Design, synthesis and evaluation of novel multi-target-directed ligands for treatment of Alzheimer's disease based on coumarin and lipoic acid scaffolds

Article abstract of DOI:10.1016/j.ejmech.2018.04.058

A novel series of coumarin-lipoic acid conjugates were synthesized via cycloaddition click reaction to find out new multi-target-directed ligands (MTDLs) for treatment of Alzheimer's disease (AD). All of synthesized compounds were screened for neuroprotec

Full text of DOI:10.1016/j.ejmech.2018.04.058

Accepted Manuscript
Design, synthesis and evaluation of novel multi-target-directed ligands for treatment
of Alzheimer's disease based on coumarin and lipoic acid scaffolds
Leili Jalili-Baleh, Hamid Forootanfar, Tuba Tüylü Küçükkılınç, Hamid Nadri, Zahra
Abdolahi, Alieh Ameri, Mandana Jafari, Beyza Ayazgok, Maryam Baeeri, Mahban
Rahimifard, Syed Nasir Abbas Bukhari, Mohammad Abdollahi, Mohammad Reza
Ganjali, Saeed Emami, Mehdi Khoobi, Alireza Foroumadi
PII:
S0223-5234(18)30400-8
10.1016/j.ejmech.2018.04.058
EJMECH 10407
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2018
Revised Date: 21 April 2018
Accepted Date: 28 April 2018
Please cite this article as: L. Jalili-Baleh, H. Forootanfar, Tuba.Tüü. Küçükkılınç, H. Nadri, Z. Abdolahi,
A. Ameri, M. Jafari, B. Ayazgok, M. Baeeri, M. Rahimifard, S.N. Abbas Bukhari, M. Abdollahi, M.R.
Ganjali, S. Emami, M. Khoobi, A. Foroumadi, Design, synthesis and evaluation of novel multi-target-
directed ligands for treatment of Alzheimer's disease based on coumarin and lipoic acid scaffolds,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of novel multi-target-directed ligands for
treatment of Alzheimer’s disease based on coumarin and lipoic acid scaffolds
a
b
c
d
d
Leili Jalili-Baleh, Hamid Forootanfar, Tuba Tüylü Küçükkılınç, Hamid Nadri, Zahra Abdolahi, Alieh
e
b
c
a
a
Ameri, Mandana Jafari, Beyza Ayazgok, Maryam Baeeri, Mahban Rahimifard, Syed Nasir Abbas
f
a
g,h
i
a,j,*
Bukhari, Mohammad Abdollahi, Mohammad Reza Ganjali, Saeed Emami, Mehdi Khoobi
,
k,**
Alireza Foroumadi
a
The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411,
bIran
Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical
Sciences, Kerman, Iran
c
Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, Ankara, Turkey
d
Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka 2014, Saudi
f
Arabia
g
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran
University of Medical Sciences, Tehran, Iran
h
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
i
Mazandaran University of Medical Sciences, Sari, Iran
j
Department of Pharmaceutical Biomaterials, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
k
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Abstract: A novel series of coumarin-lipoic acid conjugates were synthesized via cycloaddition click reaction to
find out new multi-target-directed ligands (MTDLs) for treatment of Alzheimer’s disease (AD). All of synthesized
compounds were screened for neuroprotective and anti-cholinesterase activities. Based on primary screening, two
compounds (5 and 11) were subjected to further biological evaluations. In particular, compound 11 which was the
most potent AChE inhibitor showed good inhibitory effect on Aβ-aggregation and intracellular ROS (reactive
oxygen species) formation, as well as the ability of selective bio-metal chelation and neuroprotection against H O -
2
2
and Aβ1-42-induced cytotoxicity. In the light of these results, the applied hybridization approach introduced new
promising lead compound with desired multifunctional properties, being useful in the treatment of Alzheimer’s
disease.
Keywords: Alzheimer’s disease; Coumarin; Lipoic acid; Neuroprotective agents; Antioxidant; MTDLs
*
Corresponding author. Tel/Fax: +98-21-64121510. E-mail address: Mehdi.khoobi@gmail.com (M. Khoobi),
and aforoumadi@yahoo.com (A. Foroumadi)
1

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1. Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system [1].
In response to increasing distribution of AD in individuals under age 65, several treatment
strategies have been explored to increase elderly population functionally and life expectancy [2].
The etiology of AD is not fully understood, but diverse factors including low levels of
acetylcholine, β-amyloid (Aβ) deposits, τ-protein aggregation, oxidative stress, inflammation,
and dyshomeostasis of bio-metals have been correlated with AD [3]. The cholinergic hypothesis
was the first theory based on the fact that neuronal degeneration is caused by the loss of
cholinergic neurotransmitters in the brains of AD patients [4,5]. This theory has led to the
development of the acetylcholinesterase (AChE) inhibitors which are still the first used
symptomatic treatments for AD by elevating cholinergic neurotransmission. The level of ACh
can be also decreased by butyrylcholinesterase (BuChE) as a co-regulator of the ACh
degradation, so inhibition of both enzymes is helpful therapeutic approach for AD [6].
The Aβ deposition is another hypothesis, suggesting prevention of the self-assembled Aβ
peptide formation is an important parameter to slow down the pathological complications of the
AD [7]. Several studies suggested that blocking of peripheral anionic binding site (PAS) of
AChE prevents the initiation of Aβ-aggregation [8].
Besides the pathological signs of the disease, AD patient brains show evidence of oxidative
damages [9]. Supported by the oxidative stress hypothesis, reactive oxygen species (ROS) play
a key role in onset and progression of AD [10]. ROS induce Aβ overproduction by elevating β-
secretase expression and promotes the amyloidogenic pathway as one of the initial events in the
AD [11,12]. ROS are originated by mitochondrial abnormalities and Aβ peptides aggregation
particularly in the presence of biometal ions (Cu(II), Fe(II), Zn(II), etc.) [13]. The abnormal high
level of metal ions which has been found at the injured locations of the AD brains, is associated
with Aβ aggregates and promotes Aβ neurotoxicity and oxidative stress through the generation
of free radicals [14]. In this context, antioxidant agents can be nominated as helpful therapeutics.
Moreover, modulation of biometals dyshomeostasis by chelation in redox-inactive forms is also
promised as an additional therapeutic strategy to inhibit metal-catalyzed oxidation of
biomolecules in AD treatment [15]. Therefore, development of multitarget-directed ligands
(MTDLs) with two or more complementary biological activities may display significant
advantages against complex diseases like AD [16,17].
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Coumarins are pharmacologically active natural compounds having antioxidant and ROS
scavenging activities [18-20]. AP2469 (Fig. 1) is one of the famous coumarin-based MTDL
having AChE and beta-secretase inhibitory activities, as well as anti-Aβ aggregating,
antioxidant, and neuroprotective properties which has been generated from lead AP2238 [21].
Furthermore, ensaculin (Fig. 1) is another example of coumarin drugs with potential use in the
treatment of AD [8].
On the other hand, Lipoic acid (LA, Fig. 1) is a naturally occurring antioxidant featured by its
disulfide bond [22,23]. Since LA as a mitochondria-targeted antioxidant is able to trap free
radicals and chelat with bio-metal ions, it has been called as universal antioxidant [24-27]. LA
has also shown a neuroprotective effect against Aβ-induced cytotoxicity [28] and can stabilize
cognitive functions in patients with AD [29]. In addition, Lipocrine as a conjugate of Tacrin and
LA has been also introduced as potent AChE inhibitor with the ability to inhibit AChE-induced
Aβ-aggregation as well as neuroprotection activity against ROS (Fig. 1) [30]. Moreover,
coumarin-3-carboxamides bearing LA have shown acceptable anti-inflammatory and antioxidant
activity [31].
In this work, on the basis of important findings about role of oxidative stress as a crucial and
early event in mediating the AD pathogenesis and in continues of our previous studies for
developing new anti-Alzheimer agents [32], we designed new MTDLs for treatment of AD
based on the LA and coumarin scaffolds. To reach this goal, LA and the 7-hydroxycoumarin
scaffolds were connected via click chemistry and formation of 1,2,3-triazole ring [33-35] (Fig.
1). We report here, synthesis of coumarin-lipoic acid conjugates 4-30, and evaluation of their
antioxidant potential for neuroprotection as well as their ability to inhibit cholinesterases,
lipoxygenase (LOX), and Aβ aggregation.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 1, alkyne-azide click chemistry was employed for the preparation of
target compounds 4-30. The key intermediates 7-hydroxycoumarins 1b-k were prepared based
on well-known methods. Briefly, the reaction of resorcinol with ethyl acetoacetate in the
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presence of H SO gave 7-hydroxy-4-methyl-coumarin (1b) [36]. The acetyl and ethyl ester
2
4
analogs (1c and 1d) were prepared form the reaction of 2,4-dihydroxybenzaldehyde and ethyl
acetoacetate or diethyl malonate, respectively. The acid analog 1e was prepared via
hydrolyzation of compound 1d in an aqueous solution of sodium hydroxide (5%) at room
temperature [37]. Also, 2,4-dihydroxybenzaldehyde underwent to cyclization with appropriate
phenylacetic acid derivatives in the presence of anhydrous CH COONa in refluxing Ac O to
3
2
afford 7-hydroxy-3-phenyl-coumarin derivatives 1f-k [38]. The coumarin derivatives 1a-k were
reacted with an excess amount of different dibromoalkanes to achieve compounds 2 [39]. On
the other hand, the N-propargylamide of LA (compound 3) was synthesized via EDCI–DMAP
mediated amide bond formation between LA and propargyl amine [40]. Finally, target
compounds 4-30 were synthesized by one-pot three-component reaction of bromoalkyl
derivatives 2, propargylated compound 3 and sodium azide [41].
2
.2. Biological screenings
Primarily, all target compounds 4-30 were screened for their neuroprotective activity against
oxidative stress in PC12 cells, anti-cholinesterase activity and inhibitory activity against 15-
LOX as possible targets for treatment of AD.
2
.2.1. Neuroprotective activity against H O -induced cell death
2 2
Neurotoxicity and oxidative damage generated by H O are key parameters in the progression
2
2
of neurodegenerative diseases [42]. Thus, the neuroprotective potential of compounds 4-30 (at
the concentrations of 0.1, 1, 5, 10, 20 and 50 µM) in PC12 cells against the H O -induced cell
2
2
death was tested using MTT assay. As seen in Table 1, except for compounds 9, 16, 24, and 26
which did not show significant effect on cell viability, all other tested compounds significantly
increased PC12 cell viability in the presence of H O in a concentration-dependent manner.
2
2
Most of the compounds significantly increased cell viability even at the low concentration of 0.1
µM ( < 0.001).
p
The higher protection was observed with compounds 5, 8, 12, 13, 15, 20, 22 and 23 as
compared in the different concentrations. The neuroprotective effect of these compounds was
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higher than that of reference drug quercetin. As seen in Table 1, structurally the target
compounds have a 3, 4 or 5 atom linker (n= 3-5). The more promising compounds were
amongst the propylene (n=3) or butylene (n=4) series. Furthermore, compounds 5 and 23
contain 4-methylcoumarin moiety, and compounds 13 and 20 derived from 3-(4-
chlorophenyl)coumarin showed the best activity among other coumarin derivatives. Notably, the
unsubstituted phenyl derivatives 9, 16 and 26 had no significant effect on the H O -induced cell
2
2
death. However, introduction of halo- or methoxy- substituents resulted in effective compounds
compare compounds 10-14 with 9, compound 17-21 with 16, and compounds 27-30 with 26).
(
2
.2.2. Anti-cholinesterase activity
It is well-known that the coumarin-containing compounds can act as PAS binder and inhibit
AChE enzyme [32]. Accordingly, all final compound 4-30 were screened for their inhibitory
activity against AChE and BuChE in comparison to donepezil as reference drug. The obtained
IC values were listed in Table 2. Although most of compounds exhibited weak or no activity
5
0
on AChE, but 3,4-dimethoxyphenyl-coumarin derivative 11 with IC values of 16.4 µM showed
50
acceptable activity against this enzyme. The latter compound contains a propylene liker (n= 3).
Elongation of linker (n= 4 and 5) led to homologs 18 and 28 with no activity on cholinesterases.
On the other hand, the 3,4-dichlorophenyl-coumarin derivatives 21 and 30 with 4C or 5C linker
showed suitable inhibitory activity against BuChE (IC50 values of 10.3 and 7.8 µM,
respectively). The 3,4-dichlorophenyl-coumarin analog 14 with propylene spacer (n= 3) showed
marginal activity against BuChE (IC = 73.5 µM). Therefore, it can be concluded that in 3,4-
5
0
dichlorophenyl-coumarin derivatives, the elongation of linker increased the anti-BuChE potency.
To investigate the AChE inhibition mechanism, the most active compound 11 was selected for
kinetic studies. Reciprocal Lineweaver-Burk plots at different concentrations of the substrate
indicated a mixed-type inhibition against AChE (Fig. 2A) which means compound 11 might be
able to bind to the PAS as well as CAS (catalytic active site). Re-plotting of slope versus
concentration of compound 11 (Fig. 2B) showed an estimate of non-competitive inhibition with
Ki = 6.91 µM.
2
.2.2.1. Docking study with AChE
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The binding mode of most active compound 11 with acetylcholinesterase (1EVE) was defined
by docking study using the MOE software. As shown in Figure 3, compound 11 located in the
entire enzymatic gorge. The dimethoxyphenyl moiety of compound 11 formed
interaction with Asp71, and also made stacking with phenyl ring of Tyr333, near the top of
the gorge (the PAS). In this situation, the pyrone part of coumarin made two interactions
π-anion
π-π
π-π
with Tyr333 and Tyr69, and the oxygen of the pyrone ring formed a hydrogen bond with Try69.
Noteworthy, in the middle of the gorge (the CAS), the oxygen of amidic group was involved in
a hydrogen bond with Tyr120 along with a van der Waals interaction with Phe330, indicating
the role of amidic group on ligand activity. Moreover, the triazole ring formed a
interaction with Ile286 near the bottom of the gorge. The 1,2-dithiolane ring makes a
π
-alkyl
π-sulfur
interaction with the imidazole ring of His439. All these facts provide an explanation for
micromolar inhibitory activity of compound 11 against AChE.
2
.2.3. 15-LOX inhibitory activity
Previous studies revealed that lipid hydroperoxides as key mediators of mitochondrial
dysfunction alter tissue inflammatory responses and contribute to the pathogenesis of
neurodegenerative disorders like AD [43]. Lipoxygenase (LOX) enzymes are the biggest
contributor to the synthesis of lipid hydroperoxides through oxidation of poly-unsaturated fatty
acids such as linoleic and arachidonic acid to hydroperoxy acids [44]. Moreover, higher levels
of LOX were found in post-mortem brain samples from Alzheimer's patients compared to
controls [45,46]. Due to the relevant of the LOX enzyme in AD, all target compounds 4-30 were
also tested for their inhibitory activity against LOX enzyme. A perusal of the inhibition values
showed that all compounds were not active against 15-LOX (IC values more than 100 µM). It
5
0
was presumed that the large scaffold of the compound banned the molecule to accommodate in
the active site.
2
.3. Complementary evaluations of the selected compounds
The initial screening of the target compounds 4-30 against oxidative stress-induced cell death
in PC12 cells as well as their activity against cholinesterases revealed that the most of the
compounds had significant neuroprotective activity while a limited number of them found to be
6

