Effects of structural modifications on the metal binding, anti-amyloid activity, and cholinesterase inhibitory activity of chalcones

Article abstract of DOI:10.1039/c5ob01478f

As the number of individuals affected with Alzheimer's disease (AD) increases and the availability of drugs for AD treatment remains limited, the need to develop effective therapeutics for AD becomes more and more pressing. Strategies currently pursued include inhibiting acetylcholinesterase (AChE) and targeting amyloid-β (Aβ) peptides and metal-Aβ complexes. This work presents the design, synthesis, and biochemical evaluation of a series of chalcones, and assesses the relationship between their structures and their ability to bind metal ions and/or Aβ species, and inhibit AChE/BChE activity. Several chalcones were found to exhibit potent disaggregation of pre-formed N-biotinyl Aβ_1-42 (bioAβ₄₂) aggregates in vitro in the absence and presence of Cu²⁺/Zn²⁺, while others were effective at inhibiting the action of AChE.

Full text of DOI:10.1039/c5ob01478f

Organic &
Biomolecular Chemistry
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Effects of structural modifications on the metal
binding, anti-amyloid activity, and cholinesterase
inhibitory activity of chalcones†
Cite this: DOI: 10.1039/c5ob01478f
a
b
a
a
Marina Y. Fosso, Harry LeVine 3rd, Keith D. Green, Oleg V. Tsodikov and
a
Sylvie Garneau-Tsodikova*
As the number of individuals affected with Alzheimer’s disease (AD) increases and the availability of drugs
for AD treatment remains limited, the need to develop effective therapeutics for AD becomes more and
more pressing. Strategies currently pursued include inhibiting acetylcholinesterase (AChE) and targeting
amyloid-β (Aβ) peptides and metal-Aβ complexes. This work presents the design, synthesis, and bio-
chemical evaluation of a series of chalcones, and assesses the relationship between their structures and
their ability to bind metal ions and/or Aβ species, and inhibit AChE/BChE activity. Several chalcones were
found to exhibit potent disaggregation of pre-formed N-biotinyl Aβ1-42 (bioAβ42) aggregates in vitro in the
Received 18th July 2015,
Accepted 31st July 2015
DOI: 10.1039/c5ob01478f
2+
2+
www.rsc.org/obc
absence and presence of Cu /Zn , while others were effective at inhibiting the action of AChE.
potential pharmacological targets for the development of AD
therapeutics.
Introduction
Alzheimer’s disease (AD) is a deadly progressive neurodegene-
rative disorder that manifests itself by a decline in cognitive monly investigated treatments for AD.
Acetylcholinesterase (AChE) inhibitors are among the com-
1
4–18
Four of the six
function and the loss of memory. It is the most common form drugs currently approved for treating AD (tacrine, donepezil,
of dementia and the sixth leading cause of death in the United rivastigmine, and galantamine) increase the amount of acetyl-
1
States, affecting one in nine elderly Americans. The socio- choline neurotransmitter in the brain by inhibiting the action
economic burden associated with AD is estimated to a stagger- of AChE. However, they are only modestly effective at alleviat-
2
,3
ing $172 billion per year in the USA. There are currently no ing AD symptoms. Aβ accumulation in the cerebral cortex has
1
9
medications that can cure AD or stop its progression; only a been suggested to be an early event in AD pathogenesis, and
few have been approved to provide temporary symptomatic attempts to prevent Aβ oligomerization or disrupt existing Aβ
4
–8
relief.
Further research efforts are thus needed to develop aggregates have attracted considerable efforts aiming at
1
0,20,21
AD therapeutics. Pathologically, AD is characterized by the slowing down the progression of AD.
deposition of amyloid β-peptide (Aβ)-rich plaques and the Cu and Zn , which concentrate in senile plaques,
Metal ions such as
2
+
2+
22,23
have
accumulation of hyperphosphorylated tau protein as neuro- been shown to interact with Aβ peptides and promote their
9
–13
24,25
fibrillary tangles in the brain of afflicted patients,
as well as assembly into toxic Aβ oligomers
as well as the formation
This suggests that small
2
6–28
the degeneration of neurons and synapses due to the increased of reactive oxygen species (ROS).
1
3
activity of cholinesterases. These hallmarks thus represent molecule metal chelators capable of targeting Aβ species and
inhibiting cholinesterases such as acetylcholinesterase (AChE)
and/or butyrylcholinesterase (BChE) could be valuable AD
therapeutics.
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of
Chalcones are 1,3-diaryl-2-propen-1-ones with a wide range
Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596, USA.
