Design, synthesis, cytotoxicity screening and molecular docking of new 3-cyanopyridines as survivin inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2019.103358

Recently, targeting survivin proved to be an attractive strategy for developing anticancer agents. Survivin overexpression is highly correlated with cancer aggressiveness, recurrence and resistance to chemotherapeutic treatment and radiotherapy. Additionally, survivin is overexpressed selectively in most cancer types with a very little expression in completely differentiated cells, which encouraged us to design and synthesize a novel series of 3-cyanopyridine derivatives targeting survivin. The newly synthesized compounds were evaluated for their cytotoxic activities against three cancer cell lines; PC-3, HepG2 and MDA-MB-231. Compounds 4a, 4b, 5c and 6c showed significant cytotoxic activities that were more potent than the reference drug, 5-FU. Hence, these compounds were selected to be further studied regarding their apoptotic potential in PC-3 cells. Interestingly, they decreased the level of Bcl-2 by 1.9–3.8 folds and increased the level of Bax by 6.1–8.8 folds compared to the control. Moreover, they elevated the level of active caspase-3 by 7.1–8.5 folds in comparison to the control. In order to estimate the cytotoxicity level of these compounds in non-tumorigenic cells, WI38 cells were treated with these compounds. They showed high IC₅₀ values (148.57–193.64 μM), indicating selective cytotoxicity to the tumor cells, and much less toxic effect to the normal ones. Additional studies on the mechanism of 4a, the most active compound, revealed that it induced cell cycle arrest at the G2/M phase in addition to an increase in the percentage of pre-G1 apoptotic cells. Furthermore, western blotting was carried out using different concentrations of 4a. Results showed that 4a markedly suppressed survivin expression in PC-3 cells and caused a decrease in the caspase-7/cleaved caspase-7 ratio and in Bcl-2/Bax ratio, in addition to an increase in the level of the cleaved PARP. Finally, docking study of the most active compound in the active site (dimerization site) of survivin was in agreement with the in vitro survivin inhibitory activity.

Full text of DOI:10.1016/j.bioorg.2019.103358

Journal Pre-proofs
Design, Synthesis, Cytotoxicity Screening and Molecular Docking of New 3-
Cyanopyridines as Survivin inhibitors and Apoptosis Inducers
Rehab Sabour, Marwa F. Harras, Ahmed B.M. Mehany
PII:
S0045-2068(19)30324-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103358
YBIOO 103358
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 February 2019
23 September 2019
9 October 2019
Please cite this article as: R. Sabour, M.F. Harras, A.B.M. Mehany, Design, Synthesis, Cytotoxicity Screening
and Molecular Docking of New 3-Cyanopyridines as Survivin inhibitors and Apoptosis Inducers, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103358
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Design, Synthesis, Cytotoxicity Screening and Molecular Docking of New 3-
Cyanopyridines as Survivin inhibitors and Apoptosis Inducers
Rehab Saboura, Marwa F. Harrasa* and Ahmed B. M. Mehanyb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo,
Egypt b Department of Zoology, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt
Abstract
Recently, targeting survivin proved to be an attractive strategy for developing anticancer agents.
Survivin overexpression is highly correlated with cancer aggressiveness, recurrence and
resistance to chemotherapeutic treatment and radiotherapy. Additionally, survivin is
overexpressed selectively in most cancer types with a very little expression in completely
differentiated cells, which encouraged us to design and synthesize a novel series of 3-
cyanopyridine derivatives targeting survivin. The newly synthesized compounds were evaluated
for their cytotoxic activities against three cancer cell lines; PC-3, HepG2 and MDA-MB-231.
Compounds 4a, 4b, 5c and 6c showed significant cytotoxic activities that were more potent than
the reference drug, 5-FU. Hence, these compounds were selected to be further studied regarding
their apoptotic potential in PC-3 cells. Interestingly, they decreased the level of Bcl-2 by 1.9-3.8
folds and increased the level of Bax by 6.1-8.8 folds compared to the control. Moreover, they
elevated the level of active caspase-3 by 7.1-8.5 folds in comparison to the control. In order to
estimate the cytotoxicity level of these compounds in non-tumorigenic cells, WI38 cells were
treated with these compounds. They showed high IC50 values (148.57-193.64 µM), indicating
selective cytotoxicity to the tumor cells, and much less toxic effect to the normal ones.
Additional studies on the mechanism of 4a, the most active compound, revealed that it induced
cell cycle arrest at the G2/M phase in addition to an increase in the percentage of pre-G1
apoptotic cells. Furthermore, western blotting was carried out using different concentrations of
4a. Results showed that 4a markedly suppressed survivin expression in PC-3 cells and caused a
decrease in the caspase-7/cleaved caspase-7 ratio and in Bcl-2/Bax ratio, in addition to an
increase in the level of the cleaved PARP. Finally, docking study of the most active compound in
the active site (dimerization site) of survivin was in agreement with the in vitro survivin
inhibitory activity.

Keywords: Cyanopyridine; synthesis; anticancer; cell cycle; apoptosis; survivin
1. Introduction
Cancer is a disease where important mechanisms of the cell cycle are dysfunctional, associated
with either an over reproduction and/or decreased elimination of cells [1]. Physiological
processes are organized by a balance between cellular proliferation, differentiation and apoptosis
[2]. Programmed cell death (apoptosis), is a dynamic process that can be stimulated by DNA-
damaging agents such as radiation and chemotherapeutic drugs. Studies have shown a great
relationship between increased resistance to anticancer drugs and decreased capability to
undergo apoptosis [3]. Accordingly, susceptibility to apoptosis is an important determinant of
response to anti-neoplastic therapy [4]. Therefore, suppression of apoptosis throughout
carcinogenesis is supposed to play a principal role in the progression of cancer. There are
different types of molecular mechanisms that cancer cells use to suppress apoptosis. They
acquire resistance to apoptosis via the expression of anti-apoptotic proteins such as Bcl-2 and
survivin or by down-regulation of different caspases and pro-apoptotic proteins such as Bax [5].
The relation between pro-apoptotic and anti-apoptotic levels is an important determinant of cell
survival [2].
The anti-apoptotic protein (survivin) is one of the inhibitors of apoptosis family, which is
expressed in the G2/M phases of the cell cycle almost solely in cancer cells, but absent in most
normal adult differentiated tissues. It promotes the survival of cancer cells through suppression
of caspase-mediated apoptosis [6-12]. Overexpression of survivin is positively correlated with
cancer cell resistance to chemotherapy and poor patient prognosis. Therefore, survivin is
considered one of the important targets aiming to produce new candidates of promising
anticancer activity [13, 14].
By searching literatures for chemical scaffolds effective as survivin inhibitors, we found that 2-
oxo-3-cyanopyridine derivative I has significant anti-survivin activity [15]. Hence great interest
has been focused on 3-cyanopyridine scaffold owing to the notable anticancer activity against a
wide range of cell lines [16-19]. Interestingly different members belonging to this scaffold have
been shown promising activity through interfering survivin [20]. Furthermore, some derivatives
of 2-oxo-3-cyanopyridine with higher lipophilic properties can inhibit survivin [17]. Thus, our
structure-based design strategy is originated on structural modification of compound I. This is
achieved via sparing the pharmacophoric group responsible for survivin inhibition, 3-

