Benzofuran-chalcone hybrids as potential multifunctional agents against Alzheimer's disease: Synthesis and in vivo studies with transgenic Caenorhabditis elegans

Article abstract of DOI:10.1002/cmdc.201402291

In the search for effective multifunctional agents for the treatment of Alzheimer's disease (AD), a series of novel hybrids incorporating benzofuran and chalcone fragments were designed and synthesized. These hybrids were screened by using a transgenic Ca

Full text of DOI:10.1002/cmdc.201402291

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DOI: 10.1002/cmdc.201402291
Benzofuran–Chalcone Hybrids as Potential Multifunctional
Agents against Alzheimer’s Disease: Synthesis and in vivo
Studies with Transgenic Caenorhabditis elegans
[
a]
[a]
[b]
[a]
Koneni V. Sashidhara,* Ram K. Modukuri, Pooja Jadiya, Ranga Prasad Dodda,
[a]
[c]
[d]
[b]
Manoj Kumar, Balasubramaniam Sridhar, Vikash Kumar, Rizwanul Haque,
Mohammad Imran Siddiqi, and Aamir Nazir*
[d]
[b]
This paper is dedicated to Prof. Chhitar Mal Gupta (Former Director) on the occasion of his 70th birthday and to honor him for his
dedicated services to Central Drug Research Institute, in his long and illustrious career.
In the search for effective multifunctional agents for the treat-
ment of Alzheimer’s disease (AD), a series of novel hybrids in-
corporating benzofuran and chalcone fragments were de-
signed and synthesized. These hybrids were screened by using
a transgenic Caenorhabditis elegans model that expresses the
human b-amyloid (Ab) peptide. Among the hybrids investigat-
ed, (E)-3-(7-methyl-2-(4-methylbenzoyl)benzofuran-5-yl)-1-phe-
nylprop-2-en-1-one (4 f), (E)-3-(2-benzoyl-7-methylbenzofuran-
significantly decreased Ab aggregation and increased acetyl-
choline (ACh) levels along with the overall availability of ACh
at the synaptic junction. These compounds were also found to
decrease acetylcholinesterase (AChE) levels, reduce oxidative
stress in the worms, lower lipid content, and to provide protec-
tion against chemically induced cholinergic neurodegenera-
tion. Overall, the multifunctional effects of these hybrids quali-
fy them as potential drug leads for further development in AD
therapy.
5-yl)-1-phenylprop-2-en-1-one (4i), and (E)-3-(2-benzoyl-7-
methylbenzofuran-5-yl)-1-(thiophen-2-yl)prop-2-en-1-one (4m)
Introduction
[
2]
Alzheimer’s disease (AD) is one of the most common fatal
taining hyperphosphorylated tau protein). Moreover, oxida-
tive stress, mitochondrial dysfunction, and impairment of pro-
[1]
neurodegenerative disorders for which no cure is known. Irre-
versible memory loss, disorientation, language impairment,
and cognitive decline are the most common clinical symptoms
associated with AD. The pathological hallmarks of AD include
extracellular b-amyloid (Ab) plaque formation (containing
mainly Ab1–42) and the formation of neurofibrillary tangles (con-
[3]
tein degradation accelerate disease progression significantly.
Current therapeutic treatments for AD are focused on the
symptomatic aspects of the pathology, as they are limited pri-
marily to acetylcholinesterase (AChE) inhibitors, namely done-
[4]
pezil, rivastigmine, and galantamine. These drugs modulate
a single target, and therefore enable only palliative treatment
[5]
+
rather than a cure or prevention of AD. The complexity and
[
a] Dr. K. V. Sashidhara, R. K. Modukuri, R. P. Dodda, Dr. M. Kumar
[6]
Medicinal and Process Chemistry Division
CSIR-Central Drug Research Institute (CSIR-CDRI)
Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)
E-mail: kv_sashidhara@cdri.res.in
manifold pathogenesis of AD have prompted researchers to
move from the traditional one protein, one target, one drug
strategy to a new paradigm in drug research involving a multi-
[7]
target-directed ligand (MTDL) design strategy. Therefore, re-
searchers are now turning to the design of hybrid structures
that should be able to simultaneously interact with multiple
+
[
b] P. Jadiya, R. Haque, Dr. A. Nazir
Laboratory of Functional Genomics and Molecular Toxicology
Division of Toxicology, CSIR-Central Drug Research Institute (CSIR-CDRI)
Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)
E-mail: anazir@cdri.res.in
[8]
targets.
Benzofuran derivatives are pivotal biodynamic agents from
[c] Dr. B. Sridhar
[9]
both synthetic and natural sources. New egonol-type benzo-
Laboratory of X-ray Crystallography
CSIR-Indian Institute of Chemical Technology (CSIR-IICT)
Hyderabad, 500007 (India)
furans were recently isolated from natural sources, and have
shown significant inhibition of AChE and AChE-induced Ab ag-
[10]
[d] V. Kumar, Dr. M. I. Siddiqi
gregation. Furthermore, a literature survey of the scaffold re-
vealed that benzofuran-ring-containing derivatives (SKF-64346
CSIR-Central Drug Research Institute (CSIR-CDRI)
Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)
and aminostyrylbenzofuran derivatives) exhibited inhibitory ac-
+
[
] These authors contributed equally to this work.
[11,12]
tivity toward Ab fibril formation.
Alternatively, chalcones
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402291: details of compound synthe-
sis, characterization, HPLC analyses, X-ray crystallographic information,
and docking procedures.
(
1,3-diaryl-2-propen-1-ones) are an important class of plant sec-
ondary metabolites and possess a wide range of biological ac-
[13]
tivities. It has been reported that chalcone derivatives are
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Figure 1. Design of benzofuran–chalcone hybrids for the treatment of AD.
able to directly scavenge a variety of reactive oxygen species
the C. elegans sel-12 mutant was shown to be complemented
[
14]
[23]
(ROS) and possess strong antioxidant properties. Recent evi-
by the expression of human PS-1. C. elegans is also valuable
dence has suggested that chalcone derivatives have the ca-
to the study of neurodegenerative diseases, as it provides an
uncomplicated in vivo nervous system for the study of many
genes involved in its neuronal function—genes that have
closely related human homolgues.
pacity to inhibit Ab fibril formation and exert neuroprotec-
[
15]
tion. Interestingly, curcuminoids, a class of chalcone, have
been shown to exhibit potent inhibitory effects on Ab-induced
[
16]
oxidative stress. Therefore, introducing a chalcone pharma-
cophore into a hybrid molecule could impart it with neuropro-
tective effects and the capacity to decrease oxidative stress,
This C. elegans model provided a platform for us to examine
the effects of our novel benzofuran–chalcone hybrids on multi-
ple factors such as Ab aggregation, acetylcholine (ACh) and
AChE levels, the availability of ACh at the synaptic junction,
oxidative stress, mitochondrial content, and lipid depositions.
[17]
which are crucial properties to halt the progression of AD.
Our research group has been involved for many years in the
rational design and synthesis of several new multifunctional
compounds by using the concept of molecular hybridiza-
[
18,19]
tion.
Herein we describe the synthesis and pharmacologi- Results and Discussion
cal evaluation of novel benzofuran-based chalcone hybrids as
potential multitargeted agents for the treatment of AD.
Figure 1 shows representative potent inhibitors that contain
benzofurans and chalcones in their molecular makeup along
with structures of our designed hybrids.
Chemistry
The synthetic routes used for preparation of the target and in-
termediate compounds are depicted in Scheme 1. Although
the synthesis of 2-substituted benzofuran-based chalcones
[24]
We carried out the evaluation of these hybrids in a transgen-
were reported recently, for this study we used a new, gener-
al, and more efficient route for the synthesis of 5-substituted
benzofuran–chalcone hybrids starting from our versatile di-
[
20]
ic C. elegans model expressing “human” Ab. This model has
been used for studying Ab toxicity in relevance to age-related
disorders due to the organism’s short life cycle and experimen-
[25]
carbaldehyde intermediate 2.
Duff formylation on ortho-cresol in the presence of hexa-
[
21]
tal flexibility. This model exhibits toxic effects via aggrega-
tion of Ab and has been reported to have a proteomic expres-
sion pattern, similar to that of postmortem brain tissue from
methylenetetraamine (HMTA) and trifluoroacetic acid (TFA) at
[18]
1208C gave the aromatic dicarbaldehyde 2. Next, Rap–Stoer-
mer condensation on compound 2 with various phenacyl
bromides in the presence of potassium carbonate furnished
the benzofuran carbaldehyde derivatives 3a–c in quantitative
yields. Introduction of the chalcone scaffold was carried out by
reaction of various acetophenones in methanolic potassium
[
22]
[26]
human AD patients. C. elegans is extremely relevant in AD
research, as the first ever genetic understanding of the disease
came from this model when the presenilin-1 (PS-1) homologue
sel-12 was identified. The gene is now known to be conserved
between these nematodes and humans, as the phenotype in
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the worms leads to early paraly-
sis, which makes an easy end-
point to screen the effect of
a compound on Ab aggregation.
