Novel 3-phenylcoumarin–lipoic acid conjugates as multi-functional agents for potential treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2018.04.030

New series of triazole-containing 3-phenylcoumarin–lipoic acid conjugates were designed as multi-functional agents for treatment of Alzheimer's disease. The target compounds 4a-o were synthesized via the azide-alkyne cycloaddition reaction and their biolo

Full text of DOI:10.1016/j.bioorg.2018.04.030

Accepted Manuscript
Novel 3-phenylcoumarin–lipoic acid conjugates as multi-functional agents for
potential treatment of Alzheimer's disease
Leili Jalili-Baleh, Hamid Nadri, Hamid Forootanfar, Alireza Samzadeh-
Kermani, Tuba Tüylü Kü ç ükkılınç, Beyza Ayazgok, Mahban Rahimifard,
Maryam Baeeri, Mohsen Doostmohammadi, Loghman Firoozpour, Syed Nasir
Abbas Bukhari, Mohammad Abdollahi, Mohammad Reza Ganjali, Saeed
Emami, Mehdi Khoobi, Alireza Foroumadi
PII:
S0045-2068(18)30318-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.030
YBIOO 2353
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2018
28 April 2018
30 April 2018
Please cite this article as: L. Jalili-Baleh, H. Nadri, H. Forootanfar, A. Samzadeh-Kermani, T. Tüylü Kü ç
ükkılınç, B. Ayazgok, M. Rahimifard, M. Baeeri, M. Doostmohammadi, L. Firoozpour, S. Nasir Abbas Bukhari,
M. Abdollahi, M. Reza Ganjali, S. Emami, M. Khoobi, A. Foroumadi, Novel 3-phenylcoumarin–lipoic acid
conjugates as multi-functional agents for potential treatment of Alzheimer's disease, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.04.030
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Novel 3-phenylcoumarin–lipoic acid conjugates as multi-functional agents for potential
treatment of Alzheimer's disease
a
b
c
Leili Jalili-Baleh , Hamid Nadri , Hamid Forootanfar , Alireza Samzadeh-Kermani d, Tuba
e
e
a
a
Tüylü Küçükkılınç , Beyza Ayazgok , Mahban Rahimifard , Maryam Baeeri , Mohsen
f
g
h
Doostmohammadi , Loghman Firoozpour , Syed Nasir Abbas Bukhari , Mohammad
a
i,j
k
Abdollahi , Mohammad Reza Ganjali , Saeed Emami , Mehdi Khoobi a,l,*, Alireza
Foroumadi f,m,**
a
The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411,
Iran
b Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
c
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran
d Department of Chemistry, Faculty of Sciences, University of Zabol, Zabol, Iran
e Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, Ankara, Turkey
f
Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences,
Kerman, Iran
g
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran 1417614411, Iran
h Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka 2014, Saudi
Arabia
i
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran
University of Medical Sciences, Tehran, Iran
j Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran
k
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
l
Department of Pharmaceutical Biomaterials, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
m
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
* Corresponding authors. Tel/Fax: +98-21-64121510. E-mail address: Mehdi.khoobi@gmail.com (M. Khoobi),
and aforoumadi@yahoo.com (A. Foroumadi)
1

Abstract
New series of triazole-containing 3-phenylcoumarin–lipoic acid conjugates were designed
as multi-functional agents for treatment of Alzheimer's disease. The target compounds 4a-o
were synthesized via the azide-alkyne cycloaddition reaction and their biological activities
were primarily evaluated in terms of neuroprotection against H2O2-induced cell death in
PC12 cells and AChE/BuChE inhibition. The promising compounds 4j and 4i containing four
carbons spacer were selected for further biological evaluations. Based on the obtained results,
the benzocoumarin derivative 4j with IC50 value of 7.3 µM was the most potent AChE
inhibitor and displayed good inhibition toward intracellular reactive oxygen species (ROS).
This compound with antioxidant and metal chelating ability showed also protective effect on
cell injury induced by Aβ1-42 in SH-SY5Y cells. Although the 8-methoxycoumarin analog 4i
was slightly less active than 4j against AChE, but displayed higher protection ability against
H2O2-induced cell death in PC12 and could significantly block Aβ-aggregation. The results
suggested that the prototype compounds 4i and 4j might be promising multi-functional agents
for the further development of the disease-modifying treatments of Alzheimer's disease.
Keywords: Alzheimer’s disease; Neuroprotective activity; Coumarin; Lipoic acid; Amyloid
beta; Antioxidant
2

1. Introduction
Alzheimer's disease (AD) is certainly the most common cause of dementia and death in the
elderly population which cannot yet be prevented or cured. None of the available drugs
approved by the FDA (Food and Drug Administration) slows or stops the damages caused by
the destruction of neurons and do not address the etiology of AD. They just temporarily
improve cognitive function symptoms by increasing the cholinergic neurotransmitter,
acetylcholine (ACh), via inhibiting the acetylcholinesterase (AChE) enzyme that causes rapid
hydrolysis of acetylcholine in the brain [1].
Previous researches have shown that in addition to AChE, butyrylcholinesterase (BuChE)
as a co-regulator of the ACh degradation also plays an important role in cholinergic
neurotransmission [2]. The extracellular deposits of the protein fragment beta-amyloid (Aβ)
and accumulation of an abnormal form of tau protein inside neurons (neurofibrillary tangles)
in brain regions are two hallmark lesions of AD which provide synaptic dysfunction, pro-
inflammatory and toxic effects on neuronal cells [3-5]. According to ‘amyloid hypothesis’,
AChE impresses the Aβ aggregation via binding to the peripheral anionic binding site (PAS)
[6,7]. Besides these hallmarks, oxidative stress plays a key role in the pathogenesis of AD
and is a major mechanism underlying Aβ-induced neurotoxicity [8-10]. Several studies
suggested that Aβ aggregates with entrapped redox-active metal ions such as copper, zinc,
and iron lead to the intracellular accumulation of reactive oxygen species (ROS) [11,12].
Therefore, development of drugs with additional antioxidant and metal chelating properties
could be useful therapeutic strategies for targeting of oxidative stress and inhibiting Aβ-
induced neurotoxicity in AD. In concordance with these findings, drug design strategies
reshaped against the particular step of the neurotoxic cascade.
In overall, it is well-established that AD is associated with multiple etiologies and
pathophysiologic mechanisms. Consequently, therapeutics that could modulate
simultaneously multiple targets and have potential to modify disease course including
cholinesterases, Aβ aggregation, ROS, metal ions and neurotoxic complications may enhance
efficacy of pharmacotherapy [13-15]. With this paradigm, the “multi-target-directed ligands”
(MTDLs) strategy encouraged medicinal chemist to rationally design hybrid molecules
exhibiting various pharmacological functions. Using two or several structural features of two
compounds with specific activity into a single molecule to reach the desired dual or multiple
complementary biological activities; in most cases, at least one is directly related to an AChE
3

inhibition [16-18]. For example, tacrine-melatonin and tacrine-ferulic acid hybrids have been
designed as potent cholinesterase inhibitors with antioxidant property [19]. Lipocrine (Fig. 1)
which is a conjugate of tacrine (a potent AChE inhibitor) and lipoic acid (LA, an antioxidant
and radical scavenger), showed effective inhibition against AChE, BuChE, AChE-induced
Aβ aggregation, and ability to protect cells against ROS [20].
Coumarins with benzopyranone framework are widely distributed in nature and possess a
wide range of biological activities [21-22]. In particular, some pharmacological properties of
coumarins are associated with neurological diseases especially for AD [23-26]. It has been
observed that 3-arylcoumarins such as AP2469 (Fig. 1), beside a remarkable inhibition of
AChE, have shown anti-Aβ aggregation, antioxidant, neuroprotective, and anti-inflammatory
activities, and consequently act as disease-modifying agents [27, 28].
On the other hand, lipoic acid (LA) is naturally occurring substance which prescribed as a
supplement, possessing diverse pharmacologic and antioxidant properties. LA antioxidant
properties have been related to the mitochondria-targeted endogenous antioxidant, free
radicals scavenging in lipid and aqueous mediums, and metal-chelating properties [29].
However, there are no significant results of clinical trials for LA benefit in dementia [30], it
suggests that single antioxidants may not be sufficient for the treatment of neurodegenerative
diseases, while optimal hybridization of antioxidants with compounds which are able to
modulate different AD crucial targets may provide a synergistic effect to display a multi-
target profile. By this concept, several studies have demonstrated the beneficial properties of
LA derivatives in several models of oxidative stress and neuroprotection [31-34].
Continuing with our interest in the development of new structures for the potential
treatment of AD [35-40], we focused our efforts on the development of a novel series of 3-
arylcoumarins as new MTDLs with additive neuroprotective effects through hybridization
approach. To reach this purpose, LA as an antioxidant and the 3-arylcoumarin scaffold as
AChE inhibitor were connected together by application of click reaction (Fig. 1). Thus we
report here, the synthesis and biological evaluation of designed compounds 4 with potential
application in the AD.
2. Results and discussion
2.1. Chemistry
4

