Multifunctional Mono-Triazole Derivatives Inhibit Aβ42 Aggregation and Cu2+-Mediated Aβ42 Aggregation and Protect against Aβ42-Induced Cytotoxicity

Article abstract of DOI:10.1021/acs.chemrestox.9b00168

Amyloid beta (Aβ) peptide aggregation is considered as one of the key hallmarks of Alzheimer's disease (AD). Moreover, Aβ peptide aggregation increases considerably in the presence of metal ions and triggers the generation of reactive oxygen species (ROS)

Full text of DOI:10.1021/acs.chemrestox.9b00168

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Article
Multifunctional mono-triazole derivatives inhibit A#42 aggregation, Cu2+
mediated A#42 aggregation and protect against A#42 induced cytotoxicity
Amandeep Kaur, Simranjeet Singh Narang, Anupamjeet Kaur, Sukhmani Mann,
Nitesh Priyadarshi, Bhupesh Goyal, Nitin Kumar Singhal, and Deepti Goyal
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00168 • Publication Date (Web): 12 Aug 2019
Downloaded from pubs.acs.org on August 13, 2019
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Multifunctional mono−triazole derivatives inhibit Aβ42 aggregation, Cu2+
mediated Aβ42 aggregation and protect against Aβ42 induced cytotoxicity
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Amandeep Kaur,a Simranjeet Singh Narang,a Anupamjeet Kaur,a Sukhmani Mann,a
Nitesh Priyadarshi,c Bhupesh Goyal,*,b Nitin Kumar Singhal,*,c and Deepti
Goyal*,a
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aDepartment of Chemistry, Faculty of Basic and Applied Sciences,
Sri Guru Granth Sahib World University, Fatehgarh Sahib–140406, Punjab, India
bSchool of Chemistry and Biochemistry,
Thapar Institute of Engineering & Technology, Patiala–147004, Punjab, India
cNational Agri−Food Biotechnology Institute, S.A.S. Nagar, Punjab, India
*Corresponding authors
E-mail address: deeptig@iitbombay.org, bhupesh@iitbombay.org, nitin@nabi.res.in
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TABLE OF CONTENTS (TOC)
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ABSTRACT
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Amyloid beta (Aβ) peptide aggregation is considered as one of the key hallmarks of Alzheimer’s
disease (AD). Moreover, Aβ peptide aggregation increases considerably in the presence of metal
ions and triggers the generation of reactive oxygen species (ROS), which ultimately leads to
oxidative stress and neuronal damage. Based on the ‘multi–target–directed ligands’ (MTDLs)
strategy, we designed, synthesized and evaluated a novel series of triazole based compounds for
AD treatment via experimental and computational methods. Among the designed MTDLs [4(a-
x)], the triazole derivative 4v exhibited most potent inhibition of self induced Aβ42 aggregation
(78.02%) with an IC50 value of 4.578 ± 0.109 μM and also disassembled the preformed Aβ42
aggregates significantly. In addition, compound 4v showed excellent metal chelating ability and
maintained copper in the redox–dormant state to prevent the generation of ROS in
copper−ascorbate redox cycling. Further, 4v significantly inhibited Cu2+–induced Aβ42
aggregation and disassembled the Cu2+–induced Aβ42 protofibrils as compared to the reference
compound clioquinol (CQ). Importantly, 4v did not show cytotoxicity and was able to inhibit the
toxicity induced by Aβ42 aggregates in SH–SY5Y cells. Molecular docking results confirmed the
strong binding of 4v with Aβ42 monomer and Aβ42 protofibril structure. The experimental and
molecular docking results highlighted that 4v is a promising multifunctional lead compound for
AD.
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INTRODUCTION
Various amyloidal diseases, such as Parkinson’s disease, Alzheimer’s disease (AD), type 2
diabetes, Creutzfeldt–Jakob disease, and Huntington’s disease etc. having different etiology are
caused due to the self assembly of specific proteins or peptides to form aggregates.1 Among the
various neurodegenerative diseases, AD2 accounts for about 60–70% of cases and thus is the
most common form of dementia. AD is a progressive neurodegenerative disease, and results into
structural and functional demolition of neurons. The patients lose their ability to perform
everyday activities and finally become bed ridden at the later stages of the disease. The World
Health Organization (WHO) recently reported that about 50 million people are affected with
dementia worldwide and this number will likely to reach 152 million by 2050.3 AD is the fifth
leading cause of death among the people aged above 65 in the developed countries.4
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AD has multifactorial and heterogeneous pathology and several hypotheses like abnormally
phosphorylated and β–folded tau protein,5 amyloid plaques,6 cholinergic impairment,7 metal
dyshomeostasis8 and oxidative stress9 have been proposed to understand the pathomechanism of
the disease. The currently administered drugs (e.g. rivastigmine,10 donepezil,11 galantamine12 and
memantine13) for AD, treat symptoms rather than dealing with the core pathology of the
disease.14 In addition, almost 99.6% of AD drugs have failed in different levels of clinical trials
from 2002 to 2012.15 The recent failure of various molecular entities [NAP (NAPVSIPQ),16
LY2886721,17 tarenflurbil,18 semagacestat,19 avagacestat,20 PBT1,21 and bapineuzumab22 etc.] at
different level of clinical trials, clearly demonstrates the dire need to identify new molecules for
the treatment of AD.
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Among the various factors responsible for AD progression, deposition of amyloid plaques as
well as soluble oligomers is the cardinal feature of AD.6 The amyloid plaques predominantly
consists of Aβ peptide (39 to 43 amino acid residues). Aβ is generated via the amyloidogenic
pathway by the proteolytic cleavage of the amyloid precursor protein (APP) by β– and γ–
secretases.23 The deposition of amyloid species gradually results in neuronal loss and finally
neuron death. Hence, various molecules showing ability to inhibit amyloid aggregation has been
reported in literature as possible therapeutics against AD.24
The Aβ aggregation increases drastically on complexation of metal ions with Aβ peptide25 and
triggers the generation of reactive oxygen species (ROS).26 This eventually leads to oxidative
stress and neuron death.27 The dyshomeostasis of brain metal ions [especially copper and zinc]
greatly influence the pathogenesis of AD. It has been found that amyloid plaques were enriched
with Cu2+ and Zn2+. These metal ions coordinate with histidine residues in senile plaque core.28
As a result, metal ions start accumulating in the extracellular plaques and intracellular copper
stores are scarce in AD patients.29 In this regard, various metal chelators have been proposed in
the literature, which can modulate the concentration of these biometals.30 However, the complex,
multifactorial nature of AD encourages the design of multi−target−directed ligands (MTDLs)31
as the future drug candidates for the treatment of AD.
To this purpose, we have designed a library of mono−triazole based compounds as MTDLs for
the treatment of AD. The nitrogen heterocycles have been explored against a plethora of
biological targets32 and may be a possible starting point for the design of new molecular entities
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for AD. These ring systems have been extensively explored in the design of scanning probes and
drug candidates against various neurodegenerative diseases.33 Jones et al. have evaluated a series
of dual functional triazole–pyridine ligands as inhibitor of Aβ aggregation and metal−triggered
Aβ aggregation.34 In 2015, Vajragupta and co–workers have reported series of substituted anti–
1,2,3–triazoles (IND, PPRD, and QND) as α7 nicotinic acetylcholine receptor agonists.35 Storr
and co-workers have synthesized three bidentate quinoline−triazole derivatives and studied their
ability to inhibit amyloid aggregation.36 Recently, Jones et. al. have synthesized three 1,2,3–
triazole derivatives (POH, PMorph, PTMorph) and demonstrated that triazole derivatives
inhibited Aβ aggregation, metal induced Aβ aggregation, and displayed metal chelation and
antioxidant activity.37 Dyrager et. al. have synthesized benzothiazole−triazole and
benzothiadiazole−triazoles, and evaluated their binding specificity to amyloid aggregates in the
brain sections from transgenic mice model of AD.38 In another report, Das and Smid have carried
out virtual screening of ZINC chemical database and in vitro studies, and identified dibenzyl
imidazolidine and triazole acetamide derivatives as potent Aβ aggregation inhibitors.39 In the
present study, we have designed, synthesized and evaluated a library of mono−triazole based
compounds 4(a–x) as multifunctional ligands, which will target four major AD hallmarks: Aβ
aggregation, metal induced Aβ aggregation, metal dyshomeostasis and oxidative stress.
