Apoptosis: A target for anticancer therapy with novel cyanopyridines

Article abstract of DOI:10.1016/j.bioorg.2019.103481

One of the many methods of treating cancer is to terminate the uncontrolled growth of cancer cells. So, aiming the apoptotic pathway is an exciting approach to finding new anticancer agents. A novel series of cyanopyridines was designed and synthesized for antiproliferative evaluation. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4-(dimethylamino)phenyl) nicotinonitrile 10f was the most potent inhibitor against the growth of PC-3, and HepG-2 cancer cell lines with IC₅₀ values of 2.04 uM (selectivity index, SI = 78.63, 43, respectively). Also, 10f was safe against the growth of normal human diploid lung fibroblasts cell line (WI-38) with an IC₅₀ value of 160.04 uM. Its analogs, 10b, 10d, 10g, and 11b, were also active against the growth of PC-3, and HepG-2 while against MCF-7 cell line, they displayed good cytotoxic activity compared to the reference standard 5-FU. Remarkably, mechanistic studies indicated that compounds 10b, 10d, 10f, 10g, and 11b stimulated the level of active caspase 3 and boosted the BAX/BCL2 ratio 20–95 folds in comparison to the control. Our results have also indicated that 10b, 10d, 10f, 10g, and 11b exhibited a very potent inhibitory activity against PIM-1 kinase enzyme, where the IC₅₀ values unraveled very potent molecules in the micromolar range (0.47–1.27 μM). Further investigations have shown that 10f, the most potent PIM-1 kinase inhibitor, induced a cell cycle arrest at the G2/M phase. Moreover, in silico evaluation of ADME properties indicated that all the cyanopyridine compounds are orally bioavailable with no permeation to the blood brain barrier.

Full text of DOI:10.1016/j.bioorg.2019.103481

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Apoptosis: A Target for Anticancer Therapy with Novel Cyanopyridines
Magda M.F. Ismail, Amel M. Farrag, Marwa F. Harras, Mona H. Ibrahim,
Ahmed B.M. Mehany
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https://doi.org/10.1016/j.bioorg.2019.103481
YBIOO 103481
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Bioorganic Chemistry
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25 November 2019
Please cite this article as: M.M.F. Ismail, A.M. Farrag, M.F. Harras, M.H. Ibrahim, A.B.M. Mehany, Apoptosis:
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Apoptosis: A Target for Anticancer Therapy with Novel Cyanopyridines
Magda M.F. Ismaila, Amel M. Farraga*, Marwa F. Harrasa, Mona H. Ibrahima and
Ahmed B. M. Mehanyb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754,
Egypt.
b Department of Zoology, Faculty of Science (Boys), Al-Azhar university, Cairo11754, Egypt.
*Corresponding author
Amel M. Farrag
Associate Professor of Pharmaceutical Chemistry
Faculty of Pharmacy, Al-Azhar University
Nasr City, Cairo, Egypt
Mobile: +201006364634
E-mail: amelfarrag@ymail.com
ORCID ID: 0000-0002-8207-0859
1

Apoptosis: A Target for Anticancer Therapy with Novel Cyanopyridines
Abstract
One of the many methods of treating cancer is to terminate the uncontrolled growth of cancer cells. So, aiming
the apoptotic pathway is an exciting approach to finding new anticancer agents. A novel series of cyanopyridines
was designed and synthesized for antiproliferative evaluation. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4-
(dimethylamino)phenyl) nicotinonitrile 10f was the most potent inhibitor against the growth of PC-3, and HepG-2
cancer cell lines with IC50 values of 2.04 uM (selectivity index, SI = 78.63, 43, respectively). Also, 10f was safe
against the growth of normal human diploid lung fibroblasts cell line (WI-38) with an IC50 value of 160.04 uM.
Its analogs, 10b, 10d, 10g, and 11b, were also active against the growth of PC-3, and HepG-2 while against
MCF-7 cell line, they displayed good cytotoxic activity compared to the reference standard 5-FU. Remarkably,
mechanistic studies indicated that compounds 10b, 10d, 10f, 10g, and 11b stimulated the level of active caspase
3 and boosted the BAX/BCL2 ratio 20- 95 folds in comparison to the control. Our results have also indicated that
10b, 10d, 10f, 10g, and 11b exhibited a very potent inhibitory activity against PIM-1 kinase enzyme, where the
IC50 values unraveled very potent molecules in the micromolar range (0.47 -1.27 uM). Further investigations have
shown that 10f, the most potent PIM-1 kinase inhibitor, induced a cell cycle arrest at the G2/M phase. Moreover,
in silico evaluation of ADME properties indicated that all the cyanopyridine compounds are orally bioavailable
with no permeation to the blood brain barrier.
Keywords: Synthesis; Cyanopyridine; Anticancer; Apoptosis; PIM-1 kinase; ADME.
2

1. Introduction
Each minute of a lifetime, millions of cells in our bodies practice a planned form of cell death called apoptosis,
or programmed cell death. This altruistic cellular process plays wide-ranging and vital roles in keeping us healthy,
not least in protecting us from cancer [1]. So, apoptosis has a critical role in monitoring cell numbers in many
developmental and physiological settings. Apoptosis is diminished in many human cancers, portentous that
disruption of apoptotic function contributes substantially to the transformation of a normal cell into a tumour cell.
The switching apoptosis on and off is determined by the ratio of pro-apoptotic effector proteins (BAX and BAK),
and antiapoptotic BCL2 proteins (BCL2, BCL-xL, MCL-1, A1, BCL-B, BCL-w) [2]. Mainly, two main signalling
paths for apoptotic cell death have been specified, the first one is the intrinsic mitochondrial apoptotic pathway
that is mainly produced by cellular stress, in which mitochondrial permeability plays a crucial role. Whereas; the
second path is the extrinsic cytoplasmic pathway that is triggered via pro-apoptotic ligands binding to the cell
surface death receptor [1,3-5]. Caspase protease activity is vital for apoptosis; once active, caspases cleave
hundreds of different proteins leading to rapid cell death with characteristic biochemical and morphological
hallmarks [6-8]. In general terms, caspase activity can be initiated either via the intrinsic pathway of apoptosis or
through the extrinsic pathway of apoptosis. Cell death also plays crucial role in the treatment of cancer. Apoptosis
deregulation has been extensively documented as a hallmark of cancer throughout cancer pathogenesis.
Consequently, the initiation of apoptosis in tumor cells represents an effective tactic for fighting different human
malignancies in the current medical age [1,3-5].
On the other hand, the literature survey reveals that 4, 6-diaryl-3-cyano-2-pyridone derivatives have stood out
as a promising class of anticancer agents with efficient apoptotic activity. N'-[2-(3-cyano-4,6-bis(3,4-
dimethoxyphenyl) pyridin-2-yloxy) acetyl] benzohydrazide I, was reported to inhibit the proliferation of MCF-7
cancer cells by inducing apoptosis and arresting the cell cycle at G1 phase via inhibition of CDK2 and CDK4 [9].
3-Cyano-4,6-diaryl-2-pyridone derivative II possess anticancer activity due to its ability to act as survivin
inhibitor, which is a unique member of the inhibitors of apoptosis [10].4-Thienyl-6-arylpyridone derivative III
showed potent antiproliferative activity with high PIM-1 kinase inhibitory activity with IC50 value of 0.94 µM,
and it was founded to boost the levels of active caspase 3 and BAX and decrease the level of BCL2 [11], (Fig.1).
This indicates the potentiality of 4, 6-diaryl-3-cyano-2-substitutedpyridine skeleton as a model for further
optimization to get a more potent apoptosis inhibitor.
O
O
Cl
S
Cl
CN
O
CN
CN
O
O
OH
H
N
N
I
N
N
O
N
H
H
H
O
O
O
O
O
Br
III
II
Fig. 1: Chemical structures of reported cyanopyridines endowed with anticancer and apoptosis inducing
activities.
3

Noteworthy, Cheney et al study at 2007, proved that some cyanopyridine derivatives also acted as PIM-1
kinase inhibitors e.g., IV-VI with IC50 of 0.05, 0.43, and 0.99 µM, respectively [12] (Fig. 2)
OH
S
CN
O
CN
O
CN
O
OH
OH
Br
N
N
H
N
H
H
S
IV
V
VI
Fig. 2: Chemical structure of reported cyanopyridines as PIM-1 kinase inhibitors.
PIM-1 is an oncogenic constitutively active serine/threonine kinase, a member of a family of proteins
containing other homologues, PIM-2 and PIM-3 and is transcriptionally regulated by cytokines, mitogens and
numerous growth factors 2-4 [13-15]. PIM-1 expressed in many types of solid and haematological cancers and
almost absent in benign lesions, PIM-1 kinase proved to be a successful anti-cancer drug target of low toxicity
[16-23]. It appears to contribute to cancer development in three significant ways when it is overexpressed by
inhibiting apoptosis, promoting cell proliferation, and promoting genomic instability [24]. PIM-1 kinase
phosphorylates and regulates the activity of many proteins involved with many biological processes such as cell
cycle, cell proliferation, apoptosis, and drug resistance [25-29]. Through the cell cycle, PIM-1 regulates G1-S
phase progression and G2/M checkpoint. [ 30,31]. Though, in terms of cancer a preponderance of evidence points
to PIM-1 playing an important role in the inhibition of apoptosis and the regulation of cell proliferation [32, 33].
In vitro studies demonstrated that overexpression of PIM-1 enhances tumour growth and protection from drug-
induced apoptosis of cancer cells [34]. Additionally, several specific and potent inhibitors of PIM-1
kinase have also been shown to induce apoptotic death of cancer cells, sensitize cancer cells to chemotherapy and
synergize with other anti-tumour agents, thus making it an attractive therapeutic target [24].
Based on the findings obeve, we planned to design and synthesize novel series of cyanopyridine scaffold via
substitution at p-4 by aromatic moiety bearing hydrophilic or lipophilic group. Moreover, we introduced at p-6
of cyanopyridine scaffold either; phenyl/ 2-thienyl or the extended flexible side chain (benzyloxyphenyl). What
is more, 2-OH group was bioisosterically replaced by 2-NH2 group; these structural modifications were done to
examine their effect on the affinity of PIM-1 and to optimize their antiproliferative activity (Fig. 3). All the
synthesized hits were evaluated for their in vitro antitumor activity against human prostate carcinoma PC-3,
human hepatocellular carcinoma HepG-2, and breast adenocarcinoma MCF-7 cell lines using MTT assay.
Additionally, cell cycle analysis and apoptosis induction potential of the most active cyanopyridine derivatives
4