As illustrated in Scheme 1, the target compounds 4a-o were prepared from key
intermediates 1 and 3. Initially, 5-(1,2-dithiolan-3-yl)-N-(prop-2-yn-1-yl)pentanamide (1)
was synthesized through the coupling reaction of lipoic acid (LA) with a slight excess of
propargylamine in the presence of N,N'-dicyclohexylcarbodimide (EDCI) and 4-
dimethylaminopyridine (DMAP). The 3-(4-hydroxyphenyl)coumarins 2a-e were synthesized
by the Perkin–Oglialoro reaction of 4-hydroxyphenyl acetic acid with salicylaldehyde
derivatives or 2-hydroxy-1-naphthaldehyde in acetic anhydride and sodium acetate. In the
next step, the bromoalkoxy intermediates 3 were prepared via the reaction of 3-(4-
hydroxyphenyl)coumarins 2 with an excess amount of dibromoalkanes and anhydrous
K2CO3 in refluxing acetone. At the final step, the bromoalkoxy compounds 3 underwent to
reaction with sodium azide and subsequent click reaction with propargylamide 1 in the
presence of Cu(II) and sodium ascorbate to afford target compounds 4a-o. All synthesized
compounds were characterized by spectroscopic methods including IR, 1H and 13C NMR, and
CHN analysis.
2.2. Biological screenings
2.2.1. Neuroprotection against H2O2-induced cell death in PC12 cells
The potential effect of all compounds 4a-o to protect PC12 cells against oxidative stress
generated by H2O2 was evaluated at the concentrations of 1, 5, 10, 20 and 50 µM, and
quercetin was used as reference compound. As seen in Table 1, most of the tested compounds
especially compounds 4a, 4d, 4e, 4g, 4i and 4n significantly increased PC12 cell viability in
the presence of H2O2 in a concentration-dependent manner. Among the tested compounds,
compound 4i could significantly protect neuronal cells even at low concentration of 1 µM,
greater than quercetin and other compounds. Compound 4b, 4j and 4o showed slightly lower
protective effect than quercetin at all concentrations. Compounds 4c, 4h, 4k, and 4m did not
show any significant effect against H2O2-induced cell death in PC12 cells.
2.2.2. Anti-cholinesterase activity
All synthesized compounds 4a-o were evaluated against AChE and BuChE in comparison
with donepezil as standard drug. The results were summarized in Table 2 as IC50 values. It
5

was observed that the activity of the compounds was strongly affected by the length of the
carbon spacer. Among the tested compounds, compounds 4f-j with four carbons linker were
more active than their counterparts with three or five carbons linkers. These compounds
showed approximately close results (IC50 = 7.3-12.6 µM) which were less affected by the
substituents. The similar trend was also observed against BuChE. The most active derivative
in this series was benzo[f]coumarin 4j (IC50 values= 7.3 and 68.6 µM for AChE and BuChE,
respectively).
The enzymatic inhibition mechanism was studied by the kinetic study of compound 4j as
the most potent AChE inhibitor at three increasing concentrations of acetylthiocholine
(ATCh) and the secondary plot was used for calculation of steady-state inhibition constant of
compound 4j. The graphical steady-state inhibition data of compound 4j for AChE is shown
in Figure 2. The results showed an increase in the slope and intercept at higher
concentrations of compound 4j, which indicated a mixed type inhibitory activity for
compound 4j with binding at both CAS and PAS of AChE. To further analyze the
dissociating constant (Ki) was obtained by Lineweaver–Burk secondary plot of compound
4j (Fig. 2, Ki = 10.98 µM).
2.2.2.1. Docking study with AChE
By molecular docking study, the putative binding mode of the most active compound 4j
was simulated. As shown in Figure 3, compound 4j exhibited multiple binding modes with
both CAS and PAS along with the mid-gorge of AChE. The benzocoumarin ring was bound
to CAS via a classic π-π stacking interaction with Tyr120 and also stacked to anionic active
site residues Phe329 and Trp83. In the PAS, there was hydrogen bond interaction with the
carbonyl group of Phe287 and the oxygen atom of the phenyl ether. The triazole moiety made
a π-π stacking with Trp278 and also the 1,2-dithiolane ring bonded with Tyr69 by van der
Waals interactions. The coumarin moiety was also involved in van der Waals interactions
with CAS residues including Phe330 and Asp71.
2.3. Biological evaluation of selected compounds 4i and 4j
6

As described above, the primarily biological screening showed that most of compound can
protect PC12 neuronal cells against oxidative stress. However, only few compounds (4f-j)
containing a 4-carbons spacer (n=4) had good inhibitory potency against AChE. Among
these analogs, compound 4i with good neuroprotective activity and compound 4j with the
highest anti-AChE potency was selected for further biological evaluations.
2.3.1. Effect on Aβ peptide aggregation
The representative compounds 4i and 4j were tested for their ability to inhibit amyloid-beta
(Aβ) aggregation. The potential of compounds to inhibit self and AChE-induced Aβ-
aggregation was evaluated using thioflavin T (ThT) fluorometric assay. The obtained results
were compared with donepezil and rifampicin as reference compounds and presented in
Table 3. While the 8-methoxycoumarin derivative 4i had a good effect on self-aggregation
and AChE-induced aggregation of Aβ peptide, the benzocoumarin analog 4j showed weak or
no activity. The effect of compound 4i on Aβ-aggregation was superior to those of reference
compounds donepezil and rifampicin.
2.3.2. Neuroprotective activity against Aβ1-42-induced cytotoxicity in SH-SY5Y cells
The neuroprotective effects of compounds 4i and 4j against Aβ1-42-induced cytotoxicity in
human neuroblastoma SH-SY5Y cells were tested. According to the results presented in Fig.
4, compounds 4i and 4j showed significant protective activity with the percentage of cell
viability of 56.2 and 52.6% respectively at the concentrations of 10 µM, slightly less than
reference drug donepezil (60.8%). The results suggested that target compounds 4i and 4j can
protect neuronal cells against Aβ-induced oxidative stress.
2.3.3. Antioxidant activity
The antioxidant activity of compound 4j was evaluated by measuring its total antioxidant
power using Ferric Reducing Antioxidant Power (FRAP) assay in comparison with well-
known antioxidants such as lipoic acid, ascorbic acid, and quercetin (Table 4). Moreover, the
antioxidant activity of compound 4j in a cellular context was also evaluated. The activity
against intracellular ROS formation evoked by exposure of PC12 cells to hydrogen peroxide
7