E-mail: sylviegtsodikova@uky.edu; Fax: +859-257-7585; Tel: +859-218-1686
29
of interesting biological activities. A large number of chal-
b
cones have been reported as Aβ-imaging tracers with high
Chalcones with tertiary amines in their
structures, especially the N,N-dimethyl amino group, have also
Department of Cellular and Molecular Biochemistry, Center on Aging, University of
3
0–32
Kentucky, 800 South Limestone Street, Lexington, KY 40536-0230, USA
Electronic supplementary information (ESI) available: Experimental procedures
brain uptake.
†
for the synthesis and characterization of all novel compounds generated along
33
been shown to exhibit substantial affinities for Aβ plaques.
2+
2+
with their NMR spectra and elemental analysis data, for Cu and Zn binding
Combining these observations into a single molecule, chal-
cone 3a (Fig. 1A), containing a metal chelation site and a di-
methylamino group, was synthesized. Chalcone 3a is capable
studies at various pH, for assays for bioAβ42 oligomer assembly and dissociation,
and for cholinesterase inhibition in the presence and absence of CuCl
ZnCl studies. See DOI: 10.1039/c5ob01478f
2
and
2
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and, therefore, any additional substitution was localized on
ring I. A methyl group or a bromine atom was thus attached at
various positions of ring I of the chalcones 3b–i (Fig. 1A) to
evaluate their effects on metal binding, Aβ modulating pro-
perties, and/or cholinesterase inhibitory capabilities. To
further investigate the necessity of a metal chelation site in the
modulation of metal-Aβ complexes, chalcones 6a–h and 7a, b,
and h (Fig. 1B), with a 2-acyl phenol and a 2-acyl aniline
moiety that can form intramolecular hydrogen bonds (Fig. 1C)
and prevent metal coordination, respectively, were prepared.
The target chalcones were synthesized according to the
known Claisen-Schmidt condensation (Fig. 1). 4-(Dimethyl-
amino)benzaldehyde, 2, was reacted with various 2-acetylpyri-
dines/acetophenones (1, 4, or 5) in the presence of a base
(
KOH or NaOH) in ethanol at room temperature. This afforded
the chalcones 3a–i, in yields ranging from 42 to 81%, and the
chalcones 6a–h, 7a–b, and h in 27 to 85%.
2
+
2+
Study of the metal (Cu and Zn ) binding properties of the
synthesized chalcones
2
+
Cu binding. The ability of 3a–i, 6a–h, 7a, b, and h to bind
2
+
Cu ions was investigated by UV-Visible absorption spec-
troscopy (Fig. 2). Upon addition of 1–5 equivalents of CuCl to
2
an ethanolic solution of chalcone 3a, a distinctive shift of the
optical band from ∼440 nm to ∼560 nm was observed,
2
+
suggesting Cu binding of 3a at the metal chelation site
Fig. 1 Synthetic scheme for the formation of A. chalcones 3a–i, and
formed by the nitrogen and the oxygen atoms from the
2+
B. chalcones 6a–h, 7a, b, and h. C. Representation of metal (M ) (note
34
2
+
2+
2-pyridyl ketone moiety (Fig. 1C), as previously reported.
either Cu or Zn ) chelation by the 2-pyridyl ketone moiety of 3a–i, as
well as intramolecular hydrogen bonding preventing metal chelation to
the 2-acyl phenol and 2-acyl aniline moieties of 6a–h and 7a, b, and h.
When a methyl group was added at the R position in 3b, the
1
optical band at 440 nm did not completely shift to 560 nm,
even in the presence of 5 equivalents of CuCl
2
, implying that
the ability to chelate Cu in this case was less efficient. This
of regulating both metal-free and metal-mediated Aβ aggrega- loss of chelation can be attributed to the steric hindrance
2+
3
4
tion. However, its ability to inhibit the action of cholinester- caused by the methyl group, which perturbs the metal chela-
ases has never been investigated.
tion site. This effect was confirmed when the methyl group
Herein, we present the design, synthesis, metal binding was moved further away from the metal chelation site, as in 3c
2
+
capabilities, and in vitro metal-free and metal-induced and 3d, and the Cu binding properties of these chalcones
reactivity towards Aβ and cholinesterase enzymes of a series were restored. Also, replacing R with the methyl group in 3e
of chalcones, allowing us to evaluate the structure–activity gave similar results as 3b, although with a less profound
4
2
+
relationship of chalcones capable of interacting with metal decrease in Cu interaction.
ions and/or Aβ peptides, and inhibiting cholinesterases.
