Synthesis of new substituted pyridine derivatives as potent anti-liver cancer agents through apoptosis induction: In vitro, in vivo, and in silico integrated approaches

Article abstract of DOI:10.1016/j.bioorg.2021.104877

Liver cancer is the most common type of cancer in many countries. New studies and statistics show rising liver cancer worldwide, so it is essential to seek new agents for this type of cancer. PIM1 has an attractive target in the discovery of cancer medica

Full text of DOI:10.1016/j.bioorg.2021.104877

Bioorganic Chemistry 111 (2021) 104877
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of new substituted pyridine derivatives as potent anti-liver cancer
agents through apoptosis induction: In vitro, in vivo, and in silico
integrated approaches
Ahmed T.A. Boraei*, Elsayed H. Eltamany, Ibrahim A.I. Ali, Sara M. Gebriel, Mohamed S. Nafie*
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Liver cancer
Pyridines
Apoptosis
RT-PCR
Liver cancer is the most common type of cancer in many countries. New studies and statistics show rising liver
cancer worldwide, so it is essential to seek new agents for this type of cancer. PIM1 has an attractive target in the
discovery of cancer medications as it is very much expressed in a variety of malignancies and influences such as
tumorigenesis, cell cycle progression, cellular proliferation, apoptosis, and cell migration. Accordingly, a series of
pyridones and pyridine-amides were synthesized and tested for anti-liver cancer activity.
In vivo
In the synthetic strategy 4,6-diaryl-3-cyano-2-pyridones 3a-n were synthesized using one-pot four component
synthetic method. Structural modifications were done on 4,6-diphenyl-3-cayno-2-pyridone 3a to enhance the
activity. Alkylation in the presence of K2CO3 afforded the O-alkylated products 4–6. The acetoxy hydrazide 7 was
synthesized and cyclized into 1,3,4-oxadiazolethione 8 which alkylated on sulfur to give 10. Azide-coupling
method was used to couple the 2-(pyridin-2-yloxy)acetohydrazide 7 to different amines and amino acid esters
to furnish the products 12a-e and 13a-b. The synthesized derivatives were subjected to cytotoxic screening
against HepG2 and THLE-2 cells, Compounds 10, 12e and 13a have a remarkable cytotoxic activity with IC50
values (10.7–13.9 µM). Compound 7 was found to be more cytotoxic by showing the lowest IC50 value of 7.26
compared to 5-FU (IC50 = 6.98 µM). It inhibited cell growth by 76.76%. Additionally, it significantly stimulated
apoptotic liver cancer cell death with 49.78-fold (22.90% compared to 0.46% for the control) arresting cell cycle
Pre-G1 with 35.16% of a cell population, compared to 1.57% for the control. Moreover, it validated the intrinsic
apoptosis through upregulation of P53, and other related genes, with inhibition of anti-apoptotic genes through
PIM-1 inhibition.
1. Introduction
cellular transformation. Moreover, PIM-1 is also associated with drug
resistance and its level of expression which was reported to be upregu-
Liver cancer was found to be the sixth most commonly occurring
cancer and the fourth type of cancer leading mortality worldwide. The
estimated new cases were 841,000 and 782,000 deaths in 2018 [1,2].
The Provirus Integration in Maloney (PIM) kinases are highly expressed
in many kinds of hematological and solid cancers. PIM kinases which are
known as (protooncogenes) regulate the network of signaling pathways
that are critical for tumorigenesis and growth, making PIM kinases
attractive drug targets [3]. PIM-1 has a significant role in the regulation
of the cell cycle through phosphorylation of the Cdc25 phosphatases and
cell-cycle inhibitors [4,5] also it stimulates the transition of cells from
G1 to S phase through the phosphorylation and activation of the phos-
phatase Cdc25A [6], causing an enhancement of Cdc25A-mediated
lated in mitoxantrone- and docetaxel-resistant cancer cell lines [7,8].
PIM kinases have become attractive therapeutic targets for cancer
treatment because they are involved in cancer-specific pathways and
upregulated in many cancers [9,10]. Since, pyridine motif is found in
many biologically and pharmaceutically active agents [11,12] for
example, Etoricoxib is non-steroidal anti-inflammatory drug [13],
amlodipine, nifedipine, and clevidipine are acting as calcium channel
blockers [14].
Moreover, it was found that the pharmacophoric features of some
anticancer drugs, such as Veliparib, Niraparib, Rucaparib and Olaparib
is strongly dependent on the presence of the amide group either primary
or embedded in a heterocyclic ring that serve as hydrogen bond donors
* Corresponding authors.
E-mail addresses: ahmed_tawfeek83@yahoo.com (A.T.A. Boraei), mohamed_nafie@science.suez.edu.eg (M.S. Nafie).
https://doi.org/10.1016/j.bioorg.2021.104877
Received 7 January 2021; Received in revised form 9 March 2021; Accepted 25 March 2021
Available online 29 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
for the interaction with biological targets [15,16]. Additionally, the
most potent PIM inhibitor identified to date 6-phenyl-N-(pyridin-3-yl)
picolinamide (LGB321), its structure contains pyridine, amine, and
amide groups which are essential for hydrogen bond interactions with
the residues of the ATP-binding site of PIM-1 [17,18] Fig. 1.
Construction of a system including both pyridine and 1,3,4-oxadia-
zole moieties was achieved by synthesizing hydrazide 7 from the
hydrazinolysis of the ester 6. The hydrazide 7 was reacted with carbon
disulfide in ethanol containing aq. KOH to afford the dithioic acid 8 on
stirring and the desired the pyridinyl-oxadiazolthione 9 upon reflux.
pyridinyl-oxadiazolthione 9 was alkylated with phenacyl bromide in the
presence of K2CO3 to give the S-alkylated product 10 (Scheme 3). The 1H
NMR of the hydrazide 7 revealed the NH2 protons at 4.30 ppm and the
NH proton at 9.39 ppm. Due to cyclization the 1H NMR a broad signal at
14.64 ppm for the delocalized NH and 13C NMR displayed a new signal
at 178.7 ppm for the thiocarbonyl group. The 13C NMR of the phena-
cylated product 10 displayed a new methylene carbon signal at 39.7
ppm which suggests the alkylation at sulfur, not nitrogen.
As part of our continued efforts for synthesis and discovery of new
anticancer hits [19–22], though stepwise biology plan including cyto-
toxic screening, apoptosis induction [23–25]. This work includes the
design and synthesis of several pyridine derivatives and evaluating their
activity towards liver cancer. Results disclosed that 2-(3-cyano-4,6-
diphenylpyridin-2-yloxy)acetohydrazide has the most potent activity. It
significantly impeded cell proliferation and cell migration by interfering
with PIM-1 enzymatic activity.
Coupling of the pyridine-hydrazide scaffold 7 with various amines
and amino acid methyl esters was done through azide-coupling strategy
in which the hydrazide 7 was converted into azide 11. The latter was
allowed to react with different amines like propyl amine, benzyl amine,
tetradodecyl amine, allyl amine and piperidine to give the amides 12a-e
respectively, and with glycine methyl ester HCl and L-leucine methyl
ester HCl in the presence of Et3N to give the coupled products 13a-b
(Scheme 4). The amines and amino acid NMR showed the characteristic
signals. Compounds 13a-b NMR revealed the methoxy protons at 3.60
ppm and the respective methoxy carbons at 65.0 ppm.
2. Results and discussion
2.1. Chemistry
Four component one-pot synthesis of various substituted 4,6-diaryl-
3-cyano-2-pyridones 3a-n from the reflux of aromatic aldehydes, ke-
tones, ethyl cyanoacetate, and ammonium acetate in n-butanol for 1–3 h
was performed Scheme 1, Table 1. The NMR of pyridone 3a-n displayed
the pyridone NH around 12.50 ppm, and carbonyl carbon was detected
around 162.5 ppm.
Alkylations of 4,6-diphenyl-3-cayno-2-pyridone 3a with benzyl
bromide, phenacyl bromide, and ethyl chloroacetate were done using
K2CO3 as proton scavenger in acetone and DMF which afforded the O-
alkylated product 4–6 respectively (Scheme 2). The 13C NMR of the
alkylated products 4–6 showed the methylene carbon at 68.8, 69.3, and
64.2 ppm respectively, which recommends the alkylation at oxygen, not
nitrogen.