was investigated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay at a
concentration range of the tested compound that did not affect cell viability (1, 5, 10 and 50
µM).
The results presented in Table 4 revealed that compound 4j displays high reducing power
close to the tested antioxidants. In the intracellular ROS formation assay by DCFH-DA
method, the reducing of ROS production by compound 4j was in a concentration-independent
manner and the most effective inhibitory concentration was 5 µM. In this concentration,
intracellular ROS generation was significantly (P > 0.01) lower than that in the H2O2 group
(Fig. 5). While LA showed the same inhibitory effect at the highest concentration used (50
µM). Results indicated that the tested compound 4j could also decrease the level of
endogenous ROS which is formed as a natural byproduct of the oxygen metabolism in cells.
2.3.4. Metal-chelating properties
It has been also identified that abnormally high levels of bio-metal ions in the AD patient
brains is closely associated with the formation of Aβ aggregates, which promoted
inflammation and neurotoxicity, leading to dysfunction and cell death [41]. Therefore,
reducing the concentration of bio-metals by metal chelators is a rational therapeutic approach
against AD pathogenesis.
The metal-binding ability of the selected compound 4j was studied by UV–vis spectroscopy
to see whether our promising molecule could decrease metal ion levels and improve
antioxidant status in AD. The bio-metals such as Mg+2, Ca+2, Al+3, Cu+2, Fe+2, and Zn+2
which have been found in Aβ plaques were studied. The results in Figure 6 show that
compound 4j produces the maximum absorption peak at 374 nm. After the addition of FeSO4
or MgCl2, the maximum absorptions shifted to 368 nm and the height of the peak decreased,
demonstrating the formation of 4j–Fe(II) or 4j–Mg(II) complexes. When the same
experiment was performed using compound 4j and CuSO4, AlCl3, CaCl2 or ZnCl2, the height
of the peak slightly decreased. While, the maximum absorption at 374 nm did not show
obvious shift, suggesting that 4j could not make interaction with these metal ions. To
determine the stoichiometry of the 4j–Fe(II) complex, the absorbance change at 368 nm was
plotted versus the mole fraction of Fe+2 / 4j. As seen in Fig. 6B, two straight lines intersected
at a mole fraction of 1, revealing a 1:1 stoichiometry for complex 4j–Fe+2.
8

3. Conclusion
In summary, a series of triazole-containing 3-phenylcoumarin–lipoic acid conjugates were
synthesized as multifunctional anti-Alzheimer agents. The biological results showed that
most of the compounds can protect PC12 neuronal cells against oxidative stress. However,
only compounds with four carbons spacer had inhibitory potency against AChE. The
benzo[f]coumarin derivative 4j showed the highest inhibitory activity towards AChE (IC50 =
7.3 µM). In addition, both intracellular ROS formation and FRAP assays besides metal
chelating property revealed an excellent direct antioxidant profile of compound 4j.
Compound 4j also could significantly protect neurons against Aβ- and H2O2-induced cell
death. Besides 4j, the 8-methoxy analog 4i found to be an effective inhibitor against Aβ-
aggregation. These results make compounds 4i and 4j as promising anti-AD agents for
further developments.
4. Experimental
4.1. Chemistry
Solvents and chemical reagents were purchased from Merck and used without further
purification. Melting points were measured on a Kofler hot stage apparatus. The IR
spectra (KBr disks) were taken by Nicolet FT-IR Magna 550 spectrometer. Nuclear Magnetic
Resonance (NMR) spectra were recorded on Brucker 500 MHz spectrometer. The chemical
shifts (δ) and coupling constants (J) are given in parts per million (ppm) and Hertz (Hz),
respectively. The results of elemental analyses (C, H, N) were within ± 0.4% of the
calculated values and determined with CHN-Rapid Heraeus Elemental Analyzer.
4.1.1. Preparation of 5-(1,2-dithiolan-3-yl)-N-(prop-2-yn-1-yl)pentanamide (1)
A mixture of lipoic acid (LA, 1 mmol), EDCI (1.2 mmol), and DMAP (1.2 mmol) in
anhydrous CH2Cl2 (10 mL) was cooled at 0 ºC, then a solution of propargylamine (1.2
mmol) in dry CH2Cl2 (1 mL) was added dropwise under magnetic stirring. The mixture
was stirred at room temperature for 12 h. After filtration, the solvent was removed by
rotary evaporator and the crude product was purified by column chromatography on silica
9

gel eluting with ethyl acetate/petroleum ether (1:2), to afford compound 1 as a yellow
solid [42].
4.1.2. General procedure for the preparation of 3-(4-hydroxyphenyl)coumarin derivatives
2a-e
A solution containing 4-hydroxyphenylacetic acid (1 mmol), desired salicylaldehyde
derivative or 2-hydroxy-1-naphthaldehyde (1 mmol) and anhydrous sodium acetate (92
mg, 1.4 mmol) in acetic anhydride (1.2 mL) was refluxed. After 2 h, the reaction mixture
was cooled, neutralized with an aqueous solution of NaHCO3 (10%) and filtered off. The
obtained solid was washed with water, ethyl acetate and dried to give compounds 2a-e
[43].
4.1.3. General procedure for the preparation of 3-(4-(2-bromoalkanoxy)phenyl)coumarin
derivatives 3a-o
A mixture of substituted 3-(4-hydroxyphenyl)coumarin 2a-e (5 mmol) with suitable
dibromoalkane (50 mmol) and anhydrous K2CO3 (1.4 g, 10 mmol) in acetone (15 mL)
was refluxed for about 4 h. After completion of the reaction (TLC controlled), the
mixture was filtered and evaporated under reduced pressure. The residue was triturated
with n-hexane and the precipitated product was filtered to afford desired compounds 3a-o
as white solids [44].
4.1.4. General procedure for the preparation of final compounds 4a-o
The bromoalkanoxy intermediate 3a-o (1 mmol), NEt3 (1 mmol) and NaN3 (1 mmol)
in t-BuOH/H2O (1: 1) were heated at 70 °C for 30 min, then 5-(1,2-dithiolan-3-yl)-N-
(prop-2-yn-1-yl)pentanamide (1, 1 mmol), sodium ascorbate (0.2 mmol), and CuSO4
(0.2 mmol) were added to the mixture. After completion of the reaction monitored by
TLC (12–48 h), cold water was added to the mixture and the precipitated solid was
filtered and washed with water. The crude product was purified by flash
chromatography on silica gel eluting with ethyl acetate/petroleum ether (1:1) to give
compounds 4a-o [45].
10

4.1.4.1. 5-(1,2-Dithiolan-3-yl)-N-((1-(3-(4-(2-oxo-2H-chromen-3-yl)phenoxy)propyl)-1H-
1,2,3-triazol-4-yl)methyl)pentanamide (4a)
Off white solid; mp 138-139 ºC; IR (KBr, cm-1) νmax: 3299 (NH), 2925 (C-H), 1720
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.26 (bs, 1H, NH), 8.19 (s, 1H, H4), 7.93 (s, 1H,
triazole), 7.76 (d, 1H, J=7.5 Hz, H5), 7.69 (d, 2H, J=8.0 Hz, phenyl), 7.60 (t, 1H, J=7.5 Hz,
H7), 7.42 (d, 1H, J=7.5 Hz, H8), 7.37 (t, 1H, J=7.0 Hz, H6), 6.99 (d, 2H, J=8.0 Hz, phenyl),
4.51 (bs, 2H, CH2-N), 4.27 (d, 2H, J=5.0 Hz, CH2-NH), 4.03 (t, 2H, J=6.0 Hz, CH2-O), 3.55-
362 (m, 1H, CH-S), 3.10-3.12 (m, 2H, CH2-S), 2.36-2.40 (m, 1H, S-CH2-CH2), 2.28 (bs, 2H,
CH2, aliphatic chain), 2.08 (t, 2H, J=7.0 Hz, CH2-CO), 1.80-1.84 (m, 1H, CH2-CH2-S), 1.59-
1.65 (m, 1H, CO-CH2-CH2), 1.45-1.58 (m, 3H, aliphatic chain), 1.25-1.32 (m, 2H, aliphatic
13
chain). C NMR (DMSO-d6, 125 MHz) δ: 171.82, 159.76, 158.62, 145.02, 139.75, 131.27,
129.75, 128.36, 126.98, 124.48, 122.78, 119.57, 115.71, 114.20, 64.60, 56.05, 46.36, 38.00,
34.94, 34.06, 34.00, 29.42, 28.21, 24.87. Anal. Calcd for C29H32 N4O4S2: C, 61.68; H, 5.71;
N, 9.92. Found: C, 61.83; H, 5.52; N, 9.66.
4.1.4.2. N-((1-(3-(4-(6-bromo-2-oxo-2H-chromen-3-yl)phenoxy)propyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (4b)
Off white solid; mp 156-158 ºC; IR (KBr, cm-1) νmax: 3296 (NH), 2937 (C-H), 1722
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.27 (bs, 1H, NH), 8.14 (s, 1H, H5), 7.98 (s, 1H,
H4), 7.94 (s, 1H, triazole), 7.72-767 (m, H7 and 2H phenyl), 7.38 (d, 1H, J=7.5 Hz, H8), 7.00
(d, 2H, J=6.5 Hz, phenyl), 4.51 (bs, 2H, CH2-N), 4.27 (bs, 2H, CH2-NH), 4.02 (bs, 2H, CH2-
O), 3.56 (m, 1H, CH-S), 3.14-3.10 (m, 2H, CH2-S), 2.36 (m, 1H, CH2-CH2-S), 2.28 (bs, 2H,
CH2, CH2-CH2-N), 2.08 (bs, 2H, CH2-CO), 1.83 (bs, 1H, CH2-CH2-S), 1.62 (bs, 1H, CO-
CH2-CH2), 1.50 (bs, 3H, aliphatic chain), 1.31 (bs, 2H, aliphatic chain). 13C NMR (DMSO-
d6, 125 MHz) δ: 171.87, 158.88, 151.73, 145.06, 137.92, 133.66, 130.41, 129.85, 127.39,
126.26, 122.74, 121.52, 118.13, 116.03, 114.25, 64.62, 56.09, 46.40, 38.04, 34.97, 34.04,
29.44, 28.26, 24.91. Anal. Calcd for C29H31BrN4O4S2: C, 54.12; H, 5.16; N, 8.71. Found: C,
54.34; H, 5.19; N, 8.47.
11