Substitution of the methyl group by a bromine atom
2
+
weakened the Cu interaction of the chalcone in all cases. No
optical shift was observed for 3f and 3i, although incomplete
in the corresponding methyl counterparts 3b and 3e, respect-
Results and discussion
Integrated design and synthesis of chalcones
ively. Moreover, while it took 2 equivalents of CuCl
2
to 3c and
3
d to completely shift the optical band at 440 nm and thus
2
+
Chalcones 3a–i (Fig. 1A) were designed to contain a metal che- completely bind Cu ions, 3g and 3h required up to 5 equiva-
lation site and an Aβ-binding moiety. This was achieved via an lents of CuCl . These results stem from the electron-withdraw-
2
integrated approach, whereby a part of the known Aβ-interact- ing effect of the bromine atom. As a matter of fact, Br is an
ing chalcone scaffold was replaced by an appropriate feature, electronegative atom and it pulls electrons away from the nitro-
with minimal structural changes. The 2-pyridyl ketone moiety gen-donor atom on ring I, thus disrupting the metal chelation
(Fig. 1C) was chosen as the metal chelation site because it had site.
previously been installed in other Aβ self-assembly inhibitors
As expected, replacement of the 2-pyridyl ketone moiety in
3
4,35
with favourable results.
Moreover, installation of the N,N- chalcones 3a–i by a 2-acyl phenol (6a–h) or a 2-acyl aniline (7a,
2+
dimethylamino group at the para-position in ring II (Fig. 1A) b, and h) moiety resulted in a complete loss of Cu binding
3
1,33
has been shown to improve binding affinity to Aβ plaques,
property. Intramolecular hydrogen bonding in 6a–h, 7a, b, and
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2
+
Fig. 2 Cu binding studies by UV-Visible spectroscopy using 0, 1, 2, and 5 equivalents of CuCl
and h dissolved in EtOH at room temperature and incubated for 3 min.
2
with 20 μM of chalcones 3a–i, 6a–h, and 7a, b,
h (Fig. 1C) prevents the formation of the characteristic metal either by steric hindrance of bulky substituents (3b and 3e) or
chelation site observed in 3a–i, and hence their ability to inter- by decreasing the electron density with electron-withdrawing
2
+
2+
act with Cu . Overall, these results demonstrate Cu inter- substituents (3f and 3i), reduces or nullifies the metal binding
action of chalcones 3a–e, 3g and 3h, suggesting that the capability of chalcones.
2
+
2+
2
-pyridyl ketone moiety, or another scaffold capable of
Zn binding. We also investigated the Zn binding pro-
2
+
forming a metal chelation site, is essential for binding metal perties of 3a, 3d, 3h, 6a, and 7a. Although Cu binding was
ions. Furthermore, disruption of the metal chelation site, studied by UV-Visible spectroscopy, which enabled us to
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perform a quantitative analysis, no optical changes could oxygen atoms (Fig. 1C). Interaction of 3a, 3d, and 3h with
2
+
be observed with this assay when ZnCl was used. Therefore, Zn could also be reaffirmed by the color change of the NMR
2
1
2
the standard qualitative H NMR spectroscopy assay was per- sample from yellow to deep purple upon addition of ZnCl .
formed to study the Zn binding properties of our chalcones On the other hand, treatment of a CD
2
+
3
CN solution of 6a and
(
Fig. 3). Upon addition of 3 equivalents of ZnCl to a CD CN 7a with 3 equivalents of ZnCl2 did not exhibit any distinct
2
3
solution of 3a, 3d, and 3h, distinct downfield shifts of the chemical shift of protons (Fig. 3, panels D and E), suggesting
peaks corresponding to the pyridyl protons were observed. that 6a and 7a, with a 2-acyl phenol and a 2-acyl aniline
2
+
This implies that the nitrogen atom on ring I is involved in moiety, respectively, do not interact with Zn ions. This was
2
+
Zn binding. It is also noticeable that the peaks associated further confirmed as no discrete color change was noticeable
with protons e and f were displaced, suggesting that the upon addition of ZnCl to the CD CN solution of 6a and 7a.
