On the role of synthesized hydroxylated chalcones as dual functional amyloid-β aggregation and ferroptosis inhibitors for potential treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.01.039

In addition to amyloid cascade hypothesis, ferroptosis – a recently identified cell death pathway associated with the accumulation of lipid hydroperoxides – was hypothesized as one of the main forms of cell death in Alzheimer's disease. Herein, a series o

Full text of DOI:10.1016/j.ejmech.2019.01.039

Accepted Manuscript
On the role of synthesized hydroxylated chalcones as dual functional amyloid-β
aggregation and ferroptosis inhibitors for potential treatment of Alzheimer's disease
Lin Cong, Xiyu Dong, Yan Wang, Yulin Deng, Bo Li, Rongji Dai
PII:
S0223-5234(19)30049-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.039
EJMECH 11046
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2018
Revised Date: 15 January 2019
Accepted Date: 15 January 2019
Please cite this article as: L. Cong, X. Dong, Y. Wang, Y. Deng, B. Li, R. Dai, On the role of synthesized
hydroxylated chalcones as dual functional amyloid-β aggregation and ferroptosis inhibitors for potential
treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.039.
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On the Role of Synthesized Hydroxylated Chalcones as Dual
Functional Amyloid-β Aggregation and Ferroptosis Inhibitors for
Potential Treatment of Alzheimer’s Disease
Lin Conga, Xiyu Dongb, Yan Wanga, Yulin Denga, Bo Lia,b*, and Rongji Daia*
a Beijing Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceuticals, School of
Life Science, Beijing Institute of Technology, Beijing, P.R. China
b Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing,
P.R. China
Abstract
In addition to amyloid cascade hypothesis, ferroptosis – a recently identified cell death pathway
associated with the accumulation of lipid hydroperoxides – was hypothesized as one of the main
forms of cell death in Alzheimer’s disease. Herein, a series of hydroxylated chalcones were
designed and synthesized as dual-functional inhibitors to inhibit amyloid-β peptide (Aβ)
aggregation as well as ferroptosis simultaneously. Thioflavin-T assay indicated trihydroxy
chalcones inhibited Aβ aggregation better. In human neuroblastoma SH-SY5Y cells,
cytoprotective chalcones 14a-c with three hydroxyl substituents exhibited a significant
neuroprotection against Aβ1-42 aggregation induced toxicity. In addition, chalcones 14a-c were
found to be good inhibitors of ferroptosis induced by either pharmacological inhibition of the
hydroperoxide-detoxifying enzyme Gpx4 using (1S, 3R)-RSL4 or cystine/glutamate antiporter
−
system Xc inhibition by erastin through lipid peroxidation inhibition mechanism. Trihydroxy
chalcone 14a was also able to completely subvert lipid peroxidation induced by Aβ1-42 aggregation
in SH-SY5Y cells indicating that they can reduce the neurotoxicity involved with oxidative stress.
Compound 14a-c showed good ADMET properties and blood-brain barrier penetration in silico
simulation software. From these data, a picture emerges wherein trihydroxy chalcones are
potential candidates for the treatment of Alzheimer’s disease by simultaneously inhibition of
Aβ1-42 aggregation and ferroptosis.
Keywords: chalcone derivatives; amyloid-β aggregation inhibition; ferroptosis inhibition;
Alzheimer’s Disease
1. Introduction
Alzheimer’s disease (AD) is a chronic, progressive and irreversible neurodegenerative disease
characterized by memory loss, language disorders, severe behavioral abnormalities and learning

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deficits[1, 2]. Approximately 44 million people are affected by AD worldwide, and this number is
expected to increase to 114 million by 2050[3]. Specific cause of AD remains unclear due to the
complicated nature and diversified factors involved in the mechanism of AD, making it difficult to
find an effective cure for AD. In the last three decades, U.S. Food and Drug Administration (FDA)
have approved five drugs to enter the market for AD treatment. Most of them are cholinesterase
inhibitors including tacrine, donepezil, rivastigmine and galantamine[4]. The other FDA approved
drug to treat AD is memantine, an effective N-methyl-D-aspartic acid receptor (NMDA)
antagonist[5]. These drugs help improve the cognitive and memory function in patients, but they
can’t reverse the effects of deterioration. Therefore, it seems likely that prevention of AD or halt
the progress of AD at the early stage may provide reasonable strategies to help patients suffering
from AD.
The most recognized hypothesis of AD is amyloid cascade hypothesis, which proposes that the
aggregation and accumulation of amyloid-β (Aβ) in brain causes neuronal cell death[6]. One of
the main features of AD is senile plaques, the product of Aβ aggregation. Amyloid-β is generated
by cleavage of the amyloid precursor protein (APP) by β-secretase (BACE-1) and γ-secretase[7].
Normally, Aβ peptide is a protein consisting of 39 to 43 amino acids, which aggregates into
oligomers, protofibrils and plaques. It is suggested that oligomers consisting of Aβ1-42 peptides is
more neurotoxic than the other Aβ peptides[8]. This provides a therapeutic treatment for AD by
directly inhibition of Aβ aggregation or secretase.
Recent research has suggested a strong interplay between Alzheimer’s disease and the recently
characterized cell death pathway ferroptosis[9, 10]. Ferroptosis, identified in 2012, is a form of
programmed cell death which is morphologically, biochemically and genetically distinct from
other cell death mechanisms such as apoptosis or autophagy[11, 12]. The features of ferroptosis
include the accumulation of lipid hydroperoxides and iron dysregulation, which also exists in
majority of neurodegenerative diseases[13, 14]. In recent years, ferroptosis has been revealed to be
associated with neurodegeneration disease, (i.e., Alzheimer’s, Huntington’s, and Parkinson’s
diseases) [15] and it has been proved to be a specific target for treating senile dementia patients[9,
16].
The progress of AD is accompanied by oxidative stress which eventually drives neuronal cell
death in the brain of AD patients[17]. Numerous studies have confirmed that oxidative stress
precedes the main neuropathologic manifestation of AD and may enhance the level of disease
hallmarks, such as the development of amyloid plaques[18]. With the development of the concept
of ferroptosis in recent years, it is reasonable to infer that oxidative stress induces cellular
ferroptosis, which drives the progression of disease[13]. The development of novel ferroptosis
inhibitors are currently considered as promising treatments for neurodegenerative disease. In
particular, Aβ induces lipid peroxidation leading to increased amyloidogenesis through
up-regulating BACE-1[19, 20]. Accordingly, radical-trapping antioxidants that inhibit lipid
peroxidation will be beneficial for the treatment of AD[21, 22].
In the past decade, many researchers have devoted their efforts to develop small molecules to
treat AD by either inhibiting the aggregation of Aβ or lipid peroxidation [23, 24]. Some of the
small molecules are polyphenolic natural products such as epigallocatechin gallate (EGCG 1),
resveratrol (2) and curcumin (3) (Figure 1), which have been shown to have neuroprotective
effect through protecting neurons from the Aβ-induced damages or attenuating neuronal death by
oxidative stress[25]. Chalcones have been proved to exhibit a variety of pharmacological