4.1.4.3. 5-(1,2-Dithiolan-3-yl)-N-((1-(3-(4-(6-nitro-2-oxo-2H-chromen-3-yl)phenoxy)propyl)-
1H-1,2,3-triazol-4-yl)methyl)pentanamide (4c)
Yellow solid; mp 148-150 ºC; IR (KBr, cm-1) νmax: 3296 (NH), 2934 (C-H), 1722, 1627
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.72 (s, 1H, H5), 8.39-8.37 (m, 2H, H7, H4), 8.27
(bs, 1H, NH), 7.94 (s, 1H, triazole), 7.69 (d, 2H, J=8.0 Hz, phenyl), 7.63 (d, 1H, J=9.0 Hz,
H8), 7.02 (d, 2H, J=8.0 Hz, phenyl), 4.53 (t, 2H, J=6.5 Hz, CH2-N ), 4.27 (d, 2H, J=5.0 Hz,
CH2-NH), 4.04 (bs, 2H, CH2-O), 3.57 (quintet, 1H, J=6.0 Hz, CH-S), 3.16-3.09 (m, 2H, CH2-
S), 2.38 (sextet, 1H, J=6.0 Hz, S-CH2-CH2), 2.29 (t, 2H, J=5.5 Hz, CH2, aliphatic chain),
2.08 (t, 2H, J=6.5 Hz, CH2-CO), 1.84-1.81 (m, 1H, S-CH2-CH2), 1.63-1.61 (m, 1H, CO-CH2-
CH2), 1.51 (bs, 3H, aliphatic chain), 1.33-1.30 (m, 2H, aliphatic chain). 13C NMR (DMSO-
d6, 125 MHz) δ: 171.86, 159.05, 158.88, 156.33, 145.06, 143.59, 137.75, 129.88, 128.06,
126.27, 124.13, 122.92, 119.99, 117.33, 114.34, 64.66, 56.09, 46.40, 38.04, 34.97, 34.04,
29.44, 28.26. Anal. Calcd for C29H31N5O6S2: C, 57.13; H, 5.13; N, 11.49. Found: C, 57.33;
H, 5.22; N, 11.36.
4.1.4.4. 5-(1,2-Dithiolan-3-yl)-N-((1-(3-(4-(8-methoxy-2-oxo-2H-chromen-3-
yl)phenoxy)propyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4d)
Off white solid; mp 157-160 ºC; IR (KBr, cm-1) νmax: 3285 (NH), 2924 (C-H), 1716
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.27 (bs, 1H, NH), 8.18 (s, 1H, H4), 7.94 (s, 1H,
triazole), 7.69 (d, 2H, J=8.5 Hz, phenyl), 7.31-7.28 (m, 3H, H5, H6, H7), 6.99 (d, 2H, J=8.0
Hz, phenyl), 4.51 (t, 2H, J=6.5 Hz, CH2-N), 4.27 (d, 2H, J=5.5 Hz, CH2-NH), 4.02 (t, 2H,
J=5.5 Hz, CH2-O), 3.92 (s, 3H, OCH3), 3.58-3.55 (m, 1H, CH-S), 3.18-3.08 (m, 2H, CH2-S),
2.39-2.35 (m, 1H, CH2-CH2-S), 2.28 (t, 2H, J=6 Hz, CH2, aliphatic chain), 2.08 (t, 2H,
J=6.5Hz, CH2-CO), 1.86-1.80 (m, 1H, CH2-CH2-S), 1.65-1.61 (m, 1H, CH2-CH2-CO), 1.52
(m, 3H, aliphatic chain), 1.31 (m, 2H, aliphatic chain). 13C NMR (DMSO-d6, 125 MHz) δ:
172.17, 158.80, 146.30, 145.27, 129.88, 127.52, 125.01, 119.75, 114.26, 64.67, 56.15, 46.50,
38.04, 35.05, 34.11, 29.52, 28.36, 24.99. Anal. Calcd for C30H34N4O5S2: C, 60.59; H, 5.76;
N, 9.42. Found: C, 60.61; H, 5.91; N, 9.31.
12

4.1.4.5. 5-(1,2-Dithiolan-3-yl)-N-((1-(3-(4-(3-oxo-3H-benzo[f]chromen-2-
yl)phenoxy)propyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4e)
Off white solid; mp 140-141 ºC; IR (KBr, cm-1) νmax: 3295 (NH), 2934 (C-H), 1702
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.94 (s, 1H, H4), 8.72 (d, 1H, J=8.5 Hz,
benzo[f]coumamin), 8.27 (bs, 1H, NH), 8.16 (d, 1H, J=9.0 Hz, benzo[f]coumamin), 8.05 (d,
1H, J=8.0 Hz, benzo[f])coumamin), 7.94 (s, 1H, triazole), 7.85 (d, 2H, J=8.0 Hz, phenyl),
7.73 (t, 1H, J=7.5 Hz, H7), 7.60 (m, 1H, H8 and 1H benzo[f]coumamin), 7.03 (d, 2H, J=8.0
Hz, phenyl), 4.53 (t, 2H, J=6.5 Hz, CH2-N), 4.28 (bs, 2H, CH2-NH), 4.05 (t, 2H, J=6.0 Hz,
CH2-O), 3.56 (quintet, 1H, J=6.0 Hz, CH-S), 3.17-3.05 (m, 2H, CH2-S), 2.37 (sextet, 1H,
J=6.0 Hz, CH2-CH2-S), 2.30 (t, 2H, J=5.5 Hz, CH2, aliphatic chain), 2.09 (t, 2H, J=6.5 Hz,
CH2-CO), 1.82 (sextet, 1H, J=6.0 Hz, CH2-CH2-S), 1.62 (quinted, 1H, J=6.0 Hz, CH2-CH2-
CO), 1.51 (bs, 3H, aliphatic chain), 1.34-1.30 (m, 2H, aliphatic chain). 13C NMR (DMSO-
d6, 125 MHz) δ: 171.87, 159.84, 158.67, 152.21, 145.05, 135.09, 130.16, 128.87, 128.05,
127.20, 125.64, 122.81, 116.37, 114.17, 113.69, 64.62, 56.08, 46.40, 38.03, 34.97, 34.10,
34.03, 29.46, 28.25, 24.91. Anal. Calcd for C33H34N4O4S2: C, 64.47; H, 5.57; N, 9.11.
Found: C, 64.50; H, 5.48; N, 9.28.
4.1.4.6. 5-(1,2-Dithiolan-3-yl)-N-((1-(4-(4-(2-oxo-2H-chromen-3-yl)phenoxy)butyl)-1H-
1,2,3-triazol-4-yl)methyl)pentanamide (4f)
Off white solid; mp 128-130 ºC; IR (KBr, cm-1) νmax: 3298 (NH), 2922 (C-H), 1722
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.26 (bs, 1H, NH), 8.18 (s, 1H, H4), 7.92 (s, 1H,
triazole), 7.75 (d, 1H, J=7.5 Hz, H5), 7.67 (d, 2H, J=8.0 Hz, phenyl), 7.59 (t, 1H, J=7.0 Hz,
H7), 7.40 (d, 1H, J=8.5 Hz, H8), 7.36 (t, 1H, J=7.0 Hz, H6), 7.00 (d, 2H, J=8.0 Hz, phenyl),
4.41 (t, 2H, J=6.5 Hz, CH2-N), 4.27 (d, 2H, J=5.0 Hz, CH2-NH), 4.04 (t, 2H, J=5.5 Hz, CH2-
O), 3.57 (quintet, 1H, J=6.0 Hz, CH-S), 3.17-3.09 (m, 2H, CH2-S), 2.37 (sextet, 1H, J=6.0
Hz, CH2-CH2-SH), 2.09 (t, 2H, J=6.5 Hz, CH2-CO), 1.97 (t, 2H, J=6.5 Hz, CH2-CH2-N),
1.83 (sextet, 1H, CH2-CH2-S), 1.72-1.69 (m, 2H, CH2-CH2-O), 1.66-1.62 (m, 1H, CH2-CH2-
CO), 1.51 (bs, 3H, aliphatic chain), 1.30-1.38 (m, 2H, aliphatic chain). 13C NMR (DMSO-
d6, 125 MHz) δ: 171.84, 159.79, 158.83, 152.68, 144.96, 139.07, 129.75, 128.94, 126.78,
124.38, 122.58, 119.59, 115.85, 114.97, 66.87, 56.08, 48.91, 38.04, 34.96, 34.09, 34.04,
13