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potent AChE and/or BuChE inhibitor. Based on the results, compound
5 with significant
neuroprotective activity and marginal anti-AChE potency as well as compound 11 possessing
more potent anti-AChE activity and moderate neuroprotective property, were selected for
further biological evaluations to check the possible usefulness of compounds for Alzheimer’s
disease.
2
.3.1. Inhibitory effects on the Aβ peptide aggregation
In the light of A
potential of compounds
ThT) assay. The obtained results indicated that compounds
β
anti-aggregating properties of coumarin and LA derivatives [47,48], the
and 11 to inhibit A -aggregation was evaluated using thioflavin T
and 11 displayed good inhibitory
5
β
(
5
activity (26.6 and 51.2% inhibition, respectively) in comparison to the reference compounds
donepezil (22%) and rifampicin (27.5%, Table 3). Compound 11 was about 2-fold more
effective than compound
inhibitory activity of compound 11 than compound
dimethoxyphenyl-coumarin scaffold on the interaction with A
and 11 to inhibit A -aggregation induced by AChE were also evaluated (Table 3). Compound
showed weak inhibition activity toward AChE-induced A
5
and reference drugs in the inhibition of A
β aggregation. The more
5
suggests the great effect of
β
. The potential of compounds
5
5
β
β
aggregation (2.9% inhibition), while
the inhibitory activity of compound 11 was about 2-fold higher than that of donepezil (47.4%
compared to 26.1%). This result was in agreement with the kinetic and docking studies
confirming potential binding of compound 11 with PAS residues of AChE.
2
.3.2. Neuroprotective activity against H O - or Aβ1-42-induced cytotoxicity in SH-SY5Y cells
2 2
The viability of human neuroblastoma SH-SY5Y cells against H O in the presence of
2
2
compounds
5 and 11 were also examined. As depicted in Fig. 4B, these compounds can more
effectively (55.6 and 64%, respectively) protect SH-SY5Y cells against H O -induced
2
2
cytotoxicity compared to donepezil as a reference drug (33%). As described in section 2.2.1,
these compounds could significantly protect neuronal PC12 cells (neuroblastic and eosinophilic
cells) against the H O -induced cell death. In PC12 cells, oxidative stress and subsequently cell
2
2
death were induced by adding 150 µM of H O ; while H O was used at the higher
2
2
2
2
concentration of 250 µM in SH-SY5Y cells. Accordingly, we could not compare the obtained
7

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results between PC12 and SH-SY5Y cells. However, it should be noted that in PC12 model
compound
5 was more effective than compound 11. In contrast, the obtained results against SH-
SY5Y cells indicated that compound 11 is better than
5
.
It has been reported that A
stimulating the formation of free radicals [49]. Therefore, the neuroprotective effects of
representative compounds and 11 towards Aβ1-42-induced cytotoxicity in human
neuroblastoma SH-SY5Y cells were evaluated. According to the obtained results (Fig. 4A),
compounds and 11 had significant protective capability against Aβ1-42-induced cytotoxicity
with the percentage of cell viability of 72.9 and 64.7 % receptivity, higher than reference drug
β senile plaques have toxic effects on neuronal cells through
5
5
donepezil (39.9%). The neuroprotective behavior of compound
5
and 11 suggests that pre-
treatment of neurons with these compounds before exposure to A
β
or H O can significantly
2
2
reduce oxidative stress and increase cell survival in human SH-SY5Y neuroblastoma cells.
2
.3.3. Effect on the intracellular ROS formation
To determine the potential use of compounds
5 and 11 for the treatment of AD as a new
antioxidant MTDLs, their antioxidant activity against intracellular ROS generation in PC12 cells
after treatment with H O were investigated by dichloro-dihydro-fluorescein diacetate (DCFH-
2
2
DA) assay. We used a range of concentrations of the tested compounds that did not affect the
cell viability (1, 5, 10 and 50 µM). The results showed a significant decrease of ROS production
in a concentration-independent manner for both tested compounds (Fig. 5, for details, see
supporting information, Table S1). In particular, compound 11 in all concentrations
significantly decreased the intracellular ROS generation in treated cells compared with untreated
control cells. Moreover, compound
5 and LA showed the same results at the highest
concentration of 50 µM. The results confirmed the successful role of the compounds in the
reduction of endogenous ROS which are formed as a natural byproduct of the normal
metabolism of oxygen in cells [50].
2
.3.4. Total Antioxidant Power
The ferric reducing antioxidant power (FRAP) of the representative compounds
5 and 11 was
assessed in comparison with lipoic acid, ascorbic acid, and quercetin. The results indicated that
+
2
compounds
5
and 11 have excellent reducing power (84.7 and 87.5 mM Fe , respectively).
8

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Compound 11 showed slightly more ability to reducing ferric ion than quercetin and lipoic acid
(Table 4).
2
.3.5. Metal-chelating property
Binding to biometal ion is one of the mechanisms by which antioxidants control autoxidation
reactions and could be a potential therapeutic strategy for the treatment of AD [51]. The
+
2
+2
+3
+2
chelation activity of the selected compound 11 for biometals such as Mg , Ca , Al , Cu ,
+
2
+2
Fe , and Zn which have been found in A
increase in maximum absorption intensity at 322 nm and also a shoulder at 356 nm was
observed after addition of CuCl or FeSO (Fig. 6), indicating the formation of complex 11
β plaques, was studied by UV-vis spectrometry. An
-
2
4
+
2
+2
+3
+2
metal (II). However, no significant change was observed when Mg , Ca , Al , and Zn were
added. The chelating property of compound 11 could be attributed to the presence of amidic
group and triazole scaffold or the sulfurs at the LA structure [52]. The stoichiometry of the 11
-
Cu (II) complex was also determined by titration of the methanolic solution of compound 11
with ascending amounts of CuCl (The molar ratio method). As shown in Figure 6, initially, the
2
absorbance increased at 365 nm and then became stable. The two straight lines crossed at a mole
+2
fraction of 0.5, indicating a 2:1 stoichiometry for the complex. It has been approved that Cu
promote the formation of senile plaques and reactive oxygen species because of its superior
+
2
affinity to complex with Aβ1-42 [53]. Therefore, the selective chelation of Cu ions by
compound 11 suggest the possible capability of the compound to stop formation of amyloid
plaques and decrease oxidative stress.
3. Conclusion
We described synthesis and biological characterization of 27 new triazole-containing
coumarin-lipoic acid conjugates as novel multifunctional antioxidants for the development of
potential anti-Alzheimer agents. The in vitro assays showed that most of the compounds had
significant neuroprotective activity against H O -induced oxidative stress in PC12 cells.
2
2
Investigation on anti-cholinesterase activity of compounds revealed that some of them such as
compounds 11 and 30 have remarkable activity against AChE and BuChE, respectively. Based
on primary screening, compound
5
as a highly effective neuroprotective agent and compound 11
9

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with superior activity against AChE were selected for further studies. Based on the obtained
results, compound 11 was about 2-fold more effective than reference drug donepezil in terms of
the inhibitory activity on self-induced and AChE-induced Aβ1-42 aggregation. Moreover, cell-
based assay indicated that compounds 11 displays significant protection against Aβ1-42-induced
cytotoxicity, being superior to donepezil. The additional ability of compound 11 in the
diminution of intracellular ROS generation, reduction of ferric ion and selective metal chelation
makes compound 11 as a promising multifunctional antioxidant for further studies to the
development of new anti-Alzheimer agents.
4. Experimental
4
.1. Chemistry
7
-hydroxy coumarin (1a), lipoic acid (LA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI), 4-dimethylaminopyridine (DMAP) and all other starting materials, reagents and
solvents were purchased from commercial sources and used without further purification. Melting
points were measured on a Kofler hot stage apparatus. The IR spectra (KBr disks) were taken
1
13
by Nicolet FT-IR Magna 550 spectrometer. H and C NMR spectra were measured using
Brucker 500 MHz instrument. The chemical shifts ( ) and coupling constants ( ) are presented
δ
J
in parts per million (ppm) and Hertz (Hz), respectively. The results of elemental analyses (C,
H, and N) were within ±0.4% of the calculated values and determined with CHN-Rapid
Heraeus Elemental Analyzer.
4
.1.1. General procedure for the preparation of 7-Hydroxy-4-methyl-coumarin (1b)
Concentrated H SO (0.5 mL) was added dropwise to an ice-cold solution of resorcinol (2.0 g,
2
4
1
8.2 mmol) in dioxane. Ethyl acetoacetate (2.8 g, 21.8 mmol) was then added, and the mixture
was heated to 60 °C for 4 h. The mixture was next poured into ice and water, and the precipitate
was filtered and dried. The product was finally recrystallized from methanol [36].
4
.1.2. General procedure for the preparation of 3-substituted coumarins 1c-e
1
0