cyanopyridine group, and incorporating benzyloxy moiety at 6- position in order to exert more
lipophilic character to the compound, while substituting the 2- and 4- positions with different
moieties (Fig.1).
To explore the scope of this class of compounds, a novel series of 3-cyanopyridine was designed
and synthesized. All of the newly synthesized compounds were evaluated for their anticancer
activity against three cell lines; prostate cancer cell line (PC-3), liver cancer cell line (HepG2)
and breast cancer cell line (MDA-MB-231). Apoptotic studies were applied in order to estimate
the potential of the most potent compounds to induce apoptosis. Therefore, promising
compounds were evaluated for their effect on the level of some apoptosis key markers (Bcl-2,
Bax, caspase-3). Cell cycle analysis and Western blotting were carried out to confirm our
findings. Moreover, a molecular docking study was done to find out the probable binding
interactions of the most active compounds with survivin.
2. Results and discussion
2.1.
Chemistry
Scheme 1 outlines the steps used for the synthesis of the target compounds. In this work,
chalcones 3a-d were prepared as reported by condensation of benzyloxyacetophenone (1) with
different acetophenones 2a-d in the presence of NaOH [21]. The condensation reaction of 3a-d
with malononitrile in ethanol containing ammonium acetate [22] afforded the target 2-amino-3-
cyanopyridine derivatives 4a-d. The structures of compounds 4a-d were well established on the
basis of their micro-analytical analysis and spectral data. IR spectrum of compound 4b, taken as
a representative example of this series, showed absorption bands at 3296, 3142 cm−1
corresponding to the NH2 function and a sharp band at 2216 cm−1 for the nitrile group. Its
1HNMR spectrum (DMSO-d6) revealed the presence of singlet at 5.18 ppm for the O-CH2
protons and a singlet at 6.76 ppm due to H-5 of the pyridine ring, in addition to a singlet at δ 9.79
ppm for NH2 protons (exchangeable with D2O).
On the other hand, chalcones 3a-d were condensed with 2-cyanoacetamide and 2-
cyanothioacetamide in ethanol in the presence of pipridine [23] to produce 3-cyanopyridine-2-
ones 5a-d and 3-cyanopyridine-2-thiones 6a-d, respectively. The proposed structures of these
compounds were supported by elemental and spectral measurements. IR spectra of compounds
5a-d and 6a-d displayed absorption bands at 3200-3434 cm-1 assigned to NH groups besides the

appearance of absorption bands at 2205-2217 cm-1 comprising the nitrile group. Moreover, for
compounds 5a-d, the carbonyl function appeared as absorption bands around 1640 cm−1.
1HNMR spectra 5a-d and 6a-d revealed the presence of singlet signals at 5.12-5.23 ppm
corresponding to the O-CH2 protons and singlet signals at 6.60-6.95 ppm representing H-5 of the
pyridine ring.
2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity
The in vitro cytotoxicity of the synthesized compounds 4a-d, 5a-d and 6a-d was evaluated
against a panel of cell lines, namely; the prostate cancer cell line (PC-3), the liver cancer cell line
(HepG2) and the breast cancer cell line (MDA-MB-231). 5-FU, being broadly used in cancer
managing, was used as the reference drug. The results were represented as IC50 values and
shown in (Table 1). Interestingly, 4a was the most potent 3-cyanopyridine derivative. It showed
17.92 times the activity of 5-FU against PC-3 cell line, 6.68 times against HepG2 and 11.75
times against MDA-MB-231.
The screening results indicated that most of the tested compounds have excellent anti-
proliferative activity against PC-3 cell line. This is demonstrated by the IC50 values of
compounds 4a,b,d, 5b-d and 6a-d (IC50 = 0.42- 9.83 μM) which were more active than 5-FU
(IC50 = 7.53 μM). While compounds 4c and 5a displayed moderate activity (IC50 = 9.83 and
14.71 μM, respectively).
Scanning the IC50 values of the tested compounds against HepG2 cell line revealed that most of
the compounds exert good to moderate cytotoxic influence. Compounds 4a,b, 5c and 6b,c (IC50
= 1.22- 6.92 μM) were found to be more potent than 5-FU (IC50 = 8.15 μM). On the other hand,
compounds 4c,d, 5a,b,d and 6a,d showed moderate activity (IC50 = 10.64- 21.35 μM).
Regarding the anticancer activity of the target compounds against MDA-MB-231breast cell line,
Compounds 4a,b,d 5b,c and 6a-d displayed good antitumor activity (IC50 = 0.66- 9.34 μM)
compared to the reference drug (IC50 = 9.35 μM). While the rest of the compounds exhibited
moderate cytotoxicity (IC50 = 10.71- 18.38 μM).
Furthermore, in order to estimate the safety on normal cells, the most active compounds 4a, 4b,
5c and 6c were further screened on non-cancerous human WI-38 cells. It was found that these
compounds are selective cytotoxic agents on the cancer cells and much less toxic on the normal

