Novel nicotinonitrile-coumarin hybrids as potential acetylcholinesterase inhibitors: design, synthesis, in vitro and in silico studies

Article abstract of DOI:10.1007/s13738-020-02018-6

Abstract: Alzheimer’s disease is a degenerative brain condition that is the leading cause of dementia affecting millions of people around the world. Therapeutic development has focused on the problem of the loss of basal forebrain cholinergic function, as it is the only evidence responsible for brain neurodegeneration in patients with Alzheimer’s disease. Several attempts to improve cholinergic neurotransmission have been investigated by minimizing synaptic degradation of acetylcholine using acetylcholinesterase inhibitors. In the current study, we explore the designing of a new series of nicotinonitrile-coumarin hybrids as potential acetylcholinesterase inhibitors. The new hybrids were prepared utilizing pyridine-2(1H)-thiones as starting precursors. The in vitro acetylcholinesterase (AChE) inhibitory activities were examined for the new nicotinonitrile-coumarin hybrid molecules, when compared with donepezil as a standard drug with IC₅₀ of 14?nM. Coumarin derivative, linked to 6-(4-nitrophenyl)-4-phenylnicotinonitrile, showed more effective inhibitory activity than the reference donepezil with IC₅₀ of 13?nM. The free radical-scavenging capabilities against DPPH of the new hybrid derivatives were screened. Additionally, their in vitro cytotoxic activities have been tested against various eukaryotic cells. Furthermore, docking study showed excellent interaction between nicotinonitrile-coumarin hybrids and AChE. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13738-020-02018-6

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02018-6
ORIGINAL PAPER
Novel nicotinonitrile‑coumarin hybrids as potential
acetylcholinesterase inhibitors: design, synthesis, in vitro and in silico
studies
Sherif M. H. Sanad1 · Ahmed E. M. Mekky1
Received: 19 May 2020 / Accepted: 22 July 2020
© Iranian Chemical Society 2020
Abstract
Alzheimer’s disease is a degenerative brain condition that is the leading cause of dementia afecting millions of people
around the world. Therapeutic development has focused on the problem of the loss of basal forebrain cholinergic function,
as it is the only evidence responsible for brain neurodegeneration in patients with Alzheimer’s disease. Several attempts to
improve cholinergic neurotransmission have been investigated by minimizing synaptic degradation of acetylcholine using
acetylcholinesterase inhibitors. In the current study, we explore the designing of a new series of nicotinonitrile-coumarin
hybrids as potential acetylcholinesterase inhibitors. The new hybrids were prepared utilizing pyridine-2(1H)-thiones as
starting precursors. The in vitro acetylcholinesterase (AChE) inhibitory activities were examined for the new nicotinonitrile-
coumarin hybrid molecules, when compared with donepezil as a standard drug with IC50 of 14 nM. Coumarin derivative,
linked to 6-(4-nitrophenyl)-4-phenylnicotinonitrile, showed more efective inhibitory activity than the reference donepezil
with IC50 of 13 nM. The free radical-scavenging capabilities against DPPH of the new hybrid derivatives were screened.
Additionally, their in vitro cytotoxic activities have been tested against various eukaryotic cells. Furthermore, docking study
showed excellent interaction between nicotinonitrile-coumarin hybrids and AChE.
Graphic abstract
Keywords Alzheimer’s disease · Nicotinonitrile-coumarin hybrids · Acetylcholinesterase inhibitors · In vitro cytotoxicity
screening · In silico study
Introduction
Alzheimer’s disease (AD) is a progressive and degen-
* Ahmed E. M. Mekky
erative brain condition that primarily affects older peo-
ple and induces memory loss and the ability to perform
normal activities. AD is the leading cause of dementia
affecting about 50 million people worldwide, plus about
ataher2211@yahoo.com
1
Chemistry Department, Faculty of Science, Cairo University,
Giza 12613, Egypt
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
10 million new patients per year. AD contributes 60–70%
of dementia cases [1–3].
Results and discussion
However, the true nature or cause of AD remains
unclear, and therefore, the development of an effective
anti-Alzheimer’s is one of the promising fields of cur-
rent research in medicinal chemistry [4]. Over the past
two decades, numerous studies of anatomy within the
hippocampus, which relates to learning and perception,
have revealed that neurotransmitter acetylcholine levels in
patients with AD decrease significantly [5–7]. Nowadays,
therapeutic development has focused on the problem of
loss of cholinergic function of the basal forebrain, since it
is the only evidence responsible for cognitive decline and
neurodegeneration in the brains of AD patients [8–10].
Many approaches have been explored to improve cho-
linergic neurotransmission such as enhanced acetylcho-
line level (ACh) synthesis or pre-synaptic release and
stimulation of postsynaptic muscarinic and nicotinic
receptors, as well as to minimize ACh synaptic degra-
dation using acetylcholinesterase (AChE) inhibitors
[11–18]. AChE inhibitors approved for treatment with AD
are currently donepezil, tacrine, galantamine and rivastig-
mine (see Fig. 1) [19].
Chemistry
Recently, we reported the synthesis of new nicotinonitrile-
coumarin hybrid molecules as potent antibacterial and anti-
cancer agents [37]. Encouraged by these results, we report
herein the synthesis of new coumarin derivatives linked to
nicotinonitriles via thioethers as potential acetylcholinest-
erase inhibitors.
Our frst step toward our target was the preparation of
numerous nicotinonitriles. We are investigating the synthetic
potential of pyridine-2(1H)-thiones as efective examples
of nicotinonitriles in this study. Both benzaldehyde 1a and
anisaldehyde 1b were reacted with a series of 4-substituted
acetophenones 2a–2e in ethanolic potassium hydroxide solu-
tion under stirring at room temperature for 3 h to give the
corresponding α,β-unsaturated carbonyl compounds 3(4)
[38–42]. The starting precursors pyridine-2(1H)-thione
derivatives 6(7) were prepared by the cyclocondensation of
3(4) with 2-cyanothioacetamide 5 in ethanol in the pres-
ence of piperidine as a catalyst at refux for 5 h [43–45]
(Scheme 1).
Coumarins are natural heterocyclic derivatives used as
essential components in the inhibition and cure of numer-
ous diseases. These derivatives act as antimicrobials, [20]
anti-inflammatory, [21] anticancer, [22] anti-Alzheimer’s
disease [23, 24] and anti-Parkinson agents [25]. Examples
of chromene derivatives considered to be effective oral
anticoagulants include warfarin and dicoumarol [26]. In
addition, coumarin moiety has been included in various
effective AChE inhibitors [27–30].
