Selective VEGFR-2 inhibitors: Synthesis of pyridine derivatives, cytotoxicity and apoptosis induction profiling

Article abstract of DOI:10.1016/j.bioorg.2020.104222

VEGFR-2 is a key regulator in cancer angiogenesis. This research displays the design and synthesis of novel 3-cyano-6-naphthylpyridine scaffold-based derivatives as selective VEGFR-2 inhibitors and cytotoxic agents. In vitro percent kinase activity inhibition screening against a panel of 23 kinases at a single high dose (30 nM) affirmed that VEGFR-2 was selectively the most responsive to inhibition by the investigated chemotypes. IC₅₀ values determination demonstrated kinase inhibitory activities of the test compounds at the sub-nanomolar level. In vitro testing of the new compounds against two prostate cancer cell lines namely PC3 and DU145 and two breast cancer cell lines namely MCF-7 and MDA-MB435 confirmed their potent cytotoxic activity with IC₅₀s at the nanomolar level. The most active compound against MCF-7 viz. 11d was subjected to an in vivo examination against a xenograft mouse model and was found effective. Studying the tissue mRNA expression levels of various cell cycle controlling biomolecules in 11d-treated MCF-7 cells demonstrated (i) upregulation of p53, p21 and p27, (ii) cleavage of PARP protein, (iii) activation of caspase-3, ?8 and ?9, (iv) downregulation of the anti-apoptotic protein Bcl, (v) upregulation of the pro-apoptotic protein Bax, and (vi) decreased expression of Cdks 2, 4, 6 and cyclin D1. Additionally, 11d affected a cell cycle arrest at the G1 phase in treated MCF-7 cells and an S phase arrest in MCF-7 p53 knockdown cells. Additionally, molecular docking was performed to predict how 11d might bind to its biological target VEGFR-2. Finally, in-silico ADME and drug-likeness profiling of these derivatives demonstrated favorable properties thereof.

Full text of DOI:10.1016/j.bioorg.2020.104222

BioorganicChemistry103(2020)104222
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Selective VEGFR-2 inhibitors: Synthesis of pyridine derivatives, cytotoxicity
and apoptosis induction profiling
⁎
Amal AbdelHaleema, Amira O. Mansourb, Marwa AbdelKaderc, Reem K. Arafac,d,
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, 11562, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, MSA University, Egypt
c Drug Design and Discovery Lab, Zewail City of Science and Technology, Cairo 12578, Cairo, Egypt
d University of Science and Technology, Zewail City of Science and Technology, Cairo 12578, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
VEGFR-2 is a key regulator in cancer angiogenesis. This research displays the design and synthesis of novel 3-
cyano-6-naphthylpyridine scaffold-based derivatives as selective VEGFR-2 inhibitors and cytotoxic agents. In
vitro percent kinase activity inhibition screening against a panel of 23 kinases at a single high dose (30 nM)
affirmed that VEGFR-2 was selectively the most responsive to inhibition by the investigated chemotypes. IC50
values determination demonstrated kinase inhibitory activities of the test compounds at the sub-nanomolar
level. In vitro testing of the new compounds against two prostate cancer cell lines namely PC3 and DU145 and
two breast cancer cell lines namely MCF-7 and MDA-MB435 confirmed their potent cytotoxic activity with IC50s
at the nanomolar level. The most active compound against MCF-7 viz. 11d was subjected to an in vivo ex-
amination against a xenograft mouse model and was found effective. Studying the tissue mRNA expression levels
of various cell cycle controlling biomolecules in 11d-treated MCF-7 cells demonstrated (i) upregulation of p53,
p21 and p27, (ii) cleavage of PARP protein, (iii) activation of caspase-3, −8 and −9, (iv) downregulation of the
anti-apoptotic protein Bcl, (v) upregulation of the pro-apoptotic protein Bax, and (vi) decreased expression of
Cdks 2, 4, 6 and cyclin D1. Additionally, 11d affected a cell cycle arrest at the G1 phase in treated MCF-7 cells
and an S phase arrest in MCF-7 p53 knockdown cells. Additionally, molecular docking was performed to predict
how 11d might bind to its biological target VEGFR-2. Finally, in-silico ADME and drug-likeness profiling of these
derivatives demonstrated favorable properties thereof.
Naphthylpyridines
Cytotoxicity
Cell cycle arrest
Apoptosis
Kinase inhibition
VEGFR-2 selective inhibition
1. Introduction
are the family of protein tyrosine kinases inhibitors (PTKIs). Protein
tyrosine kinases (PTKs) are well established key players in controlling
According to WHO, cancer represents the second leading cause of
death globally, accounting for almost 9.6 million deaths in 2018 with
an estimated 18 million cases in the same year. While the most common
types of cancer in men are lung, prostate, colorectal, stomach and liver
cancer; breast, colorectal, lung, cervical and thyroid cancer represent
the most common types among women [1].
most cellular processes including metabolism, transcription, cell cycle
progression, cytoskeletal rearrangement, cell movement, apoptosis and
differentiation, both under normal and pathological cell conditions
[4–6]. Up-to-date there are > 518 identified human protein kinases
encoded within the human genome thus, representing approximately
1.7% of all the human genes [4,6].
In an attempt to avoid the side effects of standard chemotherapy,
targeted cancer therapies have been developed. Various targeted
therapies have been approved for use in cancer treatment including
signal transduction inhibitors, angiogenesis inhibitors, apoptosis in-
ducers, hormone therapies, gene expression modulators, immuno-
therapies, and toxin delivery molecules [2,3].
Amongst this kinase arsenal is the vascular endothelial growth
factor receptor-2 (VEGFR-2) kinase which is well recognized to be as-
sociated with the development and progression of many types of can-
cers and development of angiogenesis. VEGF-2 is a key growth factor in
tumor angiogenesis, transmits its angiogenic signal via cell surface re-
ceptors VEGFR-2 located on the host vascular endothelium, which have
intracellular TK activity. Over-activation of VEGFR-2 signaling
Among the first discovered and approved targeted drug therapies
Abbreviations: VEGFR-2, vascular endothelial growth factor receptor-2; PKs, protein kinases; TK, tyrosine kinase; TKIs, tyrosine kinase inhibitors
⁎ Corresponding author at: University of Science and Technology, Zewail City of Science and Technology, Ahmed Zewail Road, October Gardens, 6th of October
City, Giza 12578, Egypt.
E-mail address: rkhidr@zewailcity.edu.eg (R.K. Arafa).
https://doi.org/10.1016/j.bioorg.2020.104222
Received 16 May 2020; Received in revised form 14 August 2020; Accepted 25 August 2020
Availableonline26August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Fig. 1. General structures of the designed compounds.
positively correlates with poor prognosis and metastasis in the majority
of solid tumor patients. Consequently, the biological rational and the
effectiveness of small-molecule VEGFR-2 inhibitors in interfering with
tumor induced angiogenic signals and selectively targeting the tumor-
associated vessels has been well established [7–10]. Small molecule
ATP competitive inhihbitors of VEGFR-2 based on a pyridine nucleus
have demonstrated high activity as antitumor agents like apatinib and
motesanib, displayed in Fig. 1 [11,12].
molecular manipulations and design strategy involved varying sub-
stituent group X in position 2 of the pyridine backbone, being bioi-
sosterically altered between amino, chloro and hydrazine moieties in
model A and oxo in model B. Moreover, substituents on position 4 of
the pyridine ring have been varied between an unsubstituted phenyl
ring, mono or disubstituted analogs with different groups of diverse
lipophilicity and electronic effects.
All synthesized compounds were tested in vitro against 2 prostate
cancer cell lines and 3 breast cancer cell lines. Moreover the most active
compounds were subjected to in vivo xenograft examination against
prostate and breast cancer. In addition, the possible mechanism of ac-
tion of these compounds was explored on many levels against various
cell cycle controlling biomolecules. Further, anti-kinase activity and
selectivity of all the synthesized compounds was assessed primarily by
an in vitro inhibitory assay against a panel of 23 kinases, where after,
IC50s were determined against the most responsive kinase viz. VEGFR-2.
Emergence of tumor resistance to the effect of currently clinically
used small-molecule TKIs opens the door for investigation into the ef-
fectiveness of new chemotypes. Naphthalene-scaffold based molecules
have been employed increasingly in anticancer drug design [10,13–15].
It was demonstrated that the distinctly planar conformation of naph-
thalene ring could be integrated in special hydrophobic interaction(s)
with the hydrophobic amino acids of the ATP binding site of PKs
[16,17]. Also, chemical entities bearing a 3-cyano-4,6-diaryl-pyridin-2-
one moiety or its 2-amino bioisostere have received considerable at-
tention owing to their ability to elicit antiproliferative and anticancer
activities employing various mechanism of actions. Kinase inhibition is
among the recognized mechanisms by which these derivatives exert
their cytotoxic activities [18–20].
2. Results and discussion
2.1. Chemistry
In view of the above mentioned findings and based on our con-
tinuous efforts to develop new cytotoxic agents based on various che-
motypes [21–24], it deemed of interest to initiate a research work di-
rected at the design and synthesis of a new series of potential cytotoxic
compounds. The design of these derivatives was directed by the idea of
cross-hybridization between the pharmacophoric elements of the cy-
totoxic agents apatinib, motesanib and pyrimidinone derivative I
(Fig. 1) [25]. A naphthyl scaffold was introduced to the 6 position of
either a 3-cyano-2-aminopyridine or pyrid-2-one backbone to yield
derivatives of general structures A and B (Fig. 1). These derivatives are
designed as potential ATP competitive kinase inhibitors and anti-
proliferative agents where the pyridine ring will emulate the adenine
moiety of ATP with its hydrogen donor–acceptor system in binding to
the hinge region of the kinase. On the other hand, the naphthyl scaffold
of the target compounds introduced in position 6 of the pyridine ring
would confer better binding to the receptor and/or contribute to the
pharmacokinetics of these molecules.
The general synthetic pathways adopted for the synthesis of the
novel 3-cyano-6-naphthylpyridine derivatives are illustrated in
Schemes 1 and 2.
Scheme 1 depicts the synthesis of 4-aryl-3-cyano-6-(1 or 2-naphthyl)
pyridine-2-ones 4a-f and 5a-f via one pot multicomponent reaction.
Whereby, a mixture of the appropriate acetyl naphthalene 1 or 2 as the
ketonic component, the appropriate substituted aromatic aldehyde
from among the aldehydes 3a-f, ethyl cyanoacetate and excess ammo-
nium acetate were reacted in refluxing absolute ethanol. The structures
of the target compounds were confirmed by elemental analysis as well
as spectral data. In general, IR spectra showed characteristic peaks at
around 3400 cm−1 corresponding to the NH stretching, 2200 cm−1
corresponding to the cyanide stretching in addition to a peak at around
1630–1655 cm−1 corresponding to carbonyl stretching of the pyridone
which appeared at lower value than the classic characteristic band
owing to the lactam-lactim tautomerism characterized by weakened
double bond character [26]. The 1H NMR showed a singlet peak at
around 12 ppm which disappeared upon deuterium exchange and was
assigned to the NH group besides the characteristic set of aromatic
peaks between 6 and 8 ppm. 13C NMR also confirmed the validity of the
Multiple structural manipulations were performed to study the
structure–activity relationship implications of these transformations on
the studied pharmacological activities of these derivatives. The adopted
2

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Scheme 1. Synthesis of 4-aryl-3-cyano-6-(1- or 2-naphthyl)pyridine-2-ones 4a-f and 5a-f and 2-amino-4-aryl-3-cyano-6-(1- or 2-naphthyl)pyridines 6a-f and 7a-f.
suggested structures where peaks around 155 and 115 corresponding to
the carbonyl and cyanide groups were displayed, respectively. Mass
spectral data were all in accordance with the calculated values.
On the other hand, the 2-amino bioisosteres 6a-d and 7a-f were
only obtained by applying the one pot multicomponent approach de-
scribed above but replacing ethyl cyanoacetate by malonitrile, adding a
catalytic amount of anhydrous potassium carbonate and performing the
reaction in refluxing toluene. Attempts to obtain the target derivatives
by conducting the reaction in different solvents including n-butanol,
absolute ethanol and methoxyethanol were all unsuccessful.
Furthermore, the reaction was attempted with the aforementioned
solvents in the absence and presence of catalysts different catalysts viz.
anhydrous potassium carbonate and sodium ethoxide. However, these
trials resulted in an unidentified product. This can probably be attrib-
uted to the observed failure of reaction in all tried protic solvents (n-
butanol, ethanol, methoxyethanol) and success of the reaction only
under aprotic solvent conditions (toluene).
confirmed based on spectral and elemental data. The IR spectra of the
obtained compounds revealed the existence of characteristic peaks at
around 2200 cm−1 corresponding to the cyanide stretching, in addition
to two bands at around 3400 and 3300 cm−1 attributed to the amino
stretching. The 1H NMR showed a singlet peak at around 7.00 ppm
which disappeared upon deuterium exchange corresponding to NH2
group in addition to the characteristic set of aromatic peaks from 6 to
8 ppm. The mass spectra were also consistent with the expected
structures.
In addition, as demonstrated in scheme 2, chlorination of the
pyridn-2-one derivatives 4a-f and 5a-f was attempted by refluxing in
phosphorous oxychloride utilizing a catalytic amount of phosphorous
pentachloride yielding the 2-chloro pyridine derivatives 8a-f and 9a-f.
Subsequent reflux of the latter with hydrazine hydrate in absolute
ethanol afforded the 2-hydrazino pyridine derivatives 10a-f and 11a-f.
The spectral and elemental data supported the structures of the
obtained 2-chloro derivatives, where the IR spectra showed the dis-
appearance of the two stretching bands of the NH group and carbonyl
The postulated structures of the synthesized compounds were
Scheme 2. Synthesis of 4-aryl-2-chloro-3-cyano-6-(1- or 2-naphthyl)pyridines 8a-f and 9a-f and 4-aryl-3-cyano-2-hydrazino-6-(1- or 2-naphthyl)pyridines 10a-f and
11a-f.
3