10b, 10d, 10f, 10g, and 11b were examined in PC-3 cells, in order to attain more mechanistic understandings and
to verify and enlighten the antitumor properties of the inspected cyanopyridines. Furthermore, the effect of the
most active compounds on the genes expression of some apoptosis key markers was also investigated. Finally,
the most active compounds, 10b, 10d, 10f, 10g and, 11b were evaluated for inhibition of PIM-1 kinase.
Ar
CN
X = OH, NH2
N
X
Bioisosterism
Extension
O
CN
OH
OH
N
Privileged
cynopyridine
group is spared
Br
S
Bioisosterism
Fig. 3: Design of novel cyanopyridine derivatives as PIM-1 inhibitors and apoptosis inducers.
2. Results and discussion
2.1. Chemistry
Synthesis of the new eighteen target compounds and intermediates is depicted in schemes 1 and 2.
Initially, Knoevenagel condensation was followed to furnish the reported α,β-unsaturated ketones, 3a-e by
condensation of acetophenone or 2-acetylthiophene with different aromatic aldehydes in 10% aq. NaOH/EtOH.
Cyclocondensation of 3a-d with malononitrile and ammonium acetate afforded 2-amino-4-aryl-6-phenyl/(2-
thienyl)-3-cyanopyridines, 4a-d, while, cyclocondensation of 3d or 3e with cyanoacetamide/ pip. yielded 2-oxo-
4-aryl-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitriles 5a, 5b [35] (Scheme 1).
5

R
4a,
4b,
4c,
4d,
= C6H5, R = H
CN
= C6H5, R = 4-CH3
= C4H3 S, R= H
= C4H3S, R= 2-OH
b
c
N
NH2
O
O
a
O
4a- d
+
R
R
R
1a&b
2a-c
3a-e
= C6H5,R = H
CN
O
3a,
3b,
3c,
3d,
3e,
2a, R= H
1a,
1b,
= C6H5
= C4H3S
2b, R= 2-OH
2c, R=4-N(CH3)2
N
H
5a, R = 2-OH
5b, R = 4-N(CH3)2
= C6H5, R = CH3
S
= C4H3S, R = H
5a&b
= C4H3S, R = 2-OH
= C4H3S, R= 4-N(CH3)2
Scheme 1: Reagents and conditions: a) EtOH, 10% aq. NaOH, stirr, 3h; b) Malononitrile, NH4OAC, EtOH,
reflux, 8h; c) Cyanoacetamide, EtOH, piperidine, reflux, 6h.
Additionally, Knoevenagel condensation of starting material
aldehydes/EtOH/KOH, produced the conjugated enones, 9a-g. Cyclocondensation of the latters with
malononitrile/AcONH4/EtOH, yielded the corresponding 2-amino-6-(4-(benzyloxy) phenyl-4-
8 [36] with different aromatic
(aryl)nicotinonitriles, 10a-g [37]. Finally, condensation of 9b-d, 9g with ethyl cyanoacetate and ammonium
acetate [38] furnished the corresponding 6-(4-(benzyloxy) phenyl)-4-(aryl)-2-oxo-1,2-dihydropyridine-3-
carbonitriles 11a-d, (Scheme 2).
O
O
O
Ar`
Ar
b
Cl
a
O
O
+
9a-g
HO
6
7
8
d
c
Ar`
Ar`
N
CN
O
CN
N
H
Ar
NH2
Ar
O
O
11a-d
10a-g
11a, Ar`= 4-CH3O-
C6H4
11b, Ar`= 2-OH-C6H4
11c, Ar`= 2-Cl-C6H4
9,10a, Ar`= 4-CH3-C6H4
9,10b, Ar`= 4-CH3O-C6H4 9,10f, Ar`= 4-N(CH3)2-C6H4
9,10c, Ar`= 2-OH-C6H4
9,10d, Ar`= 2-Cl-C6H4
9,10e, Ar`= 2,4-di Cl-C6H4
9,10g, Ar`= C4H3O
11d, Ar`= C4H3O
Scheme 2: Reagents and conditions: a) Ethanol, KOH, reflux,3h; b) MeOH, ArCHO, 10% KOH, stirr, 2h;
c) EtOH, NH4OAc, malononitrile, reflux, 9 - 12 h; d) EtOH, NH4OAc,
ethylcyano acetate, reflux, 9-12 h.
6

The infrared (IR) spectra of 4d, 9a,f-g, and 10a-g products revealed bands at a stretching frequency (υ) around
3430 cm-1 corresponding to the NH2 group. Moreover, it demonstrated relatively lower values of the carbonyl
stretching (υ) around 1650 cm-1 than the typical carbonyl stretching, which is usually around 1700 cm-1. This may
be due to the single-bond character of the tautomeric enol form, leading to lower absorption frequency, in addition
to; the cyano group appeared around 2210 cm-1. Conjugated enones (9a-c, e) exhibited absorption bands at υ 1660
cm-1 representing CO functionality along with the 1H-NMR spectra, which exhibited two doublets at δ 7.64, 7.74
ppm with characteristic J = 15.6 Hz value for E isomers of CH=CH protons. Overall cyanopyridines, 9-11
displayed characteristic multiplet peak distinguished CH2 protons at the downfield region δ 5.06-5.22 ppm, being
flanked between the oxygen atom and a phenyl group. The signals observed in 13C-NMR spectra of all the products
are determined to be in accordance with the recommended molecule structures. Mass spectrometry showed
molecular ion peaks at M+, M++1, M++2 in addition, to some of important peaks.
2.2. Biological results and discussion
2.2.1. Antiproliferative screening
The antiproliferative profiles of the synthesized compounds were evaluated in vitro against human prostate
carcinoma PC-3, eleven compounds out of seventeen showed higher cytotoxic activities (IC50’s= 2.04 - 7.5 uM),
than that of the reference standard 5-FU (IC50= 7.73 uM). 2-Amino-4,6-diphenyl-3-cyanopyridine, 4a showed
better cytotoxicity (IC50’s= 7.01uM) than 5-FU. Bio-isosteric replacement with 2-thienyl at p-6 of cyanopyridine,
as in analogs, 4b-d and 5a, b gave poor anticancer activities. Besides, SAR analysis elucidated that, on introducing
benzyloxyphenyl moiety at p-6 of cyanopyridine, a dramatic increase in cytotoxicity of compounds 10 & 11
except for compound 11d. For human hepatocellular carcinoma HepG-2, seven compounds 10a-d, f, g, and 11b
elicited cytotoxicity ranging from equipotent to more than double the activity of 5-FU; however breast
adenocarcinoma MCF-7 cell line was not sensitive to cyanopyridine hits except for compound 10d which
demonstrated enhanced anticancer effect (IC50= 7.63 uM) than that of the standard. The normal human diploid
lung fibroblast cell line WI-38 was also evaluated, in order to evaluate safety of the most active synthesized
compounds 10b, 10d, 10f, 10g and 11b towards the normal cells, cytotoxic activity evaluation of these hits on
human diploid lung fibroblasts cell line WI-38 were carried out to investigate the toxicity and selectivity of these
active derivatives (Table 1). To our delight, they were found to be inactive against this normal cell (154.39-
182.89 µM), revealing that they possessed a large safety margin to selectively target cancerous cells other than
normal cells as shown by their specified IC50 values. Due to this, 10b, 10d, 10f, 10g, and 11b were chosen for
further investigations (Table 1).
7

Table 1
In vitro e cytotoxic activities of compounds 4a-d, 5a, b, 10a-g and 11a-d.
Comp. No.
IC50 (µM)a/ (SIb)
HEPG-2C
13.65±1.20
13.29±1.20
15.91±1.61
21.33±1.70
16.32±1.30
20.99±1.60
8.18±0.64
4.54±0.28
(34.01)
PC-3C
7.01±0.62
10.14±0.97
11.02±0.87
14.06±0.98
7.38±0.64
19.65±1.61
6.24±0.52
3.14±0.22
(49.17)
MCF-7C
25.12±1.80
27.43±1.90
21.14±1.72
32.15±2.41
21.39±0.94
34.85±2.52
13.06±0.95
8.64±0.63
(17.87)
WI-38C
ND
ND
ND
ND
ND
ND
ND
4a
4b
4c
4d
5a
5b
10a
10b
154.39±0.62
10c
10d
5.19±0.52
2.36±0.16
(77.50)
5.44±0.37
4.59±0.37
(39.85)
9.46±0.91
7.63±0.89
(23.97)
ND
182.89±0.39
10e
10f
8.34±0.71
2.04±0.21
(78.63)
8.52±0.75
3.73±0.18
(43.00)
18.58±0.67
8.66±0.73
(18.52)
ND
160.40±0.60
10g
2.99±0.30
(58.47)
4.71±0.22
(37.12)
9.31±0.81
(18.78)
174.82±0.44
11a
11b
7.59±0.67
3.17±0.28
(56.32)
8.7±0.72
6.62±0.61
(26.97)
15.93±1.20
9.44±0.86
(18.91)
ND
178.52±0.34
11c
11d
5 -Fu
4.34±0.56
27.9±1.71
7.53 ±0.57
10.46±0.98
39.7±2.50
8.15 ±0.61
12.74±0.96
68.34±3.11
7.76 ±0.46
ND
ND
a Cytotoxicity, IC50 for each cell line, is the concentration of compound which reduced by 50% the optical
density of treated cells with respect to untreated cells using the MTT assay. b SI: Selectivity index = (IC50 of
WI-38)/(IC50 of cancer cell line). C Cell lines include human prostate carcinoma (PC-3) hepatocellular carcinoma
(HepG-2), breast adenocarcinoma (MCF-7), and normal human diploid lung fibroblastscell line (WI-38). d ND:
Not determined. eData represent the mean values of three independent determinations.
2.2.2. Apoptosis detection studies
2.2.2.1. Effect on the active caspase 3 level
Activation of the caspase 3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify
activators, and inhibitors of the “death cascade.” the most active cyanopyridine analogs 10b, 10d, 10f, 10g, and
11b were evaluated for their effect on caspase 3. Our investigation results reflected the elevated level of the active
caspase 3, which is considered as a marker for apoptosis, (Table 2).
2.2.2.2. Effect on mitochondrial apoptosis pathway proteins BAX and BCL2
The mitochondrial apoptotic pathway is harmonized by a family of proteins called BCL2. Among these, BCL2
and BAX finely set this programmed process as BCL2 suppresses apoptosis (anti-apoptotic) whereas BAX
induces it (pro-apoptotic). Accordingly, the tuned balance between these two characteristic proteins is
judgmental for the cell potential to undergo apoptosis. Several direct BAX activators have been identified to
hold promise for cancer therapy with the advantages of specificity and the potential of overcoming chemo- and
radio resistance. The most active cyanopyridine analogs 10b, 10d, 10f, 10g, and 11b were evaluated for their
8

