Synthesis and neuroprotective effects of novel chalcone-triazole hybrids

Article abstract of DOI:10.1016/j.bioorg.2020.104384

The development of novel neuroprotective agents is urgently needed for the treatment of neurodegenerative diseases, affecting aging individuals worldwide. In this study, a new set of chalcone-triazole hybrids (6a-g) was synthesized and evaluated for their

Full text of DOI:10.1016/j.bioorg.2020.104384

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Synthesis and neuroprotective effects of novel chalcone-triazole hybrids
Pichjira Sooknual, Ratchanok Pingaew, Kamonrat Phopin, Waralee
Ruankham, Supaluk Prachayasittikul, Somsak Ruchirawat, Virapong
Prachayasittikul
PII:
S0045-2068(20)31682-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104384
YBIOO 104384
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
27 June 2020
16 September 2020
14 October 2020
Please cite this article as: P. Sooknual, R. Pingaew, K. Phopin, W. Ruankham, S. Prachayasittikul, S. Ruchirawat,
V. Prachayasittikul, Synthesis and neuroprotective effects of novel chalcone-triazole hybrids, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104384
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Synthesis and neuroprotective effects of novel chalcone-triazole hybrids
Pichjira Sooknuala, Ratchanok Pingaewa,*, Kamonrat Phopinb,c,*, Waralee Ruankhamb,
Supaluk Prachayasittikuld, Somsak Ruchirawate,f,g, Virapong Prachayasittikulc
aDepartment of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110,
Thailand
bCenter for Research and Innovation, Faculty of Medical Technology, Mahidol University,
Bangkok 10700, Thailand
cDepartment of Clinical Microbiology and Applied Technology, Faculty of Medical
Technology, Mahidol University, Bangkok 10700, Thailand
dCenter of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol
University, Bangkok 10700, Thailand
eChulabhorn Research Institute, Bangkok 10210, Thailand
fProgram in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
gCenter of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry
of Education, Thailand
* Corresponding authors
Tel: 662-649-5000 ext 18253
E-mail: ratchanok@g.swu.ac.th (R. Pingaew)
Tel: 662-441-4376
E-mail: kamonrat.php@mahidol.ac.th (K. Phopin)

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Abstract
The development of novel neuroprotective agents is urgently needed for the treatment
of neurodegenerative diseases, affecting aging individuals worldwide. In this study, a new
series of chalcone-triazole hybrids (6a-g) were synthesized and evaluated for their biological
properties including cytotoxicity, antioxidant, anti-apoptosis, and neuroprotection using SH-
SY5Y cells. The results showed that 6a and 6e provided neuroprotection in oxidative stress-
induced neuronal cell damage. Both compounds significantly improved the morphology of
neurons and obviously increased cell survival rate of neuronal cells induced by oxidative
stress. Additionally, 6a and 6e counteracted H2O2‑induced mitochondrial dysfunction, which
was supported by maintaining mitochondrial membrane potential, attenuating BAX protein,
and increasing BCL‑2 protein within the mitochondria as well as upregulating SOD2
mitochondrial antioxidant enzyme. Interestingly, these compounds promoted neuroprotection
via SIRT-FOXO3a signaling pathway similar to resveratrol. The data indicated that the
chalcone-triazole derivatives (6a and 6e) could be considered to be promising compounds
toward the discovery of disease-modifying candidates for a neurodegenerative therapy.
Keywords: Chalcone; Triazole; Antioxidant; Oxidative stress; Neurodegeneration