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Table 1
(IC50 = 8.7, 8.6 and 8.6 nM, respectively) against DU145 cell line.
Regarding the 2-pyridone derivatives 4a-f possessing 1-naphthyl
ring at position 6, results showed that substitution on phenyl ring with
4-chloro group in derivative 4e increased the potency against PC3 cell
line compared to the parent unsubstituted derivative 4a (IC50 = 25,
45 nM, respectively). Meanwhile, all adopted substitutions in this series
enhanced the activity against DU145 cell line compared to the un-
substituted derivative 4a (IC50 = 0.98 nM) with the highest activity
observed with 4d bearing 4-hydroxy-3-methoxy groups on phenyl ring
with IC50 value of 0.09 nM. As for their 2-naphthyl counterparts 5a-f,
substitutions on phenyl ring led to an increase in potency
(IC50 = 13–52 nM) against both cell lines. The highest activity against
PC3 and DU145 cell lines was noticed with the derivatives 5c
(IC50 = 13 nM) and 5d (IC50 = 0.3 nM), respectively.
In vitro antitumor activity of the newly synthesized derivatives against prostate
and breast cancer cell lines.
Compd #
IC50 (nM)*
IC50 (nM)*
Prostate cancer cell lines
Breast cancer cell lines
PC3
DU145
MCF-7
MDA-MB435
4a
45
55
45
84
25
44
54
24
13
23
52
43
45
46
37
54
44
44
44
35
23
33
38
42
33
49
35
34
44
23
34
55
43
34
59
1.3
2.3
1.3
6.2
1.3
2.2
3.7
1.9
0.51
1.1
2.4
3.1
3.3
3.4
1.9
2.8
2.1
1.3
1.6
2.9
1.4
2.4
1.8
2.3
1.9
3.2
1.4
2.7
1.6
1.5
1.7
3.1
0.9
0.8
2.1
0.98
0.76
0.76
0.08
0.34
0.54
8.7
0.03
0.02
0.02
0.04
0.01
0.03
4.3
6.4
5.5
5.4
3.2
7.3
4.4
3.5
2.4
5.6
7.9
5.6
5.5
2.3
2.3
0.03
0.04
0.03
0.02
0.01
0.05
0.03
0.03
0.02
0.01
0.03
0.04
0.03
0.01
0.02
56
45
56
53
43
54
75
22
34
53
65
34
33
44
46
44
47
94
7
0.32
0.08
0.48
0.24
0.31
0.2
4b
4c
4d
4e
4f
5a
0.32
0.5
5b
5.4
0.12
0.22
0.01
0.44
0.32
0.15
0.12
0.34
0.44
0.22
0.11
0.23
0.21
0.34
0.26
0.23
With respect to the 2-amino-6-(1-naphthyl)pyridine derivatives 6a-
d, while the parent unsubstituted derivative 6a showed IC50 value of
45 nM against PC3 cell line, only the 4-chloro substitution on the
phenyl ring in derivative 6c enhanced the potency (IC50 = 37 nM).
Regarding the screening results against DU145, the unsubstituted de-
rivative 6a was the most potent with IC50 value of 0.35 nM. While with
respect to the 2-amino-6-(2-naphthyl)pyridine derivatives 7a-f, various
substitutions on phenyl ring resulted in equipotent or more potent de-
rivative against both PC3 and DU145 cell lines compared to the un-
substituted parent derivative 7a (IC50 = 23–44, 6.6–7.7 nM, respec-
tively). Compound 7e was the most potent against PC3 cell line
(IC50 = 23 nM) while both compounds 7e and 7f displayed the highest
activity against DU145 cell line with IC50 = 6.6 nM.
5c
4.3
5d
0.3
5e
6.5
5f
4.2
6a
0.35
0.45
0.43
0.67
8.6
0.02
6b
0.03
0.03
0.05
6c
6d
6
0.06
7a
0.63
2.3
6.5
9.6
3.3
3.3
5.4
2.3
3.6
3.5
2.4
2.4
2.3
9.4
1.3
2.3
0.5
7.2
5.3
11
0.03
0.02
0.08
0.01
0.01
0.02
0.01
0.02
0.01
0.02
0.03
0.01
0.07
0.01
0.02
0.001
0.05
0.03
0.37
7b
7.5
0.52
0.59
0.33
0.43
0.343
0.03
0.01
0.01
0.034
0.06
0.03
0.66
0.02
0.78
0.43
0.02
0.67
0.01
7c
7.4
0.31
7d
7.7
58
45
73
37
53
43
48
35
43
85
46
34
96
69
57
69
0.41
0.23
0.51
0.24
0.33
0.28
0.13
0.29
0.41
1.1
7e
6.6
7f
6.6
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
DOX
0.87
0.23
0.12
0.65
0.89
0.46
8.6
Finally, within the 2-hydrazino-6-(1-naphthyl)pyridine class 10a-f,
the most potent derivative against both PC3 and DU145 cell lines was
10c possessing 4-chloro group on the phenyl ring with IC50 values of 33
and 0.12 nM, respectively. Within their 2-naphthyl congeners 11a-f, the
highest potency against PC3 was displayed by the 4-nitro derivative
11b (IC50 = 23 nM) which also exhibited the highest potency as along
with the 4-methoxy derivative 11e against DU145 cell line
(IC50 = 0.8 nM).
0.8
0.91
0.27
1.6
9.7
8.5
0.8
1.1
9.9
0.98
1.37
1.8
2.2.2. Breast cancer, in vitro antitumor activity
Results of the in vitro evaluation against breast cancer cell lines
MCF-7 and MDA-MB435, illustrated in Table 1, revealed that all the
newly synthesized compounds possessed high inhibitory activity to-
wards the two cell lines with IC50 ranges of 0.5–9.6 and 7–96 nM, re-
spectively. It was observed that the activity was generally higher
against MCF-7 breast cancer cell line than MDA-MB435.
* Data presented are the average of three independent runs
1
SD.
group. Furthermore, the H NMR spectra revealed the absence of the
characteristic singlet peak of the NH proton. The mass spectra were in
accordance with the proposed structures and showed the isotopic peaks
at M+ +2 in addition to the molecular ion peak. The structures of the
desired 2-hydrazino derivatives were confirmed by the elemental and
spectral data, where the IR spectra displayed a broad absorption band
at 3200–3500 cm−1 attributed to the hydrazine group. The 1H NMR
spectra revealed broad singlet peaks at the ranges 4.57–4.94 and
12.37–12.86 ppm that disappeared upon deuteration assigned for NH2
and NH protons, respectively. Moreover, the mass spectra were com-
plying with the structure of the synthesized derivatives.
Regarding the 6-(1-naphthyl)pyridine-2-ones series 4a-f, substitu-
tion of phenyl ring with 4- methoxyphenyl group in 4e enhanced the
activity (IC50 = 3.2 nM) than the parent unsubstituted derivative 4a
(IC50 = 4.3 nM) against MCF-7 cell line. Yet, all implemented mod-
ifications on phenyl ring led to equipotent or more potent derivatives
against MDA-MB435 than 4a (IC50 = 56 nM), of which the highest was
4-methoxy derivative 4e (IC50 = 43 nM).
As regards to their 2-naphthyl counterparts 5a-f, the parent un-
substituted derivative 5a exhibited IC50 values of 4.4, 75, 34 nM against
MCF-7, MDA-MB435 and BT549 cell lines, respectively. Only sub-
stitution with 4-chloro, 4-nitro groups on phenyl ring resulted in more
potent derivatives 5c (IC50 = 2.4 nM) and 5b (IC50 = 3.5 nM) against
MCF-7 cell line, respectively. Meanwhile, on MDA-MB435, 5b dis-
played higher cytotoxic activity than the parent 5a by 3.5 fold
(IC50 = 22, 75 nM, respectively). Other employed substitutions pro-
vided an increase in activity with IC50 ranges of 34–65 nM than the
parent 5a.
2.2. Biological screening
2.2.1. Prostate cancer, in vitro antitumor activity
All newly synthesized compounds were tested in vitro against 2
prostate cancer cell lines namely PC3 and DU145. The results shown in
Table 1 revealed that the synthesized compounds showed high activity
against both cell lines with IC50 values in the upper nanomolar range
against PC3 and low or subnanomolar range against DU145 cell line. In
addition, SAR studies revealed that with reference to the biological
activity displayed by the 1-naphthyl and 2-naphthyl analogs against
PC3 cell line, there was no preferentiality with regards to these posi-
tional analogs. However, the 1-naphthyl analogs such as 4a and 6a
displayed higher potency (IC50 = 0.98 and 0.35, 0.89 nM, respectively)
than that observed with their 2-naphthyl counterparts 5a, 7a and 11a
As for the 6-(1-naphthyl)-2-aminopyridine class 6a-d, in vitro anti-
proliferative screening results against MCF-7 cell line revealed that the
p-substitution on phenyl ring with nitro or chloro group as in case of 6b
and 6c improved the inhibitory activity by 2.3 fold (IC50 = 2.3 nM)
compared to the unsubstituted counterpart 6a (IC50 = 5.5 nM). On the
contrary, the unsubstituted phenyl derivative 6a displayed the highest
4

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
potency against both MDA-MB435 cell lines (IC50 = 33 nM).
With reference to the biological activity displayed by their 2-
naphthyl analogs 7a-f, the parent compound 7a with unsubstituted
phenyl ring exhibited the highest activity against MCF-7 cell line
(IC50 = 2.3 nM, respectively). Other substitutions decreased the po-
tency with IC50 range of 3.3–9.6 nM with the 4-chloro derivative 7c
being the least potent. On the contrary, for MDA-MB435 cell line,
screening results showed that compound 7c (IC50 = 7 nM) displayed a
6.7 fold improvement in activity against MDA-MB435 cell line com-
pared to the parent 7a (IC50 = 47 nM). Moreover, compound 7e
showed better activity (IC50 = 45 nM) than 7a, while other derivatives
displayed less potent inhibitory activity against MDA-MB435 cell line
(IC50 = 58–94 nM).
Table 3
Apoptotic index of 11d at different doses.
Compd #
Test concentration
Apoptotic Index (% of control)
MCF-7
MCF-7 p53Knockdown
11d
0.1 µM
0.5 µM
0.75 µM
1.10
1.34
4.30
5.46
12.34
17.80
cancer cell lines exhibited an increase in apoptosis following exposure
treatment with different concentrations (0.1, 0.5, 0.75 nM) of 11d. This
increase in apoptotic index was more evident in MCF-7 cells. The cy-
totoxic effects of 11d appeared to be dose dependent in all cases.
To gain a better understanding of the mechanism that underlies the
anticancer activity of 11d, the cell cycle progression was analyzed using
flow cytometry in MCF-7 and MCF-7 p53 knockdown cells. The effects
of various 11d concentrations on the cell cycle and percentage of cells
in each phase are illustrated in Table 4 and Fig. 2.
With respect to 6-(1-naphthyl)-2-hydrazinopyridine 10a-f, the best
activities against MCF-7 cell line appeared with the parent derivative
10a and its equipotent 3,4-dimethoxyphenyl derivative 10f having
IC50 = 2.3 nM. In addition, derivatives 10d and 10e bearing 4-meth-
oxyphenyl, 4-hydroxy-3-methoxyphenyl were found to be nearly equi-
potent with IC50 = 2.4 nM. Regarding MDA-MB435, modifications on
the phenyl ring did not improve the potency compared to the parent
The data indicated that 11d interfered with the cell cycle progres-
sion through induction of cell cycle arrest in the G1 phase as indicated
by the dose dependent accumulation of cells in G1 phase. MCF-7 cells
treated with 11d showed 57%, 62%, 68% of cells in G1 phase at doses
0.1, 0.5 and 0.75 nM, respectively. This was accompanied by a corre-
sponding reduction of cells in the S and G2/M phases in a dose de-
pendent manner in MCF-7 cells. However, S phase cell cycle arrest was
observed in MCF-7 p53 knockdown cells in a dose dependent manner.
The frequencies of cells in the S phase following treatment with 0.1, 0.5
and 0.75 µM of 11d were 11%, 23% and 24% in MCF-7 p53 knockdown
cells.
compound 10a (IC50
= 37 nM) except for derivative 10e
(IC50 = 35 nM).
Lastly in this regards, concerning their 2-naphthyl analogs 11a-f, it
was found that compound 11d possessing 4-hydroxy-3-methoxy sub-
stituents on the phenyl ring exhibited the highest potency in this series
against MCF-7 cell line which was shown to be 18.8 fold more potent
than the parent compound 11a (IC50 = 0.5, 9.4 nM, respectively). It is
also noteworthy that other derivatives in this series displayed notice-
able inhibitory activity against MCF-7 cell line compared to the parent
compound 11a with IC50 range 1.3–7.2 nM. Similarly, increased po-
tency was observed with different substitutions on phenyl ring against
MDA-MB435 cell line with IC50 ranges 34–69 nM compared to the
parent 11a (IC50 = 85 nM) with the only exception being compound
11d which showed lower potency than 11a (IC50 = 94 nM).
2.2.5. Apoptotic markers analysis
Additionally, to provide insight into the molecular mechanisms
underlying the cell cycle arrest induced by derivative 11d, western
blotting was used to assess its effects on the expression levels of various
proteins relevant to cell cycle progression and apoptosis in human
breast cancer cells. The three cell lines MCF-7 and MCF-7 p53 knock-
down were treated with various concentrations of 11d for 24 h. All
treated cells showed increased CdK inhibitors p21 and p27 expression
in a dose dependent manner. Whereas, MDM2 (a negative regulator of
tumor suppressor gene p53), E2F1, Cdk2, Cdk4, Cdk6 and cyclin D1
were downregulated (Fig. 3). Similar effects on cell cycle/proliferation-
related proteins were observed in MCF-7, MCF-7 p53 knockdown and
MDA-MB468 cells despite their different p53 status suggested that the
activity of 11d is p53 independent.
2.2.3. Breast cancer, in vivo antitumor activity and in vitro mechanistic
study
Derivative11d was selected for further evaluation owing to its no-
table in vitro cytotoxic properties to determine whether it can also exert
anticancer effects in vivo. The tested compound as administrated in-
dividually by intraperitoneal injection at doses of 5 and 10 mg/Kg to
nude mice bearing MCF-7 xenograft tumors. The effect of the com-
pounds on tumor growth was studied (Table 2).
Generally, the tested compound decreased the growth of MCF-7
xenograft tumors in a time dependent manner. There was nearly a 6-
fold reduction in tumor growth on day 20 after treatment with 11d
when compared to the control group (receiving vehicle only).
Furthermore, the levels of Bax, cleaved PARP1 and cleaved caspases
3, 8 and 9 were analyzed following exposure of MCF-7 and MCF-7 p53
knockdown cells to 11d. The pro-apoptotic Bax expression level was
increased, while the anti-apoptotic protein Bcl2 level was depressed in a
dose dependent manner. Additionally, caspases 3, 8 and 9 were acti-
vated, indicating that the tested compound induces apoptosis via both
the intrinsic and extrinsic pathways. Analysis of MCF-7 tumors col-
lected at the end of the in vivo experiment showed that the pattern of
protein expression in the treated xenograft tissues was the same as that
observed in vitro (Fig. 3).
2.2.4. Detection of apoptosis and effect on cell cycle progression
Induction of apoptosis in tumor cells is an important determinant in
the outcome of therapy. Accordingly, derivative 11d was selected as
being the best representative of the synthesized compounds to be fur-
ther investigated for its effect on induction of apoptosis in human breast
cancer cells. Two human breast cancer cell lines representing two dif-
ferent genetic backgrounds MCF-7 (p53 wild type) and MCF-7 p53
knockdown were used. As illustrated in Table 3, the examined breast
Table 2
Effect of 11d on growth of MCF-7 xenograft tumor model.
Compd #
Tumor growth Vt/Vo in MCF-7 for compounds at time (in days)
0
2
4
6
8
10
12
14
16
18
20
Control
11d
1.00
1.00
1.38
1.13
1.81
1.30
4.75
1.48
9.88
1.93
12.64
2.66
24.75
3.73
27.66
4.31
29.00
3.86
38.90
5.66
40.21
6.40
5

