Antitumor activity of pyrrolizines and their Cu(II) complexes: Design, synthesis and cytotoxic screening with potential apoptosis-inducing activity

Article abstract of DOI:10.1016/j.ejmech.2018.01.009

Two novel series including Schiff bases of the pyrrolizine-5-carboxamides and their Cu(II) complexes were designed, synthesized and analysed using spectral and analytical techniques. The analytical results indicated the formation of the complexes in 1:1 o

Full text of DOI:10.1016/j.ejmech.2018.01.009

Accepted Manuscript
Antitumor activity of pyrrolizines and their Cu(II) complexes: Design, synthesis and
cytotoxic screening with potential apoptosis-inducing activity
Ahmed M. Gouda, Hoda A. El-Ghamry, Tahani M. Bawazeer, Thoraya A. Farghaly,
Ashraf N. Abdalla, Akhmed Aslam
PII:
S0223-5234(18)30009-6
10.1016/j.ejmech.2018.01.009
EJMECH 10086
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2017
Revised Date: 1 January 2018
Accepted Date: 4 January 2018
Please cite this article as: A.M. Gouda, H.A. El-Ghamry, T.M. Bawazeer, T.A. Farghaly, A.N. Abdalla,
A. Aslam, Antitumor activity of pyrrolizines and their Cu(II) complexes: Design, synthesis and cytotoxic
screening with potential apoptosis-inducing activity, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.01.009.
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Graphical abstract
Antitumor Activity of Pyrrolizines and their Cu(II) Complexes:
Design, Synthesis and Cytotoxic Screening with potential Apoptosis-
Inducing Activity
Ahmed M. Gouda, Hoda A. El-Ghamry, Tahani M. Bawazeer, Thoraya A. Farghaly*,
Ashraf N. Abdalla, Akhmed Aslam

ACCEPTED MANUSCRIPT
Antitumor Activity of Pyrrolizines and their Cu(II) Complexes: Design,
Synthesis and Cytotoxic Screening with potential Apoptosis-Inducing Activity
1
,2
3,4
4,5
Ahmed M. Gouda , Hoda A. El-Ghamry , Tahani M. Bawazeer , Thoraya A.
6
7,8
9
Farghaly *, Ashraf N. Abdalla , Akhmed Aslam
1
2
Medicinal chemistry department, Faculty of pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Umm Al-Qura University, Makkah
3
2
1955, Saudi Arabia; Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt;
4
ChemistryDepartment, Faculty of Applied Science, Umm Al–Qura University, Makkah, Saudi Arabia;
5
Medical Applications of Nanobiotechnology Research Group, King Fahad Medical Research Center,
6
King Abdulaziz University, Jeddah, Saudi Arabia; Department of Chemistry Faculty of Science, Cairo
7
University, Giza, Egypt; Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-
8
,
Qura University, Makkah 21955, Saudi Arabia; Departmentof Pharmacology Medicinal and Aromatic
9
plants research institute, National center for research, Khartoum 2404, Sudan; Department of Laboratory
Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah 21955, Saudi Arabia.
*
Corresponding author email address: thoraya-f@hotmail.com
_
_________________________________________________________________________
Abstract:
Two novel series including Schiff bases of the pyrrolizine-5-carboxamides and their Cu(II)
complexes were designed, synthesized and analyzed using spectral and analytical techniques.
The analytical results indicated the formation of the complexes in 1:1 or 1:2 (Metal:Ligand)
ratio. The geometry around the Cu centers was confirmed to be tetrahedral or octahedral. The
cytotoxic activity of the new compounds was evaluated using MCF-7 (human breast
adenocarcinoma), A2780 (human ovary adenocarcinoma) and HT29 (human colon
adenocarcinoma), in addition to MRC5 (normal human fetal lung fibroblast)cells using the MTT
cytotoxicity assay. The Schiff base 12c and the Cu complex 13b were the most active in the two
series with IC values in the range of 0.14-2.54 µM against the three cell lines. Also, the Cu
5
0
complex 13e showed excellent activity against HT29 with IC50 = 0.05µM. 7-Cyano-N-(4-
methoxyphenyl)-6-((3-phenylallylidene) amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide
12c) showed high selectivity (6-13 folds) for cancerous cells over normal cells; and it induced
(
marginal increases in the G1 and S phases of MCF-7 cells during cell cycle analysis, while
1

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compound 13b increased the MCF-7 Sub-G1 proapoptotic population, and blocked cells in the
G2-M phase in a dose dependent manner. The annexin V apoptosis assay revealed the ability of
compounds 12c and 13b to increase the early apoptotic MCF-7 cell populations two and three
fold, respectively. Furthermore, these findings were supported by data showing that the two
compounds (12c and 13b) elicit cytotoxic activity. Taken together, the data presented in this
study warrants further in vitro and in vivo investigations.
Keywords: Pyrrolizine; Cinnamaldehyde; Schiff bases; Copper complexes; Cytotoxicity; Cell
cycle; Apoptosis
_
1
_____________________________________________________________________________
. Introduction
Cancer is amongst the terrifying diseases occurring worldwide, being the second cause of
illness caused death, and accounting for one-sixth of the global deaths in 2015 [1]. Currently, the
evolution of resistance to some of the clinically available anticancer drugs has been reported
[2,3]. These factors encourage research in this fast-moving area to discover and develop new
potent and selective anticancer agents. Induction of apoptosis has a crucial role in development
and treatment of cancer [4]. The complicated signaling pathway of apoptosis involves many
regulatory and inhibitory proteins and pathways. Large numbers of anticancer agents target these
proteins and exert their anticancer action through induction of apoptosis in cancer cells [5].
Of these potential anticancer agents, several pyrrolizines bearing diarylfragments (Fig. 1)
have been developed, that have displayed diverse biological activities [6-8]. Licofelone 1 displayed
potent anticancer activity against breast, colon and prostate cancers [9-11]. Mechanistic studies
revealed the ability of licofelone 1 to activate caspase 3/9 and trigger apoptosis through a
mitochondrial pathway [10]. Compound 2 (Fig. 1) was also reported to induce a potent anticancer
activity against MCF-7 and PC-3 cancer cell lines [12]. Replacement of the 4-nitrophenyl moiety
in compound 2 with the bulky naphthalenyl generated compound 3, which displayed potent
anticancer activity and exhibited selective inhibition of cyclooxygenase 2 (COX-2), activation of
caspase3/7 and induction apoptosis [13].
2