effect on some apoptosis key markers, BAX, and BCL2. Our results confirmed the elevated level of BAX/BCL2
ratios that regulate cell death (apoptosis) (Table 2).
Table 2
Effect of compounds 10b, 10d, 10f, 10g and 11b on the genes expression of some apoptosis key markers.
Comp. No.
Caspase 3
Pg/ml
BAX
Pg/ml
199.1
189.4
215.9
166.4
259.4
18.14
BCL2
Pg/ml
1.724
2.356
1.476
2.731
0.8138
5.418
10b
10d
10f
10g
11b
277.7
266.3
363.8
285.2
398.2
55.3
Control
2.2.3. PIM-1 kinase inhibitory activity
PIM-1 is emerging as a cancer drug target, particularly in prostate cancer [39]. PIM-1 inhibitory activity was
evaluated for 10b, 10d, 10f, 10g and 11b. The obtained results revealed that 10f was the most active hit as it
displayed 80.22% inhibition of PIM-1 expression at 10 µM. (Table 3).
Table 3
PIM-1 kinase inhibitory activity of compound 10b, 10d, 10f, 10g and 11b.
Comp. No.
% Inhibition
(Con.10µM)
IC50 (µM)
10b
10d
49.60
71.79
80.22
78.85
72.99
64.15
11.71
1.27
0.47
0.56
0.84
3.98
10f
10g
11b
Colchicine
2.2.4. Cell cycle analysis
To investigate the probable mechanism underlying the antiproliferative effect exerted by the most potent
PIM-1 inhibitor compound 10f, PC-3 cell cycle progression was assessed following treatment with compound 10f
, that also had the marked highest antiproliferative activity (IC50 = 2.04 μM), on human prostate carcinoma PC-3
cell line for 24 h using propidium iodide flow cytometry analysis. As seen in (Fig. 4, Fig. 5, Fig 6 & Table 4) in
comparison with control cells, it was obvious that 10f induced a significant increase in the percentage of cells at
Pre-G1 phase by 18.9 folds, which could be indicative of apoptosis, and increase in G2/M by 4.9 folds. Such an
increase was accompanied by a decrease in the percentage of cells at the S-phase of the cell cycle. The Pre-G1
and G2/M phase results indicated that compound 10f induced apoptosis and arrest the cell cycle at the G2/M
phase.
9

Fig. 4: Cell cycle analysis of PC-3 cells treated with compound 10f at 0.86 μg/ml concentration.
Fig. 5: Cell cycle analysis of PC-3 cells treated with DMSO.
10

Fig. 6: Cell cycle analysis of PC-3 cells treated with DMSO.
Table 4
Effect of compound 10f, DMSO and 5-FU on the cell cycle of PC-3 cell line.
Sample
Conc. μM
%G0-G1
%S
%G2/M
%Pre-G1
10f / PC-3
2.04
27.44
29.28
43.28
33.28
DMSO / PC-3
(negative control)
0
54.77
29.88
36.41
27.96
8.82
1.76
5-FU / PC-3
(positive control)
7.53
42.16
15.22
2.2.5. Annexin V-FITC apoptosis assay
One of the earliest cellular changes evident during apoptosis is the translocation of phosphatidylserine (PS)
from the inner to the outer side of the plasma membrane. Fluorescently labeled Annexin V, which binds to
phosphatidylserine [40], can be used as a sensitive probe for PS in the outer leaflet of the plasma membrane [41].
Also, PI, a fluorescent molecule binding to nucleic acids, but which does not enter live cells, can be used to
counterstain dead cells. To differentiate between apoptosis and necrosis, both Annexin V-FITC and PI staining
usually performed. Annexin V/PI staining was performed in cells exposed to 10f for 24h. The results revealed
that application of compound 10f on PC-3 cells for 24 h, increases the early apoptosis ratio (lower right quadrant
of the cytogram) from 0.82% to 7.29%, and increases the late apoptosis ratio (higher right quadrant of the
cytogram) from 0.43% to 23.56%. This means that compound 10f induced almost up to 24.68 folds, both early
and late, cellular apoptosis when compared with the control. These data suggest that compounds 10f triggered
apoptosis via the programmed cell death pathway rather than a necrotic pathway (Fig. 6 & Table 5).
B
A
11

C
Figure 6: Effect of DMSO (A) , 10f (B), and 5-FU on the percentage of annexin V-FITC-positive staining in
PC-3 cells.
Table 5
12

Effect of 10f, DMSO and 5-FU on the percentage of annexin V-FITC-positive staining in PC-3cells.
Apoptosis
Sample /
cell line
Necrosis
Total
Early
Late
10f / PC-3
33.28
7.29
23.56
2.43
DMSO / PC-3
(negative control)
1.76
0.82
0.43
0.51
5-FU / PC-3
(positive control)
15.22
4.94
8.12
2.16
2.3. In silico evaluation of physicochemical and ADME properties
Computational study of the synthesized compounds 4a-d, 5a,b, 10a-g, and 11a-d was performed to evaluate
physicochemically and ADME properties using SwissADME [42]. With respects to physicochemical properties
(Table 6), all the synthesized compounds have zero violation for Lipinski´s rule for oral drugs, except for 10e,
which has only one violation (M log P > 4.15) but it still stays in the orally bioavailable compounds. All the
topological polar surface areas (TPSA) are less than 112 A0 2. Additionally, absorption (% ABS) was estimated
by using the equation % ABS = 109 – (0.345 x TPSA) [43], found that the calculated % ABS of all these hits
ranged between 70.64 % and 87.37 %, demonstrating that these synthesized derivatives may have the required
cell membrane permeability and bioavailability. All compounds have rotatable bonds between 2 and 6, which
indicating molecular flexibility to their bio target. Regards to pharmacokinetic and medicinal chemistry
parameters of the synthesized compounds (Table S, Supplementary data ), it was found that, all the derivatives
having high gastro-intestinal absorption and most of them have no permeation to the blood brain barrier except
for the reported compounds 4a and 4b. Thus ensuring that these systemically targeted molecules will have low to
no CNS side effects. Bioavailability is an index of the amount of drug present in the plasma and is considered as
the most crucial factor affecting absorption.
Table 6
Physicochemical properties based on Lipinski´s rule of five, TPSA, % ABS and number of rotatable bonds.
Comp. No.
HBD
HBA
MlogP
MW
Lipinski´s
TPSA
% ABS
No. of
Violations
Rot. bonds
4a
4b
4c
4d
5a
1
1
1
2
2
1
1
1
2
1
1
1
1
1
2
1
1
2
2
2
2
3
3
2
3
4
4
3
3
3
4
4
4
3
4
3
2.49
2.73
2.05
1.47
1.53
2.83
3.44
2.88
2.67
3.71
4.17
3.84
2.02
2.94
2.74
3.77
2.09
-0.32
271.32
285.34
277.34
293.34
294.33
320.41
391.46
407.46
393.44
411.88
446.33
419.52
367.4
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
62.7
62.7
87.37
87.37
77.63
70.65
72.73
79.71
84.18
80.99
77.20
84.18
84.18
84.18
79.65
83.09
79.29
86.27
81.74
86.33
2
2
2
2
2
3
5
6
5
5
5
6
5
6
5
5
5
0
90.94
111.17
105.12
84.89
71.93
81.16
92.16
71.93
71.93
71.93
85.07
75.11
86.11
65.88
79.02
65.72
5b
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
5-FU
408.45
394.42
412.87
368.38
130.08
13

