Synthesis and in vitro study of new coumarin derivatives linked to nicotinonitrile moieties as potential acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/jhet.4134

The appropriate pyridine-2(1H)-thiones were reacted with an equivalent amount of 5-(chloromethyl)-2-hydroxybenzaldehyde in ethanol in the presence of potassium hydroxide to give the corresponding 2-hydroxybenzaldehyde derivatives in excellent yields. The latter derivatives were taken as key synthons for the preparation of the target hybrids. Therefore, 2-hydroxybenzaldehydes were reacted with benzoylglycine in acetic anhydride in the presence of fused sodium acetate at 100°C for 6 hours to afford a new series of nicotinonitrile-coumarin hybrids. The in vitro acetylcholinesterase inhibitory activities were estimated for the new coumarins. The results were expressed as the inhibition percentage of the tested hybrids at concentration of 25 nM, compared to donepezil as a reference (inhibition percentage of 70.5). Coumarin hybrids linked to 6-(4-nitrophenyl) or 6-(4-chlorophenyl)-4-phenylnicotinonitrile exhibited more effective inhibitory activities than donepezil with inhibition percentages of 94.1 and 72.3, respectively. The new coumarins were tested for their free radical-scavenging capabilities against DPPH. Furthermore, some new coumarins were tested for in vitro cytotoxic activity against each MCF-10A, MCF-7, Caco2, and HEPG2. The new hybrids showed cytotoxicity in micromolar range (IC₅₀ of 3.5-13.9 μM) against all tested cell lines. These results clearly demonstrated that the hybrids being tested are not cytotoxic at the concentration required to inhibit acetylcholinesterase effectively.

Full text of DOI:10.1002/jhet.4134

Synthesis and in vitro Study of new Coumarin Derivatives Linked to Nicotinonitrile
Moieties as Potential Acetylcholinesterase Inhibitors
Ahmed E. M. Mekky and Sherif M. H. Sanad*
Chemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt
E-mail: sherif_hamed1980@yahoo.com
Abstract
The appropriate pyridine-2(1H)-thiones were reacted with an equivalent amount of 5-
(chloromethyl)-2-hydroxybenzaldehyde in ethanol in the presence of potassium hydroxide to
give the corresponding 2-hydroxybenzaldehyde derivatives in excellent yields. The latter
derivatives were taken as key synthons for the preparation of the target hybrids. Therefore, 2-
hydroxybenzaldehydes were reacted with benzoylglycine in acetic anhydride in the presence
o
of fused sodium acetate at 100 C for 6 h to afford a new series of nicotinonitrile-coumarin
hybrids. The in vitro acetylcholinesterase inhibitory activities were estimated for the new
coumarins. The results were expressed as the inhibition percentage of the tested hybrids at
concentration of 25 nM, compared to donepezil as a reference (inhibition percentage of 70.5).
Coumarin hybrids linked to 6-(4-nitrophenyl) or 6-(4-chlorophenyl)-4-phenylnicotinonitrile
exhibited more effective inhibitory activities than donepezil with inhibition percentages of
94.1 and 72.3, respectively. The new coumarins were tested for their free radical-scavenging
capabilities against DPPH. Furthermore, some new coumarins were tested for in vitro
cytotoxic activity against each MCF-10A, MCF-7, Caco2, and HEPG2. The new hybrids
showed cytotoxicity in micromolar range (IC50 of 3.5-13.9 µM) against all tested cell lines.
These results clearly demonstrated that the hybrids being tested are not cytotoxic at the
concentration required to inhibit acetylcholinesterase effectively.
Keywords: Alzheimer's disease, nicotinonitrile-coumarin hybrids, acetylcholinesterase
inhibitors, antioxidant activity, in vitro cytotoxicity screening.
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1. Introduction
Alzheimer's disease (AD) is a progressive and degenerative brain condition that
mainly affects the elderly and contributes to memory loss and everyday activity capacity. AD
is the main cause of dementia that affects around 50 million people around the world, with
nearly 10 million new patients per year, accounting for 60-70 per cent of dementia cases.[1-3]
The real origin of AD remains an open question, however, and thus one of the
exciting areas of current research in medicinal chemistry is the development of an effective
treatment of AD.[4] During the last 2 decades, several hippocampal anatomy studies, which
relate to learning and perception, have shown a substantial decrease in the rates of
neurotransmitter acetylcholine in AD patients.[5-7] Nowadays, clinical research has centered
on the issue of loss of basal forebrain cholinergic activity, as it is the only evidence
responsible for cognitive impairment and neurodegeneration in AD patients' brains.[8-10]
Many strategies have been introduced to improve cholinergic neurotransmission such
as improved acetylcholine level (ACh) synthesis or pre-synaptic release and stimulation of
post-synaptic muscarinic and nicotinic receptors, and to reduce ACh synaptic degradation by
inhibitors of acetylcholinesterase (AChE).[11-18] Donepezil, tacrine, galantamine, and
rivastigmine are currently approved AChE inhibitors (see Figure 1).[19]
Figure 1. Structure of some inhibitors of AChE.
Coumarin derivatives are natural heterocycles which are included in many diseases'
inhibition and cure. Derivatives bearing coumarin moiety act as antimicrobials,[20] anti-
inflammatory agents,[21] anticancer agents,[22] anti-Alzheimer's disease,[23,24] and anti-
Parkinson agents.[25] Dicoumarol and warfarin are examples of chromenes considered
effective oral anticoagulants.[26] Furthermore, the moiety of coumarin was used in several
successful inhibitors of AChE.[27-30]
In the current study, we report the preparation of a new series of nicotinonitrile-
coumarin hybrids, incorporating arene groups. These hybrids have been subjected to in vitro
study as possible inhibitors of AChE.
2
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2. Results and discussion
2.1. Chemistry
AChE is an enzyme present in cholinergic neurons and its key role is the rapid
breakdown of the neurotransmitter ACh released throughout neurotransmission.[31] The active
site of the enzyme is a 20 Å length gorge that contains a catalytic anionic site (CAS) and a
peripheral anionic binding site (PAS). Both sites play a central role in the enzyme
catalysis,[32] where CAS is responsible for the correct orientation and stabilization of the
trimethylammonium group of ACh. In addition, PAS promotes allosteric control at the
entrance to the cavity. Donepezil had been reported to bind to both PAS and CAS [32]
simultaneously, where indanone moiety forms several H-bonding and π–π interactions with
PAS aminoacid residues. In addition, its piperidine moiety forms multiple π–π stacking
interactions with CAS aminoacid residues.[33-35]
Coumarin group is typically integrated in PAS because of its planarity, steric
complementarity, and potential π-π stacking, whereas a net positive charge normally allows
for strong cation-π interactions in CAS (see Figure 2).[29, 30]
Figure 2. Structure of some potent AChE inhibitors with nanomolar affinities.