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Fig. 1. Representative pyrrolizineswith anticancer activity against diverse cancer cell lines.
On the other hand, several small molecules of the cinnamaldehyde scaffold have been
reported as apoptosis inducing anticancer agents (Fig. 2). Cinnamaldehyde compound 4 inhibited
the proliferation of several types of cancer cells and induced apoptosis through the activation of
reactive oxygen species (ROS) in HL-60 leukemia cells [14]. Moreover, compound 5 (CB403)
inhibited the growth of ten types of cancer cell lines with GI50 in the range of 1.61 to 10.77
µg/mL. This compound also initiated the cell cycle arrest of MCF-7 cells in the G2-M phase
[15]. In addition, compound 6 was also to arrest the cell cycle of the cis-platin-resistant ovarian
A2780cis cells in the G2-M phase and to induce apoptosis [16].
Fig. 2. Cinnamaldehyde-based anticancer compoundswith apoptosis inducing/cell cycle blocking activity.
In this study, new pyrrolizines (Fig. 3) were designed using the following design strategy,
(1) Cinnamaldehyde was selected for the preparation of the new Schiff bases based on previous
reports showing its apoptosis inducing anticancer activity [14-16]. (2) Scaffold A was designed as
a modification of compound 2, with elongation of the spacer between phenyl ring and the
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pyrrolizine nucleus. (3) Several Cu(II) complexes with Schiff bases have displayed potent
antiproliferative activities mediated by the induction of apoptosis [17-19]. Accordingly, it was of
interest to design scaffold B as Cu(II) complexes with the designed Schiff bases. (4) Various
3 3
substituents including electron donating (R = CH /OCH ), electron withdrawing (R = Cl/Br) and
the unsubstituted phenyl ring (R = H) were used to study their impact on activity.
Fig. 3. Design strategies of scaffold A and B
2
. Results and discussion
2
.1. Chemistry
Preparation of compounds 8 [20], 10a-e [21] and 11a-e [22] was outlined in Scheme 1
according to the reported methods. Condensation of compounds 11a-e with cinnamaldehyde in
absolute ethanol afforded the five Schiff bases 12a-e, Scheme 1.
4

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NC
NC
NC
O
N
NH2
N
c
a
d
O
N
O
NH
NH
N
(
75%)
(63-73%)
(64-75%)
HN
HN
7
8
11
12
R
R
Cl
9
9
a, 10a, 11a, 12a: R = H;
b, 10b, 11b, 12b: R = CH3;
O
H N
2
HN
b
9c, 10c, 11c, 12c: R = OCH3;
9d, 10d, 11d, 12d: R = Cl;
9e, 10e, 11e, 12e: R = Br
(
78-84%)
R
R
9
10
3 2 4 2 2 2
Scheme 1. Reagents and conditions: (a) (CH ) SO , benzene, CH (CN) ; (b) ClCH COCl, g.
acetic acid, CH COONa; and (c) acetone, K CO , reflux, 24 h; (d) trans-cinnamaldehyde,
2
2
3
absolute ethanol, glacial acetic acid, reflux, 6 h.
Characterization of compounds 12a-e was done using spectral and elemental analyses.
1
The H-NMR of all derivatives 12a-e revealed doublet signals at 8.85-8.91 ppm, indicating the
N=CH, and another singlet signal at the range of 10.61-10.88 ppm, which disappeared on
1
3
deuteration and indicated the presence of amide NHs. The C-NMR revealed two signals at the
range of 157.92-162.14 ppm, indicating the presence of N=CH and the carbonyl carbon. Mass
spectra of compounds 12a-e revealed the molecular ion peaks at m/z = 380, 394, 410, 414, and
4
58, respectively, with 72-100% intensity. The fragmentation patterns of compounds 12a-e
were matched with their chemical structures.
2
.2. Synthesis and investigation of Cu(II) complexes
Preparation of Cu(II) chelates was performed according to Scheme 2. The ligands
(
12a-e) were reacted with CuCl
2
.2H
2
O in a refluxing acetonitrile medium in the molar ratio
1
:1 (metal: ligand) to obtain the desired Cu(II) complexes (13a-e), which are readily
soluble in DMSO and DMF solvents. All the complexes are non-hygroscopic and stable in
air for a long period of time. The elemental analyses data for Cu (II) complexes are in
5

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agreement with the proposed molecular formulae and all metal complexes are deep brown
in color.
Scheme 2.Reagents and conditions: (e) CuCl .2H O, acetonitrile
2
2
2
.2.1 Elemental analysis and molar conductance
The elemental analysis results of the metal chelates are consistent with the postulated
molecular formulae and confirm the formation of 1:1 (M:L) complexes for compounds 13b, 13d
and 13e, and 1:2 (M:L) complexes for compounds 13a and 13c. The measured molar
-1
2
-1
-3
conductance values (12.20–16.8 Ω cm mol ) of all chelates in 10 M DMF solvent assured
their non-electrolytic type [23].
2
.2.2. FT-IR spectra
In order to detect the coordination sites in the ligand that contribute in bond formation
with the central metal ion, the FTIR spectra of the metal chelates are compared with that of the
free ligand. There are some peaks in the ligand spectrum that guide us to understand this issue.
These peaks change either in their positions and/or their intensities upon chelation. Some other
peaks disappear after complexation.
In the ligands spectra, the carbonyl and azomethine stretching vibration bands appeared
-1
within the 1673-1616 & 1610-1578 cm ranges, respectively. In the spectra of compounds 13a
6