Next, a new series of 2-hydroxybenzaldehydes 9(10),
bearing nicotinonitrile moieties, was prepared in excel-
lent yields. Thus, each of pyridine-2(1H)-thiones 6(7) was
stirred with an equivalent amount of potassium hydroxide in
ethanol at room temperature for 10 min. Then, an equivalent
amount of 5-(chloromethyl)-2-hydroxybenzaldehyde 8 [46]
was added with continuous stirring for 2 h to aford the cor-
responding 2-hydroxybenzaldehydes 9(10) in 88–94% yields
(Scheme 2). The structures of the isolated products 9(10)
were confrmed via their elemental analyses and spectral
data. The IR spectrum of 10a, as a representative example,
revealed the presence of OH, CN and CO groups at 3437,
2233 and 1675 cm−1, respectively. Its mass spectrum gave
In connection with our efforts to prepare heterocyclic
derivatives of potent biological activities [31–36], we
report herein the synthesis of novel series of nicotinoni-
trile-coumarin hybrid molecules, bearing arene moieties
and linked via thioethers. The new compounds were sub-
jected to in vitro as well as in silico study to shade the
light on their capability as potential acetylcholinesterase
inhibitors.
1
a molecular ion peak at m/z = 452. Its H-NMR spectrum
revealed fve singlet signals at δ 3.85, 4.66, 7.87, 10.20 and
10.68 attributed to OCH3, SCH2, pyridine-H5, CHO and
OH protons, respectively. In addition, it showed 12 aromatic
protons in the range of 6.95–8.27. Its 13C-NMR spectrum
Fig. 1 Structure of AChE inhibitors approved for treatment of AD
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Journal of the Iranian Chemical Society
showed the presence of fve signals at δ 33.4, 55.9, 115.8
and 191.6 assigned to SCH2, OCH3, CN and C=O carbons,
respectively, in addition to 19 signals corresponding to pyri-
dine and aromatic carbons (see experimental section).
Subsequently, Knoevenagel synthesis of novel coumarins
12(13) was carried out using 2-hydroxybenzaldehydes 9(10)
and ethyl acetoacetate 11 in ethanol in the presence of piper-
azine citrate (1:1, 10 mol%) at 80 °C for 3 h to give the cor-
responding 2-(((3-acetyl-2-oxo-2H-chromen-6-yl)methyl)
thio)nicotinonitriles 12(13) in 84–90% yields (Scheme 3)
[37]. The structures of 12(13) were confrmed via their ele-
mental analyses and spectral data. The IR spectrum of 12d,
Scheme 1 Synthesis of
pyridine-2(1H)-thione deriva-
tives 6(7)
Scheme 2 Synthesis of
2-hydroxybenzaldehydes 9(10)
Scheme 3 Synthesis of nico-
tinonitrile-coumarin hybrids
12(13)
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Journal of the Iranian Chemical Society
as a representative example, showed the presence of CN and
two CO groups at 2233, 1720, 1676 cm−1, respectively. Its
mass spectrum gave a molecular ion peak at m/z=518. The
1H-NMR spectrum of 12d revealed four singlet signals at δ
2.55, 3.85, 4.76 and 7.87 assigned to COCH3, OCH3, SCH2
and pyridine-H5 protons, respectively. Moreover, it reveals
two doublet signals at δ 7.40 and 7.84 and two singlet sig-
nals at δ 8.04 and 8.50 corresponding to four chromene-H8,
H7, H5 and H4 protons, respectively. Its 13C-NMR spectrum
revealed the presence of fve signals at δ 30.4, 33.4, 55.3,
116.8 and 195.1 corresponding to CH3, SCH2, OCH3, CN
and C=O carbons, respectively, in addition to 22 signals
corresponding to pyridine, aromatic and chromene carbons
(see experimental section).
6-aryl moiety, respectively, exhibited the least potent activi-
ties with IC50 of 1162 and 1544 nM, respectively.
Regarding the series 13a–13e, bearing 4-(4-methoxy-
phenyl) group at nicotinonitrile moiety, this series exhibited
generally less efective inhibitory activities toward AChE
enzyme. Furthermore, the inhibitory activities of the hybrid
derivatives examined were observed in the same order as in
the series 12a–12e. Thus, compounds 13b and 13e, with p-
Cl and p-NO2 linked to 6-aryl moiety, respectively, exhibited
the best AChE inhibitory activities with IC50 of 324 and
226 nM, respectively. Other hybrid derivatives 13a, 13c and
13d, with p–H, p-Me and p-OMe linked to 6-aryl group,
respectively, exhibited the least activities with IC50 values
in the range of 1762 and 2338 nM.
In order to elucidate the relationship between electronic
properties of the target nicotinonitrile-coumarin hybrids and
their AChE inhibitory activities, we compare the values of
electronic substituent constant (σp) [49] with the IC50 val-
ues (Table 2). After examining Table 2, a clear relation is
observed between the values of σp and IC50 values. In each
series 12 or 13, we observed that the AChE inhibitory activ-
ity increased as the substituent, linked to 6-aryl moiety at
nicotinonitrile, became more electron withdrawal.
Biology
The in vitro AChE inhibitory activity
The in vitro AChE inhibitory activities were estimated for
the new nicotinonitrile-coumarin hybrid molecules 12a–12e
as well as 13a–13e using Ellman method [47]. The inhibi-
tory activity fndings are expressed as the concentration of
the tested derivative 12(13) needed to inhibit 50% of AChE
enzyme (IC50, nM). Donepezil was used as a standard drug
with IC50 of 14 nM [48]. The fndings are listed in Table 1.
Among the series 12a–12e, bearing 4-phenyl group at
nicotinonitrile moiety, compound 12e, with p-NO2 linked to
6-aryl moiety, exhibited the most efective AChE inhibitory
activity. Thus, 12e showed more potent inhibitory activity
than the reference donepezil with IC50 of 13 nM. Compound
12b, linked to 6-(4-chlorophenyl) group, was the second in
inhibitory strength toward AChE enzyme. Compound 12b
showed IC50 of 25 nM when compared with donepezil. Other
hybrid derivatives 12a, 12c and 12d exhibited decreased
activities toward AChE. Therefore, compound 12a, linked to
6-phenyl group, showed IC50 of 767 nM. On the other hand,
compounds 12c and 12d, with p-Me and p-OMe linked to
DPPH antioxidant activity
The study of the antioxidant activities of the new hybrid
compounds is considered to add value for the diagnosis of
Alzheimer’s disease. Hence, the free radical-scavenging
capabilities of the new hybrid derivatives were examined
against DPPH (1,1-diphenyl-1-picrylhydrazyl). Ascorbic
acid was used as a reference drug, and the fndings are
shown in Table 3. Compounds 12b and 12e exhibited the
most efective free radical quenching results in DPPH assay
with IC50 of 35.4 and 28.5 μM, respectively, when compared
with ascorbic acid (IC50 of 20.4 μM). Other tested deriva-
tives displayed decreased quenching abilities in DPPH assay
with IC50 more than 45 μM.