The effect of test compounds on
the paralysis phenotype of trans-
genic CL4176 nematodes is pre-
sented in Figure 3. Out of the 16
compounds evaluated, seven de-
creased Ab aggregation, leading
to a delay in paralysis by 2- (p<
0
0
0
.01), 1.4- (p<0.05), 2.3- (p<
.01), 1.7- (p<0.01), 1.4- (p<
.05), 1.8- (p<0.01), and 2.1-fold
(
p<0.01) in worms fed with
compounds 3a, 4c, 4d, 4 f, 4i,
l and 4m, respectively. In this
4
study, all compounds were
screened in the presence of con-
trol as well as vehicle control,
and compounds that did not
affect Ab-induced paralysis were
not selected for further studies.
Scheme 1. Synthesis of substituted benzofuran–chalcone hybrids. Reagents and conditions: a) HMTA, TFA, 1208C,
3
2 4 2 3 3
h; b) H SO (aq. 10%), 1008C, 2 h; c) various substituted phenacyl bromides, K CO /CH CN, 1108C, 3 h; d) various
substituted acetophenones, KOH (10% in MeOH), 3 h, RT.
hydroxide at room temperature to give the final benzofuran–
chalcone hybrids 4a–m. The structures of the compounds
1
13
were verified by H NMR, C NMR, mass spectrometry, and IR
spectroscopy. Additionally, the structure of a representative
compound, 4d, was distinctly confirmed by single-crystal X-ray
analysis (Figure 2; see the Supporting Information for further
details).
Figure 3. Ab-induced paralysis assay in the CL4176 strain of C. elegans treat-
ed with the indicated test compounds. Control (C)= OP50 only; vehicle con-
trol (VC)=ethanol. Data are normalized with respect to control, and values
are the meanÆSEM of n=3 experiments performed in triplicate; *p<0.05,
*
*p<0.01.
It is well known that AChE is the key enzyme responsible for
the metabolic and catalytic hydrolysis of ACh and for promot-
[27]
ing the aggregation and deposition of Ab peptide.
This
prompted us to speculate that the reduced Ab aggregation
observed is due to the effect of our hybrids on the aggrega-
tion of Ab and its associated toxicity. This decrease in Ab ag-
gregation could also be associated with the binding affinity of
the benzofuran scaffold to Ab, as it has been reported to inhib-
it the polymerization process that leads to amyloid neurotoxic-
ity.
Figure 2. X-ray crystallographic structure of compound 4d.
Biological assays
Benzofuran–chalcone hybrids decrease Ab-induced toxicity in
a transgenic C. elegans model expressing human Ab
AD is characterized by aggregation of the Ab peptide. There-
fore, we performed this experiment with a transgenic C. ele-
gans strain (CL4176), which exhibits temperature-inducible ex-
pression of human Ab. The strain has been designed to ex-
press Ab specifically in the muscles; therefore, increased Ab in
Benzofuran–chalcone hybrids enhance ACh availability/excita-
tory neurotransmission in wild-type C. elegans
AD is tightly associated with impaired synthesis of the excita-
tory neurotransmitter, ACh. Therefore, we endeavored to study
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the role of our test compounds in enhancing ACh levels in the
C. elegans model. A pharmacological assay was performed to
determine the effect of hybrids on the release of ACh in the
synaptic cleft. AChE, present at the synaptic cleft, is known to
catalyze the hydrolysis of ACh into choline and acetate, thus
eliminating the neurotransmitter from the synapse. In the pres-
ence of an AChE inhibitor, aldicarb, the hydrolysis of ACh is
prevented, thus leading to its continued accumulation at the
synapse. This, in turn, causes flexing of muscles thereby para-
lyzing the worms. This assay is known as the aldicarb assay,
which gives a rapid assessment of the effect of pharmacologi-
cal and toxicological agents on the ACh content in nematodes.
As shown in Figure 4, exposure of worms with compounds 4 f,
crease the availability of ACh, thus aiding in improved excitato-
ry neurotransmission. Because 4 f, 4i, and 4m showed their
potential both in blocking Ab aggregation and in enhancing
ACh availability, we carried out further biological studies with
these three compounds to explore their multifactorial potential
against AD.
Benzofuran–chalcone hybrids increase ACh and inhibit AChE
activity in C. elegans
We further estimated the levels and activity of ACh and AChE
in control and test-compound-treated worms along with nega-
tive and positive controls (provided with the assay kit; see Ex-
perimental Section below). As depicted in Figure 5A, treatment
of worms with compounds 4 f, 4i, and 4m gave significantly
higher ACh levels than untreated worms. Exposure of worms
with compounds 4 f, 4i, and 4m showed respective 1.3- (p<
0.001), 1.5- (p<0.001), and 1.1-fold (p<0.05) increased ACh
levels. In further quantification of AChE inhibition, 4 f- and 4i-
treated worms showed significant inhibition of AChE activity.
Inhibition of AChE activity was significantly (p<0.001) de-
creased in 4 f- (1.7-fold) and 4i- (2.3-fold) treated worms, rela-
tive to control. Worms raised on compound 4m exhibited
maximum inhibition of AChE, showing 10.7-fold decrease in
comparison with untreated worms (p<0.001; Figure 5B).
Figure 4. Effect of the test compounds on acetylcholine availability as deter-
mined by the aldicarb assay in the N2 strain of C. elegans. C=vehicle control
(
ethanol); Don=donepezil. Data are normalized with respect to control, and
values are the meanÆSEM of n=3 experiments performed in triplicate;
p<0.05, **p<0.01.
*
4
i, and 4m exhibited a significant (p<0.05) increase in the
availability of ACh at the synapse, which in turn led to early al-
dicarb-induced paralysis. Compound 4 f showed a 1.6-fold in-
crease in ACh levels relative to the standard AChE inhibitor do-
nepezil. Compounds 4i and 4m exhibited an increase in the
availability of ACh nearly equal to that of donepezil. Similarly,
donepezil-, 4 f-, 4i-, and 4m-treated worms displayed 1.8-, 2.8-,
1.6-, and 1.9-fold increased ACh availability respectively, in
comparison with control worms. This experiment showed that
the hybrid molecules 4 f, 4i, and 4m are potent, vis-ꢁ-vis in-
creasing ACh availability, than the parent benzofuran com-
pound 3a.
In the central nervous system (CNS), ACh, released from
cholinergic neurons, plays a significant role in maintaining cog-
nition, memory, and learning. Impaired neurotransmission and
[
28]
decreased levels of ACh contribute to the pathology of AD.
Figure 5. Effect of test compounds on A) acetylcholine and B) acetylcholines-
terase activities, as measured by the Amplexꢂ Red assay in wild-type C. ele-
Therefore, compounds that enhance the release of ACh could
be further explored as potential treatments for the cognitive
symptoms of AD. In our study, compounds 4 f, 4i, and 4m in-
creased the levels of ACh, demonstrating that the hybrids in-
gans. Control (C)= OP50 only; positive control (PC)=for ACh activity 10 mm
À1
H
2
O
2
and for AChE activity 0.2 UmL AChE solution. Data are normalized
with respect to control, and values are the meanÆSEM of n=2 experiments
performed in triplicate; *p<0.05, ***p<0.001.
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These studies gave evidence that the benzofuran–chalcone hy-
brids inhibit AChE and also increase the levels of ACh via other
mechanisms, probably by increasing biosynthesis of the neuro-
transmitter or by blocking AChE activity.
death via a number of possible mechanisms, including oxida-
[35]
tive stress.
Benzofuran–chalcone hybrids inhibit Ab deposits in transgenic
C. elegans
Benzofuran–chalcone hybrids afford neuroprotection in a
C. elegans model of cholinergic neurodegeneration
Ab aggregate formation plays a central role in the pathogene-
sis of AD. To determine whether the benzofuran–chalcone hy-
brids have any effect on Ab oligomerization in the transgenic
C. elegans strain CL2006, which expresses human Ab under
control of the unc-54 promoter (encodes a muscle myosin clas-
s II heavy chain (MHC B)), worms were fed with the test com-
pound and subjected to Ab staining by thioflavin S. Thiofla-
vin S is used for staining of amyloid plaques; it binds to Ab fi-
brils (but not monomers) and gives a distinct spectral shift
In this study, we employed transgenic LX929 (unc-17: green flu-
orescent protein (GFP)) strain of C. elegans that expresses GFP
specifically in cholinergic neurons. In C. elegans more than
a third of neuronal cells are cholinergic neurons, and thus ACh
is the main excitatory neurotransmitter to release at neuromus-
cular junctions. The organophosphate pesticide chlorpyrifos
(CP) is known to induce cholinergic damage; therefore, we
[36]
upon binding. The Ab deposits (highlighted by arrows) with-
out test compound can clearly be seen in Figure 7A. In con-
trast, worms fed with benzofuran–chalcone hybrids 4 f, 4i, and
used this model to study the effects of our hybrids for the pre-
vention of CP-induced cholinergic neuronal damage. As shown
in Figure 6A, control worms displayed complete cholinergic
neuroanatomy, whereas treatment with CP resulted in a loss of
the GFP expression pattern due to CP-induced cholinergic neu-
rodegeneration (Figure 6B). This neurodegeneration was signif-
icantly (p<0.001) prevented if worms were fed on test com-
pound CP–4 f; a 1.6-fold increase in cholinergic neuroprotec-
tion was observed relative to that of CP-treated worms (Fig-
ure 6C). Similarly, if raised on CP–4i and CP–4m, worms exhib-
ited a 1.3- (p<0.05) and 1.5-fold (p<0.01) increase in
cholinergic neuroprotection relative to that of worms from the
CP-treated group (Figure 6D,E), this evaluation led us to con-
clude the potential of these compounds in improving choliner-
4
m showed a significant decrease in Ab deposits (Figure 7B–
D).