We recently reported the promising antibacterial and anticancer activities of novel
nicotinonitrile-coumarin hybrids.[36] Stimulated by these findings as well as the resemblance
of these hybrids and donepezil (see Figure 3), we report herein the preparation of new hybrids
as potent AChE inhibitors. To promote the interaction of new hybrids with the active gorge,
N-(2-oxo-2H-chromen-3-yl)benzamides linked to nicotinonitriles via thioethers were
included in their structures. These moieties replace indanone and piperidine moieties in
donepezil respectively.
3
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Figure 3. Structure of new hybrids and donepezil.
To achieve our target, we prepare initially a diverse series of nicotinonitriles.
Therefore, each of acetophenones 2 was reacted with benzaldehyde 1a or anisaldehyde 1b in
ethanolic potassium hydroxide solution under stirring at room temperature (rt) for 3 h to
afford a series of α,β-unsaturated carbonyl derivatives 3(4).[37-41] Each of the latter derivatives
were cyclocondensed with 2-cyanothioacetamide 5 in ethanol in the presence of piperidine at
[42-44]
reflux for 5 h afforded the key intermediates 2-thioxopyridine-3-carbonitriles 6(7)
(Scheme 1).
Scheme 1. Preparation of 2-thioxopyridine-3-carbonitriles 6(7).
The preparation of a new series of 2-hydroxybenzaldehyde-nicotinonitrile derivatives
9(10), in excellent yields, was our next step. Therefore, each of 2-thioxopyridine-3-
carbonitriles 6(7) was stirred in ethanolic potassium hydroxide solution at rt for 10 minutes.
[45]
To the mixture, 5-(chloromethyl)-2-hydroxybenzaldehyde 8
was added. For further 2 h
stirring, the reaction was continued to afford the 2-hydroxybenzaldehyde-nicotinonitriles 9a-
9e and 10a-10e in 88-94% yields (Scheme 2). The IR spectrum of 9d, as a representative
4
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example, showed three bands at 3414, 2231 and 1674 cm-1 corresponding to OH, CN and CO
groups, respectively. A molecular ion peak at m/z = 452 appeared in its mass spectrum. Its
1H-NMR spectrum showed four singlet signals at δ 3.83, 4.64, 10.22 and 10.63 attributed to
protons of CH3, CH2, CHO and OH, respectively. Its 13C-NMR spectrum revealed three
signals at δ 33.5, 55.3 and 191.7 assigned to carbons of CH2, CH3 and CO, respectively (see
experimental section).
Scheme 2. Preparation of 2-hydroxybenzaldehyde-nicotinonitrile derivatives 9(10).
Subsequently, 2-hydroxybenzaldehyde-nicotinonitriles 9(10) were taken as
intermediates for the preparation of the target coumarin hybrids 12(13). Thus, compounds
9(10) were reacted with benzoylglycine 11. The reaction was carried out in acetic anhydride
o
containing a catalytic amount of fused sodium acetate. The mixture was heated at 100 C for
6 h to afford N-(6-(((3-cyanopyridin-2-yl)thio)methyl)-2-oxo-2H-chromen-3-yl)benzamides
12(13) in 74-85% yields (Scheme 3).[46] The IR spectrum of 13c, as a typical example,
showed NH, CN and two CO bands at 3336, 2230, 1687 and 1641 cm-1, respectively. At m/z
1
= 609, a molecular ion peak appeared in its mass spectrum. The H-NMR spectrum of 13c
revealed four singlet signals at δ 2.36, 3.84, 4.69 and 9.62 corresponding to protons of CH3,
OCH3, CH2 and NH, respectively. Moreover, it revealed two doublets as well as two singlet
signals at δ 7.38, 7.82, 8.02 and 8.33, respectively, corresponding to protons of chromene. Its
13C-NMR spectrum showed five signals at δ 21.5, 33.8, 55.7, 160.5 and 165.3 assigned for
carbons of CH3, CH2, OCH3, chromene-CO and CO, respectively (see experimental section).
5
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Scheme 3. Preparation of nicotinonitrile-coumarin hybrids 12(13).
The above reaction may proceed through the initial intramolecular cyclization of
benzoylglycine 11 to give [14], followed by its in-situ Knoevenagel condensation to give
[15]. The above intermediate was subjected to the subsequent nucleophilic addition to form
intermediate [16], followed by ring opening to give 12(13) (Scheme 4).[47]
Scheme 4. The proposed mechanism for the synthesis of coumarins 12(13).
2.2. Biology
2.2.1. The in vitro AChE inhibitory activity
The new coumarins 12(13) were tested for their AChE inhibitory activity using
Ellman method.[48] The inhibition percentage of the tested hybrids 12(13) against AChE at
concentration of 25 nM were determined. At this concentration, the reference donepezil gave
inhibition percentage of 70.5.[49] The results are recorded in Figure 4.
With regards to coumarins 12a-12e, linked to 6-aryl-4-phenylnicotinonitrile, hybrids
12b and 12e displayed the best powerful AChE inhibitory activity. Thus, 12e, linked to 6-(4-
6
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nitrophenyl) group, exhibited more effective activity than donepezil with inhibition
percentage of 94.1. The second in AChE inhibitory strength was 12b, bearing 6-(4-
chlorophenyl) moiety with inhibition percentage of 72.3. Other hybrids 12a, 12c, and 12d
showed reduced efficacies. 12a, bearing 6-phenyl moiety, exhibited inhibition percentage of
14.6. Furthermore, hybrids 12c and 12d, bearing 6-(p-tolyl) and 6-(4-methoxyphenyl) moiety
respectively, exhibited the weakest efficacies with inhibition percentages of 10.7 and 7.4,
respectively.