Table 1 The AChE inhibitory
Cpd
12a 12b 12c
12d
12e
13a
OMe OMe OMe OMe OMe
Cl Me OMe NO2
2218 2338 226
13b
13c
13d
13e
Donepezil
activities of new nicotinonitrile-
coumarin hybrids as expressed
in IC50 values (nM)
X
Y
H
H
H
Cl
H
Me
H
H
–
–
14
OMe NO2
H
IC50 (nM) 767 25
1162 1544
13
1762 324
Table 2 The relation between
electronic substituent constants
σp and IC50 values
Cpd
12a 12b
Cl
767 25
12c
12d
12e
13a
13b
13c
13d
13e
Y
H
Me
1162
OMe
1544
NO2
13
H
Cl
Me
2218
OMe
2338
NO2
226
IC50 (nM)
σp (Y substituents) 0.0
1762 324
0.0
0.23 −0.17 −0.27 0.78
0.23 −0.17 −0.27 0.78
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Journal of the Iranian Chemical Society
Table 3 DPPH scavenging activity of the synthesized compounds
Cpd
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
Ascorbic acid
IC50 in μM
(in μg/mL)
> 45
35.4
> 45
> 45
28.5
> 45
> 45
> 45
> 45
> 45
20.4
(3.6)
(>25)
(18.5)
(>25)
(>25)
(15.2)
(>25)
(>25)
(>25)
(>25)
(>25)
Table 4 The cell inhibition percentage and IC50 values of some new
hybrid derivatives against each of MCF-10A, MCF-7, Caco2 and
HEPG2, and using Doxorubicin as a reference
The in silico study: molecular modeling
AChE enzyme is expressed in cholinergic neurons and
neuromuscular junctions. Its main function is the fast
breaking down of the neurotransmitter acetylcholine that
is released during cholinergic neurotransmission [50].
Molecular docking was performed to test the capability
of some new nicotinonitrile-coumarin hybrids as poten-
tial AChE inhibitors. Thus, compounds 12b and 12e were
docked as ligand molecules with human acetylcholinest-
erase (PDB ID: 4EY7) as the target protein to achieve
their optimum conformation, with reduced free energy [51,
52]. Computational docking studies have been used to esti-
mate the binding energies of the interactions between each
ligand and the amino acid residues in the target enzyme
[53]. The docking fndings of 12b and 12e are shown in
Table 5.
Compound
IC50 (µM)
MCF-10A MCF-7
Caco2
HEPG2
12b
11.6 0.2 10.2 0.3 15.9 0.3 10.7 0.3
10.5 0.3 8.6 0.2 7.5 0.2 6.2 0.2
16.8 0.4 18.1 0.3 19.6 0.4 18.4 0.4
14.6 0.3 15.4 0.3 17.8 0.3 14.3 0.3
12.7 0.3 12.2 0.2 14.5 0.3 13.7 0.3
12e
13b
13e
Doxorubicin
Cytotoxicity against eukaryotic cells
New hybrid derivatives 12b, 12e, 13b and 13e have been
reported for in vitro cytotoxic activities against each human
breast epithelial cell line (MCF-10A), human breast carci-
noma cell line (MCF-7), colon cancer cell line (Caco2) and
liver hepatocellular carcinoma cell line (HEPG2) (Table 4).
The results of cytotoxicity are expressed as the concentration
of the tested derivatives needed to inhibit 50% of cell growth
(IC50, μM). Doxorubicin was used as a standard drug with
IC50 of 12.2 0.2, 12.7 0.3, 13.7 0.3 and 14.5 0.3 µM
against each of MCF-7, MCF-10A, HEPG2 and Caco2,
respectively (see Table 4).
Both experimental and docking results demonstrated
capability of coumarin 12e as a potent AChE inhibitor.
Compound 12e demonstrated strong AChE binding inter-
actions (PDB ID: 4EY7) as shown in Fig. 2. The figure
revealed a strong network of hydrogen bonding interac-
tions of cyano-N, acetyl-O and thioether-S atoms with
aminoacids residues of ASN 233 (2.81 Å, −5.8 kcal/mol),
ARG 247 (3.05 Å, − 4.5 kcal/mol) and GLU 313 (3.46 Å,
− 2.4 kcal/mol), respectively. In addition, nitro group
linked to 6-arylnicotinonitrile moiety showed strong
hydrogen bonding interactions with aminoacids resi-
dues of both ARG 296 (2.76 Å, − 4.3 kcal/mol; 2.90 Å,
− 3.2 kcal/mol) and GLN 369 (3.00 Å, − 2.7 kcal/mol). It
showed also strong π–H interactions between coumarin
moieties with residue of VAL 239 (2.93 Å, − 4.0 kcal/
mol).
Derivatives 12b and 12e showed more powerful cytotoxic
efects against each of the MCF-7, MCF-10A and HEPG2
cell lines than the reference Doxorubicin. Therefore, 12b and
12e showed IC50 of 6.2–11.6 μM against these cell lines. On
the other hand, compounds 13b and 13e exhibited decreased
cytotoxic activity with IC50 of 14.3–18.4 μM relative to Dox-
orubicin against the above cell lines (Table 4).
With regard to the cytotoxicity against Caco2 cells, com-
pound 12e exhibited more efective cytotoxic activity than
the reference Doxorubicin with IC50 of 7.5 0.2 µM. In
addition, derivatives 12b, 13b and 13e exhibited decreased
activities with IC50 of 15.9–19.6 µM, when compared with
Doxorubicin (Table 4).
Docking results of coumarin 12b are shown in Fig. 3.
The fgure revealed a good network of hydrogen bond-
ing interactions of cyano-N and coumarin-O atoms with
aminoacids residues of VAL 239 (2.85 Å, − 5.7 kcal/mol)
and ASN 233 (3.15 Å, − 4.2 kcal/mol; 3.27 Å, − 3.9 kcal/
mol), respectively. In addition, chlorine atom linked to
6-arylnicotinonitrile moiety showed strong hydrogen
bonding interactions with aminoacids residues of both
PRO 537 (2.82 Å, − 3.6 kcal/mol) and SER 541 (2.96 Å,
− 3.3 kcal/mol). It showed also strong π–H interactions
between coumarin moieties with residue of GLU 313
(3.06 Å, − 3.7 kcal/mol).
It is worth noting that cytotoxicity of the new hybrids
12b, 12e, 13b and 13e showed IC50 in micromolar range
(IC50 of 6.2–19.6 µM) against all the cell lines tested, while
these hybrids showed efective AChE inhibitory activities
in nanomolar range (IC50 of 13–324 nM). These fndings
clearly showed that the new hybrids are not cytotoxic at the
concentration needed to efectively inhibit AChE.
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Journal of the Iranian Chemical Society
Table 5 Energy values obtained
in docking calculations of
new nicotinonitrile-coumarin
hybrids 12b and 12e with AChE
(PDB ID: 4EY7)
Compound
Ligand moiety
Site
Interaction
Distance (Å)
E (kcal/mol)
12b
CL 54
CL 54
O 47
O 47
N 56
6-ring
S 8
O 49
N 55
O 57
O 57
O 58
6-ring
O PRO 537 (A)
H-donor
H-donor
H-acceptor
H-acceptor
H-acceptor
Pi-H
H-donor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
Pi-H
2.82
2.96
3.15
3.27
2.85
3.06
3.46
3.05
2.81
2.76
2.90
3.00
2.93
−3.6
−3.3
−4.2
−3.9
−5.7
−3.7
−2.4
−4.5
−5.8
−4.3
−3.2
−2.7
−4.0
OG SER 541 (A)
CB ASN 233 (A)
ND2 ASN 233 (A)
CA VAL 239 (A)
CB GLU 313 (A)
OE1 GLU 313 (A)
NH2 ARG 247 (A)
CB ASN 233 (A)
NE ARG 296 (A)
NH2 ARG 296 (A)
CB GLN 369 (A)
CA VAL 239 (A)
12e
Fig. 2 The 2D and 3D ligand
interactions of 12e with AChE
(PDB ID: 4EY7)
Fig. 3 The 2D and 3D ligand
interactions of 12b with AChE
(PDB ID: 4EY7)
to 6-(4-nitrophenyl)-4-phenylnicotinonitrile showed more
efective inhibitory activity than the reference donepezil.