Interestingly, all the synthesized derivatives were found to have high bioavailability scores 0.55. Pan
Assay Interference Compounds (PAINS), was performed by SwissADME [42] and PAINS-Remover [44]
displayed zero alerts to all the hits. Synthetic accessibility scores of all the analogs were found to be between 2.56
and 3.33, indicating that they can be readily synthesized on a large scale. (Table S, Supplementary data)
2. Conclusion
Herein we designed and synthesized a small library of cyanopyridines that resulted in cytotoxic derivatives
against prostate carcinoma PC-3, human hepatocellular carcinoma HepG-2, and breast adenocarcinoma MCF-7
cell lines. The most active derivatives 10b, 10d, 10f, 10g, and 11b were evaluated against the normal human
diploid lung fibroblasts cell line WI-38, indicated that they were inactive against this normal human cell.
Apoptosis studies for these hits were conducted to evaluate the pro-apoptotic ability of our compounds. The result
showed that they all boosted both the level of active caspase 3 and the BAX/BCL2 ratio in comparison to control.
Furthermore, the PIM-1 kinase inhibitory activity for 10b, 10d, 10f, 10g, and 11b was evaluated where 10f showed
marked PIM-1 inhibitory activity 80.22%. Additionally, 10f was found to arrest PC-3 cells at the G2/M and
increase the percentage of cells at Pre-G1 phase by 18.9 folds. Moreover, all the derivatives are systemically
targeted molecules that may have low to no CNS side effects. Our findings report a well-scalable cyanopyridine
scaffold by applying extension tactic at p-6 that may be useful for clinical applications in cancer treatment and
may contribute to the design of other molecules to investigate and develop antitumor chemotherapeutics.
4. Experimental section
Reagents and solvents were purchased from usual commercial suppliers and were used without further
purification. Yields reported refer to purified products. All reactions were routinely checked by thin-layer
chromatography (TLC) on Merck Silica Gel 60 F254 (0.25mm thick) and visualization was performed with UV
lamp. Melting points were measured in open capillary tubes using Stuart SMP3 apparatus. IR spectra (KBr) were
measured on a Shimadzu FT/IR 1650 (Perkin Elmer) spectrometer. 1H and 13C NMR spectra were recorded on a
1
Brüker Advance-400 instrument (400 MHz for H and 100 MHz for 13C) in DMSO-d6 Chemical shifts (δ) are
reported in ppm relative to TMS as an internal standard, or to the solvent in which the spectrum was recorded.
Mass spectra were performed on a Shimadzu GS/MS-QP 2010 plus spectrometer at 70 eV. The intermediates,
3a-e [45-48], and 9b-d [49,50] are prepared according to the reported method [51], as well as compounds 4a-c
[52,53] were prepared for cytotoxic screening and SARs study.
4.1. Chemistry
4.1.1. General procedure for the synthesis of 2-amino(un, substituted phenyl)-6-(aryl)-nicotinonitrile
derivatives.(4a-d)
A solution of chalcone 3a -d (0.01 mol) with malononitrile (0.66 g, 0.08 mol) and ammonium acetate
(6.16 g, 0.08 mol) in ethanol were refluxed for 8 h. The progress of the reaction was monitored by TLC. The
precipitate was filtered off and washed with cold methanol. Recrystallization from ethanol yielded pure
compounds with 50-70% yields.
14

o
4.1.1.1. 2-Amino-4-(2-hydroxyphenyl)-6-(thiophen-2-yl)nicotinonitrile (4d) Yield 60%; m.p. 310 - 312 C; IR
(KBr, cm-1): 3432, 3348, 3238 (NH2, OH), 3184 (CH aromatic), 2965 (CH aliphatic), 2197 (C≡N), 1613 (C=N);
1H NMR (DMSO-d6) δ (ppm): 6.88 – 6.92 (t, H, H5 Ar-H), 6.96 (d, H, H3 Ar-H), 7.14 – 7.16 (t, H, H4 Ar-H), 7.28
– 7.32 (t, H, H4 thiophene), 7.57 (d, 2H, H6 Ar-H), 7.71 (d, H, H3 thiophene), 7.79 (d, H, H5 thiophene), 8.41 (s,
1H, Pyridine-H), 9.89, 10.10 (2s, 3H, NH2, OH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 85.48,
111.22, 113.54, 116.34, 116.92, 122.88, 127.26, 128.78, 129.96, 143.04, 154.54, 155.99, 156.89, 161.34; MS,
m/z: 293 (M+, 5.01 %), 294 [(M+1)+, 3.82 %]; Anal. Calcd. For C16H11N3OS (293.06): C, 65.51; H, 3.78; N,
14.32; S, 10.93, Found: C, 65.32; H, 4.12; N, 14.59; S, 11.06.
4.1.2. General procedure for the synthesis of 4-(substituted phenyl)-2-oxo-6-thiophen-2-yl)-1,2-
dihydropyridine-3-carbonitrile.(5a,b)
A mixture of chalcone 3d or 3e (0.01 mol) with 2-cyanoacetamide (0.84 g 0.01 mol) was refluxed for 6
h in ethanol containing drops of piperidine as a catalyst. The progress of the reaction was monitored by TLC. The
precipitate formed was filtered off and washed with cold methanol. Recrystallization from ethanol yielded the
pure product 5a,b.
4.1.2.1. 4-(2-Hydroxyphenyl)-2-oxo-6-(thiophen-2-yl)-1,2 dihydropyridine-3-carbonitrile (5a). Yield 67%; m.p.
380-382 oC; IR (KBr, cm-1): 3440-3200 (broad, NH, OH), 3102 (CH aromatic), 2969 (CH aliphatic), 2205 (C≡N),
1699 (C=O), 1601 (C=C); 1H NMR (DMSO-d6) δ (ppm): 7.16 - 7.18 (t, H, H5 Ar-H), 7.24 (d, H, H3 Ar -H), 7.29
- 7.33 (t, H, H4 Ar -H), 7.36 (s, 1H, Pyridon-H) 7.51 – 7.54 (t, H, H4 thiophene), 7.59 (d, H, H6 Ar -H), 7.96 (d,
H, H3 thiophene), 8.29 (d, H, H5 thiophene) 9.79, 10.60 (2s, 2H, NH, OH; exchangeable with D2O; 13C NMR
(DMSO-d6) δ (ppm): 111.74, 116.94, 117.23, 124.04, 125.01, 126.53, 128.52, 129.16, 131.52, 136.03, 152.78,
156.79, 168.35; MS, m/z: 294 (M+, 20.44 %), 295 [(M+1)+, 100 %]; Anal. Calcd. For C16H10N2O2S (294.05): C,
65.29; H, 3.42; N, 9.52; S, 10.89 Found: C, 65.47 H, 3.68; N, 9.78; S,11.09.
4.1.2.2. 4-(4-(Dimethylamino)phenyl)-2-oxo-6-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (5b). Yield
70%; m.p. 330-332 oC; IR (KBr, cm-1): 3433 (NH), 3090 (CH aromatic), 2920 (CH aliphatic), 2212 (C≡N), 1637
(C=O), 1610 (C=C); 1H NMR (DMSO-d6) δ (ppm): 3.01 (s, 6H, 2 CH3), 6.74 (d, 2H, H3,5 Ar-H, J = 8.4 Hz), 7.24
– 7.29 (t, H, H4 thiophene), 7.55 – 7.62 (m, 2H, H3,5 thiophene), 7.64 (s, H, NH; exchangeable with D2O), 7.69
(d, 2H, H2,6 Ar-H, J = 8.4 Hz), 8.23 (s, 1H, Pyridon-H); 13C NMR (DMSO-d6) δ (ppm): 41.02, 111.09, 112.21,
116.37, 122.26, 125.99, 129.18, 131.28, 132.83, 134.86, 144.73, 146.80, 152.51, 161.76; MS, m/z: 321 (M+,
83.14%); Anal. Calcd. For C18H15N3OS (321.09): C, 67.27; H, 4.70; N, 13.07; S, 9.98, Found: C, 67.54; H, 4.89;
N, 13.35; S, 9.95.
4.1.3. Synthesis of 4`-(Benzyloxy)acetophenone (8)
A mixture of 4-hydroxyacetophenone (1.08 g, 0.01 mol), benzyl chloride (1.26 g, 0.01 mol) and KOH
(0.04 g, 0.01 mol) in ethanol (30 ml) was refluxed for 3 h, then poured onto ice/water. The solid formed was
collected, washed with water, dried, and recrystallized from ethanol. Yield 98%; m.p. 96-97 oC as reported [36].
4.1.4. General procedure for the synthesis of chalcon derivatives.(9a-g)
4`-Benzyloxy acetophenone (2.26 g, 0.01 mol) and different aryl aldehydes (0.01 mol) were dissolved in
methanol (25 ml) and 5 ml of 10% alc. KOH solution was slowly added to it under stirring at 15-20°C. Stirring
15