oxygen atom might participate in metal chelation. The effect This is in agreement with the observations made in the pres-
2
3
2
+
of the involvement of the oxygen atom in the coordination of ence of Cu and reaffirms that the intramolecular hydrogen
metal ions was further extended to ring II as protons g and h bonding in 6a and 7a (Fig. 1C) prevents the formation of the
also underwent some chemical shifts (Fig. 3; panels A, B, and characteristic metal chelation site. In light of these results,
C). These results confirm that the 2-pyridyl ketone moiety chalcone 3a, with a 2-pyridyl ketone moiety, and any of its
bears a metal chelation site formed by the nitrogen and the derivatives bearing a substituent at a position that does not
2
+
1
Fig. 3 Zn binding studies of selected chalcones by H NMR spectroscopy in CD
3
CN at room temperature. NMR spectra of chalcone A. 3a without
(red, 12 mM), B. 3d without (black, 4 mM) and with ZnCl (red, 12 mM), C. 3h without (black, 4 mM) and with ZnCl
red, 12 mM), D. 6a without (black, 4 mM) and with ZnCl (red, 12 mM), and E. 7a without (black, 4 mM) and with ZnCl (red, 12 mM). Note: pictures
(
(
black, 4 mM) and with ZnCl
2
2
2
2
2
of NMR tubes for each chalcone without and with ZnCl
2
are presented above their respective spectra. The change in color from yellow to purple is
2+
observed for chalcones that bind Zn
.
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perturb the metal chelation site, such as 3d and 3h, are from 3a to 3d. Similarly, an electron-withdrawing group
2
+
2+
capable of binding Cu and Zn .
such as Br would decrease the pKa1 value, as it was observed
in chalcone 3h (pKa1 = 2.73 ± 0.04 and pKa2 = 5.89 ± 0.04).
These results suggest that 3a, 3d, and 3h will be present
Solution speciation studies
To further evaluate the metal ion interaction with our chal- as neutral species at physiological pH (7.4). On the other
cones, we performed UV-Visible variable-pH titration of chal- hand, the pK values of 6a and 7a were determined as follows:
cones 3a, 3d, 3h, 6a, and 7a in the absence and presence of 6a; pKa1 = 5.09 ± 0.07 and pKa2 = 10.38 ± 0.01; and 7a, pKa1
a
=
2
+
Cu . The acidity constants (pK s) of 3a, 3d, 3h, 6a, and 7a 4.93 ± 0.02 and pKa2 = 10.56 ± 0.04. In this case, the pKa1 value
a
were first determined from the solution speciation diagrams was relevant to the protonation of the N,N-dimethylamino
obtained from the corresponding UV-Visible variable-pH titra- nitrogen, while the pKa2 value was a measure of the ability to
2
+
tion spectra in the absence of Cu (Fig. 4). For chalcone 3a, protonate the hydroxyl oxygen and aniline nitrogen.
the pKa1 (protonation of the pyridyl nitrogen) was found to be
Once the acidity constants of chalcones 3a, 3d, 3h, 6a, and
.87 ± 0.03 and the pKa2 (protonation of the N,N-dimethyl- 7a were determined, we then performed their spectrophoto-
amino nitrogen) was 4.33 ± 0.03, which match previously metric titrations in the presence of CuCl (Fig. 5 and S53†).
2
2
3
4
reported values (pKa1 = 3.23 and pKa2 = 4.00). The solution While various differences were noticeable in the UV-visible
speciation diagram we obtained for 3a was also in agreement variable-pH titration spectra of 3a, 3d, and 3h obtained in the
3
4
with the literature, and indicated the presence of three absence (Fig. 4) and presence (Fig. 5) of CuCl
species in solution: neutral (S), monoprotonated (SH), and 6a and 7a were similar in both cases (Fig. 4 and S53†). For 3a,
diprotonated (SH
). For chalcone 3d, pKa1 = 3.11 ± 0.07 and we noticed a decrease in value of the maximum absorbance (at
pKa2 = 3.33 ± 0.05 were obtained. Indeed, as the methyl group 440 nm) from 0.49 to 0.40 in the presence of CuCl . A new
donates electrons, the basicity of the pyridyl ring increases peak appeared at 570 nm in the pH-titration spectrum of 3a in
from 3a to 3d, and thus the pKa1 value should also increase the presence of CuCl . Moreover, there was a subtle shift of the
, the spectra for
2
2
2
2
Fig. 4 Solution speciation studies of chalcones (S) 3a, 3d, 3h, 6a, and 7a. UV-visible variable-pH titration spectra (left) and solution speciation dia-
grams (right) of chalcones (S) 3a (20 μM), 3d (50 μM), 3h (50 μM), 6a (20 μM), and 7a (20 μM) at room temperature. Titrations were done from pH 12-2.
F
S
represents the fraction of species present in solution at the given pH. Acidity constants (pK
a
s) of chalcones 3a, 3d, 3h, 6a, and 7a are summarized
in the table at the bottom right.