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properties including antioxidant activity, anti-inflammatory, antitumor and neuroprotection
activity[26, 27]. Chalcone derivatives are the main ingredients in traditional Chinese medicine,
Dragon’s Blood, which has been proved to be a potential therapeutic agent of neurodegenenerative
diseases [28, 29]. In addition, we have found that the extract of Dragon’s Blood could stimulate
neurogenesis and improve learning capacity in our lab (unpublished data). These results suggest
that chalcone derivatives are promising candidates for the prevention and treatment of AD, which
has yet to be studied.
Figure 1. Literature reported Aβ aggregation inhibitors 1-3 and chalcone
Given the essential role of Aβ peptide aggregation and ferroptosis in the pathology of AD,
herein, we synthesized a small library of chalcone derivatives in order to inhibit Aβ peptide
aggregation and ferroptosis simultaneously, which has never been reported. Their ability to inhibit
Aβ peptide aggregation in solution was assayed using the Thioflavin-T method[30]. The
promising compounds were studied in human SH-SY5Y neuroblastoma cell lines for their anti-Aβ
aggregation activity and cytotoxicity property. Their ferroptosis inhibition activity was also
assayed in cells. The preferential compounds have shown better drug-like properties than the lead
compound EGCG and Curcumin in virtual simulation, which supports their potential to pass
through blood-brain barrier and perform better in vivo. To the best of our knowledge, it is for the
first time that the concept of inhibition ferroptosis and Aβ aggregation simultaneously is brought
into the molecular design for the treatment of AD. Compared to other lead compounds, we expect
that these hydroxylated chalcone derivatives provide better cytoprotection of neurons for AD
treatment in animal models in the near future.
2. Result and Discussion
2.1 Synthesis of chalcone derivatives
We focused on a series of chalcone derivatives 6a-b, 10a-c, 13a-c and 14a-c as the targeted
dual-functional compounds to inhibit Aβ aggregation and ferroptosis. The synthetic routes are
shown in Scheme 1. The targeted compounds 6a-b in Scheme 1 (A) with one hydroxyl
substitution group were directly obtained from the corresponding acetophenones and
benzaldehydes through the Claisen–Schmidt condensation reaction[31]. The above-described
reaction was performed in 10 % KOH aqueous solution with good yields. To afford chalcones with
more hydroxyl groups (Scheme 1 (B) and (C)), the hydroxyl groups were protected first by
reaction with methoxymethyl chloride before reaction with the corresponding benzaldehyde

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derivatives[32]. Methoxymethoxy (MOMO)-protected acetopheones (8 and 12) were then reacted
with the corresponding benzaldehydes in ethanol to obtain the desired compounds (9a-b and
13a-c). Deprotection of the MOMO group was performed using 10 % HCl in refluxing methanol
to yield the targeted chalcones with two or three hydroxyl groups with good yields (10a-b and
14a-c). These chalcones are stable at room temperature.
Scheme 1. Synthesis of chalcones 6a-b, 10a-b, 13a-c and 14a-c. Reaction conditions: (a)
CH3OCH2Cl, K2CO3, acetone, reflux, 4 h; (b) 10 % KOH aqueous, ethanol, rt, 48-96 h; (c) 10 %
HCl, methanol, reflux, 20-40 min.
2.2 Inhibition of self-mediated Aβ1-42 aggregation
Amyloid-β aggregation is one of the prominent pathological features of Alzheimer’s Disease.
The amyloid hypothesis suggests that accumulation of aggregated Aβ in brain causes neuronal cell

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death [33]. Effectively inhibition of Aβ aggregation plays key role in relieving and retarding the
symptom of Alzheimer’s Disease. With the synthesized chalcones 6a-b, 10a-b, 13a-c and 14a-c in
hands, their ability to inhibit Aβ aggregation in solution was first assayed using the thioflavin-T
(ThT) fluorescence method wherein EGCG and curcumin were used as reference standards[25].
ThT is a benzothiazole salt which is widely used to visualize and quantify the presence of
misfolded Aβ protein aggregates. When ThT binds to β-rich structures, such as those in Aβ
aggregates, it displays enhanced fluorescence (λex=440 nm; λem=485 nm).
Fluorescence of the aqueous solution of Aβ peptide in the presence and absence of the tested
chalcones at 25 µM was monitored. Inhibitory activities shown as the percent of fluorescence
compared to untreated samples are summarized in Figure 2. Compounds 14a-c with three
hydroxyl substitutions in ring A exhibited higher inhibitory activity against Aβ aggregation, with
percentages of inhibition ranging from 76.3 % to 70.3 %, compared to EGCG at 52.9 % and
curcumin at 51.4 %, respectively. We also examined the inhibitory activities of MOMO-protected
chalcones 13a-c to highlight the importance of additional hydroxyl groups in chalcones. Results
indicated that 13a-c could barely inhibit Aβ aggregation with only 16.4 % inhibition or less, which
indicated the key role of three hydroxyl groups in ring A. Compounds 10a-b with two hydroxyl
substitutions exhibited a tiny better inhibition potency (with 27.2 % and 19 % inhibition,
respectively) than compounds 6a-b having only one hydroxyl substitution. In summary, the Aβ
aggregation inhibitory activities of compounds we studied follow the trend 14a-c > 10a-b > 6a-b
≈ 13a-c, which is consistent with the decreasing number of hydroxyls in chalcones.
Figure 2. Inhibition activities of compounds on self-mediated Aβ1-42 aggregation tested using ThT
at 1:1 ratio with compounds (25 µM) at 37 for 2 days. The extent of aggregation is shown as a
percentage of the untreated Aβ1-42 control. Data are reported as the mean ± SD of at least three
independent experiments, (∗∗) p < 0.01, (∗∗∗) p < 0.001 vs Aβ1-42 group; EGCG and curcumin
were assayed as positive controls.
Hydrophobicity, π-stacking and hydrogen bonding interactions are all important factors for the
anti-Aβ aggregation activities[34, 35]. Coplanarity of aromatic rings and rigid planar structure also
attribute to intercalate with Aβ peptides[36]. We assumed that two aromatic rings and one
hydrogen bond acceptor carbonyl group in chalcones provided their potency to inhibit Aβ peptide
aggregation. Aromatic rings in chalcones could provide hydrophobic and π-stacking interactions

