A small bifunctional chelator that modulates Aβ42 aggregation

Article abstract of DOI:10.1139/cjc-2017-0623

Multifunctional compounds that can modulate amyloid-β (Aβ) aggregation and interact with metal ions hold considerable promise as therapeutic agents for Alzheimer's disease (AD). Using the copper-catalyzed azide-alkyne cycloaddition reaction, a novel bifun

Full text of DOI:10.1139/cjc-2017-0623

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A small bifunctional chelator that modulates Aβ42 aggregation
Chaofeng Zhang,a,*Luiza M.F. Gomes,b Tonglu Zhang,a and Tim Storrb,*
Abstract: Multifunctional compounds that can modulate amyloid-β (Aβ) aggregation and interact with metal ions hold
considerable promise as therapeutic agents for Alzheimer’s disease (AD). Using the copper-catalyzed azide-alkyne
cycloaddition reaction, a novel bifunctional chelator 2-(1-(4-(dimethylamino)benzyl) -1H-1,2,3-triazol-4-yl)phenol (L1)
was synthesized. L1 contains a bidentate metal-binding unit, and a pendant dimethylamino moiety. The product was
characterized by 1H NMR, 13C NMR, and MS. The metal-binding properties of L1 were probed by UV-visible
spectroscopy to determine Cu:L stoichiometry. L1 was determined to limit Aβ aggregation at 48 h via a ThT assay. In
addition, L1 complies with Lipinski’s rules and calculated logBB values for potential drug-likeness and BBB
permeability. These results suggest that L1 is a suitable candidate for further study as a multifunctional compound to
treat AD.
Keywords: β-Amyloid, Alzheimer’s disease, click chemistry, copper ions
Introduction
With an aging population and an increase in life span, Alzheimer’s disease (AD) is of major concern all over the
world, and there is a lack of effective pharmacotherapy options.1 There are many factors that have been shown to play
an important role in AD, such as amyloid-β (Aβ) aggregation,2-3 hyperphosphorylation of tau protein,4-5 metal
dyshomeostasis,6-7 oxidative stress,8-10 mitochondrial dysfunction and reduced levels of acetylcholine (ACh). The
available therapies for AD focus mainly on cholinesterase inhibition.1 However, the development of new therapies has
shifted from single-target molecules to multi-target compounds due to the complexity and multiple etiologies of AD.11
Many “multi-target” agents have been reported recently, such as AChE inhibitors with metal chelation properties, and
AchE-induced Aβ aggregation inhibitory activity,12 BACE1 inhibitors bearing AChE inhibitory activity,13 BACE 1
inhibition and metal chelation,14 and cholinesterase inhibitors with both antioxidant and neuroprotective properties.15
Aβ aggregation and metal dysregulation are two examples of promising targets for next-generation drug therapies that
are under investigation in current studies.11 Examples of promising target molecules are shown in Fig.1.16-21 Aβ is
derived from the proteolytic cleavage of APP by β- and γ-secretases.22 The aggregation of Aβ leads to the formation of
senile plaques (SP), which is one of the hallmarks of AD brains,2 where the plaques cause neuronal destruction and are
associated with oxidative stress, mitochondrial dysfunction, loss of membrane integrity, abnormal calcium homeostasis,
and induced apoptosis.23 There is a change in metal levels in the brain of an AD patient, with high levels of metals (ca.