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1. Introduction
Neurodegenerative disorders are characterized by memory loss, mental abilities, and
physical changes in the brain. Recently, the most considered neurodegenerative diseases are
Alzheimer’s disease (AD)
and Parkinson’s disease (PD). The incidence of patients with AD and PD have dramatically
increased around the world particularly in aging population. The medicines are available, but
with no effective treatment and side effects. Mostly, therapeutic medicines for
neurodegenerative diseases were developed based on the existing drugs to relieve symptoms
of neuronal damage (1). Addressing the global health concerns, new strategies that are able to
counteract the substantial increase of cases are still needed (2, 3).
Many lines of evidence have indicated that neurodegeneration is prone to oxidative
damage sharing common pathophysiologic mechanisms including mitochondrial dysfunction,
protein aggregation, and neuroinflammation, leading to neuronal death (1, 3). Excessive
production of reactive oxygen species (ROS) results in the damage of proteins, lipids, and
DNA, causing oxidative stress (4).
One of the neuroprotective strategies for the treatment is to discover multipotential
treatments i.e., antioxidant, anti-apoptotic, anti-inflammation, and target different mechanisms
of neurodegeneration, which could stop the progression of diseases or delay the transition from
the preclinical to the clinical stage (5). Therefore, the study has been directed to the discovery
of promising natural and synthetic compounds for treatment of neurodegenerative diseases.
Various natural and synthetic compounds i.e., chalcone and 1,2,3-triazole analogs have been
reported to exert neuroprotective effects (6-9).
Chalcone (1,3-diaryl-2-propen-1-one, Fig. 1), the flavonoid family of natural product
commonly found in vegetables, fruits, and spices, possesses a broad spectrum of biological
activities including anticancer, anti-inflammatory, antioxidant, antimicrobial, antidiabetic, and
anti-neurodegeneration (6, 7, 10-13). Chalcone based-compounds have been extensively
studied with the aim to prevent the neurodegenerative disorders (12), and suggested to be used
as a multi-functional candidate against multi-resistant AD in the future (12-29). Among them,
naturally-occurring chalcones such as licochalcones (A and E) and xanthohumol (Fig. 1) could
activate Nrf2, which is responsible for the oxidative properties (27-29).
1,2,3-Triazoles are nitrogen heterocycles existing in a diverse range of biologically
active molecules due to their distinct properties. They are capable of forming hydrogen bonds

4
with many biomolecular targets, highly stable to metabolic degradation, and less undesired
effects (8, 9, 30). The 1,2,3-triazole can be conjugated with various bioactive compounds such
as quinolone (31), triazine (32), chromenone (33), lipoic acid (34), and tacrine (35) rendering
them as multi-functional molecules for treatment of neurodegenerative disorders.
Various chalcone-triazole hybrids showing different biological activities have been
reported. The linking of chalcone and 1,2,3-triazole units leads to the hybrids with better
biological activities than their parent pharmacophoric units (36-41). Considering the
pharmacophore of bioactive chalcones which bear 4-hydroxyphenyl keto group (Fig. 1), thus,
novel series of chalcone-triazole hybrids were designed by replacing the 4-hydroxyl group with
1,2,3-triazole ring containing 4-substituents (scheme 1). The chalcone-triazole hybrids (6a-g)
were synthesized and evaluated for their neurotoxicity, anti-apoptosis, antioxidant, and
molecular mechanisms underlying neuroprotection, which could represent valid candidates for
the development of new potential therapeutics.
2. Results and discussion
2.1 Chemistry
The chalcone-triazole hybrids 6a-g were synthesized as illustrated in scheme 1 (42).
Aminochalcone 3 was prepared by the base-catalyzed Claisen-Schmidt condensation of 3,4-
dimethoxybenzaldehyde 1 and 4-aminoacetophenone 2 . Subsequently, diazotization reaction
of the aminochalcone 3 using sodium nitrite and sodium azide in a mixture of glacial acetic
acid and concentrated hydrochloric acid afforded azidochalcone 4. In the final step, the
copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) of the azido compound 4 and
alkynes 5 was performed to afford the novel hybrids 6a-g in good yields (73-89%). The
alkynes 5a-g were derived from the alkylation of the corresponding phenol derivatives with
propargyl bromide.
Structures of all chalcones (6a-g) were confirmed by 1H NMR, 13C NMR and HRMS
data. The synthesized chalcones displayed molecular ion peaks corresponding to their
molecular formulas. 1H NMR spectra showed a methine proton of the triazole ring as the
singlet at  9.0-9.2 ppm, and a methylene proton attached to triazole group displayed as the
singlet at  5.2-5.5 ppm. The chalcone moiety showed two doublets of olefinic protons with
the coupling constant (J) values of 15-16 Hz indicating that the trans-configuration was
represented. In addition, 13C NMR spectra showed carbonyl carbon at  in the range of 188-