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with Aβ peptides. The hydroxyl groups in ring A and carbonyl groups between two benzene rings
also provide hydrogen bonding interactions between the compounds and Aβ peptides. Our results
with ThT fluorescent probes supported that hydrogen bonding between the compounds and Aβ
peptide is key in the inhibitory ability. More hydroxyl groups in chalcones provide additional
hydrogen bonding donor positions, resulting in enhanced inhibition.
2.3 Neuronal cell protection activities
To transit results to cellular models, we further examined whether chalcone derivatives could
protect cells from Aβ1-42-induced toxicity in SH-SY5Y cells. EGCG and curcumin were again
used as reference compounds. Aβ1-42 (10 µM) was added to the growth medium and incubated
with SH-SY5Y cells for 2 days. As indicated in Figure 3A, cell viability was reduced to 60.4 %
compared to untreated cells upon treatment with Aβ peptide. Chalcone derivatives 10a-b, 13a-c
and 14a-c at 20 µM and 10 µM (6a-b was excluded due to their little activity shown in ThT assays)
were added to the medium for 4 h prior to the addition of Aβ1-42. Cell viability results indicated
that compounds 14a-c exhibited better inhibition of Aβ aggregation than curcumin and EGCG
(76.0 % and 92.5 %, respectively), which protect neural cells almost completely from
Aβ1-42-induced toxicity at 10 µM (106.2 % ~ 96.5 %). Compounds 14a-c provided much better
protection than other compounds, which were in line with the results of ThT experiments. Given
the foregoing results, it is concluded that compounds 14a-c protected neural cells from
Aβ1-42-induced cytotoxicity by inhibiting the aggregation of Aβ1-42. Compared to 14a-c,
compounds 13a-c with three MOMO-protected hydroxyl groups maintained only 72.0 % - 84.9 %
cell viability at 10 µM, indicating the importance of hydroxyl groups as hydrogen bonding donors
in the inhibition activities. Treatment of SH-SY5Y cells with compounds 10a-b provided less
neuroprotective activities compared to 14a-c with three hydroxyl groups, supporting the ThT
results.
Figure 3. Neuroprotection of selected compounds at different concentrations (range 20-0.5 µM)
against Aβ1-42-induced toxicity with 48 h incubation. The results are reported as a percentage of
vehicle-treated cells. Values are shown as the mean ± SD of at least three independent experiments,
(∗∗) p < 0.01 vs vehicle-treated cells group; EGCG and curcumin were assayed as positive
controls.
Since compounds 14a-c could completely protect neural cells from Aβ1-42-induced toxicity at 10
µM, we tried to examine the protective activities of 14a-c at lower concentrations. Compounds
14a-c at 1 µM showed a moderate protection (with cell viability at 77.5 % ~ 70.8 %) compared to

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Aβ1-42 treated group (with cell viability at 55.1 %), which was better than EGCG (with cell
viability at 63.0 %). When the concentration of compounds 14a-c was decreased to 0.5 µM, the
protective effect declined slightly (with cell viability at 66.7 % ~ 73.8 %). However, cell viability
of cells treated with EGCG at the same concentration was only 60.1 % (Figure 3B). Micrographs
of cells demonstrated that compounds 14a-c at 1 µM indeed protected neural cells from
Aβ1-42-induced cytotoxicity (Figure 4). Cells treated with Aβ1-42 peptide tended to aggregate which
indicated that Aβ1-42 peptide aggregation induced cytotoxicity (Figure 4B). Cells treated with Aβ
peptide but in the presence of compounds 14a-c protected cells from aggregation (Figure 4C, D,
E).
Figure 4. Representative micrographs of cells incubated with Aβ1-42 alone or with both Aβ1-42 (10
µM) and selected compounds (1 µM): (A) Vehicle, (B) Aβ1-42, (C) Aβ1-42 and 14a, (D) Aβ1-42 and
14b, (E) Aβ1-42 and 14c.
To further investigate neurotoxicity of chalcones, cell viabilities of human neuroblastoma
SH-SY5Y cells exposed to each of compounds for 24 h at 40 µM or 10 µM were examined using
the MTS assay. To reference the cytotoxicity of chalcones, doxorubicin was chosen as an
anticancer drug which exhibited higher cytotoxicity at 40 µM and 10 µM with cell viability at
37.3 % and 36.6 %, respectively [37]. As shown in Figure 5, mono-hydroxylated 6a had little
toxicity at both 40 µM or 10 µM, with cell viability at 73.6 % ~ 77.4 %. All the rest of tested
compounds did not exhibit observed cytotoxic effect.