With
regards
to
coumarins
13a-13e,
linked
to
6-aryl-4-(4-
methoxyphenyl)nicotinonitrile moiety, this series showed in general less efficacies against
AChE. Moreover, the inhibitory efficacies of the hybrids examined was in the same order as
in coumarins 12. Therefore, 13b and 13e, with 6-(4-chlorophenyl) and 6-(4-nitrophenyl)
moiety respectively, were the most effective hybrids with inhibition percentages of 23.9 and
36.6, respectively. Other hybrids 13a, 13c, and 13d, bearing 6-phenyl, 6-(p-tolyl) and 6-(4-
methoxyphenyl) moiety respectively, showed the weakest efficacies (inhibition percentages
of 2.8-5.7).
Figure 4. The relation between σp and inhibition percentages of the new nicotinonitrile-
coumarin hybrids 12(13) at concentrations of 25 nM. Inhibition percentages were calculated
taking negative control as 100% inhibition.
In order to explain the relationship between the electronic properties of the tested
coumarins and their AChE inhibitory efficacies, we compare the values of electronic
[50]
substituent constant (σp)
with the inhibition percentages of the new hybrids (Figure 4).
The examination of Figure 4 showed an obvious relation between the inhibition percentages
7
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and the values of σp. In each series, we found that the efficacies of AChE inhibition improved
as the substituent, linked to 6-arylnicotinonitrile, became more electron withdrawal.
2.2.2. DPPH antioxidant activity
Many efforts have been done to explore the success of antioxidant therapeutic
strategies in the treatment of neurodegenerative diseases as AD.[51-53] A subset of amyloid
plaques has recently been reported to develop free radicals in living, Alzheimer's models and
human Alzheimer's tissues. Such extremely reactive molecules are neutralized by antioxidant
therapy and can therefore be of therapeutic benefit in AD.[54] Depending on the above, the
assessment of the antioxidant efficacies of the tested hybrids is considered to provide value
for AD diagnostics.
Hence, the free radical quenching capabilities of the tested hybrids were studied
against DPPH. The results are expressed as the inhibition percentage of the tested hybrids
12(13) at concentration of 25 μg/mL (Figure 5). The reference ascorbic acid gave inhibition
percentage of 89.8.
Compound 12e displayed the best efficacy with inhibition percentage of 90.7.
Furthermore, compounds 12b, 13b and 13e showed free radical quenching activities with
inhibition percentages of 62.9-71.4. Other tested hybrids exhibited decreased capabilities with
inhibition percentages in the range of 4.7-17.6.
Figure 5. DPPH scavenging activity of the new hybrids at concentrations of 25 μg/mL.
Inhibition percentages were calculated taking negative control as 100% inhibition.
2.2.3. Cytotoxicity against eukaryotic cells
The in vitro cytotoxic activities of some hybrids were assessed. For this purpose, each
of MCF-10A, MCF-7, Caco2 and HEPG2 cell lines were selected. The findings of IC50 (in
8
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μM) are listed in Table 1. The reference Doxorubicin exhibited IC50 of 12.2-14.5 µM against
the tested cell lines.
Hybrids 12b, 12e and 13e exhibited more effective activities against all tested cell
lines when compared with Doxorubicin. 12e displayed the best efficacies with IC50 of 3.5 ±
0.1, 4.4 ± 0.2, 4.9 ± 0.2, and 6.2 ± 0.2 μM against each of the Caco2, HEPG2, MCF-7, and
MCF-10A cell lines, respectively. Furthermore, 12b and 13e exhibited IC50 of 4.9-7.9 μM
against the above tested cell lines (Table 1).
With regards to the MCF-7 and Caco2 cell lines, 13b displayed more powerful
efficacies than Doxorubicin with 11.9 ± 0.2 and 12.9 ± 0.3 μM, respectively. Moreover, 13b
showed less powerful efficacies against each of the MCF-10A and HEPG2 cell lines with
13.2 ± 0.3 and 13.9 ± 0.3 μM, respectively, when compared with Doxorubicin (Table 1).
Table 1. The IC50 of some new hybrids against each of MCF-10A, MCF-7, Caco2 and
HEPG2. Doxorubicin was used as a reference.
IC50 (µM)
MCF-10A
7.6 ± 0.2
6.2 ± 0.2
13.2 ± 0.3
7.9 ± 0.2
12.7 ± 0.3
MCF-7
5.6 ± 0.2
4.9 ± 0.2
11.9 ± 0.2
6.2 ± 0.2
12.2 ± 0.2
Caco2
HEPG2
4.9 ± 0.1
4.4 ± 0.2
13.9 ± 0.3
7.2 ± 0.3
13.7 ± 0.3
Compound
12b
5.1 ± 0.1
3.5 ± 0.1
12.9 ± 0.3
5.7 ± 0.2
14.5 ± 0.3
12e
13b
13e
Doxorubicin
It is a noteworthy to mention that cytotoxicity of the new hybrids 12b, 12e, 13b, and
13e showed IC50 in micromolar range (IC50 of 3.5-13.9 µM) against all tested the cell lines,
while some of these tested derivatives showed effective AChE inhibition in nanomolar range
(inhibition percentage up to 94.1 at concentration of 25 nM). These findings clearly showed
that the new nicotinonitrile-coumarin hybrid derivatives are not cytotoxic at the concentration
required to inhibit AChE enzyme effectively.
3. Conclusion
The 2-hydroxybenzaldehyde-nicotinonitriles were synthesized in excellent yields. These
hybrids were used as key intermediates for the preparation of coumarin derivatives. The new
hybrids were evaluated for their AChE inhibitory efficacies. Coumarin hybrids with 6-(4-
9
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chlorophenyl) and 6-(4-nitrophenyl) groups, linked to 4-phenylnicotinonitrile moiety,
displayed more powerful efficacies than donepezil. Some coumarins exhibited strong free
capacity to quench radicals against DPPH. Some new hybrids showed cytotoxicity in
micromolar range.
4. Experimental
“All solvents were acquired from commercial sources and used as received unless otherwise
stated. All other chemicals were acquired from Merck or Aldrich. These chemicals were used
without further purification. 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 reflectance (ATR) sampling accessory on the
Nicolet iS10 FT-IR spectrometer manufactured. NMR spectra were recorded on Bruker
1
Avance III 400 MHz spectrophotometer (400 MHz for H 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”.[36] 2-Thioxopyridine-3-carbonitriles 6(7) were prepared according to
literature procedures.[42-44]
Preparation of nicotinonitriles 9(10).
