Identification of ortho catechol-containing isoflavone as a privileged scaffold that directly prevents the aggregation of both amyloid β plaques and tau-mediated neurofibrillary tangles and its in vivo evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.105022

In this study, polyhydroxyisoflavones that directly prevent the aggregation of both amyloid β (Aβ) and tau were expediently synthesized via divergent Pd(0)-catalyzed Suzuki-Miyaura coupling and then biologically evaluated. By preliminary structure–activity relationship studies using thioflavin T (ThT) assays, an ortho-catechol containing isoflavone scaffold was proven to be crucial for preventing both Aβ aggregation and tau-mediated neurofibrillary tangle formation. Additional TEM experiment confirmed that ortho-catechol containing isoflavone 4d significantly prevented the aggregation of both Aβ and tau. To investigate the mode of action (MOA) of 4d, which possesses an ortho-catechol moiety, ¹H-¹⁵N HSQC NMR analysis was thoroughly performed and the result indicated that 4d could directly inhibit both the formation of Aβ₄₂ fibrils and the formation of tau-derived neurofibrils, probably through the catechol-mediated nucleation of tau. Finally, 4d was demonstrated to alleviate cognitive impairment and pathologies related to Alzheimer's disease in a 5XFAD transgenic mouse model.

Full text of DOI:10.1016/j.bioorg.2021.105022

Bioorganic Chemistry 113 (2021) 105022
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of ortho catechol-containing isoflavone as a privileged
scaffold that directly prevents the aggregation of both amyloid β plaques
and tau-mediated neurofibrillary tangles and its in vivo evaluation
,
,
,d,
Seung Hwan Sona 1, Ji Min Dob 1, Ji-Na Yooc 1, Hyun Woo Leea, Nam Kwon Kime,
Hyung-Seok Yooa, Min Sung Geea, Jong-Ho Kima, Ji Hye Seongf, Kyung-Soo Inna
,
,
e
Min-Duk Seoc *, Jong Kil Leea *, Nam-Jung Kima
,
d
,
,
,e,*
a College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
b Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
c College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon 16499, Republic of Korea
d Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea
e Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
f Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, polyhydroxyisoflavones that directly prevent the aggregation of both amyloid β (Aβ) and tau were
expediently synthesized via divergent Pd(0)-catalyzed Suzuki-Miyaura coupling and then biologically evaluated.
By preliminary structure–activity relationship studies using thioflavin T (ThT) assays, an ortho-catechol con-
taining isoflavone scaffold was proven to be crucial for preventing both Aβ aggregation and tau-mediated
neurofibrillary tangle formation. Additional TEM experiment confirmed that ortho-catechol containing isofla-
vone 4d significantly prevented the aggregation of both Aβ and tau. To investigate the mode of action (MOA) of
4d, which possesses an ortho-catechol moiety, 1H-15N HSQC NMR analysis was thoroughly performed and the
result indicated that 4d could directly inhibit both the formation of Aβ42 fibrils and the formation of tau-derived
neurofibrils, probably through the catechol-mediated nucleation of tau. Finally, 4d was demonstrated to alleviate
cognitive impairment and pathologies related to Alzheimer’s disease in a 5XFAD transgenic mouse model.
Polyhydroxyisoflavone
Amyloid β
Tau
Alzheimer’s disease
Catechol
1. Introduction
breakthrough is urgently needed for the treatment of this disease. The
cause of AD is largely unknown, but it is worsened by abnormal accu-
Alzheimer’s disease (AD) is a progressive and irreversible neurode-
generative disorder characterized by cognitive impairment, memory
loss, and continuous decline in behavioral and social skills [1,2]. In
2018, prevalence of the disease was reached to about 50 million patients
worldwide, and it is expected that this number will more than triple to
152 million by 2050 [3]. A few drugs are currently approved and are
used clinically to alleviate the symptoms of AD [4,5]. These medications
are generally classified into 2 groups that attenuate symptoms in
different ways: acetylcholinesterase (AchE) inhibitors and N-methyl-ᴅ-
aspartic acid receptor (NMDAR) antagonists. Unfortunately, while both
types of drugs help reduce the symptoms of AD, neither drugs cure the
mulation of pathological proteins such as amyloid β (Aβ) and tau-
mediated neurofibrillary tangles (NFT) [6–11]. However, various tar-
geting strategies for pathological proteins, especially Aβ, using mono-
clonal antibodies (mAbs) have not been successful [12,13]. Recently,
oligomeric Aβ and its plaques have been considered to play crucial roles
in AD-associated pathological toxicity [14–19]. The tau monomer is also
known to be nontoxic, but its aggregated insoluble form, NFT, has been
found to be neurotoxic [20–22].
Therefore, chemical prevention of the transformation of Aβ and tau
monomers into toxic polymers would be valuable as a novel therapeutic
strategy as well as a research tool for AD [23,24]. Isoflavones, which
possess 3-phenyl-4H-chromen-4-one as their main skeleton, are widely
disease nor effectively delay its progression [1]. Therefore,
a
* Corresponding authors.
E-mail addresses: mdseo@ajou.ac.kr (M.-D. Seo), jklee3984@khu.ac.kr (J.K. Lee), kimnj@khu.ac.kr (N.-J. Kim).
1
These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2021.105022
Received 5 November 2020; Received in revised form 3 April 2021; Accepted 23 May 2021
Available online 26 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
occurring natural flavonoids and synthetic derivatives [25–29]. Inter-
estingly, a series of polyhydroxyisoflavones such as daidzein 4a were
recently identified as novel therapeutic scaffolds for AD [30–39].
Especially, they exhibited to reduce the amount of Aβ and tau that are
sometimes pathologically aggregated in AD model. However, a detailed
study on polyhydroxyisoflavones has not yet been performed to inves-
tigate their biological ability to directly prevent pathologically aggre-
gated forms of both Aβ and tau in vitro or alleviate AD in vivo.
With polyhydroxyisoflavones in hand, we investigated whether the
compounds could prevent Aβ42 aggregation using the biochemical ThT
assay (Fig. 1A). A solution of monomeric Aβ42 (10 μM) in HEPES buffer
was incubated in the absence or presence of polyhydroxyisoflavones (30
M), and the extent of aggregation was monitored by ThT fluorescence.
μ
In the experiment, we found that 4c, 4d and 4e, 3′,4′-dihydroxyiso-
flavones possessing ortho-catechol moiety, could prevent Aβ42 aggre-
gation in both of the metal-free condition and metal (Cu2+ or Zn2+)-
treated conditions (Fig. 1 and S1). In addition, Cu2+-mediated Aβ ag-
gregation (Fig. 1A. Right Panel) was more likely to be prevented,
compared with metal-free Aβ aggregation (Fig. 1B. Right Panel) in the
normalized fluorescence intensity of Aβ ThT assay (Fig. S2) at 24 h after
the treatment of 3′,4′-dihydroxyisoflavones such as 4d. It seems that the
compounds can prevent metal free Aβ aggregation and more signifi-
cantly prevent Cu2+-mediated Aβ aggregation by the additional
enforcement. To confirm whether the additional interacting activity of
3′,4′-dihydroxyisoflavones would be involved with its metal binding
potential, we performed the additional experiment using UV/Vis spec-
trophotometry. In the result, we observed that 4d, one of 3′,4′-dihy-
droxyisoflavones could have potential interacting with Cu2+. When
Cu2+ was added to the aqueous solution of 4d, spectral change was
detected (Fig. S3) and this result was in coincidence with the previous
reports on the interaction between metal and its potential binders [41].