28.24, 26.56, 25.63, 24.91. Anal. Calcd for C30H34N4O4S2: C, 62.26; H, 5.92; N, 9.68.
Found: C, 62.37; H, 6.07; N, 9.65.
4.1.4.7. N-((1-(4-(4-(6-bromo-2-oxo-2H-chromen-3-yl)phenoxy)butyl)-1H-1,2,3-triazol-
4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (4g)
Off white solid; mp 164 ºC; IR (KBr, cm-1) νmax: 3295 (NH), 2934 (C-H), 1722 (C=O). 1H
NMR (DMSO-d6, 500 MHz) δ: 8.27 (bs, 1H, NH), 8.14 (s, 1H, H5), 7.99 (s, 1H, H4), 7.92 (s,
1H, triazole), 7.72 (d, 1H, J=8.5 Hz, H7), 7.66 (d, 2H, J=7.5 Hz, phenyl), 7.38 (d, 1H, J=8.5
Hz, H8), 7.01 (d, 2H, J=7.5 Hz, phenyl), 4.40 (t, 2H, J=6.5 Hz, CH2-N), 4.27 (d, 2H, J=5.0
Hz, CH2-NH), 4.03 (t, 2H, J=6.0 Hz, CH2-O), 3.58 (m, 1H, CH-S), 3.16-3.08 (m, 2H, CH2-
S), 2.38 (m, 1H, CH2-CH2-S), 2.09 (bs, 2H, CH2-CO), 1.96 (t, 2H, J=6.5 Hz, CH2-CH2-N),
1.82-1.84 (m, 1H, CH2-CH2-S), 1.70-1.63 (m, 2H, O-CH2-CH2, 1H, CH2-CH2-CO), 1.51 (bs,
3H, aliphatic chain), 1.32 (bs, 2H, aliphatic chain). 13C NMR (DMSO-d6, 125 MHz) δ:
171.93, 159.35, 158.99, 151.73, 144.96, 137.80, 133.44, 129.85, 127.42, 126.53, 122.70,
121.43, 118.00, 116.11, 114.27, 66.90, 56.09, 48.92, 45.69, 38.05, 34.97, 34.05, 28.26, 26.56,
25.62, 24.92. Anal. Calcd for C30H33BrN4O4S2: C, 54.79; H, 5.06; N, 8.52. Found: C, 54.49;
H, 5.31; N, 8.27.
4.1.4.8. 5-(1,2-Dithiolan-3-yl)-N-((1-(4-(4-(6-nitro-2-oxo-2H-chromen-3-yl)phenoxy)butyl)-
1H-1,2,3-triazol-4-yl)methyl)pentanamide (4h)
Yellow solid; mp 155-158 ºC; IR (KBr, cm-1) νmax: 3289 (NH), 2936 (C-H), 1721, 1627
(C=O). 1H NMR (DMSO-d6, 500 MHz) δ: 8.72 (s, 1H, H5), 8.38 (d, 1H, J=9.5 Hz, H7), 8.36
(s, 1H, H4), 8.26 (bs, 1H, NH), 7.91 (s, 1H, triazole), 7.69 (d, 2H, J=8.0 Hz, phenyl), 7.62 (d,
1H, J=9.0 Hz, H8), 7.03 (d, 2H, J=8.0 Hz, phenyl), 4.41 (t, 2H, J=6.5 Hz, CH2-N), 4.27 (d,
2H, J=5.0 Hz, CH2-NH), 4.05 (t, 2H, J=6.0 Hz, CH2-O), 3.57 (quintet, 1H, J=6.5 Hz, CH-S),
3.17-3.09 (m, 2H, CH2-S), 2.38 (sextet, 1H, J=6.0 Hz, CH2-CH2-S), 2.09 (t, 2H, J=6.5 Hz,
CH2-CO), 1.97 (t, 2H, J=6.5 Hz, CH2-CH2-N), 1.83 (sextet, 1H, CH2-CH2-S), 1.71 (sextet,
2H, CH2-CH2-O), 1.66-1.62 (m, 1H, CH2-CH2-CO), 1.52 (bs, 3H, aliphatic chain), 1.34-1.31
(m, 2H, aliphatic chain). 13C NMR (DMSO-d6, 125 MHz) δ: 171.86, 159.25, 158.87,
156.30, 144.96, 143.58, 129.84, 128.07, 126.05, 124.00, 122.71, 119.99, 117.32, 114.30,
14

