Synthesis and biological evaluation of isoliquiritigenin derivatives as a neuroprotective agent against glutamate mediated neurotoxicity in HT22 cells

Article abstract of DOI:10.1016/j.bmcl.2020.127058

Glutamate-induced neurotoxicity is characterized by cellular Ca²⁺ uptake, which is upstream of reactive oxygen species (ROS)-induced apoptosis signaling and MAPKs activation. In the present study, we synthesized isoliquiritigenin analogs with electron-donating and electron-withdrawing functional groups. These analogs were evaluated for neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells. Among these analogs, compound BS11 was selected as a potent neuroprotective agent. Cellular Ca²⁺ concentration, ROS level, MAPKs activation and AIF translocation to the nucleus were increased upon treatment with 5 mM glutamate. In contrast, we identified that compound BS11 reduced the cellular Ca²⁺ concentration and ROS level upon glutamate exposure. Western blot analysis showed that MAPK activation was decreased by treatment with compound BS11. We further identified that cotreatment of compound BS11 and glutamate inhibited translocation of AIF to the nucleus.

Full text of DOI:10.1016/j.bmcl.2020.127058

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Synthesis and Biological Evaluation of Isoliquiritigenin Derivatives as a Neu-
roprotective Agent against Glutamate mediated Neurotoxicity in HT22 Cells
Baskar Selvaraj, Dae Won Kim, Gyuwon Huh, Heesu Lee, Kyungsu Kang, Jae
Wook Lee
PII:
S0960-894X(20)30136-0
DOI:
https://doi.org/10.1016/j.bmcl.2020.127058
BMCL 127058
Reference:
Bioorganic & Medicinal Chemistry Letters
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26 December 2019
12 February 2020
21 February 2020
Please cite this article as: Selvaraj, B., Won Kim, D., Huh, G., Lee, H., Kang, K., Wook Lee, J., Synthesis and
Biological Evaluation of Isoliquiritigenin Derivatives as a Neuroprotective Agent against Glutamate mediated
Neurotoxicity in HT22 Cells, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127058
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Synthesis and Biological Evaluation of
Isoliquritigenin Derivatives as a protective
Agent against Glutamate mediated
Neurotoxicity in HT22 cells
Leave this area blank for abstract info.
Baskar Selvaraj, Dae Won Kim, Gyuwon Huh, Heesu Lee, Kyungsu Kang, Jae Wook Lee
ROS
[Ca2+
Glutamate
]
MAPK
phosphorylation
HT22 cells
cell death
OH
O
HO
N
BS11

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and Biological Evaluation of Isoliquiritigenin Derivatives as a
Neuroprotective Agent against Glutamate mediated Neurotoxicity in HT22 Cells
Baskar Selvaraja,d,f,†, Dae Won Kimb,†, Gyuwon Huhd, Heesu Leec, Kyungsu Kange,f and Jae Wook Leea,d,f,
a Convergence Research Center for Dementia, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea.
b Department of Biochemistry, College of Dentistry, Institute of Oral Science, Gangneung Wonju National University, Gangneung, 25457, Republic of Korea.
c Department of Oral Anatomy, College of Dentistry, Institute of Oral Science, Gangneung Wonju National University, Gangneung, 25457, Republic of Korea.
d Natural Product Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea.
e Natural Informatics Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea.
f Department of Biological Chemistry, Korea University of Science and Technology, Daejun, 34113, Republic of Korea.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Glutamate-induced neurotoxicity is characterized by cellular Ca2+ uptake, which is upstream of
reactive oxygen species (ROS)-induced apoptosis signaling and MAPKs activation. In the
present study, we synthesized isoliquiritigenin analogs with electron-donating and electron-
withdrawing functional groups. These analogs were evaluated for neuroprotective effect against
glutamate-induced neurotoxicity in HT22 cells. Among these analogs, compound BS11 was
selected as a potent neuroprotective agent. Cellular Ca2+ concentration, ROS level, MAPKs
activation and AIF translocation to the nucleus were increased upon treatment with 5 mM
glutamate. In contrast, we identified that compound BS11 reduced the cellular Ca2+
concentration and ROS level upon glutamate exposure. Western blot analysis showed that
MAPK activation was decreased by treatment with compound BS11. We further identified that
cotreatment of compound BS11 and glutamate inhibited translocation of AIF to the nucleus.
Keywords:
Isoliquirtigenin
Glutamate induced neurotoxicity
HT22 mouse hippocampal neuronal cells
MAPKs
AIF
2009 Elsevier Ltd. All rights reserved.
———
 Jae Wook Lee. Tel.: +82-033-650-3514; fax: +82-033-650-3419; e-mail: jwlee5@kist.re.kr
† These authors contribute equally to this work.