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RESULTS AND DISCUSSION
Rational design of multifunctional ligands
We undertook a rational pharmacophore directed design to develop a drug candidate to target
multiple pathological factors of AD.40 The proposed pharmacophore model consists of two parts:
hydrophobic part and the metal chelator part (Figure 1). The phenyl groups comprises the
hydrophobic part, was introduced to target the hydrophobic pocket of Aβ aggregates. The
designed MTDLs will form hydrophobic contacts with Aβ peptide and disrupt the peptide–
peptide interaction and produce an anti–aggregation effect. On the other hand, triazole moiety
represents the metal chelating part, which chelate metal ions via the donation of lone pair of
electron on one of the π–bonded nitrogen atoms present in the triazole ring to the metal ion.41
The metal chelating ability of designed molecules enables them to modulate the metal mediated
Aβ42 aggregation. The metal chelating property of the designed triazole based compounds helps
to reduce the oxidative stress induced by the generation of ROS, by disrupting the copper–
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ascorbate redox cycle. The designed triazole based compounds are synthesized by using the
highly regioselective Cu(I) catalysed Huisgen 1,3–dipolar cycloaddition reaction.42 Further, a
library of triazole based compounds having R group with variable electronic properties are easily
assembled. The designed library is evaluated for the amyloid aggregation inhibition, which
highlights the effect of R–group variation on the ligand–Aβ42 interactions.
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Figure 1: Design strategy for mono−triazole based compounds as MTDLs in AD.
The designed MTDLs 4(a−x) were synthesized according to the synthetic sequence depicted in
Scheme 1. The library of mono−triazole based compounds 4(a−x) were synthesized using copper
catalysed 1,3−dipolar cycloaddition reaction between alkyne based building block 2 and various
azides 3(a−x). In order to realize the designed strategy, the alkyne building block 2 was
assembled from diethyl acetamidomalonate 1.43 Propargylation of 1 in the presence of Cs2CO3
furnished the mono−alkyne building block 2. Simultaneously, various azides 3(a−x) were
prepared from the corresponding amines by following the literature procedure.44 Finally, the
mono−alkyne building block 2 was reacted with differently substituted phenyl azides 3(a−x) to
deliver the library of designed mono−triazole based compounds 4(a−x), listed in Table 1. The
formation of compounds 4(a−x) were confirmed by complementary spectral data (1H NMR, 13C
NMR and HRMS). Having the library of designed compounds in our hands we next carried out
the biophysical studies to investigate the efficacies of synthesized compounds to inhibit Aβ42
aggregation, disaggregation of preformed Aβ42 fibrils, and inhibition of metal induced Aβ42
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aggregation, disaggregation of preformed Aβ42 fibrils in the presence of metal, metal chelating
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properties and prevention of ROS generation.
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R
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R
N
EtO2C
EtO2C
EtO2C
EtO2C
3(a x)
a

N
N
EtO2C
b
NHAc
NHAc
EtO2C NHAc
1
2
4(a x)

Scheme 1: Synthetic route to generate compounds 4(a−x): Reagents and conditions: (a)
Propargyl bromide, CS2CO3, CH3CN, 18 h, rt, 100%; (b) Cu(OAc)2, sodium−ascorbate,
t−BuOH/H2O (1:1), rt.
Table 1: List of mono−triazole derivatives 4(a−x)
Mono−triazole
derivatives
Yield
(%)a
Mono−triazole
derivatives
Yield
(%)a
Cpd
4a
R
Cpd
4m
R
F
N
N
H
78
o−F
66
EtO2C
EtO2C
N
N
N
N
EtO2C NHAc
H3C
EtO2C NHAc
F
N
4b
4c
o−CH3
m−CH3
40
4n
4o
m−F
p−F
62
N
EtO2C
EtO2C
N
N
N
N
EtO2C NHAc
EtO2C NHAc
F
H3C
N
EtO2C
76
75
60
N
N
N
EtO2C
N
EtO2C NHAc
N
EtO2C NHAc
CH3
Cl
N
N
N
4d
4e
p−CH3
53
51
4p
4q
o−Cl
EtO2C
EtO2C
EtO2C NHAc
N
N
N
EtO2C NHAc
Cl
H3CO
N
o−OCH3
m−Cl
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EtO2C
EtO2C NHAc
N
N
EtO2C
N
N
N
EtO2C NHAc
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Cl
H3CO
4
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N
N
EtO2C
EtO2C NHAc
4f
m−OCH3
51
4r
p−Cl
66
N
N
N
EtO2C
EtO2C NHAc
N
9
OCH3
Br
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N
N
EtO2C
EtO2C NHAc
4g
4h
4i
p−OCH3
o−CF3
61
56
88
4s
4t
o−Br
m−Br
p−Br
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46
83
EtO2C
EtO2C NHAc
N
N
N
N
Br
F3C
N
EtO2C
EtO2C NHAc
N
N
N
EtO2C
N
N
EtO2C NHAc
Br
F3C
N
EtO2C
m−CF3
4u
N
N
N
N
EtO2C
EtO2C NHAc
EtO2C NHAc
N
CF3
I
N
4j
4k
4l
p−CF3
m−NO2
p−NO2
53
52
80
4v
4w
4x
o−I
m−I
p−I
71
45
95
N
EtO2C
EtO2C
EtO2C NHAc
N
N
N
N
EtO2C NHAc
O2N
I
N
N
N
N
EtO2C
EtO2C
N
EtO2C NHAc
N
EtO2C NHAc
I
NO2
N
N
EtO2C
EtO2C
EtO2C NHAc
N
N
N
N
EtO2C NHAc
aYields were of isolated and purified products.
Inhibition of self mediated Aβ42 aggregation by designed mono−triazole derivatives
Thioflavin T (ThT) fluorescence assay was used to study the effect of compounds 4(a−x) on the
aggregation propensity of Aβ42. ThT, a benzothiazole dye, has been used for the in vitro
identification of amyloid fibrils and was used to monitor the kinetics of fibril formation.45 ThT
binds selectively to amyloid fibrils and undergoes a huge increase of its fluorescence emission. A
known amyloid aggregation inhibitor, curcumin, was used as reference compound in the present
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assay.46 The experimental conditions such as concentration of Aβ42 and ThT to be used, and time
of incubation were optimized and were later used in the studies. The results shown in Figure 2A
indicated that 4f, 4j, 4p and 4v in the series exhibited more than 50% inhibition of Aβ42
aggregation. The results highlighted that 4v was the most potent inhibitor of Aβ42 aggregation
(78.02% inhibition) among the series of compounds featuring o−iodo group on the phenyl ring.