2.2. Biology
2.2.1. Cytotoxicity using the MTT assay
To estimate their selectivity, selected compounds were screened for
their cytotoxic activity against the liver cancer cell lines (HepG2), and
the normal liver cells. As shown in Table 2, and Fig. 2, which summa-
rizes the IC50 values of all tested derivatives. Among them, compound 7
Fig. 1. Structures of selected FDA-approved anticancer drugs showing the significance of pharmacophoric amide group in designing the target PIM-1inhibitors.
2

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Scheme 1. Four component one-pot synthesis of 2-pyridinones 3a-n.
death with 49.78-fold; it increased apoptosis ratio by 22.90% (2.67% for
early and 20.23% for late apoptosis) compared to 0.46% (0.39% for
early and 0.07% for late apoptosis) for the control. Hit compound 7
treatment induced the apoptotic cell death more than the necrotic one, it
stimulated necrotic cell death with 11.04-fold (12.26%, compared to
1.11% for the control). Moreover, treated HepG2 cells with compound 7
was subjected to DNA flow cytometry to determine at which cell cycle,
the cell proliferation was arrested. As seen in Fig. 3 (lower panel). The
compound treatment indignantly increased Pre-G1 with 35.16% popu-
lation, compared to 1.57% for the control. While cell population at other
cell phases are not significantly changed. Our results agreed with
Bachmann et al. [26] who reported that the PIM-1 inhibition would have
apoptosis-inducing activity through cell cycle arrest.
Table 1
The aromatic ketones and aldehydes used for the synthesis of 2-pyridinones 3a-
n.
m.p
Ar1
Ar
Entry
313
C6H5-
C6H5-
3a
3b
3c
313–315
312
C6H5-
3-NH2-C6H4-
4-NH2-C6H4-
4-Br-C6H4-
4-OCH3-C6H4-
Cyclohexel
C6H5-
C6H5-
294–296
260–262
271–273
312
C6H5-
3d
3e
3f
C6H5-
C6H5-
4-OCH3-C6H4-
4-NO2-C6H4-
3-OCH3-C6H4-
4-N(CH3)2-C6H4-
2-furyl
3 g
3 h
3i
302
C6H5-
240
C6H5-
310–312
300
C6H5-
3j
C6H5-
3 k
3 l
3 m
3n
304
3-Cl-C6H4
4-OCH3-C6H4
4-OCH3-C6H4
4-Cl-C6H4-
4-Cl-C6H4-
3-NH2-C6H4-
2.2.2.2. RT-PCR. HepG2 cells were treated with compound 7 (IC50
=
275–277
300–302
7.26 M for 72 h), following the routine work of RT-PCR test to inves-
μ
tigate its apoptotic pathway. The apoptotic pathway was investigated by
following the mRNA expression of P53, BAX, PUMA, Caspases 3, 8 9 as
pro-apoptotic, Bcl-2 as antiapoptotic gene, and the PIM-1 kinase genes.
As shown in Fig. 4, compound 7 significantly activated the level of
P53 gene (≈5.74-fold) and PUMA and BAX levels with ≈6.09-fold and
7.12-fold, respectively. The compound increased the mRNA levels of
casp-3 (4.64-fold), casp-8 (9.09-fold), casp-9 gene (6.27-fold), while it
significantly inhibited the anti-apoptotic Bcl-2 gene with 0.41-fold, and
the PIM-1 kinase gene expression with 0.45-fold. Thus, up-regulation of,
P53, Bax, PUMA, Caspases-3, -8, -9 and downregulation of BCL2, PIM-1
kinase genes were induced by compound 7 relative to control, propose
induction of intrinsic and extrinsic apoptosis pathway in the treated
liver cancer cell line, The results are following the decreased levels of
PIM-1 kinase activates apoptosis pro-apoptotic proteins as previously
reported in [27], and inhibits the antiapoptotic genes as reported in
2020 [22].
was found to be more cytotoxic by showing the lowest IC50 value of 7.26
µM compared to 5-FU (IC50 = 6.98 µM). It inhibited the cell growth by
76.76%. Compounds 10, 12e, and 13a have remarkable cytotoxic ac-
tivity with IC50 values (10.7–13.9 µM). Additionally, most of the tested
derivatives were safe (non-toxic) against the normal liver cells. The re-
sults recommended hit 7 for further studies.
2.2.2. Apoptotic investigation
2.2.2.1. Annexin V/PI with cell cycle analysis. For the investigation, the
apoptosis-inducing activity of compound compounds 7 (IC50 = 7.26 µM)
in HepG2, cells after treatment were subjected to flow cytometric
analysis of Annexin V/PI staining with cell cycle analysis to determine
the cell population in different cell cycle phases. As seen in Fig. 3 (upper
panel), compound 7 significantly stimulated apoptotic liver cancer cell
Scheme 2. Alkylation of 4,6-diphenyl-3-cyano-2-pyridones 3a with benzyl bromide, phenacyl bromide, and ethyl chloroacetate.
3

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Scheme 3. Hydrazinolysis of the ester 6 and heterocyclization of the respective hydrazide 7.
2.2.3. In vivo
While WBC’s count was significantly increased to be 5.04 (103/µL)
compared to the normal control levels. Reduced levels of hemoglobin,
RBC’s and elevation of WBC’s counts are routine consequences of tumor
propagation [28,29] Upon treatment with compound 7, CBC levels were
nearly retained to their normal values, where it elevated the Hb (6.32 g/
dL), RBC’s (4.87 106/µL) and reduced the WBC’s (3.98 103/µL) levels.
Interestingly our results following that of Gad et al. 2020 [29] study
illustrated the ameliorative effect in the hematological parameters after
the treatment with the tested compound.
2.2.3.1. Antitumor potentiality. After ten days of tumor cell inoculation,
masses of solid Ehrlich carcinoma (SEC) began to appear. Mice were
treated with compound 7 at its LD50 dose (5 mg/kg BW, IP) for evalu-
ation of its in vivo activity against SEC development. A total of seven
doses of the compound started on day 7 after tumor inoculation. At the
end of the procedure, weight and volume of the solid tumor masses were
measured. During the experimental duration, an increase in solid tumor
weight of around 130 mg was observed via tumor development. The
antitumor effect of compound 7 and 5-FU was elucidated; there was a
significant decrease in the solid tumor mass by 47.92% (67.87 mg) and
62.68% (48.63 mg), respectively compared to control (130.32 mg)
Fig. 5. Accordingly, treatments of compound 7significantly inhibited the
percentage of tumor inhibition by 36.05% in tumor volume (32.1 mm3)
relative to 5-FU treatment with tumor inhibition ratio 64.14% (18 mm3),
compared to control (50.2 mm3). One of the valuable results, is worthy
to be mentioned that in 7-treated-SEC group, one or two mice had not
formed remarkable tumor mass compared to the SEC control group in
which, mice were found to move very difficult due to the large tumor
mass in their legs.
Regarding biochemical parameters, due to hepatocellular damage
following tumor inoculation, liver enzymes (ALT and AST) were
significantly elevated to be significantly elevated to 74.56, 89.46 (U/L),
respectively, as compared to normal mice 45.63 and 48.49, respectively.
Treatment with compound 7, substantially reduced liver enzymes to be
57.41, 51.11 U/L, respectively indicating a remarkable enhancement in
the hepatocellular toxicity induced by SEC proliferation. Additionally,
for kidney parameters, compound 7 treatment retained the deteriorated
urea and creatinine levels to be 28.01 and 0.74 (mg/dL) nearly as
normal control 27.32 and 0.83, respectively.
2.2.3.3. Histopathological examinations. These findings were supported
by the hepatic histopathological examinations as shown in Fig. 6. In
which the compound 7 treatment enhanced their architectures near-
normal than in positive control cancer group.
2.2.3.2. Hematological and biochemical investigations. At the end of the
experiment, animals from different groups were sacrificed, and blood
samples were collected for hematological parameters including Hb,
RBC’s and WBC’s levels, and serum for determination of liver enzymes
ALT, AST levels, and kidney parameters urea and creatinine levels. As
summarized in Table 3, in SEC-bearing mice. All CBC parameters were
changed in the SEC control, where Hb content and RBC’s were signifi-
cantly decreased to be 4.02 (g/dL) and 3.01 (106/µL), respectively.
Taken together, treatment of SEC mice compound 7 was found to
improve in the results of antitumor results of tumor weight and volumes,
hematological, biochemical parameters, and histopathological
examinations.
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A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Scheme 4. Azide Coupling of 7 with amines and amino acid esters HCl.
2.2.4. In silico
Table 2
Cytotoxic activity of tested derivatives with serial concentrations against HepG2
(liver cancer cell line), and THLE2 (normal liver cells) using the MTT assay.