was obtained by Claisen-Schmit reaction using various
benzaldehydes under basic condition. Deprotection of the methyl
ether group of intermediate 3 using 3N HCl afforded final
compounds 4 with high yields (Scheme 1). All final compounds
Oxidative stress has been known as the primary cause of
neuronal cell death, which is implicated in neurodegenerative
condition1,2 such as ischemic stroke3, amotrophic lateral sclerosis
1
were analyzed by H-NMR, 13C-NMR, and LC-MS. We initially
(ALS)4,5,6, Parkinson’s disease7,8 and Alzheimer’s disease9,10
.
introduced various aromatic aldehydes that possess electron-
withdrawing or electron-donating group. We assumed that the
electronic properties of aldehydes would influence the protective
properties of these derivatives.
Glutamate is an endogenous excitatory neurotransmitter in the
mammalian central nervous system. High concentration of
glutamate in the central nervous system induce excitotoxicity and
oxidative stress, resulting in neuronal loss.11 In glutamate-
mediated neurotoxicity, glutamate induces the accumulation of
intracellular reactive oxygen species (ROS) by inhibition of
cysteine uptake into cells via the cysteine/glutamate
antiporter.12,13 Therefore, elimination of intracellular ROS by
antioxidants, such as N-acetyl cysteine, can reduce glutamate-
induced neuronal cell death.14,15
These synthetic isoliquiritigenin derivatives were evaluated
for protective effects against HT22 cell death by glutamate
induced oxidative stress. HT22 cells were treated with various
concentration of glutamate to identify the optimal concentration
that induces about 50-60% of cell viability. In the cell based
assay, 5mM glutamate was selected as the best condition to
induce cell death by glutamate toxicity in HT22 cells. Previous
study reported that 5mM glutamate treatment seems optimal
condition in HT22 cells.36 To compare protective effects of
compounds against glutamate-induced cell death, we carried out
cell viability assay using calcein AM and propidium iodide (PI)
double staining.
Glutamate-induced oxidative stress activates various signaling
pathways including the c-Jun N-terminal kinase (JNK) and p38
mitogen-activated protein kinase (MAPK) pathways, which
affect inflammation, differentiation, proliferation and cell
death.16,17 The prolonged activation of MAPKs including p38,
ERK, and JNK can induce neuronal cell death.18 Although the
increase in intracellular ROS is implicated in neuronal cell death
via MAPK activation, this is not the only mechanism of
glutamate-induced neuronal cell toxicity; caspase-independent
apoptotic pathways mediated by translocation of apoptosis
inducing factor (AIF) from the mitochondria to the nucleus are
also invovled.19,20,21,22 Therefore, preventing elevated ROS levels
OH
O
O
O
O
i)
ii)
HO
O
O
2
1
O
O
O
O
OH
O
by chemical agents can provide
a potential therapeutic
iii)
R
R
opportunity for treating neuronal diseases.
O
HO
3
Isoliquiritigenin is a natural chalcone and is known as a major
biologically active component of Glycyrrhizae radix (licorice
roots).23,24 Isoliquiritigenin has various biological activities
including anticancer25,26, antiinflammatory27,28, neuroprotective
effects29,30and NRF2 activation effects.31,32,33 Recent studies
reported that isoliquiritigenin attenuates 6-hydroxydopamine-
induced neurotoxicity and shows neuroprotective effects against
glutamate-mediated oxidative stress in a mouse hippocampal
neuronal cell line (HT22).34 A recent study reported that
metabolites of isoliquiritigenin also potently protect against
glutamate-induced neuronal cell death.35 Therefore, we believed
that analogs of isoliquiritigenin may have potent protective
effects against glutamate-induced neurotoxicity in HT22 cells.
Despite the promising pharmacology of isoliquiritigenin, the
structure activity relationships of isoliquiritigenin analogs have
not been thoroughly studied yet. Therefore, we decided to
synthesize isoliquiritigenin analogues and evaluate their
protective effects against glutamate-induced neurotoxicity in a
mouse hippocampal neuronal cell line.
4
Scheme 1. The synthetic scheme of isoliquiritigenin derivatives. Reagent
and condition; i) DIEA 3.0eq, MOMCl 3.0eq, CH2Cl2, RT, overnight ii) 8N
NaOH, CH3CH2OH, RT, overnight iii) 3N HCl, CH3OH, 60 oC, 3h
OH
O
OH
O
OH
O
HO
OCH3
OCH3
OCH3
HO
OH
HO
BS3
O
BS2
O
BS1
O
OH
OH
OH
OH
Br
OH
OH
HO
HO
Cl
HO
HO
OCH3 HO
OCH3
BS4
O
BS5
O
BS6
O
OCH3
OCH3
OCH3
Br
OH
HO
HO
BS7
O
BS8
O
OCH3
OCH3
BS9
O
OCH3
OH
OH
OH
OH
OH
OH
O
OH
O
N
HO
N
HO
BS10
O
BS12
O
BS11
O
HO
HO
OH
R1
OH
OH
OH
OH
OCH3
HO
HO
HO
CF3
HO
HO
Figure 1. Structural modification of isoliquiritigenin.
BS13
O
BS15
O
BS14
O
OH
OH
OH
Isoliquiritigenin has a chalcone structure with 2’-,4’-,4-
hydroxyl group.23 We synthesized eighteen isoliquiritigenin
analogs including isoliquiritigenin BS1 as shown in Figure 2. At
N
H
HO
BS16
BS17
BS18
Figure 2. The structure of synthetic isoliquiritigenin derivatives
first,
intermediate
2
was
synthesized
from
2,4-
dihydroxyacetophenone using chloromethyl methyl ether and
diisopropylethylamine (DIEA) with high yield. Intermediate 3