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&13c, the carbonyl bands have disappeared revealing the involvement of this band in
mesomerism (i.e. keto-amine/enol-imine structures); the mesomeric form containing –N=C-OH
type of arrangement, most probably involved in the bond formation coordinating to the metal
centers through deprotonated hydroxyl groups [24, 25]. This type of mesomerism was confirmed
-1
by the appearance of two C=N peaks at 1694 & 1579 cm for compound 13a and at 1689 &
607 for compound 13c, one of these two C=N bands was involved in coordination to the metal
1
center through a nitrogen atom. Analytical results strongly supported the formation of such
mesomeric structures. For compounds 13b, 13d & 13e, the position of the two bands (i.e. C=O
and C=N) underwent a shift in their position to higher or lower wavenumbers, proving their
coordination to the metal centers. These observations are confirmed by the outcrop of non-ligand
-1
bands at 594–520 and 494-476 cm , which were assigned to the stretching vibrations of Cu-O
and Cu-N [26].
−
1
The ligand band, which was found within the 3278-3226 cm range, was assigned to
υ(NH). The shift in position of this band, in the spectra of metal chelates, is most probably due to
the involvement of this group in H-bond formation with other electronegative atoms in the ligand
-1
[
26]. The appearance of a broad band at 3449 and 3447 cm in the spectra of hydrated
complexes (13a and 13e) are assigned to the stretching vibrations of OH of water molecules
attached to the copper center.
2
.2.3. Thermogravimetric analysis
Thermogravimetric analysis (TGA) of the metal chelates is a helpful tool to understand
the coordination structure of the complexes [27, 28], through affording exemplary knowledge on
their thermal properties, intermediate nature, and final products of their thermal decomposition
stages [29]. It is essential to realize the nature and contents of water and/or other solvent
molecules and also the anion groups associated with the central atoms. Subsequently, all the
synthesized metal chelates were subjected to TGA. The possible pyrolysis reaction, nature of
intermediates, temperature ranges, and final decomposition products, as well as the experimental
and calculated percentage mass losses in each decomposition step are collected in Table S1,
while the TG thermograms are illustrated in Fig. S21-25.
The metal chelates under study were found to degrade through two (compound 13d),
three (compounds 13b & 13c) or four (compounds 13a & 13e) stages of decomposition.
7

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The thermal decomposition of compounds 13a and 13e, as representative examples, can
be illustrated by the following schemes. For compound 13a, the decomposition stages are:
o
2
3-87 C
1
2a
12a
[
Cu(L ) (H O) ].CH CN
[Cu(L ) (H O) ]
2 2 2
2
2
2
3
-3.81 (calcd. 4.55 %)
o
8
7-125 C
1
2a
12a
[
Cu(L ) (H O) ]
[Cu(L ) ]
2
2
2
2
-
4.73 (calcd. 4.00 %)
o
1
25-326 C
1
2a
[
Cu(L ) ]
Intermediate 1
2
-
28.97 (calcd. 28.68 %)
o
3
26-783 C
Intermediate 1
CuO
-54.39 (calcd. 54.32 %)
For compound 13e, the decomposition stages are:
o
2
5-95 C
CuL12e Cl (CH CN) ].H O
[CuL12e Cl (CH CN) ]
[
2
3
2
2
2
3
2
-1.73 (calcd. 2.59 %)
o
9
5-328 C
[CuL12e Cl]
[
CuL12e Cl (CH CN) ]
2 3 2
-
17.12 (calcd. 16.94 %)
o
3
28-452 C
Intermediate 1
[
CuL12e Cl]
-
20.72 (calcd. 20.37 %)
o
4
25-749 C
Intermediate 1
CuO
-48.91 (calcd. 48.62 %)
As shown previously and as indicated in Table S1, the results of thermal analysis for the
complexes 13a-e strongly confirmed their proposed molecular composition.
2
.2.4. Magnetic moment, UV-visible and ESR spectroscopy
The information about geometrical structures of the metal chelates is gained from their
electronic spectra and magnetic moment data.
The magnetic moment values of Cu(II) complexes 13a-e were found to be 1.92, 1.86, 1.92, 2.01
and 2.13 B.M. for complexes 13a, 13b, 13c, 13d and 13e, respectively, which are close to the
spin only value of one unpaired electron expected for Cu(II) compounds (theoretical value is
8

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1
.72 B.M). These values indicated that the Cu(II) complexes are monomeric in nature without
Cu–Cu interaction [30, 31].
UV-Vis spectra of the metal complexes were measured in DMF solution from 200 to 900
nm. Cu(II) complexes 13a and 13e displayed broad absorption bands centered at 16949 and
-1
2
2
1
6619 cm , respectively, assignable to E → T transition reported for octahedral configuration
g 2g
[32, 33]. The spectra of complexes 13b, 13c and 13ddisplayed broad spectral bands at 550, 578
-1
2
2
2 2
and 548 cm , respectively, that assigned to T → E transition, which is characteristic for
tetrahedral Cu(II) complexes [34]. The possibility of square planar geometry at the metal center
is ruled out hence, in general, square planar complexes are known to exhibit two bands in the
visible region, around 700–712 and 533–550 nm as previously reported [34].
Solid state ESR spectra of complexes 13a-e (Figs. S26-30), recorded at room
⊥
||
temperature, displayed axially symmetric g-tensor parameters with g > g > 2.0023 (g = 2.46,
||
=
2
.28, 2.179, 2.187 & 2.45; g⊥ꢀ 2.11, 2.028, 2.034, 2.038 & 2.107 for complexes 13a, 13b, 13c,
1
3d and 13e, respectively) representing that the unpaired electron of Cu(II) ion is located in the
dx2−y2orbital. Such behavior is characteristic for tetrahedrally distorted [35] or octahedral
stereochemistry [36]. The spin-Hamiltonian parameters of this complex were also calculated. In
⊥
axial symmetry the g-values are related by the expression, G = (g −2)/(g −2), where G is the
||
exchange interaction parameter. According to Hathaway, if the value of G is greater than 4, the
exchange interaction between Cu(II) centers in the solid state is negligible, whereas when it is
less than 4, a considerable exchange interaction is indicated in the solid complex [37]. G values
were calculated to be within the range 4.18-10, supporting the fact that there is no copper–copper
exchange interaction between the metal centers in the investigated compounds, which also
supports the increased values of the magnetic moment of the metal chelates from the spin only
value of the soul unpaired electron of Cu(II) centers. No hyperfine splitting was observed in the
spectra.
2
.3. Pharmacology
2.3.1. Cytotoxic activity
The target compounds (12a-e and 13a-e) were evaluating for their cytotoxic activities
against human breast MCF-7, ovarian A2780 and colon HT29 cancer cell lines using the MTT
9