2.2.4.1. Molecular docking. For elucidation, the virtual mechanism of
binding, a molecular docking study was carried out to investigate the
binding interactions towards PIM-1 kinase 4K0Y and 2OBJ proteins. The
co-crystallized ligands of the studied proteins form hydrogen bonds with
Lys 67 as the key interactive amino acid. All biologically tested com-
pounds were screened for the molecular docking simulation inside both
proteins and most of all exhibited good binding affinities towards both
PIM-1 kinases by good interaction mode with the interactive key amino
acids and with high binding energies (Full results are supported as
supplementary).
Compounds
Working
% of inhibition
IC50 SD (µM)*
concentrations
at 100 µM
HepG2
THLE2
3b
3d
3e
0.01, 0.1, 1, 10,
27.52
36.48
39.53
ND
≥50
100 µM
≥50
≥50
ND
46.7 ±
1.76
3 l
43.21
71.43
43.7 ±
1.23
19.2 ±
1.01
ND
48.2 ±
0.98
3n
44.11 ±
0.89
Among which, as seen in Table 4, compound 7 was docked inside the
protein active site of the two studied proteins and formed one hydrogen
bond as HBA with bond lengths 2.39 and 2.03 Å, through its cyano group
moiety with the key amino acid Lys 67 like the co-crystallized ligand
with binding energy ꢀ 20.86 and ꢀ 20.70 Kcal/mol, respectively. Addi-
tionally, compound 7 formed arene-cation interaction with Lys 67 inside
the 2OBJ protein, and it forms good lipophilic interactions with the
nonpolar amino acids Leu 44, Ala 65, Val 52, Ile 185, Ile 104, Leu 120,
Leu 174, and Phe 49 inside both receptor pockets. Docking results
indicated that the designed compound 7 showed promising binding
activity as PIM-1 inhibitors, and this may be the proposed mode of ac-
tion for anti-liver cancer activity.
5
6
7
41.72
45.56
76.76
≥50
ND
≥50
7.26
0.34
ND
≥50
8
34.2.3
62.76
74.96
68.83
69.53
84.72
39.88 ±
0.59
9
17.6 ±
0.97
≥50
10
13.4 ±
0.76
≥50
≥50
ND
12e
13a
5-FU
12.4 ±
0.83
13.9 ±
0.98
6.98
ND
* “Values are expressed as Mean ± SD of triplet trials, ND is non-determined, and
calculated using GraphPad Prism 7 software using nonlinear regression Dose-
Inhibition curve fit”.
2.2.4.2. ADME pharmacokinetics. Compounds with the highest cyto-
toxic activity against HepG2 cells were subjected to bioinformatics study
to predict the physicochemical properties and drug-likeness scores. The
candidate drugs that are in line with Lipinski’s “five rule” (Ro5) are seen
as promising for the future [30–32]. Topological polar area of the
5

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Fig. 2. A. Sigmoidal dose-inhibition curve fit of the percentage of cell survival vs log [conc.] for IC50 calculation of compound 7 using GraphPad prism, and B:
Cytotoxic activity of compound 7 against HepG2 cells.
Fig. 3. “FITC/Annexin-V-FITC/PI differential apoptosis/necrosis” (Upper panel) and DNA content-flow cytometry aided cell cycle analyses with bar chart repre-
sentation (Lower panel) of both untreated and treated HepG2 treated with 7 (IC50 = 7.26 μM, 48 h).
surface TPSA values for drug absorption through intestine should be as
low as 140, and the blood–brain barrier should be reached as low as 90
Å2 [33,34] The tested compounds were all well-permeable and absorbed
as shown in Fig. 7. As seen in Table 5, compounds had 1–4 HB donor and
5–6 acceptors for HB acceptor. Besides, all compounds tested were well
tolerated by cell membranes (log P is between 1.18 and 3.38). Veber et al
revealed that the rotabile bond number (nrotb) should be ≤10 for
controlling conformational changes and for good oral bioavailability
[30,35]. All the tested compounds had 1–9 nrotb. Regarding drug-
likeness scores, compounds that showed positive values should be
considered as drug-like molecules. All the investigated compounds dis-
played positive values of 0.06–0.12, so they could be considered as drug-
like (see Fig. 8).
the drug-likeness score towards GPCR ligand, ion channel modulator, a
kinase inhibitor, nuclear receptor ligand, protease inhibitor, enzyme
inhibitor. The larger the value of the score is, the higher is also the
probability that the molecule will be active, so compounds 7 exhibited a
positive value of 0.11 against kinase protein as seen in Table 6.
3. Experimental section
3.1. General
Melting points were determined in open capillaries on a Temp-melt II
melting-point apparatus and the values are uncorrected. Thin-layer
chromatography (TLC) was carried out on silica gel 60 F254
Aluminium plates (E. Merck, layer thickness 0.2 mm). The spots were
Finally, Molinspiration virtual screening toolkit was used to calculate
6

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Fig. 4. RT-PCR analysis of the apoptosis-related genes was performed after the HepG2 cells were treated with compound 7 (IC50 = 7.26 μM) for 72 h.
Fig. 5. Antitumor activity of treatment of solid Erlich carcinoma (SEC) with compound 7 in mice, compared solid tumor mass, volume, tumor inhibition ratio (TIR
%). Values are expressed as Mean ± SEM values of mice in each group (n = 6). Signs of * and # are values with significant differences in tumor volume and tumor
weight, respectively compared to SEC control using unpaired t-test (P ≤ 0.05) using GraphPad prism.
detected by UV lamp. The 1H, 13C NMR spectra were recorded on Bruker
400 MHz-NMR spectrometer operating at 400 and 100 MHz respectively
using DMSO‑d6 solvent, at the Microanalytical Laboratory, Sohag Uni-
versity, Egypt. All chemical shifts are reported in ppm values relative to
TMS as internal standard. IR spectra were recorded on Fourier Trans-
form Infrared (FT-IR) spectrophotometry (Bruker) a Perking Elmer 1430
ratio recording infrared spectrophotometer using KBR.
DMSO‑d6): δ 106.6, 116.9 (CN), 128.2, 128.7, 129.3, 129.4, 130.9,
131.6, 132.8, 136.6, 160.3, 162.5; IR (KBr/cmꢀ 1): 1641 (C O), 2215
–
–
–
–
(C N), 2685 (NH); CHN Calcd. For C18H12N2O (272.31): C, 79.39; H,
–
4.44; N, 10.29; Found: C, 79.20; H, 4.53; N, 10.24%.
3.1.1.2. 6-(3-Aminophenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-car-
1
◦
bonitrile (3b). Yield: 47 %acetic acidas yellow powder, m.p. 313–315 C. H
NMR (400 MHz, DMSO‑d6): δ 5.27 (s, 2H, NH2), 6.64 (s, 1H), 6.76 (d, J
= 7.6 Hz, 1H), 7.00 (d, J = 9.2 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H),
7.58–7.71 (m, 5H), 12.54 (s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ
106.1, 113.0, 115.5, 117.0, 117.1, 128.6, 129.3, 130.0, 130.8, 133.4,
3.1.1. Preparation of2-oxo-4,6-diaryl-1,2 dihydropyridine-3-carbonitrile
derivatives (3a-n)
A mixture of the selected aromatic aldehyde (0.01 mol.), ketone
(0.01 mol.), ethyl cyanoacetate (0.01 mol.), and ammonium acetate
(0.08 mol.) was refluxed in n-butanol (20.0 mL) for 1–3 h. A precipitate
is formed on heating. The mixture is left to cool, filtered, dried, and
recrystallized from the appropriate solvent.
136.7, 149.6, 152.9, 160.2, 162.3; IR (KBr/cmꢀ 1): 1599 (C O), 2218
–
–
–
–
(C N), 2864 (NH), 3355 (NH2); CHN Calcd. For C18H13N3O (287.11):
–
C, 75.25; H, 4.56; N, 14.63. Found: C, 75.30; H, 4.53; N, 14.75%.
3.1.1.3. 6-(4-Aminophenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-car-
bonitrile (3c). Yield: 57%DMF, drops ethanol as yellow powder, m.p. 312 ◦C.1H
NMR (400 MHz, DMSO‑d6): δ 5.93 (s, 2H, NH2), 6.64 (d, J = 7.6 Hz, 3H),
7.55–7.69 (m, 7H), 12.30 (s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ
103.7, 113.9, 113.98, 117.5, 118.3, 128.5, 129.2, 129.6, 130.6, 137.0,
3.1.1.1. 2-Oxo-4,6-diphenyl-1,2-dihydropyridine-3-carbonitrile
(3a).