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Table 4
Effects of 11d on cell cycle distribution among the tested cell lines.
Cell Line
% of cellular population in the various phases of the cell cycle
0.0 nM
G1
0.1 nM
G1
0.5 nM
G1
0.75 nM
S
G2/M
S
G2/M
S
G2/M
G1
S
G2/M
MCF-7
58
42
22
12
18
37
57
43
20
11
12
35
62
44
19
23
11
29
68
45
17
24
9
MCF-7 p53 knockdown
26
Fig. 2. Dose-dependent cell cycle analysis of MCF-7 and MCF-7 p53 knockdown cells by flow cytometry after treatment with 11d at zero, 0.1, 0.5 and 0.75 nM
concentration.
2.2.6. VEGFR-2 kinase inhibition assay
parent unsubstituted phenyl derivative 6a showed good inhibitory ac-
tivity with IC50 value of 0.63 nM. All adopted substitutions on phenyl
ring showed higher activity against VEGFR-2 (IC50 = 0.46–0.57 nM).
While among their 2-naphthyl analogs 7a-f, only the 4-methoxyphenyl
7e derivative increased the potency (IC50 = 0.45 nM) compared to the
unsubstituted counterpart 7a (IC50 = 0.56 nM).
All synthesized compounds were subjected to a kinase profiling
assay against a panel of 23 kinases at a single screening high dose of
30 nM (data presented in the supplementary materials, Table S1). It
was found that VEGFR-2 was the most responsive to the kinase in-
hibitory activity of the synthesized compounds. Thus, respective IC50
values of the novel pyridine derivatives were determined against
VEGFR-2.
Among the 6-(1-naphthyl)-2-hydrazine series 10a-f, the parent
compound 10a with unsubstituted phenyl exhibited a good inhibitory
activity of IC50 = 0.64 nM. Increased activity was observed with sub-
stituted derivatives with IC50 range 0.45–0.55 nM except in the case of
compound 10e with 4-methoxyphenyl substitution (IC50 = 0.65 nM).
However, in their 2-naphthyl analogs 11a-f, all adopted substitutions
In general, all compounds showed good kinase inhibitory activity
compared to Delphinidin as a reference drug with IC50 values in the
submicromolar range against EGFR and VEGFR-2 (results shown in
Table 5).
Profiling results for VEGFR-2 kinase revealed that within the 6-(1-
naphthyl)pyridin-2-ones class 4a-f, while the parent derivative with
resulted in a noticeable increase in activity with IC50 range
0.19–0.55 nM compared to the parent unsubstituted derivative 11a
(IC50 = 0.77 nM). Compounds bearing 4-chlorophenyl (11c) and 4-
nitrophenyl (11b) were found to be 3 and 2 fold more potent than 11a
with IC50 of values 0.25, 0.34 nM, respectively. Finally, 11d was found
to be the most potent VEGFR-2 inhibitor in this series and among all
tested compounds (IC50 = 0.19 nM)
unsubstituted
phenyl
group
(4a)
showed
high
activity
(IC50 = 0.46 nM), in all cases p-substitution did not display much ad-
vantage (IC50 values of 0.69–0.94 nM) except in the case of 3,4-di-
methoxy derivative (4f) which elicited a slight increase in potency
(IC50 = 0.35 nM). Among their 2-naphthyl analogs 5a-f, the parent
unsubstituted phenyl derivative 5a displayed a good activity of
IC50 = 0.69 nM. Better inhibitory activity was observed by 5b, 5f with
IC50 values of 0.44 and 0.55 nM, respectively. However, compound 5c
with 4-chlorophenyl substitution showed the highest activity among
this series with a 3 fold increase in potency (IC50 = 0.23 nM) compared
to the parent.
2.3. Molecular docking of 11d with VEGFR-2
A docking study was performed to get an insight about the mode of
interaction of 11d to its biomolecular target VEGFR-2 kinase. Thus,
VEGFR-2 kinase domain crystal structure PDB ID: 3B8Q in complex
with a naphthamide inhibitor was adopted for this study. First the
Regarding the 6-(1-naphthyl)-2-aminopyridine derivatives 6a-d, the
6

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Fig. 3. Effects of 11d on: the expression of proteins involved in cell cycle progression in in vitro treated cells (top left panel), the expression of proteins involved in
apoptosis in in vitro treated cells (top right panel); protein expression in the treated MCF-7 xenograft tissues (lower panel).
protein was prepared and protonated, then the used docking protocol
was validated by carrying out redocking of the bound naphthamide
inhibitor. The redocking validation step successfully reproduced the
experimental binding mode of the co-crystallized ligand with good ef-
ficiency pointing the suitability of the adopted protocol for the intended
docking study (figure S1 supplementary material) as indicated by the
small RMSD of 0.5944 Å between the docked pose and the co-crystal-
lized ligand. The docking pose was able to reproduce the key interac-
tions accomplished by the co-crystallized ligand with the hot spots in
the active site, Cys919 in the hinge region, Asp1046 of the DFG motif
and Glu885 in the α-C helix.
Table 5
VEGFR-2 enzymatic inhibition by the synthesized compounds.
Compd #
IC50 (nM)
Compd #
IC50 (nM)
4a
4b
4c
4d
4e
4f
0.46
0.94
0.88
0.69
0.73
0.35
0.69
0.44
0.23
0.85
0.86
0.55
0.63
0.57
0.46
0.54
0.56
0.74
0.02
7c
0.83
0.67
0.45
0.65
0.64
0.45
0.46
0.55
0.65
0.54
0.77
0.34
0.25
0.19
0.55
0.44
5.09
—
0.04
0.07
0.03
0.04
0.03
0.01
0.04
0.01
0.01
0.07
0.03
0.02
0.04
0.02
0.01
0.03
0.02
0.06
7d
0.03
0.02
0.03
0.02
0.01
0.02
0.05
0.05
0.03
0.05
0.02
0.01
0.01
0.04
0.03
0.42
7e
7f
10a
10b
10c
10d
10e
10f
5a
5b
5c
5d
5e
5f
As displayed in Fig. 4, compound 11d was found to be able to re-
cognize the ATP binding site of VEGFR-2 kinase and interact with key
amino acids thereof. Thus, 11d elicited non-bonded interactions be-
tween its cyano group and Cys919 amino group, and through the NH of
its pyridone ring with the hydroxyl group of Thr916 through hydrogen
bonds (binding energy score [S] = −9.9868 Kcal/mol).
11a
11b
11c
11d
11e
11f
6a
6b
6c
6d
7a
7b
Delphinidin, which is known for its inhibitory activity on VEGFR-2,
was used as a reference compound in both the experimental in vitro
assays and in the docking study as well. It was found to interact by 2 of
its hydroxyl groups with Cys919 of the hinge region and Glu917
through hydrogen bonding interactions and does not extend into the
Delphinidin
—
* Data presented are the average of three independent runs
SD.
7

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Fig. 4. Docking of 11d and delphinidin with VEGFR-2 (PDB ID: 3B8Q); upper right panel: interactions of compound 11d in 2D representation; upper left panel:
Interactions of compound 11d in 3D representation; lower right panel: interactions of delphinidin in 2D representation; lower left panel: interactions of delphinidin in
3D representation.
back cleft similar to the synthesized compounds (Fig. 4) displaying a
binding energy score [S] = −8.2655 Kcal/mol.
possess promising pharmacokinetic properties.
Thus, the ability of the new compound 11d to interact with the key
amino acid, Cys919 in the hinge region and an additional hydrogen
bond with Thr916 at the ATP binding pocket of VEGFR-2 rationalizes
its good enzyme inhibiting activity as indicated by its docking pattern
and docking score compared with the positive reference compound,
delphinidin.
3. Conclusion
In this study, novel derivatives based on 3-cyano-6-naphthylpyr-
idine scaffold were designed and synthesized as potential cytotoxic
agents and kinase inhibitors. The cytotoxic activity assessment in-
dicated that all the newly synthesized compounds exhibited high ac-
tivity against prostate cancer cell lines PC3 and DU145 and breast
cancer cell lines MCF-7 and MDA-MB435 in the nanomolar range. The
in vitro results were confirmed by in vivo examinations in MCF-7 xeno-
graft mouse model where the tested compound 11d displayed a re-
markable reduction in tumor growth. The cytotoxic effect of 11d was
found to be related to a G1 phase cell cycle arrest and induction of
intrinsic and extrinsic apoptosis as indicated by increased expression of
the pro-apoptotic proteins and down regulation of anti-apototic mar-
kers and activation of apoptotic caspases 3, 8 and 9. Finally, kinase
inhibition assay results revealed that all compounds showed good in-
hibitory activity against VEGFR-2. Molecular docking demonstrated the
ability of 11d to recognize the ATP binding pocket of VEGFR-2 and to
elicit significant interactions with its key amino acids in a fashion
comparable to that of the well-known VEGFR-2 inhibitor delphinidin.
Physicochemical assessment of the synthesized compounds showed that
they have favorable properties with satisfactory drug-like profiles. To
conclude, the compounds presented herein exemplify, to our knowl-
edge, the first encounter of cytotoxic hits presented by these new
2.4. In-silico physicochemical parameters and pharmacokinetics properties
determination
Physicochemical parameters and pharmacokinetics properties of all
compounds were determined (Table 6). Results showed that all syn-
thesized compounds obey the Lipinski’s rule of five criteria with no to
one violation to the rule. With regards to compounds’ lipophilicity, logP
of all compounds lied in the range of 4.3–7.0. Considering the topolo-
gical polar surface area which is a commonly used medicinal chemistry
metric for the optimization of a drug's ability to permeate cells, all
compounds had TPSA < 140 Å2, indicating that they can permeate
cellular membranes efficiently. Molecular weights of all compounds
were less than 500 Daltons. The synthesized compounds showed
variability in their calculated solubility in terms of logS at physiological
pH. In addition, all the compounds do not have affinity to P-glyco-
protein. From the above-mentioned data, it is clear that the newly
synthesized compounds not only display promising activity but also
8

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Table 6
Physicochemical properties and pharmacokinetic parameters of the synthesized compounds.
Compd #
b_rotN
lip_acc
lip_don
lip_violation
logP(o/w)
lip_druglike
TPSA
Weight
hERG pIC50
P-gp category
4a
4b
4c
3
4
3
4
4
5
3
4
3
4
4
5
3
4
3
4
3
4
3
4
4
5
3
4
3
4
4
5
4
5
4
5
5
6
3
6
3
5
4
5
3
6
3
5
4
5
3
6
3
5
3
6
3
5
4
5
2
5
2
4
3
4
4
7
4
6
5
6
1
1
1
2
1
1
1
1
1
2
1
1
2
2
2
3
2
2
2
3
2
2
0
0
0
1
0
0
3
3
3
4
3
3
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
1
0
1
1
1
1
1
1
1
1
0
0
1
0
0
0
4.72
4.66
5.31
4.40
4.68
4.42
4.76
4.69
5.35
4.44
4.72
4.46
5.20
5.14
5.79
4.88
5.24
5.18
5.83
4.92
5.20
4.94
6.47
6.40
7.06
6.15
6.42
6.16
4.68
4.61
5.27
4.36
4.64
4.38
Yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
52.89
98.71
52.89
82.35
62.12
71.35
52.89
98.71
52.89
82.35
62.12
71.35
62.70
108.52
62.70
92.16
62.70
108.52
62.70
92.16
71.93
81.16
36.68
82.50
36.68
66.14
45.91
55.14
74.73
120.55
74.73
104.19
83.96
93.19
322.37
367.36
356.81
368.39
352.39
382.42
322.37
367.36
356.81
368.39
352.39
382.42
321.38
366.38
355.83
367.41
321.38
366.38
355.83
367.41
351.41
381.44
340.81
385.81
375.26
386.84
370.84
400.86
336.40
381.39
370.84
382.42
366.42
396.45
5.20
5.22
5.37
5.15
5.36
5.23
5.21
5.23
5.37
5.15
5.37
5.24
4.93
4.93
5.10
4.87
4.94
4.94
5.10
4.88
5.10
4.96
5.26
5.28
5.41
5.22
5.42
5.29
4.92
4.91
5.08
4.83
5.06
4.91
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
4d
4e
4f
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
11a
11b
11c
11d
11e
11f
b_rotN: Number of rotatable bonds; lip_acc: Number of hydrogen bond acceptors according to Lipinski’s rule of 5; lip_don: Number of hydrogen bond donors
according to Lipinski’s rule of 5; lip_druglike: Obeying Lipinski’s rule of 5; TPSA: topological polar surface area; The Human Ether-a-go-go-related Gene pIC50: hERG
pIC50; and P-glycoprotein affinity.
chemotype compounds as selective VEGFR-2 kinase inhibitors with
potent anticancer efficacy that can be further adopted for hit optimi-
zation and/or lead discovery.
4.1.1. General procedure for preparation of 4-aryl-3-cyano-6-(1- or 2-
naphthyl)pyridine-2-ones 4a-f and 5a-f
To a solution of the appropriate acetylnaphthalene (1 mmol, 0.5 g)
and the appropriate aromatic aldehyde (1 mmol) in absolute ethanol
(20 mL), ammonium acetate (8 mmol, 2.17 g) and ethyl cyanoacetate
(1 mmol, 0.4 g) were added. The mixture was refluxed for 8 h, cooled to
room temperature and the precipitated compound was filtered, washed
with distilled water (2 × 10 mL) then left to dry. The obtained solid
was crystallized from ethanol.
4. Experimental
4.1. Chemistry
Starting materials and reagents were purchased from Sigma-Aldrich
and used without purification. Melting points were determined on
Electrothermal Stuart SMP3 digital melting point apparatus and were
uncorrected. Infrared spectra were determined (KBr) using Shimadzu
Infrared spectrometer (IR-435) and FT-IR 1650 (Perkin Elmer), Faculty
of Pharmacy, Cairo University. 1H NMR spectra were performed in
DMSO‑d6 using Joel, 300 MHz NMR spectrometer, Faculty of Science,
Cairo University, and Bruker, 400 MHz NMR spectrometer,
Microanalytical unit, Faculty of Pharmacy, Cairo University. 13C NMR
spectra were recorded using Joel, 75 MHz NMR spectrometer, Faculty
of Science, Cairo University, and Bruker, 100 MHz NMR spectrometer,
Microanalytical unit, Faculty of Pharmacy, Cairo University. Mass
spectra were carried out using Finnigan SSQ 7000 Gas chromatograph-
Mass, at the Microanalytical unit, Faculty of Science, Cairo University.
Elemental microanalyses were performed at the Regional Center for
Mycology and Biotechnology, Al-Azhar University. Reactions were
monitored using TLC plates with silica gel 60 matrix foil support,
fluorescent indicator F254 using hexane:ethyl acetate (7:3) as solvent
system.
4.1.1.1. 1,2-Dihydro-6-(naphthalen-1-yl)-2-oxo-4-phenylpyridine-3-
carbonitrile 4a. C22H14N2O (322.36), yield %: 40; m.p. 237–239 °C; I.R.
(KBr, cm−1): ῦ 3435 (NH), 3015 (CH aromatic), 2216 (CN), 1655 (C]
1
O). H NMR (DMSO‑d6, 300 MHz): δ 6.60 (s, 1H, pyridyl), 7.54–7.75
(m, 4H naphthyl + 5H phenyl), 7.96–8.12 (m, 3H, naphthyl), 12.96
(br.s, 1H, NH, D2O exchangeable). 13C NMR (DMSO‑d6, 75 MHz): δ
161.4, 159.7, 135.8, 133.0, 130.9, 130.5, 130.4, 130.0, 128.7, 128.4,
128.1, 127.7, 127.3, 126.5, 125.1, 124.6, 116.4, 108.7. Mass (m/z, rel.
int.): 321.80 (M+, 100). Anal. for C22H14N2O, Calc. C: 81.97, H: 4.38,
N: 8.69; Found C: 82.09, H: 4.41, N: 8.85.
4.1.1.2. 1,2-Dihydro-6-(naphthalen-1-yl)-4-(4-nitrophenyl)-2-
oxopyridine-3-carbonitrile 4b. C22H13N3O3 (367.36), yield %: 30; m.p.
212–214 °C; I.R. (KBr, cm−1): ῦ 3440 (NH), 3071 (CH aromatic), 2218
(CN), 1653 (C]O). 1H NMR (DMSO‑d6, 400 MHz): δ 6.67 (s, 1H,
pyridyl), 7.61–7.67 (m, 4H, naphthyl), 7.72 (d, 1H, J = 6.2 Hz,
naphthyl), 8.01 (d, 2H, J = 8.7 Hz, phenyl), 8.05–8.07 (m, 1H,
naphthyl), 8.13 (d, 1H, J = 8.0 Hz, naphthyl), 8.39 (d, 2H,
J = 8.7 Hz, phenyl), 13.16 (br.s, 1H, NH, D2O exchangeable). Mass
9