Calcein AM is well-characterized fluorescent probe that is
permeable to live cells; cellular esterase in live cells cleaves
acetyl group off the fluorescent probe, which results in the
emission of green fluorescence. In contrast, PI is impermeable to
live cells and is commonly used for staining dead cells. Based on
the cell viability assay, a compound BS11 was selected as a
protective agent against glutamate toxicity in HT22 cells. The
other compounds show less or no protective effect in indicated
concentration or slight cytotoxicity at high concentration (Figure
3).
A compound BS1 (ISL) showed potent neuroprotective effects.
High concentration (50 μ M to 100 μ M) of BS1 showed
cytotoxicity. However,
a
compound BS11 showed
neuroprotective effects in high concentration. The compounds
(BS2 and BS3) with p-methoxy phenyl and m-, p-dimethoxy
phenyl group shows almost no neuroprotection effects. The
compounds BS5 with m-, p-dimethoxy, o-bromo phenyl group
shows neuroprotective effects at 3 μM to 6 μM. A compound
BS16 with m-hydroxy phenyl group, which has similar structure
with ISL shows no neuroprotective effects. The compound BS18
with indole group shows potent neuroprotective effects.
We next investigated whether and how the synthesized
compound BS11 could play a role in protecting HT22 cells
against excessive oxidative stress. As shown in Figure 4,
glutamate treatment significantly decreased cell viability, while
the additional compound BS11 under the same experimental
condition increases survival ratio of HT22 cells. An additional
experiment was performed using MTT. In the MTT assay data,
compound BS11 treated HT22 cells exhibited dramatically
increased cell viability against oxidative stress (Figure 4).
Figure 4. Neuroprotection effect of compound BS11 on glutamate
induced cell death on HT22 cells. HT22 cells were treated with 5 mM of
glutamate for 24h with or without compound BS11. A) Cell viability was
determined by calcein AM and propidium iodide (PI). Live cells were stained
with green color fluorescence and dead cells were stained with yellow color
fluorescence. Scale bar 200 μm. B) The protective effect of compound BS11
was determined using live and dead assay. 5 mM of glutamate treatment
induced neurotoxicity. 100 μM of BS11 treatment shows neuroprotective
effect against glutamate induced neurotoxicity. C) Cell viability was
measured by MTT. BS11 shows neuroprotective effects between 25 μM to
100 μM range. D) Microscopic images show neuroprotective effect of BS11.
5 mM of glutamate treatment induced cytotoxicity in HT22 hippocampal
neuronal cells. 100 μM of BS11 treatment shows neuroprotective effects
against glutamate induced neurotoxicity. Scale bar 100 μm.
Figure 3. The protective effects of isoliquiritigenin (ISL) derivatives were
evaluated in glutamate induced HT22 cell death. The indicated concentration
of compounds were treated to HT22 cells in the presence of 5 mM of
glutamate for 24 h. Cell viability was determined by image analysis using
calcein AM. BS1 is ISL. NAC is N-acetylcustein.
To confirm whether a compound BS11 could reduce the level
of cellular ROS, we utilized a DCFDA fluorescent probe for
FACS analysis of glutamate-treated HT22 cells. DCFDA is
usually non-fluorescent at lower ROS levels. ROS oxidize DCF