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cones. For 3a, log β
1
= 4.63 and log β
2
= 4.66; for 3d, log β
1
=
4
5
.06 and log β = 4.17; and for 3h, log β = 5.10 and log β =
.13. The similarity in log β and log β values explains the fact
1 2
2
1
2
that protonation of the neutral chalcone species first occurs at
2
+
the N,N-dimethylamino nitrogen and should not prevent Cu
from binding at the proposed chelation site of the 2-pyridyl
ketone moiety. Based on these log β values, the stability of the
2
+
Cu -chalcone complex increased in the order 3d, 3a, and 3h,
although the variation was not substantial. We were also able
to plot the solution speciation diagrams based on these log β
2
+
values (Fig. 5, right columns) and found out that free Cu ,
SCu, and SHCu were present in all the chalcone solution. At
pH 7, the SCu complexes were present in solution at about
40%, 40%, and 75% for 3a, 3d, and 3h, respectively.
In vitro study of the effect of chalcones on Aβ assembly and
dissociation in the absence and presence of metal ions
The ability of 3a to regulate metal-free and metal-induced Aβ
aggregation and dissociation has previously been demon-
3
4
strated. In these studies, Aβ fibrils, which have been pro-
posed to be an important causal agent of AD, were employed.
However, soluble Aβ oligomeric species have recently been
3
6
shown to cause more potent neurotoxicity than fibrils. This
inspired us to evaluate the effects of our chalcones on Aβ oligo-
mers assembly and disaggregation (Fig. 6). Although Aβ1-40
and Aβ1-42 are the major forms of Aβ peptides, Aβ
is the
1-42
3
7,38
39–41
most abundant
and neurotoxic.
Aβ1-42 in its N-biotiny-
lated form (bioAβ ) was thus used in our anti-oligomer assays
4
2
because it readily assembles into oligomers at near-physiologi-
Fig. 5 Solution speciation studies of chalcones (S) 3a, 3d, and 3h in the cal nanomolar concentrations, while Aβ1-40 forms oligomers
4
2
presence of CuCl
solution speciation diagrams (right) of Cu -3a, Cu -3d, and Cu -3h.
2
. UV-visible variable-pH titration spectra (left) and
only very weakly.
2+
2+
2+
2+
2+
As previously noted, metal ions such as Cu and Zn have
2
3
0 μM (3a) or 50 μM (3d and 3h) of the chalcone (S) was incubated for
2+
been observed to interact with Aβ and promote peptide
2
0 minutes with CuCl ([Cu ]/[S]). Titrations were then performed at
2
2–25
aggregation.
Therefore, the ability of chalcones 3a–i, 6a–h,
room temperature from pH 7-2 for 3a and 3d, and from pH 12-2 for 3h.
F
Cu represents the fraction of free Cu and SCu complexes present in 7a, b, and h to modulate not only Aβ species, but also Aβ-metal
2+
2+
solution at the given pH. The stability constants (log β) of Cu -3a, Cu
3
-
complexes was examined.
2
+
d, and Cu -3h complexes are summarized in the table at the bottom.
BioAβ42 oligomer aggregation. The influence of the syn-
thesized chalcones on the self-assembly of bioAβ42 monomers
into oligomers in the absence and presence of metal ions was
first examined (Fig. 6A). The inhibition experiment was per-
formed to determine whether chalcones 3a–i, 6a–h, 7a, b, and
spectrum when the chalcone 3d was titrated in the presence of
CuCl . Finally, the shape of the pH-titration curve of 3h
2
changed, resulting from a more pronounced decline in absor-
bance of the chalcone in the presence of CuCl2 as the pH
decreased. Indeed, the absorbance of 3h decreased from 0.45
to 0.28 at 280 nm, and from 0.37 to 0.20 at 470 nm in the
absence of CuCl . Meanwhile, the absorbance of 3h decreased
2
from 0.46 to 0.13 at 280 nm, and from 0.36 to 0.06 at 470 nm
in the presence of CuCl
2
. All these results support our previous
observations that chalcones with a 2-pyridyl ketone moiety,
2
+
such as 3a, 3d, and 3h, interact with Cu ions, while chal-
cones with a 2-acylphenol moiety (6a) or a 2-acylaniline moiety
2
+
(
7a) do not bind Cu .
a
Based on the determined pK values of chalcones 3a, 3d,
Fig. 6 Schematic representation of the inhibition experiments of
assembly and dissociation of bioAβ42 in the presence or absence of
and 3h above, we were able to find the stability constants
2
+
(
log β) of the complexes formed between Cu and our chal- metal salts.