continued for 2 h at the same temperature. The progress of the reaction was monitored by TLC. The precipitate
was filtered off and washed with cold methanol. The obtained product was crystallized from methanol to give the
chalcone derivatives 9a-g.
o
4.1.4.1. (E)-1-(4-(Benzyloxy)phenyl)-3-(p-tolyl)prop-2-en-1-one (9a). Yield 70%; m.p. 166-168 C; IR (KBr,
cm-1): 3030 (CH aromatic), 2912 (CH aliphatic), 1651 (C=O), 1592 (C=C); 1H NMR (DMSO-d6) δ (ppm): 2.33
(s, 3H, CH3), 5.21 (s, 2H, OCH2), 7.13 (d, 2H, H3,5 Ar-H, J = 8.8 Hz), 7.24 (d, 2H, H3,5 Ar`-H, J = 8 Hz), 7.31-
7.47 (m, 5H, phenyl-Hs), 7.64 (d, 1H, olefinic, J = 15.6 Hz), 7.74 (d, 2H, H2,6 Ar`-H, J = 8 Hz), 7.85 (d, 1H,
olefinic, J = 15.6 Hz), 8.13 (d, 2H, H2,6 Ar-H, J = 8.8 Hz); MS, m/z: 328 (M+, 4.58 %); Anal. Calcd. For C23H20O2
(328.15): C, 84.12; H, 6.14; Found: C, 84.38; H, 6.29.
4.1.4.2. (E)-1-(4-(Benzyloxy)phenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (9e). Yield 75%; m.p. 136-138 oC;
1
IR (KBr, cm-1): 3063 (CH aromatic), 2925 (CH aliphatic), 1663 (C=O), 1595 (C=C); H NMR (DMSO-d6) δ
(ppm): 5.15 (m, 2H, OCH2), 7.10 (d, 2H, H3,5 Ar-H), 7.29 -7.45 (m, 6H, 5H, phenyl-Hs, 1H, olefinic), 7.62 (s, 1H,
H3 Ar`), 7.82 (d, 1H, olefinic), 8.00 - 8.06 (m, 4H, H5,6 Ar`, H2,6 Ar); MS, m/z: 383 [(M+1)+ 20.59 %]; Anal.
Calcd. For C22H16Cl2O2 (382.05): C, 68.94; H, 4.21, Found: C, 69.12; H, 4.37.
4.1.4.3. (E)-1-(4-(Benzyloxy)phenyl)-3-(4(dimethylamino)phenyl) prop-2-en-1-one (9f). Yield 95%; m.p. 124-
126 oC; IR (KBr, cm-1): 3032 (CH aromatic), 2927 (CH aliphatic), 1672 (C=O), 1594 (C=C); 1H NMR (DMSO-
d6) δ (ppm): 2.98 (s, 6H, 2 CH3), 5.20 (s, 2H, OCH2), 6.71 (d, 2H, H3,5 Ar`-H), 7.11 (d, 2H, H3,5 Ar-H), 7.33-7.47
(m, 7H, H2,6 Ar`-H, phenyl-Hs), 7.66 (d, 1H, olefinic), 8.08 -8.11 (m, 3H, 1H olefinic, H2,6 Ar-H); 13C NMR
(DMSO-d6) δ (ppm): 41.77, 69.96, 112.22, 115.14, 116.55, 122.63, 128.28, 128.47, 128.97, 130.97, 131.09,
131.77, 137.06, 144.78, 152.34, 162.32, 187.44; MS, m/z: 357 (M+, 100 %); Anal. Calcd. For C24H23NO2 (357.17):
C, 80.64; H, 6.49, Found: C, 80.91; H, 6.70.
o
4.1.4.4. (E)-1-(4-(Benzyloxy)phenyl)-3-(furan-2-yl)prop-2-en-1-one (9g). Yield 78 %; m.p. 124-126 C; IR
(KBr, cm-1): 3037 (CH aromatic), 2930 (CH aliphatic), 1646 (C=O), 1591 (C=C); 1H NMR (DMSO-d6) δ (ppm):
5.07 (s, 2H, OCH2), 6.83-6.87 (m, 1H, H4 Ar`-H), 7.08 (d, 2H, H3,5 Ar-H), 7.30-7.47 (m, 6H, 5H, phenyl-Hs,1H
olefinic), 7.57 (d, 1H, H3 Ar`-H), 7.77 (d, 1H, olefinic), 7.89 – 7.91 (m, 3H, H2,6 Ar-H, H5 Ar`-H); MS, m/z: 304
(M+, 96.46 %); Anal. Calcd. For : C20H16O3 (304.11) : C, 78.93; H, 5.30, Found: C, 79.21; H, 5.44.
4.1.5. General procedure for the synthesis of 2-amino-6-(4-(benzyloxy)phenyl-4-(aryl)nicotinonitrile
derivatives (10a-g).
A mixture of 9a-g (2 mmol), ammonium acetate (6.16 g, 16 mmol) and malononitrile (0.132 g, 2
mmol), in ethanol (20 ml) was refluxed with stirring for 9-12 h After completion the reaction mixture was
poured into ice cold water; the crude product was filtered, dried and recrystallized from 96% ethanol with 47 -
77% yields.
4.1.5.1. 2-Amino-6-(4-benzloxy)phenyl_-4-tolylnicotinonitrile (10a). Yield 50%; m.p. 200-202 oC; IR (KBr, cm-
1
1): 3350 , 3220 (NH2), 3102 (CH aromatic), 2910 (CH aliphatic), 2202 (C≡N), 1610 (C=N), 1595 (C=C); H
NMR (DMSO-d6) δ (ppm): 2.33 (s, 3H, CH3), 5.06 - 5.21 (m, 2H, OCH2), 6.86 (s, 2H, NH2; exchangeable with
D2O), 6.92 (s, H, pyridine- H), 7.06 -7.55 (m, 9H, H3,4,5 phenyl-H, Ar`-Hs, H3,5 Ar-H ), 7.90 (d, 2H, H2.6 phenyl-
H), 8.07 (d, 2H, H2,6 Ar-H); 13C NMR (DMSO-d6) δ (ppm): 21.40, 69.94, 85.94, 113.73, 115.11, 115.32, 127.19,
16

128.17, 128.23, 128.39, 128.96, 129.69, 130.94, 136.65, 154.89, 156.99, 159.62, 162.79; MS, m/z: 391 (M+, 14.41
%), 392 [(M+1)+, 4.54%]; Anal. Calcd. For C26H21N3O (391.17) C, 79.77; H, 5.41; N, 10.73, Found: C, 80.02; H,
5.63; N, 10.94.
4.1.5.2. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4-methoxyphenyl) nicotinonitrile (10b). Yield 51 %; m.p. 190 -
192 oC; IR (KBr, cm-1): 3369, 3200 (NH2), 3095 (CH aromatic), 2919 (CH aliphatic), 2208 (C≡N), 1607 (C=N),
1
1590 (C=C); H NMR (DMSO-d6) δ (ppm): 3.80 (s, 3H, OCH3), 5.15 – 5.20 (m, 2H, OCH2), 7.03 (d, 2H, H3,5
Ar`-H, J = 8.8 Hz), 7.12 (d, 2H, H3,5 Ar-H, J = 8.8 Hz), 7.33 - 7.48 (m, 5H, phenyl-H), 7.70 (d, 2H, H2,6 Ar`-H, J
= 8.8 Hz), 8.06 (s, H, pyridine- H), 8.27 (d, 2H, H2,6 Ar-H, J = 8.8 Hz), 8.89 (s, 2H, NH2; exchangeable with
D2O); 13C NMR (DMSO-d6) δ (ppm): 56.07, 69.94, 85.61, 113.55, 114.40, 127.09, 128.99, 129.12 130.68, 135.20,
154.62, 156.99, 157.85,161.22, 161.95; MS, m/z: 407 (M+, 26.08 %), 408 [(M+1)+, 8.07 %], 409 [(M+2)+, 1.93
%]; Anal. Calcd. For C26H21N3O2 (407.16): C, 76.64; H, 5.19; N, 10.31, Found: C, 76.88; H, 5.34; N, 10.62.
4.1.5.3. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(2-hydroxyphenyl) nicotinonitrile (10c). Yield 70 %; m.p. 305 -307
oC; IR (KBr, cm-1): 3421, 3290, 3220 (OH, NH2), 3150 (CH aromatic), 2913 (CH aliphatic), 2201 (C≡N), 1628
(C=N), 1584 (C=C); 1H NMR (DMSO-d6) δ (ppm): 5.22 (s, 2H, OCH2), 7.19 (d, 2H, H3,5 Ar-H, J = 8.8 Hz), 7.33
– 7.50 (m, 7H, 5H, phenyl-Hs, 2H, H3,5Ar`-H), 7.65 – 7.69 (m, 2H, H4,6Ar`-H), 8.29 (s, H, pyridine- H), 8.38 (d,
2H, H2,6 Ar-H, J = 8.8 Hz), 8.88, 11.60 (2s, 3H, NH2, OH; exchangeable with D2O); 13C NMR (DMSO-d6) δ
(ppm): 69.91, 85.09, 111.50, 113.49, 117.88, 121.42, 126.68, 128.34, 128.44, 128.96, 130.85, 133.52, 137.24,
155.98, 156.23, 157.59, 161.14; MS, m/z: 393 (M+, 5.94 %), 395 [(M+2)+, 4.10 %]; Anal. Calcd. For C25H19N3O2
(393.14): C, 76.32; H, 4.87; N, 10.68, Found: C, 76.47; H, 4.98; N, 10.89.
4.1.5.4. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(2-chlorophenyl) nicotinonitrile(10d). Yield 66 %; m.p. 115 -117
oC; IR (KBr, cm-1): 3321,3210 (NH2), 3062 (CH aromatic), 2921 (CH aliphatic), 2210 (C≡N), 1610 (C=N), 1596
(C=C); 1H NMR (DMSO-d6) δ (ppm): 5.12 – 5.22 (m, 2H, OCH2), 6.99 (s, 2H, NH2; exchangeable with D2O),
7.08 (d, 2H, H3,5 Ar-H, J= 8.8 Hz), 7.14 (s, H, pyridine- H), 7.30 -7.73 (m, 9H, 5H, phenyl-Hs, 4H, Ar`-Hs), 8.06
(d, 2H, H2,6 Ar-H, J= 8.8 Hz); 13C NMR (DMSO-d6) δ (ppm):70.04, 85.69, 111.55, 114.31, 115.34, 127.76,
128.16, 128.25, 128.40, 128.95, 130.14, 130.53, 130.58, 130.94, 131.90, 132.16, 136.98, 137.28, 156.23, 157.89,
161.54; MS, m/z: 411 (M+, 10.18 %), 413 [(M+2)+ 3.76 %]; Anal. Calcd. For C25H18ClN3O (411.11): C, 72.90;
H, 4.40; N, 10.20; Found: C, 72.84; H, 4.63; N, 10.46.
4.1.5.5. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(2,4-dichlorophenyl) nicotinonitrile (10e). Yield 50%; m.p. 105 -
107 oC; IR (KBr, cm-1): 3382, 3220 (NH2), 3032 (CH aromatic), 2915 (CH aliphatic), 2199 (C≡N), 1623 (C=N),
1
1571 (C=C); H NMR (DMSO-d6) δ (ppm): 5.01 (s, 2H, NH2; exchangeable with D2O) 5.08 – 5.20 (m, 2H,
OCH2), 6.80 (s, H, pyridine- H), 7.06 (d, 2H, H3,5 Ar-H, J= 8.8 Hz), 7.15 (d, 1H, H5 Ar`-H), 7.18 - 7.56 (m, 5H,
phenyl-Hs), 7.80 (d, 1H, H6 Ar`-H), 7.89 (d, 2H, H2,6 Ar-H, J= 8.8 Hz), 8.10 (s, 1H, H3 Ar`-H); 13C NMR (DMSO-
d6) δ (ppm): 69.94, 85.52, 111.98, 113.55, 115.11, 127.16, 128.26, 128.39, 128.47, 128.76, 128.96, 130.94,
131.52, 133.75, 137.30, 156.55, 159.47, 160.83; MS, m/z: 445 (M+, 12.78%), 447 [(M+2)+, 5.44 %], 449 [(M+4)+,
0.74 %]; Anal. Calcd. For C25H17Cl2N3O (445.07): C, 67.28; H, 3.84; N, 9.41; Found: C, 67.50; H, 4.01; N, 9.67.
4.1.5.6. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4 (dimethylamino)phenyl) nicotinonitrile (10f). Yield 47 %; m.p.
145 -147 oC; IR (KBr, cm-1): 3325 , 3209 (NH2), 3031 (CH aromatic), 2914 (CH aliphatic), 2204 (C≡N), 1619
17