The inhibitory effect of these compounds was scored by
counting the number of plaque deposits per worm. We found
that the mean number of Ab deposits was significantly re-
duced in Ab-expressing transgenic C. elegans strain fed with 4 f
(
p<0.01), 4i (p<0.05), and 4m (p<0.01) relative to unfed
controls. These results suggested that the benzofuran–chal-
cone hybrids inhibit Ab oligomerization efficiently.
Dose–dependent antioxidant properties
[
29–34]
gic neurophysiology.
The neuroprotection observed in our
studies could also be due to decreased aggregation of Ab, as
it is well known that increased Ab deposition leads to neuronal
To further explore the role of compounds 4 f, 4i, and 4m,
which effected a significant delay in Ab-induced paralysis as
Figure 6. GFP expression pattern in cholinergic neurons of the transgenic LX929 strain of C. elegans. A) Control, B) chlorpyrifos (CP)-treated worms, and CP-
treated worms raised on C) compound 4 f, D) compound 4i, and E) compound 4m.
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Figure 8. Effect of compounds 4 f, 4i, and 3a on the relative formation of
reactive oxygen species as measured by H DCFDA assay in a transgenic
2
CL2006 strain of C. elegans. C=untreated control, RFU=relative fluorescence
units. Data are normalized with respect to control, and values are the
meanÆSEM of n=3 experiments performed in triplicate;*p<0.05,
*
*p<0.01, ***p<0.001.
4
i decreased oxidative stress in worms; the effect was moder-
ate in wild-type worms, whereas there was a pronounced de-
crease in oxidative stress in the Ab-expressing C. elegans strain.
This is an interesting observation, as this further corroborates
the notion that benzofuran moieties possess Ab binding prop-
erties, thus reducing the toxic effects of these aggregates. The
Figure 7. Thioflavin S staining to show Ab deposits on body wall muscle
(indicated by arrows). Images of Ab deposits in A) the transgenic C. elegans
strain CL2006 alone, and after treatment with test compounds B) 4 f, C) 4i,
and D) 4m. E) Quantitative analysis of Ab deposits. Control (C)= OP50 only.
Data are the meanÆSEM of n=3 experiments performed in triplicate;
[13]
antioxidant properties of chalcone and benzofuran moiet-
[38]
*
p<0.05, **p<0.01.
ies have been reported previously; some benzofuran deriva-
tives have also been reported to exhibit protection against
[39]
sulfhydryl group oxidation and lipid peroxidation.
well as an increase in the content and availability of ACh, we
studied antioxidant properties using the CL2006 strain of C. el-
egans. In the case of compound 4m, antioxidant effects were
not observed in the CL2006 strain, whereas compounds 4 f
and 4i led to a significant decrease in ROS levels relative to
those of untreated worms. We next studied the effect of test
compound dose (4 f, 4i, and 3a) on antioxidant properties
using three different concentrations (20, 200, and 2000 mm)
and the human-Ab-expressing transgenic strain CL2006.
Worms treated with compound 4 f showed significant 1.8- (p<
The rather specific and pronounced effect of the test com-
pounds in our study provided evidence that the hybrid nature
of these benzofuran–chalcone molecules leads to the specific
decrease in oxidative stress, probably through the binding of
benzofuran molecules with Ab. Intriguingly, compound 4m did
not exhibit antioxidant properties; this implies that the ob-
served effects of this compound are independent of effects on
ROS levels and are mediated by a different pathway.
Lowering of lipid deposition in an Ab-expressing C. elegans
strain
0.05), 1.9- (p<0.001), and 3.1-fold (p<0.001) decreases in ROS
levels at 20, 200, and 2000 mm, respectively (Figure 8). In deter-
mining the antioxidant effects of compound 4i, ROS levels
were found to decrease 1.4- (p<0.05), 1.7- (p<0.01), and 2.6-
fold (p<0.001) in worms treated respectively at 20, 200, and
High levels of lipids and altered lipid metabolism are associat-
[40]
ed with many neurodegenerative diseases, including AD. We
therefore studied the lipid-modulating properties of the test
compounds. Using a lipid-specific fluorescent dye, Nile red, to
track lipid deposits within the worms, we carried out fluores-
cence imaging studies using the CL2006 strain. As depicted in
Figure 9, worms of the control group exhibited an optimum
Nile red staining pattern (Figure 9A), whereas worms treated
with compounds 4 f (Figure 9B), 4i (Figure 9C), and 4m (Fig-
ure 9D) showed reduced staining, thus reflecting the effect of
these compounds on lipid deposition. The fluorescence inten-
sity, as quantified by Image J analysis, for the worms of the
control group was found to be 14.16Æ1.174 arbitrary units,
whereas worms treated with 4 f, 4i, and 4m showed fluores-
2000 mm. On the other hand, worms treated with the parent
benzofuran compound 3a at 20 mm did not show changes in
ROS content, whereas at 200 and 2000 mm, worms displayed
respective 1.4- (p<0.05) and 1.8-fold (p<0.01) decreases in
ROS levels. Our results demonstrate that the hybrid molecules
4
f and 4i exert a dose-dependent antioxidant effect across
the concentration range tested.
It has been recognized for a long time that the presence of
[
37]
ROS increases Ab production. Therefore, use of an antioxi-
dant can alleviate this ROS-mediated toxicity and lead to a re-
duction in Ab aggregation. In our studies, compounds 4 f and
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Reduced mitochondrial content of CL2006 worms is moderate-
ly restored by compound 4 f
There is much evidence to suggest that mitochondrial dysfunc-
tion plays a causative role in the pathogenesis of AD. Further-
more, the accumulation of Ab in the mitochondrial compart-
ment is known to result in impairment of mitochondrial func-
[3,42]
tion.
Therefore, therapies targeted at protecting mitochon-
dria may improve the clinical outcome of AD pathogenesis. In
this context, we studied the effect of test compounds on mito-
chondrial content using Mitotracker staining. Figure 10 shows
that CL2006 worms exhibited a lower mitochondrial content
than wild-type worms; this was moderately restored by com-
pound 4 f, whereas compounds 4i and 4m did not induce any
significant change in mitochondrial content. We further quanti-
fied the fluorescence intensity of mitochondrial content using
Image J software. In the case of worms treated with 4 f, a 1.2-
fold increase (p<0.05) in the fluorescence intensity of mito-
Figure 9. Nile red staining in the CL2006 strain of C. elegans grown on
A) control diet, and treatment with test compounds B) 4 f, C) 4i, and D) 4m.
E) Relative fluorescence intensity of nematode lipid content as quantified by
Image J software. C=control, RFU=relative fluorescence units. Data are
normalized with respect to control, and values are the meanÆSEM of n=3
experiments performed in triplicate; **p<0.01, ***p<0.001.
cence intensities of 3.35Æ0.31, 3.83Æ0.19, and 4.53Æ0.36 ar-
bitrary units, thus exhibiting 4.3- (p<0.001), 3.7- (p<0.001),
and 3.1-fold (p<0.01) decreases in fluorescence intensity, re-
spectively, relative to untreated worms (Figure 9E). Compound
4
f induced a rather pronounced lipid-lowering effect relative
to the other two compounds, which is in agreement with the
enhanced beneficial effects of this compound vis-ꢁ-vis other
reported parameters of this study as well. There is sufficient
published evidence linking high levels of lipids and cholesterol
[
41]
to amyloid fibrillogenesis and its related toxicity; thus the
decrease in lipid content caused by the test compounds re-
lates well to the reduced amyloid aggregation and associated
effects. Therefore, it could be assumed that the benzofuran–
chalcone hybrids do affect multiple factors associated with AD.
Figure 10. Mitotracker staining in the CL2006 strain of C. elegans fed with
A) OP50, and treated with test compounds B) 4 f, C) 4i, and D) 4m. E) Rela-
tive fluorescence intensity of nematode mitochondrial content as quantified
by Image J software. C=control, RFU=relative fluorescence units. Data are
normalized with respect to control, and values are the meanÆSEM of n=3
experiments performed in triplicate; *p<0.01.
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chondrial content was observed relative to the untreated
transgenic CL2006 strain (Figure 10E).