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assay [38].The cytotoxicity of compounds 12a-e and 13a-e against breast (MCF-7), ovarian
(A2780) and colon(HT29) cancer cell lineswere expressed as half maximal inhibitory
concentration (IC ) values and presented in Table 1. Moreover, cytotoxicity of the most active
5
0
compounds (12c and 13b) was also evaluated against a normal human lung fibroblast cell line
MRC5).
(
In the first series 12a-e, the MTT assay results revealed that compound 12c exhibited the highest
activity against the breast, ovarian and colon cancer cell lines, with IC50 values in the range of
0
.14-0.30 µM. Compound 12c was 6-13 fold more selective for the three cancer cell lines
compared to MRC5 normal cells. The second and third most active compounds were 12a and
2d respectively. Both compounds 12a and 12c showed submicromolar activity against the three
cancer cells, while compound 12b showed an IC value ≤ 2 µM against MCF-7 and HT29 cells.
1
5
0
1
0

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Table 1. Cytotoxic activity (MTT assay) of of compounds 12a-e and 13a-e against the three
cancer cell lines (MCF7, A2780, HT29) and MRC5 normal fibroblasts. The cells were incubated
for 72h, followed by 3h with MTT. The IC ±SD (µM) is shown for every compound tested
5
0
against each cell line.
IC (µM)
5
0
Compound
MCF-7
A2780
HT29
MRC5
1
1
1
1
1
1
1
1
1
1
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
0.72±0.15
1.94±0.06
0.14±0.04
1.46±0.24
0.50±0.29
1.27±0.19
0.69±0.11
3.88±1.14
20.84±6.41
22.12±4.70
6.80± 1.20
0.04±0.02
24.66±1.00
0.14±0.01
2.97±0.32
3.50±0.13
3.62±2.28
0.81±0.38
0.82±0.10
0.16±0.01
4.17±1.25
10.40± 0.80
0.60±0.04
0.13±0.01
0.30±0.03
3.71±0.81
7.27±1.18
5.31±0.13
2.54±0.89
0.68±0.02
1.20±0.01
0.05±0.06
12.67±1.33
nt
nt
1.88±0.08
nt
nt
nt
0.23±0.08
nt
nt
nt
Lapatinib
12.89±3.24
Cells were treated with the new compounds or vehicle for 72 h; the results were presented as
mean ± SD, n = 3; nt, not tested.
The MTT assayresults of compounds 13a-e revealed IC50 values in the range of 0.05-
2
2.12 µM against the three cancer cell lines, with compound 13b showing the highest activity
(
IC values in the range of 0.69-2.54 µM). Compounds 13d and 13e showed potent cytotoxic
5
0
activity against the A2780 and HT29 cell lines, with IC50 values≤ 5 µM. Interestingly, compound
1
1
3b showed less selectivity for MRC5 compared to compound 12c. Both compounds 12c and
3b showed better cytotoxicity compared to lapatinib, Table 1.
2
.3.2. Determination of cell cycle perturbations
Cell cycle analysis was carried out using flow cytometry by staining of DNA within cells
using propidium iodide (PI). The cell cycle distribution assay of MCF-7 cells treated with
compound 12c (24 h) was doneaccording to the reported protocols [39]. The results revealed
1
1

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marginal increases in the G1 and S phases, Table 2 and Fig. 3. In contrast, compound 13b
increased the MCF-7 Sub-G1 pro-apoptotic population and blocked cells in the G2-M phase in a
dose dependent manner, which was at the expense of a decrease in the G1 phase (Table 3, Fig.4).
Table 2: Cell cycle analysis of compound 12c. The distribution of the different phases was
analysed using the cell cycle algorithm of the Novo Express software and expressed as
percentage of total gated single events.
Concentration
Stage
Control
5 µM
10 µM
0.46%
20 µM
0.46%
Sub-G1
G1
0.31%
0.47%
33.41%
24.95%
35.02%
36.41%
27.26%
32.94%
39.47%
27.20%
30.81%
37.09%
29.27%
28.62%
S
G2-M
1
2

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Fig. 4. Flow cytometry histograms showing the effect of compound 12c on cell cycle distribution after 24
h treatment in MCF-7 cells. X-axis: DNA content of 20,000 events, y axis: cell number. A; 0µM; B:
5
µM; C: 10µM; D: 20µM. The distribution of the different phases was analysed using the cell cycle
algorithm of the NovoExpress software and the results expressed in Table 2.
1
3

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Table 3: Cell cycle analysis of compound 13b.The distribution of the different phases was
analysed using the cell cycle algorithm of the Novo Express software and expressed as
percentage of total gated single events.
Concentration
Stage
Control
5 µM
10 µM
20 µM
0.91%
Sub-G1
G1
0.31%
0.40%
0.41%
33.41%
24.95%
35.02%
33.16%
30.00%
31.39%
34.52%
26.36%
34.60%
20.51%
31.20%
44.87%
S
G2-M
Fig. 5. Flow cytometry histograms showing the effect of compound 13b on cell cycle distribution after 24
h treatment in MCF-7 cells. X-axis: DNA content of 20,000 events, y axis: cell number. A; 0µM; B:
5
µM; C: 10µM; D: 20µM. The distribution of the different phases was analysed using the cell cycle
algorithm of the Novo Express software and the results expressed in Table 2.
2.3.3. Annexin V FITC/PI apoptosis assay
1
4

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The target compounds were designed based on merging the chemical structure of the
apoptosis inducers 1-4.Accordingly, compounds 12c and 13b were observed to have the highest
activity against the three cancer cell lines (MCF-7, A2780, HT29), and were therefore selected,
representing the two newly synthesized series, for further investigations to identify their
mechanisms of action. The Annexin V Fluorescein Isothiocyanate/Propidium Iodide (FITC/PI)
FITC/PI assay was used to evaluate their ability to induce apoptosis. For this,MCF-7 cells were
treated with compound 12c and 13b for 24 h. The cells were then double stained with both
Annexin V-FITC and PI, followed by analysis using flow cytometry (Beckman Coulter FC500
flow cytometer) to detect apoptosis using a method as previously described [40].Each of the
compounds 12c and 13b increased the early apoptotic MCF-7 cell populations in a dose
dependent manner (two and three fold, respectively, compared to control) (Fig. 6 and 7). Data
analysis to determine the different phases of apoptosis was carried out using the Expo 32
software.
80
60
40
20
A
live cells
early apoptotic cells
late apoptotic cells
necrotic cells
0
control
5uM
10uM
20uM
Fig. 6. Apoptotic responses of MCF7 cells to 24h treatment with compound 12c, using annexin
V-FITC/PI dual staining. Live cells: PI-/annexin V- ; Early apoptotic cells: PI-/ annexin V+; Late
apoptotic cells: PI+/ annexin V+; Necrotic cells: PI+/ annexin V-. Data shown is mean % cell
number ± SD (n=2)
.
1
5