Yield: 70%
powder, m.p. 310–311 ◦C Lit.1,2
>
Ethanolas yellowish-brown
300 ◦C.1H NMR (400 MHz, DMSO‑d6): δ 6.84 (s, 1H), 7.57–7.58 (m, 6H),
7.74 (s, 2H), 7.91 (s, 2H), 12.77 (s, 1H, NH);13C NMR (100 MHz,
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A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
132.3, 136.5, 151.2, 160.0, 162.6, 172.4; IR (KBr/cmꢀ 1): 693 (C-Br),
Table 3
–
–
–
Hematological and biochemical investigations in the treated groups.
1609 (C O), 2221 (C N), 2806 (NH); CHN Calcd. For C18H11BrN2O
–
–
(351.2): C, 61.56; H, 3.16; Br, 22.75; N, 7.98. Found: C, 61.62; H, 3.27;
TreatmentsParameter
Normal
control
SEC
SEC + comp
7(5 mg/kg
BW)
SEC + 5-FU
(5 mg/kg
BW)
Br, 22.66; N, 7.83%.
control
Hb*# (g/dL)
9.4 ± 0.56
4.02 ±
0.31
6.32 ± 0.50
4.87 ± 0.51
3.98 ± 0.45
57.41 ± 1.54
51.11 ± 1.65
28.01 ± 0.91
0.74 ± 0.06
7.09 ± 0.82
5.02 ± 0.24
3.74 ± 0.29
3.1.1.5. 6-(4-Methoxyphenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-car-
bonitrile (3e). Yield: 47 %Ethanol
powder, m.p. 260–262 ◦C.1H
NMR (400 MHz, DMSO‑d6): δ 3.85a(ss,y3eHllo,wOCH3), 6.78 (s, 1H), 7.07 (d, J
= 6.8 Hz, 2H), 7.57 (s, 3H), 7.72 (s, 2H), 7.90 (d, J = 6.8 Hz, 2H), 12.58
(s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ 56.0, 97.7, 105.7, 114.9,
RBC’s count*#(×106/
6.31 ±
0.64
3.01 ±
0.62
μ
L)
WBC’s count*#(×103/
3.75 ±
0.62
5.04 ±
0.51
μ
L)
ALT*# (I/U)
45.63 ±
1.16
74.56 ±
1.99
49.65 ±
1.71
117.1, 124.8, 128.6, 129.2, 129.9, 130.7, 136.8, 151.7, 160.2, 162.3,
162.5; IR (KBr/cmꢀ 1): 1596 (C O), 2217 (C N), 2838 (NH); CHN
–
–
–
–
–
AST*# (I/U)
48.49 ±
0.98
89.46 ±
1.45
49.85 ±
0.87
Calcd. For C19H14N2O2 (302.11): C, 75.48; H, 4.67; N, 9.27. Found: C,
75.54; H, 4.73; N, 9.08%.
Urea*# (mg/dL)
Creatinine*# (mg/dL)
27.32 ±
1.76
33.76 ±
1.01
27.98 ±
0.85
0.83 ±
0.04
1.06 ±
0.17
0.62 ± 0.05
3.1.1.6. 2-Oxo-4-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-carbonitrile
1
◦
(3f). Yield: 40 %Ethanol as yellow powder, m.p. 271–273 C. H NMR (400
MHz, DMSO‑d6): δ1.58 (s, 2H), 1.69 (s, 2H), 2.04 (s, 2H), 2.65 (s, 2H),
7.32 (s, 2H), 7.50 (s, 3H), 12.30 (s, 1H, NH);13C NMR (100 MHz,
DMSO‑d6): δ 21.1, 22.3, 25.3, 27.7, 100.9, 112.8, 116.5, 127.9, 129.1,
*Mean ± SEM values of mice in each group (n = 6).
#
Values are significantly different (P ≤ 0.05), un-paired test using GraphPad
prism.
129.4, 136.1, 150.7, 160.3, 162.3. IR (KBr/cmꢀ 1): 1594 (C O), 2224
–
–
152.3, 152.8, 160.0, 162.7; IR (KBr/cmꢀ 1): 1592 (C O), 2209 (C N),
–
–
–
–
–
–
–
(C N), 2841 (NH), 2945 (Cyclohexyl); CHN Calcd. For C16H14N2O
–
2899 (NH), 3442 (NH2); CHN Calcd. For C18H13N3O (287.11): C, 75.25;
H, 4.56; N, 14.63. Found: C, 75.18; H, 4.47; N, 14.42%.
(250.3): C, 76.78; H, 5.64; N, 11.19. Found: C, 76.84; H, 5.77; N,
11.04%.
3.1.1.4. 6-(4-Bromophenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-car-
bonitrile (3d). Yield: 66%DMF-Acetic acid (1:1) as white powder, m.p. 294–296
◦C.1H NMR (400 MHz, DMSO‑d6): δ 6.9 (s, 1H), 7.58 (s, 3H), 7.74 (s,
4H), 7.87 (d, J = 7.6 Hz, 2H), 12.3 (s, 1H, NH); 13C NMR (100 MHz,
DMSO‑d6): δ 98.8, 107.3, 116.8, 125.3, 128.7, 129.2, 130.3, 130.9,
3.1.1.7. 4-(4-Methoxyphenyl)-2-oxo-6-phenyl-1,2-dihydropyridine-3-car-
bonitrile (3 g). Yield: 38 %DMF-Acetic
powder, m.p.
310–312 ◦C.1H NMR (400 MHz, DMSO‑da6c)i:dδ3(1.8:17) (ass, 3yHell,oOwCH3), 6.80 (s,
1H), 7.12 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 6.4 Hz, 3H), 7.73 (d, J = 8.0
Hz, 2H), 7.89 (d, J = 6.0 Hz, 2H), 12.60 (s, 1H, NH);13C NMR (100 MHz,
Fig. 6. Hepatic examinations of (A) Normal control group shows normal hepatic lobule architecture. The central vein is surrounded by hepatocyte cords. A narrow
blood sinusoid is often seen between the strands of hepatocytes. (B) SEC control group shows hydropic degeneration of the hepatocytes, loss of cell boundaries and
degeneration of the balloon, others hepatic cells had nuclear pyknosis (arrowheads) and karyolysis (arrows). (C) SEC mice treated with compound 4 group: shows
that the hepatic lobule appears to be normal, also few hepatocytes show hydropic degeneration and activated Kupffer cells. (D) SEC mice treated with 5-FU: still
exhibits hydropic degeneration of hepatocytes and nuclear pyknosis (arrowheads), and karyolysis (arrows).
8

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Table 4
Analysis of ligand-receptor interactions with binding energies (Kcal/mol) of docked compound 7 inside two studied 4K0Y and 2OBJ as PIM-1 kinase proteins.
Protein (PDB code)
Binding affinity(Kcal/mol)
Type on interaction
Bond length (A◦)
Interaction moiety involved
Amino acid
4K0Y
¡20.86
H-acceptor
2.39
Lys 67
2OBJ
¡20.70
H-acceptor
2.033
Lys 67
It forms arene-cation interaction with Lys 67
*Superimposed compound 7 (green), and the co-crystalized ligand (Orange) inside the two studied 4K0Y and 2OBJ PIM-1 kinase protein.
152.2, 159.9, 162.5; IR (KBr/cmꢀ 1): 1573 (C O), 2216 (C N), 2836
–
–
–
–
DMSO‑d6): δ 55.9, 106.5, 114.7, 117.2, 128.2, 128.6, 129.3, 130.5,
–
131.5, 132.9, 151.6, 159.7, 161.6, 162.6; IR (KBr/cmꢀ 1): 1574 (C O),
(NH); CHN Calcd. For C19H14N2O2 (302.33): C, 75.48; H, 4.67; N, 9.27.
–
–
–
–
2218 (C N), 2843 (NH); CHN Calcd. For C19H14N2O2 (302.11): C,
Found: C, 75.51; H, 4.63; N, 9.32%.
–
75.48; H, 4.67; N, 9.27. Found: C, 75.44; H, 4.62; N, 9.33; O, 10.61%.
3.1.1.10. 4-(4-(Dimethylamino)phenyl)-2-oxo-6-phenyl-1,2-dihydropyr-
idine-3-carbonitrile (3j). Yield: 44 %Ethanol as yellow powder, m.p. 260–262
◦C.1H NMR (400 MHz, DMSO‑d6): δ 3.03 (s, 6H, N(CH3)2), 6.84 (s, 1H),
6.85 (d, J = 7.6 Hz, 2H), 7.55 (s, 3H), 7.69 (d, J = 8.0 Hz, 2H), 7.87 (s,
2H), 12.50 (s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ ≈ 39.6, 105.9,
112.1, 117.8, 122.7, 128.0, 129.3, 130.1, 131.1, 133.1, 152.4, 159.8,
3.1.1.8. 4-(4-Nitrophenyl)-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbon-
itrile (3h). Yield: 41 %Acetic acid glacial + DMF-Ethanol (1:1) as yellow powder, m.