66.92, 56.08, 48.91, 43.35, 38.04, 34.96, 34.09, 34.03, 28.23, 26.54, 25.61, 24.91. Anal.
Calcd for C30H33N5O6S2: C, 57.77; H, 5.33; N, 11.23. Found: C, 57.42; H, 5.13; N, 10.95.
4.1.4.9. 5-(1,2-Dithiolan-3-yl)-N-((1-(4-(4-(8-methoxy-2-oxo-2H-chromen-3-
yl)phenoxy)butyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4i)
Off white solid; mp 129 ºC; IR (KBr, cm-1) νmax: 3295 (NH), 2931 (C-H), 1717 (C=O). 1H
NMR (DMSO-d6, 500 MHz) δ: 8.26 (bs, 1H, NH), 8.17 (s, 1H, H4), 7.91 (s, 1H, triazole),
7.69 (d, 2H, J=8.0 Hz, phenyl), 7.31-7.28 (m, 3H, H5, H6, H7), 7.00 (d, 2H, J=8.0 Hz,
phenyl), 4.40 (t, 2H, J=6.5Hz, CH2-N), 4.27 (d, 2H, J=5.0 Hz, CH2-NH), 4.04 (t, 2H, J=6Hz,
CH2-O), 3.92 (s, 3H, O-CH3), 3.52-3.51 (m, 1H, CH-S), 3.20-3.07 (m, 2H, CH2-S), 2.40-2.35
(m, 1H, CH2-CH2-S), 2.09 (t, 2H, J=6.5 Hz, CH2-CO), 1.97 (t, 2H, J=6.5 Hz, CH2-CH2-N),
1.83 (sextet, 1H, CH2-CH2-S), 1.70 (t, 2H, O-CH2-CH2), 1.64-160 (m, 1H, CH2-CH2-CO),
1.59-1.51 (m, 3H, aliphatic chain), 1.32 (m, 2H, aliphatic chain). 13C NMR (DMSO-d6, 125
MHz) δ: 172.28, 160.92, 157.30, 150.56, 148.09, 145.22, 139.94, 139.38, 132.05, 129.69,
126.12, 122.36, 119.21, 115.26, 114.13, 66.64, 56.07, 48.87, 45.72, 38.07, 34.95, 34.08,
29.51, 28.23, 26.54, 25.62, 24.89. Anal. Calcd for C31H36N4O5S2: C, 61.16; H, 5.96; N, 9.20.
Found: C, 60.87; H, 5.77; N, 9.19.
4.1.4.10. 5-(1,2-Dithiolan-3-yl)-N-((1-(4-(4-(3-oxo-3H-benzo[f]chromen-2-
yl)phenoxy)butyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4j)
Off white solid; mp 141-143 ºC; IR (KBr, cm-1) νmax: 3290 (NH), 2924 (C-H), 1720
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.94 (s, 1H, H4), 8.73 (d, 1H, J=7.5 Hz,
benzo[f]coumamin), 8.28 (bs, 1H, NH), 8.16 (d, 1H, J=8.0 Hz, benzo[f]coumamin), 8.05 (d,
1H, J=7.0 Hz, benzo[f]coumamin), 7.92 (s, 1H, triazole), 7.84 (d, 2H, J=7.0 Hz, phenyl),
7.73 (bs, 1H, H7), 7.62-7.59 (m, 1H, H8 and 1H benzo[f]coumamin), 7.03 (d, 2H, J=7.5 Hz,
phenyl), 4.42 (bs, 2H, CH2-N), 4.28 (bs, 2H, CH2-NH), 4.06 (bs, 2H, CH2-O), 3.57 (m, 1H,
CH-S), 3.15-3.10 (m, 2H, CH2-S), 2.38-2.37 (m, 1H, CH2-CH2-S), 2.09 (bs, 2H, CH2-CO),
1.96 (bs, 2H, CH2-CH2-N), 1.83 (m, 1H, CH2-CH2-S), 1.72 (bs, 2H, O-CH2-CH2), 1.63 (bs,
1H, CH2-CH2-CO), 1.52 (bs, 3H, aliphatic chain), 1.32 (bs, 2H, aliphatic chain). 13C NMR
(DMSO-d6, 125 MHz) δ: 171.90, 159.87, 158.88, 152.20, 144.99, 135.02, 130.20, 129.98,
15

128.88, 128.08, 127.00, 125.69, 122.78, 116.26, 114.14, 113.72, 66.91, 56.10, 48.96, 38.06,
34.99, 34.06, 28.27, 26.59, 25.67, 24.94. Anal. Calcd for C34H36N4O4S2: C, 64.94; H, 5.77;
N, 8.91. Found: C, 65.19; H, 5.85; N, 9.11.
4.1.4.11. 5-(1,2-Dithiolan-3-yl)-N-((1-(5-(4-(2-oxo-2H-chromen-3-yl)phenoxy)pentyl)-1H-
1,2,3-triazol-4-yl)methyl)pentanamide (4k)
Off white solid; mp 123-125 ºC; IR (KBr, cm-1) νmax: 3292 (NH), 2923 (C-H), 1721
(C=O). 1H NMR (DMSO-d6, 500 MHz) δ: 8.26 (t, 1H, J=5.5 Hz, NH), 8.18 (s, 1H, H4), 7.88
(s, 1H, triazole), 7.75 (d, 1H, J=7.0 Hz, H5), 7.67 (d, 2H, J=8.0 Hz, phenyl), 7.59 (t, 1H,
J=7.5 Hz, H7), 7.40 (d, 1H, J=8.0 Hz, H8), 7.36 (t, 1H, J=7.5 Hz, H6), 6.98 (d, 2H, J=8.0 Hz,
phenyl), 4.34 (t, 2H, J=7.0 Hz, CH2-N ), 4.26 (d, 2H, J=5.5 Hz, CH2-NH ), 3.99 (t, 2H, J=6.0
Hz, CH2-O), 3.57 (m, 1H, CH-S), 3.11-3.06 (m, 2H, CH2-S), 2.38 (sextet, 1H, J=6 Hz, CH2-
CH2-S), 2.08 (t, 2H, J=7 Hz, CH2-CO), 1.89-1.72 (m, 2H, N-CH2-CH2 and 2H, O-CH2-CH2
and 1H, CH2-CH2-S), 1.67-1.61 (m, 1H, CH2-CH2-CO), 1.53-1.47 (m, 3H, aliphatic chain),
1.41-1.29 (m, 4H, aliphatic chain). 13C NMR (DMSO-d6, 125 MHz) δ: 171.88, 159.83,
158.95, 152.69, 144.93, 139.12, 131.32, 129.77, 128.41, 126.37, 126.37, 124.60, 119.61,
115.68, 114.16, 67.32, 56.10, 49.13, 45.72, 38.05, 34.98, 34.10, 34.05, 29.47, 28.25, 27.97,
24.93, 22.52. Anal. Calcd for C31H36N4O4S2: C, 62.81; H, 6.12; N, 9.45. Found: C, 63.04;
H, 6.20; N, 9.54.
4.1.4.12. 5-(1,2-Dithiolan-3-yl)-N-((1-(5-(4-(6-bromo-2-oxo-2H-chromen-3-
yl)phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4l)
Off white solid; mp 164-165 ºC; IR (KBr, cm-1) νmax: 3298 (NH), 2933 (C-H), 1719
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.26 (bs, 1H, NH), 8.14 (s, 1H, H5), 7.99 (s, 1H,
H4), 7.89 (s, 1H, triazole), 7.72 (d, 1H, J=8.5 Hz, H7), 7.66 (d, 2H, J=7.5 Hz, phenyl), 7.39
(d, 1H, J=8.5 Hz, H8), 7.00 (d, 2H, J=7.5 Hz, phenyl), 4.35 (bs, 2H, CH2-N), 4.27 (bs, 2H,
CH2-NH), 4.01 (bs, 2H, CH2-O), 3.56 (m, 1H, CH-S), 3.16-3.11 (m, 2H, CH2-S), 2.40-2.38
(m, 1H, CH2-CH2-S), 2.09 (bs, 2H, CH2-CO), 1.87-1.83 (m, 2H, N-CH2-CH2 and 1H, CH2-
CH2-S), 1.76 (bs, 2H, CH2-CH2-O), 1.64 (bs, 1H, CH2-CH2-CO), 1.52-1.32 (m, 7H, aliphatic
13
chain). C NMR (DMSO-d6, 125 MHz) δ: 171.82, 159.30, 151.69, 144.90, 137.61, 133.47,
16