Some of the new hybrids showed good free radical-scav-
enging capabilities against DPPH as well as cytotoxic
activities against various eukaryotic cells. Docking study
predicted the capability of new coumarins as potential
AChE inhibitors.
Conclusion
The 2-hydroxybenzaldehydes, bearing nicotinonitrile
moieties, were prepared in excellent yields and used as
starting precursors for the synthesis of a new series of
nicotinonitrile-coumarin hybrid derivatives. The in vitro
acetylcholinesterase inhibitory activities were examined
for the new hybrid derivatives. Coumarin derivative linked
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Journal of the Iranian Chemical Society
Experimental
6‑(4‑Chlorophenyl)‑2‑((3‑formyl‑4‑hydroxybenzyl)
thio)‑4‑phenylnicotinonitrile (9b) Colorless solid (diox-
ane/ethanol, 91%); m.p. 120–122 °C; IR (υ cm−1): 3425
(OH), 2230 (CN), 1677 (CO); 1H-NMR (DMSO-d6): δ 4.64
(s, 2H, SCH2), 6.96 (d, 1H, Ar–H), 7.50 (d, 2H, Ar–H’s),
7.56 (t, 1H, Ar–H), 7.60 (t, 2H, Ar–H’s), 7.66 (d, 1H, Ar–H),
7.78 (s, 1H, Ar–H), 7.81 (s, 1H, pyridine-H5), 7.95 (d, 2H,
Ar–H’s), 8.12 (d, 2H, Ar–H’s), 10.22 (s, 1H, CHO), 10.65
(s, 1H, OH); 13C-NMR (DMSO-d6): δ 33.4 (SCH2), 103.5,
115.8, 116.4, 117.9, 122.6, 127.8, 128.1, 128.2, 128.9,
129.0, 129.4, 130.6, 135.0, 135.7, 136.2, 137.4, 153.7,
158.7, 161.5, 162.4, 191.9 (CO); Anal. for C26H17ClN2O2S
(456.9): C, 68.34; H, 3.75; N, 6.13; found: C, 68.69; H, 3.51;
N, 6.10%.
Introduction
All solvents were acquired from commercial sources and
used as received unless otherwise stated. Piperazine citrate
(1:1) was acquired from Boc Sciences (CAS 14396-16-8).
All other chemicals were acquired from Merck or Aldrich.
These chemicals were used without further purifcation.
The melting points were measured on a Stuart melting point
apparatus and are uncorrected. IR spectra were recorded on
a Smart iTR, which is an ultra-high-performance, versatile
attenuated total refectance (ATR) sampling accessory on
the Nicolet iS10 FT-IR spectrometer manufactured. NMR
spectra were recorded on Bruker Avance III 400 MHz spec-
trophotometer (400 MHz for 1H and 100 MHz for 13C) using
TMS as an internal standard and DMSO-d6 as solvent, and
chemical shifts were expressed as δ ppm units. Mass spectra
were recorded on a GC–MS-QP1000EX spectrometer using
inlet type at 70 eV. Elemental analyses were carried out on
a EuroVector instrument C, H, N analyzer EA3000 Series.
2‑((3‑Formyl‑4‑hydrox yb enz yl)thio)‑4‑phe ‑
nyl‑6‑(p‑tolyl)nicotinonitrile (9c) Colorless solid (diox-
ane/ethanol, 88%); m.p. 120–121 °C; IR (υ cm−1): 3432
(OH), 2231 (CN), 1675 (CO); δ 2.38 (s, 3H, p-CH3), 4.64
(s, 2H, SCH2), 6.96 (d, 1H, Ar–H), 7.33 (d, 2H, Ar–H’s),
7.54 (t, 1H, Ar–H), 7.59 (t, 2H, Ar–H’s), 7.64 (d, 1H, Ar–H),
7.79 (s, 1H, Ar–H), 7.82 (s, 1H, pyridine-H5), 7.96 (d, 2H,
Ar–H’s), 8.14 (d, 2H, Ar–H’s), 10.21 (s, 1H, CHO), 10.65
(s, 1H, OH); 13C-NMR (DMSO-d6): δ 21.5 (p-CH3), 33.6
(SCH2), 103.3, 116.2, 116.4, 117.8, 122.4, 127.7, 127.8,
128.1, 128.8, 129.1, 129.4, 130.7, 134.6, 136.0, 137.2,
141.3, 154.8, 158.1, 161.3, 162.1, 191.7 (CO); Anal. for
C27H20N2O2S (436.5): C, 74.55; H, 4.69; N, 6.27; found: C,
74.29; H, 4.62; N, 6.42%.
General procedures and spectral data
General procedure for the synthesis of 2‑((3‑for‑
myl‑4‑hydroxybenzyl)thio)nicotinonitrile deriva‑
tives 9(10) A mixture of pyridine-2(1H)-thiones 6(7)
(5 mmol) and potassium hydroxide (5 mmol) in ethanol
(15 mL) was stirred at room temperature for 10 min. Then,
5-(chloromethyl)-2-hydroxybenzaldehyde 8 was added and
the stirring was continued for 2 h. The product was collected
by fltration, washed with each of water and cold ethanol,
dried and recrystallized from the proper solvent.
2 ‑ ( ( 3 ‑ F o r m y l ‑ 4 ‑ h y d r o x y b e n z y l )
thio)‑6‑(4‑methoxyphenyl)‑4‑phenylnicotinonitrile
(9d) Colorless solid (dioxane/ethanol, 94%); m.p. 182–
184 °C; IR (υ cm−1): 3414 (OH), 2231 (CN), 1674 (CO);
1H-NMR (DMSO-d6): δ 3.83 (s, 3H, OCH3), 4.64 (s, 2H,
SCH2), 6.96 (d, 1H, Ar–H), 7.06 (d, 2H, Ar–H’s), 7.52
(t, 1H, Ar–H), 7.57 (t, 2H, Ar–H’s), 7.62 (d, 1H, Ar–H),
7.71 (s, 1H, Ar–H), 7.78 (s, 1H, pyridine-H5), 7.95 (d, 2H,
Ar–H’s), 8.07 (d, 2H, Ar–H’s), 10.22 (s, 1H, CHO), 10.63
(s, 1H, OH); 13C-NMR (DMSO-d6): δ 33.5 (SCH2), 55.3
(OCH3), 102.8, 114.2, 116.0, 116.7, 118.0, 122.4, 127.7,
128.1, 128.2, 128.8, 129.1, 129.4, 130.7, 136.0, 137.2,
154.8, 158.1, 158.2, 161.3, 162.1, 191.7 (CO); MS (m/z):
452 (M+, 63.6%); Anal. for C27H20N2O3S: C, 71.66; H, 4.45;
N, 6.19; found: C, 71.52; H, 4.29; N, 6.30%.