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Ethyl acetoacetate (0.128 g, 1 mmol) or diethyl malonate (0.176 g, 1 mmol) was added to a
solution of 2,4-dihydroxybenzaldehyde (0.122 g, 1 mmol) in absolute ethanol. Two drops of
piperidine were then added and the solution was heated under reflux for 3 h. After completion of
the reaction, the hot solution was diluted with water and stored overnight in a refrigerator. The
crystalline product filtered off, washed with a cold solution of ethanol/water (1:1.5) and dried in
the air. The acid analog 1e was prepared via hydrolyzation of compound 1d in an aqueous
solution of sodium hydroxide (5%) at room temperature for 12 h. After completion of the
reaction, the solution was acidified (pH=4) with HCl (10%) and the precipitated product was
washed with water and dried on air [37].
4
.1.3. General procedure for the preparation of 3-phenyl-coumarin derivatives 1f-k
Anhydrous CH COONa (92 mg, 1.4 mmol), appropriate phenylacetic acid derivative (1
3
mmol), and 2,4-dihydroxybenzaldehyde (1 mmol) was refluxed in Ac O (1.2 mL) for 2 h. The
2
reaction mixture was cooled, neutralized with 10% aqueous NaHCO , and filtered. The solid
3
product was washed with water and ethyl acetate and dried [38].
4
.1.4. General procedure for the preparation of bromoalkoxy intermediate 2
A mixture of substituted 7-hydroxycoumarin (5 mmol), appropriate dibromoalkane (50
mmol) and anhydrous K CO (1.4 g, 10 mmol) was refluxed in acetone (15 mL) for 4 h. After
2
3
cooling, the reaction mixture was filtered and the organic phase was evaporated under reduced
pressure. The obtained residue was triturated with -hexane to give the product as white solid
n
2
[
39].
4.1.5. General procedure for the preparation of 1,2-dithiolane-3-pentanoic acid-N-
propargylamide (3)
Lipoic acid (LA, 1 mmol), EDCI (1.2 mmol), and DMAP (1.2 mmol) were dissolved in
anhydrous CH Cl (10 mL). The reaction mixture was cooled to 0 °C and propargylamine (1.2
2
2
mmol) in dry CH Cl (1 mL) was added dropwise. Then the mixture was stirred at room
2
2
1
1

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temperature for 12 h. After completion of the reaction (monitored by TLC), the mixture was
filtered and the organic phase was evaporated. The obtained crude product was purified by
column chromatography on silica gel using ethyl acetate/petroleum ether (1:2) as the eluent, to
give yellow solid [40].
4
.1.6. General procedure for the preparation of compounds 4-30
A mixture of the intermediate
2
(1 mmol), NaN (1 mmol) and NEt (1 mmol) in
t
-
-
3
3
BuOH/H O (1:1) were heated at 70 °C for 30 min. 1,2-Dithiolane-3-pentanoic acid-
N
2
propargylamide (3, 1 mmol), sodium ascorbate (0.2 mmol), and CuSO (0.2 mmol) were then
4
added to the latter mixture. After completion of the reaction (12-48 h, monitored by TLC), the
mixture was diluted with cold water and the precipitate was filtered off and washed with water.
The resulting crude product was purified by flash chromatography on silica gel eluting with
ethyl acetate/petroleum ether (1:1) to give compounds 4-30 [41].
4.1.6.1. 5-(1,2-dithiolan-3-yl)-N-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-
-1
yl)methyl)pentanamide (4). Yeild 99%; Off white solid; mp 126-128 ºC; IR (KBr, cm ) νmax:
1
3
284 (NH), 2926 (C-H), 1732 and 1627 (C=O). H NMR (DMSO-d , 500 MHz) δ: 8.29 (t, 1H,
6
J=5.5 Hz, NH), 7.98 (d, 1H, J=9.5 Hz, H ), 7.94 (s, 1H, triazole), 7.62 ( d, 1H, J=8.5 Hz, H ), 6.97
4
5
(
d, 1H, J=2.0 Hz, H ), 6.92 (dd, 1H, J=8.5 Hz, J=2.0 Hz, H ), 6.30 (d, 1H, J=9.5 Hz, H ), 4.51 (t,
8
6
3
2
H, J=6.5 Hz, CH -N), 4.27 (d, 2H, J=6.0 Hz, CH -NH), 4.08 (t, 2H, J=6.0 Hz, CH -O), 3.58
2 2 2
(
quintet, 1H, J=6.0 Hz, CH-S), 3.18-3.09 (m, 2H, CH S), 2.38 (sextet, 1H, J=6.7 Hz, S-CH -CH2),
2
2
2
.28 (t, 2H, J=6.0 Hz, aliphatic), 2.08 (t, 2H, J=7.5 Hz, COCH ), 1.84 (sextet, 1H, J=7.0 Hz, S-
2
CH -CH2), 1.58-1.65 (m, 1H, aliphatic), 1.54-1.49 (m, 3H, aliphatic), 1.33-1.30 (m, 2H, aliphatic).
2
1
3
C NMR (DMSO-d , 125 MHz) δ: 171.8, 167.4, 161.4, 160.2, 155.3, 145.0, 144.3, 129.5, 122.9,
6
112.6, 112.4, 101.2, 65.3, 56.1, 46.3, 38.0, 34.9, 34.0, 29.2, 28.3, 24.9. Anal. Calcd for
C H N O S : C, 56.54; H, 5.78; N, 11.47. Found: C, 56.70; H, 5.86; N, 11.71.
2
3
28
4
4
2
4
.1.6.2.
5-(1,2-dithiolan-3-yl)-N-((1-(3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-
1
,2,3-triazol-4-yl)methyl)pentanamide (
5
). Yeild 95%; Off white solid; mp 83-85 ºC; IR (KBr,
-1
1
cm ) νmax: 3295 (NH), 2930 (C-H), 1721 and 1616 (C=O). H NMR (DMSO-d6, 500 MHz)
δ:
8
.29 (bs, 1H, NH-CO), 7.93 (s, 1H, triazole), 7.67 (d, 1H,
J=8.5 Hz, H ), 6.96-6.93 (m, 2H,
5
H ), 6.21 (s, 1H, H ), 4.51 (t, 2H, =7.0 Hz, CH -N-), 4.26 (d, 2H, J=5.5 Hz, CH -NH), 4.08 (t,
J
6
,8
3
2
2
1
2

ACCEPTED MANUSCRIPT
2
2
1
H,
J=6.0 Hz, CH -O), 3.58 (quintet, 1H, J=6.5 Hz, CH-S), 3.16-3.11 (m, 2H, CH -S), 2.39-
2 2
.36 (m, 4H, CH and 1H, S-CH -CH ), 2.28 (quintet, 2H, aliphatic), 2.08 (m, 2H, COCH ),
3
2
2
2
.84 (sextet, 1H, J=7.0 Hz, S-CH -CH ), 1.61-1.69 (m, 1H, aliphatic), 1.53-1.48 (m, 3H,
2 2
1
3
aliphatic), 1.26-1.35 (m, 2H, aliphatic). C NMR (DMSO-d6, 125 MHz)
δ
: 172.1, 161.4,
60.1, 154.7, 153.4, 445.1, 126.4, 122.8, 113.2, 112.4, 111.2, 101.2, 65.3, 56.1, 46.3, 34.9, 34.0,
9.2, 28.2, 24.9, 18.1. Anal. Calcd for C H N O S : C, 57.35; H, 6.02; N, 11.15. Found: C,
1
2
5
2
4
30
4
4 2
7.72; H, 6.36; N, 11.29.
4
5
.1.6.3. N-((1-(3-((3-acetyl-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methyl)-
-(1,2-dithiolan-3-yl)pentanamide (
6
). Yield 90%; Off white solid; mp 115-117 ºC; IR (KBr,
-1
1
cm ) νmax: 3489 (NH), 2927 (C-H), 1715 and 1627 (C=O). H NMR (DMSO-d6, 500 MHz)
δ:
8
1
.63 (s, 1H, H ), 8.25 (bs, 1H, NH), 7.93 (s, 1H, triazole), 7.87 ( d, 1H,
J
=8.5 Hz, H ), 7.03 (s,
4
5
H, H ), 6.98 (d, 1H,
J
=8.5 Hz, H ), 4.51 (t, 2H,
J
=7.0 Hz, CH -N), 4.27 (d, 2H,
J=5.5 Hz, CH2-
8
6
2
NH), 4.13 (t, 2H, J=6.5 Hz, CH -O), 3.57 (quintet, 1H,
J
=6.0 Hz, CH-S), 3.16-3.09 (m, 2H,
=6.7 Hz, S-CH -CH ), 2.30 (t, 2H, =6.0 Hz,
=7.0 Hz, S-CH -CH ), 1.64-1.62
2
CH -S), 2.56 (s, 3H, COCH ), 2.39 (sextet, 1H,
J
J
2
3
2
2
aliphatic), 2.08 (t, 2H, J=7.5 Hz, COCH ), 1.85 (sextet, 1H,
J
2
2
2
1
3
(
m, 1H, aliphatic), 1.51 (bs, 3H, aliphatic), 1.31-1.33 (m, 2H, aliphatic). C NMR (DMSO-d6,
125 MHz)
δ: 194.6, 171.7, 163.7, 158.7, 156.9, 147.4, 145.0, 132.1, 122.7, 120.42, 113.6,
1
11.8, 100.6, 65.6, 56.0, 46.2, 39.8, 34.9, 34.0, 33.9, 29.8, 29.1, 28.1, 24.8. Anal. Calcd for
C H N O S : C, 56.59; H, 5.70; N, 10.56. Found: C, 56.81; H, 5.99; N, 10.85.
2
5
30
4
5
2
4
.1.6.4.
ethyl
7-(3-(4-((5-(1,2-dithiolan-3-yl)pentanamido)methyl)-1H-1,2,3-triazol-1-
yl)propoxy)-2-oxo-2H-chromene-3-carboxylate (
7
). Yield 85%; Off white solid; mp 121-123
-1
1
ºC; IR (KBr, cm ) νmax: 3317 (NH), 2925 (C-H), 1737, 1641 and 1606 (C=O). H NMR
(
DMSO-d6, 500 MHz)
.82 (d, 1H, =8.5 Hz, H ), 6.99 (s, 1H, H ), 6.97 (d, 1H, J=8.5 Hz, H ), 4.51 (t, 2H, J=7.0 Hz,
6
δ: 8.70 (s, 1H, H ), 8. 22 (t, 1H, J=5.5 Hz, NH), 7.91 (s, 1H, triazole),
4
7
J
5
8
CH -N), 4.27 (m, 4H, CH -NH and O-CH -CH ), 4.13 (t, 2H, J=6.5 Hz, CH -O), 3.58 (quintet,
2
2
2
3
2
1
H,
J
=6.0 Hz, CH-S), 3.19-3.10 (m, 2H, CH -S), 2.39 (sextet, 1H,
J
=6.7 Hz, S-CH -CH ), 2.30
2
2
2
(t, 2H,
J=6.0 Hz, aliphatic), 2.08 (t, 2H, J=7.5 Hz, COCH ), 1.83 (sextet, 1H, J=7.0 Hz, S-CH2-
2
1
3
CH ), 1.52-1.49 (m, 4H aliphatic), 1.33-1.29 (m, 2H aliphatic and 3H CH ). C NMR (DMSO-
2
3
d6, 125 MHz)
δ: 172.4, 164.1, 156.9, 145.7, 132.0, 113.6, 111.4, 101.7, 89.4, 65.6, 60.8, 56.0,
4
6.2, 37.9, 34.9, 33.9, 29.1, 18.1, 24.8, 14.0. Anal. Calcd for C H N O S : C, 55.70; H, 5.75;
26 32 4
6 2
N, 9.99. Found: C, 55.99; H, 5.66; N, 9.81.
1
3