Therefore, it is plausible that 3′,4′-dihydroxyisoflavones might bind
Cu2+ and consequently interfere with the interaction between Aβ42 and
Cu2+, leading to the prevention of Aβ aggregation. In addition, 3′,4′-
dihydroxyisoflavone 4d showed the Aβ aggregation preventive activity
more or equipotent to epigallocatechin gallate (EGCG), a known com-
pound having anti-Aβ aggregation effect (Fig. S4) [53–56].
2. Results and discussion
The structurally rigid features of polyhydroxyflavonoids and their H-
bonding formation abilities make the privileged structures prevent Aβ
aggregation through their direct binding and destabilizing aggregating
conformation. Recently, a series of pioneering works that metal-
associated Aβ aggregation could be inhibited by isoflavone with ortho-
catechol supported the significance of the skeleton as a template for the
novel inhibitor preventing the aggregation of pathological proteins such
as Aβ [40–48]. In addition, ortho-catechol moiety can directly bind tau
proteins and prevent their aggregation in AD [49]. Inspired by the
previous reports, we rationally designed and synthesized a series of
isoflavones, focusing on the incorporation of an ortho-catechol moiety
that make H-bonding or coordinate binding with metal such as Cu2+
.
And we evaluated their ability to prevent the aggregation of Aβ and tau
using in vitro biochemical assay, and elucidated that the ortho-catechol
containing polyhydroxyisoflavones could inhibit the aggregation of both
pathological proteins. Finally, we showed that isoflavone 4d, which
possesses the pharmacophore, alleviates both cognitive impairment and
the pathologies related to AD in an in vivo 5XFAD transgenic mouse
model. Thus, this small molecule compound that directly prevents ag-
gregation of both pathological proteins was identified in vitro and
shown to alleviate pathology in vivo [50,51].
Next, we investigated the preventive effect of polyhydroxyiso-
flavones on tau aggregation. Similar to the Aβ42 aggregation assay, re-
combinant tau (2N4R, 10
μM) was treated with polyhydroxyisoflavones
Polyhydroxyisoflavones 3f-g and 4a-e were expediently synthesized
via 2-step reactions from 3-bromo-4H-chromen-4-one 1, involving
Suzuki-Miyaura coupling [52] and sequential demethylation (Scheme
1). 3-bromo-4H-chromen-4-one 1a and 1b was prepared from
commercially available compound 5a and 5b via sequential acetylation
and bromination (Scheme 2).
(30 μM) (Fig. 2). We confirmed that catechol containing isoflavones (4c,
4d and 4e) also caused significant reductions in heparin-induced ThT
fluorescence. Among the 3′,4′-dihydroxyisoflavones, 4d showed the best
inhibitory activity for tau aggregation in the biochemical assay. Based
on the inhibitory effect on both Aβ42 and tau aggregation, we chose 4d
as a chemical probe for further study. In addition, the anti-aggregation
Scheme 1. Design and synthesis of polyhydroxyisoflavones 3f-g and 4a-e. Reagents and reaction conditions: a) 2, Pd(OAc)2, SPhos, K2CO3, DMF/H2O (3:1), 90 ◦C; b)
Pyr∙HCl, reflux.
2

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
Scheme 2. Synthetic scheme of 3-bromochromone 1a and 1b.
Fig. 1. Effects of polyhydroxyisoflavones on the formation of Aβ aggregates. (A and B) For Aβ ThT assays examining the effect of compounds on aggregation, Aβ (10
µM) was incubated in the absence or presence of CuCl2 or ZnCl2 (10 µM) and compounds (30 µM) in a HEPES solution containing ThT (20 μM). The time course of
aggregation (left) and simplified bar plot representation of ThT data (right) for Aβ42 in the metal-free condition (A) and the presence of Cu2+ (B) (n = 6 per group).
Data are shown as the mean ± SEM. One-way ANOVA. **P < 0.01 and ***P < 0.001 vs. the nontreated group (N).
Fig. 2. Effects of polyhydroxyisoflavones on the formation of tau aggregates. For tau ThT assays examining the effect of compounds on aggregation, recombinant
wild-type 2N4R tau (10 μM), compounds (30 μM) and ThT (20 μM) were mixed in the a HEPES solution. Time-dependent ThT fluorescence of heparin-induced tau
aggregation (left) and simplified bar plot representation of ThT data (right) for 2N4R tau (n = 6 per group). Data are shown as the mean ± SEM. One-way ANOVA.
***P < 0.001 vs. the nontreated group (N).
effect of 4d on Aβ and tau was shown in a concentration-dependent
manner (Fig. S5).
analysis, the images of metal-free Aβ42 and Cu2+-added Aβ42 samples
treated with 4d revealed that pathology-related Aβ42 aggregates,
including fibrils, formed rarely (Fig. 3). In addition, tau fibril aggregates
were barely observed in the sample incubating with 4d in the
Next, we investigated whether 4d could prevent pathological ag-
gregation of both Aβ42 and tau using TEM analysis (Figs. 3 and 4). In the
3

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
were slightly reduced but there were no disappearing peaks (Fig. 5D-F),
indicating that the formation of Aβ42 fibrils was inhibited [59]. Given
that all cross peaks of Aβ42 completely disappeared after 12 h of incu-
bation with shaking (data not shown), the NMR results suggest that 4d
± 30 μM compound
10 μM Aβ42
± 10 μM Cu(II)
Aβ42 aggregates
24 hours, 37
can inhibit or slow down the aggregation of Aβ42
.
Next, we employed NMR to elucidate the role of 4d in the aggrega-
tion of tau. The effect of 4d on tau aggregation was more complicated
and ambiguous than that on Aβ42 aggregation. It is known that heparin-
induced tau aggregation results in decreased signal intensities for tau,
similar to the shaking experiment with Aβ42 [60]. The time course NMR
spectra of heparin-induced tau were obtained in the presence or absence
of 4d (Fig. 6). The NMR signal intensities for many peaks of tau without
4d gradually decreased, and those peaks completely disappeared. Sur-
prisingly, the aggregation kinetics of tau in the presence of 4d showed
significantly different patterns. Most of the perturbed peaks in the
presence of 4d showed more rapidly decreasing intensities than those in
the absence of 4d (Fig. 6A and 6B). As a result, the peaks of tau in the
presence of 4d disappeared much more quickly than those of tau
without 4d (Fig. 6C and 6D). However, a few cross-peaks of tau shown in
Fig. 6E showed reverse patterns, that is, more slowly decreasing in-
tensities and longer survival of resonances during incubation in the
presence of 4d. It was assumed that there are regional differences be-
tween the sequences that easily aggregated or were involved in the ag-
gregation process and the other sequences, probably due to the
relatively large size and random coil structure of monomeric tau. As the
intensities of HSQC spectra are proportional to the tau monomer con-
centration, the different decreasing patterns of cross peaks of tau
strongly implied that aggregation or fibrillation of tau was affected by
4d. Tau is aggregated through either cysteine-dependent polymerization
or cysteine-independent polymerization. These two types of polymeri-
zation occur and sometimes have synergistic effects on the aggregation
process [61].