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189 ppm. The newly synthesized hybrids (6a-g) contain various substituents (R) such as
phenyl, substituted phenyl, and naphthalenyl groups.
2.2 Biological activities
2.2.1 Chalcone-triazole derivatives display no cytotoxicity in Vero cells
These compounds (6a-g) were evaluated for cytotoxicity using healthy cell line
(Vero) derived from African green monkey kidney. The cytotoxicity (IC50) values are shown
in Table1. All chalcone-triazole derivatives were low cytotoxic toward the normal Vero cell
line (IC50 > 50 µg/mL).
2.2.2 Chalcone-triazole derivatives improve cell viability of SH-SY5Y cells treated with H2O2
With all the target compounds in hands, their neuroprotective properties were
assessed with the hope of preventing neuronal cell death. Firstly, the neurotoxicity of
chalcone-triazole derivatives (6a-g) in neuroblastoma SH-SY5Y cells was evaluated. The
neuronal cells were treated with various concentrations (0.1-100 µM) of the synthetic
chalcone-triazole compounds, which needed to present toxicity less than 10% for maintaining
the cell growth. Hydrogen peroxide (H2O2) can easily generate exogenous free radicals and
highly react as ROS, which is a suitable model for studying an oxidative stress in
neurodegenerative diseases. There is a wide variability of H2O2 concentration-induced
toxicity ranging from 0.1 to 1 mM that has been observed in different primary cell cultures or
cell lines. Specifically, neurons are particularly sensitive to H2O2-induced cytotoxicity (43,
44). In order to represent the oxidative stress model, 400ꢀµM of H2O2 was added to SH-SY5Y
cells for 24 h incubation, accompanied by compounds 6a and 6e pretreatment.
As shown in Table 1, treatment with 400ꢀµM H2O2 significantly reduced cell viability
(63.94 ± 2.06%), in comparison with H2O2 untreated cells, which indicated that H2O2 model
was successfully employed. Among chalcone-triazole derivatives, the results showed that 6b,
6c, 6d, 6f, and 6g at 1 µM could not significantly improve the survival rate of H2O2-treated
neuronal cells. On the other hand, pretreatment with 6a (87.82 ± 5.08%) and 6e (87.16 ±
1.99%) reversed the reduction of cell viability compared with H2O2 treatment alone. Similar
result was noted for resveratrol (86.08 ± 4.53%), which is a well-known antioxidant (Fig. 2a).
Resveratrol (3,5,4-trihydroxy-trans-stibene, Fig. 1) is a natural polyphenolic compound
mainly found in fruit and vegetables, especially grape seed. It can directly scavenge free
radical and upregulate endogenous antioxidant enzymes, implying the protection of cell

6
viability indirectly. Many reports documented that resveratrol can cross blood-brain barrier
and exerts neuroprotective properties in different in vitro and in vivo models of
neurodegeneration (4, 44, 45). Moreover, the morphological changes of neurons were
observed under a microscope. The cells were shrunk, detached, and become round shape in
H2O2-treated cells in contrast to the pretreatment with 6a and 6e (Fig. 2b).
Amyloid cascade hypothesis is proposed for the main neuropathological hallmark of
AD and dementia which is responsible for several molecular mechanisms leading to oxidative
stress, synaptic plasticity, mitochondrial alteration, membrane permeability, and
inflammation. Amyloid-β (Aβ) oligomer is widely regarded as the most harmful and major
pathogenic form of amyloid plaques (46-48). In order to evaluate the ability of 6a and 6e
against amyloid aggregation to prevent neurodegenerative diseases outstandingly AD
patients, the cells were pretreated with 6a and 6e in the presence or absence of 10ꢀµM Aβ1-42
oligomer for 3 h. The Aβ1-42 oligomer was performed according to a previous procedure (49).
The confirming toxicity of the 10ꢁμM Aβ1-42 oligomer to neurons was investigated (80.73 ±
1.96%) as previously reviewed (47, 50). The results indicated that 6a (96.32 ± 5.26%) and 6e
(98.02 ± 6.57%) prevent Aβ1-42 oligomer-induced cytotoxicity similar to resveratrol (93.32 ±
3.35%) (Fig. 2c). Therefore, compounds 6a and 6e at 1ꢀµM were selected for the subsequent
experiments to determine neuroprotective effects against the oxidative damage.
2.2.3 Pretreatment with 6a and 6e diminishes apoptotic profiles in H2O2-treated SH-SY5Y
cells
Both synthesized compounds (6a and 6e) were evaluated as anti-apoptotic agents
against H2O2-treated SH-SY5Y cells. Resveratrol was used as a reference compound. The
results showed that H2O2-treated cells led to higher apoptosis (28.07 ± 2.03%) (Fig. 3a).
Pretreatment with 6a and 6e did not affect the proportion of total apoptotic cells.
Interestingly, 6a and 6e displayed anti-apoptotic property against oxidative damage showing
apoptotic decline (20.93 ± 2.17% and 17.53 ± 2.57%) similar to the resveratrol (16.8 ±
1.02%) compared with the H2O2 group (Fig. 3a). It has been documented that BCL-2 family
including anti-apoptotic BCL-2 and pro-apoptotic BAX proteins, which plays an important
role in apoptotic system of the mitochondria, so the levels of BAX and BCL-2 proteins, were
evaluated by Western blotting (Fig. 6a and 6b). Normally, the anti-apoptotic BCL-2 protein
modulates the pro-apoptotic BAX activation at the mitochondria to inhibit cell damage.