1
◦
p. 300–302 C. H NMR (400 MHz, DMSO‑d6): δ 6.93 (s, 1H), 7.57 (s,
3H), 7.94 (s, 2H), 8.00 (d, J = 7.6 Hz, 2H), 8.39 (d, J = 7.6 Hz, 2H),
12.30 (s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ 98.8, 106.7, 116.5,
124.2, 128.2, 129.3, 130.3, 131.7, 133.1, 142.9, 148.9, 153.1, 157.8,
162.9; IR (KBr/cmꢀ 1): 1598 (C O), 2213 (C N), 2792 (NH); CHN
–
–
–
–
–
162.6; IR (KBr/cmꢀ 1): 1344, 1522 (C-NO ), 1576 (C O), 2218 (C N),
Calcd. For C20H17N3O (315.14): C, 76.17; H, 5.43; N, 13.32. Found: C,
76.21; H, 5.47; N, 13.39%.
–
–
–
–
–
2
2773 (NH); CHN Calcd. For C18H11N3O3 (317.3): C, 68.14; H, 3.49; N,
13.24. Found: C, 68.19; H, 3.53; N, 13.35%.
3.1.1.11. 4-(Furan-2-yl)-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbon-
itrile (3 k). Yield: 35 %Ethanol-acetic acid (1:1) as brown crystal, m.p. 300 ◦C.1H
NMR (400 MHz, DMSO‑d6): δ 6.85 (s, 1H), 7.05 (s, 1H), 7.57 (s, 3H),
7.71 (s, 1H), 7.88 (s, 2H), 8.09 (s, 1H), 12.54 (s, 1H, NH);13C NMR (100
MHz, DMSO‑d6): δ 93.0, 101.5, 113.8, 116.7, 117.1, 128.1, 129.4,
131.6, 132.8, 145.6, 147.5, 148.3, 151.8, 162.6; IR (KBr/cmꢀ 1): 1570
3.1.1.9. 4-(3-Methoxyphenyl)-2-oxo-6-phenyl-1,2-dihydropyridine-3-car-
bonitrile (3i). Yield: 69 %Acetic acid glacial as yellow crystal, m.p. 240–242
◦C.1H NMR (400 MHz, DMSO‑d6): δ 3.86 (s, 3H, OCH3), 6.85 (s, 1H),
7.13 (d, J = 7.2 Hz, 1H), 7.29 (s, 2H), 7.49–7.56 (m, 4H), 7.91 (s, 2H),
12.71 (s, 1H, NH);13C NMR (100 MHz, DMSO‑d6): δ 55.9, 98.8, 106.8,
114.3, 116.6, 116.7, 120.9, 128.2, 129.3, 130.4, 131.5, 133.1, 138.0,
–
–
–
(C O), 2773–2892 furyl ring, 2214 (C N), 2773 (NH); CHN Calcd. For
–
–
9

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Fig. 7. A: BOILED-Egg model for compound 7 using SwissADME “points located in the BOILED-Egg‘s yolk are molecules predicted to passively permeate through the
blood–brain barrier (BBB), while points located in the BOILED-Egg‘s white are molecules predicted to be passively absorbed by Gastrointestinal (GI) tract GI”, B: Drug
likeness score of compound 7 using MolSoft “The green color means non-drug like behavior and those fall under blue color area are considered as drug-like. Those
compounds having negative or zero value should not be considered as drug like” (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
(s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.73 (d, J =
Table 5
8.4 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 12.65 (s, 1H, NH);13C NMR (100
Molecular properties of and drug-likeness.
MHz, DMSO‑d6): δ 56.0, 114.8, 129.3, 130.0, 130.4; IR (KBr/cmꢀ 1): 689
–
Website
CompoundADME
7
13a
13c
–
–
(C-Cl), 1656 (C O), 2200 (C N), 3413 (NH); CHN Calcd. For
–
–
C
19H13ClN2O2 (336.78): C, 67.76; H, 3.89; Cl, 10.53; N, 8.32. Found: C,
molinspiration 2018.10
Mwt (D)
344.37
308.02
101.04
2.07
5
401.42
358.07
101.32
3.38
8
415.45
374.87
101.32
3.35
9
MV (A3)
67.71; H, 3.91; Cl, 10.49; N, 8.25%.
PSA (A2)
Log p
3.1.1.14. 6-(3-Aminophenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyr-
idine-3-carbonitrile (3n). Yield: 59 %Acetic acid glacial as white powder, m.p.
300–302 ◦C.1H NMR (400 MHz, DMSO‑d6): δ 3.86 (s, 3H), 5.27 (s, 1H),
6.61 (s, 1H), 6.76 (d, J = 6.8 Hz, 1H), 7.01 (s, 2H), 7.11–7.17 (m, 4H),
7.70 (d, J = 6.8 Hz, 2H), 12.30 (s, 1H, NH);13C NMR (100 MHz,
DMSO‑d6): δ 98.8, 106.8, 114.2, 116.6, 116.7, 120.9, 128.2, 129.3,
130.4, 131.5, 133.1, 138.0, 152.2, 159.9, 162.5; IR (KBr/cmꢀ 1): 1600
nrotb
nviolations
HBA
0
0
0
MolSoft
5
6
6
HBD
3
1
1
Solubility (mg/L)
Drug-likeness score
78.72
0.09
29.63
0.08
42.77
0.12
MWt: Molecular Weight, MV: Molecular Volume, PAS: Polar Surface Area, Log p:
Log P: Octanol-water partition coefficient, nrotb: number of rotatable bond,
nviolations: number of violations, HBA: Hydrogen Bond Acceptor, HBD:
Hydrogen Bond Donor.
–
–
–
(C O), 2218 (C N), 2903 (NH), 3140 (NH2); CHN Calcd. For
–
–
C
19H15N3O2 (317.35): C, 71.91; H, 4.76; N, 13.24. Found: C, 71.87; H,
4.72; N, 13.29%.
C16H10N2O2 (262.26): C, 73.27; H, 3.84; N, 10.68. Found: C, 73.73; H,
3.91; N, 10.49%.
3.1.2. Coupling of 3a with a set of alkyl halides:
To a mixture of pyridone 3a (1.0 mol), K2CO3 (1.2 mol) in acetone
10 mL and DMF 1.0 mL, the appropriate alkyl halide (1.1 mol) (named
benzyl chloride, phenacyl bromide and ethyl chloroacetate) was added.
The mixture was refluxed for 5 h, cooled, poured into ice water filtered,
dried and recrystallized in ethanol.
3.1.1.12. 4-(3-Chlorophenyl)-6-(4-chlorophenyl)-2-oxo-1,2-dihydropyr-
idine-3-carbonitrile (3l). Yield: 55%Ethanol as yellow powder,m.p. 302–304
◦C.1H NMR (400 MHz, DMSO‑d6): δ 7.08 (s, 1H), 7.61 (d, J = 8.0 Hz,
2H), 7.87 (t, J = 8.0 Hz, 1H), 7.98 (d, J = 7.2 Hz, 2H), 8.19 (d, J = 8.0
Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 8.56 (s, 1H), 12.9 (s, 1H, NH);13C NMR
(100 MHz, DMSO‑d6): δ 116.4, 123.7, 125.4, 129.4, 130.2, 131.0, 135.4,
3.1.2.1. 2-(benzyloxy)-4,6-diphenylnicotinonitrile (4). Yield: 70%
yellowish white powder, m.p. 140–142 ◦C.1H NMR (400 MHz, DMSO‑d6):aδs
5.70 (s, 2H), 7.36–7.82 (m, 14H), 8.27 (s, 2H); 13C NMR (100 MHz,
DMSO‑d6): δ 68.8, 93.0, 114.5, 115.7, 128.0, 128.3, 128.5, 129.0, 129.1,
129.3, 129.4, 130.5, 131.2, 136.3, 137.0, 137.1, 157.0, 157.8, 164.2; IR
136.6, 137.9, 148.4; IR (KBr/cmꢀ 1): 689 (C-Cl), 1600 (C O), 2216
–
–
–
–
(C N), 2956 (NH); CHN Calcd. For C18H10Cl2N2O (341.19): C, 63.37;
–
H, 2.95; Cl, 20.78; N, 8.21. Found: C, 63.33; H, 3.01; Cl, 20.83; N,
8.29%.