The complete dose–response curve of compound 4v was evaluated. The curve indicated that 4v
inhibited Aβ42 aggregation with an IC50 value of 4.578 ± 0.109 μM as compare with the control,
curcumin (95% inhibition with IC50 value= 1.482 ± 0.01 μM). In addition, transmission electron
microscopy (TEM) assay was performed to observe the morphologies of Aβ42 aggregates. TEM
images obtained after incubating fresh Aβ42 monomer showed complex network of long
rope−like amyloid fibrils (Figure 2Bb). However, a considerable reduction in the formation of
amyloid fibrils was observed in the presence of compound 4v (Figure 2Bc). The results of the
ThT assay and TEM clearly proved the potential of 4v to inhibit Aβ42 aggregation. The ability of
4v to inhibit Aβ42 aggregation may be attributed to the presence of halogen on the phenyl ring.
The substitution of halogen on the phenyl ring is known to influence the aromatic interaction
mediated self assembly processes in amyloid fibril formation.47
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Figure 2: Inhibition of self mediated Aβ42 aggregation: (A) The results of ThT fluorescence
assay. Statistical significance was analyzed by one−way analysis of variance (ANOVA) followed
by Dunnett’s test: (**) p < 0.01 and (*) p < 0.5, versus Aβ42 alone. [Aβ42]= 10 µM, [4(a−x)]=
[Curcumin]= 50 µM. (B) TEM images: [Aβ42]= 10 µM, [4v]= 20 µM, 37 °C, 24 h, constant
agitation (a) Aβ42, 0 h; (b) Aβ42, 24 h; (c) Aβ42 + 4v, 24 h. Scale bar: 0.2 µm.
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Disaggregation of self mediated Aβ42 aggregation fibrils by compound 4v
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The ability of compound 4v to disaggregate self mediated Aβ42 aggregation fibrils was
investigated using ThT fluorescence assay. In this regard, Aβ42 monomer was incubated at 37 °C
for 24 h to generate Aβ42 fibrils. The test compound 4v and the reference compound curcumin
were then added separately to the samples before incubating for another 24 h at 37 ˚C. The ThT
binding assay highlighted that 4v could disaggregate Aβ42 fibrils by 71.23% at 20 µM, as
compared to curcumin (82.75% at 20 µM, Figure 3A). Further, TEM assay was performed to
support the results obtained in ThT assay. A bundle of amyloid fibrils can be seen in a TEM
image (Figure 3Bb) obtained after incubation fresh Aβ42 monomer for 24 h. However, addition of
4v to a sample of the preformed fibrils resulted in significant reduction in the number of Aβ42
fibrils (Figure 3Bc). The results of ThT assay and TEM analysis clearly indicated that compound
4v can effectively disassemble the self mediated Aβ42 fibrils.
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Figure 3: Disaggregation of self mediated Aβ42 aggregation: (A) The results of ThT binding
assay. Statistical significance was analyzed by one−way analysis of variance (ANOVA) followed
by Dunnett’s test: (**) p < 0.01, versus Aβ42 alone. Aβ42= 10 μM, [4v]= [Curcumin]= 20 μM.
(B) TEM images: [Aβ42]= 10 µM, [4v]= 20 µM, 37 °C, 24 h, constant agitation (a) Aβ42, 0 h; (b)
Aβ42, 24 h; (c) Aβ42 fibrils + 4v, 24 h. Scale bar: 0.2 µm.
Metal chelating characteristics of compound 4v
The UV−Vis spectroscopy was used to investigate the metal chelating ability of 4v.48 The
observed spectral peaks of 4v alone and in the presence of metal ions were shown in Figure 4a.
The specific absorbance peaks of 4v were observed at 226 nm and 270 nm as shown in Figure
4a. After addition of CuSO4 to a solution of 4v, there is a considerable decrease in the
absorbance at 226 nm indicating an interaction between 4v and Cu2+. On the contrary, the
absorbance at 226 nm increases on addition of FeSO4 and ZnCl2 to a solution of 4v, suggesting
that 4v binds to Fe2+ and Zn2+. The metal chelating ability of 4v could be due to the donation of
lone pair of electron on one of the π–bonded nitrogen atoms present in the triazole ring. Further,
the stoichiometry of the 4v–Cu2+ complex was determined using Job’s method by preparing a
series of solutions that maintained the total concentration of 4v and CuSO4 constant but vary
their proportion.49 The absorbance of the 4v–Cu2+ complex at varying concentrations was
recorded on UV−Vis spectrophotometer (Figure S1, Supporting Information). The absorbance
readings at 226 nm vs mole fraction of Cu2+ was plotted in Figure 4b. The break at 0.3 in the Job
plot for 4v and CuSO4 indicated a 2:1 stoichiometry for 4v–Cu2+ complex.
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Figure 4: Metal chelating properties: (a) The UV−Vis spectrum of compound 4v alone or in
presence of 20 µM CuSO4, ZnCl2, or FeSO4 in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4);
(b) Determination of stoichiometry of 4v−Cu2+ complex using Job’s method.
Suppression of copper−based redox activity in the presence of compound 4v
A number of studies have highlighted that the generation of excess free radicals play a major role
in causing neuronal cell death in AD patients.26 Among the several biometals, Cu2+ play an
important role in catalyzing the generation of ROS, leading to an aggravated neuron toxicity and
oxidative stress.8 Therefore, investigating the redox silencing property of 4v is important in
assessing its antioxidant property. To estimate the redox silencing property of 4v copper–
ascorbate redox system described in Scheme 2 has been used as a model system.50 In the
presence of ascorbate (reducing agent), Cu2+ is reduce to Cu+, which being unstable oxidize
back to Cu2+ and thus generates •OH (major form of ROS accounting to the toxicity in AD) in the
process. Coumarin−3−carboxylic acid (CCA) was used to detect the •OH in vitro, as •OH convert
CCA to a fluorescent 7−hydroxyl−CCA (emission at 450 nm). As seen in Figure 5, the
fluorescence intensity increases steadily with time and reached a plateau at nearly 12 min. This
indicate that the •OH produced by copper and ascorbate increases with time and then achieved a
plateau. However, solutions treated with 4v showed negligible fluorescence, highlighting that
4v−bound Cu2+ (redox dormant state) was not involved in the copper–ascorbate redox cycle to
generate •OH even under highly reducing conditions.
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Scheme 2: The generation of hydroxyl radical (•OH) in the presence of ascorbate via
copper−ascorbate redox cycling.
Figure 5: Fluorescence intensity of 7−hydroxy−coumarin−3−carboxylic acid in
copper−ascorbate and copper−ascorbate−4v system. CCA [50 μM] and ascorbate [150 μM] were
incubated in each system. [Cu2+]= [5 μM], [4v]= 15 μM. PBS buffer, pH= 7.4. Excitation
wavelength= 395 nm and emission wavelength= 450 nm.
The effects of compound 4v on Cu2+−mediated Aβ42 aggregation
Next, we investigated the effect of compound 4v on Cu2+−mediated Aβ42 aggregation by using
ThT fluorescence assay. A know metal chelator, clioquinol48a (CQ), was used as a reference
compound in the present study. Aβ42 was first treated with one equivalent of Cu2+ for 2 min at rt
and later incubated with or without the test compounds (4v and CQ) for 24 h at 37 °C. According
to the ThT assay, the percentage of formation of Aβ42 fibrils is 32% higher in the presence of
Cu2+ than Aβ42 alone (Figure 6). On the contrary, the fluorescence of Aβ42 treated with Cu2+ and
test compounds decreased dramatically (fluorescence after treatment with 4v, and CQ was
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44.24% and 45.06%, respectively). These results clearly indicated the capability of 4v to inhibit
Cu2+−induced Aβ42 aggregation by effectively chelating the Cu2+.
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Figure 6: Inhibition of Cu2+−mediated Aβ42 aggregation: The results of ThT fluorescence assay.