7
Under the oxidative stress condition, the BCL-2 protein cannot modulate the activation of
BAX leading to outer mitochondrial membrane disruption. Pro-apoptotic cytochrome c
(Cytc) escapes from BAX oligomeric pores and promotes caspase cascade. An obvious
apoptotic series of biochemical and morphological characteristics were occurred, comprising
nuclear condensation, chromosomal DNA fragmentation, cytoplasmic membrane blebbing,
phosphatidylserine flipping, and cell shrinkage (51, 52).
As expected, the level of BAX protein expression was significantly increased up to
121.9 ± 1.34% upon H2O2 induced oxidative stress, while the BCL-2 protein expression was
significantly decreased (69.84 ± 4.55%) compared with the control. Surprisingly, the changes
of BAX and BCL-2 protein expressions were restored by pretreatment with 6a (BAX; 106.7
± 3.93% and BCL-2; 91.56 ± 5.10%) and 6e (BAX;110.4 ± 2.26% and BCL-2; 89.5 ± 6.03%)
in comparison with the H2O2 group. The results suggested that derivatives 6a and 6e promote
anti-apoptotic activities similar to the resveratrol (BAX; 99.43 ± 3.55% and BCL-2; 89.67 ±
2.62%) as outlined in Fig. 6a and 6b.
2.2.4 Pretreatment with 6a and 6e potentiates mitochondrial membrane potential
Mitochondrial membrane potential is one of the sensitive indicators of functional
mitochondrial status, which is declined due to an excessive ROS accumulation. Several lines
of research have reported that measurement of mitochondrial membrane potential
prominently provides an early stage of apoptosis (51-54). To identify further biological
properties of chalcone-triazole derivatives (6a and 6e) in neuroprotection, mitochondrial
membrane potential was investigated using SH-SY5Y cells treated with 6a and 6e under
oxidative stress. Briefly, the cells were incubated in the medium containing 1 μM of 6a and
6e for 3 h prior to 400 μM H2O2 for a further 24 h. As shown in Fig. 4a, the cells treated with
H2O2 significantly decreased the percentage of mitochondrial membrane potential (87.28 ±
0.83%), compared with the control. The levels of mitochondrial membrane potential were not
affected by 6a and 6e treatments compared with the unexposed cells. However, for 6a and
6e-pretreated cells, the mitochondrial membrane potential induced by H2O2 was markedly
increased to 108.1 ± 3.79% and 101.6 ± 4.45%, respectively in comparison with the H2O2
alone. The spectrometric results showed that the fluorescence intensity of H2O2 group was
observed to be markedly lower than the control, however, it was attenuated by pretreatment
with either 6a or 6e in the same manner as the resveratrol (Fig. 4b). The data indicated that 6a