ACCEPTED MANUSCRIPT
4
.1.6.5. 7-(3-(4-((5-(1,2-dithiolan-3-yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)propoxy)-2-
oxo-2H-chromene-3-carboxylic acid (
8
). Yield 80%; Off white solid; mp 140-143 ºC; IR
-1
1
(
KBr, cm ) νmax: 3289 (NH, OH), 2932 (C-H), 1749, 1677 and 1612 (C=O). H NMR (DMSO-
d6, 500 MHz)
d, 1H, =8.5 Hz, H ), 7.01 (s, 1H, H ), 6.97 (d, 1H,
N), 4.27 (d, 2H,
Hz, CH-S), 3.17-3.09 (m, 2H, CH -S), 2.38 (sextet, 1H,
δ: 13 (s, 1H, OH), 8.72 (s, 1H, H ), 8.26 (s, 1H, NH), 7.93 (s, 1H, triazole), 7.82 (
4
J
J
=8.5 Hz, H ), 4.51 (t, 2H,
J
=7.0 Hz, CH2-
=5.5 Hz, CH -NH), 4.12 (t, 2H, J=6.5 Hz, CH -O), 3.58 (quintet, 1H, =6.0
=6.7 Hz, S-CH -CH ), 2.30 (t, 2H,
5
8
6
J
J
2
2
J
2
2
2
J
=6.0 Hz, COCH ), 2.08 (t, 2H, J=7.5 Hz, aliphatic), 1.83 (sextet, 1H,
J
=7.0 Hz, S-CH -CH ),
2
2
2
1
3
1
.55-165 (m, 1H, aliphatic), 1.54-1.49 (m, 3H, aliphatic), 1.33-1.30 (m, 2H, aliphatic).
, 125 MHz)
13.8, 113.4, 111.6, 100.7, 65.6, 56.0, 46.2, 38.0, 34.9, 34.0, 29.1, 28.2, 24.8. Anal. Calcd for
C
NMR (DMSO- 6
d
δ: 171.8, 163.9, 163.6, 157.1, 156.7, 148.9, 145.0, 131.5, 122.8,
1
C H N O S : C, 54.12; H, 5.30; N, 10.52. Found: C, 54.08; H, 5.15; N, 10.31.
2
4
28
4
6
2
4
1
.1.6.6.
5-(1,2-dithiolan-3-yl)-N-((1-(3-((2-oxo-3-phenyl-2H-chromen-7-yl)oxy)propyl)-1H-
,2,3-triazol-4-yl)methyl)pentanamide (
9
). Yield 75%; Off white solid; mp 125-127 ºC; IR
-1
1
(
KBr, cm ) νmax: 3295 (NH), 2946 (C-H), 1719 (C=O). H NMR (DMSO-d6, 500 MHz)
δ:
8
.28 (t, 1H, J=5.5 Hz, NH), 8.21 (s, 1H, H ) 7.94 (s, 1H, triazole), 7.70 (m, 3H, phenyl), 7.44 (
4
m, 2H, phenyl ), 7.39 (d, 1H,
J
=7.0 Hz, H ), 7.02 (s, 1H, H ), 6.96 (d, 1H, J=9.0 Hz, H ), 4.52
5
8
6
(
t, 2H, J=7.0 Hz, CH -N ), 4.27 (d, 2H, J=6.0 Hz, CH -NH ), 4.10 (t, 2H, J=6.5 Hz, CH -O),
2 2 2
3
.57 (m, 1H, CH-S), 3.18-3.07 (m, 2H, CH -S), 2.38 (sextet, 1H,
J
=6.0 Hz, S-CH -CH ), 2.30
2
2
2
(
m, 2H, CH , aliphatic chain), 2.08 (t, 2H,
J
=7.0 Hz, CH -CO), 1.82 (sextet, 1H, S-CH -CH ),
2
2
2
2
1
.65-1.60 (m, 1H, CO-CH -CH ), 1.55-1.49 (m, 3H, aliphatic chain), 1.33-1.30 (m, 2H,
2 2
1
3
aliphatic chain). C NMR (DMSO- 6
28.2, 128.0, 123.2, 122.9, 113.1, 65.3, 56.0, 46.3, 34.7, 34.0, 29.2, 28.2, 24.9. Anal. Calcd for
C H N O S : C, 61.68; H, 5.71; N, 9.92. Found: C, 61.80; H, 5.89; N, 10.18.
d , 125 MHz) δ: 171.8, 161.4, 159.9, 140.7, 134.8, 129.7,
1
2
9
32
4
4 2
4
.1.6.7.
5-(1,2-dithiolan-3-yl)-N-((1-(3-((3-(4-methoxyphenyl)-2-oxo-2H-chromen-7-
yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (10
). Yield 75%; Off white solid; mp
-
1
1
1
5
43-145 ºC; IR (KBr, cm ) νmax: 3291 (NH), 2933 (C-H), 1721 (C=O). H NMR (DMSO-d6
,
00 MHz) δ: 8.26 (t, 1H, J=5.5 Hz, NH), 8.13 (s, 1H, H ) 7.93 (s, 1H, triazole), 7.66 (m, 3H,
4
phenyl and H ), 7.04-6.99 ( m, 3H, phenyl and H ), 6.94 (d, 1H,
J
=9.0 Hz, H ), 4.52 (t, 2H,
5
8
6
J
=6.5 Hz, CH -N), 4.27 (d, 2H,
J
=5.0 Hz, CH -NH), 4.09 (t, 2H,
J
=5.5 Hz, CH -O), 3.80 (s,
2
2
2
3
H, OMe), 3.56 (quintet, 1H, J=6.0 Hz, CH-S), 3.18-3.08 (m, 2H, CH -S), 2.37 (sextet, 1H,
2
1
4

ACCEPTED MANUSCRIPT
J
=6.0 Hz, S-CH -CH), 2.30 (q, 2H, CH , aliphatic chain), 2.08 (t, 2H, J=7.0 Hz, CH -CO), 1.83
2
2
2
(
sextet, 1H, S-CH -CH), 1.65-1.55 (m, 1H, CO-CH -CH ), 1.54-1.48 (m, 3H, aliphatic chain),
2 2 2
1
3
1
.33-1.30 (m, 2H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
59.26, 154.4, 145.0, 139.3, 139.2, 130.3, 129.5, 128.6, 127.0, 122.9, 113.2, 112.9, 100.6, 65.3,
6.0, 55.2, 46.3, 38.0, 34.9, 34.0, 29.2, 28.2, 24.9. Anal. Calcd for C H N O S : C, 60.59; H,
δ: 171.8, 161.0, 160.0,
1
5
3
0
34
4
5 2
5.76; N, 9.42. Found: C, 60.46; H, 5.37; N, 9.23.
4
.1.6.8. N-((1-(3-((3-(3,4-dimethoxyphenyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (11
). Yield 80%; Off white solid; mp
-
1
1
1
5
36-138 ºC; IR (KBr, cm ) νmax: 3295 (NH), 2931 (C-H), 1719 (C=O). H NMR (DMSO-d6
,
00 MHz) δ: 8.27 (t, 1H, J=5.5 Hz, NH), 8.17 (s, 1H, H ), 7.93 (s, 1H, triazole), 7.66 (d, 1H,
4
J
=9.0 Hz, H ), 7.32 (m, 2H, phenyl), 7.02-7.00 ( m, 1H, phenyl and 1H ), 6.95 (d, 1H,
J=9.0
5
8
Hz, H ), 4.52 (t, 2H,
J
=6.5 Hz, CH -N), 4.26 (d, 2H,
J
=5.0 Hz, CH -NH), 4.09 (t, 2H,
J=5.5 Hz,
6
2
2
CH -O), 3.80 (s, 6H, 2MeO), 3.56 (quintet, 1H, J=6.0 Hz, CH-S), 3.22-3.08 (m, 2H, CH -S),
2
2
2
.38 (sextet, 1H,
J=6.0 Hz, S-CH -CH), 2.29 (q, 2H, CH , aliphatic chain), 2.08 (t, 2H, J=7.0
2 2
Hz, CH -CO), 1.75-165 (m, 1H, S-CH -CH), 1.65-160 (m, aliphatic), 1.53-1.50 (m, 3H,
2
2
1
3
aliphatic chain), 1.33-1.29 (m, 2H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
δ
: 171.9,
66.1, 161.1, 160.0, 154.4, 149.0, 145.1, 142.0, 127.3, 122.9, 113.3, 112.9, 112.2, 111.4, 100.7,
5.3, 56.1, 55.5, 46.3, 38.0, 35.0, 34.0, 29.3, 28.2, 24.9. Anal. Calcd for C H N O S : C,
1
6
3
1
36
4
6 2
5
9.60; H, 5.81; N, 8.97. Found: C, 59.64; H, 5.83; N, 9.07.
4
.1.6.9. N-((1-(3-((3-(4-bromophenyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (12
). Yield 87%; Off white solid; mp 150-152
-1
1
ºC; IR (KBr, cm ) νmax: 3282 (NH), 2928 (C-H), 1720 (C=O). H NMR (DMSO-d6, 500
MHz)
δ
: 8.26 (m, 1H, NH and 1H, H ), 7.93 (s, 1H, triazole) 7.70-7.64 (m, 5H, phenyl and H ),
4
5
7
.01( s, 1H, H ), 6.96 (d, 1H,
J
=9.0 Hz, H ), 4.52 (t, 2H,
J
=6.5 Hz, CH -N), 4.27 (d, 2H,
J=5.0
8
6
2
Hz, CH -NH), 4.01 (t, 2H,
J
=5.5 Hz, CH -O), 3.56 (quintet, 1H,
J
=6.0 Hz, CH-S), 3.20-3.08
m, 2H, CH -S), 2.37 (sextet, 1H, J=6.0 Hz, S-CH -CH), 2.30 (q, 2H, CH , aliphatic chain),
2
2
2
(
2
2
2
.08 (t, 2H,
.53-1.49 (m, 3H, aliphatic chain), 1.34-1.30 (m, 2H, aliphatic chain). C NMR (DMSO- 6
25 MHz) : 176.1, 171.8, 161.9, 159.7, 156.8, 154.8, 145.1, 140.8, 133.9, 131.1, 130.3, 121.4,
13.0, 100.7, 65.4, 56.0, 46.3, 38.0, 34.9, 34.0, 29.2, 28.2, 24.9. Anal. Calcd for
J=7.0 Hz, CH -CO), 1.85-1.80 (m, 1H, S-CH -CH), 1.67-1.60 (m, 1H, aliphatic),
2
2
1
3
1
d
,
1
1
δ
C H BrN O S : C, 54.12; H, 4.86; N, 8.71. Found: C, 54.32; H, 5.02; N, 8.33.
2
9
31
4
4 2
1
5