2‑((3‑Formyl‑4‑hydroxybenzyl)thio)‑4,6‑diphenylnico‑
tinonitrile (9a) Colorless solid (ethanol, 90%); m.p. 114–
115 °C; IR (υ cm−1): 3422 (OH), 2236 (CN), 1679 (CO);
1H-NMR (DMSO-d6): δ 4.63 (s, 2H, SCH2), 6.96 (d, 1H,
Ar–H), 7.46 (t, 1H, Ar–H), 7.50 (t, 2H, Ar–H’s), 7.54 (t,
1H, Ar–H), 7.59 (t, 2H, Ar–H’s), 7.65 (d, 1H, Ar–H), 7.73
(d, 2H, Ar–H’s), 7.80 (s, 1H, Ar–H), 7.83 (s, 1H, pyridine-
H5), 7.95 (d, 2H, Ar–H’s), 10.22 (s, 1H, CHO), 10.67 (s, 1H,
OH); 13C-NMR (DMSO-d6): δ 33.7 (SCH2), 103.9, 115.4,
116.2, 117.7, 122.5, 127.7, 128.0, 128.2, 128.5, 129.1,
129.5, 129.9, 130.5, 134.9, 136.3, 137.1, 152.8, 157.6,
161.2, 162.0, 191.8 (CO); Anal. for C26H18N2O2S (422.5):
C, 73.91; H, 4.29; N, 6.63; found: C, 73.74; H, 4.42; N,
6.50%.
2 ‑ ( ( 3 ‑ F o r m y l ‑ 4 ‑ h y d r o x y b e n z y l )
thio)‑6‑(4‑nitrophenyl)‑4‑phenylnicotinonitrile
(9e) Colorless solid (dioxane, 92%); m.p. 132–135 °C;
IR (υ cm−1): 3425 (OH), 2230 (CN), 1677 (CO); 1H-NMR
1 3

Journal of the Iranian Chemical Society
(DMSO-d6): δ 4.66 (s, 2H, SCH2), 6.94 (d, 1H, Ar–H), 7.53
(t, 1H, Ar–H), 7.59 (t, 2H, Ar–H’s), 7.64 (d, 1H, Ar–H),
7.80 (s, 1H, Ar–H), 7.84 (s, 1H, pyridine-H5), 7.96 (d, 2H,
Ar–H’s), 8.05 (d, 2H, Ar–H’s), 8.38 (d, 2H, Ar–H’s), 10.22
(s, 1H, CHO), 10.65 (s, 1H, OH); 13C-NMR (DMSO-d6):
δ 33.9 (SCH2), 104.2, 115.8, 116.5, 117.8, 122.4, 122.8,
124.4, 127.7, 128.2, 129.1, 129.5, 130.7, 136.3, 137.1,
138.5, 144.6, 153.5, 157.5, 161.0, 162.3, 191.5 (CO); Anal.
for C26H17N3O4S (467.5): C, 66.80; H, 3.67; N, 8.99; found:
C, 66.88; H, 3.86; N, 8.73%.
137.3, 141.1, 154.2, 158.3, 160.4, 161.3, 162.4, 191.5 (CO);
Anal. for C28H22N2O3S (466.5): C, 72.08; H, 4.75; N, 6.00;
found: C, 72.23; H, 4.61; N, 6.09%.
2‑((3‑Formyl‑4‑hydroxybenzyl)thio)‑4,6‑bis(4‑meth‑
oxyphenyl)nicotinonitrile (10d) Colorless solid (diox-
ane/ethanol, 90%); m.p. 120–122 °C; IR (υ cm−1): 3431
(OH), 2228 (CN), 1671 (CO); 1H-NMR (DMSO-d6): δ 3.83
(s, 6H, 2 OCH3), 4.63 (s, 2H, SCH2), 6.95 (d, 1H, Ar–H),
7.05 (d, 2H, Ar–H’s), 7.12 (d, 2H, Ar–H’s), 7.67 (d, 1H,
Ar–H), 7.75–7.79 (m, 4H, 3 Ar–H’s and pyridine-H5), 8.22
(d, 2H, Ar–H’s), 10.21 (s, 1H, CHO), 10.69 (s, 1H, OH);
13C-NMR (DMSO-d6): δ 32.8 (SCH2), 55.3 (2 OCH3),
101.2, 114.2, 114.3, 115.0, 116.0, 117.4, 122.0, 127.7,
128.3, 128.9, 129.0, 129.1, 130.1, 136.7, 153.5, 157.5,
159.8, 160.7, 161.4, 161.9, 191.9 (CO); MS (m/z): 482 (M+,
70.4%); Anal. for C28H22N2O4S: C, 69.69; H, 4.60; N, 5.81;
found: C, 69.77; H, 4.59; N, 5.70%.
2 ‑ ( ( 3 ‑ F o r m y l ‑ 4 ‑ h y d r o x y b e n z y l )
thio)‑4‑(4‑methoxyphenyl)‑6‑phenylnicotinonitrile
(10a) Colorless solid (dioxane/ethanol, 88%); m.p. 194–
196 °C; IR (υ cm−1): 3437 (OH), 2233 (CN), 1675 (CO);
1H-NMR (DMSO-d6): δ 3.85 (s, 3H, OCH3), 4.66 (s, 2H,
SCH2), 6.95 (d, 1H, Ar–H), 7.13 (d, 2H, Ar–H’s), 7.53 (t,
1H, Ar–H), 7.57 (t, 2H, Ar–H’s), 7.63 (d, 1H, Ar–H), 7.73
(d, 2H, Ar–H’s), 7.78 (s, 1H, Ar–H), 7.81 (s, 1H, pyridine-
H5), 8.27 (d, 2H, Ar–H’s), 10.20 (s, 1H, CHO), 10.68 (s, 1H,
OH); 13C-NMR (DMSO-d6): δ 33.4 (SCH2), 55.9 (OCH3),
102.6, 114.7, 115.2, 115.8, 117.9, 122.5, 128.0, 128.5,
128.9, 129.0, 129.0, 130.5, 130.7, 135.1, 137.2, 153.4,
158.1, 160.3, 161.1, 161.8, 191.6 (CO); MS (m/z): 452 (M+,
74.6%); Anal. for C27H20N2O3S: C, 71.66; H, 4.45; N, 6.19;
found: C, 71.79; H, 4.31; N, 6.04%.