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Figure 5. Cytotoxicity of selected compounds at different concentrations (40 µM and 10 µM) with
24 h incubation. The results are reported as a percentage of control cells. Values are shown as the
mean ± SD of at least three independent experiments, (∗∗) p < 0.01 vs vehicle-treated cells group;
EGCG and curcumin were assayed as positive controls.
Although it has been shown that chalcones with three hydroxyl groups could effectively inhibit
Aβ peptides aggregation, on the other hand, three hydroxyl groups may render the compound
more difficult to pass through BBB and enter into the brain of patients[38, 39]. We used the
ADMET Tool of DS to predict the ability of the designed compounds to penetrate BBB[40]. As
shown in Table 1, compared to the lead compounds EGCG and curcumin, level of BBB
penetration of most designed chalcones ranged from medium to high, which means the
Brain-Blood ratio was between 5:1 and 0.3:1. In addition, all the designed compounds satisfy
Lipinsiki and Veber’s rule, indicating that they fulfill the drug-like necessity.
Table 1. Physical properties and ADMET prediction of the selected compounds.
Compound
MWa
ClogPa
HBAa
HBDa
PSAa
Absorption
Solubility
BBB
Levelb
Levelb
Levelb
EGCG
Curcumin
6a
458.372
368.38
3.097
3.554
3.946
3.427
3.462
3.616
2.959
3.113
3.64
11
6
2
4
4
6
5
7
4
6
3
4
8
2
1
1
3
1
3
1
3
1
2
2
197.36
93.06
37.29
55.76
77.76
74.22
86.99
83.45
77.76
74.22
57.53
66.76
3
0
0
0
0
0
0
0
0
0
0
0
1
3
2
3
3
3
3
3
3
2
3
3
4
3
1
1
2
2
3
2
2
2
1
2
238.281
284.307
270.28
6b
14a
13a
358.385
286.279
374.384
290.698
378.804
254.281
270.28
14b
13b
14c
3.794
3.704
3.201
13c
10a
10b
a MW: molecular weight; Clog P: calculated logarithm of the octanol-water partition coefficient; HBA:
hydrogen-bond acceptor atoms; HBD: hydrogen-bond donor atoms; PSA: polar surface area;
b Absorption level (0: good, 3: very poor); Solubility level (0: extremely low, 3: good); BBB level(1: high, 2:

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medium, 3: low and 4: undefined);
2.4 Inhibition of cellular lipid peroxidation and ferroptosis
The accumulation of lipid hydroperoxides (LOOH) has long been implicated in cell death and
dysfunction, initiating or deteriorating neurodegenerative disease including Alzheimer’s
disease[19]. However, only recently has a novel cellular death form – ferroptosis been directly
linked to the accumulation of lipid peroxidation associated with neurodegenerative disease[41]. It
has been recently elucidated that ferroptosis is one of the main pathogenic mechanism of AD.
Effective anti-ferroptotic molecules are protective in animal models to treat neurodegenerative
disease, and anti-ferroptotic iron chelator deferiprone is currently being tested in a phase 2
randomized controlled trial for AD[42].
Lipid peroxidation is a free radical chain reaction involving with initiation (Scheme 2 Eq1) and
propagation steps (Scheme 2 Eq 2-3). The hydroxyl groups in chalcone derivatives could react
with lipid peroxyl radicals by H-atom transfer to inhibit lipid peroxidation through the mechanism
shown in Scheme 2 Eq4-5[43].
Scheme 2. Mechanism of lipid peroxidation and its inhibition by chalcone (e.g., compound 6a)
To provide more insight on the potencies of chalcones as lipid peroxidation inhibitors, we
carried out experiments to examine whether chalcones with best Aβ1-42. aggregation inhibitory
activities 14a-c could inhibit lipid peroxidation in cellular models. We chose compound 14a as an
example. To corroborate that Aβ1-42-induced cells express higher rate of lipid peroxidation,
commercial available fluorescent probe C11-BODIPY581/591 was used as a lipid peroxidation
indicator[44]. Upon lipid peroxidation, the fluorescence of C11-BODIPY581/591 (λex = 488 nm; λem
= 525 nm) is increased (Scheme 3).

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Scheme 3. The reaction of C11-BODIPY581/591 with peroxyl radicals.
As shown in Figure 6A, incubating cells with Aβ1-42 (10 µM) for two days induced higher level
of lipid peroxidation by 54.0 %. The fluorescence decreased to untreated control level when the
cells were incubated with Aβ1-42 (10 µM) in the presence of compound 14a (10 µM) for 48 h,
indicating that increased level of lipid peroxidation induced by Aβ aggregation was inhibited by
compound 14a. These results revealed that 14a can not only inhibit the aggregation of Aβ1-42, but
also inhibit lipid peroxidation derived therefrom.
We further replaced Aβ1-42 with (1S,3R)-RSL3 to examine the ability of 14a-c to inhibit
ferroptosis – a recently recognized cell death form associated with lipid peroxidation.
(1S,3R)-RSL3 stimulated cellular ferroptosis by pharmacological inhibition of the
hydroperoxide-detoxifying enzyme Gpx4. The results were in agreement with the above analysis.
When cells were treated with RSL3 (500 nM) for one day, the fluorescence of the sample
increased indicating the level of lipid peroxidation increased compared to the untreated cells.
Whereas compounds 14a-c could reduce the level of lipid peroxidation to the normal level as
untreated cells (Figure 6B). These results served as more direct affirmation that 14a-c could
inhibit lipid peroxidation derived from ferroptosis effectively.
Figure 6. Histogram obtained from flow cytometry (3 × 105 cells/ml; λex = 488 nm, λem = 525 ±
25 nm; 10,000 events) following induction of oxidative stress with Aβ (15 µM) in SH-SY5Y cells
(A) or RSL3 (5 µM) in HEK-293 cells (B) grown in media containing 14a-c (10 µM) for 24~48 h.
Cells treated with Vehicle were used as positive control (red).
Given that chalcones 14a-c with three hydroxyl groups could inhibit lipid peroxidation revealed
by flow cytometry, we assayed their inhibitory activity of ferroptosis in cellular models thoroughly.