Trp84, Trp279, Trp290, and Phe330 play important roles in the
[43]
binding of E2020. Trp84 and Trp279 are especially signifi-
cant, as both undergo a p–p stacking interaction with E2020.
It has been reported that E2020 is an active site gorge span-
ning AChE inhibitor, interfering with both the catalytic active
Benzofuran–chalcone hybrids do not induce tissue damage in
C. elegans at tested concentrations
[45]
site (CAS) and peripheral active site (PAS) simultaneously. In-
terestingly, docking of hybrids 4 f, 4i, and 4m into the active
site gave almost identical positioning with respect to E2020
(Figure 12B–D), indicating that these compounds use a similar
network of interactions that stabilize E2020. Figure 12 shows
an ensemble of a few top-docked conformations for each com-
pound out of 20 poses that were generated to determine any
conformational preference in the active site pocket. All docked
conformations were found to be similar, indicating the pre-
ferred binding mode of these compounds. At the bottom site
of the binding pocket, the indole ring of Trp84 appeared to
undergo p–p stacking with one of the aromatic rings of hy-
brids 4 f, 4i, and 4m. We have not observed any p–p stacking
interactions with Trp279 near the entrance of active site; how-
ever the lack of this interaction was found to be compensated
by hydrogen bonding of the carbonyl group of compounds
with the main-chain NH group of Phe288. Residues Tyr121 and
Trp279 belong to the PAS, and in the docking study we ob-
served that the benzofuran moieties of 4 f, 4i, and 4m are
present in close proximity to these residues. The role of PAS
residues in the formation of amyloid fibrils has been report-
Next, to examine the gross toxic effect of the test compounds,
we carried out a Trypan-blue-based dye exclusion test in
worms treated with compounds 4 f, 4i, and 4m. Trypan blue is
a critical dye that is taken up by damaged cells and is excluded
by cells with healthy morphology; hence the test provides a re-
liable assessment of cell/tissue damaging potential. We used
N,N’-dimethyl-4,4’-bipyridinium dichloride (Paraquat, PQ) as
a positive control for inducing cellular damage. As shown in
Figure 11A, control worms exhibited a very mild staining pat-
tern (largely background), whereas worms treated with PQ not
only stained intensely (Figure 11B), but also exhibited a strong
developmental effect, as the worms were developmentally im-
paired and smaller. Worms treated with compounds 4 f (Fig-
ure 11C), 4i (Figure 11D), and 4m (Figure 11E) exhibited
a staining pattern similar to that of the control worms, hence
depicting no cell damage.
Molecular modeling studies
[43]
The structure of Torpedo californica AChE (TcAChE)
with
[46]
E2020 (donepezil) has been used for many docking studies of
ed. Parent compounds 3a, 3b, and 3c showed a lack of p–p
stacking interactions with Trp84 in the active site of TcAChE
(Supporting Information Figure S1). We hypothesize that the
addition of a chalcone moiety increases the stacking interac-
tions, allowing a better positioning into the active site of
TcAChE. From these interaction studies, we propose that both
benzofuran and chalcone moieties are essential for ACh activi-
ty and AChE inhibitory activity. These compounds also reduce
[
44]
AChE inhibitors, implying that it can serve as a good model
for the evaluation of newly identified compounds. In the pres-
ent study, we used the TcAChE–E2020 complex for analysis of
the binding mode of hybrids 4 f, 4i, and 4m in the active site
of TcAChE. Re-docking with E2020 resulted in a conformation
close to the co-crystal structure, with an RMSD of 0.66 ꢃ. This
validates our docking approach adopted in this study. Residues
Figure 11. Cytotoxicity assay by Trypan blue staining in the N2 strain of C. elegans. A) Control worms, and B) PQ-treated worms raised on C) compound, 4 f
D) compound 4i, and E) compound 4m.
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Ab aggregation which may be
due to either steric blockage of
ligand entry into the active site,
or inducing
a conformational
change in the catalytic triad.
SAR of benzofuran–chalcone hy-
brids against Ab aggregation
The preceding molecular model-
ing studies that were performed
to gain insight into the binding
mode revealed that both benzo-
furan and chalcone moieties are
essential to show similar p—p
stacking interactions to fit into
the active site of TcAChE, which
stabilizes E2020. In terms of the
structure–activity
relationship
(SAR) of the hybrids, decreased
Ab peptide aggregation was
found to depend on both the
substituent patterns on the
1
phenyl ring of chalcones (R ) and
benzofuran (R). In general, on
Figure 12. A) Superimposed co-crystallized (magenta) and docked conformation (blue) of E2020 in the active site
1
the chalcone phenyl ring (R ), of TcAChE (grey). An ensemble of docked conformations, each for compounds B) 4 f, C) 4i, and D) 4m are shown.
electron-donating groups (such Only polar hydrogen atoms of TcAChE are shown; other hydrogen atoms are omitted for clarity. Hydrogen bonds
are indicated by blue solid lines.
as methoxy or methyl) seem to
enhance activity. Replacement of
the phenyl ring with smaller het-
erocyclic rings (thiophene, as in
4
l and 4m) did not influence ac-
tivity, suggesting that they share
or have overlapping binding
sites. On the other hand, the
substitution on the phenyl ring
of benzofuran (R) with a halogen
(Cl; in 3a and 4d) increases the
inhibitory potency over that of
unsubstituted benzofuran hy-
brids (4h and 4k). A summary of
the SAR is depicted in Figure 13.
Figure 13. SAR of the synthesized hybrids against Ab aggregation.
Experimental Section
Chemistry
Conclusions
General: All reagents were obtained commercially and were used
without further purification. Chromatography was carried out on
silica gel (60–120 and 100–200 mesh). All reactions were monitored
by thin-layer chromatography (TLC); silica gel plates with fluores-
cence F254 were used. Melting points were taken in open capillaries
on a Complab melting point apparatus and are uncorrected. IR
spectra were recorded on a PerkinElmer FT-IR RXI spectrophotome-
In summary, novel benzofuran–chalcone hybrids were synthe-
sized and characterized as multifunctional agents against AD.
Among the synthesized compounds, 4 f, 4i, and 4m signifi-
cantly decreased disease effects in a transgenic C. elegans
model of AD. The compounds exerted their beneficial effects
by reducing Ab aggregation, decreasing oxidative stress (4 f
and 4i), increasing ACh levels, and decreasing lipid deposition,
thereby preventing cholinergic neuronal degeneration. These
properties highlight the therapeutic potential of these novel
prototypes to be developed as new multifunctional drugs in
the treatment of Alzheimer’s disease.
1
13
ter. H NMR and C NMR spectra were recorded using a Bruker Su-
percon Magnet DRX-300 spectrometer (operating at 300 MHz for
1
13
H NMR and 75 MHz for C NMR), with CDCl as solvent and tetra-
3
methylsilane (TMS) as internal standard. Chemical shifts (d) are re-
ported in parts per million. ESIMS data were recorded on a Thermo
Lcq Advantage Max-IT. HRMS data were collected on a 6520 Agi-
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À1
1
lent Q ToF LC–MS/MS instrument (Accurate mass). HPLC analyses
for purity (>95% area) of synthesized compounds were performed
on a Shimadzu instrument equipped with a Shimadzu ODS (C ) re-
1595, 1550, 1285, 768 cm
;
H NMR (CDCl , 300 MHz): d=8.06–
3
8.01 (m, 4H), 7.85 (d, J=15.6 Hz, 1H), 7.77 (s, 1H), 7.61 (s, 1H),
7.57–7.50 (m, 4H), 6.98 (d, J=8.85 Hz, 2H), 3.88 (s, 3H), 2.63 ppm
(s, 3H); C NMR (CDCl , 75 MHz): d=188.5, 182.7, 163.6, 156.4,
18
13
versed-phase column (4.6 mmꢄ250 mm, 2 mm). The mobile phase
3
was a mixture of iPrOH and CH CN. The purity of the compounds
152.9, 143.7, 139.7, 135.4, 131.8, 131.2, 131.0, 130.9, 129.0, 128.6,
3
was determined by integrating peak areas of the liquid chromato-
127.1, 123.6, 121.8, 121.7, 116.6, 114.0, 55.6, 15.3 ppm; ESIMS (m/z):
431 [M+H] ; HRMS m/z calcd for C H ClO [M+H] 431.1050,
26 19 4
found 431.1036.
+
+
gram, monitored at l 280 and 325 nm. Parameters were as follows:
À1
flow rate: 0.4 mLmin ; gradient system, from 60% iPrOH and 40%
CH CN to 90% iPrOH and 10% CH CN. The solvent ratio was main-
tained for 30 min. All final compounds were found to have >95%
3
3
(E)-3-(2-(4-Chlorobenzoyl)-7-methylbenzofuran-5-yl)-1-(4-chloro-
phenyl)prop-2-en-1-one (4b): This compound was synthesized by
the method described for compound 4a, employing 2-(4-chloro-
purity.