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
(m/z, rel. int.): 367 (M+, 40.82). Anal. for C22H13N3O3, Calc. C: 71.93,
4.1.1.9. 4-(4-Chlorophenyl)-1,2-dihydro-6-(naphthalene-2-yl)-2-
H: 3.57, N: 11.44; Found C: 72.09, H: 3.61, N: 11.67.
oxopyridine-3-carbonitrile 5c [28,29].. C22H13ClN2O (356.81), yield %:
34; m.p.
>
300 °C; I.R. (KBr, cm−1): ῦ 3433 (NH), 3035 (CH
1
4.1.1.3. 4-(4-Chlorophenyl)-1,2-dihydro-6-(naphthalen-1-yl)-2-
aromatic), 2218 (CN), 1635 (C]O). H NMR (DMSO‑d6, 300 MHz): δ
7.02 (s, 1H, pyridyl), 7.62–7.69 (m, 4H, naphthyl), 7.80 (d, 2H,
J = 7.7 Hz, phenyl), 7.99–8.08 (m, 4H, naphthyl + phenyl), 8.57
(br.s, 1H, C-1 naphthyl), 12.93 (br.s, 1H, NH, D2O exchangeable). Mass
(m/z, rel. int.): 358 (M+ +2, 36.50), 356 (M+, 100). Anal. for
oxopyridine-3-carbonitrile 4c. C22H13ClN2O (356.8), yield %: 51; m.p.
230–232 °C; I.R. (KBr, cm−1): ῦ 3443 (NH), 3011 (CH aromatic), 2214
(CN), 1648 (C]O). 1H NMR (DMSO‑d6, 300 MHz): δ 6.6 (s, 1H,
pyridyl), 7.60–7.79 (m, 8H, naphthyl + phenyl), 7.92–8.13 (m, 3H,
naphthyl), 13.00 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 358 (M++2, 28.7), 356 (M+, 81.47). Anal. for C22H13ClN2O,
Calc. C: 74.06, H: 3.67, N: 7.85; Found C: 74.11, H: 3.72, N: 7.91.
C
22H13ClN2O, Calc. C: 74.05, H: 3.67, N: 7.85; Found C: 73.93, H:
3.84, N: 8.11.
4.1.1.10. 1,2-Dihydro-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-2-
yl)-2-oxopyridine-3-carbonitrile 5d. C23H16N2O3 (368.38), yield %: 50;
m.p. > 300 °C; I.R. (KBr, cm−1): ῦ 3502 (OH), 3444 (NH), 3055 (CH
aromatic), 2947 (CH aliphatic), 2218 (CN), 1651 (C]O). 1H NMR
(DMSO‑d6, 300 MHz): δ 3.87 (s, 3H, OCH3), 6.94–6.97 (m, 2H,
phenyl + pyridyl), 7.28 (dd, 1H, J = 7.5, 2.1 Hz, phenyl), 7.38 (d,
1H, J = 2.1 Hz phenyl), 7.60–7.64 (m, 2H, naphthyl), 7.99–8.08 (m,
4H, naphthyl), 8.54 (s, 1H, C-1 naphthyl), 9.69 (br.s, 1H, OH, D2O
exchangeable), 12.71 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 368 (M+, 2.10). Anal. for C23H16N2O3, Calc. C: 74.99, H: 4.38, N:
7.60; Found C: 75.05, H: 4.36, N: 7.72.
4.1.1.4. 1,2-Dihydro-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-1-
yl)-2-oxopyridine-3-carbonitrile 4d. C23H16N2O3 (368.38), yield %: 36;
m.p. 279–281 °C; I.R. (KBr, cm−1): ῦ 3441 (NH), 3387 (OH), 3012 (CH
aromatic), 2920 (CH aliphatic), 2225 (CN), 1635 (C]O). 1H NMR
(DMSO‑d6, 300 MHz): δ 3.82 (s, 3H, OCH3), 6.62 (s, 1H, pyridyl), 6.92
(d, 1H, J = 8.1 Hz, phenyl), 7.25 (dd, 1H, J = 8.1, 1.8 Hz, phenyl),
7.32 (d, 1H, J = 1.8 Hz, phenyl), 7.58–7.71 (m, 4H, naphthyl), 7.93
(dd, 1H, J = 6.3, 3.6 Hz, naphthyl), 8.05 (dd, 1H, J = 6.3, 3.6 Hz,
naphthyl), 8.11 (d, 1H, J = 8.1 Hz, naphthyl), 9.69 (br.s., 1H, OH, D2O
exchangeable), 12.79 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 368 (M+, 24.7). Anal. for C23H16N2O3, Calc. C: 74.99, H: 4.38, N:
7.60; Found C: 75.05, H: 4.41, N: 7.72.
4.1.1.11. 1,2-Dihydro-4-(4-methoxyphenyl)-6-(naphthalen-2-yl)-2-
oxopyridine-3-carbonitrile 5e [29].. C23H16N2O2 (352.39), yield %: 45;
m.p. 282–284 °C; I.R. (KBr, cm−1): ῦ 3433 (NH), 3055 (CH aromatic),
2931 (CH aliphatic), 2218 (CN), 1643 (C]O). 1H NMR (DMSO‑d6,
300 MHz): δ 3.86 (s, 3H, OCH3), 6.98 (s, 1H, pyridyl), 7.14 (d, 2H,
J = 7.5 Hz, phenyl), 7.6–7.66 (m, 2H, naphthyl), 7.77 (d, 2H,
J = 7.5 Hz, phenyl), 7.98–8.07 (m, 4H, naphthyl), 8.55 (s, 1H, C-1
naphthyl), 12.77 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 352 (M+, 90.72). Anal. for C23H16N2O2, Calc. C: 78.39, H: 4.58, N:
7.95; Found C: 78.41, H: 4.62, N: 8.02.
4.1.1.5. 1,2-Dihydro-4-(4-methoxyphenyl)-6-(naphthalen-1-yl)-2-
oxopyridine-3-carbonitrile 4e. C23H16N2O2 (352.39), yield %: 37; m.p.
212–214 °C; I.R. (KBr, cm−1): ῦ 3451 (NH), 3010 (CH aromatic), 2933
(CH aliphatic), 2216 (CN), 1646 (C]O). 1H NMR (DMSO‑d6, 300 MHz):
δ 3.83 (s, 3H, OCH3), 6.54 (s, 1H, pyridyl), 7.09 (d, 2H, J = 9.0 Hz,
phenyl), 7.59–7.74 (m, 6H, naphthyl + phenyl), 7.94–8.12 (m, 3H,
naphthyl), 12.85 (br.s, 1H, NH, D2O exchangeable). 13C NMR
(DMSO‑d6, 75 MHz): δ 161.6, 161.1, 159.1, 150.5, 133.0, 131.0,
130.4, 130.1, 129.9, 128.4, 127.8, 127.6, 127.3, 126.4, 125.1, 124.6,
116.8, 114.2, 108.4, 55.3. Mass (m/z, rel. int.): 352 (M+, 98.27). Anal.
for C23H16N2O2, Calc. C: 78.39, H: 4.58, N: 7.95; Found C: 78.47, H:
4.59, N: 7.98.
4.1.1.12. 1,2-Dihydro-4-(3,4-dimethoxyphenyl)-6-(naphthalene-2-yl)-2-
oxopyridine-3-carbonitrile 5f. C24H18N2O3 (382.41), yield %: 51; m.p.
271–273 °C; I.R. (KBr, cm−1): ῦ 3446 (NH), 3051 (CH aromatic), 2935
(CH aliphatic), 2210 (CN), 1635 (C]O). 1H NMR (DMSO‑d6, 300 MHz):
δ 3.86 (s, 6H, 2 × OCH3), 7.16 (d, 1H, J = 8.5 Hz, phenyl), 7.19 (d, 1H,
J = 8.5 Hz, phenyl), 7.39 (s, 1H, pyridyl), 7.41 (d, 1H, J = 2.0 Hz,
phenyl), 7.61–7.66 (m, 2H, naphthyl), 7.78 (d, 1H, J = 2.0 Hz,
naphthyl), 8.00–8.08 (m, 3H, naphthyl), 8.55 (s, 1H, C-1 naphthyl),
12.71 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel. int.): 382 (M+,
3.69). Anal. for C24H18N2O3, Calc. C: 75.38, H: 4.74, N: 7.33; Found C:
75.41, H: 4.81, N: 7.49.
4.1.1.6. 1,2-Dihydro-4-(3,4-dimethoxyphenyl)-6-(naphthalen-1-yl)-2-
oxopyridine-3-carbonitrile 4f. C24H18N2O3 (382.41), yield %: 49; m.p.
265–267 °C; I.R. (KBr, cm−1): ῦ 3444 (NH), 3056 (CH aromatic), 2994
(CH aliphatic), 2212 (CN), 1644 (C]O). 1H NMR (DMSO‑d6, 300 MHz):
δ 3.80 (s, 6H, 2 × OCH3), 6.65 (s, 1H, pyridyl), 7.13 (d, 1H, J = 8.4 Hz,
phenyl), 7.33–7.38 (m, 2H, naphthyl), 7.61–7.72 (m, 4H,
naphthyl + phenyl), 7.92–8.07 (m, 2H, naphthyl), 8.11 (d, 1H,
J = 8.4 Hz, naphthyl), 12.84 (br.s, 1H, NH, D2O exchangeable). Mass
(m/z, rel. int.): 382 (M+, 59.79). Anal. for C24H18N2O3, Calc. C: 75.38,
H: 4.74, N: 7.33; Found C: 75.43, H: 4.80, N: 7.35.
4.1.2. General synthesis procedure for preparation of 2-amino-4-aryl-3-
cyano-6-(1- or 2-naphthyl)pyridines 6a-d and 7a-f
A mixture of the chosen acetylnaphthalene (1 mmol, 0.5 g), the
desired aromatic aldehyde (1 mmol) was solubilized in dry toluene
(20 mL). Ammonium acetate (8 mmol, 2.17 g) and malononitrile
(1 mmol, 0.4 g) were added. The reaction mixture was refluxed for 8 h
in the presence of catalytic amount of anhydrous potassium carbonate,
and then cooled to room temperature. The separated compound was
collected by filtration, washed with distilled water (2 × 10 mL) then
left to dry. The obtained precipitate was crystallized from ethanol.
4.1.1.7. 1,2-Dihydro-6-(naphthalen-2-yl)-2-oxo-4-phenylpyridine-3-
carbonitrile 5a [27]. C22H14N2O (322.36), yield %: 44; m.p. > 300 °C;
I.R. (KBr, cm−1): ῦ 3442 (NH), 3051 (CH aromatic), 2222 (CN), 1654
(C]O). 1H NMR (DMSO‑d6, 300 MHz): δ 7.02 (s, 1H, pyridyl),
7.58–7.66 (m, 5H, naphthyl + phenyl), 7.76–7.79 (m, 2H, naphthyl),
7.99–8.05 (m, 4H, naphthyl + phenyl), 8.57 (s, 1H, C-1 naphthyl),
12.88 (br.s, 1H, NH, D2O exchangeable).
4.1.1.8. 1,2-Dihydro-6-(naphthalene-2-yl)-4-(4-nitrophenyl)-2-
4.1.2.1. 2-Amino-6-(naphthalen-1-yl)-4-phenylpyridine-3-carbonitrile 6a
[30].. C22H15N3 (321.37), yield %: 30; m.p. 150–152 °C; I.R. (KBr,
cm−1): ῦ 3367 (NH2), 3032 (CH aromatic), 2222 (CN).
oxopyridine-3-carbonitrile 5b. C22H13N3O3 (367.36), yield %:65;
m.p. > 300 °C; I.R. (KBr, cm−1): ῦ 3390 (NH), 3055 (CH aromatic),
2218 (CN), 1654 (C]O). 1H NMR (DMSO‑d6, 300 MHz): δ 7.11 (s, 1H,
pyridyl), 7.55–7.64 (m, 3H, naphthyl + phenyl), 8.01–8.05 (m, 5H,
naphthyl + phenyl), 8.41 (d, 2H, J = 7.7 Hz, phenyl), 8.66 (s, 1H, C-1
naphthyl), 12.98 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 367 (M+, 39.23). Anal. for C22H13N3O3, Calc. C: 71.93, H: 3.57, N:
11.44; Found C: 72.08, H: 3.52, N: 11.62.
4.1.2.2. 2-Amino-6-(naphthalen-1-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 6b. C22H14N4O2 (366.37), yield %: 24; m.p. 203–205 °C;
I.R. (KBr, cm−1): ῦ 3481, 3375 (NH2), 3049 (CH aromatic), 2208 (CN).
1H NMR (DMSO‑d6, 400 MHz): δ 7.02 (s, 1H, pyridyl), 7.26 (br.s, 2H,
NH2, D2O exchangeable), 7.53–7.63 (m, 3H, naphthyl), 7.72 (d, 1H,
10