1
(C=N), 1597 (C=C); H NMR (DMSO-d6) δ (ppm): 3.01 (s, 6H, CH3), 5.09 (s, 2H, OCH2), 6.71, (d, 2H, H3,5
Ar`-H, J = 8.8 Hz), 6.81 (d, 2H, H3,5 Ar-H, J = 8.8 Hz), 7.33 - 7.46 (m, 5H, phenyl-Hs), 7.72 (d, 2H, H2,6 Ar`-H,
J = 8.8 Hz), 7.78 (s, H, pyridine- H), 7.87 (d, 2H, H2,6 Ar-H, J = 8.8 Hz), 8.29 (s, 2H, NH2; exchangeable with
D2O); 13C NMR (DMSO-d6) δ (ppm): 41.75, 69.85, 85.66, 112.25, 115.29, 115.42, 116.00, 116.75, 119.21 128.22,
128.41, 128.96, 129.39, 129.53, 130.52, 130.77, 134.06, 137.28, 154.79, 159.30; MS, m/z: 420 (M+, 6.76 %), 421
[(M+1)+, 3.32 %]; Anal. Calcd. For C27H24N4O (420.20): C, 77.12; H, 5.75; N, 13.32;, Found: C, 77.34; H, 5.89;
N, 13.49.
4.1.5.7. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(furan-2-yl)nicotinonitrile (10g). Yield 52 %; m.p. 182 -184 oC; IR
(KBr, cm-1): 3337 , 3201 (NH2), 3050 (CH aromatic), 2926 (CH aliphatic), 2207 (C≡N), 1620 (C=N), 1590
(C=C); 1H NMR (DMSO-d6) δ (ppm): 5.16 – 5.20 (m, 2H, OCH2), 6.75 – 6.80, (m , H, H4 Ar`-H), 6.90 (s, 2H,
NH2; exchangeable with D2O), 6.97 (s, H, pyridine- H), 7.10 (d, 2H, H3,5 Ar-H, J = 8.8 Hz), 7.25 -7.52 (m, 5H,
phenyl-Hs), 7.65 (d, H, H5 Ar`-H), 7.83 (d, H, H3 Ar`-H), 8.06 (d, 2H, H2,6 Ar-H, J = 8.8 Hz); 13C NMR (DMSO-d6)
δ (ppm): 69.92, 84.09, 104.10, 112.24, 113.38, 114.28, 127.99, 128.25, 128.93, 128.97, 129.12 130.47, 137.09,
141.59, 149.30, 158.88, 160.60, 161.60; MS, m/z: 367 (M+, 100 %), 368 [(M+1)+, 27.77 %], 369 [(M+2)+, 4.25
%]; Anal. Calcd. For C23H17N3O2 (367.13): C, 75.19; H, 4.66; N, 11.44, Found: C, 75.45; H, 4.80; N, 11.62.
4.1.6. General procedure for the synthesis of 6-(4-(benzyloxy)phenyl)-4-(aryl)-2-oxo-1,2dihydropyridine-3-
carbonitrile derivatives.(11a-d)
A mixture of 9b–d, 9g (0.01 mol), ammonium acetate (6.16 g, 0.08 mol) and ethylcyanoacetate (1.13g,
0.01 mol) was refluxed in ethanol (20ml) for 9-12 h. After completion of the reaction, it was further cooled and
filtered the solid. The solid was purified by ethanol to give derivatives 11a-d.
4.1.6.1. 6-(4-(Benzyloxy)phenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (11a). Yield
79 %; m.p. 239-241oC; IR (KBr, cm-1): 3240 (NH), 3023 (CH aromatic), 2906 (CH aliphatic), 2216 (C≡N), 1641
1
(C=O), 1598 (C=C); H NMR (DMSO-d6) δ (ppm): 3.82 (s, 3H, OCH3), 5.19 (s, 2H, OCH2), 6.57 (s, H, NH;
exchangeable with D2O), 6.73 (s, H, pyridone- H), 7.08- 7.15 (m, 4H, H3,5 Ar`-H, H3,5 Ar-H), 7.32 - 7.45 (m, 5H,
phenyl-Hs), 7.69 (d, 2H, H2,6 Ar`-H), 7.85 (d, 2H, H2,6 Ar-H) ); 13C NMR (DMSO-d6) δ (ppm): 55.91, 69.92,
105.45, 114.65, 115.63, 117.56, 125.06, 127.22, 127.46, 128.68, 128.96, 129.89, 130.47, 137.08, 158.40, 161.18,
161.51, 162.78; MS, m/z: 408 (M+, 7.18%), 409 [(M+1)+, 2.01 %]; Anal. Calcd. For C26H20N2O3 (408.15): C,
76.45; H, 4.94; N, 6.86, Found: C, 76.80; H, 4.91; N, 7.12.
4.1.6.2. 6-(4-(Benzyloxy)phenyl)-4-(2-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (11b). Yield
85 %; m.p. 290 -292oC; IR (KBr, cm-1): 3375 – 3268 (br. OH, NH), 3032 (CH aromatic), 2929 (CH aliphatic),
2202 (C≡N), 1698 (C=O), 1601 (C=C) ; 1H NMR (DMSO-d6) δ (ppm): 5.20-5.22 (m, 2H, OCH2), 6.81 (s, H, NH;
exchangeable with D2O), 7.14 (d, 2H, H3,5 Ar-H, J = 8.8 HZ), 7.33 – 7.48 (m, 5H, phenyl-Hs), 7.62-7.70 (m, 2H,
H3,5 Ar`-H), 7.94 (s, H, Pyridone- H), 7.99 (d, H, H4 Ar`-H), 8.26 (d, 2H, H2.6 Ar-H, J = 8.8 HZ), 8.48 (d, H, H6
Ar`-H), ), 11.02 (s, H, OH; exchangeable with D2O) ; 13C NMR (DMSO-d6) δ (ppm): 69.93, 105.02, 115.08,
115.25, 115.57, 116.17, 116.72, 117.48, 117.91, 121.22, 124.70, 126.52, 128.22, 128.96, 129.56, 130.01, 137.29,
158.93, 160.20, 166.75; MS, m/z: 394 (M+, 3.40 %), 395 [(M+1)+, 7.38 %]; Anal. Calcd. For C25H18N2O3
(394.13): C, 76.13; H, 4.60; N, 7.10, Found: C, 76. 34; H, 4.73; N, 7.36.
18

4.1.6.3.6-(4-(Benzyloxy)phenyl)-4-(2-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (11c). Yield 70
%; m.p. 260 -262 oC; IR (KBr, cm-1): 3272 (NH), 3030 (CH aromatic), 2904 (CH aliphatic), 2214 (C≡N), 1650
(C=O ), 1603 (C=C); 1H NMR (DMSO-d6) δ (ppm): 5.18 (s, 2H, OCH2), 6.77 (s, H, NH; exchangeable with D2O),
7.11 (d, 2H, H3,5 Ar-H, J = 8.8 HZ), 7.30 – 7.55 (m, 9H, 5H, phenyl-Hs, 4H, Ar`-Hs), 7.65 (s, H, pyridone- H),
7.85 (d, 2H, H2,6 Ar-H, J = 8.8 HZ); 13C NMR (DMSO-d6) δ (ppm): 70.05, 104.72, 114.85, 115.70, 123.33, 127.44
128.24, 128.47, 128.96, 129.93, 131.50, 135.73, 136.99, 158.33, 159.93, 160.87, 166.54; MS, m/z: 412 (M+, 100
%), 414 [(M+2)+, 35.38%]; Anal. Calcd. For C25H17ClN2O2 (412.10): C, 72.73; H, 4.15; N, 6.79, Found: C, 72.51;
H, 4.38; N, 6.98.
4.1.6.4. 6-(4-(Benzyloxy)phenyl)-4-(furan-2-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (11d). Yield 85%;
o
m.p. 214 -216 C; IR (KBr, cm-1): 3221 (NH), 3050 (CH aromatic), 2926 (CH aliphatic), 2205 (C≡N), 1720
(C=O), 1595 (C=C); 1H NMR (DMSO-d6) δ (ppm): 5.16 – 5.20 (m, 2H, OCH2), 6.81 – 6.83, (m , H, H4 Ar`-H),
7.14 (d, 2H, H3,5 Ar-H, J = 8.8 HZ), 7.28 (d, H, H5 Ar`-H), 7.37-7.52 (m, 5H, phenyl-Hs), 7.67 (d, H, H3 Ar`-H),
7.83 (d, 2H, H2,6 Ar-H, J = 8.8 HZ), 8.01 (s, H, NH; exchangeable with D2O), 8.08 (s, H, pyridone- H); 13C NMR
(DMSO-d6) δ (ppm): 69.93, 104.55, 110.57, 114.09, 115.68, 121.29, 123.99, 128.23, 128.47, 129.79, 136.10,
143.65, 149.87, 158.32, 159.42, 160.21, 169.95; MS, m/z: 368 (M+, 100 %), 369 [(M+1)+, 26.93 %], 370 [(M+2)+,
4.44 %]; Anal. Calcd. For C23H16N2O3 (368.12): C, 74.99; H, 4.38; N, 7.60, Found: C, 75.16; H, 4.51; N, 7.79.
4.2. Biological evaluation
4.2.1. Methodology: cell culture
Cancer cells from different cancer cell lines; Human prostate carcinoma cell lines (PC-3), hepatocellular
carcinoma cell lines (HEPG-2), and human breast adenocarcinoma cell line (MCF-7) were obtained from
VACSERA- Cell Culture Unit, Cairo, Egypt. Also, the cytotoxic evaluation of all compounds against normal cells
WI-38 was carried out to explore the toxicity and selectivity of the tested compounds. For comparison, 5-FU (5-
Fluro uracil) was used as a standard reference drug.
Anti-proliferative activities of the synthesized compounds were carried out based on MTT assay [54]. MTT
assay is a laboratory experiment, and a standard colorimetric assay (an assay measures changes in color) for
measuring cellular growth, yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a
tetrazole] was reduced to purple formazan in the mitochondria of living cells. Briefly, human cancer cell lines
were dropped in 96-well plates at a density of 3–8×103 cells/well. Next, the wells were incubated for 12 h in a
5% CO2 incubator at 37 °C. Then, for each well, the growth medium was exchanged with 0.1 mL of fresh
medium containing graded concentrations of the test compounds to be or equal DMSO and incubated for two
days. Then 10 μl MTT solution (5 μg/ml) was added to each well, and the cells were incubated for an additional
4 h. The crystals of MTT-formazan were dissolved in 100 μl of DMSO; the absorbance of each well was
measured at 490 nm using an automatic ELISA reader system (TECAN, CHE). The IC50 values were calculated
using the nonlinear regression fitting models (Graph Pad, Prism Version 5). The means of at least three separate
experiments gave the reported results. Statistical differences were analyzed according to a one-way ANOVA
test wherein the differences were significant at p < 0.05.
19

