphthalenyl proton), 8.04 (d, 1H, J = 8.2 Hz, naphthalenyl
proton), 8.24 (d, 1H, J = 7.2 Hz, naphthalenyl proton), 8.67 (d, 1H, J = 8.4 Hz, naphthalenyl
proton), 9.94 (s, H, methylene amino (N=CH)), 10.71 (s, H, CONH). 13C-NMR (CDCl3-100
MHz) δ (ppm): 24.59, 25.46, 50.18, 116.52, 117.98, 119.69, 119.78, 123.17, 123.86, 125.31,
126.56, 127.95, 128.17, 129.01, 129.06, 130.99, 131.72, 133.03, 133.99, 138.34, 140.11,
148.26, 158.35, 158.50. MS (EI): m/z (%) 407 (M++3, 1), 406 (M++2, 5), 405 (M++1, 31),
404 (M+, 100), 403 (M+-1, 5), 375 (3), 312 (99), 283 (43), 277 (38), 256 (19), 243 (6), 229
(12), 202 (14), 184 (13), 174 (14), 159 (14), 146 (4), 139 (16), 127 (38), 92 (12), 80 (53).
Anal. Calcd. for C26H20N4O (404.46): C, 77.21; H, 4.98; N, 13.85. Found: C, 76.83; H, 5.21; N,
13.80.
4.1.1.11. (EZ)-7-Cyano-6-((naphthalen-2-ylmethylene)amino)-N-(4-tolyl)-2,3-dihydro-1H-
pyrrolizine-5-carboxamide (12b)
Compound 8b was refluxed with 2-naphthaldehyde to afford compound 12b as yellow crystals,
m.p. 262-5°C, yield 83%. IRꢄmax/cm-1 3277, 3179 (NHs), 3059 (Ar C-H), 2211 (CN), 1660
1
(C=O), 1605, 1546 (C=C). H-NMR (CDCl3-500 MHz) δ (ppm): 2.35 (s, 3H, CH3), 2.61 (m,
2H, pyrrolizine CH2-2), 3.12 (t, 2H, J = 7.3 Hz, pyrrolizine CH2-1), 4.60 (t, 2H, J = 7.0 Hz,
pyrrolizine CH2-3), 7.15 (d, 2H, J = 7.7 Hz, phenyl CH-3′+CH-5′), 7.52 (d, 2H, J = 7.4 Hz,
phenyl CH-2′+CH-6′), 7.65 (m, 3H, naphthalenyl CH-1ꢁ+CH-7ꢁ+CH-8ꢁ), 7.99 (d, 1H, J = 8.0
Hz, naphthalenyl CH-9ꢁ), 8.08 (d, 1H, J = 8.1 Hz, naphthalenyl CH-6ꢁ), 8.27 (d, 1H, J = 7.2
29