A mixture of 2-thioxopyridine-3-carbonitriles 6(7) (5 mmol) in ethanol (20 mL) containing
KOH (5 mmol) was stirred at rt for 10 min. To the mixture, a solution of benzyl chloride
derivative 8 (5 mmol) in 10 mL of ethanol was added and the stirring was continued for 2 h.
The obtained nicotinonitriles were collected by filtration, washed with each of water, dried
and recrystallized from dioxane / ethanol mixture.
2-((3-Formyl-4-hydroxybenzyl)thio)-4,6-diphenylnicotinonitrile (9a).
o
Colorless solid (91%); m.p. 114 C; IR: υ 3422 (OH), 2236 (CN), 1679 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 4.63 (s, 2H, CH2), 6.96 (d, 1H, ArH), 7.46 (t, 1H, ArH), 7.50 (t, 2H, ArH’s),
7.54 (t, 1H, ArH), 7.59 (t, 2H, ArH’s), 7.65 (d, 1H, ArH), 7.73 (d, 2H, ArH’s), 7.80 (s, 1H,
13
ArH), 7.83 (s, 1H, H5), 7.95 (d, 2H, ArH’s), 10.22 (s, 1H, CHO), 10.67 (s, 1H, OH); C-
NMR (DMSO-d6): δ 33.7, 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; MS (m/z):
10
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422 (M+, 48.2%); Anal. for C26H18N2O2S: C, 73.91; H, 4.29; N, 6.63; found: C, 73.75; H,
4.41; N, 6.52%.
6-(4-Chlorophenyl)-2-((3-formyl-4-hydroxybenzyl)thio)-4-phenylnicotinonitrile (9b).
Colorless solid (90%); m.p. 122 oC; IR: υ 3425 (OH), 2230 (CN), 1677 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 4.64 (s, 2H, CH2), 6.96 (d, 1H, ArH), 7.50 (d, 2H, ArH’s), 7.56 (t, 1H, ArH),
7.60 (t, 2H, ArH’s), 7.66 (d, 1H, ArH), 7.78 (s, 1H, ArH), 7.81 (s, 1H, H5), 7.95 (d, 2H,
ArH’s), 8.12 (d, 2H, ArH’s), 10.22 (s, 1H, CHO), 10.65 (s, 1H, OH); 13C-NMR (DMSO-d6): δ
33.4, 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; Anal. for C26H17ClN2O2S
(456.9): C, 68.34; H, 3.75; N, 6.13; found: C, 68.69; H, 3.51; N, 6.10%.
2-((3-Formyl-4-hydroxybenzyl)thio)-4-phenyl-6-(p-tolyl)nicotinonitrile (9c).
Colorless solid (87%); m.p. 120 oC; IR: υ 3432 (OH), 2231 (CN), 1675 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 2.38 (s, 3H, CH3), 4.64 (s, 2H, CH2), 6.96 (d, 1H, ArH), 7.33 (d, 2H, ArH’s),
7.54 (t, 1H, ArH), 7.59 (t, 2H, ArH’s), 7.64 (d, 1H, ArH), 7.79 (s, 1H, ArH), 7.82 (s, 1H, H5),
7.96 (d, 2H, ArH’s), 8.14 (d, 2H, ArH’s), 10.21 (s, 1H, CHO), 10.65 (s, 1H, OH); 13C-NMR
(DMSO-d6): δ 21.5, 33.6 (CH2), 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; MS (m/z):
436 (M+, 38.0%); Anal. for C27H20N2O2S: C, 74.55; H, 4.69; N, 6.27; found: C, 74.27; H,
4.65; N, 6.40%.
2-((3-Formyl-4-hydroxybenzyl)thio)-6-(4-methoxyphenyl)-4-phenylnicotinonitrile (9d).
Colorless solid (93%); m.p. 184 oC; IR: υ 3414 (OH), 2231 (CN), 1674 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 3.83 (s, 3H, CH3), 4.64 (s, 2H, CH2), 6.96 (d, 1H, ArH), 7.06 (d, 2H, ArH’s),
7.52 (t, 1H, ArH), 7.57 (t, 2H, ArH’s), 7.62 (d, 1H, ArH), 7.71 (s, 1H, ArH), 7.78 (s, 1H, H5),
7.95 (d, 2H, ArH’s), 8.07 (d, 2H, ArH’s), 10.22 (s, 1H, CHO), 10.63 (s, 1H, OH); 13C-NMR
(DMSO-d6): δ 33.5, 55.3, 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; MS (m/z): 452
(M+, 63.6%); Anal. for C27H20N2O3S: C, 71.66; H, 4.45; N, 6.19; found: C, 71.54; H, 4.28; N,
6.33%.
2-((3-Formyl-4-hydroxybenzyl)thio)-6-(4-nitrophenyl)-4-phenylnicotinonitrile (9e).
11
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Colorless solid (94%); m.p. 132 oC; IR: υ 3425 (OH), 2230 (CN), 1677 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 4.66 (s, 2H, CH2), 6.94 (d, 1H, ArH), 7.53 (t, 1H, ArH), 7.59 (t, 2H, ArH’s),
7.64 (d, 1H, ArH), 7.80 (s, 1H, ArH), 7.84 (s, 1H, H5), 7.96 (d, 2H, ArH’s), 8.05 (d, 2H,
ArH’s), 8.38 (d, 2H, ArH’s), 10.22 (s, 1H, CHO), 10.65 (s, 1H, OH); 13C-NMR (DMSO-d6): δ
33.9, 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; MS (m/z): 467 (M+, 62.4%);
Anal. for C26H17N3O4S: C, 66.80; H, 3.67; N, 8.99; found: C, 66.85; H, 3.83; N, 8.75%.
2-((3-Formyl-4-hydroxybenzyl)thio)-4-(4-methoxyphenyl)-6-phenylnicotinonitrile (10a).
Colorless solid (87%); m.p. 196 oC; IR: υ 3437 (OH), 2233 (CN), 1675 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 3.85 (s, 3H, CH3), 4.66 (s, 2H, CH2), 6.95 (d, 1H, ArH), 7.13 (d, 2H, ArH’s),
7.53 (t, 1H, ArH), 7.57 (t, 2H, ArH’s), 7.63 (d, 1H, ArH), 7.73 (d, 2H, ArH’s), 7.78 (s, 1H,
13
ArH), 7.81 (s, 1H, H5), 8.27 (d, 2H, ArH’s), 10.20 (s, 1H, CHO), 10.68 (s, 1H, OH); C-
NMR (DMSO-d6): δ 33.4, 55.9, 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; MS (m/z):
452 (M+, 74.6%); Anal. for C27H20N2O3S: C, 71.66; H, 4.45; N, 6.19; found: C, 71.78; H,
4.30; N, 6.07%.