0.4 mM Cu; 1.0 mM Zn; 0.9 mM Fe) being found within SP from AD brain tissue compared to healthy tissue.24-26
Metal ions have been shown to accelerate aggregation of Aβ; stabilizing neurotoxic oligomers and showing
Fenton-like reactions of Aβ-bound redox active metal ions.27 Furthermore, extensive evidence supports the connection
between these two targets, probably due to the role played by trace elements during the misfolding process that occurs
in Aβ aggregation.11
Considering the crucial roles of Aβ and metal ions in AD pathology, we synthesized a multifunctional ligand that
can simultaneously impact on Aβ, metal ions and metal-Aβ species. The ligand was designed based on the direct
incorporation of metal-binding atom donors into the structural framework of an Aβ-interacting compound. Click
chemistry can be used effectively to connect various functional entities through a copper-catalyzed Huisgen-type
1,3-dipolar cycloaddition of terminal alkynes and organic azides. A series of dual-function triazole-pyridine ligands16
and multifunctional quinoline-triazole ligands17 have been previously reported by our group (Fig.1). Both these series
were designed to have two nitrogen (N) donor atoms and different side chains that may modulate the affinity for the
metal ion and/or the Aβ-peptide. The triazole-pyridine series interacts with hydrophilic N-terminal residues of Aβ and
demonstrate an ability to limit metal-induced Aβ aggregation.16 The quinoline-triazole series weakly interact with the
Aβ peptide, and only modify the aggregation profile of the Aβ peptide in the presence of excess Cu ions (1.4:1).17 In
this paper, we focused on the design of a multifunctional ligand with increased peptide interactions, and a higher
affinity for Cu. In structure-reactivity investigations of multifunctional ligands in both the absence and presence of
metal ions for their antiamyloidogenic characteristics, it was evident that the dimethylamino group is a key component
for the activity of the ligand.28-29 Based on a structure-mechanism design strategy18 and the previous work,16-17, 28-29
a
novel triazole-phenol derivative was designed (Scheme 1.) inspired by the metal chelator clioquinol (CQ) and a known
Aβ imaging agent, a p-I-stilbene derivative.30 The metal-binding properties of the new ligand, its interaction with Aβ,
as well as its potential blood-brain barrier (BBB) permeability were evaluated.
Received
aCollege of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China.
bDepartment of Chemistry, Simon Fraser University, Burnaby, V5A1S6, Canada.
Corresponding author: Chaofeng Zhang (email: zhangchaofeng@tyut.edu.cn) and Tim Storr (email: tim_storr@sfu.ca).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.

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Fig.1. Examples of multifunctional metal-binding agents.
Scheme1. Incorporation approach to design small molecule to target metal-Aβ species and regulate their aggregation.
Experimental
Some azides are hazardous, and to avoid injury, follow safe laboratory practices, wear appropriate protective
equipment and use appropriate shielding equipment. Azides can decompose violently upon heating, shock and/or
friction. Only small quantities should be prepared at a single time. All common chemicals were purchased as reagent
grade from Aldrich and used without further purification. All of the solvents were treated according to general methods.
1H and 13C NMR spectra were recorded on Bruker AV-400 or AV-500 instruments. Mass spectra were obtained on an
Agilent 6210 time-of-flight electrospray ionization mass spectrometer. UV-vis measurements were performed on a
TU-1901 double-beam UV-Vis spectrophotometer. The Thioflavin T assay was measured using a Synergy 4
Fluorometer plate reader from BioTek.
Synthesis of 2-(1-(4-(dimethylamino)benzyl)-1H-1,2,3-triazol-4-yl)phenol (L1)
4-(N,N-dimethylamino)benzylalcohol 2 was synthesized according to a reported method.31 1H NMR, (400MHz,
CDCl3): δ 7.31-7.26 (m, 1H), 6.80-6.73(m, 1H), 4.60(s, 1H), 2.98(s, 3H). 4-(chloromethyl)-N,N-dimethylaniline 3
and 4-(azidomethyl)-N,N-dimethylaniline 4 were synthesized according to previously reported procedures.32
4-(chloromethyl)-N,N-dimethylaniline: 1H NMR (400 MHz, CDCl3): δ 7.85–7.78 (m, 1H), 7.57–7.51 (m, 1H), 4.58 (s,
1H), 3.21 (s, 3H). 4-(azidomethyl)-N,N-dimethylaniline: 1H NMR (400 MHz, CDCl3): δ 7.26–7.22 (m, 1H),
6.79–6.75 (m, 1H), 4.27 (s, 1H), 3.01 (s, 3H). o-((Trimethylsilyl)ethynyl)phenol 6 was synthesized from
2-iodophenol 5 according to previously reported procedure33 to afford the title compound as a red brown oil in 56%
yield . 1H NMR (400 MHz, Chloroform-d): δ 7.37 (dd, J =7.7, 1.6 Hz, 1H), 7.29–7.24 (m, 1H), 6.97 (dd, J = 8.3, 1.0
Hz, 1H), 6.88 (td, J = 7.6, 1.1 Hz, 1H), 5.86 (s, 1H), 0.31 (s, 9H); 13C NMR (400 MHz, Chloroform-d): δ 98.92,
102.39, 109.49,114.52, 120.21, 130.66, 131.57, 157.07.