cells WI-38 as demonstrated from their IC50 values (193.15, 193.64, 169.32 and 148.57 µM,
respectively) (Table 2)
Compounds 4a, 4b, 5c and 6c, exhibiting significant and selective cytotoxic effects, were chosen
to be further studied regarding their potential to induce apoptosis in PC-3 cells.
2.2.2. Apoptosis studies
Apoptosis is the physiologically important way of programmed cell death that compensates cell
growth and tissue homeostasis in the normal prostate gland [24]. The ability of 4a, 4b, 5c and 6c
to induce apoptosis in PC-3 cells was estimated by observing the levels of some apoptosis key
markers; Bcl-2, Bax and active caspase-3 that give clear evidence for apoptosis induction.
2.2.2.1. Effects on the levels of Bax and Bcl-2 proteins
Apoptosis is regulated by different factors including the Bcl-2 family: suppressors (Bcl-2
protein), inducers (Bax protein) and by stimulation of the apoptotic pathway through caspase-3
[25]. Many studies on human cancers have revealed the presence of a relationship between
increased levels of Bcl-2, decreased levels of Bax and uncontrolled cell growth [24,26]. Here, the
levels of Bcl-2 and Bax after treatment of PC-3 cells with 4a, 4b, 5c and 6c were determined.
The results as illustrated in Table 3 demonstrated that 4a, 4b, 5c and 6c decreased the level of
Bcl-2 by 3.8, 2.3, 3.3 and 1.9 folds, respectively and increased the level of Bax by 8.8, 7.2, 7.7
and 6.1 folds, respectively compared to the control. These results indicated that these compounds
moved the prostate cancer cells towards apoptosis.
2.2.2.2. Effects on the level of active caspase-3
The principal constituents of apoptotic pathways are the caspase family of cysteine proteases
[24]. Up to now, several earlier studies have proved that caspase-3 is an executioner of apoptosis
and that its activation results in the induction of apoptosis [25,27]. In this study, PC-3 cells were
treated with compounds 4a, 4b, 5c and 6c and the level of caspase-3 was determined. Results
revealed that the active caspase-3 was significantly overexpressed by 8.5, 7.2, 7.5 and 7.1 folds,
respectively compared to the control indicating the apoptosis induction potential of these
compounds (Table 3).

2.2.3. Cell cycle analysis:
The mechanism of the cytotoxic effect of many antitumor agents involves the combination of
induction of apoptosis together with cell cycle arrest [28, 29]. Furthermore, regulation of the cell
cycle and induction of apoptosis are suggested to be effective methods in the development of
anticancer therapeutics [30]. Thus, it was significant to know if the arrest of the cell cycle was a
part of the cytotoxic mechanism of our most active compound 4a. Flow cytometry analysis was
done on PC-3 cells after incubation with 4a for 24 h and the results were shown in Fig. 2. The
outcome proposed that 4a could induce cell cycle arrest in the G2/M phase as it increased the
DNA content in G2/M phase by 3.1 folds with subsequent reduction in G0/G1 and S phases
compared to the control cells. Moreover, it caused a notable increase in the amount of pre-G1
apoptotic cells.
The apoptotic effect of 4a was further estimated by Annexin V-FITC assay. As displayed in Fig.
3, the total apoptotic cells percentage significantly increased after treatment by this compound
indicating the capability of 4a to induce apoptosis which were consistent with the previous
results.
2.2.4. Western blotting:
Survivin is a proto-oncogene biomarker known for its anti-apoptotic properties. Increased
expression of survivin is associated with a decrease in the overall survival in cancer patients [31].
To determine whether our new compound 4a causes survivin degradation, western blotting
analysis was performed in PC-3 cell line using different concentrations of 4a. As shown in Fig.
4-A, 4a markedly inhibited survivin expression in PC-3cells in a concentration dependent
manner. Moreover, Fig. 4-B displayed the effect of 4a on the expression of caspase-7 and
cleaved caspase-7 and showed that 4a caused a significant increase of cleaved caspase-7 with a
subsequent decrease in the caspase-7/cleaved caspase-7 ratio. In addition, the apoptosis inhibitor
Bcl-2 expression was effectively decreased, while the apoptosis inducer Bax protein expression
was increased as shown in Fig. 4-C.
PARP (poly ADP-ribose polymerase), the substrate of caspases, is cleaved during apoptosis by
caspases. [32]. Consistent with survivin inhibition, 4a elevated the level of cleaved PARP (PARP
85 KD) as a further indication of the apoptosis induction potential of this compound (Fig. 4-D).

2.3. Molecular docking study
In most cancer cells survivin exists as a homodimer, which is considered necessary for its
physiological function. Destabilization and degradation of the protein is encouraged by targeting
of the small molecule into a domain that interfere with the dimerization hydrophobic interface,
thereby further leading to cancer cells apoptosis [33].
To understand the obtained biological data on a structural basis, compound I together with the
most active compound; 4a were evaluated through docking techniques (Table 4). In the present
study, the ligand-receptor interactions of both compounds with (survivin) were investigated by
performing docking studies using Molecular Operating Environment (MOE). The X-ray crystal
structure of the survivin protein (PDB code 3UIH) was obtained from the Protein Databank.
Docking of compound I with survivin showed marked binding interaction with binding energy -
6.79 Kcal/mol, the apparent H-bonding resulted from the interaction of the NH group of the
pyridine ring with the catalytic residue Gln92. Additionally, the phenolic oxygen made H-
bonding interaction with Glu40. Furthermore, arene-cation interaction was observed between the
substituted phenolic group at position 6 of the pyridine ring and Arg18 amino acid (Fig. 5).
Docking study of compound 4a (Fig. 6) showed that it was able to fit in the binding pocket of
survivin with higher binding energy - 8.478 Kcal/mol,
Results obtained indicated that 4a exhibited strong interactions forming appealing hydrogen
bonding and π-π stacking interactions with the survivin protein that include: (1) H-bonding
interaction between nitrogen atom of NH2 group at position 2 of the pyridine ring and the side
chain acidic group of Glu40; (2) H-bonding interaction between the cyano group and the side
chain basic group of Arg18; (3) H-bonding interaction between the cyano group and the side
chain of Lys15; (4) π-π stacking interaction between the phenyl ring at position 4 of the pyridine
ring and residue Phe93.
Moreover compounds 4b, 5c and 6c displayed high binding affinities with very close binding
modes in the survivin active site (Fig. 7).
Additionally, the docking of protein-protein interactions of chain A and chain B wild-type and
complex-type were carried out to predict affinity and survivin binding free energy (∆G). The
wild-type ∆G was found to be -59.3 kcal/mol, while the complex-type displayed higher ∆G value
(-58.7 kcal/mol). The complex dimer has rmsd value of 0.47 Å relative to wild-type dimer.