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100
B
live cells
early apoptotic cells
late apoptotic cells
necrotic cells
80
60
40
20
0
control
5uM
10uM
20uM
Fig.7. Apoptotic responses of MCF7 cells to 24h treatment with compound 13b, using annexin
V-FITC/PI dual staining. Live cells: PI-/annexin V- ; Early apoptotic cells: PI-/ annexin V+; Late
apoptotic cells: PI+/ annexin V+; Necrotic cells: PI+/ annexin V-. Data shown is mean % cell
number ± SD (n=2)
3
. Conclusion
Two new series including Schiff bases of pyrrolizine-5-carboxamide and their Cu(II) complexes
were synthesized and analyzed using spectral and elemental analysis. The cytotoxic activity of
the new compounds was evaluated against the cancer cell linesMCF-7 (human breast
adenocarcinoma), A2780 (human ovarian adenocarcinoma) and HT29 (human colon
adenocarcinoma) in this study. The responses observed were compared to normal human fetal
lung fibroblasts (MRC5), and the tested compounds showed IC50 values in the range of 0.04-
2
4.66 µM. Compounds 12c and 13b were the most active in the two series, with IC values in
50
the sub-micromolar range against MCF-7 and A2780 cancer cells. Compound 12c showed high
selectivity (6-13 fold) for the three cell lines over MRC5 normal cells. Furthermore, compound
1
2c, induced an increase in the G1 and S cell cycle phases of MCF-7 cells after cell cycle
analysis. In contrast, compound 13b increased MCF-7 sub-G1 pro-apoptotic populations, and
blocked cells in the G2-M phase in a dose dependent manner, at the expense of a decrease in G1.
1
6

ACCEPTED MANUSCRIPT
Moreover, using the Annexin V apoptosis assay, compounds 12c and 13b were shown to
increase the early apoptotic MCF-7 cell populations in a dose dependent manner (two and three
fold, respectively).As such, the two compounds could be promising cytotoxic drug candidates,
and warrant further in vitro and in vivo studies.
4
. Experimental
4
.1. Chemistry
Chemical reagents and solvents were purchased from Sigma-Aldrich. Thin-layer
chromatography (TLC) was used to follow-up the reactions and to check the purity of the
compounds. Melting points (m.p.) are uncorrected and were determined by digital melting point
apparatus (IA 9100MK). A BRUKER TENSOR 37 spectrophotometer was used to determine the
-1
Infrared spectra (IR) using KBr disc. The IR spectra were expressed in wave number (cm ). The
proton magnetic spectra were recorded on a BRUKER AVANCE III at 500 MHz (faculty of
pharmacy, Umm Al-Qura University, KSA) in chloroform, and j constants are given in Hz. The
1
3
C-NMR spectra of the new compounds in chloroform were done at 125 MHz. The electron
impact mass spectral data have been measured by a Finnigan MAT8222 instrument at 70 eV.
Elemental analyses were done in the microanalytical center, Cairo University. Shimadzu TG-
5
0 thermogravimetric analyzer was used to study the thermal behavior (TGA) of the solid
°
°
-1
chelates within 25-800 C range; heating rate 10 Cmin under inert atmosphere. Compounds 8,
1
0a-e and 11a-e were prepared according to previous reports [20-22].
4
.2. General procedure for the preparation of compounds 12a-e.
A mixture of compounds 11a-e (2 mmol), cinnamaldehyde (2.2 mmol) and glacial acetic
acid (0.5 ml) in absolute ethanol (30 mL) was refluxed for six hours. The solvent was evaporated
under reduced pressure and crude product was recrystallized from chloroform-acetone (1:1).
4
.2.1. 7-Cyano-N-phenyl-6-((3-phenylallylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-
carboxamide (12a).
Compound 11a was refluxed with cinnamaldehyde to generate compound 12a as yellow
-1
crystals, m.p. 245-8 °C, yield 66%. IR (cm , KBr phase): 3270 (ν ), 3030 (νAr-H), 2210
NH
1
7