8
and 6e are able to modulate the levels of mitochondrial membrane potential, which can
clarify the molecular mechanism underlying the inhibitory effect of 6a and 6e against the
apoptosis (Fig. 3a).
2.2.5 Treatment of 6a and 6e attenuates the ROS levels in H2O2-induced SH-SY5Y cells
ROS has been reported to be closely associated with the neurodegeneration. When
free radical production and detoxification are unbalance, the over accumulation of ROS
damages the neurons by attacking several biological components including nucleic acids,
proteins, and lipids resulting in oxidative stress and cellular apoptosis. Antioxidants can be
used as potential therapeutics to scavenge the ROS production (1, 4, 5). To determine the
inhibition of ROS production associated with 6a and 6e in H2O2-induced oxidative damage in
SH-SY5Y cells, the cellular DCFDA assay was performed. Previous publications focused on
the possible role of H2O2, which can mediate ROS production in SH-SY5Y cells by
disturbing oxidant and antioxidant defenses (55-58). We also provided that H2O2 evoked
ROS-induced neurotoxicity in in vitro model. After the neurons were exposed to 400ꢀµM of
H2O2 for 24 h, ROS production was significantly increased (130.7 ± 3.93%) compared with
the unexposed cells (Fig. 5a). The experiments also exhibited that pretreatment with 6a and
6e did not alter the intracellular ROS levels, on the other hand, pretreatment with 6a and 6e
further declined the induction of ROS caused by H2O2 (110.4 ± 5.12% and 109.9 ± 6.88%,
respectively) in comparison with the H2O2 exposure. Similarly, the fluorescence intensity was
strongly reduced in the cells exposed to 6a and 6e compared with the H2O2 group (Fig. 5b).
A set of antioxidant enzymes, including glutathione (GSH), catalase (CAT), and
superoxide dismutase (SOD), plays a predominant role in mitigating extensive oxidative
damage, impairment of the cellular functions, and mitochondrial dysfunction. SOD is one of
the first effective intracellular antioxidant defense against ROS. Especially, SOD2 is located
inside the mitochondria which catalyzes the reaction of superoxide into the less reactive O2
and H2O2 (54, 59, 60). Additionally, Western blot analysis further confirmed the reduction of
SOD2 mitochondrial antioxidant enzyme in H2O2-treated cells (66.56 ± 2.96%) in accordance
with no treatment (Fig. 6f). Following pretreatment with 6a and 6e, the protein expression
levels of SOD2 were significantly increased (86.77 ± 5.49% and 88.27 ± 5.79%) compared
with the H2O2 treatment and greater than the resveratrol (81.4 ± 2.07%). The association of
chalcone hybrids and AD progression is also supported by both in vitro and in vivo models.

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Chalcone-O-alkylamine and -O-carbamate derivatives displayed good antioxidant activity,
inhibited Aβ1-42 aggregation, and provided neuroprotective effect against H2O2-induced
PC12ꢀcell (14) as well as xanthohumol from hops and chalcone analogues revealed an up-
regulation of antioxidant genes through Kelch-like ECH-associated protein 1
(Keap1)/Nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element
(ARE) enhancement in PC12 cells (13, 17, 29). Therefore, the presence of chalcone-triazole
derivatives (6a and 6e) attenuated H2O2-induced neuronal damage by a reduction of the
excessive ROS. In view of structural feature, presumably, the α, β-unsaturated keto group
(electrophilic center) of 6a and 6e could mimic the SOD activity by interacting with the ROS
leading to the significant increase level of SOD2 protein. Phenolic compound bearing
aldehyde group was also reported to upregulate the SOD2 protein level (58). In addition, the
1,2,3-triazole scaffold of the compounds may exhibit bio-metal chelating effect, which could
protect against the intracellular ROS formation (8).
2.2.6 Involvement of the SIRT1/2/3-FOXO3a pathway by 6a and 6e exhibits neuroprotection
in H2O2-treated SH-SY5Y cells
Sirtuin (SIRT) family is well known to confer resistance of cells to oxidative stress,
longevity, DNA damage repair, mitochondrial function, and metabolism (61, 62). Apart from
this, SIRT1, SIRT2, and SIRT3 have also been found to mediate beneficial effects in
neurological diseases. SIRT1 is mainly localized in the nucleus of the cells, SIRT2 is found
in the cytoplasm, and SIRT3 is predominant in the mitochondria. Several oxidative stress
models were shown to decrease the excessive ROS in part due to the activation of SIRT1 and
SIRT3, whereas inhibit SIRT2. This mechanism is highlighted through the SIRT1/2/3-
FOXO3a pathway in which the transcription factor mitochondrial FOXO3a became
upregulated and further stimulated downstream antioxidant enzymes (SOD2 and CAT) to
detoxify ROS and promote DNA repairing (63, 64). Moreover, FOXO3a requires SIRT
deacetylase activity for its transcriptional protection of cell death from the oxidative damage
(61, 65).
The well-known SIRT1 activator, resveratrol, was administered to determine its
biological properties on several in vitro and in vivo models. The neuroprotection afforded by
resveratrol against oxidative stress was reported as a robust candidate for AD therapeutic