6-(4-Chlorophenyl)-2-((3-formyl-4-hydroxybenzyl)thio)-4-(4-
methoxyphenyl)nicotinonitrile (10b).
Colorless solid (93%); m.p. 132 oC; IR: υ 3422 (OH), 2233 (CN), 1676 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 3.83 (s, 3H, CH3), 4.65 (s, 2H, CH2), 6.95 (d, 1H, ArH), 7.10 (d, 2H, ArH’s),
7.52 (d, 2H, ArH’s), 7.65 (d, 1H, ArH), 7.71 (d, 2H, ArH’s), 7.79 (s, 1H, ArH), 7.82 (s, 1H,
H5), 8.16 (d, 2H, ArH’s), 10.21 (s, 1H, CHO), 10.68 (s, 1H, OH); 13C-NMR (DMSO-d6): δ
33.5, 55.9, 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; Anal. for C27H19ClN2O3S
(486.9): C, 66.59; H, 3.93; N, 5.75; found: C, 66.47; H, 3.84; N, 5.83%.
2-((3-Formyl-4-hydroxybenzyl)thio)-4-(4-methoxyphenyl)-6-(p-tolyl)nicotinonitrile
(10c).
Colorless solid (91%); m.p. 102 oC; IR: υ 3439 (OH), 2232 (CN), 1677 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 2.38 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.65 (s, 2H, CH2), 6.95 (d, 1H, ArH),
7.13 (d, 2H, ArH’s), 7.34 (d, 2H, ArH’s), 7.62 (d, 1H, ArH), 7.71 (d, 2H, ArH’s), 7.80 (s, 1H,
13
ArH), 7.82 (s, 1H, H5), 8.16 (d, 2H, ArH’s), 10.21 (s, 1H, CHO), 10.68 (s, 1H, OH); C-
12
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NMR (DMSO-d6): δ 21.4, 33.4, 55.9, 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, 137.3, 141.1, 154.2, 158.3, 160.4, 161.3, 162.4, 191.5; MS
(m/z): 466 (M+, 44.5%); Anal. for C28H22N2O3S: C, 72.08; H, 4.75; N, 6.00; found: C, 72.26;
H, 4.60; N, 6.07%.
2-((3-Formyl-4-hydroxybenzyl)thio)-4,6-bis(4-methoxyphenyl)nicotinonitrile (10d).
Colorless solid (91%); m.p. 122 oC; IR: υ 3431 (OH), 2228 (CN), 1671 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 3.83 (s, 6H, 2 CH3), 4.63 (s, 2H, CH2), 6.95 (d, 1H, ArH), 7.05 (d, 2H, ArH’s),
7.12 (d, 2H, ArH’s), 7.67 (d, 1H, ArH), 7.75-7.79 (m, 4H, 3 ArH’s and H5), 8.22 (d, 2H,
ArH’s), 10.21 (s, 1H, CHO), 10.69 (s, 1H, OH); 13C-NMR (DMSO-d6): δ 32.8, 55.3 (2 C),
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; MS (m/z): 482 (M+, 70.4%); Anal. for
C28H22N2O4S: C, 69.69; H, 4.60; N, 5.81; found: C, 69.74; H, 4.58; N, 5.71%.
2-((3-Formyl-4-hydroxybenzyl)thio)-4-(4-methoxyphenyl)-6-(4-
nitrophenyl)nicotinonitrile (10e).
Colorless solid (93%); m.p. 130 oC; IR: υ 3427 (OH), 2232 (CN), 1679 (CO) cm-1; 1H-NMR
(DMSO-d6): δ 3.84 (s, 3H, CH3), 4.64 (s, 2H, CH2), 6.95 (d, 1H, ArH), 7.08 (d, 2H, ArH’s),
7.62 (d, 1H, ArH), 7.78 (s, 1H, ArH), 7.80 (s, 1H, H5), 8.05 (d, 2H, ArH’s), 8.25 (d, 2H,
ArH’s), 8.38 (d, 2H, ArH’s), 10.22 (s, 1H, CHO), 10.65 (s, 1H, OH); 13C-NMR (DMSO-d6): δ
33.8, 55.6, 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; MS (m/z): 497 (M+, 70.3%);
Anal. for C27H19N3O5S: C, 65.18; H, 3.85; N, 8.45; found: C, 65.16; H, 3.88; N, 8.46%.
Preparation
of
N-(6-(((3-cyanopyridin-2-yl)thio)methyl)-2-oxo-2H-chromen-3-
yl)benzamides 12(13).
A mixture of benzoylglycine 11 and 2-hydroxybenzaldehydes 9(10) (5 mmol) in 20 mL of
o
acetic anhydride containing 8 mmol of fused sodium acetate was heated at 100 C. After
heating the reaction for 6 h, the volume of the mixture was reduced to its half by evaporation.
After cooling the mixture, the mixture product was filtered off, washed with ethanol and
recrystallized from dioxane.
N-(6-(((3-Cyano-4,6-diphenylpyridin-2-yl)thio)methyl)-2-oxo-2H-chromen-3-
yl)benzamide (12a).
13
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o
Yellow solid (81%, cLogP 7.47); m.p. 224 C; IR: υ 3338 (NH), 2230 (CN), 1692, 1654 (2
CO) cm-1; 1H-NMR (DMSO-d6): δ 4.70 (s, 2H, CH2), 7.41 (d, 1H, H8), 7.46 (t, 1H, ArH),
7.50-7.54 (m, 3H, ArH’s), 7.57-7.61 (m, 3H, ArH’s), 7.64 (t, 2H, ArH’s), 7.80 (s, 1H,
pyridine-H), 7.84 (d, 1H, H7), 7.92-7.95 (m, 4H, ArH’s), 8.06 (s, 1H, H5), 8.25 (d, 2H,
ArH’s), 8.32 (s, 1H, H4), 9.72 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ 33.6, 103.8, 115.8,
116.4, 116.6, 118.3, 124.2, 126.6, 127.7, 127.9, 128.1, 128.3, 128.6, 129.2, 129.6, 129.8,
130.5, 131.5, 133.6, 135.3, 136.3, 136.5, 144.8, 152.7, 157.4, 157.9, 159.8, 161.2, 165.6; MS
m/z (%): 565 (M+, 51.4); Anal. for C35H23N3O3S: C, 74.32; H, 4.10; N, 7.43; found: C, 74.17;
H, 3.97; N, 7.58%.