ACCEPTED MANUSCRIPT
1
(
νC≡N), 1616 (νC=O), 1600 (νC=N). H-NMR (CDCl -500 MHz) δ (ppm): 2.48-2.54 (m, 2H,
3
pyrrolizine CH
2 2
-2), 2.95 (t, 2H, J = 7.6 Hz, pyrrolizine CH -1), 4.49 (t, 2H, J = 7.2 Hz,
pyrrolizine CH -3), 7.11-7.18 (m, 2H, Ph-CH=CH), 7.33-7.39 (m, 3H, aromatic CH-3ʺ+CH-
2
4
ʺ+CH-5ʺ), 7.43-7.46 (m, 3H, aromatic CH-3ʹ+ CH-4ʹ+ CH-5ʹ), 7.59 (d, 2H, J = 7.1 Hz,
aromatic CH-2ʺ+ CH-6ʺ), 7.70 (d, 2H, J = 8.0 Hz, aromatic CH-2ʹ+ CH-6ʹ), 8.91 (d, H, J = 9.0
1
3
Hz, N=CH), 10.81 (s, H, CONH). C-NMR (CDCl -100 MHz) δ (ppm):24.46 (CH -2), 25.37
3
2
(
CH
29.09, 130.67, 135.01, 137.89, 138.43, 147.93, 148.30, 152.81, 158.20 (N=C), 161.25 (C=O).
DEPT135 (CDCl -100 MHz) δ (ppm): 24.46, 25.37, 50.12, 119.84, 123.93, 126.77, 128.23,
2 2
-1), 50.12 (CH
-N), 116.18, 117.92 (C≡N), 119.85, 123.93, 126.76, 128.24, 129.05,
1
3
+
+
1
2
29.05, 129.08, 130.66, 147.92.MS (EI): m/z (%) 381 (M +1, 27), 380 (M , 100), 287 (61),
77 (19), 260 (67), 250 (67), 184 (14), 130 (11), 115 (24), 103 (8) 91 (5), 77 (15). Anal.
O (380.44):C, 75.77; H, 5.30; N, 14.73. Found: C, 76.07; H, 5.36; N, 14.37.
20 4
Calcd. for C24H N
4
.2.2. 7-Cyano-6-((3-phenylallylidene)amino)-N-(p-tolyl)-2,3-dihydro-1H-pyrrolizine-5-
carboxamide (12b).
Compound 11b was refluxed with cinnamaldehyde to generate compound 12b as yellow
-1
crystals, m.p. 256-9 °C, yield 71%. IR (cm , KBr phase): 3268 (νNH), 2998 (νAr-H), 2214
1
(
ν
C≡N), 1664 (νC=O), 1608 (νC=N). H-NMR (CDCl
3
-500 MHz) δ (ppm): 2.34 (s, 3H, CH
3
),
2
4
7
7
6
1
1
1
.43-2.49 (m, 2H, J = 8.1 Hz, pyrrolizine CH -2), 2.87 (t, 2H, J = 7.6 Hz, pyrrolizine CH -1),
2 2
2
.44 (t, 2H, J = 7.1 Hz, pyrrolizine CH -3), 7.05-7.10 (dd,H, J = 15.1, 9.9 Hz, Ph-CH=CH),
.16 (d, 2H, J = 8.1 Hz, aromatic CH-3ʹ+CH-5ʹ), 7.24 (d, H, J = 16.0 Hz, aromatic Ph-CH),
.42-7.45 (m, 3H, aromatic CH-3ʺ+CH-4ʺ+CH-5ʺ), 7.54-7.57 (m, 4H, aromatic CH-2ʹ+CH-
1
3
ʹ+ CH-2ʺ+ CH-6ʺ), 8.88 (d, H, J = 8.9 Hz, N=CH), 10.71 (s, H, CONH). C-NMR (CDCl
-
3
00 MHz) δ (ppm):20.94 (CH ), 24.34 (CH -2), 25.28 (CH -1), 50.06 (CH -N), 116.40,
3
2
2
2
17.88(C≡N),, 119.51, 119.61, 127.18, 128.09, 129.01, 129.54, 130.36, 133.34, 135.13,
35.95, 138.32, 146.93, 148.26, 158.07 (N=C),, 160.59 (C=O). DEPT135 (CDCl -100 MHz) δ
3
(
ppm): 20.94, 24.34, 25.28, 50.06, 119.61, 127.19, 128.09, 129.01, 129.54, 130.35, 146.92.MS
+
+
(
EI): m/z (%) 395 (M +1, 27), 394 (M , 100), 287 (69), 260 (88), 184 (23), 130 (13), 115
35), 103 (12), 91 (9), 77 (21). Anal. Calcd. for C H N O (394.47):C, 76.12; H, 5.62; N,
(
2
5
22
4
1
4.20. Found: C, 75.89; H, 5.66; N, 14.38.
1
8

ACCEPTED MANUSCRIPT
4
5
.2.3. 7-Cyano-N-(4-methoxyphenyl)-6-((3-phenylallylidene)amino)-2,3-dihydro-1H-pyrrolizine-
-carboxamide (12c).
Compound 11c was refluxed with cinnamaldehyde to generate compound 12c as yellow
-1
crystals, m.p. 225-7 °C, yield 73%. IR (cm , KBr phase): 3273 (ν ), 2932 (νAr-H), 2209
NH
1
(
νC≡N), 1671 (νC=O), 1578 (νC=N). H-NMR (CDCl -500 MHz) δ (ppm):2.39-2.45 (m, 2H,
3
pyrrolizine CH
2
-2), 2.82 (t, 2H, J = 7.2 Hz, pyrrolizine CH
2 3
-1), 3.79 (s, 3H, OCH ), 4.39-4.41
(
t, 2H, J = 6.9 Hz, pyrrolizine CH -3), 6.88 (d, 2H, J = 8.3 Hz, aromatic CH-3ʹ+CH-5ʹ), 7.00-
2
7
3
2
.05 (dd, H, Ph-CH=CH), 7.18 (d, H, J = 15.8 Hz, Ph-CH), 7.39-7.41 (m, 3H, aromatic CH-
ʺ+CH-4ʺ+CH-5ʺ), 7.51 (d, 2H, aromatic CH-2ʹ+CH-6ʹ), 7.57-7.59 (d, 2H, aromatic CH-
1
3
ʺ+CH-6ʺ), 8.85 (d, H, J = 8.8 Hz, N=CH), 10.63 (s, H, CONH). C-NMR (CDCl -100 MHz)
3
δ (ppm): 24.27 (CH
2
-2), 25.22 (CH
2 2 3
-1), 50.03 (CH -N), 55.47 (OCH ), 114.19, 116.49, 117.79
(
C≡N),, 120.90, 121.00, 127.30, 128.03, 128.98, 130.24, 131.80, 135.15, 138.43, 146.58,
1
2
48.23, 155.96, 157.92 (N=C), 160.31 (C=O). DEPT135 (CDCl -100 MHz) δ (ppm): 24.28,
3
5.23, 50.04, 55.48, 114.20, 120.91, 121.00, 127.31, 128.04, 128.99, 130.26, 146.59.MS (EI):
+
+
m/z (%) 411 (M +1, 29), 410 (M , 100), 307 (16), 287 (36), 260 (81), 232 (11), 205 (9),
84 (17), 130 (8), 115 (29), 103 (8), 88 (14). Anal. Calcd. for C25 (410.47): C, 73.15;
H, 5.40; N, 13.65. Found: C, 73.22; H, 5.44; N, 14.09.
1
22 4 2
H N O
4
.2.4. N-(4-Chlorophenyl)-7-cyano-6-((3-phenylallylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-
carboxamide (12d)
Compound 11d was refluxed with cinnamaldehyde to generate compound 12d as yellow
-1
crystals, m.p. 253-5 °C, yield 64%. IR (cm , KBr phase): 3278 (νNH), 2996 (νAr-H), 2210
1
(
ν
C≡N), 1637 (νC=O), 1610 (νC=N), H-NMR (CDCl
3
-500 MHz) δ (ppm):2.53-2.59 (m, 2H,
pyrrolizine CH -2), 3.02 (t, 2H, J = 7.4 Hz, pyrrolizine CH -1), 4.49 (t, 2H, J = 7.2 Hz,
2
2
pyrrolizine CH
ʹ+CH-5ʹ), 7.41-7.47 (m, 4H, Ph-CH + aromatic CH-3ʺ+CH-4ʺ+CH-5ʺ), 7.61 (broad s, 2H,
aromatic CH-2ʺ+CH-6ʺ), 7.68 (d, 2H, J = 8.8 Hz, aromatic CH-2ʹ+ CH-6ʹ), 8.88 (d, H, J = 8.9
2
-3), 7.22-7.25 (dd, H, Ph-CH=CH), 7.33 (d, 2H, J = 8.6 Hz, aromatic CH-
3
1
3
Hz, N=CH), 10.88 (s, H, CONH). C-NMR (CDCl
3
-100 MHz) δ (ppm): 24.58 (CH
2
-2),
2
1
5.43(CH -1), 50.16 (CH -N), 115.78, 117.86(C≡N), 121.18, 125.94, 128.51, 128.86,
2
2
29.00, 129.18, 131.19, 134.76, 136.98, 148.36, 149.58, 152.81, 158.01 (N=C),, 162.14
(
C=O). DEPT135 (CDCl -100 MHz) δ (ppm): 24.58, 25.43, 50.16, 121.18, 125.95, 128.50,
3
1
9