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BioorganicChemistry103(2020)104222
J = 6.9 Hz, naphthyl), 7.99 (d, 2H, J = 8.6 Hz, phenyl), 8.04 (d, 2H,
J = 8.9 Hz, naphthyl), 8.21 (d, 1H, J = 8.9 Hz, naphthyl), 8.38 (d, 2H,
J = 8.6 Hz, phenyl). Mass (m/z, rel. int.): 366 (M+, 100). Anal. for
3-carbonitrile 7f. C24H19N3O2 (381.43), yield %: 28; m.p. 188–190 °C;
I.R. (KBr, cm−1): ῦ 3462, 3360 (NH2), 3092 (CH aromatic), 2920 (CH
aliphatic), 2202 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.87 (s, 6H,
2 × OCH3), 7.00 (br.s, 2H, NH2, D2O exchangeable), 7.15 (d, 1H,
J = 8.2 Hz, phenyl), 7.30–7.33 (m, 2H, phenyl), 7.48 (s, 1H, pyridyl),
7.57–7.59 (m, 2H, naphthyl), 7.97 (t, 1H, J = 5.1 Hz, naphthyl), 8.03
(d, 2H, J = 8.6 Hz, naphthyl), 8.30 (dd, 1H, J = 8.6, 1.6 Hz, naphthyl),
8.74 (s, 1H, naphthyl). Mass (m/z, rel. int.): 381 (M+, 1.80). Anal. for
C
22H14N4O2, Calc. C: 72.12, H: 3.85, N: 15.29; Found C: 72.29, H: 3.84,
N: 15.37.
4.1.2.3. 2-Amino-4-(4-chlorophenyl)-6-(naphthalen-1-yl)pyridine-3-
carbonitrile 6c. C22H14ClN3 (355.82), yield %: 31; m.p. 120–125 °C; I.R.
(KBr, cm−1): ῦ 3468, 3367 (NH2), 3051 (CH aromatic), 2210 (CN). 1H
NMR (DMSO‑d6, 400 MHz): δ 6.96 (s, 1H, pyridyl), 7.15 (br.s, 2H, NH2,
D2O exchangeable), 7.51–7.64 (m, 5H, naphthyl + phenyl), 7.70 (dd,
1H, J = 7.1, 1.1 Hz, naphthyl), 7.74 (d, 2H, J = 8.5 Hz, phenyl),
8.00–8.04 (m, 2H, naphthyl), 8.20 (d, 1H, J = 7.1 Hz, naphthyl). Mass
(m/z, rel. int.): 355 (M+, 10.41). Anal. for C22H14ClN3, Calc. C: 74.26,
H: 3.97, N: 11.81; Found C: 74.39, H: 4.02, N: 11.98.
C24H19N3O2, Calc. C: 75.57, H: 5.02, N: 11.02; Found C: 75.72, H: 5.09,
N: 11.18.
4.1.3. General procedure for preparation of 4-Aryl-2-chloro-3-cyano-6-(1-
or 2-naphthyl)pyridines 8a-f and 9a-f
Compound 4a-f or 5a-f (15 mmol) and a catalytic amount of
phosphorous pentachloride (5 mmol) in 5 mL phosphorous oxychloride
was refluxed for 5 h on a water bath. The reaction mixture was then
cooled to room temperature and poured onto crushed ice portion wise
with continuous stirring with scratching till a precipitate started
forming. The obtained solid was filtered and crystallized from ethanol.
4.1.2.4. 2-Amino-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-1-yl)
pyridine-3-carbonitrile 6d. C23H17N3O2 (367.4), yield %: 48; m.p.
108–110 °C; I.R. (KBr, cm−1): ῦ 3462, 3446 (NH2), 3367 (OH), 3049
(CH aromatic), 2920 (CH aliphatic), 2208 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 3.84 (s, 3H, OCH3), 6.92–6.96 (m, 2H, phenyl + pyridyl),
6.99 (br.s, 2H, NH2, D2O exchangeable), 7.18 (d, 1H, J = 2.0 Hz,
phenyl), 7.27 (d, 1H, J = 2.0 Hz, phenyl), 7.53–7.62 (m, 3H, naphthyl),
7.68 (d, 1H, J = 6.2 Hz, naphthyl), 8.00 (t, 2H, J = 3.8 Hz, naphthyl),
8.16–8.18 (m, 1H, naphthyl), 9.53 (s, 1H. OH, D2O exchangeable). Mass
(m/z, rel. int.): 367 (M+, 100). Anal. for C23H17N3O2, Calc. C: 75.19, H:
4.66, N: 11.44; Found C: 75.26, H: 4.69, N: 11.56.
4.1.3.1. 2-Chloro-6-(naphthalen-1-yl)-4-phenylpyridine-3-carbonitrile
8a. C22H13ClN2 (340.81), yield %: 62; m.p. 138–140 °C; I.R. (KBr,
cm−1): ῦ 3045 (CH aromatic), 2223 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.55–7.68 (m, 7H, naphthyl + phenyl), 7.84 (d, 2H,
J = 6.8 Hz, naphthyl), 7.97 (s, 1H, pyridyl), 8.06 (t, 1H, J = 5.6 Hz,
naphthyl), 8.12 (d, 1H, J = 8.2 Hz, naphthyl), 8.20 (t, 1H, J = 5.6 Hz,
naphthyl). Mass (m/z, rel. int.): 342 (M+ +2, 24.95), 340 (M+, 100).
Anal. for C22H13ClN2, Calc. C: 77.53, H: 3.84, N: 8.22; Found C: 77.60,
H: 3.91, N: 8.35.
4.1.2.5. 2-Amino-6-(naphthalen-2-yl)-4-phenylpyridine-3-carbonitrile 7a
[27].. C22H15N3 (321.37), yield%: 38; m.p. 226–228 °C as reported;
I.R. (KBr, cm−1): ῦ 3473, 3304 (NH2), 3051 (CH aromatic), 2204 (CN).
4.1.3.2. 2-Chloro-6-(naphthalen-1-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 8b. C22H12ClN3O2 (385.8), yield %: 95; m.p. 148–150 °C;
I.R. (KBr, cm−1): ῦ 3049 (CH aromatic), 2223 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.61–7.68 (m, 3H, phenyl + pyridyl), 7.86 (d, 1H,
J = 6.4 Hz, naphthyl), 8.02 (d, 1H, J = 6.4 Hz, naphthyl), 8.04–8.13
(m, 4H, naphthyl + phenyl), 8.21 (dd, 1H, J = 6.4, 3.8 Hz, naphthyl),
8.39 (d, 1H, J = 8.8 Hz, naphthyl), 8.46 (d, 1H, J = 8.8 Hz,
naphthyl).Mass (m/z, rel. int.): 387 (M+ +2, 18.71), 385 (M+,
59.74). Anal. for C22H12ClN3O2, Calc. C: 68.49, H: 3.14, N: 10.89;
Found C: 68.61, H: 3.10, N: 11.03.
4.1.2.6. 2-Amino-6-(naphthalen-2-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 7b. C22H14N4O2 (366.37), yield %: 24; m.p. 258–260 °C;
I.R. (KBr, cm−1): ῦ 3469, 3307 (NH2), 3076 (CH aromatic), 2202 (CN).
1H NMR (DMSO‑d6, 400 MHz):
δ 7.22 (br.s, 2H, NH2, D2O
exchangeable), 7.55 (s, 1H, pyridyl), 7.59 (t, 2H, J = 4.0 Hz,
naphthyl), 7.96–8.05 (m, 5H, naphthyl + phenyl), 8.30 (dd, 1H,
J = 8.6, 1.5 Hz, naphthyl), 8.42 (d, 2H, J = 8.7 Hz, phenyl), 8.76 (s,
1H, naphthyl). Mass (m/z, rel. int.): 366 (M+, 100). Anal. for
C22H14N4O2, Calc. C: 72.12, H: 3.85, N: 15.29; Found C: 72.31, H:
3.87, N: 15.38.
4.1.3.3. 2-Chloro-4-(4-chlorophenyl)-6-(naphthalen-1-yl)pyridine-3-
carbonitrile 8c. C22H12Cl2N2 (375.25), yield %: 97; m.p. 160–162 °C;
I.R. (KBr, cm−1): ῦ 3055 (CH aromatic), 2222 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.60–7.67 (m, 4H, naphthyl), 7.71 (d, 2H, J = 8.5 Hz,
phenyl), 7.88 (d, 2H, J = 8.5 Hz, phenyl), 8.02 (s, 1H, pyridyl),
8.05–8.08 (m, 1H, naphthyl), 8.13 (dd, 1H, J = 8.1, 3.3 Hz, naphthyl),
8.20 (t, 1H, J = 6.4 Hz, naphthyl). Mass (m/z, rel. int.): 375 (M+, 100).
Anal. for C22H12Cl2N2, Calc. C: 70.42, H: 3.22, N: 7.47; Found C: 70.53,
H: 3.19, N: 7.58.
4.1.2.7. 2-Amino-4-(4-chlorophenyl)-6-(naphthalen-2-yl)pyridine-3-
carbonitrile 7c [29].. C22H14ClN3 (355.82), yield %: 41; m.p.
261–263 °C; I.R. (KBr, cm−1): ῦ 3495, 3392 (NH2), 3035 (CH
aromatic), 2204 (CN).
4.1.2.8. 2-Amino-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-2-yl)
pyridine-3-carbonitrile 7d. C23H17N3O2 (367.4), yield%: 48; m.p.
225–227 °C; I.R. (KBr, cm−1): ῦ 3518, 3466, (NH2), 3309 (OH), 3172
(CH aromatic), 2930 (CH aliphatic), 2202 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 3.88 (s, 3H, OCH3), 6.95 (m, 3H, phenyl + NH2, D2O
exchangeable), 7.19 (dd, 1H, J = 8.1, 2.0 Hz, phenyl), 7.31 (d, 1H,
J = 2.0 Hz, phenyl), 7.46 (s, 1H, pyridyl), 7.57–7.59 (m, 2H, naphthyl),
7.96–8.05 (m, 3H, naphthyl), 8.29 (dd, 1H, J = 8.6, 1.5 Hz, naphthyl),
8.73 (s, 1H, naphthyl), 9.53 (br.s, 1H, OH, D2O exchangeable). Mass
(m/z, rel. int.): 367 (M+, 100). Anal. for C23H17N3O2, Calc. C: 75.19, H:
4.66, N: 11.44; Found C: 75.28, H: 4.71, N: 11.53.
4.1.3.4. 2-Chloro-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-1-yl)
pyridine-3-carbonitrile 8d. C23H15ClN2O2 (386.83), yield %: 57; m.p.
130–132 °C; I.R. (KBr, cm−1): ῦ 3385 (OH), 3049 (CH aromatic), 2953
(CH aliphatic), 2223 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.86 (s, 3H,
OCH3), 6.98 (d, 1H, J = 8.2 Hz, phenyl), 7.26 (dd, 1H, J = 8.2, 2.0 Hz,
phenyl), 7.44 (d, 1H, J = 2.0 Hz, phenyl), 7.58–7.71 (m, 3H, naphthyl),
7.82 (d, 1H, J = 7.1 Hz, naphthyl), 7.98 (s, 1H, pyridyl), 8.06 (t, 1H,
J = 5.3 Hz, naphthyl), 8.12 (d, 1H, J = 8.3 Hz, naphthyl), 8.15 (t, 1H,
J = 5.3 Hz, naphthyl), 9.42 (br.s, 1H, OH, D2O exchangeable). Mass
(m/z, rel. int.): 388 (M+ +2, 24.50), 386 (M+, 100). Anal. for
4.1.2.9. 2-Amino-4-(4-methoxyphenyl)-6-(naphthalen-2-yl)pyridine-3-
carbonitrile 7e [27].. C23H17N3O (351.4), yield %: 41; m.p. 280–282 °C;
I.R. (KBr, cm−1): ῦ 3473, 3307 (NH2), 3110 (CH aromatic), 2960 (CH
aliphatic), 2204 (CN).
C
23H15ClN2O2, Calc. C: 71.41, H: 3.91;, N: 7.24; Found C: 71.52, H:
3.89, N: 7.31.
4.1.3.5. 2-Chloro-4-(4-methoxyphenyl)-6-(naphthalen-1-yl)pyridine-3-
4.1.2.10. 2-Amino-4-(3,4-dimethoxyphenyl)-6-(naphthalen-2-yl)pyridine-
carbonitrile 8e. C23H15ClN2O (370.83), yield %: 96; m.p. 118–120 °C;
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BioorganicChemistry103(2020)104222
I.R. (KBr, cm−1): ῦ 3040 (CH aromatic), 2954 (CH aliphatic), 2222
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.86 (s, 3H, OCH3), 7.17 (d, 2H,
J = 8.8 Hz, phenyl), 7.60–7.68 (m, 2H, naphthyl), 7.66 (t, 1H,
J = 8.0 Hz, naphthyl), 7.81–7.84 (m, 3H, naphthyl + phenyl), 7.94
(s, 1H, pyridyl), 8.06 (t, 1H, J = 5.2 Hz, naphthyl), 8.12 (d, 1H,
J = 8.2 Hz, naphthyl), 8.18 (t, 1H, J = 5.2 Hz, naphthyl). Mass (m/z,
rel. int.): 372 (M+ +2, 32.83), 370 (M+, 100). Anal. for C23H15ClN2O,
Calc. C: 74.49, H: 4.08;, N: 7.55; Found C: 74.65, H: 4.13, N: 7.64.
I.R. (KBr, cm−1): ῦ 3059 (CH aromatic), 2960 (CH aliphatic), 2223
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.88 (s, 3H, OCH3), 7.19 (d, 2H,
J = 8.7 Hz, phenyl), 7.61–7.64 (m, 2H, naphthyl), 7.83 (d, 2H,
J = 8.7 Hz, phenyl), 8.00 (d, 1H, J = 8.6 Hz, naphthyl), 8.07–8.12
(m, 2H, naphthyl), 8.34 (dd, 1H, J = 8.6, 1.4 Hz, naphthyl), 8.37 (s,
1H, pyridyl), 8.89 (s, 1H, C-1 naphthyl). Mass (m/z, rel. int.): 372 (M+
+2, 39.80), 370 (M+, 100). Anal. for C23H15ClN2O, Calc. C: 74.49, H:
4.08, N: 7.55; Found C: 74.57, H: 4.13, N: 7.68.
4.1.3.6. 2-Chloro-4-(3,4-dimethoxyphenyl)-6-(naphthalen-1-yl)pyridine-
3-carbonitrile 8f. C24H17ClN2O2 (400.86), yield %: 90; m.p. 135–137 °C;
I.R. (KBr, cm−1): ῦ 3005 (CH aromatic), 2976 (CH aliphatic), 2223
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.81 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 7.19 (d, 1H, J = 8.6 Hz, phenyl), 7.44–7.46 (m, 1H, naphthyl),
7.60–7.69 (m, 2H, naphthyl), 7.74 (dd, 1H, J = 8.6, 1.9 Hz, phenyl),
7.77 (d, 1H, J = 1.9 Hz, phenyl), 7.83 (d, 1H, J = 6.3 Hz, naphthyl),
8.01 (s, 1H, pyridyl), 8.06 (t, 1H, J = 5.5 Hz, naphthyl), 8.13 (d, 1H,
J = 8.2 Hz, naphthyl), 8.16 (t, 1H, J = 5.5 Hz, naphthyl). 13C NMR
(DMSO‑d6, 100 MHz): δ 162.8, 155.3, 154.0, 151.3, 149.1, 133.8,
130.8, 130.4, 129.1, 127.7, 127.2, 126.8, 125.8, 124.4, 122.4, 116.8,
113.4, 112.9, 112.3, 106.6, 98.9, 56.4, 56.1. Mass (m/z, rel. int.): 402
(M+ +2, 32.16), 400 (M+, 100). Anal. for C24H17ClN2O2, Calc. C:
71.91, H: 4.27; N: 6.99; Found C: 72.03, H: 4.32, N: 7.12.
4.1.3.12. 2-Chloro-4-(3,4-dimethoxyphenyl)-6-(naphthalen-2-yl)pyridine-
3-carbonitrile 9f. C24H17ClN2O2 (400.86), yield %: 86; m.p. 188–190 °C;
I.R. (KBr, cm−1): ῦ 3005 (CH aromatic), 2927, 2841 (CH aliphatic),
2223 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.81 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 7.19 (d, 1H, J = 8.4 Hz, phenyl), 7.61–7.64 (m, 2H,
naphthyl), 7.74 (dd, 1H, J = 8.4, 1.8 Hz, phenyl), 7.77 (d, 1H,
J = 1.8 Hz, phenyl), 8.00–8.13 (m, 3H, naphthyl), 8.35 (dd, 1H,
J = 8.6, 1.5 Hz, naphthyl), 8.40 (s, 1H, pyridyl), 8.89 (s, 1H, C-1
naphthyl). 13C NMR (DMSO‑d6, 100 MHz): δ 162.8, 155.3, 154.0,
151.2, 149.1, 134.5, 133.4, 129.5, 129.1, 128.5, 128.1, 127.2, 124.4,
122.4, 120.0, 116.8, 113.4, 112.9, 112.4, 106.5, 98.9, 56.3, 56.2. Mass
(m/z, rel. int.): 402 (M+ +2, 36), 400 (M+, 100). Anal. for
C
22H13ClN2O, Calc. C: 71.91, H: 4.27, N: 6.99; Found C: 72.09, H:
4.25, N: 7.13.
4.1.3.7. 2-Chloro-6-(naphthalen-2-yl)-4-phenylpyridine-3-carbonitrile 9a
[31].. C22H13ClN2 (340.81), yield %: 94; m.p. 197–199 °C; I.R. (KBr,
cm−1): ῦ 3055 (CH aromatic), 2220 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.59–7.65 (m, 5H, naphthyl + phenyl), 7.83–7.85 (m,
2H, naphthyl), 7.99–8.12 (m, 3H, naphthyl + phenyl), 8.35 (dd, 1H,
J = 8.6, 1.7 Hz, naphthyl), 8.42 (s, 1H, pyridyl), 8.91 (s, 1H, C-1
naphthyl). Mass (m/z, rel. int.): 342 (M+ +2, 33.72), 340 (M+, 100).
Anal. for C22H13ClN2, Calc. C: 77.53, H: 3.84, N: 8.22; Found C: 77.59,
H: 3.87, N: 8.34.
4.1.4. General procedure for preparation of 4-Aryl-3-cyano-2-hydrazino-6-
(1- or 2-naphthyl)pyridines 10a-f and 11a-f
A mixture of the appropriate chloro derivative 8a-f or 9a-f
(15 mmol) and excess hydrazine hydrate (30 mL, 98%) was refluxed in
absolute ethanol for 4 h. The mixture was then cooled to room tem-
perature, poured on ice-water. The precipitated compound was ob-
tained by filtration, left to dry and recrystallized from ethanol to give
derivatives 10a-f and 11a-f.
4.1.4.1. 2-Hydrazinyl-6-(naphthalen-1-yl)-4-phenylpyridine-3-carbonitrile
4.1.3.8. 2-Chloro-6-(naphthalen-2-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 9b. C22H12ClN3O2 (385.8), yield %: 86; m.p. > 300 °C; I.R.
(KBr, cm−1): ῦ 3057 (CH aromatic), 2222 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.60–7.66 (m, 2H, naphthyl), 7.99–8.13 (m, 4H,
naphthyl + phenyl), 8.36 (dd, 1H, J = 8.6, 1.7 Hz, naphthyl), 8.42
(d, 1H, J = 8.8 Hz, naphthyl), 8.48 (d, 2H, J = 8.8 Hz, phenyl), 8.53 (s,
1H, pyridyl), 8.93 (s, 1H, C-1 naphthyl). Mass (m/z, rel. int.): 387 (M+
+2, 7.48), 385 (M+, 27.98). Anal. for C22H13ClN2O, Calc. C: 68.49, H:
3.14, N: 10.89; Found C: 68.62, H: 3.18, N: 11.03.
10a. C22H16N4 (336.39), yield %: 89; m.p. 135–137 °C; I.R. (KBr,
1
cm−1): ῦ 3433, 3309 (NH2, NH), 3057 (CH aromatic), 2223 (CN). H
NMR (DMSO‑d6, 400 MHz): δ 4.64 (br.s, 2H, NH2, D2O exchangeable),
7.17 (s, 1H, pyridyl), 7.55–7.64 (m, 6H, naphthyl + phenyl), 7.69–7.76
(m, 3H, naphthyl + phenyl), 8.03 (d, 2H, J = 7.2 Hz, naphthyl), 8.24
(d, 1H, J = 8.0 Hz, naphthyl), 12.46 (br.s, 1H, NH, D2O exchangeable).
Mass (m/z, rel. int.): 336 (M+, 100). Anal. for C22H16N4, Calc. C: 78.55,
H: 4.79, N: 16.66; Found C: 78.59, H: 4.86, N: 16.74.
4.1.4.2. 2-Hydrazinyl-6-(naphthalen-1-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 10b. C22H15N5O2 (381.39), yield %: 72; m.p. 180–182 °C;
I.R. (KBr, cm−1): ῦ 3419, 3360 (NH2, NH), 3057 (CH aromatic), 2220
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 4.78 (br.s, 2H, NH2, D2O
exchangeable), 7.28 (s, 1H, pyridyl), 7.52–7.58 (m, 2H, naphthyl),
7.61–7.66 (m, 4H, naphthyl + phenyl), 7.78 (d, 1H, J = 6.7 Hz,
phenyl), 7.99–8.06 (m, 2H, naphthyl), 8.24 (d, 1H, J = 8.2 Hz,
naphthyl), 8.39 (dd, 1H, J = 8.2, 3.2 Hz, naphthyl), 12.59 (br.s, 1H,
NH, D2O exchangeable). Mass (m/z, rel. int.): 381 (M+, 90.52). Anal.
for C22H15N5O2, Calc. C: 69.28, H: 3.96, N: 18.36; Found C: 69.34, H:
3.98, N: 18.50.
4.1.3.9. 2-Chloro-4-(4-chlorophenyl)-6-(naphthalen-2-yl)pyridine-3-
carbonitrile 9c. C22H12Cl2N2 (375.25), yield %: 98; m.p. 256–258 °C;
I.R. (KBr, cm−1): ῦ 3057 (CH aromatic), 2220 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 7.59–7.65 (m, 4H, naphthyl + phenyl), 7.77–7.79 (m, 2H,
phenyl), 8.00–8.13 (m, 3H, naphthyl), 8.37 (dd, 1H, J = 8.6, 1.7 Hz,
naphthyl), 8.43 (s, 1H, pyridyl), 8.93 (s, 1H, C-1 naphthyl). Mass (m/z,
rel. int.): 375 (M+, 100). Anal. for C22H13ClN2O, Calc. C: 70.42, H:
3.22, N: 7.47; Found C: 70.51, H: 3.20, N: 7.56.
4.1.3.10. 2-Chloro-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-2-yl)
pyridine-3-carbonitrile 9d. C23H15ClN2O2 (386.83), yield %: 95;
m.p. > 300 °C; I.R. (KBr, cm−1): ῦ 3387 (OH), 3055 (CH aromatic),
2938 (CH aliphatic), 2220 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.88
(s, 3H, OCH3), 7.28–7.39 (m, 3H, phenyl), 7.59–7.64 (m, 2H, naphthyl),
8.00–8.12 (m, 3H, naphthyl), 8.33–8.35 (m, 1H, naphthyl), 8.36 (s, 1H,
pyridyl), 8.90 (s, 1H, C-1 naphthyl), 9.42 (br.s, 1H, OH, D2O
exchangeable). Mass (m/z, rel. int.): 388 (M+ +2, 33.82), 386 (M+,
100). Anal. for C23H15ClN2O2, Calc. C: 71.41, H: 3.91, N: 7.24; Found C:
71.50, H: 3.97, N: 7.32.
4.1.4.3. 4-(4-Chlorophenyl)-2-hydrazinyl-6-(naphthalen-1-yl)pyridine-3-
carbonitrile 10c. C22H15ClN4 (370.83), yield %: 98; m.p. 166–168 °C;
I.R. (KBr, cm−1): ῦ 3433, 3309 (NH2, NH), 3049 (CH aromatic), 2220
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 4.71 (br.s, 2H, NH2, D2O
exchangeable), 7.19 (s, 1H, pyridyl), 7.51–7.59 (m, 2H, naphthyl),
7.61–7.65 (m, 3H, naphthyl + phenyl), 7.76 (dd, 2H, J = 8.4, 1.8 Hz,
phenyl), 8.03 (dd, 2H, J = 8.4, 4.4 Hz, naphthyl), 8.11 (d, 1H,
J = 8.4 Hz, naphthyl), 8.23 (d, 1H, J = 8.4 Hz, naphthyl), 12.49
(br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel. int.): 372 (M+ +2,
23.87), 370 (M+, 100). Anal. for C22H15ClN4, Calc. C: 71.25, H: 4.08, N:
15.11; Found C: 71.33, H: 4.12, N: 15.27
4.1.3.11. 2-Chloro-4-(4-methoxyphenyl)-6-(naphthalen-2-yl)pyridine-3-
carbonitrile 9e. C23H15ClN2O (370.83), yield %: 84; m.p. 222–224 °C;
12