N-(6-(((6-(4-Chlorophenyl)-3-cyano-4-phenylpyridin-2-yl)thio)methyl)-2-oxo-2H-
chromen-3-yl)benzamide (12b).
Yellow solid (84%, cLogP 8.03); m.p. 236-238 oC; IR: υ 3334 (NH), 2231 (CN), 1688, 1652
1
(2 CO) cm-1; H-NMR (DMSO-d6): δ 4.66 (s, 2H, CH2), 7.40 (d, 1H, H8), 7.47 (d, 2H,
ArH’s), 7.50-7.54 (m, 2H, ArH), 7.57-7.61 (m, 4H, ArH’s), 7.77 (s, 1H, pyridine-H), 7.81 (d,
1H, H7), 7.91 (d, 2H, ArH’s), 7.95 (d, 2H, ArH’s), 8.07 (s, 1H, H5), 8.12 (d, 2H, ArH’s), 8.30
(s, 1H, H4), 9.64 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ 33.7, 104.1, 115.7, 116.2, 116.4,
118.0, 124.7, 126.4, 127.9, 128.0, 128.2, 128.4, 129.1, 129.2, 129.7, 130.5, 131.6, 133.8,
135.1, 135.3, 135.8, 136.5, 146.3, 153.5, 158.0, 158.2, 160.0, 161.7, 165.8; Anal. for
C35H22ClN3O3S (600.0): C, 70.05; H, 3.70; N, 7.00; found: C, 70.24; H, 3.86; N, 6.84%.
N-(6-(((3-Cyano-4-phenyl-6-(p-tolyl)pyridin-2-yl)thio)methyl)-2-oxo-2H-chromen-3-
yl)benzamide (12c).
Yellow solid (79%, cLogP 7.96); m.p. 216-219 oC; IR: υ 3347 (NH), 2233 (CN), 1689, 1648
1
(2 CO) cm-1; H-NMR (DMSO-d6): δ 2.37 (s, 3H, CH3), 4.71 (s, 2H, CH2), 7.34 (d, 2H,
ArH’s), 7.39 (d, 1H, H8), 7.51-7.55 (m, 2H, ArH), 7.59-7.64 (m, 4H, ArH’s), 7.81 (s, 1H,
pyridine-H), 7.84 (d, 1H, H7), 7.90 (d, 2H, ArH’s), 7.94 (d, 2H, ArH’s), 8.02 (s, 1H, H5),
13
8.13 (d, 2H, ArH’s), 8.27 (s, 1H, H4), 9.65 (br s, 1H, NH); C-NMR (DMSO-d6): δ 21.5,
33.6, 104.3, 115.9, 116.5, 116.7, 118.1, 124.7, 126.8, 127.8, 128.1, 128.4, 128.8, 129.0,
129.3, 130.9, 131.7, 133.5, 134.5, 135.3, 135.4, 135.8, 141.2, 146.6, 153.7, 158.2, 158.6,
160.1, 161.4, 165.6; MS (m/z): 579 (M+, 45.6%); Anal. for C36H25N3O3S: C, 74.59; H, 4.35;
N, 7.25; found: C, 74.30; H, 4.52; N, 7.19%.
14
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N-(6-(((3-Cyano-6-(4-methoxyphenyl)-4-phenylpyridin-2-yl)thio)methyl)-2-oxo-2H-
chromen-3-yl)benzamide (12d).
o
Yellow solid (74%, cLogP 7.34); m.p. 214 C; IR: υ 3342 (NH), 2230 (CN), 1691, 1649 (2
CO) cm-1; 1H-NMR (DMSO-d6): δ 3.82 (s, 3H, CH3), 4.67 (s, 2H, CH2), 7.06 (d, 2H, ArH’s),
7.41 (d, 1H, H8), 7.47-7.52 (m, 2H, ArH), 7.55-7.59 (m, 4H, ArH’s), 7.82 (s, 1H, pyridine-
H), 7.88 (d, 1H, H7), 7.91 (d, 2H, ArH’s), 7.98 (d, 2H, ArH’s), 8.10 (s, 1H, H5), 8.22 (d, 2H,
13
ArH’s), 8.38 (s, 1H, H4), 9.76 (br s, 1H, NH); C-NMR (DMSO-d6): δ 33.5, 55.3, 102.8,
114.4, 116.0, 116.4, 116.7, 119.2, 124.6, 125.8, 127.9, 128.1, 128.4, 129.0, 129.2, 129.5,
130.4, 130.7, 131.7, 133.8, 135.5, 136.4, 146.4, 155.2, 157.2, 158.3, 158.9, 160.2, 162.3,
165.5; MS m/z (%): 595 (M+, 42.4); Anal. for C36H25N3O4S: C, 72.59; H, 4.23; N, 7.05;
found: C, 72.67; H, 4.18; N, 6.94%.
N-(6-(((3-Cyano-6-(4-nitrophenyl)-4-phenylpyridin-2-yl)thio)methyl)-2-oxo-2H-
chromen-3-yl)benzamide (12e).
Yellow solid (80%, cLogP 6.07); m.p. 244-247 oC; IR: υ 3349 (NH), 2233 (CN), 1694, 1653
(2 CO) cm-1; 1H-NMR (DMSO-d6): δ 4.71 (s, 2H, CH2), 7.39 (d, 1H, H8), 7.45 (t, 1H, ArH),
7.49 (t, 1H, ArH), 7.53-7.58 (m, 4H, ArH’s), 7.79 (s, 1H, pyridine-H), 7.82 (d, 1H, H7), 7.88
(d, 2H, ArH’s), 7.93 (d, 2H, ArH’s), 8.04 (s, 1H, H5), 8.23 (d, 2H, ArH’s), 8.35 (s, 1H, H4),
8.42 (d, 2H, ArH’s), 9.82 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ 33.7, 103.7, 116.1, 116.9,
117.2, 119.6, 122.1, 124.6, 124.9, 125.7, 127.8, 128.1, 128.3, 129.1, 129.7, 130.6, 131.7,
133.6, 135.7, 136.6, 138.5, 144.6, 146.8, 156.0, 158.0, 158.6, 160.4, 161.8, 165.7; MS m/z
(%): 610 (M+, 37.9); Anal. for C35H22N4O5S: C, 68.84; H, 3.63; N, 9.18; found: C, 69.04; H,
3.51; N, 9.03%.