ACCEPTED MANUSCRIPT
+
+
1
29.00, 129.18, 131.19, 149.56. MS (EI): m/z (%) 416 (M +2, 36), 415 (M +1, 44), 414
+
(
M , 100), 311 (13), 287 (73), 260 (96), 184 (13), 130 (12), 115 (27), 103 (9), 91 (5), 77
11). Anal. Calcd. for C H ClN O (414.89):C, 69.48; H, 4.62; N, 13.50. Found: C, 70.17; H,
(
2
4
19
4
4.48; N, 13.67.
4
.2.5. N-(4-Bromophenyl)-7-cyano-6-((3-phenylallylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-
carboxamide (12e).
Compound 11e was refluxed with cinnamaldehyde to generate compound 12e as yellow
-1
crystals, m.p. 253-6 °C, yield 75%. IR (cm , KBr phase): 3226 (νNH), 2988 (νAr-H), 2210
1
(
νC≡N), 1673 (νC=O), 1578 (νC=N). H-NMR (CDCl -500 MHz) δ (ppm):2.50-2.55 (m, 2H,
3
pyrrolizine CH -2), 2.97 (t, 2H, J = 7.4 Hz, pyrrolizine CH -1), 4.46 (t, 2H, J = 7.0 Hz,
2
2
pyrrolizine CH
2
-3), 7.09 (dd,H, Ph-CH=CH), 7.33 (d, H, J = 15.8 Hz, Ph-CH), 7.45-7.47 (m,
5
6
1
1
1
2
H, aromatic CH-3ʹ+CH-5ʹ+CH-3ʺ+CH-4ʺ+CH-5ʺ), 7.57-7.60 (m, 4H, aromatic CH-2ʹ+CH-
1
3
ʹ+CH-2ʺ+CH-6ʺ), 8.90 (d, H, J = 9.1 Hz, N=CH), 10.86 (s, H, CONH). C-NMR (CDCl3-
00 MHz) δ (ppm): 24.49(CH -2), 25.35(CH -1), 50.11(CH -N), 116.08, 116.29,
17.67(C≡N), 121.25, 126.68, 128.21, 129.11, 130.74, 131.96, 134.93, 137.57, 138.18,
48.04, 148.46, 152.79, 158.16(N=C), 161.28 (C=O). DEPT135 (CDCl -100 MHz) δ (ppm):
4.49, 25.35, 50.11, 121.24, 126.71, 128.21, 129.11, 130.73, 131.96, 147.98.MS (EI): m/z
2
2
2
3
+
+
+
(
(
%) 460 (M +2, 71), 459 (M +1, 55), 458 (M , 72), 355 (12), 328 (12), 287 (85), 260
100), 232 (12), 184 (16), 130 (14), 115 (29), 103 (10),91 (11), 77 (12). Anal. Calcd. for
C H BrN O (459.34):C, 62.75; H, 4.17; N, 12.20. Found: C, 63.47; H, 4.27; N, 12.87.
24
19
4
4
.3. General procedure for preparation of compounds 13a-e
Copper(II) chloride dihydrate (2mmol; 0.34 g) andpyrrolizine-5-carboxamides ligands
(12a-e) (2mol; 0.76 g of 12a, 0.78 g of 12b, 0.82 g of 12c, 0.83 g of 12d and 0.92 g of 12e) were
refluxed in dry acetonitrile (50 mL) for 10 h. The deep brown complexes formed on heat were
filtered off and washed with hot acetonitrile. The crude product was dried and recrystallized
from acetonitrile.
12a
4
.3.1. [Cu(L ) (H O) ].CH CN (13a).
2 2 2 3
°
Yield 54 %, deep brown solid, m.p.229-31 C; Anal. Calcd. for C H N O Cu (899.5):C,
5
0
45
9
4
−1
6
6.76; H, 5.04; N, 14.01, Cu, 7.06. Found: C, 67.27; H, 4.57; N, 14.47, Cu, 6.90.Λ (/Ω
m
2
0