(KBr /cmꢀ 1): 2195 (C N); CHN Calcd. For C25H18N2O (362.14): C,
–
–
–
82.39; H, 5.53; N, 7.69. Found: C, 82.32; H, 5.57; N, 7.61%.
3.1.1.13. 6-(4-Chlorophenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihy-
dropyridine-3-carbonitrile (3m). Yield: 51%Ethanol
as yellowish white pow-
3.1.2.2. 2-(2-Oxo-2-phenylethoxy)-4,6-diphenylnicotinonitrile (5). Yield:
der,m.p. 275–277 ◦C.1H NMR (400 MHz, DMSO‑d6): δ 3.87 (s, 3H), 6.87
Table 6
Molinspiration bioactivity scores for compounds 7.
Compound
GPCR ligand
Ion channel modulator
Kinase inhibitor
Nuclear receptor ligand
Protease inhibitor
Enzyme inhibitor
0.02
7
ꢀ 0.11
ꢀ 0.34
0.11
ꢀ 0.28
ꢀ 0.15
10

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
90%
powder, m.p.166–168 ◦C.1H NMR (400 MHz,
white
N, 14.41; S, 8.36%.
as
DMSO‑d6y)e:lδlow6.is0h(s, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H),
7.61–7.82 (m, 9H), 7.98 (d, J = 7.2 Hz, 2H), 8.08 (d, J = 7.2 Hz, 2H); 13C
NMR (100 MHz, DMSO‑d6): δ 69.3, 92.7, 114.8, 115.6, 127.8, 128.2,
128.3, 128.7, 129.1, 129.2, 129.4, 130.6, 130.8, 131.1, 132.9, 135.0,
136.2, 136.6, 136.8, 157.1, 157.5, 163.6, 194.3; IR (KBr/cmꢀ 1): 1587
3.1.3.5. 2-((5-((2-Oxo-2-phenylethyl)thio)-1,3,4-oxadiazol-2-yl)
methoxy)-4,6-diphenylnicotinonitrile (10). A mixture of oxadiazolethione
9 (1.0 mmol), potassium carbonate and phenacyl bromide in acetone/
DMF 10:1 was heated under reflux for 3 h cooled, poured into water
filtered, dried and recrystallized from MeOH to give (10);
–
–
–
(C O), 2219 (C N); CHN Calcd. For C26H18N2O2 (390.44): C, 79.98; H,
–
–
Yield: 70% as yellow brown powder, m.p. 92–94 ◦C.1H NMR (400 MHz,
DMSO‑d6): δ 5.1 (s, 2H), 5.9 (s, 2H), 7.54–7.68 (m, 8H), 7.76 (s, 3H),
7.89 (s, 1H), 7.99 (d, J = 7.2 Hz, 2H), 8.24 (s, 2H);13C NMR (100 MHz,
DMSO‑d6): δ 39.7, 58.6, 93.1, 115.1, 115.4, 128.0, 128.8, 129.1, 129.3,
130.6, 131.3, 134.3, 135.6, 136.2, 136.8, 157.4, 157.7, 163.0, 164.1,
164.6, 192.8; CHN Calcd. For C29H20N4O3S (504.56): C, 69.03; H, 4.00;
N, 11.10; S, 6.35. Found: C, 69.09; H, 3.97; N, 11.15; S, 6.32%.
4.65; N, 7.17. Found: C, 79.93; H, 4.59; N, 7.23%.
3.1.2.3. Ethyl 2-(3-cyano-4, 6-diphenylpyridin-2-yloxy)acetate (6).
Yield: 60%
powder, m.p.138–140 ◦C.1H NMR (400 MHz,
as white
DMSO‑d6): δ 1.22 (t, J = 7.0 Hz, 3H, CH3), 4.2 (q, J = 6.9 Hz, 2H, CH2),
5.18 (s, 2H), 7.53 (s, 3H), 7.60 (s, 3H), 7.77 (s, 2H), 7.87 (s, 1H), 8.18 (s,
2H); 13C NMR (100 MHz, DMSO‑d6): δ 14.5, 61.2, 64.2, 92.7, 114.9,
115.4, 127.8, 129.1, 129.3, 130.6, 131.3, 136.1, 136.6, 157.1, 157.3,
163.4, 168.7; IR (KBr/cmꢀ 1): 1590 (C O), 2224 (C N); CHN Calcd.
–
–
–
–
–
3.1.3.6. Coupling of 2-(3-cyano-4,6-diphenylpyridin-2-yloxy)aceto hydra-
For C22H18N2O3 (358.40): C, 73.73; H, 5.06; N, 7.82. Found: C, 73.79; H,
4.99; N, 7.85%.
zide(7) with amines (12a-e) and α-Amino acid ester (13a,b):. To a cold
solution (~ꢀ 5◦C) of hydrazide 7 (0.01 mmol) in 1.0 N HCl (20 mL) and
acetic acid 96% (3 mL), 5 mL aqueous solution of NaNO2 (0.02 mmol)
was added to the mixture dropwise, then the mixture was stirred at 0 ◦C
for 30 min. The reaction mixture was extracted three times with 30.0 mL
ethyl acetate. The extract was washed several times with 3% aqueous
solution of Na2CO3 till it became neutral then washed with water. The
azide extract 11 was dried over Na2SO4. The azide solution 11 was
allowed to react with appropriate amines (N-propyl amine, benzyl
amine, tetradecyl amine, allyl amine and pipyridine) or selected amino
ester hydrochloride (0.01 mol) (glycine and L-leucine) in the presence of
triethyl amine (1.0 mL, 0.01 mol) after complete addition, the reaction
mixture was kept at ꢀ 5◦C for 24 h, and then at room temperature for
another 24 h. The reaction mixture washed with 0.5 N HCl, water, 3%
solution of Na2CO3, and dried over Na2SO4. The solution was evaporated
to dryness, and the residues were recrystallized by ethyl acetate/pe-
troleum ether 1:1 to give the desired product.
3.1.3. Hydrazinolysis of the ester 6:
3.1.3.1. 2-(3-Cyano-4,6-diphenylpyridin-2-yloxy)acetohydrazide (7). To
the ester 6 (1.0 mol) in MeOH (20 mL), hydrazine hydrate (3.0 mol) was
added and the mixture was refluxed for 3.5 h then left to cool. The
formed precipitate was filtered, washed with water, dried and recrys-
tallized from ethanol.
1
◦
Yield: 50% as yellowish white powder, m.p. 204 C. H NMR (400 MHz,
DMSO‑d6): δ 4.30 (s, 2H), 5.03 (s, 2H), 7.50–8.28 (m, 11H), 9.39 (s, 1 H);
13C NMR (100 MHz, DMSO‑d6): δ 64.9, 93.2, 114.7, 115.7, 128.0, 129.1,
129.4, 130.6, 131.2, 136.3, 136.9, 156.9, 157.5, 163.8, 167.0; IR (KBr/
cmꢀ 1): 1604 (C O), 2216 (C N), 2731 (NH), 3291 (NH2); CHN Calcd.
–
–
–
–
–
For C20H16N4O2 (344.37): C, 69.76; H, 4.68; N, 16.27. Found: C, 69.80;
H, 4.65; N, 16.35%
3.1.3.2. Heterocyclization of hydrazide (7). To a mixture of hydrazide 7
(1.1 mmol, 0.5 g) in ethanol 10 mL, KOH (0.4 g in 0.5 mL water) and
carbon disulphide (2.0 mL) were added and stirred overnight then
diethyl ether 10.0 mL was added and stirred for additional 6 h, the
excess solvent was evaporated, distilled water was added and the
mixture was acidified with conc. HCl, the formed solid was filtered,
washed with water, dried and recrystallized from ethanol to give 8.
When the mixture was refluxed with stirring for 7 h followed by solvent
evaporation, acidification and recrystallization compound 9 was
obtained.
3.1.3.7. 2-(3-Cyano-4,6-diphenylpyridin-2-yloxy)-N-propylacetamide
(12a). Yield: 80% as white powder, m.p. 180–182 ◦C.1H NMR (400 MHz,
DMSO‑d6): δ 0.82 (t, J = 6.0 Hz, 3H), 1.42–1.45 (m, 2H), 3.20 (m, 2H),
5.00 (s, 2H), 7.53–8.23 (m, 12H); 13C NMR (100 MHz, DMSO‑d6): δ 11.8,
22.9, 65.9, 93.0, 114.5, 115.7, 128.0, 129.1, 129.3, 129.4, 130.6, 131.2,
136.3, 136.9, 156.9, 157.4, 163.9, 167.5;IR (KBr/cmꢀ 1): 1589 (C O),
–
–
–
–
2219 (C N), 2875 (NH); CHN Calcd. For C23H21N3O2 (371.44): C,
–
74.37; H, 5.70; N, 11.31. Found: C, 74.31; H, 5.75; N, 11.29%.