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Cellular ferroptosis was induced in HEK-293 cells by either pharmacological inhibition of
glutathione peroxidase 4 (Gpx4) using (1S,3R)-RSL3 or glutathione (GSH) depletion by the
system xc- inhibitor erastin[45, 46]. The results shown in Table 2 demonstrated that compounds
14a-c were effective inhibitors of (1S,3R)-RSL3 induced ferroptosis. In contrast, EGCG didn’t
show any ferroptosis inhibitory activity at concentrations up to 20 µM. Compound 14a (IC50 =
0.45 µM) exhibited the highest inhibitory activity than 14b and 14c (IC50 = 1.77 µM ~ 0.86 µM).
Compounds 14a-c also inhibited erastin-induced ferroptosis with IC50 at 3.15 µM ~ 3.88 µM.
These results indicated that 14a is a better dual-functional inhibitor for ferroptosis and Aβ
aggregation simultaneously associated with AD.
Table 2. Inhibition of (1S,3R)-RSL3 and erastin induced ferroptosis by the selected compoundsa.
Compounds
Inhibition of ferroptosis
IC50 ± SD (µM) for (1S,3R)-RSL3
IC50 ± SD (µM) for erastin
induced ferroptosisb
3.81 ± 0.11
induced ferroptosisb
0.45 ± 0.05
1.77 ± 0.01
0.86 ± 0.01
Nc
14a
14b
3.88 ± 0.15
14c
3.15 ± 0.08
Nc
EGCG
aCell viabilities were determined 24 h post induction by MTS assay.
bIC50: 50 % inhibition concentration. Results are shown as the mean of at least three experiments.
cN means no active. Compounds defined “not active” meant that percent inhibition was less than
10 % at 20 µM.
3. Conclusions
Herein we have presented a series of chalcone derivatives functionalized with various number
of hydroxyl groups in ring A and different substitutions in ring B, which were synthesized in a
expeditious manner in order to achieve the first time reported candidates for the treatment of AD
by inhibition of Aβ aggregation and ferroptosis simultaneously. We determined how systematic
changes in the number of hydroxyl groups and substitution groups impact their inhibitory
activities of Aβ aggregation and ferroptosis. It was found that chalcones with different number of
hydroxyl substitutions have a quite different inhibitory activities of Aβ1-42 aggregation, with more
hydroxyl groups providing increasing activities. Chalcone derivatives 14a-c exhibited low toxicity
and better neuroprotection against Aβ1-42-induced neurotoxicity than the lead compounds EGCG
and curcumin in the SH-SY5Y cellular assays. In addition, they also displayed potent
anti-ferroptotic cell death activities by lipid peroxidation inhibition in cellular assays. Among
them, compound 14a with three hydroxyl groups in ring A and methyl group in ring B prevented
ferroptosis with the lowest IC50 values. Due the predicted good BBB permeability, we expect that
compound 14a is a potent molecule for further evaluation in vivo in the near future. Given the
recent hypothesis that ferroptosis is the main form of cell death in Alzheimer’s disease, our results
may indicate a novel idea using dual functional inhibitors for Aβ peptide aggregation and
ferroptosis in molecular design.
4. Experimental Section
Materials
Reagents and solvents were purchased from commercial sources and used as received.

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Reactions were monitored by thin layer chromatography on precoated silica gel GF254 plates
under 254 nm UV light. Column chromatography was performed with silica gel (200-300 mesh).
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded using Bruker Avance III 400
spectrometer at 700 MHz, 400 MHz and 175 MHz with tetramethylsilane (TMS) as the internal
standard at Analysis & Testing Center of Beijing Institute of Technology. High resolution mass
spectra (HRMS) were obtained with an Agilent 6520 LCMS-Q-TOF mass spectrometer at
Analysis & Testing Center of Beijing Institute of Technology.
General procedure for the synthesis of intermediates 8 and 12[32]
Hydroxyacetophenone（1 mmol）and K2CO3（3 mmol）were added to dry acetone (30 mL). The
reaction mixture was cooled on ice bath for 30 min until the solid was dissolved completely.
Methoxymethyl chloride (4 mmol) was added dropwise when stirring, and the reaction mixture
was subsequently refluxed for 4 h. Upon completion of the reaction as indicated by TLC, the
resulting solution was cooled to room temperature and then filtered to remove the precipitated
salts. Acetone was evaporated under reduced pressure and the crude product was purified by silica
gel column chromatography using ethyl acetate/petroleum ether (1:5) as eluent to afford the pure
product.
General procedure for the synthesis of intermediates 9a-b, 13a-c and chalcones 6a-b
Acetophenone (0.5 mmol) was dissolved in ethanol (20 mL), and an aqueous solution of KOH
（10 % w/v, 10 mL）was added. After the solid was dissolved completely, aryl aldehyde (0.6
mmol) was added to the mixture. The reaction mixture was then stirred at room temperature for
48-96 h. Upon completion of the reaction as indicated by TLC, the mixture was acidified with HCl
(10 % v/v aqueous solution) to pH 7, and then extracted with 3×50 mL ethyl acetate. The
combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated to
yield crude product which was purified by silica gel chromatography (gradient ethyl
acetate/petroleum ether from 1/10 to 1/3).
General procedure for the synthesis of chalcones 10a-b and 14a-c
Methoxymethoxy-protected chalcones (0.5 mmol) was added to methanol (20 mL), and HCl
(10 % v/v aqueous solution, 5 mL) was added dropwise when the mixture was refluxed for 20-40
min. After cooled down to room temperature, the mixture was diluted with water (40 mL), and
extracted with 3×50 mL of ethyl acetate. The product was recrystallized from ethyl acetate in
petroleum ether (1:1) to yield the products as yellow solid.
(E)-1-(3-hydroxyphenyl)-3-(m-tolyl)prop-2-en-1-one (6a)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:3) as
yellow powder with 74.2 % yield; mp 87 – 89 ; 1H NMR (700 MHz, CDCl3) δ 7.82 (d, J = 15.7
Hz, 1H, Hβ), 7.72 (dd, J = 2.3, 1.7 Hz, 1H, H2), 7.59 (d, J = 7.7 Hz, 1H, H6), 7.52 (d, J = 15.7 Hz,
1H, Hα), 7.44 (s, 1H, H8), 7.43(d, J = 7.7 Hz, 1H, H12), 7.39 (t, J = 7.9 Hz, 1H, H5), 7.31 (t, J = 7.7
Hz, 1H, H11), 7.24 (d, J = 7.4 Hz, 1H, H10), 7.18 (dd, J = 8.1, 2.5Hz, 1H, H4), 2.40 (s, 3H, CH3);