Furthermore, docking study was done to show the affinity of 4a for some other BIR domain
containing proteins (XIAP-BIR2 (PDB code 1I3O, chain E) and ML-IAP (PDB code 3f7g, chain
E) and they showed lower binding affinities with binding scores ranging from -3 to -2 Kcal/mol).
2.4. Molecular dynamic simulation
Molecular dynamic simulation was performed in this study in order to understand the dynamics
of survivin interaction sites. The results are reported in Table 5 which include the amino acid
residues involved in interface binding of wild-type and the amino acid residues which is
disrupted in wild-type and involved in ligand complex.
Crystallographic studies have indicated that survivin forms homodimers across the interface (the
amino acid residues of 6-10, 89-102) of dimerization [8,34], which is mediated by protein-
protein interactions. The dimerization is stabilized by several typical interactions. However,
these interactions are supposed to be broken or disrupted during docking of chemical
compounds, these are:
A. The backbone amino acid residues of Glu94, Leu96, Leu98 and Gly99 which interacted by
hydrogen bonding interactions between chain A and chain B and these interactions remain
unchanged during the molecular dynamic simulations of the docked compound. (Fig. 8).
B. Aromatic stacking network interactions between Trp10, and Phe101 located in the
dimerization interface, which suggested to form intramolecular constraint to keep survivin in
dimer form or alternatively to be disrupted in ligand binding site conformation that destabilize
surviving dimer [8,34]. As shown in Fig. 9, Trp10 of chain A is involved in aromatic network
stacking interactions with Trp10 of chain B and Phe101 in wild-type conformation. However,
upon docking, the interaction between Trp10 of chain A and Trp10 of chain B is disorganized
as Trp10 of chain A (docked form) is rotated inside the chain A to stabilize complex
conformation by forming hydrogen bonding mediated interactions between Trp10, Arg108,
and Asp105.
C. Hydrogen bonding interaction of Thr5 and Asp105 between chain A and chain B.
respectively is essential for survivin protein-protein interactions. However, in complex
conformation, the network of hydrogen bonding interactions is formed among Phe58,

Asp105, Arg108, Trp10, and Ala9 (Fig. 10). This network is suggested to stabilize the
complex via enhancing the aromatic stacking interaction between the ligand and Phe58.
D. The dimeric structure is stabilized by the formation of hydrophobic interactions between
Phe93 and Leu98 which are considered core residues of the dimerization interface [35] and a
common feature in survivin protein-protein interactions. While, in case of complex, Phe93
facilitates complex binding by forming hydrophobic interaction with the ligand (Fig. 11).
Moreover it was found that the ligand formed hydrophobic interaction with leu96, and
phe101.
Results analysis indicated that characteristic differences between the wild-type and complex
conformation, which correlated well with published results.
This modeling study revealed that 4a restricts the dimerization hydrophobic domain of survivin
which was in agreement with western plotting analysis that showed that 4a can considerably
decrease the amount of survivin in PC-3 cells, proving the possible proposed mechanism in the
molecular docking. Moreover, high selectivity of 4a on cancer cells versus normal cells further
supported our suggested mechanism of inhibiting the function of survivin protein that is mainly
expressed in cancer cells.
3. Conclusion
Based on the structural features of the survivin inhibitor I, a novel series of 3-cyanopyridines
were designed and synthesized. Their cytotoxic activities against PC-3, HepG2 and MDA-MB-
231 cell lines were investigated through MTT screening. Most of the new compounds exhibited
high in vitro anticancer activities, especially against the prostatic PC-3 cell line. The best
activities were observed in 4a, 4b, 5c and 6c. These compounds were subjected to apoptosis
studies in order to estimate their apoptotic potentials. They caused a decrease in the expression of
the apoptosis suppressor Bcl-2 and an increase in the level of apoptosis inducer Bax in addition
to increasing the amount of active caspase-3. Cell cycle analysis of the most potent compound 4a
revealed cell cycle arrest at the G2/M phase with an increase in the amount of pre-G1 apoptotic
cells as a further indication of apoptosis potential of this compound. Additionally, western
blotting analysis was carried out in PC-3 cell line and the results indicated that 4a caused potent
suppression in the expression of survivin protein. At last, molecular docking study showed that
4a can fit the survivin active site with a higher binding score than the inhibitor I.

4. Experimental
All chemicals were purchased from VWR International Merck, Germany or Sigma-Aldrich and
used without further purification. Melting Points were carried out by open capillary tube method
using Stuart SMP3 Melting Point apparatus and they are uncorrected. Elemental Microanalysis
was carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University.
Infrared spectra were recorded using potassium bromide discs on Schimadzu FT/IR 1650 (Perkin
Elmer) at the faculty of science, Al-Azhar University. 1HNMR and 13CNMR Spectra were
recorded on a Varian Gemini 300 MHz Spectrophotometer, the spectra were run at 300 MHz in
deuterated dimethylsulfoxide (DMSO-d6) at the Armed Forces Laboratories or on Agilent
Technologies 400 MHz NMR spectrophotometer at Microanalytical Unit, faculty of pharmacy,
Cairo University. Chemical shifts were expressed in δ units and were related to that of the
solvents. As for the proton magnetic resonance, D2O was carried out for NH and OH
exchangeable protons. Mass Spectra were recorded using Shimadzu Gas Chromatograph Mass
spectrometer-Qp 2010 plus (Japan) at the Regional Center for Mycology and Biotechnology, Al-
Azhar University. All the reactions were followed by thin layer chromatography using silica gel
F254 plates (Merck) and were visualized by UV-lamp. 1-(4-(benzyloxy)phenyl)ethanone (1) was
prepared according to reported procedure [36], chalcones 3a-d were prepared as reported
[37,38].
4.1. Synthesis
4.1.1. General procedure for the synthesis of compounds 4a-d
A mixture of chalcones 3a-d (3 mmol), malononitrile (0.2 g, 3 mmol) and ammonium acetate
(0.7 g, 9 mmol) in 20 ml absolute ethanol was heated under reflux for 8 hours during which the
product was separated out. Then the reaction mixture was cooled and the precipitate was filtered,
washed with cold ethanol, air dried and crystallized from ethanol.
2-amino-6-(4-(benzyloxy)phenyl)-4-phenylnicotinonitrile (4a)
Yield 65%; m.p. 125-127 oC; IR (KBr, cm-1): 3248, 3197 (NH2), 3067 (CH aromatic), 2935 (CH
aliphatic), 2216 (CN), 1604 (C=C &C=N); 1HNMR (DMSO-d6) δ (ppm): 5.19 (s, 2H, OCH2),