2‑((3‑Formyl‑4‑hydroxybenzyl)thio)‑4‑(4‑methoxyph
enyl)‑6‑(4‑nitrophenyl)nicotinonitrile (10e) Colorless
solid (dioxane, 92%); m.p. 128–130 °C; IR (υ cm−1): 3427
(OH), 2232 (CN), 1679 (CO); 1H-NMR (DMSO-d6): δ 3.84
(s, 3H, OCH3), 4.64 (s, 2H, SCH2), 6.95 (d, 1H, Ar–H), 7.08
(d, 2H, Ar–H’s), 7.62 (d, 1H, Ar–H), 7.78 (s, 1H, Ar–H),
7.80 (s, 1H, pyridine-H5), 8.05 (d, 2H, Ar–H’s), 8.25 (d, 2H,
Ar–H’s), 8.38 (d, 2H, Ar–H’s), 10.22 (s, 1H, CHO), 10.65
(s, 1H, OH); 13C-NMR (DMSO-d6): δ 33.8 (SCH2), 55.6
(OCH3), 104.4, 114.8, 115.6, 116.3, 117.9, 122.4, 122.9,
124.5, 128.8, 129.1, 130.5, 130.7, 137.1, 138.7, 144.7,
153.5, 157.5, 160.2, 161.0, 162.3, 191.5 (CO); Anal. for
C27H19N3O5S (497.5): C, 65.18; H, 3.85; N, 8.45; found: C,
65.18; H, 3.85; N, 8.45%.
6‑(4‑Chlorophenyl)‑2‑((3‑formyl‑4‑hydroxybenzyl)
thio)‑4‑(4‑methoxyphenyl)nicotinonitrile (10b) Color-
less solid (dioxane/ethanol, 94%); m.p. 130–132 °C; IR
1
(υ cm−1): 3422 (OH), 2233 (CN), 1676 (CO); H-NMR
(DMSO-d6): δ 3.83 (s, 3H, OCH3), 4.65 (s, 2H, SCH2),
6.95 (d, 1H, Ar–H), 7.10 (d, 2H, Ar–H’s), 7.52 (d, 2H, Ar–
H’s), 7.65 (d, 1H, Ar–H), 7.71 (d, 2H, Ar–H’s), 7.79 (s,
1H, Ar–H), 7.82 (s, 1H, pyridine-H5), 8.16 (d, 2H, Ar–H’s),
10.21 (s, 1H, CHO), 10.68 (s, 1H, OH); 13C-NMR (DMSO-
d6): δ 33.5 (SCH2), 55.9 (OCH3), 102.8, 114.6, 115.8, 116.4,
117.8, 122.6, 128.1, 128.9, 129.0, 129.1, 130.5, 130.7,
135.0, 135.7, 137.4, 153.7, 158.7, 160.3, 161.5, 162.4, 191.9
(CO); Anal. for C27H19ClN2O3S (486.9): C, 66.59; H, 3.93;
N, 5.75; found: C, 66.44; H, 3.87; N, 5.82%.
General procedure for the synthesis of 2‑(((3‑acetyl‑2‑o
xo‑2H‑chromen‑6‑yl)methyl)thio)nicotinonitrile deriv‑
atives 12(13) A mixture of 2-hydroxybenzaldehydes 9(10)
(5 mmol) and ethyl acetoacetate 11 (5 mmol) in ethanol
(15 mL) in the presence of piperazine citrate (10 mol%) was
heated at 80 °C for 3 h. The reaction mixture was cooled, fl-
trated, washed with each of water and cold ethanol, and the
reaction product was recrystallized from the proper solvent.
2‑((3‑Formyl‑4‑hydroxybenzyl)thio)‑4‑(4‑methoxyp
henyl)‑6‑(p‑tolyl)nicotinonitrile (10c) Colorless solid
(dioxane/ethanol, 93%); m.p. 100–102 °C; IR (υ cm−1): 3439
(OH), 2232 (CN), 1677 (CO); δ 2.38 (s, 3H, p-CH3), 3.84 (s,
3H, OCH3), 4.65 (s, 2H, SCH2), 6.95 (d, 1H, Ar–H), 7.13
(d, 2H, Ar–H’s), 7.34 (d, 2H, Ar–H’s), 7.62 (d, 1H, Ar–H),
7.71 (d, 2H, Ar–H’s), 7.80 (s, 1H, Ar–H), 7.82 (s, 1H, pyri-
dine-H5), 8.16 (d, 2H, Ar–H’s), 10.21 (s, 1H, CHO), 10.68
(s, 1H, OH); 13C-NMR (DMSO-d6): δ 21.4 (p-CH3), 33.4
(SCH2), 55.9 (OCH3), 102.4, 114.7, 116.1, 116.4, 118.0,
122.5, 127.9, 128.7, 128.9, 129.4, 130.5, 130.9, 134.4,
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑4,6‑diphenylnicotinonitrile (12a) Yellow solid
(dioxane, 84%); m.p. 230–233 °C; IR (υ cm−1): 2232 (CN),
1
1725, 1677 (2 CO); H-NMR (DMSO-d6): δ 2.55 (s, 3H,
COCH3), 4.77 (s, 2H, SCH2), 7.37 (d, 1H, chromene-
H8), 7.48 (t, 1H, Ar–H), 7.52 (t, 2H, Ar–H’s), 7.56 (t, 1H,
Ar–H), 7.60 (t, 2H, Ar–H’s), 7.80 (d, 1H, chromene-H7),
7.83 (s, 1H, pyridine-H5), 7.94 (d, 2H, Ar–H’s), 8.02 (s,
1H, chromene-H5), 8.24 (d, 2H, Ar–H’s), 8.50 (s, 1H,
1 3

Journal of the Iranian Chemical Society
chromene-H4); Anal. for C30H20N2O3S (488.5): C, 73.75;
H, 4.13; N, 5.73; found: C, 73.92; H, 4.24; N, 5.55%.