ACCEPTED MANUSCRIPT
13C NMR (100 MHz, CDCl3) δ 191.54, 156.76, 146.06, 139.29, 138.61, 134.54, 131.68, 129.89,
129.28, 128.83, 125.82, 121.63, 120.86, 120.77, 115.42, 21.28; HRMS (EI) m/z calcd for
C16H14O2 (M+) 239.0994, found 239.1056.
(E)-1-(3,4-dimethoxyphenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one (6b)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:2) as
1
yellow powder with 61.9 % yield; mp 120 – 122 ; H NMR (700 MHz, CDCl3) δ 7.75 (d, J =
15.5 Hz, 1H, Hβ), 7.67 (s, 1H, H3), 7.54 (d, J = 7.6 Hz, 1H, H2), 7.34(d, J = 15.3 Hz, 1H, Hα), 7.31
(d, J = 7.7 Hz, 1H, H5), 7.16 (d, J = 8.2 Hz, 1H, H12), 7.13 (d, J = 8.0 Hz, 1H, H4), 7.10 (s, 1H, H8),
6.84 (d, J = 8.3 Hz, 1H, H11), 3.89 (s, 3H, OCH3), 3.88 (s, 3H, OCH3); 13C NMR (175 MHz,
CDCl3) δ 191.30, 156.83, 151.61, 149.18, 145.92, 139.59, 129.86, 127.69, 123.46, 120.69, 120.52,
119.80, 115.39, 111.16, 110.31, 55.99, 55.97; HRMS (EI) m/z calcd for C17H16O4 (M+) 285.1049,
found 285.1113.
(E)-1-(3,5-bis(methoxymethoxy)phenyl)-3-(m-tolyl)prop-2-en-1-one (9a)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:10) as
1
yellow powder with 74.5 % yield; mp 78 – 80 ; H NMR (400 MHz, CDCl3) δ 13.31 (s, 1H,
OH), 7.83(d, J = 15.3 Hz, 1H, Hβ), 7.82 (d, J = 9.1 Hz, 1H, H6), 7.53 (d, J = 15.5 Hz, 1H, Hα),
7.42 (d, J = 6.1 Hz, 2H, H8, H12), 7.29 (t, J = 7.8 Hz, 1H, H11), 7.22 (d, J = 7.5 Hz, 1H, H10), 6.63
(d, J = 2.4 Hz, 1H, H3), 6.57 (dd, J = 8.9, 2.4 Hz, 1H, H5), 5.20 (s, 2H, CH2), 3.47 (s, 3H, OCH3),
2.38 (s, 3H, CH3); 13C NMR (175 MHz, CDCl3) δ 192.07, 166.25, 163.66, 144.84, 138.69, 134.69,
131.64, 131.40, 129.16, 128.90, 119.97, 114.97, 108.24, 103.94, 94.03, 56.42, 21.36; HRMS (EI)
m/z calcd for C20H22O5(M+) 343.1467, found 343.1518.
(E)-1-(3,5-bis(methoxymethoxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (9b)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:10) as
yellow powder with 64.2 % yield; mp 221 – 223 ; 1H NMR (400 MHz, CDCl3) δ 13.28 (s, 1H,
OH), 7.81(d, J = 15.4 Hz, 1H, Hβ), 7.80 (d, J = 9.0 Hz, 1H, H6), 7.52 (d, J = 15.4 Hz, 1H, Hα),
7.31 (t, J = 7.9 Hz, 1H, H12), 7.23 (d, J = 7.7 Hz, 1H, H8), 7.12 (s, 1H, H11), 6.95 (dd, J = 8.1, 2.4
Hz, 1H, H10), 6.62 (d, J = 2.4 Hz, 1H, H3), 6.57 (dd, J = 8.9, 2.4 Hz, 1H, H5), 5.20 (s, 2H, CH2),
3.83 (s, 3H, OCH3), 3.47 (s, 3H, OCH3); 13C NMR (175 MHz, CDCl3) δ 191.97, 166.24, 163.70,
159.97, 144.51, 136.09, 131.40, 130.00, 121.17, 120.48, 116.39, 114.92, 113.68, 108.27, 103.93,
94.02, 56.42, 55.34; HRMS (EI) m/z calcd for C20H22O6(M+) 359.1416, found 359.1497.
(E)-1-(3,5-dihydroxyphenyl)-3-(m-tolyl)prop-2-en-1-one (10a)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:5) as
1
yellow powder with 67.4 % yield; mp 126 – 128 ; H NMR (400 MHz, MeOD) δ 7.94 (d, J =
8.9 Hz, 1H, H6), 7.76 (d, J = 15.5 Hz, 1H, Hβ), 7.70 (d, J = 15.5 Hz, 1H, Hα), 7.49 (s, 1H, H8),
7.48 (d, J = 8.7 Hz, 1H, H12), 7.28 (t, J = 7.5 Hz, 1H, H11), 7.21 (d, J = 7.5 Hz, 1H, H10), 6.42 (dd,
J = 8.9, 2.4 Hz, 1H, H5), 6.31 (d, J = 2.4 Hz, 1H, H3), 2.36 (s, 3H, CH3); 13C NMR (175 MHz,