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Table 1 EC50 values (in μM) for chalcones in the absence or presence to disassemble pre-formed bioAβ42 oligomers in the absence
2 2
of metal (ZnCl or CuCl ) for prevention of bioAβ42 oligomers self-
assembly and for dissociation of pre-formed bioAβ42 oligomers
or presence of metal ions. BioAβ42 oligomers, of size similar to
that of Aβ42 oligomers from AD brain, were used in this
45
a
study. In general, all the chalcones were better at disassem-
bling bioAβ42 oligomers than at preventing their formation
from monomers (Table 1). Chalcones 3a–i, which bear the
Prevention of assembly of bioAβ42 oligomers
Cpd
No metal
ZnCl
2
CuCl
2
2
-pyridyl ketone moiety, showed greater reactivity in the
3
6
6
d
f
g
19.25 ± 0.96
>50
8.93 ± 0.38
20.00 ± 0.01
>50
9.25 ± 0.96
19.88 ± 0.63
16.63 ± 5.68 absence of metal ions. Incubation of these chalcones with
9.83 ± 0.62
metal ions prior to the addition of pre-formed bioAβ42 oligo-
mers may allow them to first interact with the metal ions,
Dissociation of pre-formed bioAβ42 oligomers
which could then distort the structural backbone known to
bind Aβ species, and thus disassemble less effectively pre-
formed bioAβ42 oligomers. Furthermore, while the EC50 values
Cpd
No metal
ZnCl
2
CuCl
2
3
3
3
3
3
3
3
3
3
6
6
6
6
6
6
6
6
7
7
7
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
a
b
h
1.04 ± 0.09
3.13 ± 0.61
1.25 ± 0.10
1.70 ± 0.41
1.53 ± 0.39
6.50 ± 1.05
9.33 ± 1.67
10.50 ± 2.52
10.25 ± 0.29
1.15 ± 0.06
2.05 ± 0.06
2.68 ± 0.49
2.33 ± 0.15
2.85 ± 0.31
2.35 ± 0.44
1.28 ± 0.17
2.58 ± 0.39
>50
1.10 ± 0.07
4.30 ± 0.84
1.58 ± 0.29
4.14 ± 1.47
1.45 ± 0.30
5.38 ± 0.81
11.13 ± 0.63
11.50 ± 2.52
11.00 ± 0.71
1.88 ± 0.15
2.25 ± 0.19
3.68 ± 0.62
2.43 ± 0.26
2.55 ± 0.17
2.40 ± 0.08
1.45 ± 0.10
2.50 ± 0.36
>50
2.63 ± 0.93
>50
2.45 ± 0.44
>50
of the chalcones obtained in the presence of ZnCl were com-
2
parable (although slightly higher) to those in metal-free con-
ditions, there was a significant increase in EC50 values of the
3.70 ± 0.49
2
+
2+
8.25 ± 0.50 chalcones in Cu -treated bioAβ oligomer dissociation. Cu
4
2
18.50 ± 2.38
33.25 ± 0.96
13.75 ± 1.50
ions thus seem to provide a greater barrier to chalcones than
2
+
Zn ions in the dissociation of bioAβ42 oligomers, suggesting
2
+
2+
1.93 ± 0.30 that these chalcones may interact better with Cu than Zn .
2.08 ± 0.10
3.20 ± 0.58
2.53 ± 0.46
2+
This may result from the fact that chelation of Zn is more
4
6,47
pronounced in nitrogen-rich environment.
The metal ions
2.93 ± 0.41 could also be binding to the Aβ oligomers stabilizing their
2.38 ± 0.62
3.78 ± 1.30
structure.
In both the absence and presence of metal ions, chalcones
2.53 ± 0.42
>50
>50
>50
3f–i, with a Br substituent on the pyridyl moiety, were less
efficient at dissociating bioAβ42 oligomers than their methyl
counterparts 3b–e. Since the former are poorer metal chelating
agents than their methyl counterparts, they would be expected
to bind bioAβ42 oligomers better in accordance with previous
observations. This reinforces the fact that the assembly of Aβ
species is a complex process. Finally, no direct correlation was
>50
15.50 ± 2.38
>50
>50
a
All compounds not presented displayed EC50 values >50 μM.
h were able to control the formation of metal-free and metal- observed between the substitution pattern on ring I of these
associated bioAβ42 oligomers using a quantitative bioAβ42 chalcones and their ability to dissociate bioAβ42 oligomers.
4
3,44
single-site streptavidin-based assay.