130.22, 127.41, 126.33, 122.50, 121.52, 118.14, 117.89, 115.99, 114.26, 67.31, 56.07, 49.10,
45.19, 38.02, 34.95, 34.03, 29.44, 28.23, 27.93, 24.90, 22.49. Anal. Calcd for
C31H35BrN4O4S2: C, 55.44; H, 5.25; N, 8.34. Found: C, 55.34; H, 5.31; N, 8.53.
4.1.4.13. 5-(1,2-Dithiolan-3-yl)-N-((1-(5-(4-(6-nitro-2-oxo-2H-chromen-3-
yl)phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4m)
Yellow solid; mp 150-152 ºC; IR (KBr, cm-1) νmax: 3292 (NH), 2935 (C-H), 1725 (C=O).
1H NMR (DMSO-d6, 500 MHz) δ: 8.72 (s, 1H, H5), 8.38 (d, 1H, J=9.0 Hz, H7), 8.36 (s, 1H,
H4), 8.26 (bs, 1H, NH), 7.89 (s, 1H, triazole), 7.68 (d, 2H, J=8.0 Hz, phenyl), 7.63 (d, 1H,
J=9.0 Hz, H8), 7.02 (d, 2H, J=8.0 Hz, phenyl), 4.35 (t, 2H, J=6.5 Hz, CH2-N), 4.27 (d, 2H,
J=5.5 Hz, CH2-NH), 4.01 (t, 2H, J=6.0 Hz, CH2-O), 3.56 (quintet, 1H, J=6.5 Hz, CH-S),
3.17-3.09 (m, 2H, CH2-S), 2.41-2.36 (sextet, 1H, J=6.0 Hz, CH2-CH2-S), 2.09 (t, 2H, J=6.5
Hz, CH2-CO), 1.88-1.75 (m, 2H, N-CH2-CH2 and 2H, O-CH2-CH2 and 1H, CH2-CH2-S),
1.65-1.62 (m, 1H, CH2-CH2-CO), 1.51-1.30 (m, 7H, aliphatic chain). 13C NMR (DMSO-d6,
125 MHz) δ: 171.85, 159.34, 158.89, 156.30, 144.92, 143.58, 129.84, 128.09, 125.97,
122.65, 120.00, 117.31, 114.29, 67.35, 56.09, 49.11, 44.98, 38.04, 34.96, 34.04, 29.45, 28.24,
27.94, 24.99, 22.55. Anal. Calcd for C31H35N5O6S2: C, 58.38; H, 5.53; N, 10.98. Found: C,
58.17; H, 5.49; N, 11.01.
4.1.4.14. 5-(1,2-Dithiolan-3-yl)-N-((1-(5-(4-(8-methoxy-2-oxo-2H-chromen-3-
yl)phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4n)
Off white solid; mp 130-132 ºC; IR (KBr, cm-1) νmax: 3292 (NH), 2935 (C-H), 1725
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.26 (bs, 1H, NH), 8.17 (s, 1H, H4), 7.89 (s, 1H,
triazole), 7.68 (d, 2H, J=8.0 Hz, phenyl), 7.30-7.28 (m, 3H, H5, H6, H7), 6.99 (d, 2H, J=8.5
Hz, phenyl), 4.35 (t, 2H, J=6.5 Hz, CH2-N), 4.26 (d, 2H, J=5.5 Hz, CH2-NH), 4.00 (t, 2H,
J=6.0 Hz, CH2-O), 3.92 (s, 3H, O-CH3), 3.57 (quintet, 1H, J=6.5 Hz, CH-S), 3.17-3.10 (m,
2H, CH2-S), 2.38 (sextet, 1H, J=6.0 Hz, CH2-CH2-S), 2.09 (t, 2H, J=6.5 Hz, CH2-CO), 1.88-
1.63 (m, 2H, N-CH2-CH2 and 2H, O-CH2-CH2, 1H, CH2-CH2-S and 1H, CH2-CH2-CO),
1.51-1.31 (m, 7H, aliphatic chain). 13C NMR (DMSO-d6, 125 MHz) δ: 172.58, 166.07,
163.32, 159.58, 158.97, 151.29, 149.96, 146.37, 139.11, 132.71, 129.76, 126.90, 124.66,
17

119.69, 114.16, 67.32, 56.08, 49.13, 45.69, 38.05, 34.97, 34.05, 29.48, 28.26, 27.97, 24.93,
22.52. Anal. Calcd for C32H38N4O5S2: C, 61.71; H, 6.15; N, 9.00. Found: C, 61.93; H, 6.22;
N, 9.01.
4.1.4.15. 5-(1,2-Dithiolan-3-yl)-N-((1-(5-(4-(3-oxo-3H-benzo[f]chromen-2-
yl)phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (4o)
Off white solid; mp 158-160 ºC; IR (KBr, cm-1) ν
: 3297 (NH), 2927 (C-H), 1717
max
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ: 8.93 (s, 1H, H4), 8.72 (d, 1H, J=8.5 Hz, H5
benzo[f]coumarin), 8.27 (t, 1H, NH), 8.15 (d, 1H, J=9.0 Hz, benzo[f]coumarin), 8.05 (d, 1H,
J=8.0 Hz, benzo[f]coumarin), 7.90 (s, 1H, triazole), 7.84 (d, 2H, J=8.5 Hz, phenyl), 7.734-
7.71 (m, 1H, J=7.5 Hz, H7), 7.64-7.56 (m, 1H, H8 and benzo[f]coumarin), 7.02 (d, 2H, J=8.0
Hz, phenyl), 4.36 (t, 2H, J=7.0 Hz, CH2-N), 4.27 (d, 2H, J=5.5 Hz, CH2-NH), 4.02 (t, 2H,
J=6.5 Hz, CH2-O), 3.56 (m, 1H, CH-S), 3.15-3.07 (m, 2H, CH2-S), 2.36 (sextet, 1H, J=6.0
Hz, S-CH2-CH2), 2.09 (t, 2H, J=7.0 Hz, CH2-CO), 1.89-1.74 (m, 2H, N-CH2-CH2 and 2H, O-
CH2-CH2, 1H, S-CH2-CH2), 1.63-1.61 (m, 1H, CH2-CH2-CO), 1.52-1.30 (m, 7H, aliphatic
13
chain). C NMR (DMSO-d6, 125 MHz) δ: 172.69, 171.85, 159.86, 158.96, 152.17, 144.93,
134.97, 130.12, 129.96, 128.87, 128.04, 126.90, 125.68, 122.75, 116.37, 114.11, 113.71,
67.33, 56.09, 49.13, 45.10, 38.04, 34.97, 34.10, 34.05, 29.49, 28.26, 27.98, 24.92, 22.53.
Anal. Calcd for C35H38N4O4S2: C, 65.40; H, 5.96; N, 8.72. Found: C, 65.44; H, 6.04; N,
8.82.
4.2. Docking study
The Molecular Operating Environment (MOE) software (Chemical Computing Group,
Montreal, Canada) was used for docking simulations. The crystal structure of Torpedo
californica acetylcholinesterase (PDB cod: 1EVE) was used for docking, Autodock
Tools (4.2) [46]. The minimization of the ligand 3D structure was conducted by
Openbabel (2.3.1) [47]. The dimensions of the active site were set as box size: 15×15×15
Å, the center of box: x = 2.023, y = 63.295, z = 67.062 (geometrical center of co-
crystallized ligand). The calculated geometries were established in terms of free energy
18

binding and the best poses were selected for further analysis. All molecular
visualizations were carried out by PyMol molecular graphics system [48].
4.3. Biological evaluations
4.3.1. In vitro AChE/BuChE inhibition assay
The cholinesterases inhibitory activity of compounds 4a-o was evaluated against eelAChE
and eqBuChE using Ellman’s method [49]. A mixture of phosphate buffer (0.1 M, pH 8.0, 2
mL), 5,5´-dithiobis-2-nitrobenzoic acid (DTNB, 60 µL), different concentration of compound
solution (30 µL) and acetylcholinesterase or butyrylcholinesterase (20 µL, 5 IU/mL) was pre-
incubated in 24-well plates for 3 min followed by the addition of the substrate
(acetylthiocholine iodide or butyrylthiocholine iodide, 20 µL). The assay was measured at
412 nm by using a multi-mode reader (BioTek Synergy HT). To evaluate the IC50, the log
concentration versus percentage of inhibition curve was plotted graphically and compared
with donepezil as a reference drug. Experiments were performed in triplicate.
4.3.2. Determination of the inhibitory effect on Aβ1-42 self and hAchE-induced aggregation
The thioflavin T (ThT)-based fluorescence assay was used to investigate the self and
AChE-induced Aβ1-42 aggregation [50]. Amyloid-β protein 1-42 (Sigma A9810, 50 µM) was
dissolved in phosphate buffer Saline (pH = 7.4, PBS, HyClone Thermo Scientific) containing
1% ammonium hydroxide and incubated for 72 h at 37 °C to Aβ prefibrillation. Aβ1-42 (10
µL) with or without human recombinant AChE (0.01 u/mL, Sigma C1682) were incubated in
phosphate buffer (0.05 M, pH = 7.4) at 37 C for 48 h in the absence and presence of the
compounds (100 μM). After incubation, 50 μL of thioflavin T (200 μM, in 50 mM glycine-
NaOH buffer, pH = 8.5) was added to the mixture. The amyloid aggregation was detected by
Microplate Reader (Spectra Max) using excitation/emission at 448 nm/490 nm. Rifampicin
and donepezil were used as reference agents. The percent of inhibitory potency of the tested
compounds on self or AChE-induced Aβ-aggregation was determined by the following
formula: [(IFi/IFo) × 100] where IFi and IFo are the fluorescence intensities obtained for Aβ
± AChE in the presence and in the absence of inhibitors.
19