Statistical significance was analyzed by one–way analysis of variance (ANOVA) followed by
Dunnett’s test: (**) p < 0.01 and (***) p < 0.001, versus Aβ42 + Cu2+ alone. Aβ42= 10 μM, Cu2+=
10μM, [4v]= [CQ]= 20 μM.
The effect of compound 4v on Cu2+−mediated Aβ42 aggregation fibrils
ThT fluorescence assay was used to investigate the ability of compound 4v to disaggregate
Cu2+−mediated Aβ42 aggregation fibrils. In this assay, Aβ42 monomer was incubated with one
equivalent of Cu2+ for 24 h at 37 °C to generate the Aβ42 fibrils. Later, 4v and CQ (20 µM) were
added separately to Aβ42 fibrils and incubated for another 24 h at 37 °C. The results (Figure 7)
indicated that compound 4v manifested excellent disaggregation potency (64.02%) as compared
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to the reference CQ (58.95%). Therefore, it is concluded that compound 4v has the ability to
inhibit Cu2+−mediated Aβ42 aggregation and disrupt well structured Aβ42 fibrils.
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Figure 7: Disaggregation of Cu2+−mediated Aβ42 aggregation fibrils by 4v: The results of ThT
binding assay. Statistical significance was analyzed by one–way analysis of variance (ANOVA)
followed by Dunnett’s test: (**) p < 0.01, Aβ42= 10 μM, Cu2+= 10 μM, [4v]= [CQ]= 20 μM.
The effect of compound 4v on neuronal cell viability and on Aβ42 mediated neurotoxicity
After establishing the potential of 4v as an effective MTDL in AD, a study was conducted to
check the cytotoxicity of compound 4v and its efficiency to ameliorate the neurotoxicity induced
by Aβ42 aggregates in cellular model. In this regard, 3–(4,5–Dimethylthiazol–2–yl)–2,5–
diphenyltetrazolium bromide (MTT) reduction assay51 using SH–SY5Y cells were performed.
The results shown in Figure 8a, highlighted that the incubation of the SH–SY5Y cells with upto
25 µM concentration of the compound 4v did not influence the cell viability of the stain
significantly (cell viability in the presence of 4v: 91% at 5 µM and 90% at 25 µM).
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Figure 8: Cell viability assay: (a) The cell viability in the presence of 4v alone at 5 µM, 25 µM,
50 µM and 100 µM after 48 h of incubation; (b) Cell viability in the presence of Aβ42 [5µM]
alone and mixture of Aβ42–4v at molar ratios 1:1, 1:5, 1:10 and 1:100. Data is expressed as mean
values ± SEM from at least three independent experiments. Statistical significance was analyzed
by one–way analysis of variance (ANOVA) followed by Dunnett’s test: (**) p < 0.01, In both
panel (a) and (b) error bars represent the average of three replicate experiments.
Further, the ability of 4v to rescue SH−SY5Y cells from Aβ42 mediated toxicity was studied by
MTT assay. As shown in Figure 8b, the cell viability reduces to 58% as compared to the control
(100%) in the presence of Aβ42 aggregates. The reduction in cell viability indicates that Aβ42
aggregates were toxic to the SH–SY5Y cells. However, the cell viability significantly improved
(72%) when cells were treated with compound 4v (5 µM). The cell viability increases with
increasing the molar ratio of 4v to Aβ42. The cell viability increases to 78% at Aβ42/4v = 1:10;
however, a further increase in the concentration of 4v (> 50 µM) did not affect the cell viability
significantly. These results highlighted the potential of compound 4v to rescue SH–SY5Y cells
from Aβ42 mediated toxicity.
Molecular docking analysis of compound 4v with Aβ42 monomer and Aβ42 protofibril
Molecular docking is an indispensable tool to understand the binding regions and key
interactions of ligand with protein.52 After getting the satisfactory results in various biophysical
studies, we further explored the possible modes of interaction of 4v with Aβ42 monomer and
Aβ42 protofibril structure using molecular docking analysis, via AutoDock 4.253 In this respect,
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4v was docked to Aβ42 monomer (PDB ID: 1IYT) and the observed binding energy was −5.12
kcal/mol (Table 2). The negative binding energy highlights the favorable binding of 4v with Aβ42
monomer. Compound 4v formed two hydrogen bonds with Aβ42 monomer. The first one was
formed between the nitrogen atom of triazole ring of 4v with NH of the backbone of Gln15 of
Aβ42 monomer. The second hydrogen bond is formed between the carbonyl oxygen atom of ethyl
acetate moiety of 4v with NH of the backbone of Lys16 residue of Aβ42 monomer (Figure 9a and
Table 2). Hydrophobic contacts also play an important role in the effective binding of 4v with
Aβ42. The hydrophobic contacts were analyzed using LigPlot+ software. The 2D interaction map
displayed the hydrophobic contacts of 4v with Ser8, Glu11, Val12, Gln15, Lys16, Phe19 and
Phe20 residues of Aβ42 monomer (Figure 9b).
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Table 2: Molecular docking analysis of 4v with Aβ42 monomer and Aβ42 protofibril structure.
Protein
AutoDock
binding
Aβ42 residues involved in
intermolecular hydrogen bonding in intermolecular
Aβ42 residues involved
structurea
energy
with 4v
hydrophobic contacts
(kcal/mol)
with 4v
Residue Atomsb Distance
(nm)
Aβ42
monomer
−5.12
−6.54
Gln 15
Lys16
N : HN 0.28
O : HN 0.17
Ser8, Glu11, Val12,
Gln15, Lys16, Phe19,
Phe20
Leu17 (C, D, E), Val18
(C, E), Phe19 (C, D, E),
Gly37 (E), Gly38 (E),
Val39 (D), Val40 (E)
Aβ42
protofibril
aThe PDB ID for Aβ42 monomer and Aβ42 protofibril used in the present study are 1IYT and 2BEG, respectively.
bThe atoms on left represent ligand atoms, and on the right represent Aβ42 residue atoms.
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Figure 9: (a) The docked complex of 4v with Aβ42 monomer (PDB ID: 1IYT) highlights the best
docked conformations. The Aβ42 monomer is shown in cartoon representation and compound 4v
is shown in stick representation; (b) The 2D interaction maps of 4v displays the formation of
hydrogen bond (green line) and hydrophobic contacts with 1IYT. The maps are generated using
LigPlot+ software.
To elucidate the binding regions of 4v with Aβ42 protofibril, 4v was docked to Aβ42 protofibril
(PDB ID: 2BEG). The Aβ42 protofibril (PDB ID: 2BEG) model consist of two β−strand regions
(Leu17−Ser26 and Ile31−Ala42) linked with the bend region (Asn27−Ala30). The binding
energy of −6.54 kcal/mol was observed in best docked pose of 4v with Aβ42 protofibril structure
(Figure 10a, Table 2). Compound 4v shows hydrophobic contacts with Leu17 (C, D, E), Val18
(C, E), Phe19 (C, D, E), Gly37 (E), Gly38 (E), Val39 (E) and Val40 (E) residues of Aβ42
protofibril (Figure 10b). The molecular docking studies highlighted that 4v binds preferably to
the central hydrophobic core (CHC) of Aβ42 monomer via the hydrogen bond and through
hydrophobic contacts. The CHC of Aβ42 is known to play a pivotal role in Aβ42 aggregation.54
Further, the docking results indicated that 4v binds to the even edge (chain E) of the Aβ42
protofibril. The fibril structure grows faster from the even edge of Aβ42 protofibril as compared
to the odd edge.55
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Figure 10: (a) The docked complex of 4v with protofibril (PDB ID: 2BEG) Aβ42 protofibril
(PDB ID: 2BEG); (b) The 2D interaction maps displaying the hydrophobic contacts of 4v with
Aβ42 protofibril (PDB ID: 2BEG). The maps are generated using LigPlot+ software.