7.08-7.45 (m, 7H, 7 aromatic H), 7.71 (s, 1H, CH of pyridine), 7.85-8.17 (m, 7H, 7 aromatic H),
10.19 (2H, NH2; exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 69.98, 90.75, 115.12 ,
115.21 (2C), 122.47, 128.27 (2C), 128.48 (2C), 128.72 (2C), 128.95 (2C), 129.24 (2C), 129.63,
130.91, 131.04, 136.95, 138.29, 143.63, 162.41, 162.78, 162.96; MS, m/z: 377 (M+, 1.42%);
Anal. Calcd. For C25H19N3O (377.44): C, 79.55; H, 5.07; N, 11.13, Found: C, 79.82; H, 5.21; N,
11.28.
2-amino-6-(4-(benzyloxy)phenyl)-4-(4-fluorophenyl)nicotinonitrile (4b)
Yield 57%; m.p. 140-42 oC; IR (KBr, cm-1): 3296, 3142 (NH2), 3032 (CH aromatic), 2931 (CH
aliphatic), 2216 (CN), 1601 (C=C &C=N); 1HNMR (DMSO-d6) δ (ppm): 5.18 (s, 2H, OCH2),
6.76 (s, 1H, CH of pyridine), 7.04-7.92 (m, 13H, 13 aromatic H), 9.79 (2H, NH2; exchangeable
with D2O); 13CNMR (DMSO-d6) δ (ppm): 69.88, 81.07, 115.11, 115.58 (2C), 116.13 (2C),
128.02, 128.21 (2C), 128.43 (2C), 128.86 (2C), 128.94, 129.59 (2C), 129.86, 130.63, 131.24,
133.25, 137.07, 161.13, 162.38, 164.85; MS, m/z: 395 (M+, 45.77%); Anal. Calcd. For
C25H18FN3O (395.43): C, 75.93; H, 4.59; N, 10.63, Found: C, 76.12; H, 4.70; N, 10.89.
2-amino-6-(4-(benzyloxy)phenyl)-4-(4-chlorophenyl)nicotinonitrile (4c)
Yield 68%; m.p. 102-104 oC; IR (KBr, cm-1): 3350, 3222 (NH2), 3064 (CH aromatic), 2977 (CH
aliphatic), 2212 (CN), 1595 (C=C &C=N); 1HNMR (DMSO-d6) δ (ppm): 5.22 (s, 2H, OCH2),
7.12 (d, 1H, aromatic H), 7.33 (s, 1H, CH of pyridine), 7.34-7.98 (m, 13H, 13 aromatic H), 8.20
(2H, NH2; exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 70.00, 87.38, 113.54,
113.82 (2C), 115.24, 127.60, 128.25 (2C), 128.50 (2C), 128.82 (2C), 128.97 (2C), 129.45,
129.58 (2C), 130.65, 130.94, 133.38, 136.88, 161.00, 162.13, 163.02; MS, m/z: 413 [ (M+2)+,
3.46%], 411 (M+, 1.36%); Anal. Calcd. For C25H18ClN3O (411.88): C, 72.90; H, 4.40; N, 10.20,
Found: C, 73.12; H, 4.57; N, 10.48.
2-amino-6-(4-(benzyloxy)phenyl)-4-(thiophen-2-yl)nicotinonitrile (4d)
Yield 72%; m.p. 225-227 oC; IR (KBr, cm-1): 3307, 3212 (NH2), 3102 (CH aromatic), 2925 (CH
aliphatic), 2216 (CN), 1600 (C=C &C=N); 1HNMR (DMSO-d6) δ (ppm): 5.22 (s, 2H, OCH2),
7.00 (s, 1H, CH of pyridine), 7.04-8.12 (m, 12H, 9 aromatic H+ 3H of thiophene), 8.35 (2H,
NH2; exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 70.03, 84.51, 113.49, 113.82,

115.29 (2C), 126.45 (2C), 127.58 (2C), 128.27, 128.51 (2C), 128.98 (2C), 129.50, 130.66,
130.98, 136.87, 140.58, 161.5, 162.66, 163.08; MS, m/z: 383 (M+, 0.88%); Anal. Calcd. For
C23H17N3OS (383.47): C, 72.04; H, 4.47; N, 10.96, Found: C, 72.31; H, 4.64; N, 11.23.
4.1.2. General procedure for the synthesis of compounds 5a-d and 6a-d
A mixture of chalcones 3a-d (3 mmol) and cyanoacetamide/ cyanothioacetamide (3 mmol) in 20
ml absolute ethanol containing few drops of piperdine was refluxed for 6 hours, allowed to cool,
poured on ice-cold water and acidified with HCl. The solid formed was filtered and
recrystallized from ethanol.
6-(4-(benzyloxy)phenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-carbonitrile (5a)
Yield 74%; m.p. 154-156 oC; IR (KBr, cm-1): 3284 (NH), 3061 (CH aromatic), 2952 (CH
aliphatic), 2215 (CN), 1638 (CO), 1570 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.22 (s, 2H,
OCH2), 6.78 (s, 1H, CH of pyridine), 7.15-7.92 (m, 14H, aromatic H), 12.69 (1H, NH;
exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 69.92, 115.60 (2C), 115.83, 123.22,
127.12 (2C), 128.24, 128.46 (2C), 128.68 (2C), 128.97, 129.22 (2C), 129.93 (2C), 130.75 (2C),
136.54, 137.09, 151.89, 158.76, 161.22, 163.20; MS, m/z: 378 (M+, 100.00%); Anal. Calcd. For
C25H18N2O2 (378.42): C, 79.35; H, 4.79; N, 7.40, Found: C, 79.48; H, 4.87; N, 7.67.
6-(4-(benzyloxy)phenyl)-4-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (5b)
Yield 58%; m.p. 260-262 oC; IR (KBr, cm-1): 3200 (NH), 3071 (CH aromatic), 2945 (CH
aliphatic), 2217 (CN), 1640 (CO), 1571 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.19 (s, 2H,
OCH2), 6.65 (s, 1H, CH of pyridine), 7.12-7.89 (m, 13H, aromatic H), 8.10 (1H, NH;
exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 69.91, 105.91, 115.30 (2C), 115.65,
116.33, 128.21 (2C), 128.44, 128.56 (2C), 128.95 (2C), 129.95 (2C), 131.20 (2C), 130.39,
131.29, 133.03, 133.06, 137.04, 160.26, 162.45, 164.92; MS, m/z: 396 (M+, 100.00%); Anal.
Calcd. For C25H17FN2O2 (396.41): C, 75.75; H, 4.32; N, 7.07, Found: C, 76.01; H, 4.45; N, 7.24.
6-(4-(benzyloxy)phenyl)-4-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (5c)
Yield 81%; m.p. 245-247 oC; IR (KBr, cm-1): 3383 (NH), 3067 (CH aromatic), 2936 (CH
aliphatic), 2205 (CN), 1641 (CO), 1600 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.14 (s, 2H,