10
according to its abilities for preventing Aβ accumulation, attenuating tau aggregation,
enhancing antioxidant defense, reciprocally reducing inflammation and decreasing
cytotoxicity through SIRT family (62, 66). Further investigation into SIRT1, SIRT2, and
SIRT3 roles may provide promising molecular targets underlying neurodegenerative
disorders.
In order to understand whether the modulation of SIRT pathway by 6a and 6e was
related to neuroprotective effect, the protein expressions of SIRT1, SIRT2, SIRT3, and
FOXO3a were examined by Western blotting. As illustrated in Fig. 6c-6g, H2O2 treatment
exhibited the reduction of SIRT1, SIRT3, FOXO3a (59.75 ± 3.68%, 41.93 ± 2.94%, 60.58 ±
4.47%, respectively) and the induction of SIRT2 protein levels (134.50 ± 6.04%) compared
to the control. However, no obvious differences were identified in the SIRT1, SIRT2, SIRT3,
and FOXO3a between the untreated cells and compounds 6a and 6e-pretreated cells. By
contrast, the decreased levels of SIRT1, SIRT3, and FOXO3a proteins caused by H2O2 were
markedly improved by pretreatments with 6a (SIRT1; 90.52 ± 8.20%, SIRT3; 76.48 ± 3.96%,
and FOXO3a; 87.17 ± 7.19%, respectively) and 6e (SIRT1; 91.51 ± 2.04%, SIRT3; 68.0 ±
3.09%, and FOXO3a; 88.93 ± 4.58%, respectively), while SIRT2 protein was reduced by 6a
(111.60 ± 5.18%) and 6e pretreatments (99.25 ± 2.44%), in accordance with the H2O2 alone.
Particularly, 6a and 6e demonstrated slightly stronger effects on SIRT1 protein expression
compared with the resveratrol (90.03 ± 4.62%), whereas the levels of SIRT2, SIRT3, and
FOXO3a protein expressions by resveratrol pretreatment were more potent than 6a and 6e
(94.50 ± 5.43%, 85.64 ± 4.21%, and 94.13 ± 2.90%, respectively). The resveratrol has been
highlighted to influence mitochondrial biogenesis, cytoprotection, amyloid aggregation, and
ROS production via activating SIRT1 (4). Similar to the previous studies, butein and
isoliquiritigenin are natural chalcones found in Hydnophytum formicarum Jack. Both
compounds were considered to be used as an alternative AD treatment, which promoted
SIRT1 and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)
activities in modulation of Aβ shedding and attenuation of neuronal cell death (55, 67).
Another model of oxidative stress mediated by Aβ1-42 against human fetal neurons was
reported that phenol–triazole ligands can target multiple factors associated with AD
comprising antioxidant capacity, copper-binding affinity, interaction and modulation of Aβ
peptide aggregation (68) as well as anti-aggregation towards Aβ1-42 and anticholinesterase
activities by 2-arylbenzofuran derivative bearing a substituted triazole (69). These results