which then emit fluorescent. In this experiment, glutamate
treatment increased fluorescence intensity. However, the
compound BS11 treated HT22 cells exhibited decreased
fluorescent intensity in both FACS and image-based analysis
(Figure S1). Thus, our data demonstrated that compound BS11
can prevent excessive cellular ROS in HT22 cells.
Because glutamate-induced neuronal cell death was
dramatically suppressed by treatment with compound BS11, it is
expected that compound BS11 would decrease Ca2+ influx, which
is an upstream signal of apoptosis. HT22 cells were stained with
Fluo-3 AM to measure intracellular Ca2+. Compared with control
cells, 5mM glutamate exhibited increased green fluorescence
intensity. On the contrary, cells cotreated with compound BS11
and glutamate (5mM) exhibited decreased calcium concentration
by 50%, indicated that compound BS11 blocks Ca2+ influx
(Figure 5).
Figure 6. The effect of compound BS11 on MAPK, Bcl-2, Bax, and AIF
translocation in HT22 with glutamate induced cell death. A) Protein
expression level of p-JNK, p-ERK, and p-p38 was increased by glutamate.
Compound BS11 blocked phosphorylation of JNK, ERK, and p38. B)
Glutamate decreases Bcl-2 level and increases Bax level. However,
compound BS11 treatment increases Bcl-2 level and decreases Bax level. C)
Translocation of AIF induced by glutamate was blocked by compound BS11.
To gain insight into the mechanism of protective effect of
compound BS11, we investigated the effects of compound BS11
treatment on MAPKs and apoptosis signaling pathway proteins
using western blotting (Figure 6A).37 MAPKs and apoptosis
signaling pathways are mainly involved in the neuronal and
epithelial cell death caused by ROS.38 We therefore decided to
analyze cellular levels of JNK, p-JNK, ERK, p-ERK, p38, and p-
p38. In Figure 6A, glutamate treatment increased p-JNK, p-ERK,
and p-p38 in HT22 cells, which resulted in the activation of
MAPK signaling. In contrast, compound treatment followed by
5mM glutamate treatment decreased the level of p-JNK, p-ERK,
and p-p38 in the densitometry analysis. Therefore, we assumed
that compound BS11 could ameliorate oxidative stress-induced
HT22 cell death. In the additional western blot analysis,
glutamate treatment increased Bax level and decreased Bcl-2
level in HT22 cells. The addition of compound BS11
dramatically decreased level of Bax, pro-apoptotic protein.
Furthermore, the level of the anti-apoptotic protein Bcl-2 was
Figure 5. Compound BS11 blocked cellular Ca2+ influx induced by
glutamate. A) HT22 cells were treated with compound BS11 with or without
glutamate for 8h and then Flu-3 was treated to determine cellular Ca2+
concentration. The images were obtained by fluorescent microscope. Scale
bar 200 μ m. B) The fluorescent intensity was quantitatively analyzed and
presented as a fold change comparing with the control.

restored by cotreatment with compound BS11 (Figure 6B). In
addition, translocation of AIF to the nucleus is significantly
induced by 5mM glutamate treatment, and cotreatment with
compound BS11 decreased translocation of AIF to the nucleus
(Figure 6C).
To analyze the population of dead and apoptotic cells, we
performed FACS analysis using annexin V and PI staining. In the
process of apoptosis, phospho-lipids inside the membrane are
moved to the outer membrane and it can be stained by annexin V.
PI can permeate dead cells. Glutamate-treated cells showed a
large population of apoptotic and dead cells. However,
compound BS11 treated cells showed a reduction in the number
of apoptotic and dead cells (Figure 7).
In conclusion, we measured the protective effects of
isoliquiritigenin analogs against glutamate-induced HT22
hippocampal neuronal cell death. In cell-based assays, we
identified compound BS11 as the most potent protective agent. In
addition, western blotting analysis revealed that treatment with
compound
BS11
attenuated
glutamate-induced
HT22
hippocampal neuronal cell death by blocking MAPKs signaling.
The MAPKs signaling pathway is involved in modulating
inflammation, differentiation, proliferation, and cell death.
MAPK signaling pathway is activated by ROS, which can cause
cell death. N-acetyl cysteine (NAC), a known antioxidant, can
also reduce activation of the MAPK signaling pathway and
prevent cell death by ROS. A recent report demonstrated that
treatment with known chemical MAPK inhibitor shows
protective effects in glutamate-treated HT22 cells.39 In addition,
natural compounds also shows protective effects by inhibiting
MAPK signal cascades.37,40,41 In previous research, Jeong and et.
al. reported that NRF2/ARE pathway is involved in protecting
cells.42 However, BS11 showed no NRF2 activation effects
(Figure S2). As the efforts to develop more potent protective
agent for neuronal cells, further structure-activity relationship
studies are underway.
Figure 7. Compound BS11 prevents apoptotic cell death induced by
glutamate in HT22 cells. HT22 cells were exposed with glutamate in the
presence or in the absence of compound BS11 for 8h and then stained with
annexin V and propidium Iodide (PI). Glutamate treated cells increased the
number of apoptotic or dead cells. Cotreatment of compound BS11 reduced
the number of apoptotic or dead cells.
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