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
4.1.4.4. 2-Hydrazinyl-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-1-
yl)pyridine-3-carbonitrile 10d. C23H18N4O2 (382.41), yield %: 79; m.p.
170–172 °C; I.R. (KBr, cm−1): ῦ 3422, 3329 (NH2, NH), 3201 (OH),
carbonitrile 11c. C22H15ClN4 (370.83), yield %: 95; m.p. 240–242 °C;
I.R. (KBr, cm−1): ῦ 3454, 3286 (NH2, NH), 3055 (CH aromatic), 2218
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 4.59 (br.s, 2H, NH2, D2O
exchangeable), 7.48–7.64 (m, 4H, naphthyl), 7.69 (s, 1H, pyridyl),
7.73–7.79 (m, 2H, phenyl), 7.96–8.09 (m, 3H, phenyl + naphthyl),
8.38 (dd, 1H, J = 8.8, 1.6 Hz, naphthyl), 8.78 (s, 1H, C-1 naphthyl),
12.86 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel. int.): 372 (M+
+2, 0.08), 370 (M+, 0.22). Anal. for C22H15ClN4, Calc. C: 71.25, H:
4.08, N: 15.11; Found C: 71.33, H: 4.12, N: 15.24.
1
3059 (CH aromatic), 2922, 2851 (CH aliphatic), 2220 (CN). H NMR
(DMSO‑d6, 400 MHz): δ 3.86 (s, 3H, OCH3), 4.73 (br.s, 2H, NH2, D2O
exchangeable), 6.94 (d, 1H, J = 8.1 Hz, phenyl), 7.14 (dd, 1H, J = 8.1,
1.7 Hz, phenyl), 7.17 (s, 1H, pyridyl), 7.33 (d, 1H, J = 1.7 Hz, phenyl),
7.51–7.58 (m, 2H, naphthyl), 7.63 (t, 1H, J = 7.4 Hz, naphthyl), 7.74
(d, 1H, J = 6.8 Hz, naphthyl), 8.02–8.04 (m, 2H, naphthyl), 8.21 (d,
1H, J = 7.8 Hz, naphthyl), 9.42 (br.s, 1H, OH, D2O exchangeable),
12.46 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel. int.): 382 (M+,
56.09). Anal. for C23H18N4O2, Calc. C: 72.24, H: 4.74, N: 14.65; Found
C: 72.35, H: 4.78, N: 14.79.
4.1.4.10. 2-Hydrazinyl-4-(4-hydroxy-3-methoxyphenyl)-6-(naphthalen-2-
yl)pyridine-3-carbonitrile 11d. C23H18N4O2 (382.41), yield %: 59; m.p.
296–298 °C; I.R. (KBr, cm−1): ῦ 3331–3288 (OH, NH2, NH), 3053 (CH
aromatic), 2970 (CH aliphatic), 2218 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 3.87 (s, 3H, OCH3), 4.68 (br.s, 2H, NH2, D2O
exchangeable), 7.04 (d, 1H, J = 8.1 Hz, phenyl), 7.16 (d, 1H,
J = 6.7 Hz, naphthyl), 7.37 (s, 1H, phenyl), 7.55–7.57 (m, 2H,
naphthyl + phenyl), 7.69 (s, 1H, pyridyl), 7.96–8.08 (m, 3H,
naphthyl), 8.37 (d, 1H, J = 8.4 Hz, naphthyl), 8.76 (s, 1H, C-1
naphthyl), 9.58 (s, 1H, OH, D2O exchangeable), 12.37 (br.s, 1H, NH,
D2O exchangeable). Mass (m/z, rel. int.): 382 (M+, 100). Anal. for
4.1.4.5. 2-Hydrazinyl-4-(4-methoxyphenyl)-6-(naphthalen-1-yl)pyridine-
3-carbonitrile 10e. C23H18N4O (366.42), yield %: 59; m.p. 275–277 °C;
I.R. (KBr, cm−1): ῦ 3454, 3363 (NH2, NH), 3043 (CH aromatic), 2927
(CH aliphatic), 2220 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.85 (s, 3H,
OCH3), 4.67 (br.s, 2H, NH2, D2O exchangeable), 7.13–7.15 (m, 3H,
pyridyl + phenyl), 7.51–7.58 (m, 2H, naphthyl), 7.62 (t, 1H,
J = 7.9 Hz, naphthyl), 7.67 (d, 2H, J = 8.7 Hz, phenyl), 7.74 (d,
1H, J = 6.2 Hz, naphthyl), 8.02–8.04 (m, 2H, naphthyl), 8.23 (d, 1H,
J = 8.1 Hz, naphthyl), 12.39 (br.s, 1H, NH, D2O exchangeable). Mass
(m/z, rel. int.): 366 (M+, 100). Anal. for C23H18N4O, Calc. C: 75.39, H:
4.95, N: 15.29; Found C: 75.48, H: 4.97, N: 15.42.
C
23H18N4O2, Calc. C: 72.24, H: 4.74, N: 14.65; Found C: 72.34, H: 4.78,
N: 14.73.
4.1.4.11. 2-Hydrazinyl-4-(4-methoxyphenyl)-6-(naphthalen-2-yl)pyridine-
3-carbonitrile 11e. C23H18N4O (366.42), yield %: 64; m.p. 219–221 °C;
I.R. (KBr, cm−1): ῦ 3446, 3348 (NH2, NH), 3061 (CH aromatic), 2956
(CH aliphatic), 2220 (CN). 1H NMR (DMSO‑d6, 400 MHz): δ 3.87 (s, 3H,
OCH3), 4.63 (br.s, 2H, NH2, D2O exchangeable), 7.17 (d, 2H,
J = 8.6 Hz, phenyl), 7.55–7.58 (m, 2H, naphthyl), 7.65 (s, 1H,
pyridyl), 7.70 (d, 2H, J = 8.6 Hz, phenyl), 7.79 (t, 1H, J = 3.3 Hz,
naphthyl), 8.06 (d, 1H, J = 5.5 Hz, naphthyl), 8.08 (t, 1H, J = 3.3 Hz,
naphthyl), 8.37 (dd, 1H, J = 8.6, 1.5 Hz, naphthyl), 8.77 (s, 1H, C-1
naphthyl), 12.37 (br.s, 1H, NH, D2O exchangeable). Mass (m/z, rel.
int.): 366 (M+, 100). Anal. for C23H18N4O, Calc. C: 75.39, H: 4.95, N:
15.29; Found C: 75.45, H: 4.99, N: 15.40.
4.1.4.6. 2-Hydrazinyl-4-(3,4-dimethoxyphenyl)-6-(naphthalen-1-yl)
pyridine-3-carbonitrile 10f. C24H20N4O2 (396.44), yield %: 42; m.p.
155–157 °C; I.R. (KBr, cm−1): ῦ 3421, 3329 (NH2, NH), 3055 (CH
aromatic), 2927, 2850 (CH aliphatic), 2223 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 3.84 (s, 6H, 2 × OCH3), 4.74 (br.s, 2H, NH2, D2O
exchangeable), 7.15 (d, 1H, J = 8.2 Hz, phenyl), 7.20 (s, 1H,
pyridyl),7.26 (dd, 1H, J = 8.2, 1.8 Hz, phenyl), 7.31 (s, 1H, phenyl),
7.51–7.58 (m, 2H, naphthyl), 7.63 (t, 1H, J = 7.6 Hz, naphthyl), 7.75
(d, 1H, J = 7.6 Hz, naphthyl), 8.02 (d, 2H, J = 7.6 Hz, naphthyl), 8.22
(d, 1H, J = 7.6 Hz, naphthyl), 12.4 (br.s, 1H, NH, D2O exchangeable).
13C NMR (DMSO‑d6, 100 MHz): δ 157.9, 153.5, 149.9, 149.2, 147.8,
145.5, 138.7, 133.9, 131.1, 129.7, 129.1, 128.7, 126.9, 126.4, 126.1,
125.8, 121.7, 116.9, 113.0, 112.3, 102.1, 56.06, 56.01. Mass (m/z, rel.
int.): 396 (M+, 100). Anal. for C24H20N4O2, Calc. C: 72.71, H: 5.08, N:
14.13; Found C: 72.88, H: 5.15, N: 14.21.
4.1.4.12. 2-Hydrazinyl-4-(3,4-dimethoxyphenyl)-6-(naphthalen-2-yl)
pyridine-3-carbonitrile 11f. C24H20N4O2 (396.44), yield %: 53; m.p.
143–145 °C; I.R. (KBr, cm−1): ῦ 3431, 3344 (NH2, NH), 3055 (CH
aromatic), 2931 (CH aliphatic), 2222 (CN). 1H NMR (DMSO‑d6,
400 MHz): δ 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.69 (br.s, 2H,
NH2, D2O exchangeable), 7.18 (d, 1H, J = 8.2 Hz, phenyl), 7.29 (dd,
1H, J = 8.2, 1.7 Hz, phenyl), 7.34 (d, 1H, J = 1.7 Hz, phenyl),
7.55–7.58 (m, 2H, naphthyl), 7.70 (s, 1H, pyridyl), 7.97 (t, 1H,
J = 5.4 Hz, naphthyl), 8.04 (d, 1H, J = 8.6 Hz, naphthyl), 8.08 (t,
1H, J = 5.4 Hz, naphthyl), 8.38 (dd, 1H, J = 8.6, 1.4 Hz, naphthyl),
4.1.4.7. 2-Hydrazinyl-6-(naphthalen-2-yl)-4-phenylpyridine-3-carbonitrile
11a. C22H16N4 (336.39), yield %: 65; m.p. 137–139 °C; I.R. (KBr,
1
cm−1): ῦ 3433, 3298 (NH2, NH), 3053 (CH aromatic), 2218 (CN). H
NMR (DMSO‑d6, 300 MHz): δ 4.57 (br.s, 2H, NH2, D2O exchangeable),
7.53–7.76 (m, 8H, phenyl + naphthyl + pyridyl), 7.97 (t, 1H,
J = 3.3 Hz, naphthyl), 8.01–8.08 (m, 2H, naphthyl), 8.36 (d, 1H,
J = 8.7 Hz, naphthyl), 8.77 (s, 1H, C-1 naphthyl), 12.41 (br.s,1H, NH,
D2O exchangeable). Mass (m/z, rel. int.): 336 (M+, 100). Anal. for
C22H16N4, Calc. C: 78.55, H: 4.79, N: 16.66; Found C: 78.69, H: 4.87, N:
16.82.
8.77 (s, 1H, C-1 naphthyl), 12.37 (br.s, 1H, NH, D2O exchangeable). 13
C
NMR (DMSO‑d6, 100 MHz): δ 155.6, 153.9, 149.9, 149.2, 147.8, 145.9,
136.8, 133.5, 130.0, 129.1, 128.5, 128.1, 127.4, 127.2, 126.9, 125.3,
121.7, 117.5, 113.0, 112.6, 102.5, 56.2, 56.1. Mass (m/z, rel. int.): 396
(M+, 100). Anal. for C24H20N4O2, Calc. C: 72.71, H: 5.08, N: 14.13;
Found C: 72.89, H: 5.13, N: 14.25.
4.1.4.8. 2-Hydrazinyl-6-(naphthalen-2-yl)-4-(4-nitrophenyl)pyridine-3-
carbonitrile 11b. C22H15N5O2 (381.39), yield %: 47; m.p. 290–292 °C;
I.R. (KBr, cm−1): ῦ 3421, 3302 (NH2, NH), 3051 (CH aromatic), 2222
(CN). 1H NMR (DMSO‑d6, 400 MHz): δ 4.74 (br.s, 2H, NH2, D2O
exchangeable), 7.56–7.59 (m, 2H, naphthyl), 7.79 (s, 1H, pyridyl),
7.95–8.09 (m, 4H, naphthyl + phenyl), 8.13 (dd, 1H, J = 8.6, 1.3 Hz,
naphthyl), 8.37–8.39 (m, 1H, naphthyl),8.43 (d, 2H, J = 8.6 Hz,
phenyl), 8.80 (s, 1H, C-1 naphthyl), 12.37 (br.s, 1H, NH, D2O
exchangeable). Mass (m/z, rel. int.): 381 (M+, 100). Anal. for
4.2. Biological screening
4.2.1. Materials and cell lines
Human prostate and breast cancer cells as target cells were pur-
chased from American Type Culture Collection (Rockville, MD) in July
2014 and were used within 6 months of acquisition. Identity verifica-
tion of cell lines is routinely performed at ATCC by short tandem repeat
(STR) profiling so no further authentication was performed thereto.
Cells were cultured according to ATCC instructions in Dulbecco’s
modified Eagle’s medium (DMEM, GIBCO BRL, Grand Island, NY) and
supplemented with 10% fetal calf serum (GIBCO BRL), 4 mM gluta-
mine, 100 U/ mL penicillin and 100 mg/ mL streptomycin. To generate
C
22H15N5O2, Calc. C: 69.28, H: 3.96, N: 18.36; Found C: 69.45, H:
3.98, N: 18.51.
4.1.4.9. 4-(4-Chlorophenyl)-2-hydrazinyl-6-(naphthalen-2-yl)pyridine-3-
13