CONCLUSION
On the basis of knowledge that triazole moiety possesses a metal chelating properties, we have
developed a novel series (24 variants) of triazole based compounds as multitarget agents for the
treatment of Alzheimer’s disease (AD). The compounds were designed by combining the metal
chelating triazole moiety and an anti–amyloid aggregation pharmacophore into one molecule.
Among the synthesized library, compound 4v exhibited significant inhibition of Aβ42
aggregation and disaggregation of Aβ42 fibrils. Compound 4v inhibited Aβ42 aggregation with an
IC50 values of 4.578 μM. Compound 4v showed efficient metal chelating ability, due to the
donation of lone pair of electron on one of the π–bonded nitrogen atoms present in the triazole
ring to the metal ion. Compound 4v binds Cu2+ and maintained it in a redox–dormant state even
under highly reducing conditions, and thus effectively prevented the production of reactive
oxygen species (ROS). Similar to clioquinol (reference compound), 4v inhibited Cu2+–induced
Aβ42 aggregation and disassembled preformed Cu2+–induced Aβ42 aggregates. The cytotoxicity
of compound 4v and its ability to inhibit Aβ42 induced toxicity in vitro was further evaluated in
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SH–SY5Y cells. Compound 4v did not influence the cell viability of the SH–SY5Y cells
significantly even at 25 µM concentration. In addition, 4v was able to rescue the cells from the
toxicity induced by Aβ42 aggregates and the cell viability improves to 78%. Further, the
molecular docking analysis highlighted that 4v binds with the central hydrophobic core (CHC)
region of Aβ42 monomer and showed interactions with the even edge in the Aβ42 protofibril
structure. In conclusion, the ability to inhibit Aβ42 aggregation, metal−induced Aβ42 aggregation,
disaggregation of Aβ42 fibrils, chelation of Cu2+, transforming it into a redox−dormant state, and
controlling the generation of ROS proved the multifunctional nature of 4v. Furthermore, ability
of 4v to rescue SH–SY5Y cells from Aβ42 induced toxicity makes it a promising candidate for
further evaluation. Overall, the designed scaffold showed satisfactory profile in the various
assays and hence, could be important multi−target−directed ligands (MTDLs) in AD.
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EXPERIMENTAL SECTION
General Methods
Human Aβ42 was purchased from Anaspec. All the reagents were purchased from Sigma Aldrich
and were used without further purification. The progress of the chemical reactions were
monitored by thin layer chromatography (TLC) using an appropriate solvent system for
development. The reported yields of the synthesized compounds were the isolated yields. In the
1H NMR the coupling constants (J) were given in hertz (Hz) and chemical shifts were stated in
parts per million (ppm). The abbreviations s, d, t, q and m stand for singlet, doublet, triplet,
1
quartet and multiplet, respectively. H NMR (400 MHz or 500 MHz, CDCl3 or DMSO−d6) and
13C NMR (100 MHz or 125 MHz, CDCl3 or DMSO−d6) spectra were recorded on a Bruker
NMR spectrometer. I.R. spectra were recorded using Perkin Elmer. HRMS data were recorded
using electrospray ionization (ESI) on Bruker MaXis Impact spectrometer and Agilent 6520
Q−TOF instrument. Melting points were recorded with a Perfit apparatus. HPLC analysis was
recorded on Agilent Technologies 1260 Infinity series system, reverse phase C18 column eluted
with 100% acetonitrile at a flow rate of 1mL/ min.
General procedure for synthesis of aryl azides 3(a−x)
°
The solution of aryl amine (13.6 mmol) in 50 mL HCl:H2O (1:1) was cooled at −5 C using
ice−salt mixture. A solution of sodium nitrite (27.2 mmol) in water (15 mL) was added slowly at
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°
−5 C and the reaction mixture was stirred for a period of 1 h. After this, the reaction mixture
was neutralized using sodium acetate (272 mmol). Later, a solution of NaN3 (27.2 mmol) in
water (15 mL) was added slowly over a period of 30 min by maintaining the temperature at −5
°C. After stirring the reaction mixture for 30 min, the solution was allowed to warm at room
temperature. The aqueous layer was extracted with ethyl acetate (100 mL x 2), dried over sodium
sulphate and evaporated with utmost care to yield the corresponding azide.
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General procedure for synthesis of mono−triazole based derivatives 4(a-x)
The mono-alkyne precursor (1 mmol) was dissolved in t−BuOH/H2O (1:1 mL) and the azide (1.1
mmol), Cu(OAc)2 (0.1 mmol) and sodium ascorbate (0.2 mmol) were added. The resulting
mixture was stirred at rt, until TLC indicated completion of reaction. The mixture was diluted
with ethyl acetate and washed with aq NH4OH (0.2%) and brine. The aqueous phases were
extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were dried over Na2SO4
and concentrated in vacuo. The crude product was purified by flash chromatography on silica
gel.
Compound (4a). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95
(s, 3H), 3.60 (s, 2H), 4.18 (q, J= 7.0 Hz, 4H), 7.46−7.57 (m, 1H), 7.62−7.87 (m, 2H), 7.87−7.89
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(m, 2H), 8.21 (s, 1H), 8.54 (s, 1H) ppm. C NMR (100 MHz, DMSO−d6): δ= 13.79, 22.15,
28.96, 61.85, 65.88, 119.78, 122.13, 128.47, 129.85, 136.58, 141.77, 166.82, 169.48 ppm. I.R:
1187, 1513, 1638, 1736, 3115, 3216 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H23N4O5 [M+H]+
375.1624, found: 375.1660. Rf. 0.39 (30% ethyl acetate/ petroleum ether). Mp: 101−102 °C.
1
Compound (4b). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 1.63 (s,
3H), 2.00 (s, 3H), 3.87 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.80 (s, 1H), 7.41−7.44 (m, 1H),
7.49−7.53 (m, 1H), 7.68−7.70 (m, 2H), 7.75 (s, 1H) ppm. 13C NMR (100 MHz, DMSO−d6): δ=
13.79, 17.41, 22.10, 28.89, 61.83, 65.95, 125.42, 125.80, 126.89, 129.59, 131.33, 132.94, 136.28,
140.72, 166.84, 169.39 ppm. I.R: 1050, 1293, 1502, 1666, 1741, 3378 cm-1. HRMS (Q−TOF)
m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found: 389.1811. Rf. 0.33 (50% ethyl acetate/
petroleum ether). Mp: 89−90 °C.
Compound (4c). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95
(s, 3H), 2.41 (s, 3H), 3.60 (s, 2H), 4.18 (q, J= 7.0 Hz, 4H), 7.28−7.30 (m, 1H), 7.48 (m, 1H),
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7.66−7.68 (m, 1H), 7.72 (s, 1H), 8.21 (s, 1H), 8.51 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO−d6): δ= 13.79, 20.90, 22.16, 28.96, 61.84, 65.88, 116.90, 120.21, 122.08, 129.03,
129.62, 136.57, 139.56, 141.68, 166.82, 169.47 ppm. I.R: 1193, 1514, 1641, 1739, 3142, 3250
cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found: 389.1821. Rf. 0.33
(30% ethyl acetate/ petroleum ether). Mp: 130−132 °C.