2
-(4-Chlorobenzoyl)-7-methylbenzofuran-5-carbaldehyde (3a):
To a mixture of 4-hydroxy-5-methylisophthalaldehyde (2) (1.0 g,
.09 mmol), 4-chlorophenacylbromide (1.6 g, 7.31 mmol) and K CO3
benzoyl)-7-methylbenzofuran-5-carbaldehyde
3.35 mmol), 4-chloroacetophenone (0.6 g, 4.02 mmol) and 10%
methanolic KOH (15 mL) to afford compound 4b. Pale-yellow
(3a)
(1.0 g,
6
2
(
0.8 g, 6.08 mmol), CH CN (20 mL) was added. The reaction mixture
solid; yield: 65%, 0.94 g; mp: 156–1578C; n˜
1700, 1662, 1570, 1530, 1274, 756 cm ; H NMR (CDCl , 300 MHz):
(KBr)=3015, 2935,
3
max
À1
1
was held at reflux for 3 h. After completion of the reaction, K CO3
2
3
was removed in a sintered funnel, and the filtrate was concentrat-
ed under vacuum and purified by column chromatography with
EtOAc/hexane (1:9) to give pure 3a as a pale-yellow solid. Yield:
d=8.06–7.98 (m, 4H), 7.90 (d, J=15.5 Hz, 1H), 7.81 (s, 1H), 7.63 (s,
13
1H), 7.56–7.47 (m, 6H), 2.65 (s, 3H); C NMR (CDCl , 75 MHz): d=
3
188.7, 182.4, 144.8, 139.5, 139.1, 136.3, 131.0, 130.7, 129.7, 128.8,
8
1
1
8
1
1
5%, 1.54 g; mp: 168–1698C; n˜
(KBr)=3020, 1691, 1646, 1600,
128.3, 126.9, 123.5, 121.9, 120.9, 116.2, 15.0 ppm; ESIMS (m/z): 435
[M+H] ; HRMS m/z calcd for C H Cl O [M+H] 435.0555, found
25 16 2 3
435.0560.
max
À1
1
+
+
217, 777 cm ; H NMR (CDCl , 300 MHz): d=10.04 (s, 1H), 8.09 (s,
3
H), 8.03 (d, J=8.6 Hz, 2H), 7.86 (s, 1H), 7.63 (s, 1H), 7.53 (d, J=
13
.6 Hz, 2H), 2.65 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=191.4,
3
(E)-3-(2-(4-Chlorobenzoyl)-7-methylbenzofuran-5-yl)-1-phenyl-
82.5, 158.2, 153.3, 139.8, 135.0, 133.4, 131.0, 129.1, 128.9, 126.8,
+
prop-2-en-1-one (4c): This compound was synthesized by the
method described for compound 4a, employing 2-(4-chlorobenzo-
yl)-7-methylbenzofuran-5-carbaldehyde (3a) (1.0 g, 3.35 mmol),
acetophenone (0.4 g, 4.02 mmol) and 10% methanolic KOH
(15 mL) to afford compound 4c. Yellow solid; yield: 70%, 0.93 g;
24.9, 124.1, 116.7, 15.2 ppm; ESIMS (m/z): 299 [M+H] ; HRMS m/z
+
calcd for C H ClO [M+H] 299.0475, found 299.0461.
1
7
11
3
7
-Methyl-2-(4-methylbenzoyl)benzofuran-5-carbaldehyde (3b):
This compound was synthesized by the method described for
compound 3a, employing compound 2 (1.0 g, 6.09 mmol), 4-meth-
ylphenacylbromide (1.5 g, 7.31 mmol), and K CO (0.8 g, 6.0 mmol)
mp: 179–1808C; n˜
(KBr)=3030, 2925, 1730, 1613, 1561, 1548,
max
À1
1
1253, 770 cm
; H NMR (CDCl , 300 MHz): d=8.05–8.02 (m, 4H),
3
2
3
to afford compound 3b. Pale-yellow solid; yield: 80%, 1.35 g; mp:
7.88 (d, J=15.6 Hz, 1H), 7.79 (s, 1H), 7.62 (s, 1H), 7.59–7.48 (m,
À1
13
1
65–1668C; n˜ max (KBr)=3043, 1684, 1659, 1629, 1204, 755 cm
;
7H), 2.63 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=190.3, 182.6,
3
1
H NMR (CDCl , 300 MHz): d=10.05 (s, 1H), 8.08 (s, 1H), 7.99 (d,
156.4, 152.9, 144.6, 139.7, 138.3, 135.3, 132.9, 131.5, 131.0, 129.0,
128.7, 128.7, 128.6, 127.1, 123.6, 122.0, 121.8, 116.6, 15.3 ppm;
3
2
H, J=8.1 Hz), 7.84 (s, 1H), 7.60 (s, 1H), 7.35 (d, J=8.1 Hz, 2H),
13
+
+
2
1
.66 (s, 3H), 2.47 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=191.4,
ESIMS (m/z): 401 [M+H] ; HRMS m/z calcd for C H ClO [M+H]
25 17 3
3
83.5, 158.2, 153.8, 144.3, 134.2, 133.3, 128.7, 126.9, 124.8, 124.1,
401.0944, found 401.0920.
+
116.3, 21.8, 15.2 ppm; ESIMS (m/z): 279 [M+H] ; HRMS m/z calcd
+
(E)-3-(2-(4-Chlorobenzoyl)-7-methylbenzofuran-5-yl)-1-p-tolyl-
for C H O [M+H] 279.1021, found 279.1017.
1
8
14
3
prop-2-en-1-one (4d): This compound was synthesized by the
method described for compound 4a, employing 2-(4-chlorobenzo-
yl)-7-methylbenzofuran-5-carbaldehyde (3a) (1.0 g, 3.35 mmol), 4-
methylacetophenone (0.5 g, 3.95 mmol) and 10% methanolic KOH
(15 mL) to afford compound 4d. Pale-yellow solid; yield: 75%,
2
-Benzoyl-7-methylbenzofuran-5-carbaldehyde (3c): This com-
pound was synthesized by the method described for compound
a, employing compound 2 (1.0 g, 6.09 mmol), phenacyl bromide
1.4 g, 7.28 mmol) and K CO (0.8 g, 6.0 mmol) to afford compound
3
(
2
3
3
c. White solid; yield: 89%, 1.43 g; mp: 163–1648C; n˜
(KBr)=
1.03 g; mp: 160–1618C; n˜ (KBr)=3043, 2912, 1670, 1580, 1532,
max
max
À1
1
À1
1
3
028, 1677, 1665, 1615, 1229, 768 cm ; H NMR (CDCl , 300 MHz):
1215, 1246, 751 cm ; H NMR (CDCl , 300 MHz): d=8.04 (d, J=
3
3
d=10.06 (s, 1H), 8.10–8.05 (m, 3H), 7.86 (s, 1H), 7.69–7.54 (m, 4H),
8.64 Hz, 2H), 7.96 (d, J=8.0 Hz, 2H), 7.88 (d, 1H, J=15.6 Hz), 7.80
(s, 1H), 7.63 (s, 1H), 7.57–7.51 (m, 4H), 7.31 (d, J=8.0 Hz, 2H), 2.64
1
3
2
1
.68 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=191.4, 183.9, 158.3,
3
13
53.6, 136.9, 133.4, 133.3, 129.5, 128.9, 128.7, 126.9, 124.9, 124.2,
(s, 3H), 2.44 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=189.7, 182.6,
3
+
116.8, 15.2 ppm; ESIMS (m/z): 265 [M+H] ; HRMS m/z calcd for
156.4, 152.9, 144.1, 143.8, 139.7, 135.7, 135.3, 131.6, 131.0, 129.4,
+
C H O [M+H] 265.0865, found 265.0855.
129.0, 128.7, 128.6, 127.1, 123.6, 127.1, 123.6, 121.9, 121.8, 116.6,
1
7
12
3
+
2
1.7, 15.3 ppm; ESIMS (m/z): 415 [M+H] ; HRMS m/z calcd for
(
E)-13-(2-(4-Chlorobenzoyl)-7-methylbenzofuran-5-yl)-1-(4-me-
thoxyphenyl)prop-2-en-1-one (4a): To a mixture of 2-(4-chloro-
benzoyl)-7-methylbenzofuran-5-carbaldehyde (3a) (1.0 g,
.35 mmol) and 4-methoxyacetophenone (0.6 g, 4.0 mmol), 10%
+
C H ClO [M+H] 415.1101, found 415.1116.