Nontreated
+ 4d
+ 4a
Fig. 3. Representative TEM images of Aβ. Images of the aggregated Aβ42 in
samples incubated for 24 h with 4a and 4d in the presence or absence of Cu2+
(scale bar, 500 nm).
± 30 μM compound
10 μM 2N4R tau
Tau aggregates
+ heparin
24 hours, 37
Nontreated
+ 4d
Recently, it was reported that blocking cysteine covalently on tau
makes the protein a stable nucleated oligomer and does not lead to the
formation of tau-derived neurofibrillary tangles. In TEM analysis, this
nucleated form is generally observed as a globular form [62]. In
particular, our TEM results clearly showed the formation of nucleated
tau after treatment with compound 4d (Fig. 4 and S7). It was reported
that the ortho-catechol (1,2-dihydroxybenzene) moiety, a crucial phar-
macophore of 4d, can covalently bind cysteine residues on tau, pre-
venting the aggregation process [51]. It is thought that 4d might play a
role in the aggregation process of tau, such as nucleation and oligo-
merization, which results in inhibition of the formation of tau-derived
neurofibrils, probably through the catechol-mediated nucleation of
tau. To investigate whether compound 4d can bind a cysteine residue in
a covalent manner, simple experiments in which 4d was incubated with
cycteine or N-acetyl cysteine were also performed. As anticipated, co-
valent adducts of 4d with corresonding cysteine residues were detected
by LC-MS analysis, indicating that compound 4d might bind a cysteine
residue in tau through covalent bonding (data in SI).
+ 4a
Fig. 4. Representative TEM images of tau. Images of heparin-induced 2N4R tau
after a 24 h incubation with 4a and 4d (scale bar, 500 nm).
transmission electron microscopy (TEM) experiment (Fig. 4). However,
4a-treated Aβ42 samples showed a morphology similar to that of non-
treated Aβ42 samples in the presence or absence of Cu2+. Tau with
vehicle or 4a also formed fibrous aggregates after 24 h of incubation.
Taken together, these data indicated that 4d could inhibit both Aβ42 and
tau aggregation.
To understand the mechanism of action of 4d at the molecular level,
we employed nuclear magnetic resonance (NMR) spectroscopy. First, 4d
was titrated to the metal-free Aβ42 monomer or monomeric tau at
various molar ratios. In the 1H-15N heteronuclear single quantum cor-
relation (HSQC) spectra of Aβ42 and tau, no significant spectral changes
were detected when 4d was added (Fig. S6). This means that 4d might
not bind directly to monomeric Aβ42 or monomeric tau or might bind
very weakly. Then, we investigated the inhibitory effects of 4d on the
aggregation of Aβ42. After incubation at 25 ◦C with shaking for 4 h, a
reaction condition known to induce rapid protein aggregation, most of
the cross peaks disappeared, indicating the formation of Aβ42 aggregates
(Fig. 5A-C). Although several peaks of amino acid residues such as F4,
R5, V36, and I41 were still alive after 4 h incubation, the intensities of
those peaks were very low. However, coincubation of Aβ42 with 4d
resulted in the NMR signal different from that observed in the absence of
4d. The intensities of all cross peaks of Aβ42 after 4 h of coincubation
To examine the in vivo effects of 4d on AD pathology, 5XFAD mice
were treated with compound 4d daily for 2 months starting at the age of
5 months (Fig. 7A) [63,64]. Compound 4a-treated mice were used as a
control group. During the treatment period, the body weights of the
mice were not significantly changed, and no significant side effects were
observed (data not shown). The Morris water maze (MWM) test was
performed to identify defects in spatial learning and memory. The
vehicle-treated 5XFAD mice consistently reached the platform later than
the vehicle-treated wild-type (WT) mice, indicating that they had diffi-
culties with learning and memory.
Remarkably, administration of 4d to 5XFAD mice led to a significant
improvement in performance in the task at days 5, 6 and 7 (Fig. 7B). At
day 8, the hidden platform was removed, and a probe task was per-
formed. The number of platform crosses increased significantly in 4d-
treated 5XFAD mice compared with vehicle-treated 5XFAD mice
(Fig. 7C). The swim speed and total track length did not differ between
4

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
Fig. 5. Effects of 4d on the fibrillation of Aβ42. (A-C) The 1H-15N HSQC spectra of Aβ42 in its apo-states before incubation (A) and after incubation for 4 h at 25 ◦
C
with shaking at 200 rpm (B). The intensities of the corresponding cross peaks before and after incubation are plotted in (C). (D-F) The spectra of Aβ42 in the presence
of 4d before (D) and after incubation (E). The peak intensities of each condition are plotted in (F). The chemical shifts of Aβ42 residues are referred from the reported
backbone assignment data of Aβ42 [57,58]. The residues that were not observed in the HSQC spectra are marked with black asterisks in (C) and (F).
Fig. 6. Effects of 4d on the fibrillation of tau. The time course 1H-15N HSQC spectra of tau in the absence (A) and presence (B) of 4d for 21 h. Spectral measurements
were started immediately after the addition of heparin. Selected regions shown in (C-E) are represented by dotted rectangles. The selected residues shoiwng rapidly
decreasing intensities in the presence of 4d are enlarged in (C) and (D). (E) The residues showing the longer survivals of resonances in the presence of 4d are
highlighted by red asterisks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
Water maze
A
B
month
compound 10 mg/kg
p.o treat every days
5
6
7
5
4
3
2
1
0
C
WT + vehicle
5XFAD + vehicle
5XFAD + 4a
60
40
20
0
*
*
*
**
#
5XFAD + 4d
*
##
##
#
(days)
8
0
1
2
3
4
5
6
7
WT + v5eXhFicAleD + ve5hXiFclAeD +54XdFAD + 4a
Fig. 7. In vivo effects of polyhydroxyisoflavones in 5XFAD mice. (A) In vivo experimental schedule. (B) Escape latency of wild-type or 5XFAD mice treated with
polyhydroxyisoflavones (n = 9–13 per group). (C) The representative swimming path and number of platform crossings in the probe test (n = 9–13 per group). Data
are shown as the mean ± SEM. One-way ANOVA. *P < 0.05 and **P < 0.01 vs. saline-treated wild-type (WT) mice. #P < 0.05 and ##P < 0.01 vs. saline-treated
5XFAD mice.