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Compound (4d). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 2.02 (s,
3H), 2.41 (s, 3H), 3.85 (s, 2H), 4.32 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.28−7.30 (m, 2H),
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7.54−7.57 (m, 2H), 7.69 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 14.02, 20.96, 23.41,
29.39, 38.14, 63.14, 120.27, 120.97, 130.32, 142.77, 167.65, 169.47 ppm. I.R: 1197, 1465, 1668,
1739, 3142, 3375 cm-1.HRMS (Q−TOF) m/z: calcd. for C19H25N4O5 [M+H]+ 389.1780, found:
389.1813. Rf. 0.36 (50% ethyl acetate/ petroleum ether). Mp: 126−128 °C.
Compound (4e). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.94
(s, 3H), 3.61 (s, 2H), 3.85 (s, 3H), 4.18 (q, J= 7.0 Hz, 4H), 7.12−7.16 (m, 1H), 7.31−7.33 (m,
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1H), 7.49−7.54 (m, 1H), 7.58−7.61 (m, 1H), 8.12 (s, 1H), 8.20 (s, 1H) ppm. C NMR (100
MHz, DMSO−d6): δ= 13.78, 22.09, 28.81, 56.02, 61.83, 65.96, 113.03, 120.87, 125.25, 125.59,
125.68, 130.42, 140.47, 151.23, 166.87, 169.42 ppm. I:R: 1200, 1265, 1504, 1678, 1741, 3056,
3410 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O6 [M+H]+ 405.1729, found: 405.1765. Rf.
0.32 (30% ethyl acetate/ petroleum ether). Mp: 78−80 °C.
Compound (4f). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.19 (t, J= 7.0 Hz, 6H), 1.95
(s, 3H), 3.59 (s, 2H), 3.85 (s, 3H), 4.18 (q, J= 7.0 Hz, 4H), 7.03−7.06 (m, 1H), 7.45−7.52 (m,
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3H), 8.21 (s, 1H), 8.58 (s, 1H) ppm. C NMR (100 MHz, DMSO−d6): δ= 13.79, 22.15, 28.92,
55.55, 61.84, 65.87, 105.42, 111.73, 114.06, 122.29, 130.80, 137.62, 141.72, 160.13, 166.80,
169.49 ppm. I.R: 1190, 1235, 1497, 1639, 1742, 3241 cm-1. HRMS (Q−TOF) m/z: calcd. for
C19H25N4O6 [M+H]+ 405.1729, found: 405.1754. Rf. 0.24 (30% ethyl acetate/ petroleum ether).
Mp: 120−122 °C.
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Compound (4g). White solid. H NMR (500 MHz, CDCl3): δ= 1.30 (t, J= 7.1 Hz, 6H), 2.03 (s,
3H), 3.87 (s, 2H), 3.88 (s, 3H), 4.32 (q, J= 7.1 Hz, 4H), 6.82 (s, 1H), 7.01−7.03 (m, 2H),
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7.59−7.61 (m, 2H), 7.68 (s, 1H) ppm. C NMR (125 MHz, CDCl3): δ= 14.06, 23.14, 29.12,
55.68, 62.90, 66.13, 114.85, 121.13, 122.00, 130.40, 142.59, 159.76, 167.48, 169.47 ppm. I.R:
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1198, 1215, 1517, 1643, 1739, 3264 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H25N4O6 [M+H]+
405.1729, found: 405.1766. Rf. 0.26 (50% ethyl acetate/ petroleum ether). Mp: 132−134 °C.
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Compound (4h). White solid. H1 NMR (400 MHz, DMSO−d6): δ= 1.16 (t, J= 7.0 Hz, 6H), 1.94
(s, 3H), 3.63 (s, 2H), 4.17 (q, J= 7.0 Hz, 4H), 7.66−7.64 (m, 1H), 7.87−7.84 (m, 1H), 7.96−7.92
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(m, 1H), 8.04−8.02 (m, 1H), 8.13 (s, 1H), 8.26 (s, 1H) ppm. C NMR (100 MHz, DMSO−d6):
δ= 13.70, 22.42, 28.96, 62.28, 66.32, 125.02, 126.45, 127.51, 129.11, 131.02, 133.94, 134.35,
140.86, 166.74, 169.47 ppm. I.R: 1057, 1114, 1497, 1667, 1739, 3407 cm-1. HRMS (Q−TOF)
m/z: calcd. for C19H22F3N4O5 [M+H]+ 443.1498, found: 443.1537. Rf. 0.34 (50% ethyl acetate/
petroleum ether). Mp: 102−104 °C.
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Compound (4i). White solid. H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.0 Hz, 6H), 2.01 (s,
3H), 3.87 (s, 2H), 4.30 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.63−7.70 (m, 2H), 7.83 (s, 1H),
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7.89−7.91 (m, 1H), 8.00 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 14.14, 23.26, 29.26,
63.15, 66.15, 117.42, 121.14, 123.43, 125.46, 130.73, 132.82, 137.45, 143.43, 167.48, 169.60
ppm. I.R: 1120, 1159, 1509, 1657, 1740, 3301 cm-1. HRMS (Q−TOF) m/z: calcd. for
C19H22F3N4O5 [M+H]+ 443.1498, found: 443.1533. Rf. 0.37 (50% ethyl acetate/ petroleum ether).
Mp: 138−140 °C.
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Compound (4j). White solid. H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.0 Hz, 6H), 2.00 (s,
3H), 3.87 (s, 2H), 4.30 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.76−7.80 (m, 2H), 7.83-7.85 (m, 2H),
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7.86 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 14.14, 23.26, 29.26, 63.16, 66.14, 114.96,
120.39, 121.12, 127.28, 127.31, 130.99, 143.46, 144.43, 167.47, 169.61 ppm. I.R: 1123, 1160,
1505, 1660, 1750, 3074, 3403 cm-1. HRMS (Q−TOF) m/z: calcd. for C19H21F3N4O5Na [M+Na]+
465.1361, found: 465.1357. Rf. 0.33 (40% ethyl acetate/ petroleum ether). Mp: 188−190 °C.
Compound (4k). White solid. 1H NMR (400 MHz, DMSO−d6): δ= 1.20 (t, J= 7.0 Hz, 6H), 1.96
(s, 3H), 3.62 (s, 2H), 4.19 (q, J= 7.0 Hz, 4H), 7.89−7.93 (m, 1H), 8.21 (s, 1H), 8.31−8.34 (m,
1H), 8.38−8.40 (m, 1H), 8.71−8.72 (m, 1H), 8.82 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO−d6): δ= 13.79, 22.20, 28.92, 61.89, 65.82, 114.43, 122.78, 122.97, 125.81, 131.57,
137.13, 142.29, 148.56, 166.77, 169.53 ppm. I.R: 1264, 1540, 1683, 1740 cm-1. HRMS
(Q−TOF) m/z: calcd. for C18H22N5O7 [M+H]+ 420.1475, found: 420.1494. Rf. 0.32 (30% ethyl
acetate/ petroleum ether). Mp: 150−151 °C.
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Compound (4l). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0, 6H), 2.00 (s, 3H),
3.90 (s, 2H), 4.32 (q, J= 7.0, 4H), 6.81 (s, 1H), 7.89 (s, 1H), 7.92−7.96 (m, 2H), 8.39−8.43 (m,
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2H) ppm. C NMR (100 MHz, CDCl3): δ= 14.07, 23.26, 29.29, 63.13, 65.86, 120.35, 121.13,
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125.80, 141.05, 143.86, 147.28, 167.52, 169.70 ppm. I.R: 1216, 1285, 1445, 1667, 1758, 3389
cm-1. HRMS (Q−TOF) m/z: calcd. for C18H21N5O7Na [M+Na]+ 442.1338, found: 442.1336. Rf.
0.24 (50 % ethyl acetate/ petroleum ether). Mp: 186−188 °C.