3.1.3.8. N-benzyl-2-(3-cyano-4,6-diphenylpyridin-2-yloxy)acetamide
(12b). Yield: 30% as white powder, m.p. 183–185 ◦C.1H NMR (400
MHz, DMSO‑d6): δ 4.35 (d, J = 6.0 Hz, 2H), 5.11 (s, 2H), 7.10–7.15 (m,
3H), 7.20 (d, J = 7.2 Hz, 2H), 7.50–7.53 (m, 3H), 7.61–7.62 (m, 2H),
3.1.3.3. 2-(2-((3-Cyano-4,6-diphenylpyridin-2-yl)oxy)acetyl)hydrazine-
1-carbodithioic acid (8). Yield: 60%
powder, m.p. 95–97
as brownish red
◦C.1H NMR (400 MHz, DMSO‑d6): δ 3.54 (s, 1H, SH), 5.64 (s, 2H),
7.54–7.61 (m, 8H), 7.73–7.79 (m, 2H), 7.87 (s, 1H), 8.28–8.31 (m,
2H);13C NMR (100 MHz, DMSO‑d6): δ 59.0, 92.9, 115.0, 115.4, 128.1,
128.7, 129.1, 129.3, 129.4, 130.6, 130.9, 131.3, 136.2, 136.8, 136.8,
7.77 (m, 2H), 7.86 (s, 1H), 8.22 (d, J = 7.2 Hz, 1H), 8.67 (m, 1H); 13
C
NMR (100 MHz, DMSO‑d6): δ 42.4, 65.9, 93.2, 114.7, 115.7, 127.1,
127.5, 127.7, 128.0, 128.5, 129.1, 129.3, 130.6, 131.2, 136.3, 136.9,
139.6, 156.9, 157.6, 163.9, 167.8;IR (KBr/cmꢀ 1): 1584 (C O), 2217
–
–
157.3, 157.7, 159.1, 163.3, 180.5;IR (KBr/cmꢀ 1): 759 (C S), 1561
(C N), 2911 (NH); CHN Calcd. For C27H21N3O2 (419.48): C, 77.31; H,
–
–
–
–
–
–
–
–
–
(C O), 1628 (C S), 2250 (C N), 3416, 3460 (CO-NH); CHN Calcd.
5.05; N, 10.02. Found: C, 77.26; H, 4.97; N, 10.13%.
–
–
For C21H16N4O2S2 (420.51): C, 59.98; H, 3.84; N, 13.32; S, 15.25.
Found: C, 59.93; H, 3.87; N, 13.28; S, 15.34%.
3.1.3.9. 2-(3-Cyano-4,6-diphenylpyridin-2-yloxy)-N-tetradecylacetamide
(12c). Yield: 50% as white powder, m.p. 138–140 ◦C.1H NMR (400 MHz,
DMSO‑d6): δ 0.85 (t, J = 5.6 Hz, 3H), 1.15–1.25 (m, 24H), 3.28 (m, 2H),
4.98 (s, 2H), 7.52–8.24 (m, 12H); 13C NMR (100 MHz, DMSO‑d6): δ 14.3,
22.5, 26.8, 29.1, 29.4, 31.7, 40.7, 127.1, 127.5, 128.0, 129.0, 129.2,
3.1.3.4. 4,6-Diphenyl-2-((5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)
methoxy)nicotinonitrile (9). Yield: 80% as yellow orange powder, m.p. 215
◦C.1H NMR (400 MHz, DMSO‑d6): δ 5.77 (s, 2H), 7.54–7.62 (m, 6H),
7.77–7.80 (m, 2H), 7.93 (s, 1H), 8.25–8.27 (m, 2H), 14.64 (br.s, 1H,
NH);13C NMR (100 MHz, DMSO‑d6): δ 58.7, 93.2, 115.1, 115.5, 128.0,
128.6, 129.1, 129.2, 129.3, 129.4, 130.6, 130.7, 131.3, 131.5, 136.1,
129.4, 131.2;IR (KBr/cmꢀ 1): 1589 (C O), 2200 (C N), 2848 (NH);
–
–
–
–
–
CHN Calcd. For C34H43N3O2 (525.74): C, 77.68; H, 8.24; N, 7.99. Found:
C, 77.71; H, 8.19; N, 7.93%.
136.7, 157.4, 157.7, 159.9, 162.9, 178.7; IR (KBr/cmꢀ 1): 695 (C S),
–
–
–
–
–
–
–
1598 (C O), 1641 (C N), 2218 (C N); CHN Calcd. For C21H14N4O2S
3.1.3.10. N-allyl-2-(3-cyano-4,6-diphenylpyridin-2-yloxy)acetamide
–
(12d). Yield: 48% as white powder, m.p. 153–155 ◦C.1H NMR (400 MHz,
(344.37): C, 65.27; H, 3.65; N, 14.50; S, 8.30. Found: C, 65.32; H, 3.69;
11

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
DMSO‑d6): δ 3.79–3.87 (m, 3H), 4.99–5.18 (m, 4H), 5.77–5.84 (m, 1H),
7.53–8.23 (m, 11H); 13C NMR (100 MHz, DMSO‑d6): δ 41.2, 65.8, 93.1,
114.6, 115.8, 128.0, 129.0, 129.3, 130.6, 131.2, 135.5, 136.3, 136.9,
and Cell cycle distribution were then acquired by Cytoflex FACS ma-
chine. Flow cytometric methodologies (annexin V/PI staining with cell
cycle analyses) are carried out as previously described in [22,28,29].
157.0, 157.5, 163.9, 167.5;IR (KBr/cmꢀ 1): 1588 (C O), 2220 (C N),
–
–
–
–
–
3287 (NH); CHN Calcd. For C23H19N3O2 (369.42): C, 74.78; H, 5.18; N,
3.2.3. RT-PCR
11.37. Found: C, 74.71; H, 5.23; N, 11.33%.
HepG2 cells were treated with compound7 (IC50 = 7.26 μM, 48 h). At
the end of treatments, cells were collected, RNA was extracted, cDNA
was synthesized and Real-time RT-PCR reactions were performed
following the manufacturer’s instructions and our previous publications
[39–42]. Then, The Ct values were collected, and the relative folds of
changes between all the samples. Primers sequnces in both forward and
reverse directions are summarized in Table 7.
3.1.3.11. 2-(2-Oxo-2-(piperidin-1-yl)ethoxy)-4,6-diphenylnicotinonitrile
(12e). Yield: 75% as white powder, m.p. 208–210 ◦C.1H NMR (400 MHz,
DMSO‑d6): δ 1.47–1.66 (m, 6H), 3.48 (d, J = 8.0H, 4H), 5.35 (s, 2H),
7.53–8.20 (m, 11H); 13C NMR (100 MHz, DMSO‑d6): δ 24.5, 25.8, 26.5,
42.8, 45.5, 64.6, 92.7, 114.4, 115.7, 127.9, 129.1, 129.3, 129.4, 130.6,
131.2, 136.3, 137.0, 156.9, 157.4, 164.1, 165.2;IR (KBr/cmꢀ 1): 1587
–
–
–
3.2.4. In vivo (SEC animal model)
(C O), 2218 (C N), 2856 (NH); CHN Calcd. For C25H23N3O2 (397.48):
–
–
All protocols and procedures employed in animal studies were suu-
marized in Fig. 8, and were ethically reviewed and approved by Suez
Canal University’s Ethical Committee (REC-07-2020).
C, 75.55; H, 5.83; N, 10.57. Found: C, 75.51; H, 5.86; N, 10.55%.