Among these chal-
Chalcones 6a–h exhibited EC50 values that were quite con-
cones, 3d, 6f, and 6g were the only ones to demonstrate oligo- stant both in metal-free and metal-treated studies. Also, substi-
mer assembly inhibition (EC50 > 50 μM; Table 1). However, in tution on ring I only seems to slightly reduce the efficacy of
the absence of metals, they displayed EC50 values that were 6-, the chalcones (6a versus 6b–h). When compared to 3a–i, chal-
>
36-, and 14-fold greater than that of the known assembly cones 6b, 6f, 6g, and 6h demonstrated better reactivity at dis-
4
4
inhibitor clioquinol (EC50 = 1.4 μM). Interestingly, 6g, which assembling bioAβ42 oligomers than their 2-pyridyl ketone
2
+
does not bind Cu ions (Fig. 2), exhibited the greatest reactiv- counterparts in the absence and presence of metal ions.
ity with Aβ monomers, both in the absence or presence of
Chalcones 7a, b, and h were the least potent of the syn-
metal ions. The presence of ZnCl or CuCl does not seem to thesized molecules, as they showed poor reactivity (EC50
affect the inhibitory capability of 3d and 6g. CuCl , on the con- 50 μM). 7h was only able to dissociate bioAβ42 oligomers in the
2
2
>
2
2
+
trary, increases the reactivity of 6f while metal-free or Zn - absence of metal ions.
induced Aβ oligomer assembly were unaffected by the com-
Overall, chalcones 3a, 3c, 3d, 3e, 6a, and 6g were the best
pound. Moreover, chalcone 3a, which was previously found to dissociators in the absence of metal ions, and they displayed
3
4
exhibit antifibrillogenic activity, appears not to be efficacious EC50 values that were within 2-fold of the EC value of the dis-
50
4
8
at the oligomeric level. This suggests that the ability of chal- sociator 2,5-dihydroxybenzoic acid (0.7 μM). 6b, 6f, 6g, and
cones to interact with metal ions might not be an indispensa- 6h also had great effect on the in vitro modulation of metal-
ble feature in the prevention of bioAβ42 oligomers formation.
Aβ42 oligomer dissociation. The influence of the syn-
free and metal-induced bioAβ42 dissociation.
Taken together, the results from bioAβ42 oligomer aggrega-
thesized chalcones on the dissociation of pre-formed Aβ oligo- tion and bioAβ42 oligomer dissociation studies reveal that 3d
mers in the absence and presence of metal ions was also and 6g are better than the assembly inhibitor clioquinol (that
4
4
investigated (Fig. 6B). The experiment was performed, in this does not work on dissociation) and the dissociator 2,5-di-
4
8
case, to assess the ability of chalcones 3a–i, 6a–h, 7a, b, and h hydroxybenzoic acid (that has no effect on assembly).
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Organic & Biomolecular Chemistry
In vitro cholinesterase inhibition
tion lowers its IC50 value by 8-fold (7a: IC50 = 4.00 ± 1.20 μM
versus 7h: IC50 = 0.503 ± 0.154 μM). Overall, chalcones with the
AChE and BChE inhibition. Cholinesterases represent
another valuable target in the discovery of AD therapeutics. To
that effect, we evaluated the potential cholinesterase inhibitory
activity of our chalcones 3a–i, 6a–h, 7a, b and h by determin-
ing their individual IC50 values against AChE from Electro-
phorus electricus (EeAChE) and BChE from equine serum
2
-acyl aniline moiety (7a, 7b, and 7h) were better in vitro AChE
inhibitors than the chalcones with the 2-acyl phenol moiety
6a–h), which in turn were more potent than 3a–i. Further-
more, when compared to other chalcones reported in the lit-
(
5
0
erature as potent AChE inhibitors, 6e, 6g, 7a, 7b, and 7h
displayed improved inhibitory activities with IC50 ≤ 4 μM.
Since BChE is another cholinesterase enzyme associated
4
9
(
esBChE) (Table 2, Fig. S54†) according to Ellman’s method.
All our tested chalcones displayed micromolar IC50 values that
were in the range of the IC50 value previously observed for the
FDA-approved AChE inhibitor rivastigmine. The IC50 values
ranged from 9.55 ± 1.95 μM to >200 μM for derivatives 3a–i
with the 2-pyridyl ketone moiety, and 1.61 ± 0.62 μM to
1
8
with Aβ plaques, we tested the inhibitory activity of our syn-
thesized chalcones towards BChE. Unfortunately, our tested
compounds were in general less effective against BChE than
AChE. Nevertheless, 3a, 3c, 3e, 3h, 6d, 6f, 6g, and 7b exhibited
some potency against BChE, with 3c, 3e, and 6f even showing
some improvement in activity when compared to AChE.
5
0
>
200 μM for derivatives 6a–h with the 2-acylphenol moiety,
while 7a, 7b, and 7h, with the 2-acyl aniline moiety, had IC50
values of 4.00 ± 1.20 μM, 1.44 ± 0.48 μM, and 0.503 ±
Our synthesized chalcones thus appear to be more active
against AChE than BChE, and, as a result, their inhibitory effect
on AChE was also examined in the presence of metal ions.