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
MCF-7 p53 knockdown cells, parental cells were transfected with an
expression plasmid containing siRNA directed against p53 and were
grown in the same media as MCF-7 cells but supplemented with 0.5 µg/
mL puromycin (Sigma, St. Louis, MO). For detection of apoptosis, an-
tibodies against human Bax (N-20), Bcl-2 (100), Cdk2 (M2), Cdk4 (H-
22), Cdk6 (C-21), Cyclin D1 (DCS-6), E2F1 (KH95), MDM2 (SMP14),
PARP1 (H-250), p21 (C19) and p27 (C19) were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). The anti-human p53 antibody
(Ab-6) was from EMD Chemicals, Inc. (Gibbstown, NJ). The Caspase-3
(9662), Caspase-8 (9746) and Caspase-9 (9502) antibodies were from
Cell Signaling Technology, Inc. (Danvers, MA).
4.2.1.2.4. VEGFR-2 kinase activity assay by ELISA. The assay was
performed in 96-well plates pre-coated with 20 µg/mL poly(Glu,Tyr)
4:1 (Sigma) as a substrate. In each well, 85 µL of an 8 µM ATP solution
and 10 µL of the test compounds were added at varying concentrations.
Delphinidin was used as positive control for VEGFR-2 kinase, and 0.1%
(v/v) DMSO was used as negative control. Experiments at each
concentration were performed in duplicates. The reaction was
initiated by adding 5 µL of VEGFR-2 kinase. After incubation for 1 h
at 37 °C, the plate was washed three times with PBS containing 0.1%
Tween 20 (T-PBS). Next, 100 µL of anti-phosphotyrosine (PY99; 1:500
dilution) antibody was added. After 1 h of incubation at room
temperature, the plate was washed three times. Goat anti-mouse IgG
horseradish peroxidase (100 µL; 1:2000 dilution) diluted in T-PBS
containing 5 mg/mL BSA was added. The plate was reincubated at room
temperature for 1 h, and washed as before. Finally, 100 µL of
developing solution (0.03% H2O2, 2 mg/mL o-phenylenediamine in
citrate buffer 0.1 M, pH 5.5) was added and incubated at room
temperature until color emerged. The reaction was terminated by the
addition of 100 µL of 2 M H2SO4 and absorbance at 492 nm (A492) was
measured using a multiwell spectrophotometer (VERSAmaxTM). The
inhibition rate (%) was calculated using the equation: inhibition rate
(%) = [1−(A492/A492control)] × 100% [38].
4.2.1.1. In vitro cytotoxic activity. In vitro growth inhibition was
determined by the microculture tetrazolium (MTT) assay according to
Mosmann’s method as previously reported [31,32]. The cells were
grown (4–5
×
103 per well) and were exposed to various
concentrations of the tested compounds (0–1.0 µM) for 72 h. The
results were expressed as IC50 representing compound concentration
inducing a 50% inhibition of cell growth of the treated cells when
compared to the growth of control cells. Each experiment was
performed 3 times and the average of the 3 experiments was
determined and used for data presentation.
4.2.1.2. In vivo antitumor activity and mechanistic studies
4.2.1.2.1. Human breast cancer xenograft model. This study was
performed following the Cairo University guidelines for the care and
use of animals after the approval of Committee on Animal Research and
Ethics.
4.3. Molecular docking of 11d with VEGFR-2
Molecular modeling studies were carried out using Molecular
Operating Environment (MOE, 2019.01) software. Amber12: EHT force
field was used in preparing the crystal structure. The X-ray crystal-
lographic structure of VEGFR2 in complex with a naphthamide in-
hibitor (PDB ID: 3B8Q) was downloaded from the protein data bank.
The enzyme was prepared first by removal of chain B of its dimer as it
has more outliers than chain B, followed by removing water molecules
as they have no contributing role in binding. The enzyme was then
protonated at temperature of 277 Kelvin and pH of 8 (the experimental
conditions of its crystallization) using Amber 12:EHT force field. The
co-crystalized ligand was used to define the active site for docking.
Triangle Matcher placement method and London dG scoring function
were used for the docking protocol. Docking protocol was first vali-
dated by carrying out redocking of the bound naphthamide inhibitor.
The same protocol was then used for predicting the binding mode and
binding interactions of the newly synthesized ligand 11d at the active
site of VEGFR-2. In addition, delphinidin was also used as a reference
compound to compare its binding score and interactions to that of 11d.
Female athymic pathogen-free nude mice (nu/nu, 4–6 weeks old)
were used for this experiment and MCF-7 human breast cancer xeno-
graft tumor cancer model was established according to the reported
methods [33,34].
Animals bearing human cancer xenografts were randomly divided
into treatment and control groups (7–10 mice/ group). The untreated
control group received the vehicle only. The test compound was dis-
solved in the vehicle, PEG400:ethanol:saline (57.1:14.3:28.6, v/v/v)
and administrated by intraperitoneal injection at doses of 5 or 10 mg/
Kg/d, 3 d/wk for 3 weeks. At the end of the experiments, xenograft
tumors were removed and homogenized, the resultant supernatants
were used for Western blotting analysis of several proteins.31
Effect of 11d on apoptosis and cell cycle progression was de-
termined. To assess apoptosis the kit from BioVision (Mountain View,
CA) was used where 2–3 × 105 cells per well were exposed to the test
compound (0, 0.1, 0.5 and 0.75 nM) and incubated for 24 h prior to
analysis. Cells in early stages (positive for Annexin V-FITC alone) and
late stages (positive for Annexin V-FITC and PI) of apoptosis were de-
tected [31,35].
4.4. In-silico physicochemical parameters and pharmacokinetics properties
To determine the effect of 11d on cell cycle distribution, 2–3 × 105
cells per well were exposed to the compound (0, 0.1, 0.5 and 0.75 nM)
and incubated for 24 h prior to analysis. Cells were then trypsinized,
washed with PBS and fixed in 1.5 mL of 95% ethanol at 4 °C overnight,
followed by incubation with RNAse and staining with PI. The DNA
content was determined by flow cytometry [33].
Computational study for prediction of the physicochemical para-
meters and pharmacokinetics properties of the synthesized compounds
has been conducted. Molecular weight, number of hydrogen bond ac-
ceptor and donor atoms of Lipinski’s ‘rule of five’, drug likeness ac-
cording to Lipinski’s rule of 5, Lipinski’s rule number of violations, in
addition, number of rotatable bonds and topological polar surface area,
logP (o/w) were calculated using Molecular Operating Environment
(MOE, 2019.01) software. Finally, hERG, pIC50, logS at physiological
pH and affinity to P-glycoprotein were calculated using Optibrium
implemented in SeeSAR 6.0 of BiosolveIT suite.
4.2.1.2.2. Apoptotic markers analysis. In the in vitro studies, cells
were exposed to various concentrations of 11d for 24 h. In vivo tissue
homogenates were prepared in RIPA buffer (100 mg tumor tissue/1 mL
RIPA) for immunoblotting analysis as reported previously [31].
4.2.1.2.3. Kinase inhibition assay by western blot. To determine the
enzyme inhibition ability of the synthesized compounds against protein
kinase targets, profiling of all synthesized compounds in this study
against kinase panel was performed using a commercially available
microfluidic assay format. The % kinase activity inhibition was
determined for the tested compounds at single concentration of
30 nM. Microfluidic mobility shift assays to determine kinase
inhibition were carried out using ProfilerPro assay kits (Caliper
LifeSciences) according the manufacturer’s procedure [36,37].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
14