ACCEPTED MANUSCRIPT
2
−1
-1
cm mol )= 12.2;IR (cm , KBr phase): 3449 (νH2O), 3030 (νAr-H), 2212 (νC≡N), 1694 (νC=N),
1
579 (νC=N), 594 (νCu-O), 480 (νCu-N)
12b
4
.3.2. [CuL Cl ](13b)
2
°
Yield 60 %, deep brown solid, m.p.343-5 C; Anal. Calcd. for C25
22 4 2
H N OCuCl (528.92):
−1
C, 56.77; H, 4.19; N, 10.59, Cu, 12.01. Found: C, 56.07; H, 4.33; N, 10.89; Cu, 11.89.Λ
m
(/Ω
2
−1
-1
cm mol )= 16.8;IR (cm , KBr phase) 3235 (νNH), 2975 (νAr-H), 2218 (νC≡N), 1694 (νC=O),
1
601 (νC=N), 520 (νCu-O), 491 (νCu-N).
12c
4
.3.3. [Cu(L ) ] (13c)
2
°
Yield 58 %, deep brown solid, m.p.334-7 C; Anal. Calcd. for C H N O Cu (882.47): C,
5
0
42
8
4
−1
2
6
8.05; H, 4.80; N, 12.70; Cu, 7.20. Found: C, 68.67; H, 4. 73; N, 13.47;Cu, 7.91; Λ (/Ω cm
m
−
1
-1
mol )= 13.9;IR (cm , KBr phase): 2931 (νAr-H), 2223 (νC≡N), 1689 (νC=N), 1607 (νC=N), 590
(νCu-O), 494 (νCu-N)
1
2d
4
.3.4.[CuL Cl ] (13d)
2
°
Yield 60 %, deep brown solid, m.p.247-9 C; Anal. Calcd. for C H N OCuCl (549.34): C,
2
4
19
4
3
−
1
2
5
2.47; H, 3.49; N, 10.20; Cu, 11.57. Found: C, 52.19; H, 4.46; N, 11.76, Cu, 11.50; Λ (/Ω cm
m
−
1
-1
mol )= 15.3;IR (cm , KBr phase): 3281 (νNH), 2997 (νAr-H), 2211 (νC≡N), 1646 (νC=O), 1619
(ν
C=N), 573 (νCu-O), 481 (νCu-N).
1
2e
4
.3.5.[CuL Cl
Yield 60 %, deep brown solid, m.p .> 350 C; Anal. Calcd. for C28
C, 48.46; H, 3.92; N, 12.11; Cu, 9.16. Found: C, 48.43; H, 3.66; N, 11.96; Cu, 9.28;
2 3 2 2
(CH CN) ].H O (13e).
°
27 6 2 2
H N O CuCl Br (693.91):
−
1
2
Λ (/Ω cm
m
−
1
-1
mol )= 14.8; IR (cm , KBr phase): 3447 (ν ), 3339 (ν ), 2977 (νAr-H), 2219 (νC≡N), 1661
H2O
NH
(νC=O), 1601 (νC=N), 593 (νCu-O), 476 (νCu-N)
4
4
4
.4. Pharmacological screening
.4.1. Growth inhibition
.4.1.1. Cell culture
Three cancer adenocarcinoma cell lines, MCF-7 (human breast adenocarcinoma), A2780 (human
ovarian adenocarcinoma) and HT29 (human colon) adenocarcinoma), were used in this study.
For comparison of cancer cell responses compared to normal cells, the MRC5 normal human
2
1

ACCEPTED MANUSCRIPT
fetal lung fibroblast cell line was also used. All cell lines were obtained from the ATCC. The
three cancer cells were sub-cultured in RPMI-1640 media (supplemented with 10% FBS); while
MRC5 was maintained in Eagles minimum essential medium (EMEM, supplemented with 10%
FBS); growth was maintained in a 37°C incubator in a mixture of 5% CO
humidity of 100%.
2
and air, with a relative
4
.4.1.2. Cytotoxicity assay
As previously reported [38], the cytotoxicity of the new compounds was evaluated by the
MTT assay. The three cancer cell lines (MCF-7, A2780, HT29) and the normal fibroblasts
3
(
MRC5) were separately cultured in 96-well plates (3 ×10 /well), and incubated at 37 °C
overnight. Final compound concentrations: 0, 0.05, 0.5, 5, 25, 50 µM (DMSO 0.1%; n= 3). The
plates were incubated with the compounds for 72 h, followed by addition of MTT to each well.
The plates were incubated for a further 3 hr, supernatant was aspirated, and DMSO added to each
well. Absorbance was read on a multi-plate reader, with the optical density of the purple
formazan A550 being proportional to the number of viable cells. Compound concentration causing
5
0% inhibition (IC ) compared to control cell growth (100%) was determined. Graph Pad Prism
50
version 5.00 for Windows was used for analysis (Graph Pad Software, San Diego, California,
USA).
4.4.2. Determination of cell cycle perturbations
To determine any effects of the new compounds on the cell cycle, distribution analysis was done
according to the previous method [39]. MCF-7 cells were cultured in 6 well plates (1×105 cells)
overnight at 37 °C. The cells were then treated with either compound 12c or 13b (0, 5, 10 and 20
µM) for 24h, followed by washing with PBS. After trypsinsation, cells were collected and then
centrifuged at 400 xg, following which the resulting pellets were washed in cold PBS. After
another spin, the cells were fixed overnight in 70% ice cold ethanol. Following centrifugation to
remove the ethanol, cells were re-suspended in cold PBS and ribonuclease A added for 15 min,
which was then followed by the addition of PI (2 µl/ml). Samples were held on ice, and analysed
by flow cytometry using the Novo Cyte flow cytometer. Data analysis was done, using the Novo
Express cell cycle algorithm, comparing the DNA contents (PI bound to DNA) of 20000 events.
2
2

ACCEPTED MANUSCRIPT
2
Doublets were differentiated from single cells in the G -M phase by gating them out
manually.The results were presented in Fig. 5.
4
.4.3. Annexin V FITC/PI apoptosis assay
Apoptosis using Annexin V FITC/PI staining according to the previous report [40]. MCF-7 cells
5
were cultured in 6 well plates (1×10 cells/well) overnight at 37 °C. Each of compound 12c and
1
3b were used to treat cells (0, 5, 10 and 20 µM). After 24 h, supernatants of the treated cells
were collected in tubes and kept on ice. Consequently, cells were trypsinized, before being added
to the tubes, and then centrifuged (500 xg). After a further wash with PBS, the cells were
centrifuged again, and pellets re-suspended in annexin V binding buffer (100 µL) and annexin V
FITC (10 µL). Tubes were incubated at room temperature in dark for 20 min, before adding
binding buffer (400 µL,) and 10 µL propidium iodide (PI). Analysis was performed by flow
cytometry (Beckman Coulter Flow Cytometer, Texas, USA). Different cell populations (early
apoptotic, late apoptotic, and necrotic cells) were identified using annexin V and PI staining.
Supplementary data
1
13
Supplementary data including all spectral data and copies of IR, H-NMR, C-NMR, Mass,
thermal and ESR spectra, of all the final compounds were provided with this manuscript (Figures
S1-S30).
Conflict of Interest
All authors of this manuscript have not declared any conflict of interest.
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