(DMSO-d6): δ 2.55 (s, 3H, COCH3), 4.76 (s, 2H, SCH2),
7.39 (d, 1H, chromene-H8), 7.53 (t, 1H, Ar–H), 7.58 (t, 2H,
Ar–H’s), 7.79 (d, 1H, chromene-H7), 7.82 (s, 1H, pyridine-
H5), 7.96 (d, 2H, Ar–H’s), 8.03 (s, 1H, chromene-H5),
8.22 (d, 2H, Ar–H’s), 8.41 (d, 2H, Ar–H’s), 8.52 (s, 1H,
chromene-H4); Anal. for C30H19N3O5S (533.5): C, 67.53; H,
3.59; N, 7.88; found: C, 67.41; H, 3.72; N, 7.64%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑6‑(4‑chlorophenyl)‑4‑phenylnicotinonitrile
(12b) Yellow solid (dioxane, 88%); m.p. 236–238 °C; IR
(υ cm−1): 2232 (CN), 1723, 1676 (2 CO); 1H-NMR (DMSO-
d6): δ 2.55 (s, 3H, COCH3), 4.69 (s, 2H, SCH2), 7.38 (d, 1H,
chromene-H8), 7.47 (d, 2H, Ar–H’s), 7.57 (t, 1H, Ar–H),
7.61 (t, 2H, Ar–H’s), 7.81 (d, 1H, chromene-H7), 7.84
(s, 1H, pyridine-H5), 7.94 (d, 2H, Ar–H’s), 8.01 (s, 1H,
chromene-H5), 8.10 (d, 2H, Ar–H’s), 8.52 (s, 1H, chromene-
H4); 13C-NMR (DMSO-d6): δ 30.6 (CH3), 33.5 (SCH2),
103.9, 116.1, 116.5, 116.9, 118.2, 125.2, 127.9, 128.2,
128.3, 129.0, 129.6, 130.5, 135.2, 135.3, 135.5, 135.8,
136.4, 146.9 153.9, 157.9, 158.7, 161.7, 162.3 (chromene-
CO), 195.2 (CO); Anal. for C30H19ClN2O3S (523.0): C,
68.90; H, 3.66; N, 5.36; found: C, 68.74; H, 3.85; N, 5.47%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑4‑(4‑methoxyphenyl)‑6‑phenylnicotinonitrile
(13a) Yellow solid (dioxane, 89%); m.p. 226–228 °C; IR
(υ cm−1): 2231 (CN), 1721, 1675 (2 CO); 1H-NMR (DMSO-
d6): δ 2.55 (s, 3H, COCH3), 3.84 (s, 3H, OCH3), 4.78 (s, 2H,
SCH2), 7.13 (d, 2H, Ar–H’s), 7.42 (d, 1H, chromene-H8),
7.53 (t, 1H, Ar–H), 7.57 (t, 2H, Ar–H’s), 7.73 (d, 2H, Ar–
H’s), 7.84 (d, 1H, chromene-H7), 7.87 (s, 1H, pyridine-H5),
8.05 (s, 1H, chromene-H5), 8.24 (d, 2H, Ar–H’s), 8.50 (s,
1H, chromene-H4); 13C-NMR (DMSO-d6): δ 30.5 (CH3),
33.5 (SCH2), 55.9 (OCH3), 102.4, 114.5, 115.3, 115.6,
116.3, 117.9, 125.1, 128.0, 128.5, 128.9, 129.7, 130.6,
130.8, 135.2, 135.3, 135.5, 146.8, 153.4, 158.1, 158.4,
160.2, 161.8, 162.2, 195.1 (CO); MS (m/z): 518 (M+,
53.0%); Anal. for C31H22N2O4S: C, 71.80; H, 4.28; N, 5.40;
found: C, 71.89; H, 4.51; N, 5.27%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑4‑phenyl‑6‑(p‑tolyl)nicotinonitrile (12c) Yellow
solid (dioxane, 86%); m.p. 216–219 °C; IR (υ cm−1): 2232
(CN), 1722, 1678 (2 CO); 1H-NMR (DMSO-d6): δ 2.39 (s,
3H, p-CH3), 2.56 (s, 3H, COCH3), 4.78 (s, 2H, SCH2), 7.34
(d, 2H, Ar–H’s), 7.40 (d, 1H, chromene-H8), 7.55 (t, 1H,
Ar–H), 7.59 (t, 2H, Ar–H’s), 7.82 (d, 1H, chromene-H7),
7.85 (s, 1H, pyridine-H5), 7.94 (d, 2H, Ar–H’s), 8.05 (s, 1H,
chromene-H5), 8.13 (d, 2H, Ar–H’s), 8.52 (s, 1H, chromene-
H4); Anal. for C31H22N2O3S (502.5): C, 74.08; H, 4.41; N,
5.57; found: C, 73.85; H, 4.57; N, 5.40%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)thio)‑6‑
(4‑chlorophenyl)‑4‑(4‑methoxyphenyl)nicotinonitrile
(13b) Yellow solid (dioxane, 87%); m.p. 244–246 °C; IR (υ
cm−1): 2232 (CN), 1722, 1675 (2 CO); 1H-NMR (DMSO-
d6): δ 2.57 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 4.69 (s, 2H,
SCH2), 7.10 (d, 2H, Ar–H’s), 7.41 (d, 1H, chromene-H8),
7.52 (d, 2H, Ar–H’s), 7.81 (d, 1H, chromene-H7), 7.84 (s,
1H, pyridine-H5), 8.05 (s, 1H, chromene-H5), 8.13 (d, 2H,
Ar–H’s), 8.24 (d, 2H, Ar–H’s), 8.52 (s, 1H, chromene-H4);
13C-NMR (DMSO-d6): δ 30.4 (CH3), 33.5 (SCH2), 55.9
(OCH3), 103.2, 114.7, 116.3, 116.5, 117.1, 118.5, 125.0,
128.1, 128.8, 129.2, 130.5, 130.7, 135.1, 135.5, 135.6, 135.8,
147.1, 154.0, 158.0, 158.9, 160.3, 161.6, 162.6 (chromene-
CO), 195.2 (CO); Anal. for C31H21ClN2O4S (553.0): C,
67.33; H, 3.83; N, 5.07; found: C, 67.09; H, 3.96; N, 5.23%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑6‑(4‑methoxyphenyl)‑4‑phenylnicotinonitrile
(12d) Yellow solid (dioxane/ethanol, 84%); m.p. 220–
1
222 °C; IR (υ cm−1): 2233 (CN), 1720, 1676 (2 CO); H-
NMR (DMSO-d6): δ 2.55 (s, 3H, COCH3), 3.85 (s, 3H,
OCH3), 4.76 (s, 2H, SCH2), 7.08 (d, 2H, Ar–H’s), 7.40 (d,
1H, chromene-H8), 7.52 (t, 1H, Ar–H), 7.57 (t, 2H, Ar–H’s),
7.84 (d, 1H, chromene-H7), 7.87 (s, 1H, pyridine-H5), 7.95
(d, 2H, Ar–H’s), 8.04 (s, 1H, chromene-H5), 8.24 (d, 2H,
Ar–H’s), 8.50 (s, 1H, chromene-H4); 13C-NMR (DMSO-
d6): δ 30.4 (CH3), 33.4 (SCH2), 55.3 (OCH3), 102.4, 114.3,
116.3, 116.8, 116.9, 118.2, 124.9, 127.8, 128.3, 128.9,
129.5, 130.3, 130.4, 135.1, 135.4, 136.1, 146.6, 154.6,
157.8, 158.1, 158.3, 162.0, 162.3 (chromene-CO), 195.1
(CO); MS (m/z): 518 (M+, 54.2%); Anal. for C31H22N2O4S:
C, 71.80; H, 4.28; N, 5.40; found: C, 71.60; H, 4.37; N,
5.53%.