ACCEPTED MANUSCRIPT
4
4
.1.6.10. N-((1-(3-((3-(4-chlorophenyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-
-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (13
). Yield 92%; Off white solid; mp 163-165
-1
1
ºC; IR (KBr, cm ) νmax: 3320 (NH), 2932 (C-H), 1720 (C=O). H NMR (DMSO-d6, 500
MHz)
δ
: 8.27-8.25 (m, 1H, NH and 1H, H ), 7.94 (s, 1H, triazole), 7.75 (d, 2H,
J
=7.5 Hz,
=8.5 Hz, H ), 7.51 (d, 2H, J=8.0 Hz, phenyl), 7.01(s, 1H, H ), 6.96 (d,
5
4
phenyl), 7.68 (d, 1H,
J
8
1
2
H,
H,
J=9.0 Hz, H ), 4.52 (t, 2H, J=6.5 Hz, CH -N ), 4.27 (d, 2H, J=5.0 Hz, CH -NH), 4.01 (t,
6 2 2
J
=5.5 Hz, CH -O), 3.57 (quintet, 1H, J=6.0 Hz, CH-S), 3.16-3.09 (m, 2H, CH -S), 2.38
2
2
(sextet, 1H,
J=6.0 Hz, S-CH -CH), 2.31-2.29 (m, 2H, CH , aliphatic chain), 2.08 (bs, 2H, CH -
2 2 2
CO), 1.85-1.81 (m, 1H, S-CH -CH), 1.64-1.61 (m, 1H, CO-CH -CH ), 1.52-1.51 (m, 3H,
2
2
2
1
3
aliphatic chain), 1.33-1.31 (m, 2H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
δ
: 171.8,
61.5, 159.7, 154.7, 154.0, 141.0, 133.6, 132.8, 130.0, 128.3, 128.0, 122.7, 121.9, 113.0, 65.4,
6.0, 46.3, 38.0, 34.9, 34.0, 29.2, 28.2, 24.8. Anal. Calcd for C H ClN O S : C, 58.13; H,
1
5
5
2
9
31
4
4 2
.22; N, 9.35. Found: C, 58.35; H, 5.46; N, 9.47.
.1.6.11. N-((1-(3-((3-(3,4-dichlorophenyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-
4
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (14
). Yield 97%; Off white solid; mp
-
1
1
1
5
7
50-152 ºC; IR (KBr, cm ) νmax: 3280 (NH), 2927 (C-H), 1716 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ: 8.34 (s, 1H, H ), 8.26 (bs, 1H, NH), 8.00 (s, 1H, H ), 7.93 (s, 1H, triazole), 7.73-
4 5
.68 (m, 3H, phenyl), 7.01 (s, 1H, H ), 6.96 (d, 1H,
J
=8.5 Hz, H ), 4.52 (bs, 2H, CH -N), 4.28
8
6 2
(
bs, 2H, CH -NH), 4.10 (bs, 2H, CH -O), 3.56 (bs, 1H, CH-S), 3.15-3.10 (m, 2H, CH -S), 2.38
2
2
2
(sextet, 1H,
J
=6.0 Hz, S-CH -CH), 2.30 (bs, 2H, aliphatic), 2.08 (s, 2H, CH CO), 1.85-1.80 (m,
2
2
1
2
H, S-CH -CH), 1.63-1.57 (m, 1H, aliphatic), 1.52-1.50 (m, 3H, aliphatic chain), 1.33-1.31 (m,
2
1
3
H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
41.8, 135.4, 130.6, 130.2, 129.9, 129.8, 128.3, 122.9, 122.7, 120.4, 112.9, 100.8, 65.4, 56.0,
6.3, 38.0, 34.9, 34.0, 29.2, 28.2, 24.8. Anal. Calcd for C H Cl N O S : C, 54.97; H, 4.77; N,
δ: 171.8, 161.7, 159.5, 154.9, 145.0,
1
4
2
9
30
2
4
4 2
8
.84. Found: C, 54.76; H, 4.43; N, 8.55.
4
4
.1.6.12. 5-(1,2-dithiolan-3-yl)-N-((1-(4-((2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-
-1
-yl)methyl)pentanamide (15
)
. Yield 96%; Off white solid; mp 128-130 ºC; IR (KBr, cm )
1
νmax: 3285 (NH), 2926 (C-H), 1733, 1628 (C=O). H NMR (DMSO-d6, 500 MHz)
δ
: 8.24 (t,
=8.5 Hz, H5
=9.5 Hz, H3),
=6.0 Hz, CH -NH), 4.09 (t, 2H, J=6.0 Hz, CH -O),
1
H,
, 6.96 (d, 1H,
.40 (t, 2H, =6.5 Hz, CH -N), 4.27 (d, 2H,
J
=5.5 Hz, NH), 7.97 (d, 1H,
=2.0 Hz, H ), 6.92 (dd, 1H, J=8.5 Hz, J=2.0 Hz, H ), 6.27 (d, 1H, J
6
J=9.5 Hz, H ), 7.90 (s, 1H, triazole), 7.61 (d, 1H, J
4
)
J
8
4
J
J
2
2
2
1
6

ACCEPTED MANUSCRIPT
3
.58 (quintet, 1H,
J
=6.0 Hz, CH-S), 3.19-3.07 (m, 2H, CH -S), 2.39 (sextet, 1H,
J=6.7 Hz, CH-
2
CH -S), 2.09 (t, 2H,
J
=6.0 Hz, CH -CH -N), 1.96 (quintet, 2H, J=7.0 Hz, COCH ), 1.83 (sextet,
2
2 2 2
1
H,
m, 2H, aliphatic). C NMR (DMSO-d6, 125 MHz)
12.3, 101.1, 67.5, 56.0, 45.9, 38.0, 34.9, 34.0, 28.1, 26.4, 25.3, 24.8. Anal. Calcd for
C H N O S : C, 57.35; H, 6.02; N, 11.15. Found: C, 57.52; H, 6.29; N, 11.23.
J=7.0 Hz, CH-CH -S), 1.71-169 (m, 3H, aliphatic), 1.55-1.50 (m, 3H, aliphatic), 1.34-1.32
2
1
3
(
δ: 151.4, 144.2, 129.3, 122.6, 112.6,
1
2
4
30
4
4 2
4
1
.1.6.13.
5-(1,2-dithiolan-3-yl)-N-((1-(4-((2-oxo-3-phenyl-2H-chromen-7-yl)oxy)butyl)-1H-
,2,3-triazol-4-yl)methyl)pentanamide (16
). Yield 76%; Off white solid; mp 134-136 ºC; IR
-1
1
(
KBr, cm ) νmax: 3292 (NH), 2924 (C-H), 1722 (C=O). H NMR (DMSO-d6, 500 MHz)
δ
8.26
(
t, 1H, NH), 8.20 (s, 1H, H ), 7.91 (s, 1H, triazole), 7.71-7.68 (m, 3H, phenyl), 7.45-7.39 ( m,
4
2
H, phenyl and 1H, H ), 7.02 (s, 1H, H ), 6.97 (d, 1H,
J
=8.0 Hz, H ), 4.41 (t, 2H,
J=7.0 Hz,
5
8
6
CH -N ), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.12 (t, 2H,
J
=6.5 Hz, CH -O), 3.57 (quintet, 1H,
2
2
2
J
=6.0 Hz, CH-S), 3.19-3.10 (m, 2H, CH -S), 2.37 (sextet, 1H, J=6.0 Hz, CH-CH -S), 2.08 (t,
2
2
2
H, J=7.0 Hz, CH -CO), 1.97 (bs, 2H, CH -CH -N), 1.84-1.83 (m, 1H, CH-CH -S), 1.72-1.64
2 2 2 2
(
m, 2H, O-CH -CH and 1H, CH-CH -CO), 1.55-1.49 (m, 3H, aliphatic chain), 1.35-1.32 (m,
2 2 2
1
3
2
H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
34.7, 128.4, 128.2, 127.9, 123.2, 122.9, 112.9, 67.9, 56.1, 48.8, 37.9, 34.9, 33.9, 28.1, 26.4,
5.4, 24.8. Anal. Calcd for C H N O S : C, 62.26; H, 5.92; N, 9.68. Found: C, 62.09; H,
δ: 171.9, 162.3, 159.8, 145.0, 141.0,
1
2
3
0
34
4
4 2
6.19; N, 9.84.
4
.1.6.14.
5-(1,2-dithiolan-3-yl)-N-((1-(4-((3-(4-methoxyphenyl)-2-oxo-2H-chromen-7-
yl)oxy)butyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (17
). Yield 75%; Off white solid; mp
-
1
1
1
5
45-146 ºC; IR (KBr, cm ) νmax: 3292 (NH), 2931 (C-H), 1720 (C=O). H NMR (DMSO-d6
,
00 MHz) δ: 8.26 (t, 1H, J=5.5 Hz, NH), 8.13 (s, 1H, H ), 7.91 (s, 1H, triazole), 7.67 (m, 3H,
4
phenyl and H ), 7.00 (m, 3H, phenyl and H ), 6.94 (d, 1H,
J
=8.5 Hz, H ), 4.41 (t, 2H,
J=7.0 Hz,
5
8
6
CH -N), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.10 (t, 2H,
J
=6.5 Hz, CH -O), 3.80 (s, 3H, OMe),
2
2
2
3
2
1
1
1
.58 (m, 1H, CH-S), 3.19-3.09 (m, 2H, CH -S), 2.38 (sextet, 1H, J=6.0 Hz, S-CH -CH), 2.08 (s,
2 2
H, CH -CO), 1.98-1.94 (m, 2H, CH -CH -N), 1.83 (sextet, 1H, J=6.0 Hz, S-CH -CH), 1.73-
2
2
2
2
.70 (m, 2H, O-CH -CH ), 1.65-1.60 (m, 1H, CH-CH -CO), 1.54-1.49 (m, 3H, aliphatic chain),
2
2
2
1
3
.34-1.31(m, 2H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
δ: 171.8, 161.2, 160.0,
59.2, 154.4, 144.9, 139.4, 139.3, 129.5, 127.0, 122.7, 114.2, 113.6, 113.1, 100.7, 67.5, 56.0,
1
7