Fig. 8. Representative confocal microscopy images (A) and quantification data (B) of mouse brain slices stained with thioflavin S (n = 4–6 per group, scale bar, 200
μ
m). (C) Measurement of Aβ42 protein levels using an ELISA kit (n = 4–6 per group). Data are shown as the mean ± SEM. One-way ANOVA. #P < 0.05 and ##P < 0.01
vs. saline-treated 5XFAD mice.
groups, suggesting that our polyhydroxyisoflavones did not affect motor
function (Fig. S8). Next, the effect of 4d on Aβ aggregation was inves-
tigated by observing aggregated proteins, including amyloid plaques, in
the cortex and hippocampus of 5XFAD mice. As shown in Fig. 8A and 8B,
thioflavin S-positive Aβ plaques were observed in 7-month-old 5XFAD
mice. However, thioflavin S-positive Aβ plaques were significantly
decreased in both the cortex and hippocampus regions of 4d-treated
5XFAD mice (Fig. 8A and 8B). In the 4a-treated 5XFAD mice, thioflavin
S-positive Aβ plaques were slightly reduced, but the difference did not
reach statistical significance (Fig. 8A and 8B). The anti-Aβ aggregation
effect of 4d was further confirmed by human Aβ42 ELISA (Fig. 8C).
Immunostaining using a 6E10 antibody showed similar results,
supporting the reduction of Aβ plaques by 4d (Fig. 9). Activation of glial
cells, including astrocytes and microglia, is also considered to contribute
to AD pathology. In our experiments, the levels of gliosis induced by
astrocytes and microglia were increased in 5XFAD mice compared to WT
mice, as expected, but these levels did not change after 4d treatment,
indicating that 4d acts directly on Aβ and tau without glial cell changes
(Fig. 10 and Fig. 11). Collectively, our observations and data reveal that
4d treatment attenuates Aβ aggregation and improves memory deficits
in 5XFAD mice.
6

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
5XFAD + 4a
5XFAD + 4d
5XFAD + vehicle
5XFAD + vehicle
5XFAD + 4a
5XFAD + 4d
4
3
2
1
0
#
#
1
2
Hippocampus
Cortex
Fig. 9. Representative confocal microscopy images and quantification data of mice brain slices stained with 6E10 antibody (scale bar = 200
Data are shown as mean ± SEM. One-way ANOVA. #P < 0.05 vs. saline-treated 5XFAD mice.
μm, n = 4–6 per group).
WT + vehicle
5XFAD + vehicle
5XFAD + 4a
5XFAD + 4d
WT + vehicle
5XFAD + vehicle
5XFAD + 4a
5
4
3
2
1
0
5XFAD + 4d
**
*
*
**
**
**
1
2
Hippocampus
Cortex
Fig. 10. Representative confocal microscopy images and quantification data of glial activation in the mice brain. (B) Immunofluorescence images and quantification
of GFAP (scale bar = 200 m, n = 4–5 per group). Data are shown as mean ± SEM. One-way ANOVA. *P < 0.05 and **P < 0.01 vs. saline-treated WT mice.
μ
WT + vehicle
5XFAD + vehicle
5XFAD + 4a
5XFAD + 4d
WT + vehicle
5XFAD + vehicle
5XFAD + 4a
5XFAD + 4d
2.5
2.0
1.5
1.0
0.5
0.0
*
*
*
*
*
*
Hippocampus
Cortex
Fig. 11. Representative confocal microscopy images and quantification data of glial activation in the mice brain. Immunofluorescence images and quantification of
Iba-1 (scale bar = 200
μm, n = 4–5 per group). Data are shown as mean ± SEM. One-way ANOVA. *P < 0.05 vs. saline-treated WT mice.
3. Conclusion
4. Experimental section
A series of polyhydroxyisoflavones were expediently synthesized and
evaluated biologically. An in vitro ThT assay and TEM analysis
demonstrated that ortho-catechol containing isoflavones were identified
as a privileged scaffold that directly prevents the aggregation of both Aβ
and tau. By utilizing 1H-15N HSQC spectra, we investigated the mode of
action of the catechol-containing isoflavone, 4d, during the protein
aggregation process. Finally, 4d was shown to alleviate cognitive
impairment and pathologies related to AD in a 5XFAD transgenic mouse
model.
4.1. Chemistry
Unless stated otherwise, all chemicals were obtained from com-
mercial suppliers (Sigma Aldrich, TCI, Alfa Aesar and Acros Organics)
and used as received. All reactions were performed under argon atmo-
sphere. 1H NMR was measured and obtained using a Bruker 400 and 13
C
NMR spectra was measured on a Varian VNMRS500 spectrometer. 1H
and 13C NMR chemical shifts were determined relative to the signal of
the residual solvent peak used as an internal reference. Signals are
7

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
recorded as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d
= doublet, t = triplet, m = multiplet, br = broad). Coupling constants (J)
were reported in Hertz (Hz). High-resolution mass spectrometry (HRMS)
data were recorded using a JEOL JMS-700 instrument in the EI or ESI
mode. Flash column chromatography was performed using silica gel 60
(230-400mesh) and analytical thin layer chromatography (TLC) was
performed using TLC Silica gel 60 F254. Optical Spectral were measured
by NanoDrop One UV/VIS Spectrophotometer (Thermo Fisher
Scientific).
described above. Purification by chromatography on silica gel afforded
1b as a colorless oil (149 mg, 32% yield (79% yield brsm)); 1H NMR
(CDCl3, 400 MHz): δ 7.75 (d, 1H, J = 5.9 Hz), 7.22 (d, 1H, J = 2.3 Hz),
6.84 (d, 1H, J = 2.2 Hz), 6.21 (d, 1H, J = 6.0 Hz), 2.42 (s, 3H), 2.34 (s,
3H); 13C NMR (DMSO‑d6, 125 MHz): δ 169.97, 169.41, 168.87, 157.58,
155.52, 154.75, 149.98, 115.55, 114.41, 110.92, 110.35, 21.41, 21.32;
HR-MS: (EI + ) calcd for C13H10BrO6, 340.9661; found, 340.9662.
4.1.3. General procedure for the preparation of 3a-3g
3-Bromochromone (0.5 mmol, 1 equiv), benzeneboronic acid (1.5
equiv), palladium acetate (0.3 equiv), potassium carbonate (4 equiv)
and SPhos (0.5 equiv) were added in a round-bottomed flask. DMF/H2O
(3:1, 5 mL) was added under argon atmosphere. The reaction was stirred
at 90 ◦C under argon atmosphere until completion of the reaction. After
cooling, the solution was quenched with 2 N aqueous HCl solution and
extracted by EtOAc. The organic phase was combined and dried with
anhydrous Na2SO4, and then filtered. The filtrate was evaporated under
reduced pressure and purified by silica gel flash column
chromatography.