N-(6-(((3-Cyano-4-(4-methoxyphenyl)-6-phenylpyridin-2-yl)thio)methyl)-2-oxo-2H-
chromen-3-yl)benzamide (13a).
Yellow solid (82%, cLogP 7.34); m.p. 216-219 oC; IR: υ 3349 (NH), 2232 (CN), 1689, 1642
1
(2 CO) cm-1; H-NMR (DMSO-d6): δ 3.83 (s, 3H, CH3), 4.65 (s, 2H, CH2), 7.11 (d, 2H,
ArH’s), 7.39 (d, 1H, H8), 7.49-7.54 (m, 2H, ArH’s), 7.57-7.62 (m, 4H, ArH’s), 7.73 (d, 2H,
ArH’s), 7.85 (s, 1H, pyridine-H), 7.89 (d, 1H, H7), 7.94 (d, 2H, ArH’s), 8.11 (s, 1H, H5),
8.25 (d, 2H, ArH’s), 8.38 (s, 1H, H4), 9.81 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ 33.6,
55.6, 103.3, 114.5, 115.4, 115.9, 116.4, 118.5, 124.8, 126.2, 127.7, 127.9, 128.2, 128.8,
129.2, 129.4, 130.4, 130.6, 131.7, 133.6, 135.4, 136.3, 146.2, 153.0, 157.9, 158.3, 158.7,
15
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160.3, 162.1, 165.6; MS (m/z): 595 (M+, 62.5%); Anal. for C36H25N3O4S: C, 72.59; H, 4.23;
N, 7.05; found: C, 72.42; H, 4.08; N, 6.91%.
N-(6-(((6-(4-Chlorophenyl)-3-cyano-4-(4-methoxyphenyl)pyridin-2-yl)thio)methyl)-2-
oxo-2H-chromen-3-yl)benzamide (13b).
o
Yellow solid (75%, cLogP 7.90); m.p. 233 C; IR: υ 3352 (NH), 2233 (CN), 1695, 1647 (2
CO) cm-1; 1H-NMR (DMSO-d6): δ 3.84 (s, 3H, CH3), 4.72 (s, 2H, CH2), 7.10 (d, 2H, ArH’s),
7.41 (d, 1H, H8), 7.52 (t, 1H, ArH), 7.56-7.61 (m, 4H, ArH’s), 7.78 (s, 1H, pyridine-H), 7.83
(d, 1H, H7), 7.96 (d, 2H, ArH’s), 8.09 (s, 1H, H5), 8.14 (d, 2H, ArH’s), 8.24 (d, 2H, ArH’s),
13
8.29 (s, 1H, H4), 9.62 (br s, 1H, NH); C-NMR (DMSO-d6): δ 33.7, 55.6, 103.5, 114.6,
116.4, 116.5, 116.9, 118.4, 124.9, 126.3, 128.0, 128.1, 128.8, 129.1, 129.3, 130.6, 130.8,
131.4, 133.6, 135.2, 135.3, 135.9, 146.4, 153.8, 158.2, 158.6, 160.2, 160.4, 161.9, 165.5;
Anal. for C36H24ClN3O4S (630.1): C, 68.62; H, 3.84; N, 6.67; found: C, 68.76; H, 3.99; N,
6.54%.
N-(6-(((3-Cyano-4-(4-methoxyphenyl)-6-(p-tolyl)pyridin-2-yl)thio)methyl)-2-oxo-2H-
chromen-3-yl)benzamide (13c).
Yellow solid (85%, cLogP 7.83); m.p. 218-220 oC; IR: υ 3336 (NH), 2230 (CN), 1687, 1641
(2 CO) cm-1; 1H-NMR (DMSO-d6): δ 2.36 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.69 (s, 2H,
CH2), 7.11 (d, 2H, ArH’s), 7.34 (d, 2H, ArH’s), 7.38 (d, 1H, H8), 7.52 (t, 1H, ArH), 7.57 (t,
2H, ArH’s), 7.71 (d, 2H, ArH’s), 7.79 (s, 1H, pyridine-H), 7.82 (d, 1H, H7), 7.95 (d, 2H,
ArH’s), 8.02 (s, 1H, H5), 8.13 (d, 2H, ArH’s), 8.33 (s, 1H, H4), 9.62 (br s, 1H, NH);13C-
NMR (DMSO-d6): δ 21.5, 33.8, 55.7, 103.2, 114.5, 115.9, 116.2, 116.4, 117.9, 125.2, 126.4,
127.7, 128.0, 128.6, 128.8, 129.2, 130.4, 130.6, 131.6, 133.8, 134.6, 135.3, 141.4, 145.9,
155.2, 157.6, 157.8, 159.9, 160.5, 161.8, 165.3; MS (m/z): 609 (M+, 61.3%); Anal. for
C37H27N3O4S: C, 72.89; H, 4.46; N, 6.89; found: C, 73.07; H, 4.56; N, 7.00%.
N-(6-(((3-Cyano-4,6-bis(4-methoxyphenyl)pyridin-2-yl)thio)methyl)-2-oxo-2H-chromen-
3-yl)benzamide (13d).
Yellow solid (76%, cLogP 7.22); m.p. 204-206 oC; IR: υ 3347 (NH), 2231 (CN), 1691, 1645
(2 CO) cm-1; 1H-NMR (DMSO-d6): δ 3.81 (s, 3H, CH3), 3.85 (2 s, 3H, CH3), 4.64 (s, 2H,
CH2), 7.05 (d, 2H, ArH’s), 7.10 (d, 2H, ArH’s), 7.43 (d, 1H, H8), 7.51 (t, 1H, ArH), 7.55 (t,
2H, ArH’s), 7.66 (d, 2H, ArH’s), 7.76 (s, 1H, pyridine-H), 7.81 (d, 1H, H7), 7.93 (d, 2H,
ArH’s), 8.04 (s, 1H, H5), 8.15 (d, 2H, ArH’s), 8.29 (s, 1H, H4), 9.70 (br s, 1H, NH); 13C-
16
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NMR (DMSO-d6): δ 33.8, 55.8 (2 C), 101.8, 114.2, 114.6, 115.3, 115.8, 116.5, 118.0, 124.7,
125.8, 127.8, 128.2, 129.0, 129.4, 130.2, 130.4, 130.8, 131.4, 133.5, 135.4, 145.7, 153.8,
157.6, 157.8, 158.4, 160.2, 160.3, 161.6, 165.5; MS (m/z): 625 (M+, 33.8%); Anal. for
C37H27N3O5S: C, 71.03; H, 4.35; N, 6.72; found: C, 70.84; H, 4.51; N, 6.63%.