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Compound (4m). White solid. 1H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.2 Hz, 6H), 2.03 (s,
3H), 3.89 (s, 2H), 4.31 (q, J= 7.2 Hz, 4H), 6.77 (s, 1H), 7.28−7.32 (m, 1H), 7.33 (s, 1H),
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7.39−7.45 (m, 1H), 7.85 (s, 1H), 7.93−7.98 (m, 1H) ppm. C NMR (100 MHz, CDCl3): δ=
13.97, 22.99, 28.91, 62.94, 66.08, 116.91, 117.11, 124.07, 124.16, 124.59, 125.27, 125.31,
130.01, 130.09, 142.33, 167.32, 169.40 ppm. I.R: 1195, 1275, 1463, 1643, 1740, 3257 cm-1.
HRMS (Q−TOF) m/z: calcd. for C18H22FN4O5 [M+H]+ 393.1530, found: 393.1558. Rf. 0.31
(30% ethyl acetate/ petroleum ether). Mp: 64−66 °C.
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Compound (4n). White solid. H NMR (400 MHz, CDCl3): δ= 1.28 (t, J= 7.1 Hz, 6H), 2.00 (s,
3H), 3.85 (s, 2H), 4.29 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.09−7.14 (m, 1H), 7.45−7.50 (m, 3H),
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7.76 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 14.11, 23.21, 29.21, 63.10, 66.14, 108.08,
108.34, 115.60, 115.68, 115.71, 115.81, 121.14, 131.31, 131.40, 143.11, 164.48, 167.46, 169.58
ppm. I.R: 1191, 1234, 1509, 1641, 1740, 3253 cm-1. HRMS (Q−TOF) m/z: calcd. for
C18H22FN4O5 [M+H]+ 393.1530, found: 393.1566. Rf. 0.25 (30% ethyl acetate/ petroleum ether).
Mp: 128−130 °C.
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Compound (4o). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.3 Hz, 6H), 2.01 (s,
3H), 3.86 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.79 (s, 1H), 7.19−7.23 (m, 2H), 7.65−7.69 (m, 2H),
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7.71 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 13.98, 23.09, 29.06, 62.95, 66.01, 116.61,
116.84, 121.14, 122.21, 122.30, 142.84, 167.34, 169.40 ppm. I.R: 1206, 1283, 1510, 1670, 1726,
3394 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22FN4O5 [M+H]+ 393.1530, found: 393.1563.
Rf. 0.22 (50% ethyl acetate/ petroleum ether). Mp: 120−122 °C.
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Compound (4p). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0 Hz, 6H), 2.02 (s,
3H), 3.89 (s, 2H), 4.31 (q, J= 7.0 Hz, 4H), 6.80 (s, 1H), 7.42−7.47 (m, 2H), 7.55−7.58 (m, 1H),
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7.60−7.62 (m, 1H), 7.77 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 13.99, 23.03, 28.98,
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62.93, 66.09, 124.98, 127.66, 127.98, 128.29, 130.66, 130.78, 134.86, 141.66, 167.32, 169.33
ppm. I.R: 730, 1210, 1497, 1679, 1742 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22ClN4O5
[M+H]+ 409.1272, found: 409.1271. Rf. 0.20 (50% ethyl acetate/ petroleum ether). Mp: 84−86
°C.
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Compound (4q). White solid. H NMR (400 MHz, CDCl3): δ= 1.29 (t, J= 7.1 Hz, 6H), 2.01 (s,
3H), 3.86 (s, 2H), 4.30 (q, J= 7.1 Hz, 4H), 6.78 (s, 1H), 7.37−7.47 (m, 2H), 7.57−7.61 (m, 1H),
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7.73−7.74 (m, 1H), 7.75 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 14.01, 23.12, 29.11,
62.99, 66.00, 118.24, 120.62, 120.94, 128.73, 130.91, 135.81, 137.99, 143.16, 167.65, 169.56
ppm. I.R: 730, 1198, 1496, 1595, 1677, 1741 cm-1. HRMS (Q−TOF) m/z: calcd. for
C18H22ClN4O5 [M+H]+ 409.1272, found: 409.1276. Rf. 0.34 (50% ethyl acetate/ petroleum
ether). Mp: 107−109 °C.
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Compound (4r). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J = 7.1 Hz, 6H), 2.01 (s,
3H), 3.86 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.46−7.51 (m, 2H), 7.62−7.67 (m, 2H),
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7.73 (s, 1H) ppm. C NMR (125 MHz, CDCl3): δ= 13.98, 22.92, 23.14, 23.85, 29.08, 62.99,
65.27, 66.01, 71.39, 120.92, 121.46, 129.96, 134.45, 135.45, 142.99, 166.66, 167.34, 169.31,
169.45 ppm. I.R: 517, 1091, 1446, 1669, 1744, 3392 cm-1. HRMS (Q−TOF) m/z: calcd. for
C18H22ClN4O5 [M+H]+ 409.1272, found: 409.1272. Rf. 0.34 (50% ethyl acetate/ petroleum
ether). Mp: 107−109 °C.
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Compound (4s). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.3 Hz, 6H), 2.03 (s,
3H), 3.89 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.80 (s, 1H), 7.36−7.40 (m, 1H), 7.46−7.50 (m, 1H),
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7.52−7.55 (m, 1H), 7.36−7.59 (m, 1H), 7.72 (s, 1H), ppm. C NMR (100 MHz, CDCl3): δ=
14.00, 23.08, 28.99, 62.93, 66.09, 118.38, 125.04, 128.14, 128.54, 131.11, 133.91, 136.51,
141.57, 167.31, 169.31 ppm. I.R: 513, 1195, 1508, 1646, 1741, 3283 cm-1. HRMS (Q−TOF)
m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found: 453.0763. Rf. 0.22 (50% ethyl acetate/
petroleum ether). Mp: 100−102 °C.
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Compound (4t). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.0 Hz, 6H), 2.02 (s,
3H), 3.87 (s, 2H), 4.31 (q, J= 7.0 Hz, 4H), 6.79 (s, 1H), 7.36−7.40 (m, 1H), 7.55−7.57 (m, 1H),
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7.63−7.65 (m, 1H), 7.75 (s, 1H), 7.89−7.91 (m, 1H), ppm. C NMR (100 MHz, CDCl3): δ=
13.98, 23.10, 29.07, 62.98, 65.98, 118.72, 120.91, 123.33, 123.42, 131.08, 131.67, 137.86,
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143.02, 167.31, 169.43 ppm. I.R: 678, 1021, 1486, 1643, 1739, 3255 cm-1. HRMS (Q−TOF)
m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found: 453.0762. Rf. 0.25 (30% ethyl acetate/
petroleum ether). Mp: 116−118 °C.
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3H), 3.86 (s, 2H), 4.31 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.57−7.60 (m, 2H), 7.62−7.65 (m, 2H),
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7.74 (s, 1H) ppm. C NMR (100 MHz, CDCl3): δ= 13.98, 23.08, 29.05, 62.97, 65.99, 120.83,
121.67, 122.28, 132.91, 135.91, 143.01, 167.33, 169.42 ppm. I.R: 664, 1109, 1498, 1667, 1740,
3141, 3386 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H22BrN4O5 [M+H]+ 453.0767, found:
453.0766. Rf. 0.21 (50% ethyl acetate/ petroleum ether). Mp: 148−150 °C.
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Compound (4v). White solid. H NMR (400 MHz, CDCl3): δ= 1.31 (t, J= 7.3 Hz, 6H), 2.04 (s,
3H), 3.90 (s, 2H), 4.31 (q, J= 7.3 Hz, 4H), 6.82 (s, 1H), 7.21−7.24 (m, 1H), 7.41−7.43 (m, 1H),
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7.48−7.52 (m, 1H), 7.62 (s, 1H), 7.97−8.00 (m, 1H) ppm. C NMR (100 MHz, CDCl3): δ=
14.01, 23.18, 29.00, 29.70, 62.95, 66.10, 93.86, 124.92, 127.86, 129.29, 131.48, 140.21, 141.67,
167.29, 169.33 ppm. I.R: 1264, 1516, 1693, 1742, 3380 cm-1. HRMS (Q−TOF) m/z: calcd. for
C18H22IN4O5 [M+H]+ 501.0590, found: 501.0606. Rf. 0.21 (30% ethyl acetate/ petroleum ether).