4.2.2. Cell cycle analysis
PC-3 cells were seeded in a 6-well plate at a concentration of 1 X 105 cells per well, then incubated for 24 h. The
cells were treated with (0.1% DMSO) vehicle or 2.04 μM of 10f compound for 24 h. After that, cells were
harvested and fixed for 12h using ice-cold 70% ethanol at 4 °C. Removal of ethanol and washing cells with cold
PBS was done. Then incubated for 30 min at 37°C in 0.5 mL of PBS containing 1 mg/mL Rnase. The cells were
stained for 30 min with propidium iodide in the dark. Then flow cytometer was used to detect DNA contents.
[55].
4.2.3. Apoptosis detection studies
4.2.3.1. Annexin V-FITC assay.
PC-3 cells were seeded in a 6-well plate (1 X105 cell/well), incubated for 24 h, then treated with vehicle (0.1%
DMSO) or 2.04 µM of 10f compound for 24 h. The cells were then harvested, washed using PBS and stained for
15 min at room temperature in the dark using annexin V-FITC and PI in binding buffer (10mM HEPES, 140mM
NaCl, and 2.5mM CaCl2 at pH 7.4), then analyzed by the flow cytometer [56].
2.2.3.2. Determination of the active caspase 3
The active caspase 3 level was measured by using Quantikine-Human active Caspase 3 Immunoassay (R&D
Systems, Inc. Minneapolis, USA) according to the manufacturer protocol. Briefly, after washing the cells with
PBS, the cells were collected and lysed by adding it to the extraction buffer containing protease inhibitors (1 mL
per 1 x 107 cells.) then the lysate was diluted immediately before the assay. At the end of the assay, the optical
density of each well was determined within 30 min using a microplate reader set at 450 nm.
4.2.3.3. Determination of the genes expression of some apoptosis key markers (BAX and BCL2)
Cells were obtained from American Type Culture Collection, cells were grown in RPMI 1640 containing 10%
fetal bovine serum at 37°C, stimulated with the compounds to be tested for BAX or BCL2 and lysed with Cell
Extraction Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured
for human active BAX or BCL2 content. (cells are Plated in a density of 1.2 – 1.8 × 10,000 cells/well in a volume
of 100µl complete growth medium + 100 ul of the tested compound per well in a 96-well plate for 24 hours before
measured for human active BAX or BCL2).
4.2.4. Determination of PIM-1 kinase activity
The effects of the synthesized compounds on the activity of PIM-1 kinase were measured using Human proto-
oncogene serine/threonine- protein kinase PIM-1 (PIM-1) ELISA Kit (catalog #MBS701210). The cells were
centrifuged for 15 min at 1000x g, 2-8 °C and assayed immediately according to the manufacturer's instructions.
Shortly, the assay was performed using 100 mL of the supernatant of the cells in each well, which were incubated
for 2 h at 37°C before starting the assay procedure. Finally, the optical density of each well was determined within
5 min using a microplate reader set at 450 nm.
4.3. In silico physicochemical and ADME properties prediction
The molecular structures were converted into SMILES database using Chemdraw 12.0. Then, these SMILES
were inserted as input in SwissADME website to calculate the physicochemical descriptors, lipophilicty,
pharmacokinetics properties, ADME parameters, and medicinal chemistry friendliness.
20

Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
Declared none.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-
profit sectors.
References
1- Lopez, J.; Tait, S., W., G. Mitochondrial apoptosis: killing cancer using the enemy within. British
Journal of Cancer,2015,85,1-6.
2- Adams, J., M.; Cory, S. The Bcl-2 protein family: arbiters of cell survival. Science, 1998, 281, 1322–
26.
3- Wei, H.; John, J., K. Anticancer therapy targeting the apoptotic pathway. The Lancet Oncology, 2003,
4(12), 721–729.
4- Alex, R. D. D.; Stephanie, G.; Andreas, S.; David, L. V. Thirty years of BCL-2: translating cell death
discoveries into novel cancer therapies. Nature Reviews Cancer, 2016, 16(2), 99–109.
5- Susan, E. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol., 2007, 35(4), 495–516.
6- Howard, Y., C.; Xiaolu, Y. Proteases for Cell Suicide: Functions and Regulation of Caspases.
Microbiology and molecular biology reviews, 2000, 64, 821–884.
7- Taylor, R., C.; Cullen, S., P.; Martin, S., J. Apoptosis: controlled demolition at the cellular level. Nat
Rev Mol Cell Biol, 2008, 9(3), 231–241.
8- Xiao-He, Y.; Todd, L. S.; Xuesong, Liu.; Bryn, R., B.; Christopher, J. F.; Ann, D., T. Reconstitution of
Caspase 3 Sensitizes MCF-7 Breast Cancer Cells to Doxorubicin- and Etoposide-induced Apoptosis.
Cancer Research, 2001, 61, 348-354.
9- Ahmed, M.; Mona, M.; Hassan, A.; Ola, R.; Omaima, S.; Mohamed, E.; Zaki, A., S.; Hayam, A. New
3-Cyano-2-Substituted Pyridines Induce Apoptosis in MCF 7 Breast Cancer Cells. Molecules, 2016,
21(230), 1-25
10- Michael, D., W.; Chaohong, S.,b.; Aaron, K.; Daryl, S.; Kathy, S.; Ethan, H.; Liping, Y.; David, G., N.;
Jun, C.; Sha, J.; Kenneth, M., C.; Yihong, F.; Steven, N., A.; Binumol, I.; Edward, T., O.; Philip, J., H.;
Saul, H., R.; Steven, W., E. Discovery of a novel small molecule binding site of human surviving.
Bioorganic & Medicinal Chemistry Letters 2007, 17, 3122–3129.
11- Khaled, A., M., A.; Ghada, H., A.; Abeer, M., E. Eco-friendly synthesis of novel cyanopyridine
derivatives and their anticancer and PIM-1 kinase inhibitory activities. European Journal of Medicinal
Chemistry, 2017, 134 ,357-365.
12- Cheney, I., W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.; Hamatake, R.; Hong, Z.; Wu, J. Z.
Identification and structure-activity relationships of substituted pyridines as inhibitors of PIM-1 kinase.
Bioorg. Med. Chem. Lett. 2007, 17, 1679-1683.
13- van der Lught, N., M.; Domen, j.; Verhoeven, E.; Linders, K.; van der Gulden, H.; Allen, J.; Berns, A.
Proviral tagging in E mu-myc transgenic mice lacking the PIM-1 proto-oncogene leads to
compensatory activation of PIM-2. EMBO J., 1995, 14, 2536-2544.
14- Feldman J., D.; Vician, L; Crispino, M.; Tocco, G.; Marcheselli, V., L.; Bazan, N., G.; Baudry, M.;
Herschman, H., R. KID-1, a protein kinase induced by depolarization in brain. J Biol Chem., 1998,
273(26), 16535-43.
15- Wang, Z.; Bhattacharya, N.; Weaver, M.; Petersen, K.; Meyer, M.; Gapter, L.; Magnuson, N., S. PIM-
1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis.
J Vet Sci., 2001, 2(3), 167-79.
21