4.1.1. General procedure for the preparation of compounds 6a and 6b
To a solution of the corresponding hydroxychromone(0.5 mmol, 1
equiv) in dry CH2Cl2 (3 mL) was added triethylamine (1.5–2.5 equiv),
acetyl chloride (1.5–2.5 equiv) and a catalytic amount of 4-DMAP under
argon atmosphere. The resulting solution was stirred at room tempera-
ture for the appropriate time. After the reaction was complete, the so-
lution was then diluted with CH2Cl2 and quenched with 2 N aqueous HCl
solution. The organic phase was combined and dried with anhydrous
MgSO4. The solution was filtered and evaporated under reduced pres-
sure. The residue was purified by silica gel flash column
chromatography.
4.1.3.1. 7-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one 3a. 3a was
prepared from 1a (50 mg, 0.177 mmol) and (4-methoxyphenyl)boronic
acid (40 mg, 0.263 mmol) using the general procedure described above.
Purification by chromatography on silica gel afforded 3a as a white solid
(25 mg, 53%); 1H NMR (DMSO‑d6, 400 MHz): δ 8.33 (s, 1H), 7.97 (d, 1H,
J = 8.8 Hz), 7.51 (d, 2H, J = 8.7 Hz), 6.99 (d, 2H, J = 8.8 Hz), 6.93 (d,
1H, J = 8.8 Hz), 6.86 (s, 1H), 3.79 (s, 3H).
4.1.1.1. 4-oxo-4H-chromen-7-yl acetate 6a. 6a was prepared from 7-
hydroxy-4H-chromen-4-one (100 mg, 0.617 mmol) using the general
procedure described above. Purification by chromatography on silica gel
afforded 6a as a white solid (99 mg, 79% yield); 1H NMR (CDCl3, 400
MHz): δ 8.23 (d, 1H, J = 8.8 Hz), 8.39 (d, 1H, J = 6.1 Hz), 7.28 (d, 1H, J
= 2.2 Hz), 7.16 (dd, 1H, J = 8.7, 2.1 Hz), 6.34 (d, 1H, J = 6.0 Hz), 2.34
(s, 3H); 13C NMR (CDCl3, 125 MHz): δ 176.99, 168.67, 157.04, 155.61,
154.62, 127.36, 122.75, 119.67, 113.21, 111.24, 21.26; HR-MS: (EI + )
calcd for C11H8O4, 204.0423; found, 204.0422.
4.1.3.2. 5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one 3b. 3b
was prepared from 1b (190 mg, 0.559 mmol) and (4-methoxyphenyl)
boronic acid (127 mg, 0.836 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
3b as a pale yellow solid (29 mg, 18%); 1H NMR (CDCl3, 400 MHz): δ
12.93 (s, 1H), 7.86 (s, 1H), 7.46 (d, 2H, J = 8.9 Hz), 6.98 (d, 2H, J = 8.8
Hz), 6.37 (d, 1H, J = 2.3 Hz), 6.30 (d, 1H, J = 2.1 Hz) 3.85 (s, 3H).
4.1.1.2. 4-oxo-4H-chromene-5,7-diyl diacetate 6b. 6b was prepared
from 5,7-dihydroxy-4H-chromen-4-one (300 mg, 1.684 mmol) using the
general procedure described above. Purification by chromatography on
silica gel afforded 6b as a white solid (408 mg, 92% yield); 1H NMR
(CDCl3, 400 MHz): δ 7.75 (d, 1H, J = 5.9 Hz), 7.22 (d, 1H, J = 2.3 Hz),
6.84 (d, 1H, J = 2.2 Hz), 6.21 (d, 1H, J = 6.0 Hz), 2.42 (s, 3H), 2.34 (s,
3H); 13C NMR (CDCl3, 125 MHz): δ 175.87, 169.61, 168.10, 158.01,
154.48, 154.04, 150.30, 115.98, 114.15, 113.98, 109.26, 21.27, 21.16;
HR-MS: (EI + ) calcd for C13H11O6, 263.0556; found, 263.0557.
4.1.3.3. 3-(3,4-dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one 3c. 3c
was prepared from 1a (98 mg, 0.346 mmol) and (3,4-dimethoxyphenyl)
boronic acid (95 mg, 0.522 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
3c as a white solid (41 mg, 40%); 1H NMR (DMSO‑d6, 400 MHz): δ 8.37
(s, 1H), 7.97 (d, 1H, J = 8.7 Hz), 7.19 (s, 1H), 7.12 (dd, 1H, J = 8.3, 1.9
Hz), 7.0 (d, 1H, J = 8.3 Hz), 6.94 (dd, 1H, J = 8.8, 2.3 Hz), 6.87 (d, 1H, J
= 2.0 Hz) 3.78 (s, 6H).
4.1.2. General procedure for the preparation of 1a and 1b
To a solution of the corresponding acetoxychromones (0.5 mmol, 1
equiv) and potassium acetate (4 equiv) in AcOH (1.5 mL) was added N-
bromosuccinimide (1.2 equiv) slowly under argon atmosphere. The re-
action mixture was stirred at room temperature for the appropriate time.
After the reaction, the solution was diluted with EtOAc and carried out
aqueous workup. The organic phase was combined and dried with
anhydrous MgSO4. The solvent was filtered and evaporated under
reduced pressure. The residue was purified by silica gel flash column
chromatography.
4.1.3.4. 3-(3,4-dimethoxyphenyl)-5,7-dihydroxy-4H-chromen-4-one
3d. 3d was prepared from 1b (156 mg, 0.459 mmol) and (3,4-dime-
thoxyphenyl)boronic acid (125 mg, 0.687 mmol) using the general
procedure described above. Purification by chromatography on silica gel
afforded 3d as a white solid (87 mg, 60%); 1H NMR (DMSO‑d6, 400
MHz): δ 12.95 (s, 1H), 10.90 (s, 1H), 8.41 (s, 1H), 7.17 (s, 1H), 7.12 (d,
1H, J = 8.1 Hz), 7.02 (d, 1H, J = 8.2 Hz), 6.40 (s, 1H), 6.24 (s, 1H), 3.78
(s, 6H).
4.1.2.1. 3-bromo-4-oxo-4H-chromen-7-yl acetate 1a. 1a was prepared
from 6a (484 mg, 2.37 mmol) using the general procedure described
above. Purification by chromatography on silica gel afforded 1a as a
white solid (166 mg, 25% yield); 1H NMR (CDCl3, 400 MHz): δ 8.29 (d,
1H, J = 8.8 Hz), 8.22 (s, 1H), 7.32 (d, 1H, J = 2.1 Hz), 7.21 (dd, 1H, J =
8.8, 2.1 Hz), 2.36 (s, 3H); 13C NMR (DMSO‑d6, 125 MHz): δ 171.38,
169.17, 156.67, 156.40, 155.28, 127.47, 121.44, 120.83, 112.27,
110.13, 21.44; HR-MS: (EI + ) calcd for C11H7BrO4, 281.9528; found,
281.9526.