ACCEPTED MANUSCRIPT
MeOD) δ 191.95, 166.24, 165.21, 143.97, 138.49, 134.85, 132.19, 131.03, 128.93, 128.51, 125.49,
120.13, 113.30, 107.89, 102.46, 19.95; HRMS (EI) m/z calcd for C16H14O3(M+) 255.0943, found
255.1020.
(E)-1-(3,5-dihydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (10b)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:5) as
1
yellow powder with 73.4 % yield; mp 146 – 148 ; H NMR (400 MHz, MeOD) δ 7.92 (d, J =
8.9 Hz, 1H, H6), 7.73 (d, J = 15.5 Hz, 1H, Hβ), 7.67 (d, J = 15.5 Hz, 1H, Hα), 7.32 – 7.17 (m, 3H,
H8, H11, H12), 6.93 (dd, J = 7.8, 1.8 Hz, 1H, H10), 6.41 (dd, J = 8.9, 2.4 Hz, 1H, H5), 6.31 (d, J =
2.4 Hz, 1H, H3), 3.80 (s, 3H, OCH3); 13C NMR (175 MHz, MeOD) δ 191.86, 166.22, 165.21,
160.10, 143.71, 136.22, 132.24, 129.58, 120.94, 120.58, 116.12, 113.32, 113.12, 107.91, 102.48,
54.42; HRMS (EI) m/z calcd for C16H14O4 (M+) 271.0892, found 271.0946.
(E)-1-(2-hydroxy-3,4-bis(methoxymethoxy)phenyl)-3-(m-tolyl)prop-2-en-1-one (13a)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:7) as
yellow powder with 70.7 % yield; mp 72 – 74 ; 1H NMR (400 MHz, CDCl3) δ 13.34 (s, 1H, OH),
7.84 (d, J = 15.5 Hz, 1H, Hβ), 7.65 (d, J = 9.2 Hz, 1H, H6), 7.53 (d, J = 15.5 Hz, 1H, Hα), 7.44 –
7.40 (m, 2H, H8, H12), 7.28 (t, J = 7.5 Hz, 1H, H11), 7.21 (d, J = 7.5 Hz, 1H, H10), 6.73 (d, J = 9.2
Hz, 1H, H5), 5.28 (s, 2H, CH2), 5.22 (s, 2H, CH2), 3.65 (s, 3H, OCH3), 3.50 (s, 3H, OCH3), 2.38 (s,
3H, CH3); 13C NMR (175 MHz, CDCl3) δ 192.72, 158.63, 156.38, 145.18, 138.73, 134.62, 134.02,
131.73, 129.21, 128.93, 126.12, 125.92, 119.97, 116.13, 106.06, 98.04, 94.63, 57.31, 56.53, 21.37;
HRMS (EI) m/z calcd for C20H22O6 (M+) 359.1416, found 359.1492.
(E)-1-(2-hydroxy-3,4-bis(methoxymethoxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (13b)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:5) as
yellow powder with 68.4 % yield; mp 107 – 109 ; 1H NMR (400 MHz, Acetone-d6) δ 13.59 (s,
1H, OH), 8.01 (d, J = 9.2 Hz, 1H, H6), 7.89 (d, J = 15.4 Hz, 1H, Hβ), 7.85 – 7.79 (m, 3H, Hα, H8,
H12), 7.03 (s, 1H, H9), 7.01 (s, 1H, H11), 6.80 (d, J = 9.1 Hz, 1H, H5), 5.35 (s, 2H, CH2), 5.15 (s,
2H, CH2), 3.87 (s, 3H, OCH3), 3.60 (s, 3H, OCH3), 3.49 (s, 3H, OCH3); 13C NMR (175 MHz,
CDCl3) δ 192.67, 161.96, 158.59, 156.20, 144.84, 134.02, 130.50, 127.42, 125.94, 117.68, 116.19,
114.51, 105.97, 98.04, 94.63, 57.30, 56.52, 55.47; HRMS (EI) m/z calcd for C20H22O7 (M+)
375.1366, found 375.1434.
(E)-3-(3-chlorophenyl)-1-(2-hydroxy-3,4-bis(methoxymethoxy)phenyl)prop-2-en-1-one (13c)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was isolated by silica gel chromatography using ethyl acetate/petroleum ether (1:5) as
1
yellow powder with 65.0 % yield; mp 85 – 87 ; H NMR (400 MHz, CDCl3) δ 13.18 (s, 1H,
OH), 7.79 (d, J = 15.5 Hz, 1H, Hβ), 7.64(d, J = 9.2 Hz, 1H, H6), 7.62 (s, 1H, H8), 7.54 (d, J = 15.5
Hz, 1H, Hα), 7.49 (d, J = 7.0 Hz, 1H, H12), 7.41 – 7.32 (m, 2H, H10, H11), 6.76 (d, J = 9.2 Hz, 1H,
H5), 5.30 (s, 2H, CH2), 5.22 (s, 2H, CH2), 3.66 (s, 3H, OCH3), 3.52 (s, 3H, OCH3); 13C NMR (100
MHz, CDCl3) δ 196.65, 161.94, 160.53, 146.71, 140.83, 138.60, 137.51, 134.09, 134.03, 131.91,

ACCEPTED MANUSCRIPT
130.87, 130.66, 125.80, 119.64, 109.96, 101.66, 98.34, 60.13, 59.40; HRMS (EI) m/z calcd for
C19H19ClO6 (M+) 379.0870, found 379.0940.
(E)-3-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one (14a)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was recrystallized using ethyl acetate/petroleum ether (1:1) as yellow powder with 89.6 %
1
yield; mp 153 – 155 ; H NMR (400 MHz, Acetone-d6) δ 13.53 (s, 1H, OH), 7.93 (d, J = 15.5
Hz, 1H, Hβ), 7.84 (d, J = 15.5 Hz, 1H, Hα), 7.73 (d, J = 8.9 Hz, 1H, H6), 7.68 (s, 1H, H8), 7.62 (d,
J = 7.6 Hz, 1H, H12), 7.33 (t, J = 7.6 Hz, 1H, H11), 7.26 (d, J = 7.5 Hz, 1H, H10), 6.53 (d, J = 8.9
Hz, 1H, H5), 2.37 (s, 3H, CH3); 13C NMR (175 MHz, Acetone-d6) δ 192.51, 153.44, 152.04,
144.12, 138.58, 134.95, 132.41, 131.40, 129.26, 128.85, 126.15, 122.58, 120.55, 113.86, 107.59,
20.42; HRMS (EI) m/z calcd for C16H14O4 (M+) 271.0892, found 271.0951.
(E)-3-(4-methoxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one (14b)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was recrystallized using ethyl acetate/petroleum ether (1:1) as yellow brown powder with
88.3 % yield; mp 151 – 153 ; 1H NMR (400 MHz, CDCl3) δ 13.62 (s, 1H, OH), 7.87 (d, J = 15.4
Hz, 1H, Hβ), 7.62 (d, J = 8.7 Hz, 2H, H8, H12), 7.47 (d, J = 8.9 Hz, 1H, H6),7.39 (d, J = 15.4, 1H,
Hα), 6.95 (d, J = 8.7 Hz, 2H, H9, H11), 6.57 (d, J = 8.9 Hz, 1H, H5), 3.87 (s, 3H, OCH3); 13C NMR
(175 MHz, MeOD) δ 192.60, 161.94, 153.04, 151.96, 143.72, 132.37, 130.20, 127.51, 121.97,
117.80, 114.07, 113.76, 107.20, 54.49; HRMS (EI) m/z calcd for C16H14O5 (M+) 287.0841, found
287.0917
(E)-3-(3-chlorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one (14c)
Synthesis was completed according to the general synthetic procedure mentioned above. The
product was recrystallized using ethyl acetate/petroleum ether (1:1) as yellow powder with 88.4 %
yield; mp 180 – 182 ; 1H NMR (400 MHz, MeOD) δ 7.85 – 7.72 (m, 3H, Hα, Hβ, H8), 7.66– 7.63
(m, 1H, H12), 7.59 (d, J = 8.9 Hz, 1H, H6), 7.41 (d, J = 5.0 Hz, 2H, H10, H11), 6.49 (d, J = 8.9 Hz,
1H, H5); 13C NMR (175 MHz, MeOD) δ 192.20, 153.11, 152.44, 141.77, 137.05, 134.62, 132.38,
130.11, 129.84, 127.86, 126.67, 122.33, 122.15, 113.68, 107.40; HRMS (EI) m/z calcd for
C15H11ClO4 (M+) 291.0346, found 291.0569.
Inhibition of self-mediated Aβ1-42 aggregation assay
Aβ1-42 protein was pretreated with 1,1,1,3,3,3-hexafloro-2-propanol (HFIP) and was dissolved
in DMSO as a stock solution of 1 mM. Experiments were performed by incubating the Aβ1-42
protein in 10 mM phosphate buffer (pH 7.4) containing 100 mM NaCl at 37 for 7 days at 25 µM
with or without inhibitors (25 µM, Aβ/inhibitor = 1/1). Blanks containing the tested inhibitors
were also prepared and tested. After incubation, samples were diluted to a final volume of 2.0 mL
with 50 mM glycine-NaOH buffer (pH 8.0) containing 5 µM thioflavin T. Measurement of
fluorescence intensity was carried out using fluorescence microplate reader (spectra MAX
GeminiXS, Molecular Devices) on a 96-well plate (Greiner bio-one, Germany) (λex=440 nm;
λem=485 nm). Percentage inhibition of aggregation was calculated using the formula (1-IFi/IFc) ×
100 % in which IFi and IFc are the fluorescence intensities obtained in the presence and absence of
inhibitors after subtracting the background, respectively. Each measurement was run in triplicate.