26
19
3
(E)-3-(7-Methyl-2-(4-methylbenzoyl)benzofuran-5-yl)-1-p-tolyl-
prop-2-en-1-one (4e): This compound was synthesized by the
method described for compound 4a, employing 7-methyl-2-(4-
methylbenzoyl)benzofuran-5-carbaldehyde (3b) (1.0 g, 3.59 mmol),
4-methylacetophenone (0.5 g, 4.25 mmol) and 10% methanolic
KOH (15 mL) to afford compound 4e. Pale-yellow solid; yield: 68%,
3
methanolic KOH (15 mL) was added. The reaction mixture was
stirred at room temperature for 3 h. After completion of the reac-
tion (TLC monitoring) the reaction mixture was poured into 10%
dilute HCl solution (15 mL) and extracted with CHCl , dried over
3
Na SO ; removal of the solvent afforded crude compound. The
crude product was purified by column chromatography with
0.95 g; mp: 137–1388C; n˜ (KBr)=3038, 2942, 1689, 1598, 1549,
2
4
max
À1
1
716 cm ; H NMR (CDCl , 300 MHz): d=8.01–7.96 (m, 4H), 7.89 (d,
3
EtOAc/hexane (2:8) to provide pure 4a. Pale-yellow solid; yield:
J=15.6 Hz, 1H), 7.80 (s, 1H), 7.63 (s, 1H), 7.58–7.53 (m, 2H), 7.37–
7.31 (m, 4H), 2.66 (s, 3H), 2.48–2.45 ppm (m, 6H); C NMR (CDCl3,
13
6
9%, 0.99 g; mp: 178–1798C; n˜ max (KBr)=3021, 2927, 1705, 1645,
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7
1
1
5 MHz): d=189.8, 183.8, 156.3, 153.3, 144.3, 144.1, 143.7, 135.8,
(E)-3-(2-(4-Chlorobenzoyl)-7-methylbenzofuran-5-yl)-1-(3,4,5-tri-
methoxyphenyl)prop-2-en-1-one (4j): This compound was synthe-
sized by the method described for compound 4a, employing 2-(4-
chlorobenzoyl)-7-methylbenzofuran-5-carbaldehyde (3a) (1.0 g,
34.5, 131.4, 129.8, 129.5, 129.4, 128.7, 128.3, 127.3, 123.6, 121.9,
+
21.7, 116.2, 21.9, 21.8, 15.3 ppm; ESIMS (m/z): 395 [M+H] ; HRMS
+
m/z calcd for C H O [M+H] 395.1647, found 395.1638.
2
7
22
3
3
.35 mmol), 3,4,5-trimethoxyacetophenone (0.8 g, 4.02 mmol) and
10% methanolic KOH (15 mL) to afford compound 4j. Yellow solid;
yield: 66%, 1.08 g; mp: 140–1418C; n˜ (KBr)=3028, 2936, 1663,
(E)-3-(7-Methyl-2-(4-methylbenzoyl)benzofuran-5-yl)-1-phenyl-
prop-2-en-1-one (4 f): This compound was synthesized by the
method described for compound 4a, employing 7-methyl-2-(4-
methylbenzoyl)benzofuran-5-carbaldehyde (3b) (1.0 g, 3.59 mmol),
acetophenone (0.5 g, 4.20 mmol) and 10% methanolic KOH
max
À1
1
1566, 1218, 701 cm ; H NMR (CDCl , 300 MHz): d=8.06–8.03 (m,
3
2H), 7.90 (d, J=15.42 Hz, 1H), 7.82 (s, 1H), 7.63 (s, 1H), 7.57–7.46
(m, 4H), 7.30–7.24 (m, 2H), 3.97–3.95 (m, 9H), 2.66 ppm (s, 3H);
13
(
15 mL) to afford compound 4 f. Yellow solid; yield: 73%, 0.99 g;
C NMR (CDCl , 75 MHz): d=188.5, 184.2, 163.5, 156.3, 152.9,
3
mp: 170–1718C; n˜ max (KBr)=3012, 2938, 1645, 1588, 1527,
143.8, 138.4, 137.1, 133.1, 133.0, 131.5, 131.4, 131.1, 130.9, 129.5,
128.7, 128.4, 127.1, 123.6, 122.0, 121.3, 116.9, 113.9, 55.6, 15.4 ppm;
ESIMS (m/z): 491 [M+H] ; HRMS m/z calcd for C H ClO [M+H]
28 23 6
À1
1
7
85 cm ; H NMR (CDCl , 300 MHz): d=8.06–7.98 (m, 4H), 7.89 (d,
3
+
+
J=15.6 Hz, 1H), 7.80 (s, 1H), 7.62–7.49 (m, 6H), 7.35 (d, J=7.9 Hz,
1
3
2
1
1
2
H), 2.65 (s, 3H), 2.47 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=
491.1261, found 491.1264.
3
90.3, 183.7, 156.4, 153.4, 144.8, 144.1, 138.3, 134.5, 132.9, 131.3,
29.8, 129.4, 128.7, 128.6, 128.3, 127.3, 123.6, 121.9, 121.6, 116.2,
1.8, 15.3 ppm; ESIMS (m/z): 381 [M+H] ; HRMS m/z calcd for
+
(E)-3-(2-Benzoyl-7-methylbenzofuran-5-yl)-1-(4-chlorophenyl)-
+
prop-2-en-1-one (4k): This compound was synthesized by the
method described for compound 4a, employing 2-benzoyl-7-meth-
ylbenzofuran-5-carbaldehyde (3c) (1.0 g, 3.78 mmol), 4-chloroace-
tophenone (0.6 g, 4.54 mmol) and 10% methanolic KOH (15 mL) to
afford compound 4k. Pale-yellow solid; yield: 67%, 1.08 g; mp:
C H O [M+H] 381.1491, found 381.1479.
2
6
20
3
(E)-1-(4-Methoxyphenyl)-3-(7-methyl-2-(4-methylbenzoyl)benzo-
furan-5-yl)prop-2-en-1-one (4g): This compound was synthesized
by the method described for compound 4a, employing 7-methyl-
1
7
75–1768C; n˜ max (KBr)=3032, 2943, 1736, 1676, 1566, 1218,
2
3
-(4-methylbenzoyl)benzofuran-5-carbaldehyde
.59 mmol), 4-methoxylacetophenone (0.6 g, 4.31 mmol) and 10%
(3b)
(1.0 g,
À1
1
48 cm ; H NMR (CDCl , 300 MHz): d=8.07–8.04 (m, 2H), 7.98 (d,
3
J=8.5 Hz, 2H), 7.89 (d, J=15.6 Hz, 1H), 7.79 (s, 1H), 7.67–7.61 (m,
methanolic KOH (15 mL) to afford compound 4g. Pale-yellow solid,
yield: 65%, 0.95 g; mp: 190–1918C; n˜ (KBr)=3036, 2948, 1600,
13
2
H), 7.57–7.46 (m, 6H), 2.65 ppm (s, 3H); C NMR (CDCl , 75 MHz):
3
max
À1
1
d=189.0, 184.1, 156.5, 153.2, 145.2, 139.3, 137.2, 136.6, 133.2,
1
2
7
8
209, 720 cm ; H NMR (CDCl , 300 MHz): d=8.03 (d, J=8.7 Hz,
3
1
1
31.2, 130.0, 129.6, 128.7, 128.5, 127.3, 123.8, 122.2, 121.1, 116.6,
H), 7.94 (d, 2H, J=8.0 Hz), 7.88 (d, 1H, J=15.0 Hz), 7.79 (s, 1H),
.62 (s, 1H), 7.58–7.52 (m, 2H), 7.35 (d, J=7.92 Hz, 2H), 6.99 (d, J=
.7 Hz, 2H), 3.90 (s, 3H), 2.65 (s, 3H), 2.47 ppm (s, 3H); C NMR
+
5.3 ppm; ESIMS (m/z): 401 [M+H] ; HRMS m/z calcd for
+
13
C H ClO [M+H] 401.0944, found 401.0936.
25
17
3
(
CDCl , 75 MHz): d=188.5, 183.8, 163.5, 156.3, 153.2, 144.1, 143.9,
3
1
1
34.5, 131.4, 131.1, 130.9, 129.7, 129.4, 128.3, 127.2, 123.5, 121.9,
(
E)-3-(7-Methyl-2-(4-methylbenzoyl)benzofuran-5-yl)-1-(thio-
+
21.3, 116.3, 113.9, 55.6, 21.8, 15.4 ppm; ESIMS (m/z): 411 [M+H] ;
phen-2-yl)prop-2-en-1-one (4l): This compound was synthesized
by the method described for compound 4a, employing 7-methyl-
+
HRMS m/z calcd for C H O [M+H] 411.1596, found 411.1576.