4.1.3.5. 3-(3,4-dimethoxyphenyl)-4H-chromen-4-one 3e. 3e was pre-
pared from commercially available 3-bromo-4H-chromen-4-one (100
mg, 0.444 mmol) and (3,4-dimethoxyphenyl)boronic acid (121 mg,
0.665 mmol) using the general procedure described above. Purification
by chromatography on silica gel afforded 3e as a white solid (89 mg,
71%); 1H NMR (CDCl3, 400 MHz): δ 8.32 (dd, 1H, J = 8.0, 1.6 Hz), 8.03
(s, 1H), 7.69 (m, 1H), 7.49 (d, 1H, J = 8.5 Hz), 7.44 (m, 1H), 7.22 (d, 1H,
J = 2.0 Hz), 7.07 (dd, 1H, J = 8.2, 2.0 Hz), 6.94 (d, 1H, J = 8.3 Hz), 3.94
(s, 3H), 3.92 (s, 3H).
4.1.2.2. 3-bromo-4-oxo-4H-chromene-5,7-diyl diacetate 1b. 1b was pre-
pared from 6b (360 mg, 1.374 mmol) using the general procedure
4.1.3.6. 5,7-dihydroxy-3-phenyl-4H-chromen-4-one 3f. 3f was prepared
8

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
from 1b (48 mg, 0.141 mmol) and phenylboronic acid (26 mg, 0.213
mmol) using the general procedure described above. Purification by
chromatography on silica gel afforded 3f as a pale yellow solid (13 mg,
36%); 1H NMR (DMSO‑d6, 400 MHz): δ 12.89 (s, 1H), 10.97 (s, 1H), 8.44
(s, 1H), 7.56 (m, 2H), 7.45 (m, 3H), 6.42 (s, 1H), 6.24 (s, 1H); 13C NMR
(DMSO‑d6, 125 MHz): δ 180.45, 165.00, 162.54, 158.14, 155.55,
131.38, 129.54, 128.77, 128.58, 122.84, 104.99, 99.66, 94.34; HR-MS:
(EI + ) calcd for C15H10O4, 254.0579; found, 254.0573.
4.1.4.5. 3-(3,4-dihydroxyphenyl)-4H-chromen-4-one 4e. 4e was pre-
pared from 3e (75 mg, 0.266 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
4e as a yellow solid (30 mg, 44%); 1H NMR (DMSO‑d6, 400 MHz): δ 8.32
(dd, 1H, J = 8.0, 1.6 Hz), 8.04 (s, 1H), 7.71 (m, 1H), 7.51 (d, 1H, J = 8.9
Hz), 7.45 (t, 1H, J = 7.4 Hz), 7.23 (d, 1H, J = 1.7 Hz), 6.89 (m, 2H), 6.72
(s, 1H), 5.81 (s, 1H); 13C NMR (DMSO‑d6, 125 MHz): δ 175.89, 156.08,
154.19, 145.95, 145.39, 134.56, 126.06, 125.92, 124.55, 124.34,
123.25, 120.40, 118.89, 117.08, 115.87; HR-MS: (EI + ) calcd for
C15H10O4, 254.0579; found, 254.0578.
4.1.3.7. 3-phenyl-4H-chromen-4-one 3g. 3 g was prepared from 3-
bromo-4H-chromen-4-one 1c (100 mg, 0.444 mmol) and phenylboronic
acid (81 mg, 0.664 mmol) using the general procedure C described
above. Purification by chromatography on silica gel afforded 3 g as a
white solid (85 mg, 86%); 1H NMR (CDCl3, 400 MHz): δ 8.32 (dd, 1H, J
= 8.0, 1.6 Hz), 7.69 (m, 1H), 7.58 (m, 2H), 7.51–7.38 (m, 5H); 13C NMR
(CDCl3, 125 MHz): δ 176.36, 156.30, 153.21, 133.75, 131.94, 129.07,
128.63, 128.32, 126.53, 125.49, 125.37, 124.67, 118.17; HR-MS: (EI + )
calcd for C15H10O2, 222.0681; found, 222.0684.
4.1.5. UV/Vis spectrophotometer analysis
For UV/Vis spectrophotometer investigating the interaction of 4d
with Cu(II), the analysis was conducted in a Chelex-treated HEPES
buffer solution (10 mM HEPES, 100 mM NaCl, pH 7.4) [49]. The solu-
tion of 4d (100 µM) was titrated up to 4 equiv of CuCl2 at room tem-
perature. The spectraphotometer anylasis was conducted 5 min after the
addtion of CuCl2.
4.2. Biological evaluation
4.1.4. General procedure for the preparation of 4a-4e
Mixture of corresponding isoflavone (0.1 mmol) and pyridine hy-
drochloride (1.34 g) in a round-bottomed flask was heated and stirred at
150 ◦C for the appropriate time. After the reaction was completed, the
solution was cooled and poured into 2 N aqueous HCl solution. After
aqueous workup, extraction with EtOAc was performed and dried with
Na2SO4. The solution was filtered and evaporated under reduced pres-
sure. The residue was purified by flash chromatography on silica gel.
Thioflavin T (ThT), Thioflavin S, anhydrous dimethyl sulfoxide
(DMSO), triton X-100, paraformaldehyde, phosphate buffer, phosphate-
buffered saline (PBS, pH 7.4), Heparin sodium salt from porcine intes-
tinal mucosa, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and Guanidine
hydrochloride were purchased from Sigma Aldrich (St. Louis, MO, USA).
Fluorescence mounting medium was purchased from Dako (Santa Clara,
CA, USA). Chelex was purchased from Bio-rad (Bio-Rad Laboratories,
Hercules, CA, USA). Amyloid beta (Aβ) 1–42 peptide was purchased
from Anaspec (Fremont, CA, USA).
4.1.4.1. 7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one 4a. 4a was
prepared from 3a (20 mg, 0.075 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
daidzein 4a as a white solid (7.2 mg, 38%); All spectroscopic data were
consistent with previously reported data [51].
4.2.1. Aβ peptides preparation
To ensure the monomeric state of the peptide, Aβ peptides were
dissolved in HFIP, incubated for 3 days at room temperature. The sol-
vent was then removed by speed vacuum. The monomerized Aβ was
stored as powder at ꢀ 80 ◦C until use.
4.1.4.2. 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one 4b. 4b
was prepared from 3b (23 mg, 0.082 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
4b as a pale yellow solid (12.5 mg, 56%); 1H NMR (DMSO‑d6, 400 MHz):
δ 12.96 (s, 1H), 8.33 (s, 1H), 7.38 (d, 2H, J = 8.5 Hz), 6.82 (d, 2H, J =
8.6 Hz), 6.39 (d, 1H, J = 2.0 Hz), 6.22 (d, 1H, J = 2.0 Hz); 13C NMR
(DMSO‑d6, 125 MHz): δ 180.76, 164.83, 162.53, 158.12, 157.95,
154.55, 130.71, 122.80, 121.73, 115.59, 104.98, 99.50, 94.20; HR-MS:
(EI + ) calcd for C15H10O5, 270.0528; found, 270.0526.