Synthesis of 2-(1-(4-(dimethylamino)benzyl)-1H-1,2,3-triazol-4-yl)phenol L1: In
a
20 mL vial,
o-((trimethylsilyl)ethynyl)phenol 6 (194.4mg, 1.02 mmol) and 4-(azidomethyl)-N,N-dimethylaniline 4 (184.4 mg,
1.05 mmol) were dissolved in 6 mL of methanol. In a separate 20 mL vial, CuSO4·5H2O (125.5 mg, 0.5 mmol), K2CO3
(256.3 mg, 1.85 mmol) and L-ascorbic acid (176.12 mg, 1.0 mmol) were dissolved in 6 mL of H2O. The aqueous
solution was added dropwise to the stirring methanol solution. Pyridine (0.5 mL) was added slowly to the resulting
mixture and the solution was stirred at room temperature for overnight. Chelex resin was added and the solution was
stirred for an additional 2 h. The solution was filtered, concentrated, and the residue was diluted with H2O/ CH2Cl2
(1:1). The aqueous layer was extracted with CH2Cl2, combined organic layers were dried with Na2SO4, concentrated
under reduced pressure, purified by silica gel chromatography (CH2Cl2/MeOH 99:1) to afford a pale yellow solid (30
mg, 10% yield). 1H NMR (500 MHz, Chloroform-d): δ 10.95 (s, 1H), 7.69 (s, 1H), 7.34 (dd, J = 7.8, 1.6 Hz, 1H), 7.25
(d, J = 8.3 Hz, 2H), 7.23 –7.20 (m, 1H), 7.06 (dd, J = 8.2, 1.2 Hz, 1H),6.86 (td, J = 7.5, 1.2 Hz, 1H), 6.74 (d, J = 8.6
Hz, 2H), 5.50 (s, 2H), 3.00 (s, 6H); 13C NMR (400 MHz, Chloroform-d): δ 40.37, 54.45, 112.56, 114.08, 117.58,
118.48, 119.35, 120.93, 125.84, 129.57, 129.67, 147.81, 150.84, 155.89; ESI(-)-MS (m/z): [M-H]- Calcd for
C17H18N4O: 293.1403; Found 293.1460.
Predictability of drug-like/BBB permeability
Using a web-based program,34 several physicochemical properties were determined in order to predict the
drug-like
properties
and
blood-brain
barrier
(BBB)
permeability
of
the
2-(1-(4-(dimethylamino)benzyl)-1H-1,2,3-triazol-4-yl)phenol compound. Molecular weight (MW), calculated
logarithm of the octanol-water partition coefficient (clogP), H-bond acceptors (HBA), H-bond donors (HBD) and total
potential surface area (TPSA) were calculated to determine the drug-like properties of the
2-(1-(4-(dimethylamino)benzyl)-1H-1,2,3-triazol-4-yl)phenol.35 Clark's equation36 (Eq. (1)) was used to determine the
log BB, which is a common measure of the degree of BBB penetration.
logBB = − 0.0148 × TPSA + 0.152 × clogP + 0.139
(1)
Metal binding studies
Metal binding studies were performed by varying the molar fractions of CuCl2 from 0 to 1 (0 to 50 ꢀM) in 10%
DMSO/ HEPES Buffer pH 7.4 in the presence of ligand to obtain UV-visible spectra. An absorbance maximum was
assigned as interaction of metal and ligand for each solution, which gave the determination of the metal:ligand ratio in
the complex of ligand-Cu(II).