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)thio)‑4‑(
4‑methoxyphenyl)‑6‑(p‑tolyl)nicotinonitrile (13c) Yel-
low solid (dioxane/ethanol, 85%); m.p. 218–220 °C; IR (υ
cm−1): 2230 (CN), 1723, 1675 (2 CO); 1H-NMR (DMSO-
d6): δ 2.39 (s, 3H, p-CH3), 2.55 (s, 3H, COCH3), 3.84 (s, 3H,
OCH3), 4.77 (s, 2H, SCH2), 7.12 (d, 2H, Ar–H’s), 7.34 (d,
2H, Ar–H’s), 7.41 (d, 1H, chromene-H8), 7.71 (d, 2H, Ar–
H’s), 7.82 (d, 1H, chromene-H7), 7.87 (s, 1H, pyridine-H5),
8.04 (s, 1H, chromene-H5), 8.13 (d, 2H, Ar–H’s), 8.49 (s,
1H, chromene-H4); 13C-NMR (DMSO-d6): δ 21.3 (p-CH3),
30.6 (CH3), 33.4 (SCH2), 55.9 (OCH3), 102.6, 114.5, 116.0,
116.5, 116.7, 118.1, 125.3, 127.8, 128.8, 128.9, 130.5,
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑6‑(4‑nitrophenyl)‑4‑phenylnicotinonitrile
(12e) Yellow solid (dioxane, 90%); m.p. 244–247 °C;
1
IR (υ cm−1): 2231 (CN), 1724, 1674 (2 CO); H-NMR
1 3

Journal of the Iranian Chemical Society
130.7, 134.4, 135.0, 135.2, 141.3, 146.6, 154.3, 157.5,
158.0, 160.2, 161.7, 162.1 (chromene-CO), 195.0 (CO); MS
(m/z): 532 (M+, 66.2%); Anal. for C32H24N2O4S: C, 72.16;
H, 4.54; N, 5.26; found: C, 72.02; H, 4.66; N, 5.32%.
and then, the absorption of each well was measured at 405 nm
using the 96-well plate reader Synergy HT, Biotek, USA. Eser-
ine (0.5 mM) was used as a positive control. The inhibition
percentages were calculated by the following formula:
Inhibition%
(
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)
thio)‑4,6‑bis(4‑methoxyphenyl)nicotinonitrile
(13d) Yellow solid (dioxane, 89%); m.p. 182–184 °C; IR
(υ cm−1): 2231 (CN), 1725, 1674 (2 CO); 1H-NMR (DMSO-
d6): δ 2.53 (s, 3H, COCH3), 3.81 (s, 6H, 2 OCH3), 4.69
(s, 2H, SCH2), 7.01 (d, 2H, Ar–H’s), 7.11 (d, 2H, Ar–H’s),
7.36 (d, 1H, chromene-H8), 7.65 (d, 2H, Ar–H’s), 7.71 (s,
1H, pyridine-H5), 7.80 (d, 1H, chromene-H7), 7.98 (s, 1H,
chromene-H5), 8.16 (d, 2H, Ar–H’s), 8.46 (s, 1H, chromene-
H4); 13C-NMR (DMSO-d6): δ 29.9 (CH3), 32.8 (SCH2), 55.3
(2 OCH3), 101.2, 114.2, 114.3, 115.1, 115.9, 116.2, 117.8,
124.4, 127.7, 128.9, 129.1, 130.1, 130.4, 134.3, 134.8, 146.5,
153.5, 153.6, 157.5, 158.2, 160.5, 161.4, 161.6 (chromene-
CO) 194.9 (CO); Anal. for C32H24N2O5S (548.6): C, 70.06;
H, 4.41; N, 5.11; found: C, 70.23; H, 4.48; N, 4.99%.
)
=
Controlabsorbance − Sampleabsorbance × 100∕Controlabsorbance
Five diferent concentrations of each compound were tested
in triplicate. The IC50 values were determined using sigma
plot software.
DPPH radical‑scavenging assay
DPPH radical-scavenging activity was measured according
to Thuong et al. method [55] with slight modifcations. The
new hybrids have been examined within the range of 0–25 µg/
mL using methanol as a solvent. In 96-well plates, 1 mL of
methanolic DPPH solution (0.3 mM) was added to 2.5 mL of
tested derivative in 5 diferent concentrations. One milliliter of
methanol was added, and the solution was mixed for a minute
and incubated in a dark place at room temperature. After thirty
minutes, the absorbance of the reaction mixture was measured
at 520 nm on a microplate reader. The reading blank consisted
of 2.5 mL of tested derivative and 1 mL of methanol; mean-
while, the mixture of 1 mL DPPH and 2.5 mL of methanol
was used as negative control. The percent of the antioxidant
activity was calculated using the equation:
2‑(((3‑Acetyl‑2‑oxo‑2H‑chromen‑6‑yl)methyl)thio)‑4
‑(4‑methoxyphenyl)‑6‑(4‑nitrophenyl)nicotinonitrile
(13e) Yellow solid (dioxane, 86%); m.p. 240–241 °C; IR
(υ cm−1): 2232 (CN), 1725, 1676 (2 CO); 1H-NMR (DMSO-
d6): δ 2.55 (s, 3H, COCH3), 3.83 (s, 3H, OCH3), 4.77 (s,
2H, SCH2), 7.11 (d, 2H, Ar–H’s), 7.39 (d, 1H, chromene-
H8), 7.79 (d, 1H, chromene-H7), 7.83 (s, 1H, pyridine-H5),
8.05 (s, 1H, chromene-H5), 8.22–8.25 (m, 4H, Ar–H’s),
8.40 (d, 2H, Ar–H’s), 8.51 (s, 1H, chromene-H4); Anal. for
C31H21N3O6S (563.5): C, 66.07; H, 3.76; N, 7.46; found: C,
65.93; H, 3.86; N, 7.32%.
Inhibition%
(
)
=
Blank sampleabsorbance − Sampleabsorbance × 100∕Blank sampleabsorbance
The IC50 value was calculated and compared with ascorbic
acid as a standard drug.
AChE inhibition assay
Cytotoxicity
Assessment of the AChE inhibitory activity was carried out
using the previously stated Ellman method [47, 54] with slight
modifcations. In a mixture of DMSO and methanol (1 and
9 mL, respectively), the stock solutions of the tested compounds
were prepared, followed by dilution in the bufer KH2PO4/
K2HPO4 (50 mM, pH 7.7) to obtain fnal test concentrations.
Ten microliters of the respective assayed sample (at stock
solution of 0.5 mM) was added to 60 μL of phosphate bufer
(50 mM, pH 7.7). Then, 10 µL of 0.005 unit per well enzyme
solution (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder,
from electric eel, 1000 units, Sigma-Aldrich) was added. The
resulting content was mixed and pre-read at 405 nm and then
incubated at 37° C for 10 min. In each well, the reaction was
started by adding 10 μL of acetylthiocholine iodide (0.5 mM),
followed by adding 10 μL of 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB, 0.5 mM per well). At 37 °C, the wells were incubated,
Cell line, culture conditions and preparation of compounds
The human breast epithelial cell line (MCF-10A), human
breast carcinoma cell line (MCF-7), colon cancer cell line
(Caco2) and liver hepatocellular carcinoma cell line (HEPG2)
were obtained from ATCC via Holding company for biological
products and vaccines (VACSERA), Cairo, Egypt [56].
The selected cell lines were cultivated in Dulbecco’s Modi-
fed Eagle’s Medium (DMEM). All of the growth media were
supplemented with 10% Fetal Bovine Serum (FBS) and anti-
biotics (100 U/mL penicillin and 100 mg/mL streptomycin)
at 37 °C in a humidifed atmosphere containing 5% CO2. New
hybrid derivatives 12b, 12e, 13b and 13e as well as Doxoru-
bicin, as a positive control, were dissolved in DMSO, and fnal
concentrations were diluted in culture medium.
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Journal of the Iranian Chemical Society
Neutral red uptake assay (NRU assay)
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