2
7
22
4
2
-(4-methylbenzoyl)benzofuran-5-carbaldehyde
(3b)
(1.0 g,
(
E)-3-(2-Benzoyl-7-methylbenzofuran-5-yl)-1-p-tolylprop-2-en-1-
3
.59 mmol), 4-acetylthiophene (0.5 g, 4.31 mmol) and 10% metha-
one (4h): This compound was synthesized by the method de-
scribed for compound 4a, employing 2-benzoyl-7-methylbenzofur-
an-5-carbaldehyde (3c) (1.0 g, 3.78 mmol), 4-methylacetophenone
nolic KOH (15 mL) to afford compound 4l. Pale-yellow solid; yield:
7
1
4
1
6%, 1.04 g; mp: 130–1318C; n˜
(KBr)=3012, 2921, 2925, 1710,
max
À1
1
627, 1210, 719 cm ; H NMR (CDCl , 300 MHz): d=8.00–7.89 (m,
3
(
0.6 g, 4.54 mmol) and 10% methanolic KOH (15 mL) to afford
H), 7.80 (s, 1H), 7.69 (dd, J=1.1, 4.9 Hz, 1H), 7.62 (s, 1H), 7.53 (s,
compound 4h. Yellow solid; yield: 68%, 0.97 g; mp: 168–1698C;
n˜ max (KBr)=3008, 2915, 1648, 1570, 1559, 736 cm ; H NMR (CDCl ,
3
1
7
H), 7.46–7.40 (m, 1H), 7.37–7.34 (m, 2H), 7.21–7.18 (m, 1H), 2.66
À1
1
13
3
(s, 3H), 2.47 ppm (s, 3H); C NMR (CDCl , 75 MHz): d=183.7, 181.9,
3
00 MHz): d=8.07–8.04 (m, 2H), 7.96 (d, J=8.0 Hz, 2H), 7.88 (d, J=
1
1
56.4, 153.3, 145.7, 144.0, 134.5, 134.0, 131.9, 131.1, 129.8, 129.4,
5.6 Hz, 1H), 7.79 (s, 1H), 7.67–7.62 (m, 2H), 7.57–7.51 (m, 4H),
28.3, 127.3, 123.6, 122.0, 121.2, 116.2, 21.8, 15.3; ESIMS (m/z): 387
13
.31 (d, J=8.0 Hz, 2H), 2.65 (s, 3H), 2.44 ppm (s, 3H); C NMR
+
+
[M+H] ; HRMS m/z calcd for C H O S [M+H] 387.1055, found
24 18 3
(
CDCl , 75 MHz): d=189.7, 184.1, 156.4, 153.1, 144.2, 143.7, 137.2,
3
3
87.1051.
1
1
35.7, 133.1, 131.5, 129.5, 129.4, 128.8, 128.7, 128.5, 127.2, 123.6,
+
21.9, 121.7, 116.7, 21.7, 15.3 ppm; ESIMS (m/z): 381 [M+H] ;
+
HRMS m/z calcd for C H O [M+H] 381.1491, found 381.1503.
(E)-3-(2-Benzoyl-7-methylbenzofuran-5-yl)-1-(thiophen-2-yl)prop-
-en-1-one (4m): This compound was synthesized by the method
2
6
20
3
2
(
E)-3-(2-Benzoyl-7-methylbenzofuran-5-yl)-1-phenylprop-2-en-1-
described for compound 4a, employing 2-benzoyl-7-methylbenzo-
furan-5-carbaldehyde (3c) (1.0 g, 3.78 mmol), 2-acetylthiophene
(0.5 g, 4.54 mmol) and 10% methanolic KOH (15 mL) to afford
one (4i): This compound was synthesized by the method de-
scribed for compound 4a, employing 2-benzoyl-7-methylbenzofur-
an-5-carbaldehyde (3c) (1.0 g, 3.78 mmol), acetophenone (0.6 g,
4
4
compound 4m. Pale-yellow solid; yield: 78%, 1.09 g; mp: 110–
À1
.54 mmol) and 10% methanolic KOH (15 mL) to afford compound
111 8C; n˜
(KBr)=3027, 2944, 2858, 1736, 1623, 1248, 742 cm
;
max
1
i. Pale-yellow solid; yield: 75%, 1.03 g; mp: 180–1818C; n˜
H NMR (CDCl , 300 MHz): d=8.08–8.04 (m, 2H), 7.96–7.89 (m, 2H),
max
3
À1
1
(
KBr)=3047, 2928, 1680, 1555, 1512, 758 cm
;
H NMR (CDCl3,
7.80 (s, 1H), 7.69 (dd, J=1.1, 4.9 Hz, 1H), 7.65–7.63 (m, 2H), 7.58–
3
1
00 MHz) d=8.08–8.03 (m, 4H), 7.90 (d, J=15.6 Hz, 1H), 7.80 (s,
7.53 (m, 3H), 7.43 (d, J=15.5 Hz, 1H), 7.21–7.18 (m, 1H), 2.66 ppm
13
13
H), 7.65–7.52 (m, 9H), 2.66 ppm (s, 3H); C NMR (CDCl , 75 MHz):
(s, 3H); C NMR (CDCl , 75 MHz): d=184.1, 181.9, 156.4, 153.0,
3
3
d=190.4, 184.2, 156.5, 153.0, 144.7, 138.2, 137.1, 133.1, 132.9,
31.3, 129.5, 128.8, 128.7, 128.6, 128.5, 127.2, 123.7, 122.1, 121.6,
145.6, 143.9, 137.1, 134.0, 133.1, 131.9, 131.1, 129.5, 128.7, 128.5,
1
128.3, 127.2, 123.7, 122.2, 121.1, 116.8, 15.3 ppm; ESIMS (m/z): 373
+
+
+
1
16.8, 15.4 ppm; ESIMS (m/z): 367 [M+H] ; HRMS m/z calcd for
[M+H] ; HRMS m/z calcd for C H O S [M+H] 373.0898, found
23 16 3
+
C H O [M+H] 367.1334, found 367.1339.
373.0896.
2
5
18
3
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Biological methods
Estimation of ACh and AChE activities with Amplexꢀ Red: We quanti-
fied the levels of ACh and AChE in wild-type worms treated with
compounds 4 f, 4i, and 4m. After treatment for 48 h, worms were
washed thrice with sodium phosphate buffer (PBS) to remove ad-
hering bacteria. Worms were homogenized by sonication on ice at
15 s intervals using 25% amplitude. Samples were then centrifuged
at 14000 rpm for 30 min to remove any insoluble residue. The su-
pernatant was assayed for ACh and AChE levels using the Amplexꢂ
Red ACh/AChE assay kit (Invitrogen Cat.# A12217) according to
the manufacturer’s instructions. Absorbance was measured in a mi-
croplate reader with an excitation wavelength of 544 nm and an
C. elegans strains and maintenance: Genetic model system C. ele-
gans was employed to study the biological effects of the test com-
pounds. Wild-type strain N2 (var. Bristol) and transgenic strains
CL4176 and CL2006 expressing human Ab, LX929 expressing GFP
in all cholinergic neurons, were employed for the studies. The
strains were obtained from the Caenorhabditis Genetics Center
(
University of Minnesota, USA). The culture and maintenance of
various C. elegans strains was carried out as per standard meth-
[
47]
ods. Briefly, the strains were cultured on bacterial lawns of OP50-
seeded nutrient growth medium (NGM) plates at 228C. Strain
CL4176 was maintained at 168C. NGM was prepared by adding
[51]
emission wavelength of 590 nm. Experiments were performed in
triplicates at two different times, and final fluorescence was nor-
malized with respect to protein content as quantified using the
Bradford method.
À1
À1
5
9
1
0 mm NaCl, 2.5 gL peptone (Sigma), 17 gL agar (Hi-media) in
75 mL double-distilled water and autoclaved for 30–40 min at
5 psi. After the cooling of media to 50–608C, 5 mgmL cholester-
À1
ol solution (Sigma) prepared in ethanol, 1 mm CaCl , 1 mm MgSO ,
2
4
Estimation of oxidative stress: We carried out 2,7-dichlorodihydro-
and 25 mm KH PO were added. On the day of initiation of treat-
2
4
fluorescein diacetate (H DCFDA) (Invitrogen Cat.# D399) assays to
2
ment, worms were synchronized by hypochlorite bleaching
[52]
determine the levels of ROS using standard protocols.
After
[48]
method for the isolation of embryos.
treatment for 48 h, control and treated worms of CL2006 strains
were washed thrice with M9 buffer to remove bacteria. Approxi-
mately 100 worms per 100 mL of buffer were pipetted in four repli-
cates into the wells of a 96-well plate; then 100 mL of 100 mm
Treatment of worms with test compounds: Test compounds were ini-
tially dissolved in ethanol to prepare a stock of 20 mm. A working
concentration of 2 mm was then prepared from the stock in OP50,
followed by seeding it onto NGM plates and incubated overnight
at 378C. Embryos of worms were transferred onto OP50–test-com-
pound-seeded plates and grown for 48 h.
H DCFDA was added to each well, and basal fluorescence was
2
quantified immediately after addition of dye with an excitation
wavelength of 485 nm and an emission wavelength of 520 nm.
Plates were incubated for 1 h and then a second measurement
was quantified. The initial fluorescence was subtracted from the
second reading, and the fluorescence per 100 worms was calculat-
ed by dividing the delta by number of worms. The experiment was
repeated twice and the average was considered.