ACCEPTED MANUSCRIPT
Cell culture
SH-SY5Y cell line (human neruoblastoma cells) was cultured in DMEM/F12 media with 10 %
fetal bovine serum (FBS), 1 % non-essential amino acids and 1 % penicillin/streptomycin in a 37
incubator containing 5 % CO2 humidified atmosphere. HEK-293 cells were cultured in DMEM
media with 10 % FBS, 1 % penicillin/streptomycin and 1 mM sodium pyruvate under the same
environment with SH-SY5Y. The culture media were changed every three days.
Determination of neurotoxicity in SH-SY5Y cells
SH-SY5Y cells were seeded into 96-well plates at a density of 2×104 cells/100 µL/well. Cells
were allowed to adhere for 24 h. To test the toxicity of the compounds, cells were treated with
different concentrations of compounds for 24 h on the next day. Cell viability was determined
using the CellTiter 96 AQueous One Solution Assay (MTS assay; Promega) following the
manufacturer’s instruction. Absorbance at 490 nm was measured on a fluorescent microplate
reader 4 hours later. The cell viability was directly proportional to the absorbance of each well and
normalized by negative control group. Date are reported as the mean ± standard deviation of at
least three independent experiments.
Determination of neuroprotective activities in SH-SY5Y cells
To measure the neuroprotective activities from Aβ toxicity of the selected compounds,
SH-SY5Y cells were seeded into 96-well plates at a density of 2×104 cells/100 µL/well. Cells
were allowed to adhere for 24 h. Aβ1-42 (10 µM) was then added to the wells after addition of
different concentrations of compounds. After 48 h, cell viability was determined using MTS assay
as mentioned above. Date are reported as the mean ± standard deviation of at least three
independent experiments.
ADMET prediction
ADMET prediction was carried out using the Discovery Studio 3.1 (DS). The three-dimensional
structures of the selected compounds were constructed using ChemBio3D Ultra 12.0 software.
The ADMET properties of compounds was predicted using ADMET Tool in DS.
Determination of ferroptosis inhibition activities in HEK-293 cells
HEK-293 cells (5000 cells in 100 µL media) were seeded in a 96-well plate and incubated for
24 h. Ferroptosis was induced by (1S,3R)-RSL3 (500 nM) or erastin (10 µM) after addition of
different concentrations of compounds for 4 h. On the next day, cell viability was assessed using
the CellTiter 96 AQueous One Solution Assay (MTS assay; Promega) as mentioned above. Cell
viability was calculated by normalizing the data to vehicle group. Date are reported as the mean ±
standard deviation of at least three independent experiments.
Determination of cellular lipid peroxidation inhibition activities
SH-SY5Y (2×104 in 100 µL media) cells were seeded in a 96-well plate and incubated for 24 h.
On the next day, the cells were incubated with the tested compounds and Aβ1-42 (20 µM) or RSL3
(500 nM) for another 48 h or 24 h, respectively. Cells were treated with BODIPY-C11581/591 (1 µM)
and incubated at 37 in dark for 30 minutes. Cells were then analyzed by flow cytometry (λex =

ACCEPTED MANUSCRIPT
488 nm; λem = 525 ± 25 nm). Cells treated with diethylmaleate (DEM, 1 mM) alone but no Aβ1-42
protein or RSL3 was used as a positive control. Cells treated with Aβ1-42 or RSL3 alone but no test
compounds were used as positive control as well. Cells from untreated cultures were used as
negative control. Data were collected 10,000 events per sample and reported as the mean ±
standard deviation of at least three independent experiments.
Conflicts of Interest
There are no conflicts to declare.
Acknowledgements
We acknowledge the support by the Fund of Innovation Center of Radiation Application, China
(KFZC2018040208), National Key Scientific Instrument and Equipment Project of the Ministry of
Science and Technology of China (2013YQ03059514) and the Beijing Institute of Technology
Research Fund Program for Young Scholars. We acknowledge Prof. Hong Qing at Beijing
Institute of Technology for helpful discussions.
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Highlights
• Hydroxylated chalcones were synthesized as dual-functional inhibitors.
• Chalcones 14a-c inhibited amyloid-β aggregation at micromolar concentrations.
• Chalcones 14a-c protected neural cells against Aβ aggregation induced toxicity.
• Chalcones 14a-c protected cells from ferroptosis at micromolar concentrations.