ThT Assay
Aβ1–42 peptide was purchased from Cellmano Biotech Limited, monomerized according to a reported procedure37

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and stored in a -80˚C freezer until use. The monomeric film was dissolved in 1:1 DMSO:ddH2O to a final volume of
200 ꢀL and the concentration was determined in a Thermo Nicolet UV nanodrop instrument (ε = 1450 M-1). In a
96-well plate the peptide (5 ꢀM) was incubated at 37˚C with agitation in the presence and absence of CuCl2 (5 ꢀM)
and/or ligands (15 ꢀM) in 10% DMSO in 0.1 M PBS pH 7.4. PBT2 has been shown to influence Aβ aggregation in the
presence of Cu, 38-39 and was used as a control. Thioflavin T (ThT) was added to the wells at 5 ꢀM, and fluorescence
(λex 404 nm, λem 477 nm) measured in quadruplicate at 0, 8, 24 and 48 h.
Results and Discussion
The synthesis of a new metal-binding compound towards Alzheimer’s disease was performed in a few steps
(Scheme 2). The key reaction in the synthesis of the target compound is the click chemistry between aryl azide and terminal
alkyne. On the one hand, the aryl azide was obtained by reduction, chlorination, and azidation from
4-dimethylaminobenzaldehyde 1 in high yields. On the other hand, the protected alkyne was synthesized by a
Sonogashira coupling reaction between 2-iodophenol 5 and trimethylsilylacetylene. The coupling between the aryl
azide and the terminal alkyne was realized by a copper-catalyzed Huisigen azide-alkyne cycloaddition reaction where
the deprotection of the alkyne function and the coupling were performed in the same step. The low yield could be due
to the ligand binding the copper thus limiting the coupling reaction. In the future, an accelerating ligand could be
added to enhance the click chemistry reaction.40-41
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH, reflux, 3 h, 98%. (b) SOCl2, DCM, r.t., 12 h, 95%. (c) NaN3, DMF,
r.t., 30 h, 62%. (d) trimethylsilylacetylene, PdCl2(PPh3)2, Et3N, CuI, dioxane, 45℃, 5 h, 56%. (e) Pyridine, K2CO3,
L-ascorbic acid, CuSO4, CH3OH/H2O, r.t., 12 h, 10%.
The Lipinski rule of five35 describes parameters that predict whether a compound will exhibit favorable
pharmacokinetic properties. These parameters include MW, HBA, HBD and cLogP, and can be calculated using a
web-based program34. The values for cLogP and TPSA can be used to calculate the log BB value using Clark's
equation (Eq. (1)), which can inform potential BBB permeability. Log BB values ＞3.0 indicate readily crosses BBB
and values ＜−1.0 indicate poorly distributed to the brain.42 The predicted parameters for L1 are promising in terms
of drug-likeness, however the calculated log BB value for BBB permeability is little low suggesting moderate
distribution to the brain (Table 1). While we did not test the antioxidant properties of L1, we expect the phenol moiety
to exhibit ROS quenching effects as reported for similar derivatives.43-45
Table 1. Values (MW, clogP, HBA, HBD, TPSA, logBB) for ligand
Calculation
MWa
294.36
≤500
clogPb
2.96
≤5
HBAc
5
HBDd
1
TPSA (Å)e
54.18
logBBf
Ligand
Lipinski’s rules and others
− 0.21
≤10
≤5
≤90
> 3.0 (readily); < −1.0 (poorly)
aMW = molecular weight; bclogP = calculated logarithm of the octanol-water partition coefficient; cHBA = H-bond acceptors;
dHBD = H-bond donors; eTPSA = total potential surface area; flogBB= −0.0148TPSA + 0.152 clogP + 0.130.
The interaction of the ligand with Cu2+ was probed using UV-vis spectroscopy. Upon addition of CuCl2 to a
solution of ligand, decreased intensity and shifts of the optical band at ca. 250 nm and 290 nm were observed relative
to ligand. A new band at ca. 320 nm was observed at the same time (Fig. 2A). Changes in the intensity of the
ligand-based transition, along with the observation of new absorption indicated Cu2+ binding to ligand. To determine
the ligand-Cu(II) stoichiometry of the complex, the technique of continuous variation (also called Job’s method) was
used by preparing solutions of ligand and CuCl2 so that the sum of concentrations of both species was constant in all
samples, but the proportions of both components varied between 0% and 100%. The UV spectra were recorded and the
absorbance at 320 nm, which corresponds to a ligand to metal charge transfer absorption band, was plotted versus the
mole fraction of CuCl2. The concentration of complex (ligand-Cu(II)) increases as the concentration of CuCl2 is
increased, until it reaches a maximum which corresponds to the optimal metal:ligand ratio of the complex. As shown
in Fig.2B, the absorption at 320 nm reaches a maximum over a range of % Cu values. The broadness of the peak
suggests that a mixture of 1:1 and 1:2 metal:ligand species are present in solution at pH 7.4.46-47
Fig. 2. (A) UV-vis spectra of ligand and copper chloride at different mole fraction of CuCl2 (Ctot = 50 ꢀM) in HEPES buffer
(0.1M, PH=7.4). (B) Determination of the stoichiometry of complex ligand-Cu(II) by Job’s method.