3.1.3.12. Reaction of 2-(3-cyano-4,6-diphenylpyridin-2-yloxy)acetohy-
drazide (7) with α-amino acids esters to give(13a-b)
3.2.5. In silico studies
3.1.3.12.1. Methyl 2-(2-(3-cyano-4,6-diphenylpyridin-2-yloxy)acet-
amido)acetate (13a). Yield: 30% as white crystal, m.p. 188–190 ◦C.1H NMR
(400 MHz, DMSO‑d6): δ 3.60 (s, 3H), 3.94 (d, J = 5.6 Hz, 2H), 5.11 (s,
2H), 7.51–7.78 (m, 6H), 7.85 (s, 1H), 8.23–8.61 (m, 4H), 13.66 (s, 1H);
13C NMR (100 MHz, DMSO‑d6): δ 52.1, 55.9, 65.4, 93.0, 114.5, 114.7,
117.4, 118.2, 128.0, 129.3, 129.6, 130.0, 130.6, 131.2, 135.5, 132.3,
3.2.5.1. Molecular docking simulation. For elucidation, the virtual
mechanism of binding, the molecular docking study towards the Pim-1
kinase active site was carried out. All synthesized derivatives were
chemically and energetically optimized. Additionally, protein structures
(PDB = 2OBJ, 4K0Y) were freely accessible from the PDB and optimized
following the routine work as discussed by Nafie et al [43]. MOE 2019
was used as the validated molecular docking calculation, and Chimera
software was finally used as the visualized software for the analysis of
drug-target interactions.
136.9, 157.4, 157.5, 165.9, 169.7, 170.5; IR (KBr/cmꢀ 1): 1587 (C O),
–
–
–
–
2220 (C N), 2905 (NH); CHN Calcd. For C23H19N3O4 (401.42): C,
–
68.82; H, 4.77; N, 10.47. Found: C, 68.79; H, 4.73; N, 10.51%.
3.1.3.12.2. Methyl 2-(2-(3-cyano-4,6-diphenylpyridin-2-yloxy)acet-
amido)-4-methyl pentanoate (13b). Yield: 30% as Yellowish white powder, m.
p. 118–120 ◦C.1H NMR (400 MHz, DMSO‑d6): δ 0.71 (d, J = 6.0 Hz, 3H),
0.76 (d, J = 6.0 Hz, 3H), 1.52–1.59 (m, 1H), 3.58 (s, 3H), 4.39 (q, J =
6.0 Hz, 1H), 4.99 (s, 2H), 5.03–5.15 (m, 2H), 7.29 (s, 1H), 7.51–7.78 (m,
5H), 7.84 (s, 2H), 8.21–8.25 (m, 2H), 8.60 (d, J = 8.0 Hz, 1H); 13C NMR
(100 MHz, DMSO‑d6): δ 21.6, 23.1, 24.5, 50.4, 52.3, 65.4, 65.5, 92.9,
93.0, 114.6, 115.7, 128.0, 128.7, 129.1, 130.3, 130.7, 131.2, 132.3,
135.5, 136.8, 137.0, 156.9, 157.5, 163.8, 167.8, 169.7, 173.2;IR (KBr/
3.2.5.2. ADME pharmacokinetics. In silico ADME pharmacokinetics pa-
rameters of the most active compounds were calculated using a set of
software including “MolSoft”, “Molinspiration”, and “SwissADME”
websites as previously described by Youssef et al., 2020 [29,44].
4. Conclusion
cmꢀ 1): 1586 (C O), 2221 (C N), 2953 (NH); CHN Calcd. For
–
–
–
–
–
C
27H27N3O4 (457.53): C, 70.88; H, 5.95; N, 9.18. Found: C, 70.91; H,
In summary, a series of 14 derivatives of 4,6-diaryl-3-cyano-2-pyri-
dones 3a-n were synthesized from various aromatic aldehydes, ke-
tones, ethyl cyanoacetate, and ammonium acetate in butanol. Alkylation
of pyridones was found to proceed on oxygen. Alkylation of 1,3,4-oxa-
diazolethione was deduced to occur on sulfur. Azide-coupling method
was found to couple the hydrazide 7 with a set of amines and amino acid
esters HCl. Compound 7 give the most cytotoxic activity against HepG2
with the lowest IC50 value of 7.26 compared to 5-FU (IC50 = 6.98 µM),
5.91; N, 9.12%.
3.2. Biological assays
3.2.1. Cell culture, treatment and MTT assay
The human liver carcinoma cell line (HepG2), and normal liver cells
(THLE2) were purchased from the National Cancer Institute, Cairo,
Egypt. Cells were maintained in DMEM media supplemented with
2mML-glutamine (Lonza, Belgium) and 10% FBS (Sigma, St. Louis, MO,
USA), 1% Penicillin/Streptomycin (Lonza, Belgium) according to the
routine culture work [36]. Cells after culturing were treated with the
indicated compounds at the indicated concentrations (0.01, 0.1, 1, 10,
Table 7
Primers used for real-time RT-PCR.
Primer
Sequence
β-Actin
FOR: 5′-GCACTCTTCCAGCCTTCCTTCC-3′REV: 5′-
and 100 μM). Cell viability was assessed after 48 h using MTT solution
GAGCCGCCGATCCACACG-3′
(Promega) [37]. Absorbance was subsequently measured (570 nm)
using a plate reader, then percentages of cell viabilities at corresponding
concentrations were recorded and the IC50 values were calculated using
GraphPad Prism 7 software [38–40].
P53
FOR: 5′-CTTTGAGGTGCGTGTTTGTG-3′REV: 5′-
GTGGTTTCTTCTTTGGCTGG-3′
Bcl-2
FOR: 5′-GAGGATTGTGGCCTTCTTTG-3′REV: 5′-
ACAGTTCCACAAAGGCATCC-3′
PUMA
BAX
FOR: 5′-GAGGAGGAACAGTGGGC-3′REV: 5′-
CTAATTGGGCTCCATCTCGG-3′
FOR: 5′-TTTGCTTCAGGGTTTCATCC-3′REV: 5′-
CAGTTGAAGTTGCCGTCAGA-3′
3.2.2. Annexin V/PI and cell cycle analysis
Apoptosis rate in cells was quantified using annexin V-FITC (BD
Pharmingen, USA). Cells were seeded into 6-well culture plates (3–5 ×
105 cells/well) and cultured as routine work. Cells were then treated
Casp-3
Casp-8
Casp-9
PIM-1
FOR: 5′-TGGCCCTGAAATACGAAGTC-3′REV: 5′-
GGCAGTAGTCGACTCTGAAG-3′
FOR: 5′-AATGTTGGAGGAAAGCAAT-3′REV: 5′-
CATAGTCGTTGATTATCTTCAGC-3′
with compound 7 (IC50 = 7.26 μM, 48 h), and cells were collected and
FOR: 5′-CGAACTAACAGGCAAGCAGC-3′REV: 5′-
ACCTCACCAAATCCTCCAGAAC-3′
washed with ice-cold PBS. Next, cells were suspended in 100 µL of
annexin binding buffer solution “25 mM CaCl2, 1.4 M NaCl, and 0.1 M
Hepes/NaOH, pH 7.4, and incubated in the dark for 30 min with annexin
V-FITC solution (1:100)and PI at 10 µg/mL concentration”. Stained cells
FOR: 5′-GCAAATAGCAGCCTTTCTGG-3′REV: 5′-
CCTAGGACCCCTGGAGAGTC-3′
12

A.T.A. Boraei et al.
Bioorganic Chemistry 111 (2021) 104877
Fig. 8. In vivo experiment design [28,29]
(2004) 367–371, https://doi.org/10.1002/(ISSN)1097-004510.1002/pros.v60:
410.1002/pros.20064.
and it was nontoxic against THLE2 cells. It inhibited cell growth by
76.76%. Additionally, it significantly stimulated apoptotic liver cancer
cell death with 49.78-fold (22.90% compared to 0.46% for the control)
arresting cell cycle pre-G1 with 35.16% population, compared to 1.57%
for the control. Moreover, it validated the intrinsic apoptosis through
upregulation of P53, and other related genes, with inhibition of anti-
apoptotic genes through PIM-1 inhibition. Hence, this compound is
worthy to be tested preclinically and further developed as a chemo-
therapeutic anti-cancer drug.
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Declaration of Competing Interest
[15] G. Papeo, H. Posteri, D. Borghi, A.A. Busel, F. Caprera, E. Casale, M. Ciomei,
A. Cirla, E. Corti, M. D’Anello, M. Fasolini, B. Forte, A. Galvani, A. Isacchi,
A. Khvat, M.Y. Krasavin, R. Lupi, P. Orsini, R. Perego, E. Pesenti, D. Pezzetta,
S. Rainoldi, F. Riccardi-Sirtori, A. Scolaro, F. Sola, F. Zuccotto, E.R. Felder,
D. Donati, A. Montagnoli, Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-
yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A
Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer
Therapy, J. Med. Chem. 58 (17) (2015) 6875–6898, https://doi.org/10.1021/acs.
jmedchem.5b00680.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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