A. AbdelHaleem, et al.
BioorganicChemistry103(2020)104222
Acknowledgements
[18] K.S. Kim, L. Zhang, R. Schmidt, Z.W. Cai, D. Wei, D.K. Williams, et al., Discovery of
pyrrolopyridine-pyridone based inhibitors of Met kinase: Synthesis, x-ray crystal-
lographic analysis, and biological activities, J. Med. Chem. 51 (2008) 5330–5341.
[19] A.H. Abadi, D.A. Abouel-Ella, J. Lehmann, H.N. Tinsley, B.D. Gary, G.A. Piazza,
et al., Discovery of colon tumor cell growth inhibitory agents through a combina-
torial approach, Eur. J. Med. Chem. 45 (2010) 90–97.
The authors acknowledge BioSolveIT for offering a one year license
based on top 3 ranking in Spring 2017 scientific challenge.
[20] A.M. Serry, S. Luik, S. Laufer, A.H. Abadi, One-pot synthesis of 4,6-diaryl-2-oxo
(imino)-1,2-dihydropyridine-3-carbonitrile; a new scaffold for p38 α-MAP kinase
inhibition, J. Comb. Chem. 12 (2010) 559–565.
Appendix A. Supplementary material
[21] N.E. Abo-Elmagd, R.F. George, M.A. Ezzat, R.K. Arafa, New 1-phthalazinone
Scaffold based Compounds: Design, Synthesis, Cytotoxicity and Protein Kinase
Inhibition Activity, Mini Rev. Med. Chem. 18 (20) (2018) 1759–1774.
[22] N.A. El Sayed, A.A. Eissa, G.F. El Masry, M.M. Abdullah, R.K. Arafa, Discovery of
novel quinazolinones and their acyclic analogues as multi-kinase inhibitors: design,
synthesis, SAR analysis and biological evaluation, RSC Adv. 6 (113) (2016)
111767–111786.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104222.
References
[1] https://www.who.int/health-topics/cancer#tab=tab_1; last accessed March.
[2] https://www.cancer.gov/about-cancer/treatment/types/targeted-therapies/tar-
geted-therapies-fact-sheet; last accessed March.
[23] T.M. Eldebss, S.M. Gomha, M.M. Abdulla, R.K. Arafa, Novel pyrrole derivatives as
selective CHK1 inhibitors: design, regioselective synthesis and molecular modeling,
Med. Chem. Comm. 6 (5) (2015) 852–859.
[3] https://emedicine.medscape.com/article/1372666-overview; last accessed March.
[4] H. Chen, J. Kovar, S. Sissons, K. Cox, W. Matter, F. Chadwell, et al., A cell-based
immunocytochemical assay for monitoring kinase signaling pathways and drug
efficacy, Anal Biochem. 338 (2005) 136–142.
[24] F. Ragab, T. Yahya, M. El-Naa, R. Arafa, Design, synthesis and structure–activity
relationship of novel semi-synthetic flavonoids as antiproliferative agents, Eur. J.
Med. Chem. 82 (2014) 506–520.
[25] A.S. Mostafa, K.B. Selim, Synthesis and anticancer activity of new dihydropyr-
imidinone derivatives, Eur. J. Med. Chem. 156 (2018) 304–315.
[5] P. Traxler, P. Furet, Strategies toward the design of novel and selective protein
tyrosine kinase inhibitors, Pharmacol. Ther. 82 (1999) 195–206.
[26] A.H. Abadi, T.M. Ibrahim, K.M. Abouzid, J. Lehmann, H.N. Tinsley, B.D. Gary,
et al., Design, synthesis and biological evaluation of novel pyridine derivatives as
anticancer agents and phosphodiesterase 3 inhibitors, Bioorg. Med. Chem. 17
(2009) 5974–5982.
[6] J.J. Cui, M. Tran-Dub, H. Shen, M. Nambu, P.P. Kung, M. Pairish, et al., Structure
based drug design of Crizotinib (PF-02341066), a potent and selective dual in-
hibitor of Mesenchymal Epithelial Transition Factor (c-MET) kinase and Anaplastic
Lymphoma Kinase (ALK), J. Med. Chem. 54 (2011) 6342–6363.
[27] S.F. Mohamed, M.M. Youssed, A.E. Amr, E.R. Kotb, Antimicrobial activities of some
synthesized pyridines, oxazines and thiazoles from 3-aryl-1-(2-naphthyl)prop-2-en-
1-ones, Sci. Pharm. 76 (2008) 279–303.
[7] N. Ferrara, VEGF as a Therapeutic Target in Cancer, Oncology. 69 (2005) 11–16.
[8] S. Matsumoto, N. Miyamoto, T. Hirayama, H. Oki, K. Okada, M. Tawada, et al.,
Structure-based design, synthesis, and evaluation of imidazo[1,2-b]pyridazine and
imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase in-
hibitors, Bioorg. Med. Chem. Lett. 21 (2013) 7686–7698.
[28] E.R. Kotb, M.A. El-Hashash, M.A. Salama, H.S. Kalf, N.A.M. Abdel Wahed, Synthesis
and reactions of some novel nicotinonitrile derivatives for anticancer and anti-
microbial evaluation, Acta Chim. Slov. 56 (2009) 908–919.
[9] T. Usui, H.S. Ban, J. Kawada, T. Hirokawa, H. Nakamura, Discovery of in-
denopyrazoles as EGFR and VEGFR-2 tyrosine kinase inhibitors by in silico high-
throughput screening, Bioorg. Med. Chem. Lett. 18 (2008) 285–288.
[10] K.H. Lee, B.R. Huang, Synthesis and cytotoxic evaluation of α-methylene- γ-bu-
tyrolactone bearing naphthalene and naphtha[2,1-b]furan derivatives, Eur. J. Med.
Chem. 37 (2002) 333–338.
[29] H.A.A. Regaila, N. Latif, I.H. Ibrahim, Reactions of 2-naphthyl chalcones with active
methylene compounds and arylhydrazines, J. Chem. Soc. Pak. 9 (1987) 565–573.
[30] W. Mao, C. Guo, W. Wang, Luan C-j, Du R-j. 2-Amino-6-(naphthalen-1-yl)-4-phe-
nylpyridine-3-carbonitrile, Acta Crystallogr. Sect. E: Struct. Reports Online. 67 (4)
(2011) o853-o.
[31] E.R. Kotb, M.M. Anwar, H.S. Abbas, S.I. Abd El-Moez, A concise synthesis and
antimicrobial activity of a novel series of naphthylpyridine-3-carbonitrile com-
pounds, Acta Pol. Pharm. 70 (2013) 667–679.
[11] S. Cai, H. Deng, Y. Chen, X. Wu, X. Guan, Treatment of medullary thyroid carci-
noma with apatinib: A case report and literature review, Medicine (Baltimore). 96
(50) (2017) e8704.
[32] J. Wang, J. Zhu, J. Zhong, J. Sun, S. Tang, C. Guo, Novel 4-aryl-2-amino-6-(naph-
thalene-1-yl)-3-cyanopyridine derivates as potential organic fluorescent materials
prepared under microwave irradiation via three-component domino reactions,
Synth. Comm. 44 (2014) 2205–2214.
[12] J.N. Ho, S.S. Byun, S.E. Lee, J.I. Youn, S. Lee, Multikinase inhibitor motesanib
enhances the antitumor effect of cisplatin in cisplatin–resistant human bladder
cancer cells via apoptosis and the PI3K/Akt pathway, Oncol. Rep. 41 (4) (2019)
2482–2490.
[33] H. Wang, D. Yu, S. Agrawal, R. Zhang, Experimental therapy of human prostate
cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oli-
gonucleotides: in vitro and in vivo activities and mechanisms, Prostate 54 (2003)
194–205.
[13] S. Jiang, C.C. Grundy, J. Drewe, B. Tseng, S.X. Cai, Discovery of (naphthalene-4-yl)
(phenyl)methanones and N-methyl-N-phenylnaphthalen-1-amines as new apoptosis
inducers using a cell- and caspase-based HTS assay, Bioorg. Med. Chem. Lett. 18
(2008) 5725–5728.
[34] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63.
[35] S. Kondo, Y. Kondo, G. Li, R.H. Silverman, J.K. Cowell, Targeted therapy of human
malignant glioma in a mouse model by 2–5A antisense directed against telomerase
RNA, Oncogene 16 (1998) 3323–3330.
[14] G. Xu, Y. Zhou, S. Zhang, S. Ma, F. Xu, H. Xu, et al., Synthesis and biological
evaluation of N-(6,7-dimethoxynaphthalen-yl)sulfamide derivatives as novel in-
hibitors of angiogenesis and tumor growth, Med. Chem. 4 (2014) 598–605.
[15] T. Kaku, T. Hitaka, A. Ojida, N. Matsunaga, M. Adachi, T. Tanaka, et al., Discovery
of Orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective
17, 20-lyase inhibitor with potential utility in the treatment of prostate cancer,
Bioorg. Med. Chem. Lett. 19 (2011) 6383–6399.
[36] H. Wang, L. Nan, D. Yu, S. Agrawal, R. Zhang, Antisense anti-MDM2 oligonucleo-
tides as a novel therapeutic approach to human breast cancer: in vitro and in vivo
activities and mechanisms, Clin. Cancer Res. 7 (2001) 3613–3624.
[37] F. Wang, S.J. Ezell, Y. Zhang, W. Wang, E.R. Rayburn, D.H. Nadkarni, et al., FBA-
TPQ, a novel marine-derived compound as experimental therapy for prostate
cancer, Invest. New Drugs. 28 (2010) 234–241.
[16] F. Xu, Y. Jia, Q. Wang, L. Zhang, Y. Zhang, K. Yang, et al., Synthesis and biological
evaluation of N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides as inhibitors of an-
giogenesis and tumor growth, Eur. J. Med. Chem. 64 (2013) 337–388.
[17] F. Xu, H. Xu, X. Wang, L. Zhang, Q. Wen, Y. Zhang, et al., Discovery of N-(3-((7H-
purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamide derivatives as novel pro-
tein kinase and angiogenesis inhibitors for the treatment of cancer: synthesis and
biological evaluation. Part III, Bioorg. Med. Chem. 22 (2014) 1487–1495.
[38] D. Perrin, C. Frémaux, A. Scheer, Assay development and screening of a serine/
threonine kinase in an on-chip mode using Caliper nanofluidics technology, J.
Biomol. Screen 11 (2006) 359–368.
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