ThT shows an increase in fluorescence in the presence of Aβ1-42 fibrils48 and was used to investigate the influence
of L1 on the aggregation pattern in the presence and absence of Cu2+. PBT2 (Fig.1) was used for comparison with the

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ligand synthesized in this work, due to the promising characteristics of this ligand in AD models (Fig.3). Over time,
Aβ1-42 shows an increase in ThT fluorescence, reaching its maximum fluorescence at 48 h. Up to 24 h of incubation
both L1 and PBT2 not show any difference in aggregation pattern, however at 48 h L1 shows a significant decrease in
ThT fluorescence intensity, suggesting that the ligand interacts with the peptide to limit the formation of higher MW
fibrils. In the presence of Cu2+, there is no increase in ThT fluorescence, in accordance with results shown previously
that copper stabilizes low molecular weight species.49-50 L1 does not appear to prevent Cu2+ interaction with Aβ1-42
，
suggesting that the Cu-affinity of this derivative is lower than that of the Aβ peptide. While PBT2 does not change
the aggregation pattern of Aβ1-42 under Cu-free conditions, in the presence of Cu2+ a small increase in ThT
fluorescence is obverted, thus showing that PBT2 can influence the Cu-Aβ interaction.
Fig. 3. ThT fluorescence of (A) Aβ1-42 in the presence and absence of ligands; (B) Aβ1-42 with CuCl2 in the presence and
absence of ligands.
Conclusion
In summary, using a click reaction between o-((trimethylsilyl)ethynyl)phenol and 4-(azidomethyl)-N,N-
dimethylaniline, a novel triazole-phenol derivative L1 was synthesized. This triazole-phenol derivative conformed to
Lipinski’s rules and the calculated logBB value for potential blood-brain barrier (BBB) permeability. Metal binding
properties and Aβ interaction properties of this triazole-phenol derivative were studied by UV-vis and a ThT Assay.
The further synthesis and functional studies of similar compounds are underway in our group.
Supplementary data
Supplementary data are available with the article through the journal Web site at
http://nrcresearchpress.com/doi/suppl/.
Acknowledgment
Research project supported by Shanxi Scholarship Council of China (2017-037) (C. Z.), the Foundation of
Taiyuan University of Technology (1205-04020203) (C. Z.), the Michael Smith Foundation for Health Research (T.S.)
and Science Without Borders/0711-13-6 CAPES.
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Page 6 of 7
Figures and Schemes:
Fig.1. Examples of multifunctional metalꢀbinding agents.
Fig. 2. (A) UVꢀvis spectra of ligand and copper chloride at different mole fraction of CuCl2 (Ctot = 50 ꢁM) in HEPES buffer
(0.1M, PH=7.4). (B) Determination of the stoichiometry of complex ligandꢀCu(II) by Job’s method.
Fig. 3. ThT fluorescence of (A) Aβ1ꢀ42 in the presence and absence of ligands; (B) Aβ1ꢀ42 with CuCl2 in the presence and
absence of ligands.

Page 7 of 7
Scheme1. Incorporation approach to design small molecule to target metalꢀAβ species and regulate their aggregation.
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH, reflux, 3 h, 98%. (b) SOCl2, DCM, r.t., 12 h, 95%. (c) NaN3, DMF,
r.t., 30 h, 62%. (d) trimethylsilylacetylene, PdCl2(PPh3)2, Et3N, CuI, dioxane, 45℃, 5 h, 56%. (e) Pyridine, K2CO3,
Lꢀascorbic acid, CuSO4, CH3OH/H2O, r.t., 12 h, 10%.