Analysis of Ab aggregation: Ab-induced paralysis assays were car-
[49]
ried out as per a published method with slight modifications. In
this study, we used temperature-inducible Ab-expressing transgen-
ic CL4176 strains of C. elegans. The strain expresses Ab specifically
in muscles, and increased Ab expression leads to paralysis. This
gives an easy endpoint to carry out large scale analysis of com-
pounds for their effect against Ab aggregation. Age-synchronized
CL4176 worms were grown on NGM plates seeded with OP50 and
premixed with test compounds for 48 h at 168C. After 48 h the
temperature was increased to 258C for another 36 h to induce ex-
pression of Ab, leading to paralysis. The scoring of paralysis was
carried out by comparing each test-compound-treated group with
control. Paralysis phenotype was counted for ~50 worms per treat-
ment condition. The assays were repeated thrice, and the data for
each compound were statistically analyzed and plotted against
control values.
Analysis of cholinergic neurodegeneration: Cholinergic neurodegen-
eration was induced by treatment of worms with an organophos-
phate insecticide chlorpyrifos (CP; Riedel–de Haꢅn Cat.# 45395). It
was mixed with OP50 at 100 ppm concentration and seeded onto
NGM plates and incubated overnight at 378C. Initially, we em-
ployed four different concentrations of CP: 1, 10, 100, and
1
000 ppm in order to determine the concentration that induces
cholinergic neurodegeneration without causing mortality in
worms. Age-synchronized worms of LX929 were then grown on
normal diet or treatment plates for 48 h at 228C. After this, worms
were washed thrice using M9 buffer followed by mounting the
worms onto a 2% agar-padded glass slide using 100 mm NaN3
(
Sigma Cat.# 71289). Imaging of live (anesthetized) worms was car-
Aldicarb assay for effect on excitatory neurotransmission: The com-
pounds exhibiting a positive effect in the Ab aggregation assay
were further studied for their effect on availability of excitatory
neurotransmitter ACh, which holds immense significance in the
treatment of AD. The aldicarb assay was carried out as described
ried out to monitor the cholinergic neurodegeneration in control
and experimental conditions using a fluorescence upright micro-
scope (Nikon). Ten subjects were analyzed per group, and the ex-
periment was repeated twice. Fluorescence intensity was quanti-
fied using Image J software (Image J, US National Institutes of
Health (NIH), Bethesda, MD, USA).
[50]
previously. In brief, 1m stock solution of aldicarb (Fluka Cat.#
3386) was prepared in 70% ethanol. NGM–aldicarb plates were
prepared at least one day before to get a final concentration of
mm aldicarb in NGM and stored at 48C until use. Embryos of
3
1
Quantification of lipid content: Nile red staining of lipid deposits
[47]
wild-type (N2) worms were transferred onto OP50-seeded control
plates and test-compound-seeded NGM plates at 228C. After 48 h
treatment, the control or treated worms were washed off their
parent plates to remove the adhering bacteria and transferred
onto the aldicarb assay plates (20–30 worms per plate). The worms
were poked with a poking lash (prepared by fixing an eye lash at
the tip of a microtip). The paralysis phenotype of worms was
scored at a fixed time point, and the percentage of worms para-
lyzed under treated conditions was normalized against control
values. Assays were repeated thrice.
was performed as described previously. This lipid-specific Nile
red dye (Invitrogen Cat.# N1142) was mixed with E. coli OP50 at
a ratio of 1:250. The synchronously aged embryos of CL2006 de-
rived from axenizing by hypochlorite treatment, were fed onto
these Nile-red-containing treatment plates for 48 h at 228C. Worms
were washed off and mounted using NaN on a glass slide with
3
a cover slip. Imaging of ten live (immobilized) worms was carried
out using a fluorescence upright microscope (Nikon). Images of
Nile red staining were further quantified using Image J software
(Image J, NIH) by measuring florescence intensity.
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Mitotracker staining for mitochondrial content: Mitotracker deep red
Crystal structure data for compound 4d
6
33 stain solution (Invitrogen Cat.#: M-22426) was prepared in
CCDC-877579 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
OP50 to reach a concentration of 100 nm. OP50–stain solution was
seeded onto NGM plates and incubated overnight at 378C. Em-
bryos of N2 and CL2006 worms were transferred onto the NGM–
OP50 stain solution and grown for 48 h at 228C. Worms were
washed from plates, cleared off any adhering bacteria and immobi-
lized in 100 mm NaN on a glass slide and analyzed at room tem-
3
Abbreviations
perature at a laser excitation wavelength of 637 nm and an emis-
[53]
sion wavelength of 660 nm (confocal microscope, Carl Zeiss). We
analyzed 10 worms per group, and experiments were repeated
twice. Images were further quantified by measuring florescence in-
tensity using Image J Software (Image J, NIH).
Ab: b-amyloid peptide; ACh: acetylcholine; AChE: acetylcholines-
terase; AD: Alzheimer’s disease; CNS: central nervous system; CP:
chlorpyrifos; ESIMS: electrospray ionization mass spectrometry;
HMTA: hexamethylenetetraamine; HPLC: high-performance liquid
chromatography; HRMS: high-resolution mass spectrometry;
H DCFDA: 2,7-dichlorodihydrofluorescein diacetate; MTDL: multi-
Trypan-blue-based dye exclusion test for studies on tissue/cell
damage in C. elegans: In this assay, worms treated with different
compounds and Paraquat (PQ; positive control) were studied. In
PQ treatment, age-synchronized worms were grown onto the PQ–
OP50-seeded NGM plates at 4 mm, and the staining was performed
2
target-directed ligand; NGM: nutrient growth medium; PS-1: prese-
nilin-1; ROS: reactive oxygen species; SAR: structure–activity rela-
tionship; TFA: trifluoroacetic acid; TLC: thin-layer chromatography;
TMS: tetramethylsilane.
[54]
as described previously with slight modifications. After treatment
for 48 h, worms were washed thrice using M9 buffer to remove
Acknowledgements
any adhering bacteria and transferred onto a watch glass, wherein
À1
3
00 mL Trypan blue stain (14 mgmL ; Sigma Cat.# T6146-5g) was
added. The samples were incubated for 20 min with continuous
shaking on an orbital shaker. Worms were washed thrice with M9
buffer at 3000 rpm for 2 min and then kept in the shaker for 5 min
for de-staining in M9. Worms were finally mounted on agar-
padded slides and observed (10 worms per treatment condition)
for staining pattern under a microscope (Nikon). Each experiment
was carried out in triplicate.
The instrumentation facilities at the Sophisticated Analytical In-
strument Facility (SAIF) of the Central Drug Research Institute
(
CDRI) are gratefully acknowledged. M.K., R.K.M., and P.J. are
thankful to the Council of Scientific and Industrial Research
CSIR), New Delhi (India) for financial support. Ram Jeet (CSIR-
(
CDRI) is acknowledged for HPLC data, and Suriya P. Singh (CSIR-
CDRI) is acknowledged for technical support. We are grateful to
Dr. S. K. Puri (Director, CSIR-CDRI) for funding and for his con-
stant encouragement in the drug discovery program. Nematode
strains used in this work were provided by the C. elegans Genetics
Center (CGC), University of Minnesota, MN (USA), which is funded
by the US National Institutes of Health National Center for Re-
search Resources (NIH–NCRR). This work is part 31 in the series,
Thioflavin S staining of Ab: For thioflavin S staining, individual trans-
genic animals from treatment plates were washed thrice using M9
buffer and were fixed in 4% paraformaldehyde in PBS overnight at
4
8C. Afterward, worms were freeze-fractured by placing worms be-
tween two microscope slides, and quick-frozen at À458C. The
slides were cracked off from one another. Worms were then per-
meabilized by incubation in 200 mL permeabilization solution (5%
fresh b-mercaptoethanol, 1% Triton X-100, 125 mm Tris, pH 7.4) for
“Advances in drug design and discovery”; this is CSIR-CDRI com-
munication number 8772.
4
(
0
8 h at 378C. Permeabilized worms were finally washed with PBST
0.1% Triton in PBS) and subjected to staining by transfer to
.125% thioflavin S (Sigma) in 50% ethanol for 2 min, de-stained
Keywords: Alzheimer’s disease · benzofurans · beta-amyloid ·
Caenorhabditis elegans · chalcones
for 2 min in 50% ethanol, followed by washing twice in 50% etha-
nol and mounted on a microscope slide with 50% glycerol. Ab
fluorescence images were acquired using a 40ꢄ objective (Carl
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Received: July 16, 2014
Published online on && &&, 0000
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 15
&14
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
See elegant conjugates! To arrest multi-
faceted Alzheimer’s disease, a series of
multifunctional benzofuran–chalcone
hybrids were synthesized and bio-evalu-
ated using transgenic C. elegans. These
hybrid compounds potently reduced
the aggregation of b-amyloid peptide,
increased the acetylcholine levels, and
provided protection against neuro-
degeneration.
K. V. Sashidhara,* R. K. Modukuri,
P. Jadiya, R. P. Dodda, M. Kumar,
B. Sridhar, V. Kumar, R. Haque,
M. I. Siddiqi, A. Nazir*
&& – &&
Benzofuran–Chalcone Hybrids as
Potential Multifunctional Agents
against Alzheimer’s Disease: Synthesis
and in vivo Studies with Transgenic
Caenorhabditis elegans
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 15
&15
&
ÞÞ
These are not the final page numbers!