4.2.2. Tau purification
Wild-type human tau (2N4R isoform, 441 residues) pET29b plasmid
was purchased from Addgene (#16316). The plasmid was transformed
into Escherichia coli strain BL21 (DE3) Codon Plus cells. Cells were
grown at 37 ◦C in LB medium containing 50 mg/mL kanamycin until the
OD600 reached 0.6, then overexpression was induced by adding 0.5 mM
isopropyl β-D-thiogalactopyranoside (IPTG), followed by 4 h of incuba-
tion at 37 ◦C. Cells were harvested by centrifugation at 7500g for 10 min.
Collected pellets were resuspended in lysis buffer (25 mM Tris-HCl pH
6.8, 500 mM NaCl, 1 mM EDTA, 1 mM MgCl2, 5 mM dithiothreitol
(DTT)) and disrupted by sonication. The lysates were boiled for 20 min
and clarified by centrifugation at 36,000g for 45 min and cell debris was
removed. The supernatant was diluted to a final salt concentration of 50
mM NaCl, then purified using anion exchange column (HiTrap Q HP, 1
× 5 mL, GE Healthcare Life Sciences) equilibrated with 25 mM Tris-HCl
pH 6.8, 50 mM NaCl, 1 mM EDTA, 1 mM MgCl2, 2 mM DTT, 0.2 mM
4.1.4.3. 3-(3,4-dihydroxyphenyl)-7-hydroxy-4H-chromen-4-one 4c. 4c
was prepared from 3c (32 mg, 0.107 mmol) using the general procedure
described above. Purification by chromatography on silica gel afforded
4c as a pale yellow solid (4.2 mg, 15%); 1H NMR (DMSO‑d6, 400 MHz): δ
10.80 (bs, 1H), 8.99 (bs, 2H), 8.26 (s, 1H), 7.96 (d, 1H, J = 8.7 Hz), 7.02
(s, 1H), 6.93 (d, 1H, J = 8.6 Hz), 6.86 (s, 1H), 6.78 (m, 2H); 13C NMR
(DMSO‑d6, 125 MHz): δ 175.21, 163.00, 157.89, 153.34, 145.79,
145.31, 127.85, 124.14, 123.51, 120.36, 117.18, 117.12, 115.81,
115.63, 102.61; HR-MS: (EI + ) calcd for C15H10O5, 270.0528; found,
270.0527.
¨
phenylmethylsulfonyl fluoride (PMSF) in an AKTAprime plus (GE
Healthcare Life Sciences). Bound proteins were eluted with a salt
gradient from 50 mM to 1 M NaCl. Further purification was performed
using HiLoad 16/600 Superdex 200 prep-grade column in an
4.1.4.4. 3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one
4d. 4d was prepared from 3d (65 mg, 0.227 mmol) using the general
procedure described above. Purification by chromatography on silica gel
afforded 4d as a pale yellow solid (45.6 mg, 70%); 1H NMR (DMSO‑d6,
400 MHz): δ 13.00 (s, 1H), 10.88 (s, 1H), 9.08 (s, 1H), 9.01 (s, 1H), 8.29
(s, 1H), 6.99 (s, 1H), 6.78 (m, 2H), 6.38 (s, 1H), 6.22 (s, 1H); 13C NMR
(DMSO‑d6, 125 MHz): δ 180.78, 164.77, 162.55, 158.07, 154.49,
146.06, 145.43, 122.94, 122.16, 120.49, 117.07, 115.91, 105.00, 99.47,
94.16; HR-MS: (EI + ) calcd for C15H10O6, 286.0477; found, 286.0472.
¨
AKTApurifier (GE Healthcare Life Sciences) with PBS buffer. Purified tau
protein was concentrated by ultrafiltration (Amicon, 10 kDa cut-off,
Millipore) up to 2.6 mg/mL.
4.2.3. Recombinant tau protein preparation
Various human tau cDNA was constructed in a pRK172 vector based
on the longest form of human wild-type tau encoded 441 amino acid
(2N4R); deletion of amino acids at positions 252 to 376 (DMTBR), po-
sitions 306 to 311 (DPHF6), positions 275 to 280 (DPHF6*) and with
9

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
substitutions at C291A and C322A (C291, 322A). A construct that
encoded 2N3R tau with a mutation at C322A (2N3R-C322A) was pro-
duced. Each recombinant tau was expressed in Escherichia coli BL21
(DE3) and purified by modified method reported previously. After E. coli
expressing tau was sonicated and boiled, recombinant tau proteins in the
heat-stable fraction was purified by ion-exchange chromatography (P11;
GE Healthcare, or Cellufine Phosphate; JNC Corp.), ammonium sulfate
fractionation, gel filtration chromatography (NAP10 column; GE
Healthcare) and reverse phase-HPLC (COSMOSIL Protein-R Waters;
Nacalai Tesque Inc.). After freeze-drying, recombinant tau proteins were
dissolved in milliQ water and stored at ꢀ 80 ◦C as a stock solution.
Fibrillation of Aβ42 was accelerated by shaking the samples with 200
rpm at 25 ◦C without addition of an agent. The 15N-labeled Aβ42 (25
μM)
in the presence or absence of 4d (100 μM) were incubated with shaking
for 4 h and 12 h, respectively. After incubation, the 2D 1H-15N HSQC
spectra of Aβ42 were obtained at 5 ◦C.
For NMR titration of 4d to tau, uniformly 15N-labeled tau was pre-
pared by growing the cells in M9 minimal medium containing 1 g/L
15NH4Cl. The 15N-labeled tau was expressed and purified as described
above. Final NMR samples were dissolved in a buffer containing 50 mM
sodium phosphate pH 6.4, 50 mM NaCl, 2.5 mM DTT, 2.5 mM EDTA, 3%
d6-dimethyl sulfoxide (DMSO) and 10% D2O. 4d was added to the 50
μ
M 15N-labeled tau with molar ratios of 1:2 (100
μM), 1:4 (200 μM), 1:6
4.2.4. ThT fluorescence assay
(300
μ
M) and 1:12 (600
μ
M). The 2D 1H-15N HSQC spectra were
The ThT binding activity of some compounds was determined in
black 96-well flat-bottom plates. For Aβ ThT assays examining the effect
of compounds on aggregation, Aβ (10 µM) was incubated in the absence
or presence of CuCl2 or ZnCl2 (10 µM) and compounds (30 µM) in a
collected at 20 ◦C with 8 scans per increment and 1024 X 256 points in
the 1H and 15N dimensions, respectively.
To induce the fibrillation of tau, 0.3 mg/mL of heparin was added to
50
μ
M
15N-labeled tau in the presence or absence of 200
μM of 4d,
HEPES solution (20 mM, 150 mM NaCl, pH 7.4) containing ThT (20
μ
M)
respectively. NMR measurement was started immediately after addition
of heparin. The 2D 1H-15N HSQC experiments of 15N-labeled tau were
recorded at 37 ◦C. The HSQC spectra of 15N-labeled tau in the presence
or absence of 4d were acquired consecutively for 21 h. All NMR spectra
were measured on a Bruker AVANCE III 800-MHz spectrometer equip-
ped with a cryogenic probe, and NMR data were processed using
NMRPipe and NMRDraw software, and spectrum analysis was per-
formed by NMRView J program.