OCH2), 6.60 (s, 1H, CH of pyridine), 7.02-7.98 (m, 13H, aromatic H), 10.00 (1H, NH;
exchangeable with D2O); 13CNMR (DMSO-d6) δ (ppm): 69.80, 103.69, 112.44 (2C), 114.90,
115.42, 128.19 (2C), 128.39, 128.90 (2C), 128.95 (2C), 129.24 (2C), 129.38 (2C), 130.39,
133.82, 134.64, 136.85, 137.30, 150.94, 160.05, 164.19; MS, m/z: 414 [ (M+2)+, 1.87%], 412
(M+, 5.03%); Anal. Calcd. For C25H17ClN2O2 (412.87): C, 72.73; H, 4.15; N, 6.79, Found: C,
72.96; H, 4.37; N, 6.94.
6-(4-(benzyloxy)phenyl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (5d)
Yield 64%; m.p. 135-137 oC; IR (KBr, cm-1): 3434 (NH), 3178 (CH aromatic), 2950 (CH
aliphatic), 2206 (CN), 1641 (CO), 1587 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.21 (s, 2H,
OCH2), 6.94 (s, 1H, CH of pyridine), 7.01-7.47 (m, 7H, 7 aromatic H), 7.57 (1H, NH;
exchangeable with D2O), 7.65-8.09 (m, 5H, 2 aromatic H+ 3H of thiophene); 13CNMR (DMSO-
d6) δ (ppm): 70.00, 105.17, 115.30 (2C), 120.79, 125.69, 127.81, 128.27 (2C), 128.91, 128.95
(2C), 129.11 (2C), 130.60, 131.21, 132.93, 136.37, 136.75, 140.29, 144.53, 162.72, 163.18; MS,
m/z: 384 (M+, 41.29%); Anal. Calcd. For C25H16N2O2S (384.45): C, 71.85; H, 4.19; N, 7.29,
Found: C, 72.01; H, 4.36; N, 7.48.
6-(4-(benzyloxy)phenyl)-4-phenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (6a)
Yield 83%; m.p. 203-205 oC; IR (KBr, cm-1): 3208 (NH), 3056 (CH aromatic), 2938 (CH
aliphatic), 2213 (CN), 1569 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.12 (s, 2H, OCH2), 6.73 (1H,
NH; exchangeable with D2O), 6.85 (s, 1H, CH of pyridine), 7.01-8.09 (m, 14H, 14 aromatic H);
13CNMR (DMSO-d6) δ (ppm): 69.86, 102.61, 115.57 (2C), 128.20 (2C), 128.43, 128.92 (2C),
129.26 (2C), 129.42 (3C), 129.71, 129.89 (2C), 130.76, 130.98, 131.06, 137.01, 154.89, 158.89,
161.25, 165.65; MS, m/z: 394 (M+, 100.00%); Anal. Calcd. For C25H18N2OS (394.49): C, 76.12;
H, 4.60; N, 7.10, Found: C, 76.01; H, 4.79; N, 7.36.
6-(4-(benzyloxy)phenyl)-4-(4-fluorophenyl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (6b)
Yield 55%; m.p. 110-112 oC; IR (KBr, cm-1): 3202 (NH), 3065 (CH aromatic), 2935 (CH
aliphatic), 2207 (CN), 1568 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.19 (s, 2H, OCH2), 5.82 (1H,
NH; exchangeable with D2O), 6.95 (s, 1H, CH of pyridine), 7.06-8.27 (m, 13H, aromatic H);
13CNMR (DMSO-d6) δ (ppm): 69.83, 91.49, 110.91, 115.02 (2C), 115.47 (2C), 115.97, 121.05,

128.21 (2C), 128.89 (2C), 129.42 (2C), 130.40 (2C), 130.98, 131.79, 137.24, 151.40, 155.80,
157.32, 160.72, 162.80; MS, m/z: 412 (M+, 33.73%); Anal. Calcd. For C25H17FN2OS (412.48):
C, 72.80; H, 4.15; N, 6.79, Found: C, 73.04; H, 4.31; N, 6.88.
6-(4-(benzyloxy)phenyl)-4-(4-chlorophenyl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (6c)
Yield 75%; m.p. 233-235 oC; IR (KBr, cm-1): 3203 (NH), 3067 (CH aromatic), 2939 (CH
aliphatic), 2214 (CN), 1573 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.23 (s, 2H, OCH2), 6.74 (s,
1H, CH of pyridine), 6.82 (1H, NH; exchangeable with D2O), 7.14-8.27 (m, 13H, aromatic H);
13CNMR (DMSO-d6) δ (ppm): 69.91, 101.16, 115.58 (2C), 116.05, 124.87, 128.23 (2C), 128.41
(2C), 128.94, 129.37 (2C), 129.79 (2C), 130.01 (2C), 131.03, 135.40, 137.22, 153.39, 158.31,
161.06, 165.32, 167.54; MS, m/z: 430 [(M+2)+, 2.76%], 428 (M+, 8.41%); Anal. Calcd. For
C25H17ClN2OS (428.93): C, 70.00; H, 3.99; N, 6.53, Found: C, 69.89; H, 4.16; N, 6.74.
6-(4-(benzyloxy)phenyl)-4-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (6d)
Yield 58%; m.p. 228-230 oC; IR (KBr, cm-1): 3177 (NH), 3064 (CH aromatic), 2936 (CH
aliphatic), 2205 (CN), 1566 (C=C); 1HNMR (DMSO-d6) δ (ppm): 5.17 (s, 2H, OCH2), 6.74 (s,
1H, CH of pyridine), 6.89 (1H, NH; exchangeable with D2O), 7.13-8.26 (m, 12H, 9 aromatic H+
3H of thiophene); 13CNMR (DMSO-d6) δ (ppm): 69.86, 98.76, 109.92, 115.43 (2C), 115.52,
118.70, 128.22 (2C), 128.43, 128.82 (2C), 128.94 (2C), 129.38, 130.29, 130.36, 137.09, 137.23,
157.49, 160.78, 163.52, 166.23; MS, m/z: 400 (M+, 84.40%); Anal. Calcd. For C23H16N2OS2
(400.52): C, 68.97; H, 4.03; N, 6.99, Found: C, 69.20; H, 4.18; N, 7.23.
4.2. Biological evaluation
4.2.1. In vitro anti-tumor assay
4.2.1.1. Methodology: cell culture
Cancer cell lines; Human prostate carcinoma cell lines (PC-31), hepatocellular carcinoma cell
lines (HEPG-2) and human breast adenocarcinoma cell line (MDA-MB-231) and normal cell line
(WI-38) were obtained from American Type Cell Culture Collection (ATCC, Manassas, USA)
and grown on the appropriate growth medium Dulbecco's modified Eagle's medium (DMEM/
Life Technologies) supplemented with 10% FBS (fetal bovine serum) (Hyclone), 10 ug/ml of
insulin (Sigma) and 1% penicillin-streptomycin. All of the other chemicals and reagents were