4.3.3. Neuroprotection assay against H2O2-induced cell death in PC12 cells
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was
performed to evaluate cell viability of rat differentiated PC12 cells (purchased from the
Iranian Biological Resource Center, IBRC) [51]. Cells were seeded in 96-well plates (10,000
cells/well) and incubated at 37 ºC in a humidified atmosphere containing 5% CO2 for 24 h,
then cells treated with different concentrations (1, 5, 10, 20, and 50 µM) of the tested
compounds and incubated for 3 h followed by exposing cells with H2O2 (150 µM) for
another 2 h. After that, the medium was removed and MTT solution (20 µL, 5 mg/mL) was
added and incubated for 4 h. Then, the medium was removed and the produced formazan
crystals were solubilized using DMSO (100 µL). The assay was performed at 570 nm by a
multi-mode plate reader (Biotek, Winooski, VT). Results were expressed as the percentage of
viability compared to untreated control cells. All experiments were repeated for three
times.
4.3.4. Neuroprotection assay against Aβ1-42-induced cytotoxicity in SH-SY5Y cells
MTT assay was used to determine cell viability of human neuroblastoma SH-SY5Y cells
(purchased from the American Type Culture Collection, ATCC, USA) [51]. Cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS,
1% L-glutamine, and 1% antibiotic mix at 37 °C in a humidified atmosphere containing 5 %
CO2. SH-SY5Y cells were seeded into 96-well plates (5 × 103 cells/well) and treated with
tested compound at a concentration of 10 µM for 3 h, prior to the addition of Aβ1–42 (10 µM).
After 24 h of incubation, 10 µL of the MTT reagent (5 mg/mL) was added and DMSO (100
µL) was used to solve formazan crystals. The optical density at 570 nm was measured using a
microplate reader (Biotek Power Wave XS). Cell viability was reported as the percentage of
viability compared to untreated control cells.
4.3.5. Determination of intracellular H2O2-induced ROS production in PC12 cells
ROS-sensitive dye, 2´,7´-dichloro-fluorescein diacetate (DCFH-DA) was used as a probe to
evaluate the formation of intracellular ROS [52]. PC12 cells were seeded in 96-well plates
(1×104 cells/well) and cultured in RPMI medium supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin and 100 mg/mL streptomycin and incubated at 37 ºC in
humidified 5 % CO2. After 24 h, the cells were incubated in the presence or absence of
20

different concentrations of the tested compound (1, 5, 10 and 50 µM) for 6 h, then the
medium was removed and the cells were washed with PBS and incubated followed by
exposing with H2O2 (150 µM) for additional 18 h. After that the cells were washed with PBS
and incubated with DCFH-DA (10 µM) for 30 min at 37 ºC in the dark. At the end, the cells
were washed two times with PBS and the fluorescence of the DCF was measured
(excitation/emission at 485 nm/528 nm) by ELISA fluorimeter (BioTek). The results were
expressed as a percentage of intracellular ROS compared to untreated control cells.
4.3.6. FRAP (ferric reducing antioxidant power) assay
The method of Benzie and Strain was employed to evaluate the total antioxidant power of
the tested compound with slight modifications [53]. A 240 µL freshly prepared FRAP
solution (300 mM of acetate buffer, pH = 3.6, and 10 mM TPTZ (2,4,6-tripyridyl-s-triazine)
solution in 40 mM hydrochloric acid were mixed with 20 mM of ferric chloride solution) was
added to 10 µL of the tested compound solution (10 µM) and incubated for 15 min. The
optical densities were measured at 593 nm by a microplate reader (BioTek Synergy HT) and
FRAP values reported in mmol of ferrous, according to the plotted standard curve of ferrous
sulfate.
4.3.7. Metal binding studies
The metal binding ability of the tested compound was measured by a dual-beam (GBC
Cintra 101) spectrophotometer. The UV absorption was recorded in the absence and presence
of metal ions including CuCl2, FeSO4, ZnCl2, AlCl3, MgCl2, and CaCl2 with a wavelength
ranging from 200 to 500 nm after incubating for 30 min at room temperature. The final
concentrations of the metals and tested compound were 20 µM and the volume of the reaction
mixture was 1 mL. To determine the stoichiometry of the complex, the molar ratio method
was used. A constant volume of the ligand solution was 2.5 mL (40 µM) and titration step of
FeSO4 (1000 µM) was 0.01 mL [54].
21

Acknowledgment
This work was supported financially by a grant from Pharmaceutical Sciences Research
Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences
(grant number: 35661) and Iran National Science Foundation (INSF, grant number: 94021726).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version.
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27

Table 1. The protective effect of compounds 4a-o against H2O2-induced injury in PC12 cell
line at different concentrations.
PC12 cells viability (% of control)
Compound
n
R
H2O2
1 µM
5 µM
10 µM
20 µM
50 µM
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
-
H
33.6±1
57.4±1.7*** 61.6±0.5*** 69.4±0.6***
75.7±3.5*** 80.8±2.6***
41.5±1.4*** 43.3±1.9***
27.0±1.1ns 26.3±1.3ns
82.2±2.4*** 86.0±2***
75.7±3.5*** 80.9±2.6***
4a
4b
6-Br
6-NO2
32.6±0.9 36.6±0.9*** 38.3±1.3*** 41.1±1.3***
37.1±2.8 32.2±1.7ns 31.5±1.9ns 29.6±1.3ns
4c
8-OMe 30.2±2.3 55.9±2.8*** 62.4±2***
74.7±0.6***
57.4±1.7*** 61.6±0.5*** 69.4±0.6***
31.3±2.3 31.6±0.9ns 34.1±1.1** 36.1±1.1***
47.1±1.6*** 55.3±0.9*** 65.1±2***
36.2±2.8 35.1±2.6ns 29.7±1.4ns 27.6±1.4ns
61.1±3.7*** 64.9±4.2*** 71.1±4.5***
4d
-
33.6±1
4e
H
29.0±1.1ns
71.3±2.1*** 74.4±2.3***
22.4±1.7ns 18.8±1.2ns
26.9±1.8ns
4f
6-Br
6-NO2
33.5±1
4g
4h
8-OMe 29.41±1
76.6±4.2*** 81.04±3.9***
42.6±0.9*** 43.7±0.5***
21.7±1.9ns 19.5±1.3ns
55.8±1.6*** 58.43±1.4***
19.9±0.8ns 18.2±1.4ns
75.7±1.3*** 79.0±3.1***
50.3±2.4*** 52.6±2.8***
56.9±1.6*** 58.8±2.1***
4i
-
30.8±1.2 34.7±0.9*** 38.4±2.1*** 42.4±1.1***
30.3±3.1 28.1±1.6ns 25.9±1.5ns 24.5±2ns
26.9±2.1 42.5±2.6*** 44.3±1.6*** 54.2±2.1***
4j
H
4k
6-Br
6-NO2
4l
27.3±2.2 24.8±0.8ns 21.3±1. ns
19.0±1.1ns
4m
4n
8-OMe 26.5±1.7 52.2±1.7*** 58.3±2.9*** 71.4±2.8***
-
-
33.9±0.9 41.1±0.1*** 45.0±1.4*** 46.7±0.3***
28.6±0.9 40.7±1.4*** 47.3±0.8*** 53.7±1.2***
4o
Quercetin
Cell viability was determined using MTT assay protocol. Data are expressed as the mean ± SEM of three independent
replicates. The significant (***p < 0.001) and not significant (ns) values with respect to the H2O2 group.
28

Table 2. Inhibitory activity of synthesized compounds 4a-o against AChE and BuChE.
AChEa
IC50 (µM)
>100
>100
>100
>100
>100
11.3
BuChEa
IC50 (µM)
>100
>100
>100
>100
>100
83.2
Compound
n
R
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
-
H
4a
6-Br
6-NO2
8-OMe
-
4b
4c
4d
4e
H
4f
6-Br
6-NO2
8-OMe
-
10.1
75.8
4g
9.8
>100
86.7
4h
12.6
4i
7.3
68.6
4j
H
>100
>100
>100
>100
>100
0.023
>100
>100
>100
>100
>100
3.39
4k
6-Br
6-NO2
8-OMe
-
4l
4m
4n
4o
-
Donepezil
a Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of ChE.
29