N-(6-(((3-Cyano-4-(4-methoxyphenyl)-6-(4-nitrophenyl)pyridin-2-yl)thio)methyl)-2-oxo-
2H-chromen-3-yl)benzamide (13e).
Yellow solid (76%, cLogP 6.20); m.p. 240-241 oC; IR: υ 3346 (NH), 2232 (CN), 1688, 1651
1
(2 CO) cm-1; H-NMR (DMSO-d6): δ 3.83 (s, 3H, CH3), 4.71 (s, 2H, CH2), 7.09 (d, 2H,
ArH’s), 7.42 (d, 1H, H8), 7.55 (t, 1H, ArH), 7.59 (t, 2H, ArH’s), 7.76 (s, 1H, pyridine-H),
7.81 (d, 1H, H7), 7.93 (d, 2H, ArH’s), 8.08 (s, 1H, H5), 8.22 (d, 2H, ArH’s), 8.27 (d, 2H,
ArH’s), 8.33 (s, 1H, H4), 8.39 (d, 2H, ArH’s), 9.82 (br s, 1H, NH); 13C-NMR (DMSO-d6): δ
33.8, 55.8, 102.6, 114.6, 115.5, 115.9, 116.3, 117.8, 122.3, 124.6, 124.7, 126.2, 127.8, 128.0,
129.3, 130.2, 130.7, 131.8, 133.9, 135.4, 138.7, 144.6, 146.3, 154.2, 157.9, 158.1, 160.2,
160.5, 161.9, 165.6; MS (m/z): 640 (M+, 32.7%); Anal. for C36H24N4O6S: C, 67.49; H, 3.78;
N, 8.75; found: C, 67.32; H, 3.92; N, 8.83%.
4.3. AChE inhibition assay
“AChE inhibitory activities were assessed using Ellman procedure[48] with minor
changes. Stock solutions of the tested hybrids were prepared in a mixture of 1 mL DMSO and
9 mL methanol, followed by dilution in the buffer KH2PO4/K2HPO4 (50 mM, pH 7.7) to
acquire the final concentration. To 60 μL of 50 mM phosphate buffer (pH 7.7), 10 μL of the
respective assayed sample (at stock solution of 0.5 mM) was applied. 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 applied. The resulting content was mixed and
pre-read at 405 nm, 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,
then the absorption of each well was measured at 405 nm using the 96-well plate reader
Synergy HT, Biotek, USA. Eserine (0.5 mM) was used as a positive control. The inhibition
percentages were calculated by the following formula:” [55]
Inhibition percentage = (Controlabsorbance - Sampleabsorbance) X 100 / Controlabsorbance
Each compound was tested in triplicates at concentration of 25 nM.
17
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4.4. DPPH radical-scavenging assay
“DPPH radical-scavenging activity was assessed according to Thuong et. al.
procedure with minor changes. Methanol was used as a solvent to assess the tested
derivatives. 1 mL of methanolic DPPH solution (0.3 mM) was applied to 2.5 mL of the tested
derivative in 96-well plates. 1 mL of methanol was applied and the solution was mixed for a
minute at rt and incubated in a dark place. After 30 min, the absorbance of the reaction
mixture was calculated at 520 nm on a microplate reader. The reading blank consisted of 2.5
mL of estimated hybrid and 1 mL of methanol, meanwhile 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:”[56]
Inhibition percentage = (Controlabsorbance- Sampleabsorbance) X 100 / Controlabsorbance
The new hybrids as well as the reference ascorbic acid have been examined in triplicates at
concentration of 25 μg/mL.
4.5. Cytotoxicity
4.5.1. 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.”[57]
“The selected cell lines were cultivated in Dulbecco’s Modified Eagle’s Medium
(DMEM). All of the growth media were supplemented with 10% Foetal Bovine Serum (FBS)
and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37°C in a humidified
atmosphere containing 5% CO2. New hybrid derivatives 12b, 12e, 13b, and 13e as well as
Doxorubicin, as a positive control, were dissolved in DMSO and final concentrations were
diluted in culture medium.”[58]
4.5.2. Neutral red uptake assay (NRU assay)
“The NRU assay depends on the ability of living cells to bind neutral red, in
lysosomes.[59] The cytotoxicity of New hybrid derivatives 12b, 12e, 13b, and 13e were
evaluated against each of MCF-10A, MCF-7, Caco2 and HEPG2 cell lines using Doxorubicin
as a standard drug. Exponentially growing cells were collected using 0.25% Trypsin-EDTA,
18
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then the cell suspension counted using hemocytometer, and cell viability checked by trypan
blue (100% viability). Then, the cells suspension was diluted with complete medium to have
an approximately 1.0 x 105 cell/mL, then 200 µL of the cell suspension was dispensed by
multichannel pipette into the inner 60 wells of the 96 well plate, the peripheral wells were
filled with PBS, then the plate incubated for 24 h before treatment with the tested compounds
to allow attachment of cells to the wall of the plate. Different concentrations of the tested
derivatives (5, 25, 50 and 75 µg/mL) were prepared using DMEM media. 200 μL of
treatment media was dispensed into 4 replicates for each concentration, other wells were
filled with untreated cells only (as a negative control) and wells filled with media containing
Doxorubicin HCL as a positive control. The 96 well plate incubated at 37° C for 48 h. Then,
the medium and extracts were discarded and replaced with 100 µL of neutral red solution (50
mg/mL) and centrifuged at 1800 rpm for ten min to remove any crystals of dye. After
incubation at 37°C for 3 h, the dye medium was discarded and the microplate was washed
twice with 150 µL PBS to remove the unabsorbed neutral red dye contained in the wells. The
cellular morphology of each of the treated MCF-10A, MCF-7, Caco2 and HEPG2 cell lines
with the tested derivatives were observed using Inverted Microscope Leica DMI3000B. The
absorbance of acidified ethanol solution containing extracted neutral red dye was determined
using microplate reader (BioTek, ELX808) at 540 nm to estimate the optical density and the
cell viability percentage was measured.”[58]
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