from Sigma or Invitrogen. Plate cells (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of
100 µl complete growth medium + 100 ul of the tested compound per well in a 96-well plate for
24 h before the MTT assay.
4.2.1.2. MTT assay
Cytotoxicity was determined using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) method. Exponentially growing cells were trypsinized, counted and seeded at
the appropriate densities (2000-1000 cells/0.33 cm2 well) into 96-well microtiter plates. Cells
then were incubated in a humidified atmosphere at 37˚C for 24 h. Then, cells were exposed to
different concentrations of compounds (0.01, 0.1, 1.0, 10, 100 µM) for 48 h. Then the viability of
treated cells was determined using the MTT technique as follow. Media was removed; cells were
incubated with 200 μl of 5% MTT solution/well (Sigma Aldrich, MO) and were allowed to
metabolize the dye into colored-insoluble formazan crystals for 2 h. The remaining MTT
solution was discarded from the wells and the formazan crystals were dissolved in 200 µl/well
acidified isopropanol for 30 min, covered with aluminum foil and with continuous shaking at
room temperature. The absorbance of each well was measured at a wavelength of 450 nm using a
Robonik P2000 EIA reader. The cell viability was expressed as a percentage of control and the
concentration that induces 50% of maximum inhibition of cell proliferation (IC50) was
determined using Graph Pad Prism software (Graph Pad software Inc, CA) [39,40].
4.2.2. Determination of Bax
Human active Bax content was determined according to DRG® Human Bax ELISA (EIA-4487)
kit. PC-3 cells were grown in RPMI 1640 containing 10% fetal bovine serum at 37°C, stimulated
with the compounds to be tested for Bax, 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 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 compounds per well in a 96-well plate for 24
hours before measuring human Bax).
4.2.3. Determination of Bcl-2
Zymed® Bcl-2 ELISA Kit (Cat. No. 99-0042) was used for determination of Bcl-2 content. PC-3
cells were grown in RPMI 1640 containing 10% fetal bovine serum at 37°C, stimulated with the

compounds to be tested for Bcl-2, 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 Bcl-2
contents. (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 compounds per well in a 96-well plate for 24
hours before measuring human Bcl-2).
4.2.4. Determination of caspase-3
PC-3 cells were grown in RPMI 1640 containing 10% fetal bovine serum at 37 °C, stimulated
with the compounds to be tested for caspase-3, 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
caspase-3 content (cells are plated in a density of 1.2–1.8×10,000 cells/well in a volume of 100
μL complete growth medium+ 100 uL of the tested compound per well in a 96-well plate for 24
h before the enzyme assay) using Invitrogen ELISA Kit (Cat. No. KHO1091).
4.2.5. Cell Cycle Analysis (DNA-Flow Cytometry Analysis):
PC-3 cells at a density of 4 x106 cells by T 75 flasks were exposed to 4a at its IC50 for 24 h. The
cells then were collected by trypsinization, washed in phosphate buffered saline and fixed in ice-
cold absolute alcohol. Thereafter, cells were stained using Cycle TESTTM PLUS DNA Reagent
Kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. Cell-cycle
distribution was determined using a FACS Calibur flow cytometer (BD Biosciences, San Jose,
CA).
4.2.6. Apoptotic analysis
PC-3 cells were seeded in 24-well tissue culture plates. After 24 h, the medium was changed and
4a at IC50 concentrations were added. After treatment for 48 h, cells floating in the medium were
collected. The adherent cells were detached with 0.05% trypsin. Then culture medium having
10% FBS (and floating cells) was added to inactivate trypsin. After gentle pipetting, the cells
were centrifuged at 1500 g for 5 min. The supernatant was removed and cells were stained with
annexin V-fluorescein isothiocyanate and propidium iodide (PI) according to the manufacturer’s
instructions. Untreated cells were used as the control for double staining. Immediately after
staining the cells, were analyzed by a FACScan flow cytometer.

4.2.7. Western blotting
β-actin antibody was purchased from Sigma–Aldrich Chemical Company (Egyptian International
Center for Import Cairo, Egypt). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Co., Santa Cruz, CA, USA).
Antibodies Poly (ADP-ribose) polymerase (PARP), Bcl-2, Bax, caspase-7, cleaved caspase-7,
survivin, horseradish peroxidase-conjugated (HRP) anti-mouse and anti-rabbit secondary
antibodies were all obtained from Cell Signaling Technology (Beverly, MA, USA). Mini-protean
precast Tris-Glycine gels were from BioRad (Hercules, CA, USA) and the 8.0 μm pore size
trans-well membrane inserts was from BD Bioscience (Cat# 353097). All other chemicals were
purchased from Sigma-Aldrich (Egyptian International Center for Import Cairo, Egypt) unless
otherwise stated.
Protein lysates (approximately 20–30 μg of protein) were denatured in 2X Laemmli sample
buffer and subjected to electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels
(SDS-PAGE) or Tris–glycine gels as previously described [41,42]. The separated proteins were
transferred onto nitrocellulose membranes followed by blocking with 5% non-fat milk powder
(w/v) in Tris-buffered saline (10mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1% Tween 20) for 45
min at room temperature as earlier described [41-43]. Membranes were probed for proteins of
interest using specific primary antibodies followed by the appropriate peroxidase-conjugated
secondary antibody. The blots were exposed to enhanced chemiluminescence (ECL) and
subjected to autoradiography using a BioRad imaging system. To ensure equal protein loading,
membranes were stripped and re-probed with appropriate loading controls. Densitometric
analyses of the visualized protein bands were performed using the BioRad digitized scientific
software program Quantity One. Bands were scanned and processed with Adobe Photoshop CS
6.0 (Adobe Systems, San Jose, CA, USA). Multiple exposures were performed to ensure a linear
range of band densities. Three independent experiments were performed for each analysis and
protein expression levels were analyzed in triplicate with comparable results. Final data were
analyzed by one-way ANOVA.
4.3. Molecular docking study
Molecular docking study was done using Molecular Operating Environment (MOE-Dock
2014.09) software [44]. The structures of compounds I and 4a were drawn using the builder