Mp: 70−72 °C. HPLC purity: 100%.
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Compound (4w). White solid. H NMR (400 MHz, CDCl3): δ= 1.30 (t, J= 7.2 Hz, 6H), 2.02 (s,
3H), 3.86 (s, 2H), 4.31 (q, J= 7.2 Hz, 4H), 6.78 (s, 1H), 7.21−7.24 (m, 1H), 7.66−7.69 (m, 1H),
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7.73 (s, 1H), 7.74−7.77 (m, 1H), 8.08−8.09 (m, 1H) ppm. C NMR (100 MHz, CDCl3): δ=
13.98, 23.11, 29.06, 62.98, 65.99, 94.44, 119.42, 120.88, 129.13, 131.15, 137.66, 137.71, 142.98,
167.32, 169.43 ppm. I.R: 1047, 1265, 1498, 1678, 1741, 3383 cm-1. HRMS (Q−TOF) m/z: calcd.
for C18H22IN4O5 [M+H]+ 501.0590, found: 501.0630. Rf. 0.37 (40% ethyl acetate/ petroleum
ether). Mp: 82−84 °C.
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Compound (4x). White solid. H NMR (400 MHz, CDCl3): δ= 1.28 (t, J= 7.1 Hz, 6H), 2.00 (s,
3H), 3.85 (s, 2H), 4.29 (q, J= 7.1 Hz, 4H), 6.79 (s, 1H), 7.44−7.46 (m, 2H), 7.74 (s, 1H),
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7.81−7.83 (m, 2H) ppm. C NMR (100 MHz, CDCl3): δ= 14.13, 23.23, 29.22, 63.10, 66.14,
93.60, 120.94, 121.95, 136.73, 139.01, 143.14, 167.46, 169.55 ppm. I.R: 663, 1051, 1498, 1667,
1740, 3142, 3385 cm-1. HRMS (Q−TOF) m/z: calcd. for C18H21IN4O5Na [M+Na]+ 523.0454,
found: 523.0449. Rf. 0.29 (30% ethyl acetate/ petroleum ether). Mp: 152−154 °C.
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The Thioflavin T assay
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Aβ42 peptide was purchased from Anaspec and dissolved in 1,1,1,3,3,3−hexafluoro−2−propanol
(HFIP). HFIP treated Aβ42 peptide was further evaporated and stored at −20 ˚C. The stock
solution of Aβ42 (221.5 μM) was prepared by dissolving 0.1 mg Aβ42 in 100 μL NaOH (10 mM)
and stored at −80 ˚C for further use. Then 2 mM stock solutions of all the test compounds and
controls were prepared in DMSO. 1 mM stock solution of ThT was prepared in 50 mM PBS
buffer (pH 7.4) then diluted to 200 µM stock. For self induced aggregation assay, mixture of
Aβ42 (9 µL, 10 µM) and ThT (20 µL, 20 µM) with or without the presence of test compounds
and controls (2.5 µL, 50 µM) were diluted to final volume of 200 µL with 50 mM PBS buffer
(pH 7.4) in black 96−well plate and incubated for 24 h at 37 ˚C with constant agitation (180
rpm). For disaggregation assay, the monomer Aβ42 was incubated for 24 h at 37 ˚C to form
fibrils. Then, the test compound and control were added to the aliquots of Aβ42 fibrils and
incubated for another 24 h under same conditions. The fluorescence intensities were recorded in
SpectraMax M5e spectrophotometer with excitation and emission wavelengths at 450 nm and
485 nm respectively. Percentage inhibition was calculated by using the formula (1–
Fsample/Fcontrol)*100.
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For the inhibition of Cu2+–mediated Aβ42 aggregation, the Aβ42 stock solution was diluted in 20
μM HEPES (pH 6.6) with 150 μM NaCl. The mixture of the Aβ42 (9 μL, 10 μM) with copper
(2.5 μL, 10 μM) with or without the presence of the test compounds (5 μL, 20 μM) were
incubated at 37 °C for 24 h. Then 16.5 μL of the sample was diluted to a final volume of 200 μL
with 50 mM glycine−NaOH buffer (pH 8.0) containing thioflavin T (20 μM). For disaggregation
°
assay, the monomer Aβ42 was incubated with copper for 24 h at 37 C to form fibrils. Then, the
test compound and control were added to the aliquots of Aβ42 fibrils and incubated for another 24
h under same conditions.
Statistical Analysis
Statistical analysis was performed with one–way analysis of variance (ANOVA) followed by
Dunnett’s test on at least three different measurements (**) p < 0.01 and (***) p < 0.001; Mean±SD
for n= 3 experiments). ThT Data analysis and graph production was done by using IBM SPSS
Statistics (version 21) for Windows.
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Transmission electron microscopy assay
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Transmission electron microscopy was performed to visualise Aβ42 fibrils in the presence of
compound 4v. For self induced Aβ42 aggregation assay, Aβ42 monomer (10 μM) was incubated
alone or in the presence of 4v (20 μM) in 50 mM PBS buffer (pH 7.4) at 37 °C for 24 h. For Aβ42
disaggregation assay, Aβ42 fibrils (10 μM) was incubated in the presence of 4v (20 μM) in 50
mM PBS buffer (pH 7.4) at 37 °C for 24 h. 5 μL of the samples were placed on a carbon–coated
200 mesh (agar scientific) copper grid for 2 min and allowed to dry for 30 min. Each grid was
stained with 2% phosphotungstic acid for 45 seconds. After draining off the excess staining
solution, the specimen was loaded to grid holder and transferred for imaging by transmission
electron microscopy (JEOL JEM–2100) operating at 200 kV.
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Metal chelating studies
The chelating ability of compound 4v towards biometals such as Cu2+, Zn2+, Fe2+ was examined
by UV−Vis spectroscopy (Ultrospec 3000). The stock solution of compound 4v (4 mM), metal
ions (8 mM) were prepared in methanol. The solution of 4v (20 µM, final concentration) alone or
in the presence of CuSO4, ZnCl2 or FeSO4 (20 µM, final concentration) in (20 mM HEPES, 150
°
mM NaCl, pH 7.4) were incubated for 30 min at 25 C. The spectra of each sample were
recorded with wavelength ranging from 200 nm to 600 nm using blank containing 20 mM
HEPES, 150 mM NaCl, pH 7.4.
The stoichiometry of the 4v−Cu2+ complex was determined by Job’s method, by preparing the
separate solutions of 4v and CuSO4 in which the total concentration remain constant (40 µM) but
their proportion of each component varied from 0% to 100%. The absorbance at 226 nm was
plotted against mole fraction of Cu2+. The breakpoint displays the stoichiometry of the complex.
Ascorbate studies
The ascorbate study was performed by using SpectraMax M5e spectrophotometer. The
fluorescence intensities were recorded with excitation and emission wavelengths at 395 nm and
450 nm for the period of 16 min. The stock solution of 4v (in methanol), CuSO4 (in Milli−Q
water), CCA and ascorbate were dissolved and diluted in 50 mM PBS buffer (pH 7.4). The final
volume of the sample was 200 µL. The production of hydroxyl radical was measured as the
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