ACCEPTED MANUSCRIPT
5
5.1, 48.8, 34.9, 34.0, 28.2, 26.4, 25.4, 24.9. Anal. Calcd for C H N O S : C, 61.16; H, 5.96;
31 36 4
5 2
N, 9.20. Found: C, 61.31; H, 6.15; N, 9.54.
4
.1.6.15.
N-((1-(4-((3-(3,4-dimethoxyphenyl)-2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (18
). Yield 74%; Off white solid; mp
-
1
1
1
5
34-135 ºC; IR (KBr, cm ) νmax: 3298 (NH), 2934 (C-H), 1716 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ
: 8.28 (t, 1H, NH), 8.17 (s, 1H, H ), 7.91 (s, 1H, triazole), 7.66 (d, 1H,
J
=9.0 Hz,
4
H ), 7.31 (m, 2H, phenyl), 7.02-7.00 ( m, 2H, phenyl and H ), 6.95 (d, 1H,
J=9.0 Hz, H ), 4.41
5
8
6
(t, 2H,
J
=6.5 Hz, CH -N), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.10 (t, 2H,
J
=6.5 Hz, CH -O), 3.80
2
2
2
(
bs, 6H, 2OMe), 3.57 (quintet, 1H,
H, =6.0 Hz, CH-CH -S), 2.09 (s, 2H, CH -CO), 1.98-1.93 (m, 2H, CH -CH -N), 1.83 (sextet,
=6.5 Hz, 1H, CH-CH -S), 1.72-1.70 (m, 2H, O-CH -CH ), 1.66-1.62 (m, 1H, CH-CH -CO),
J=6.0 Hz, CH-S), 3.16-3.10 (m, 2H, CH -S), 2.38 (sextet,
2
1
J
2
2
2
2
J
1
1
1
2
2
2
2
1
3
.52-1.49 (m, 3H, aliphatic chain), 1.33-1.30 (m, 2H, aliphatic chain). C NMR (DMSO- 6
25 MHz) : 171.9, 161.3, 160.1, 154.5, 149.0, 148.3, 145.0, 127.3, 122.8, 121.1, 113.1, 112.9,
12.1, 111.4, 100.7, 67.6, 56.1, 55.6, 48.9, 38.1, 35.0, 34.1, 28.3, 26.5, 25.5, 24.9. Anal. Calcd
d ,
δ
for C H N O S : C, 60.17; H, 6.00; N, 8.77. Found: C, 59.98; H, 5.88; N, 8.39.
3
2
38
4
6 2
4
.1.6.16. N-((1-(4-((3-(4-bromophenyl)-2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (19
). Yield 83%; Off white solid; mp 158-160
-1
1
ºC; IR (KBr, cm ) νmax: 3291 (NH), 2923 (C-H), 1719 (C=O). H NMR (DMSO-d6, 500
MHz)
δ
: 8.24 (m, 1H, NH and H ), 7.91 (s, 1H, triazole), 7.69-7.63 (m, 5H, phenyl and H ),
4
5
7
.02 (s, 1H, H ), 6.97 (d, 1H,
J
=9.0 Hz, H ), 4.41 (t, 2H,
J
=7.0 Hz, CH -N), 4.27 (d, 2H,
J=6.0
8
6
2
Hz, CH -NH), 4.11 (t, 2H,
J
=6.5 Hz, CH -O), 3.58 (quintet, 1H,
J
=6.0 Hz, CH-S), 3.16-3.09
m, 2H, CH -S), 2.39 (sextet, 1H, J=6.0 Hz, S-CH -CH), 2.08 (s, 2H, CH -CO), 1.95 (m, 2H,
2
2
2
(
2
2
CH -CH -N), 1.82 (sextet, 1H, J=6.5 Hz, S-CH -CH), 1.73-1.70 (m, 2H, O-CH -CH ), 1.65-
2 2 2
2
2
1
60 (m, 1H, CH-CH -CO), 1.54-1.48 (m, 3H, aliphatic chain), 1.34-1.31 (m, 2H, aliphatic
2
1
3
chain). C NMR (DMSO- 6
d
, 125 MHz)
30.2, 122.9, 121.4, 113.0, 112.9, 100.7, 67.6, 56.0, 48.8, 38.0, 34.9, 34.0, 28.2, 26.4, 25.4,
4.8. Anal. Calcd for C H BrN O S : C, 54.79; H, 5.06; N, 8.52. Found: C, 54.80; H, 5.12;
δ: 171.8, 161.7, 159.9, 154.7, 145.1, 134.0, 131.1,
1
2
3
0
33
4
4 2
N, 8.59.
4
.1.6.17. N-((1-(4-((3-(4-chlorophenyl)-2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (20 . Yield 97%; Off white solid; mp 170-172
)
1
8

ACCEPTED MANUSCRIPT
-1
1
ºC; IR (KBr, cm ) νmax: 3320 (NH), 2932 (C-H), 1720 (C=O). H NMR (DMSO-d6, 500
MHz) : 8.27 (t, 1H, =5.0 Hz NH), 8.24 (s, 1H, H ), 7.91 (s, 1H, triazole), 7.74 (d, 2H, =8.0
Hz, phenyl), 7.67 (d, 1H,
d, 1H, =8.5 Hz, H ), 4.40 (t, 2H,
H,
sextet, 1H,
CH -N), 1.83 (sextet, 1H, S-CH -CH), 1.73-1.70 (m, 2H, O-CH -CH ), 1.67-1.62 (m, 1H, CH-
δ
J
J
4
J
=8.5 Hz, H ), 7.50 (d, 2H,
J
=8.0 Hz, phenyl), 7.02 (s, 1H, H ), 6.96
5
8
(
J
J
=7.0 Hz, CH -N), 4.26 (d, 2H,
J
=6.0 Hz, CH -NH), 4.11 (t,
6
2
2
2
J=6.5 Hz, CH -O), 3.57 (quintet, 1H, J=6.0 Hz, CH-S), 3.17-3.10 (m, 2H, CH -S), 2.37
2 2
(
J=6.0 Hz, S-CH -CH), 2.09 (t, 2H, J=7.5 Hz, CH -CO), 1.98-1.95 (m, 2H, CH -
2 2 2
2
2
2
2
1
3
CH -CO), 1.54-1.51 (m, 3H, aliphatic chain), 1.33-1.30 (m, 2H, aliphatic chain). C NMR
2
(
DMSO-d6, 125 MHz)
22.6, 121.8, 113.0, 104.4, 100.9, 67.7, 56.1, 48.8, 38.0, 34.9, 34.0, 28.2, 26.5, 25.4. Anal.
Calcd for C H ClN O S : C, 58.76; H, 5.42; N, 9.14. Found: C, 58.82; H, 5.50; N, 9.31.
δ: 171.9, 161.8, 159.8, 154.8, 145.0, 141.2, 133.7, 132.8, 130.0, 128.1,
1
3
0
33
4
4 2
4
.1.6.18.
N-((1-(4-((3-(3,4-dichlorophenyl)-2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (21
). Yield 95%; Off white solid; mp
-
1
1
1
5
7
13-115 ºC; IR (KBr, cm ) νmax: 3290 (NH), 2931 (C-H), 1718 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ: 8.35 (s, 1H, H ), 8.26 (bs, 1H, NH), 8.01 (s, 1H, H ), 7.89 (s, 1H, triazole), 7.75-
4 5
.68 (m, 3H, phenyl), 7.04-6.97 (m, 2H, H ), 4.35 (t, 2H,
J
=7.0 Hz, CH -N), 4.26 (d, 2H,
8
,6
2
J
=6.0 Hz, CH -NH), 4.07 (t, 2H, J=6.5 Hz, CH -O), 3.62-3.57 (m, 1H, CH-S), 3.15-3.10 (m,
2
2
2
H, CH -S), 2.39-2.35 (m, 1H, S-CH -CH), 2.08 (bs, 2H, CH -CO), 1.98-1.97 (m, 2H, CH -
2 2 2 2
CH -N), 1.87-1.76 (m, 1H, S-CH -CH and 2H, O-CH -CH ), 1.67-1.63 (m, 1H, CO-CH -CH),
2
2
2
2
2
1
3
1
.51-1.32 (m, 5H, aliphatic). C NMR (DMSO-d6, 125 MHz)
δ: 170.2, 161.9, 155.2, 146.8,
1
2
41.1, 135.3, 131.3, 130.0, 128.4, 122.1, 119.2, 112.8, 100.5, 67.6, 56.0, 48.9, 38.3, 35.0, 34.0,
9.2, 28.2, 27.6, 22.5. Anal. Calcd for C H Cl N O S : C, 55.64; H, 4.98; N, 8.65. Found: C,
3
0
32
2
4
4 2
5
5.98; H, 5.23; N, 8.91.
4
.1.6.19. 5-(1,2-dithiolan-3-yl)-N-((1-(5-((2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-
triazol-4-yl)methyl)pentanamide (22
). Yield 96%; Off white solid; mp 93-94 ºC; IR (KBr,
-1
1
cm ) νmax: 3295 (NH), 2929 (C-H), 1731, 1621 (C=O). H NMR (DMSO-d6, 500 MHz)
δ: 8.23
(t, 1H,
J
=5.5 Hz, NH), 7.97 (d, 1H, J=9.0 Hz, H ), 7.87 (s, 1H, triazole), 7.60 (d, 1H,
J=8.5 Hz,
4
H ), 6.96 (s, 1H, H ), 6.92 (dd, 1H,
J=8.5 Hz,
J
=2.0 Hz, H ), 6.27 (d, 1H,
J
=9.0 Hz, H ), 4.34
5
8
6
3
(t, 2H,
J
=6.5 Hz, CH -N), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.05 (t, 2H, J=6.0 Hz, CH -O), 3.57
2
2
2
(quintet, 1H,
J
=6.0 Hz, CH-S), 3.19-3.07 (m, 2H, CH -S), 2.38 (sextet, 1H,
J=6.7 Hz,
2
CHCH S), 2.09 (t, 2H,
J=6.0 Hz, COCH ), 1.88-1.73 (m, 5H, aliphatic), 1.66-1.62 (m, 1H,
2
2
1
9

ACCEPTED MANUSCRIPT
1
3
CHCH -CO), 1.53-1.31 (m, 7H aliphatic chain). C NMR (DMSO-d6, 125 MHz)
δ
: 171.7,
61.7, 160.1, 155.3, 144.8, 144.1, 129.3, 122.4, 112.5, 112.2, 112.1, 101.0, 67.9, 56.0, 49.0,
7.9, 34.9, 34.0, 33.9, 29.3, 28.1, 27.6, 24.8, 22.3. Anal. Calcd for C H N O S : C, 58.12; H,
2
1
3
2
5
32
4
4 2
6.24; N, 10.84. Found: C, 57.9; H, 6.19; N, 11.10.
4
1
.1.6.20.
5-(1,2-dithiolan-3-yl)-N-((1-(5-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-
,2,3-triazol-4-yl)methyl)pentanamide (23). Yield 98%; Off white solid; mp 66-67 ºC; IR
-
1
1
(
KBr, cm ) νmax: 3295 (NH), 2862 (C-H), 1721, 1616 (C=O). H NMR (DMSO-d6, 500 MHz)
: 8.29 (t, 1H, J=5.0 Hz NH), 7.89 (s, 1H, triazole), 7.67 (d, 1H, =8.5 Hz, H ), 6.95-6.93 (m,
H, H ), 6.20 (s, 1H, H ), 4.35 (t, 2H, =7.0 Hz, CH -N), 4.26 (d, 2H, J=5.5 Hz, CH -NH),
δ
,
J
5
2
4
2
J
6
,8
3
2
2
.04 (t, 2H,
J=6.0 Hz, CH -O), 3.58 (quintet, 1H, J=6.5 Hz, CH-S), 3.15-3.11 (m, 2H, CH -S),
2 2
.49-2.39 (m, 3H, CH and 1H CH-CH S), 2.09 (t, 2H,
J
=7.0 Hz, CH -CO), 1.86-1.76 (m, 5H,
3
2
2
1
3
aliphatic), 1.65-1.60 (m, 1H, aliphatic), 1.51-1.31 (m, 7H, aliphatic). C NMR (DMSO-d6, 125
MHz) : 171.8, 161.7, 154.7, 153.4, 144.9, 131.6, 126.4, 122.6, 113.0, 112.4, 111.0, 101.1,
8.0, 56.1, 49.1, 38.0, 34.9, 34.10, 29.4, 28.3, 27.8, 24.9, 22.4, 18.1. Anal. Calcd for
C H N O S : C, 58.84; H, 6.46; N, 10.56. Found: C, 59.09; H, 6.68; N, 10.83.
δ
6
2
6
34
4
4 2
4
.1.6.21.
N-((1-(5-((3-acetyl-2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-triazol-4-
yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (24). Yield 72%; Off white solid; mp 120-122
-1
1
ºC; IR (KBr, cm ) νmax: 3489 (NH), 2927 (C-H), 1715, 1627 (C=O). H NMR (DMSO-d6, 500
MHz) : 8.62 (s, 1H, H ), 8.22 (s, 1H, NH), 7.87 (s, 1H, triazole), 7.85 ( d, 1H, =8.5 Hz, H5),
.03 (s, 1H, H ), 6.99 (d, 1H, =7.0 Hz, CH -N), 4.26 (d, 2H, =5.5
=6.0 Hz, CH-S), 3.19-3.07
m, 2H, CH -S), 2.55 (s, 3H, COCH ), 2.39-2.37 (m, 1H, CHCH S), 2.09 (t, 2H, J=7.5 Hz,
δ
J
4
7
J
=8.5 Hz, H ), 4.34 (t, 2H,
J
J
8
6
2
Hz, CH -NH), 4.11 (t, 2H, J=6.5 Hz, CH -O), 3.58 (quintet, 1H,
J
2
2
(
2
3
2
CH CO), 1.87-1.76 (m, 5H, aliphatic), 1.65-1.62 (m, 1H, aliphatic), 1.54-1.50 (m, 3H,
2
1
3
aliphatic), 1.40-1.31 (m, 4H, aliphatic). C NMR (DMSO-d6, 125 MHz)
δ
: 194.6, 171.7,
51.7, 147.5, 144.8, 132.1, 122.4, 113.7, 100.6, 68.3, 56.0, 49.0, 37.9, 34.9, 34.0, 33.9, 29.9,
9.3, 28.1, 27.6, 24.8, 22.3. Anal. Calcd for C H N O S : C, 58.04; H, 6.13; N, 10.3. Found:
1
2
2
7
34
4
5 2
C, 58.19; H, 5.93; N, 9.98.
4
.1.6.22.
ethyl
7-((5-(4-((5-(1,2-dithiolan-3-yl)pentanamido)methyl)-1H-1,2,3-triazol-1-
yl)pentyl)oxy)-2-oxo-2H-chromene-3-carboxylate (25). Yield 83%; Off white solid; mp 108-
-1
1
1
10 ºC; IR (KBr, cm ) νmax: 3308 (NH), 2851 (C-H), 1736 and 1603 (C=O). H NMR
2
0