[52]. The plates were incubated at 37 ◦C without shaking. At specific
time points, fluorescence generated by the binding of ThT to Aβ aggre-
gates was measured using a plate reader (FLUOstar Omega, BMG lab-
tech, Ortenberg, Germany) at excitation and emission wavelengths of
430 and 492 nm, respectively [26,65]. For Tau ThT assays examining
the effect of compounds on aggregation, Recombinant wild-type 2N4R
tau (10 μM), compounds (30 μM) and ThT (20 μM) were mixed in the a
HEPES solution (10 mM, 100 mM NaCl, pH 7.4), and incubated with
heparin (0.06 mg/mL) at 37 ◦C without shaking. At specific time points,
fluorescence generated by the binding of ThT to tau aggregates was
measured using a plate reader at excitation and emission wavelengths of
430 and 492 nm, respectively [51]. When primary inner filter effect
(IFE) might be necessary to be mainly considered in the reaction con-
dition, we applied a below equation to our experimental results,
following Beer-Lambert’s law [66,67].
4.2.7. Animals and treatment
5XFAD mice were purchased from Jackson Laboratory (stock num-
ber: 034840-JAX, Bar Harbor, ME, USA). Mice were housed in plastic
containers under constant temperature (23 ± 1 ◦C) and humidity (60 ±
10%), in a 12-h light/dark cycle with free access to food and water.
5XFAD transgenic mice overexpress human APP695 with three familial
mutations (Swedish [K670N, M671L], Florida [I716V] and London
[V717I]) and human presenilin 1 with two familial mutations (M146L
and L286V) under the control of the murine Thy1 promotor (Moechars,
Lorent, De Strooper, Dewachter, & Van Leuven, 1996). Five-month-old
5XFAD female mice (22 ± 2 g) and male mice (30 ± 2 g) were injec-
ted intraperitoneally with 10 mg/kg of compound in saline (0.9% NaCl)
per day for eight weeks. All animal studies were performed in accor-
dance with the “Principles of Laboratory Animal Care” (National In-
stitutes of Health publication number 80–23, revised 1996) and
approved by the Animal Care and Use Guidelines Committee of Kyung
Hee University (approval number: KHUASP(SE)-17–126-1).
Fcorr = Fobsexp[(Aexc + Aem ) / 2]
4.2.5. Transmission electron microscopy (TEM)
Monomerized Aβ were dissolved in HEPES buffer (20 mM, 150 mM
NaCl, pH 7.4) to a final protein concentration of 10 µM and incubated in
the absence or presence compounds (30 µM) at 37 ◦C for 24 h without
agitation [42]. For Tau ThT assays examining the effect of compounds
on aggregation, recombinant wild-type 2N4R tau (10 μM), compounds
(30 μM) were mixed in the a HEPES solution (10 mM, 100 mM NaCl, pH
7.4), and incubated with heparin (0.06 mg/mL) at 37 ◦C without
shaking [51].
4.2.8. Morris water maze
Morris water maze (MWM) task was used to assess spatial memory
performance. The water maze was a white tank (1.0 m in diameter, 40
cm in height) filled to a depth of 30 cm with water (22–24 ◦C). White,
opaque, nontoxic paint was used to hinder visibility. A submerged
plexiglas platform (10 cm in diameter, placed 6–8 mm below the surface
of the water) was located at a fixed position throughout the training
session. The position of the platform varied from mouse to mouse while
being counterbalanced across experiment groups. All mice were habit-
uated to the maze one day before training. The training session consisted
of a series of three trials per day for seven consecutive days, for a total of
21 trials. In each of the three trials, the animals were placed at different
starting positions that were equally spaced around the perimeter of the
pool in a random order. The mouse was given 60 sec to find the sub-
merged platform. If the mouse did not mount the platform within 60 sec,
it was guided to the platform. The time to mount the platform was
recorded as latency for each trial. Mice were allowed to remain on the
platform for 10 sec before being returned to a home cage. On the eight
day, a single probe trial, in which the platform was removed, was per-
formed after the hidden platform task was completed. Each mouse was
placed into one quadrant of the pool and allowed to swim for 60 sec. All
trials were recorded using a charge-coupled device camera connected to
Samples were prepared for TEM studies by spotting aliquots (5 µL) of
the aggregation assay onto 200 mesh Carbon coated Cupper grid (SPI
supplies, West Chester, PA). The samples were then blotted with a filter
paper and allowed to dry. The dried samples were negatively stained
with 2% uranyl acetate in water (5 µL) for 30 s, washed with water,
blotted with a filter paper, and dried again. Samples were then analyzed
by a Tecnai G2 F20 TEM (FEI Tecnai™ G2, Hillsboro, Oregon) operated
at 200 kV [42,68].
4.2.6. NMR experiments
For NMR titration of 4d to Aβ42, uniformly 15N-labeled Aβ42 peptide
was purchased from rPeptide (A-1102–2). 100 μL of 1% NH4OH was
added to 1 mg of the lyophilized 15N-labeled Aβ42 powder, and the so-
lution was diluted with NMR buffer (20 mM Tris-HCl pH 7.4, 50 mM
NaCl and 10% D2O). 50
μ
M 15N-labeled Aβ42 in NMR buffer was titrated
by 3 (150 μM), 6 (300 μM), 9 (450 μM) and 12 (600 μM) molar equiv-
alents of 4d. The 2D 1H-15N heteronuclear single quantum correlation
(HSQC) spectra were measured at 5 ◦C with 16 scans per increment and
2048 X 128 points in the 1H and 15N dimensions, respectively.
10

S.H. Son et al.
Bioorganic Chemistry 113 (2021) 105022
a video monitor and a computer and analyzed by the free tracking
software tool Toxtrac (Rodriguez, Zhang, Klaminder, Brodin, & Ander-
sson, 2017). The software, user manual, and documentation are avail-
able at https://toxtrac.sourceforge.io. The operators responsible for
experimental procedure and data analysis were blinded and unaware of
group allocation throughout the experiments
Declaration of Competing Interest
The author declare that there is no conflict of interest.
Acknowledgments
This work was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIT) (NRF-
2020R1F1A1064755 and 2020R1A2C300888911).
4.2.9. Brain tissue preparation
Mice were euthanized after behavioral testing by administration of a
mixture of ketamine and xylazine in saline (0.9% NaCl) as the anes-
thetic, and cardiac perfusion was performed immediately using 4%
paraformaldehyde in PBS. After perfusion, brains were removed, post-
fixed overnight at 4 ◦C, and incubated in 30% sucrose at 4 ◦C until
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105022.
equilibration. Sequential 30-μm-thick coronal sections were prepared by
a cryostat (CM1850; Leica, Wetzlar, Germany) and stored at ꢀ 20 ◦C.
References
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