button. Then, these compounds were exposed to energy minimization by utilizing the default
MMFF94x force field in the MOE program. Here, low energy 3D conformers of the tested
compounds were docked into the active site of surviving protein (PDB code: 3UIH). During the
docking process, water molecules were removed. The missing hydrogen atoms were added in
order of the correct ionization states to be assigned to the protein structure. “Docking” module in
MOE was run to perform the molecular docking. Docking procedure has been applied with
default settings. The top 30 poses as ranked by London dG were kept and minimized using
MMFF94x within a rigid receptor. The GBVI/WSA dG (Generalized-Born Volume
Integral/Weighted Surface area) scoring function was then applied to score the resulting poses.
“Ligand Interactions” MOE tool was then used to analyze the molecular docking results by a
visualization of the protein–ligand interactions in the active site of the complex. The tool
presents in a diagram form an identification and visualization of the interactions between the
ligand and the receptor interacting entities, solvent molecules, and ions in the active site of the
protein. Among the main interactions included are hydrogen bonds, salt bridges, hydrophobic
and cation–π and π –π interactions.
4.4. Molecular dynamic simulation
Prior to MD simulations, the complex, in the dimerization interface was minimized stepwise
with respect to the force field energy by using the Amber package to attain a low energy
conformation. Survivin was described using the Amber Force Field, ligand was described using
the Generalized Amber Force Field (GAFF), and the Zinc Amber Force Field (ZAFF) was used.
Then the complex was subjected to MD simulations at 300 K during 400 ps. The time step of the
simulation was 2.0 fs with a cutoff 10 Å for the non-bonded interactions. The MD simulations
are performed at constant temperature and pressure. During the MD simulations all backbone
atoms of the surviving were restrained to their starting positions with harmonic force constant
2.0 Kcal/(mol Å2).
This research did not receive any specific grant from funding agencies in the public, commercial,
or not-for-profit sectors.
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Table 1. Cytotoxicity of compounds 4-6 and 5-FU evaluated in different human cancer cells a.
IC50 (µM)
Compound
PC-3
HepG2
MDA-MB-231
0.66±0.03
2.16±0.23
12.82±1.60
9.16±0.94
18.38±1.07
8.68±0.85
3.32±0.27
10.71±1.01
8.22±0.76
4.72±0.41
2.83±0.15
9.34±0.85
9.35±0.74
4a
4b
4c
0.42±0.05
1.45±0.12
9.83±0.97
7.12±0.81
14.71±1.3
4.97±0.46
1.62±0.09
6.95±0.58
6.31±0.51
2.41±0.30
1.72±0.11
7.42±0.64
7.53±0.09
1.22±0.06
3.53±0.28
14.61±1.20
12.62±1.10
21.35±1.30
12.51±1.10
3.64±0.32
13.17±0.98
10.64±0.95
6.92±0.68
3.91±0.25
11.52±0.97
8.15±0.11
4d
5a
5b
5c
5d
6a
6b
6c
6d
5-FU
a The inhibitory effects of compounds on the cancer cell lines were
determined by the MTT assay. The data are expressed as the mean ±
SD of three independent experiments.

Table 2. IC50 values of compounds 4a, 4b, 5c and 6c on the normal WI-38 cell line a.
Compound
4a
4b
5c
6c
WI-38
193.15±0.09
193.64±0.11
169.32±0.68
148.57±0.12
IC50 (µM)
a The inhibitory effects of compounds on the normal WI-38 cell line were determined by the MTT assay.
The data are expressed as the mean ± SD of three independent experiments.

Table 3. Effect of compounds 4a, 4b, 5c and 6c on the level of some apoptosis key markers a.
Compound
Bcl-2
(ng/ml)
Bax
(ng/ml)
Caspase-3
(Pg/ml)
4a
4b
1.44±0.27
301.0±0.10
393.6±0.32
2.43±0.25
1.67±0.30
2.82±0.25
5.60±0.16
244.3±0.09
263.8±0.78
208.9±0.11
33.9±0.08
336.7±0.22
346.7±0.19
329.1±0.11
46.2±0.15
5c
6c
Control
a The effects of 4a, 4b, 5c and 6c on the amount of Bcl-2, Bax and Caspase-3 were determined after
24 h using ELISA Kits. The data are expressed as the mean ± SD of three independent experiments

Table 4. Docking parameters for compounds I and 4a with survivin.
Compound Docking score
Atoms and amino acids
involved in the interaction
Type of
interaction
Distance
(A0)
ΔG (kcal/mol)
Pyridine N ….. Gln 92
Hydroxyl O ….. Glu 40
4-Phenyl ring ….. Arg 18
H-bond
H-bond
Pi-H
2.55
3.25
3.83
I
-6.790
-8.478
Amino N
Cyano N
Cyano N
…. Glu 40
…. Lys 15
…. Arg 18
H-bond
H-bond
H-bond
Pi-cation
3.15
2.84
3.63
3.87
4a
4-Phenyl ring …. Phe 93

Table 5. The interactions involved at the dimerization interface of the wild-type and the
complex-type.
Dimer type
Wild-type
H-bonding
interactions
Aromatic stacking
interactions
Hydrophobic
interactions
Thr5 and Asp105
Trp10 and Phe101
Phe93 and Leu98
Ala9, Trp10, Phe58, 1. Trp10 and Phe101
1. Ligand with Phe93
2. Ligand with Leu96
and Phe101
Complex- Asp105, and Arg108
disrupted
type
2. Ligand with Phe58