ACCEPTED MANUSCRIPT
(DMSO-d6, 500 MHz)
δ
: 8.70 (s, 1H, H ), 8.22 (t, 1H, J=5.5 Hz, NH), 7.87 (s, 1H, triazole),
4
7
2
.82 ( d, 1H,
J=8.5 Hz, H ), 7.00 (s, 1H, H ), 6.97 (dd, 1H, J=2.0 Hz, J=8.5 Hz, H ), 4.34 (t,
5 8 6
H, =7.0 Hz, CH -N), 4.28-4.25 (m, 2H, CH -NH and 2H, O-CH -CH ), 4.10 (t, 2H, J=6.5 Hz,
J
2
2
2
3
CH -O), 3.58 (quintet, 1H,
J
=6 Hz, CH-S), 3.19-3.07 (m, 2H, CH -S), 2.39 (sextet, 1H,
J=6.0
2
2
Hz, CHCH S), 2.09 (t, 2H, J=7.0 Hz, CH -CO), 1.88-1.76 (m, 5H, aliphatic), 1.67-1.61 (m, 1H,
2
2
1
3
CHCH CO), 1.53-1.50 (m, 3H, aliphatic), 1.39-1.28 (t, 4H, aliphatic and 3H, CH ). C NMR
2
3
(DMSO-d6, 125 MHz)
δ: 171.7, 164.0, 162.8, 156.1, 148.9, 144.8, 131.5, 122.4, 113.5, 111.2,
1
00.5, 68.3, 60.7, 56.0, 49.0, 37.9, 34.9, 34.0, 33.9, 29.2, 28.1, 27.6, 24.8, 22.3, 13.9. Anal.
Calcd for C H N O S : C, 57.12; H, 6.16; N, 9.52. Found: C, 56.95; H, 5.89; N, 9.25.
2
8
36
4
6 2
4
1
.1.6.23.
5-(1,2-dithiolan-3-yl)-N-((1-(5-((2-oxo-3-phenyl-2H-chromen-7-yl)oxy)pentyl)-
H-1,2,3-triazol-4-yl)methyl)pentanamide (26). Yield 77%; Off white solid; mp 128-130
-1
1
ºC; IR (KBr, cm ) νmax: 3330 (NH), 2930 (C-H), 1720 (C=O). H NMR (DMSO-d6, 500
MHz) 8.26 (t, 1H, J=5.5 Hz, NH), 8.20 (s, 1H, H ), 7.89 (s, 1H, triazole), 7.69 (m, 3H,
phenyl), 7.43 ( m, 2H, phenyl), 7.39 (t, 1H,
=8.0 Hz, H ), 4.36 (t, 2H, =7.0 Hz, CH -N ), 4.27 (d, 2H,
δ
4
J
=7.0 Hz, H ), 7.01 (s, 1H, H ), 6.95 (d, 1H,
5
8
J
J
J
=6.0 Hz, CH -NH ), 4.08 (t,
6
2
2
2
1
2
H,
H,
J
=6.5 Hz, CH -O), 3.56-3.52 (m, 1H, CH-S), 3.17-3.10 (m, 2H, CH -S), 2.39 (sextet,
2
2
J=6.0 Hz, CH-CH -S), 2.09 (t, 2H,
J
=7.0 Hz, CH -CO), 1.89-1.76 (m, 2H, N-CH -CH ,
2
2
2
2
H, O-CH -CH and 1H, CH-CH -S), 1.65-1.62 (m, 1H, CHCH CO), 1.54-1.50 (m, 3H,
2
2
2
2
1
3
aliphatic), 1.41-1.30 (m, 4H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
δ
: 171.8,
61.7, 159.9, 144.9, 140.8, 134.9, 129.6, 128. 2, 123.0, 112.9, 68.0, 56.0, 49.0, 34.9, 34.0,
9.4, 28.2, 27.7, 24.9, 22.4. Anal. Calcd for C H N O S : C, 62.81; H, 6.12; N, 9.45.
1
2
3
1
36
4
4 2
Found: C, 63.01; H, 6.30; N, 9.21.
4
.1.6.24.
5-(1,2-dithiolan-3-yl)-N-((1-(5-((3-(4-methoxyphenyl)-2-oxo-2H-chromen-7-
yl)oxy)pentyl)-1H-1,2,3-triazol-4-yl)methyl)pentanamide (27
). Yield 78%; Off white solid; mp
-
1
1
1
5
20-122 ºC; IR (KBr, cm ) νmax: 3291 (NH), 2934 (C-H), 1722 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ 8.26 (t, 1H, J=5.5Hz, NH), 8.12 (s, 1H, H ), 7.89 (s, 1H, triazole), 7.66 (m, 3H,
4
phenyl and H ), 7.01-6.98 (m, 3H, phenyl and H ), 6.93 (d, 1H,
J
=8.0 Hz, H ), 4.35 (t, 2H,
5
8
6
J
=7.0 Hz, CH -N), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.06 (t, 2H,
J
=6.5 Hz, CH -O), 3.80 (s,
2
2
2
3
H, OMe), 3.57 (m, 1H, CH-S), 3.16-3.10 (m, 2H, CH -S), 2.37 (sextet, 1H,
J=6.0 Hz, CH-
2
CH -S), 2.08 (t, 2H,
J=7.0 Hz, CH -CO), 1.89-176 (m, 2H, N-CH -CH and 2H, O-CH -CH
2
2
2
2
2
2
and 1H, CH-CH -S), 1.66-1.62 (m, 1H, CHCH CO), 1.54-1.48 (m, 3H, aliphatic), 1.41-1.30 (m,
2
2
2
1

ACCEPTED MANUSCRIPT
1
3
4
H, aliphatic chain). C NMR (DMSO-d6, 125 MHz)
44.9, 139.4, 129.5, 127.1, 122.7, 122.5, 113.6, 113.5, 113.0, 112.6, 100.6, 68.0, 56.0, 55.1,
9.0, 38.0, 34.9, 34.0, 29.4, 28.2, 27.7, 24.9, 22.4. Anal. Calcd for C H N O S : C, 61.71; H,
δ: 171.8, 161.3, 160.0, 149.2, 154.5,
1
4
3
2
38
4
5 2
6.15; N, 9.00. Found: C, 61.80; H, 6.31; N, 9.25.
4
.1.6.25. N-((1-(5-((3-(3,4-dimethoxyphenyl)-2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (28
). Yield 915; Off white solid; mp
-
1
1
1
5
14-117 ºC; IR (KBr, cm ) νmax: 3297 (NH), 2935 (C-H), 1717 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ
8.25 (t, 1H, NH), 8.16 (s, 1H, H ), 7.88 (s, 1H, triazole ring), 7.65 (d, 1H,
J
=9.0
=9.0 Hz, H6),
=6.5 Hz, CH -O),
4
Hz, H ), 7.31 (m, 2H, phenyl), 7.02-6.99 ( m, 2H, phenyl and H ), 6.94 (d, 1H,
J
5
8
4
3
1
2
.35 (t, 2H,
J
=7.0 Hz, CH -N), 4.27 (d, 2H,
J
=6.0 Hz, CH -NH), 4.06 (t, 2H,
J
2
2
2
.80 (d, 6H,
H, =6.0 Hz, CH-CH -S), 2.09 (t, 2H,
H and O-CH -CH , 1H, CH-CH -S), 1.65-1.62 (1H, CHCH CO), 1.52-1.50 (m, 3H, aliphatic
J=5.0 Hz, 2OMe), 3.57 (m, 1H, CH-S), 3.16-3.10 (m, 2H, CH -S), 2.38 (sextet,
2
J
J=7.0 Hz, CH -CO), 1.88-1.75 (m, 2H, N-CH -CH and
2 2 2
2
2
2
2
2
1
3
chain), 1.41-1.30 (4H, aliphatic). C NMR (DMSO-d6, 125 MHz)
δ
: 171.8, 161.4, 160.0,
54.4, 148.9. 148.2, 144.9, 129.3, 127.3, 122.7, 121.0, 113.0, 112.1, 111.4, 100.6, 68.0, 56.0,
5.5, 49.0, 38.0, 34.9, 34.0, 34.0, 29.4, 28.2, 27.7. Anal. Calcd for C H N O S : C, 60.72; H,
1
5
3
3
40
4
6 2
6.18; N, 8.58. Found: C, 60.54; H, 6.34; N, 8.62.
4
4
.1.6.26. N-((1-(5-((3-(4-bromophenyl)-2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-triazol-
-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (29
). Yield 82%; Off white solid; mp 136-139
-1
1
ºC; IR (KBr, cm ) νmax: 3290 (NH), 2930 (C-H), 1720 (C=O). H NMR (DMSO-d6, 500
MHz)
δ
8.26-8.24 (m, 1H, NH and H ), 7.89 (s, 1H, triazole), 7.68-7.63 (m, 5H, phenyl and H ),
4
5
7
.01 (s, 1H, H ), 6.95 (d, 1H,
J
=8.0 Hz, H ), 4.32 (t, 2H,
J
=7.0 Hz, CH -N), 4.26 (d, 2H,
J=6.0
8
6
2
Hz, CH -NH), 4.07 (t, 2H,
J
=6.5 Hz, CH -O), 3.57 (m, 1H, CH-S), 3.20-3.09 (m, 2H, CH -S),
2
2
2
2
.40-2.36 (m, 1H, CH-CH -S), 2.09 (m, 2H,
J
= 7.0 Hz, CH -CO), 1.87-1.77 (m, 2H, N-CH -
2
2
2
CH and 2H, O-CH -CH and 1H, CH-CH -S), 1.63-1.61 (m, 1H, CHCH CO), 1.51-1.31 (m,
2
2
2
2
2
1
3
7
1
3
8
H, aliphatic). C NMR (DMSO- 6
d
, 125 MHz)
δ
: 171.8, 161.9, 159.6, 154.9, 145.1, 141.0,
34.1, 131.1, 130.3, 122.7, 122.0, 121.4, 113.9, 113.0, 112.8, 100.6, 68.1, 56.1, 49.1, 38.0, 34.9,
4.1, 34.0, 29.4, 28.2, 27.7, 24.9, 22.4. Anal. Calcd for C H BrN O S : C, 55.44; H, 5.25; N,
3
1
35
4
4 2
.34. Found: C, 55.49; H, 5.30; N, 8.42.
2
2