0
.154 μM, respectively. The known chalcone 3a displayed
potent AChE inhibitory activity with IC50 = 14.8 ± 5.6 μM.
Methyl substitution at any position of the pyridyl ring does not
appear to improve the inhibitory activity of 3a against AChE.
2
+
Effect of metals on AChE inhibition. The effect of Cu and
2
+
Zn on AChE inhibition was tested and, while most of our
chalcones suffered a decrease in their activity, displaying IC50
values >200 μM, 6a, 6b, 7a, and 7b were still potent, with IC50
values ranging from 5.78 ± 1.87 μM to 22.5 ± 9.0 μM in the
Meanwhile, the attachment of a bromide atom at the R (3f:
1
IC50 = 9.67 ± 3.00 μM) or R (3g: IC50 = 9.55 ± 1.95 μM) position
2
seems to enhance its inhibitory activity. For chalcones 6a–h,
substitution tends to enhance the inhibitory activity of 6a
against AChE (IC50 = 13.5 ± 4.4 μM), except in the case of a
presence of CuCl
.28 μM in the presence of ZnCl
value of all the chalcones but 6b. On the other hand, Zn
2
, and from 0.688 ± 0.159 μM to 9.27 ±
2
+
2
2
. Cu increased the IC50
2
+
methyl substitution at the R
2
position (6c: IC50 > 200 μM) and
actually improved the inhibitory activity of 6a, 6b, and 7a. It
thus appears that chalcones bearing a 2-acyl aniline moiety
may be important AChE inhibitors.
a bromo substitution at the R position (6h: IC > 200 μM).
3
50
Finally, methyl substitution at the R position of chalcone 7a
lowers its IC50 value by 2-fold (7a: IC50 = 4.00 ± 1.20 μM versus
1
7
b: IC50 = 1.44 ± 0.48 μM) and bromo substitution at R posi-
3
Conclusions
Three main classes of chalcones were synthesized and
, and BChE alone by chalcones 3a–i, biochemically evaluated in vitro. These compounds showed
ability to chelate metal ions and/or potent dissociation of pre-
Table 2 Inhibition (IC50 values (in μM)) of the activity of AChE alone and
in the presence of ZnCl
2
and CuCl
2
6
a–h, and 7a, b, and h
formed bioAβ42 aggregates. Chalcones 3a–i showed the ability
Cpd
AChE
AChE + ZnCl
2
AChE + CuCl
2
BChE
2
+
2+
to bind metal ions such as Cu and Zn . Additionally, 3d and
6g effectively modulated the assembly and disassembly of
bioAβ42 oligomers in the presence and absence of metal ions,
showing a broader reactivity on Aβ modulation than clioquinol
and 2,5-dihydroxybenzoic acid. Chalcones 7a, 7b, and 7h, with
a 2-acyl aniline moiety, also displayed potent AChE inhibitory
activities and thus appear to be a class of compounds worthy
of additional studies. Finally, 6g appeared to be a multifunc-
tional compound as it exhibited metal-Aβ modulator capability
and inhibited AChE activity. Further optimization studies are
currently underway in our laboratory.
3
3
3
3
3
3
3
3
3
6
6
6
6
6
6
6
6
7
7
7
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
a
b
h
14.8 ± 5.6
16.3 ± 6.3
12.3 ± 1.3
>200
35.9 ± 7.6
9.67 ± 3.00
9.55 ± 1.95
14.9 ± 0.9
55.8 ± 16.8
13.5 ± 4.4
10.1 ± 3.8
>200
7.14 ± 1.60
1.61 ± 0.62
7.76 ± 2.77
2.85 ± 0.89
>200
4.00 ± 1.20
1.44 ± 0.48
0.503 ± 0.154
>200
>200
>200
>200
>200
—
>200
>200
>200
>200
>200
15.3 ± 7.7
5.78 ± 1.87
—
>200
>200
>200
>200
—
14.3 ± 2.8
>200
8.24 ± 1.20
>200
16.4 ± 3.1
>200
>200
39.0 ± 9.8
>200
>200
>200
>200
34.8 ± 5.0
>200
2.82 ± 0.29
4.32 ± 1.05
>200
>200
3.92 ± 0.90
>200
>a200
a
—
>200
>200
>200
>200
>200
9.27 ± 2.28
3.50 ± 0.81
—
>200
>200
>200
>200
—
0.688 ± 0.159
2.07 ± 0.61
>200
20.2 ± 8.9
22.5 ± 9.0
>200
Notes and references
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These were not detemined as the IC50 value against AChE alone was
already >200 μM.
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Paper
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