16- Tursynbay, Y.; Zhang, J.; Li, Z.; Tokay, T.; Zhumadilov, Z.; Wu, D.; Xie, Y. PIM-1 kinase as cancer
drug target: an update (Review). Biomed Rep, 2016, 4, 140–146.
17- Keane, N., A.; Reidy, M.; Natoni, A.; Raab, M., S.; O'Dwyer, M. Targeting the PIM kinases in
multiple myeloma. Blood Cancer J, 2015, 5, e325.
18- Foulks, J., M.; Carpenter, K., J.; Luo, B.; Xu, Y.; Senina, A.; Nix, R.; Chan, A.; Clifford, A.; Wilkes,
M.; Vollmer, D.; Brenning, B1.; Merx, S.; Lai, S.; McCullar, M., V.; Ho, K., K.; Albertson, D., J.;
Call, L., T.; Bearss, J.,J.; Tripp, S., 5.; Liu, T.; Stephens, B., J.; Mollard, A.; Warner, S. L.; Bearss, D.,
J.; Kanner, S.;B. A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial
carcinomas. Neoplasia, 2014, 16, 403–412.
19- Decker, S.; Finter, J.; Forde, A., J.; Kissel, S.; Schwaller, J.; Mack, T.,S., Kuhn, A.; Gray, N.; Follo,
M.; Jumaa, H.; Burger, M.; Zirlik, K.; Pfeifer, D.; Miduturu, C., V.; Eibel, H.; Veelken, H.; Dierks, C.
PIM kinases are essential for chronic lymphocytic leukemia cell survival (PIM2/3) and CXCR4-
mediated microenvironmental interactions (PIM1). Mol Cancer Ther, 2014, 13, 1231–1245.
20- - Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X., H.; Castillo, J.; Sim, J.; Yu, J.; Wang, Y.; Langowski, J.,
L.; Holash, J.; Shannon, K.; Garcia, P., D. PIM-2 is required for maintaining multiple myeloma cell
growth through modulating TSC2 phosphorylation. Blood, 2013, 122, 1610–1620.
21- Drygin, D.; Haddach, M.; Pierre, F.; Ryckman, D., M. Potential use of selective and nonselective PIM
kinase inhibitors for cancer therapy. J Med Chem, 2012, 55, 8199–8208.
22- Guo, S.; Mao, X.; Chen, J.; Huang, B.; Jin, C.; Xu, Z.; Qiu, S. Overexpression of PIM-1 in bladder
cancer. J Exp Clin Cancer Res, 2010, 29, 161–167.
23- Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J. PIM serine/threonine kinases in
the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica, 2010, 95,
1004–1015.
24- Nancy, S., M.; Zeping, W.; Gang, D.; Raymond, R. Why target PIM1 for cancer diagnosis and
treatment? Future Oncol., 2010, 6(9), 1461–1478.
25- Le, B., T.; Kumarasiri, M.; Adams, J., R., J.; Yu, M.; Milne, R.; Sykes, M., J.; Wang, S. Targeting PIM
kinases for cancer treatment: opportunities and challenges. Future Med Chem, 2015, 7, 35–53.
26- Narlik-Grassow, M.; Blanco-Aparicio, C.; Carnero, A. The PIM family of serine/threonine kinases in
cancer. Med Res Rev, 2014, 34, 136–159.
27- Nawijn, M., C.; Alendar, A.; Berns, A. For better or for worse: the role of PIM oncogenes in
tumorigenesis. Nat Rev Cancer, 2011, 11, 23–34.
28- Cuypers, H., T.; Selten, G.; Quint, W.; Zijlstra, M.; Maandag, E., R.; Boelens, W.; van Wezenbeek, P.;
Melief, C.; Berns, A. Murine leukemia virus-induced T-cell lymphomagenesis: integration of
proviruses in a distinct chromosomal region. Cell, 1984, 37, 141–150.
29- Wang Z, Bhattacharya N, Weaver M et al.: PIM-1: a serine/threonine kinase with a role in cell survival,
proliferation, differentiation and tumorigenesis. J. Vet. Sci. 2, 167–179 (2001).
30- Losman, J.; Chen, X., P.; Jiang, H.; Pan, P., Y.; Kashiwada, M.; Giallourakis, C.; Cowan, S.; Foltenyi,
K.; Rothman, P.IL-4 signaling is regulated through the recruitment of phosphatases, kinases, and SOCS
proteins to the receptor complex. Cold Spring Harb Symp Quant Biol, 1999, 64, 405-416.
31- Malte, B.; Tarik, M. Molecules in focus the serine/threonine kinase PIM-1. The International Journal of
Biochemistry & Cell Biology, 2005, 37, 726–730.
32- Amaravadi, R.; Thompson, C., B. The survival kinases Akt and Pim as potential pharmacological
targets. J. Clin. Invest, 2005, 115, 2618–2624.
33- Moroy, T.; Grzeschiczek, A.; Petzold, S.; Hartmann, K., U. Expression of a PIM-1 transgene
accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice. Proc. Natl Acad. Sci. USA 90,
1993, 10734–10738.
34- Fiskus, W., C.; Buckly, K., M.; Rao, R.; Yongchao, W.; Atul, J.; Daniel, G., C.; Anand, J.; Celalettin,
U.; Peter, A.; Cornelia, Q.; Kapil, N., B. Synergistic activity of co-treatment with PIM-1 kinase
inhibitor SGI-1776 and histone deacetylase inhibitor panobinostat or heat shock protein 90 inhibitor
AUY922 against human CML and myeloproliferative neoplasm (MPN) cells. Presented at: 51st ASH
Annual Meeting and Exposition. New Orleans, LA, USA, 5–8 December 2009.
22

35- Farag, M., A., A. Synthesis and Antimicrobial Evaluation of Some Novel Bis-α, β-Unsaturated
Ketones, Nicotinonitrile, 1,2-Dihydropyridine-3-carbonitrile, Fused Thieno[2,3-b] pyridine and
Pyrazolo[3,4-b]pyridine Derivatives. Int. J. Mol. Sci., 2013,14, 2967-2979.
36- Xorge, A., D.; Beatriz, G.; Erika, H.; Jorge, S. Studies on bis- (benzylisoquinolines). I. Synthesis of dl-
N-(4-hydroxyphenylacetyl)-5-iodo-4-acetoxy-3-methoxyphenyl alanine methyl ester. J. Am. Chem.
Soc., 1955, 77, 1288-1290.
37- Siham, A., L.; Faeza, H., A.; Nabilah, A., A. Synthesis, characterization and evaluation of antioxidant
activities of some novel chalcones analogues. Chem. Cent. J., 2014, 8, 1-10.
38- Eman, M., F.; Hebat-Allah, S., A.; Randa, E., A.; Wafaa, A., Z. Synthesis and Cytotoxic Effect of
Some Novel 1,2-Dihydropyridin-3 carbonitrile and Nicotinonitrile Derivatives. Molecules, 2016, 21, 1-
15.
39- Yernar T.; Jinfu Z.; Zhi L.; Tursonjan T.; Zhaxybay Z.; Denglong W.; Yingqiu X. Pim-1 kinase as cancer
drug target: An update (Review). Biomedical Reports, 2016, 4, 140-146.
40- Andree, H., A.; Reutelingsperger, C., P.; Hauptmann, R.; Hemker, H., C.; Hermens, W., T.; Willems,
G. M. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers, J. Biol.
Chem., 1990, 265, 4923–4928.
41- Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. A novel assay for apoptosis. Flow
cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein
labelled Annexin V, J. Immunol. Methods,1995, 184, 39–51.
42- SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry
friendliness of small molecules. Sci. Rep. 7, 42717. doi: 10.1038/srep42717.
43- Zhao, Y., H.; Abraham, M., H.; Le, J.; Hersey, A.; Luscombe, C., N.; Beck, G.; Cooper, I. Rate-limited
steps of human oral absorption and QSAR studies Pharmacol. Res., 2002, 19, 2002, 1446-1457
44- Jonathan, B B, Georgina A H ( 2010) New Substructure Filters for Removal of Pan Assay Interference
Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J Med Chem 53:
2719–2740. doi: 10.1021/jm901137j.
45- William, M., C.; I., V.; Sherlock, T., W. Systematic organic chemistry, third ed, D. Van Nostrand
Company, Inc, 1937, 99-100.
46- Magnus, R.; Teerawut, B.; Hannah, B.; Erli, S. Continuous-flow hydration–condensation reaction:
Synthesis of α,β-unsaturated ketones from alkynes and aldehydes by using a heterogeneous, solid acid
catalyst. Beilstein. J. Org. Chem., 2011, 7, 1680–1687.
47- Arumugam, T. Regiospecific synthesis and biological evaluation of spirooxindolopyrrolizidines via
[3+2] cycloaddition of azomethine ylide. Eur. J. Med. Chem., 2010, 45, 6120- 6126.
48- Thanh-Dao, T.; Thi-Thao-Nhu, N.; Tuong-Ha, D.; Thi-Ngoc-Phuong, H.; Cat-Dong, T.; Khac-Minh, T.
Synthesis and Antibacterial Activity of Some Heterocyclic Chalcone Analogues Alone and in
Combination with Antibiotics. Molecules, 2012, 17, 6684-6696.
49- Sunniya, I.; Sardraz, K.; Aishah, B.; Safia, M.; Muhammad, A.; Abdel-Hamid, E.; Salim, S.; Xin, G.;
Ghayoor, A.C.; Amir, F.; Rahman, S.Z.S. Synthesis and evaluation of modified chalcone based p53
stabilizing agents. Bioorg Med Chem Lett., 2017. 27, 4101-4106.
50- Nemai, C.G.; Sushmita, R.; Pallab, M. Cerium (III)-catalyzed regioselective coupling of 2-
hydroxychalcones and polyphenols: an efficient domino approach towards synthesis of novel dibenzo-
2,8dioxabicyclo [3.3.1] nonanes. RSC Adv., 2014, 4, 42078–42086.
51- Thekke, V.S.; Badiadka, N.; Hemmige, S.Y. Synthesis and characterization of some chalcones and
their cyclohexenone derivatives. Cent. Eur. J. Chem., 2010, 8, 174–181.
52- Hetal, C.S.; Vaishali, H.S.; Nirmal, D.D. A novel strategy for the synthesis of 2-amino-4,6-
diarylnicotinonitrile. ARKIVOC, 2009, (ii), 76-87.
53- Monica, M.; Olivier, d.; Jacobus, V.V.; Anikó Go¨blyo¨s, Jacobien, K. von Frijtag Drabbe Ku¨nzel,
Thea, M.; Regina, L.; Henk, de V.; Margot, W.B.; Johannes, B.; Adriaan, P.IJ. 2-Amino-6-furan-2-yl-
4-substituted Nicotinonitriles as A2A Adenosine Receptor Antagonists, J. Med. Chem., 2008, 51,
4449–4455.
54- van Meerloo, J.; Kaspers; G., J., L.; Cloos, J. Cell Sensitivity Assays: The MTT Assay. In: Cree I. (eds)
Cancer Cell Culture. Methods in Molecular Biology (Methods and Protocols). Humana Press, 2011,
731, 237-245.
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55- Wang, J.; Lenardo, M.J. Roles of caspases in apoptosis, development, and cytokine maturation
revealed b,y homozygous gene deficiencies. J. Cell Sci., 2000, 113,753-757.
56- Lo, K., K.; Lee, T., K.; Lau, J., S.; Poon, W., L.; Cheng, S., H. Luminescent biological probes derived
from ruthenium (II) estradiol polypyridine complexes, Inorg. Chem. 2008, 47, 200-208.
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Graphical abstract
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Highlights
 Synthesis of Novel 14 Cyanopyridines derivatives.
 All the analogues were tested on three different cancer cell lines (PC3, HepG2, MCF-
7).
 Assessment of their pro-apoptotic potential.
 Evaluation of PIM-1 kinase inhibitory activity.
 Cell cycle analysis of the most potent PIM-1 kinase inhibitor.
 In silico prediction of physicochemical and ADME properties.
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Dear Respected Editor,
Conflict of interest for the manuscript entitled:
" Apoptosis: A Target for Anticancer Therapy with Novel Cyanopyridines"
The authors declare no conflict of interest.
Please address all correspondence concerning this manuscript to me
and feel free to correspond with me by e-mail (amelfarrag@ymail.com).
Most Sincerely,
On behalf of the authors,
Amel M Farrag
Ass. Prof. of Pharmaceutical Chemistry.
Faculty of Pharmacy (Girls) Al-Azhar University.
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