ACCEPTED MANUSCRIPT
4
.1.6.27.
N-((1-(5-((3-(3,4-dichlorophenyl)-2-oxo-2H-chromen-7-yl)oxy)pentyl)-1H-1,2,3-
triazol-4-yl)methyl)-5-(1,2-dithiolan-3-yl)pentanamide (30
). Yield 100%; Off white solid; mp
-
1
1
1
5
32-134 ºC; IR (KBr, cm ) νmax: 3285 (NH), 2924 (C-H), 1717 (C=O). H NMR (DMSO-d6
,
00 MHz)
δ
: 8.34 (s, 1H, H ), 8.27 (t, 1H,
J
=5.5 Hz, NH), 8.00 (s, 1H, H ), 7.89 (s, 1H,
4
5
triazole), 7.74-7.66 (m, 3H, phenyl), 7.01 (s, 1H, H ), 6.96 (d, 1H,
J
=8.0 Hz, H ), 4.35 (t, 2H,
8
6
J
=7.0 Hz, CH -N), 4.27 (d, 2H, J=6.0 Hz, CH -NH), 4.08 (t, 2H, J=6.5 Hz, CH -O), 3.57
2 2 2
(quintet, 1H,
J
=6.0 Hz, CH-S), 3.17-3.10 (m, 2H, CH -S), 2.38 (sextet, 1H,
J=6.0 Hz, CH-CH2-
2
S), 2.08 (t, 2H,
J
=7.0 Hz, CH -CO), 1.89-1.75 (m, 2H, N-CH -CH and 2H, O-CH -CH and
2
2
2
2
2
1
H, CH-CH -S), 1.65-1.61 (m, 1H, CHCH CO), 1.52-1.48 (m, 3H, aliphatic), 1.39-1.30 (m, 4H,
2
2
1
3
aliphatic). C NMR (DMSO-d6, 125 MHz)
δ: 171.8, 162.1, 159.5, 155.0, 144.9, 141.9, 135.4,
1
30.9, 130.6, 129.9, 129.8, 128.3, 122.6, 120.3, 113.3, 112.7, 100.7, 68.1, 56.1, 49.1, 38.0, 34.9,
4.0, 29.4, 28.2, 27.7, 24.9, 22.4. Anal. Calcd for C H Cl N O S : C, 56.27; H, 5.18; N, 8.47.
3
3
1
34
2
4
4 2
Found: C, 56.30; H, 5.24; N, 8.71.
4
4
.2. Biological assays
.2.1. Neuroprotection assay against H O -induced cell death in PC12 cells
2
2
Cell viability of rat differentiated PC12 cells was examined by 3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyl tetrazolium bromide (MTT) assay according to the previously reported method
2
[
54]. PC12 cells (purchased from the Iranian Biological Resource Center, IBRC, Tehran, Iran)
were seeded in 96-well plates (10,000 cells/well) and incubated at 37 ºC in a humidified
atmosphere containing 5% CO . After 24 h, cells were treated with different concentrations
2
(0.1, 1, 5, 10, 20, and 50 µM) of each tested compounds and incubated for 3 h followed by
exposing cells with H O (150 µM) for another 2 h. MTT solution (20 µL, 5 mg/mL) was
2
2
consequently added after the removal of the previous medium and incubated in a CO2
incubator for 4 h. Afterward, the medium was removed and the produced formazan crystals
were solubilized using DMSO (100 µL). Finally, the related absorbance was recorded at 570
nm using a Synergy 2 multi-mode plate reader (Biotek, Winooski, VT, USA). Results were
expressed as the percentage of untreated control cells (PC12 cells in the absence of tested
compound and H O ). All above-mentioned experiments were repeated for three times and
2
2
mean±SEM of the obtained results were reported.
2
3

ACCEPTED MANUSCRIPT
4
.2.2. In vitro AChE/BuChE inhibition assay
The inhibitory activities of the compounds 4-30 towards AChE (from Electrophorus
electricus (electric eelAChE), Sigma-Aldrich) and horse serum BuChE (eqBuChE, Sigma-
Aldrich) were measured by Ellman’s method [55]. In 24-well plates, a mixture of phosphate
buffer (0.1 M, pH 8.0, 2 mL), 5,5´-dithiobis-2-nitrobenzoic acid (DTNB, 60 µL),
acetylcholinesterase or butyrylcholinesterase (20 µL, 5 IU/mL) and different concentration
of the compound solution (30 µL) was pre-incubated for 5 min followed by the addition of the
substrate (acetylthiocholine iodide or butyrylthiocholine iodide, 20 µL). Changes in absorbance
were measured at 412 nm by using a multi-mode reader (BioTek Synergy HT). To evaluate the
IC50 (concentration of compound produced 50% of enzyme activity inhibition), log
concentration versus percentage of inhibition curve was plotted and analyzed. All experiments
were performed in triplicate and donepezil was used as a positive control.
4
.2.3. Determination of the inhibitory potency on self-induced and AChE-induced A 1-42
β
aggregation
In order to investigate the self-induced and AChE-induced Aβ1-42 aggregation, a thioflavin T
ThT)-based fluorescence assay was performed [56]. For prefibrillation, amyloid- protein 1-42
Sigma A9810) 50 µM was dissolved in Phosphate Buffer Saline pH 7.4 (PBS, HyClone Thermo
C. For the assay
β1-42 (10 µL) ± human recombinant AChE (0.01 u/mL, Sigma C1682) were added to 0.05 M
(
(
β
Scientific) containing 1% ammonium hydroxide, and incubated for 72 h at 37
°
A
KP buffer pH 7.4 and incubated at 37
°
C for 48 h in the absence and presence of compounds
M, in 50 mM glycine-NaOH buffer, pH 8.5)
was added to the mixture. Fluorescence was measured by Microplate Reader (SpectraMax) at
λex=448 and λem=490 nm. Rifampicin (100 M, Sigma R-3501) and Donepezil (100 M, Sigma
(100 µM). Then, 50 µL of thioflavin T (ThT, 200 µ
µ
µ
D-6821) were also tested as reference drugs. The percent of inhibition of self or AChE-induced
aggregation for each compound was determined by the following formula: [(IFi/IFo) × 100]
where IFi and IFo are the fluorescence intensities obtained for A
the absence of inhibitors.
β ± AChE in the presence and in
2
4

ACCEPTED MANUSCRIPT
4
.2.4. 15-LOX inhibition assay
Soybean lipoxygenase (type 1-B lyophilized powder
≥
50000 units/mg solid, Sigma) inhibition
assay was performed as reported previously [57]. The tested compounds dissolved in DMSO
and diluted in phosphate buffer (0.1 M, pH=8) then incubated at room temperature with linoleic
acid in ethanol (134 mM) and enzyme solution (final concentration: 167 U/mL). The conversion
of linoleic acid to 13-hydroperoxylinoleic acid at 234 nm was recorded. The experiment was
done on Unico double beam spectrophotometer.
4
.2.5. Neuroprotection assay against H O or Aβ1-42-induced cytotoxicity in SH-
2 2
SY5Y cells
Cell viability was determined using MTT assay [54]. Human neuroblastoma SH-SY5Y cells
purchased from the American Type Culture Collection, ATCC, USA) 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% CO . SH-SY5Y cells
2
3
were seeded into 96-well plates (5 × 10 cells/well) and treated with compounds at a
concentration of 10 µM or 50 µM for 3 h, prior to the addition of 250 µM H O or 10 µM Aβ1–
2
2
42.
After 24 hours of incubation, 10 µL of the MTT reagent (5 mg/mL) was added to each well.
1
5
00 µL of DMSO was used to solve formazan crystals. Absorbance values were measured at
70 nm using a microplate reader (Biotek Power Wave XS). Cell viability was reported as the
percentage of untreated control cells as below:
Viability=100× [OD of treated wells – OD of blank wells/ OD of control wells – OD of blank wells]
4
.2.6. Determination of intracellular H O -induced ROS production in PC12 cells
2 2
Intracellular ROS formation was determined by using the ROS-sensitive dye, 2´,7´-dichloro-
fluorescein diacetate (DCFH-DA), as a probe [58]. Briefly, PC12 cells were seeded in 96-well
4
plates at 1×10 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% CO for 24 h. PC12 cells were then incubated in the presence or absence of
2
2
5

ACCEPTED MANUSCRIPT
various concentrations of the tested samples (1, 5, 10 and 50 µM, which were dissolved in
≤
0
.5% DMSO and diluted with fresh medium) for 6 h, next washed with PBS and incubated with
H O (150 µM) for additional 18 h. Treated cells were then washed with PBS and incubated
2
2
with a concentration of 10 µM DCFH-DA for 30 min at 37 ºC in the dark. At the end of
incubation, cells were washed two times with PBS. The fluorescence of the dichlorofluorescein
(DCF) was measured (λex= 485 nm, λem= 528 nm) by ELISA fluorimeter (BioTek). The results
were expressed as a percentage of intracellular ROS compared to untreated control cells.
4
.2.7. FRAP assay
To evaluate the antioxidant power of the potent compounds, the method of Benzie and Strain
was employed with slight modifications [59]. FRAP solution was prepared with a fresh mixture
of 300 mM acetate buffer (pH 3.6), 10 mM hydrochloric (40 mM) solution of TPTZ (2,4,6-
tripyridyl-s-triazine) and 20 mM ferric chloride. 240 µL FRAP solution was added to 10 µL of
the tested compound solution (10 µM) and incubated for 15 min. The reduced ferric ion formed
a colored ferrous-TPTZ complex at low pH. The absorbance was measured at 593 nm using a
microplate reader (BioTek Synergy HT). FRAP values were reported in mmol of ferrous
according to the plotted standard curve of ferrous sulfate.
4
.2.8. Metal binding studies
The metal binding studies were carried out in a dual-beam GBC Cintra 101 spectrophotometer
with 1-cm quartz cells. To investigate the metal binding ability of compound 11, the UV
absorption was recorded in the absence and presence of CuCl , FeSO , ZnCl , AlCl , MgCl and
2
4
2
3
2
CaCl with a wavelength ranging from 200 to 500 nm after incubating for 30 min at room
2
temperature. Final volume of the reaction mixture was 1 mL, and final concentrations of the
tested compound and metals were 20 µM. Numerical subtraction of the spectra of the metal
alone and the compound alone from the spectra of the mixture gave the difference UV-vis
spectra indicating the complex formation. The molar ratio method was performed to determine
the stoichiometry of the complex
. The volume of the ligand solution was kept constant (2.5 mL,
4
0 µM) and titrated by CuCl (0.01 mL, 1000 µM) [60].
2
2
6

ACCEPTED MANUSCRIPT
4
.3. Docking simulations
Docking studies were performed using the Molecular Operating Environment (MOE) software
Chemical Computing Group, Montreal, Canada) The crystal structure of acetylcholinesterase
complexed with donepezil (1EVE) was retrieved from the Protein Data Bank (PDB . The
preparation and optimization of the receptor were done by Autodock Tools (4.2) [61]. The ligand
D structure and minimization prepared by Openbabel (2.3.1) [62]. The dimensions of active site
box were set as 15×15×15 Å, the center of grid box was placed at x = 2.023, y = 63.295, z =
(
.
)
3
6
7.062. The 3D molecular visualization of the strongest cluster was done by Discovery Studio
.0 software [63].
2
4
.4. Statistical analysis
Data are reported as mean ±SEM of at least 3 independent experiments. Statistical analysis
was performed using one-way ANOVA with Dunnett post hoc test and p-values less than 0.05
were considered statistically significant. Analyses were performed using PRISM 6 software on a
Windows platform.
Acknowledgments
This work was supported by a grant from Pharmaceutical Sciences Research Center (PSRC),
Tehran University of Medical Sciences (grant number: 35661) and Iran National Science
Foundation (INSF, grant number: 94003365).
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