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indicated that chalcone-triazole hybrids 6a and 6e display anti-neurodegenerative effects by
modulation of the SIRT1/2/3-FOXO3a signaling pathway.
In conclusion, the data demonstrated that chalcone-triazole derivatives-treated
neuronal cells maintain anti-apoptotic, cellular antioxidant status, and mitochondrial function
without neurotoxicity. Moreover, these pretreatments also completely protect the neurons
against H2O2-mediated oxidative damage through SIRT1/2/3-FOXO3a signaling pathway
(Fig. 7). Notably, the alternative strategy for the design of novel neuroprotective compounds
equivalent to the well-known antioxidant (resveratrol) was provided. Thus, chalcone-triazole
derivatives could be developed as lead candidates in the prevention or treatment of
neurodegenerative diseases.
3. Experimental
3.1 General
Column chromatography was carried out using silica gel 60 (70-230 mesh ASTM).
Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F254 aluminum
sheets. 1H- and13C-NMR spectra were recorded on a Bruker AVANCE 300 NMR spectrometer
1
(operating at 300 MHz for H and 75 MHz for 13C). FTIR spectra were obtained using a
universal attenuated total reflectance attached on a Perkin–Elmer Spectrum One spectrometer.
High resolution mass spectra (HRMS) were recorded on a Bruker Daltonics (microTOF).
Melting points were determined using a Griffin melting point apparatus and were uncorrected.
3.2 Synthesis of (E)-1-(4-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3) (70)
To a mixture of 3,4-dimethoxybenzaldehyde 1 (6 mmol) and 4-aminoacetophenone 2
(6 mmol) in ethanol (15 mL) was stirred at 4 oC, then 40% KOH (5 mL) was added dropwise,
and stirred at room temperature for 4 h. The reaction mixture was neutralized with 2M HCl,
then the precipitate was filtered, washed with cold water and recrystallized from ethanol to give
compound 3.
Yellow solid. 85%. mp 121-122 oC. IR (UATR) cm-1: 3456, 3360, 3232, 1628, 1598, 1510. 1H
NMR (300 MHz, DMSO-d6) δ 3.79 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.09 (s, 2H, NH2), 6.61
(d, J = 8.6 Hz, 2H, C3-ArH and C5-ArH), 6.98 (d, J = 8.3 Hz, 1H, C5-ArH), 7.30 (d, J = 8.2
Hz, 1H, C6-ArH), 7.47 (s, 1H, C2-ArH), 7.56 (d, J = 15.4 Hz, 1H, CH=CHCO), 7.74 (d, J =
15.4 Hz, 1H, CH=CHCO), 7.92 (d, J = 8.6 Hz, 2H, C2-ArH and C6-ArH). 13C NMR (75 MHz,

12
DMSO-d6) δ 56.1, 56.2, 111.0, 112.1, 113.2, 120.5, 123.7, 126.1, 128.5, 131.5, 142.3, 149.5,
151.2, 154.1, 186.4. HRMS-TOF: m/z [M + H]+ 284.1293 (Calcd for C17H18NO3: 284.1281).
3.3 Synthesis of (E)-1-(4-azidophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4)
To a cold solution of aminochalcone 3 (3 mmol) in HCl:CH3COOH (3:3 mL) at 0 oC,
a solution of sodium nitrite (9 mmol) in water (5 mL) was added. The stirred reaction mixture
was maintained for 15 min and then added dropwise a solution of sodium azide (9 mmol) in
water (5 mL). The reaction mixture was allowed to stir at room temperature for 0.5 h, then the
precipitate was filtered, washed with cold water and recrystallized from methanol to provide
compound 4.
Yellow solid. 77%. mp 120-121 oC. IR (UATR) cm-1: 2124, 1655, 1598, 1510. 1H NMR (300
MHz, CDCl3) δ 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.88 (d, J = 8.3 Hz, 1H, C5-ArH),
7.11 (d, J = 8.6 Hz, 2H, C3-ArH and C5-ArH), 7.13 (d, J = 1.5 Hz, 1H, C2-ArH), 7.24 (dd, J
= 8.3, 1.5 Hz, 1H, C6-ArH), 7.35 (d, J = 15.6 Hz, 1H, CH=CHCO), 7.75 (d, J = 15.6 Hz, 1H,
CH=CHCO), 8.02 (d, J = 8.6 Hz, 2H, C2-ArH and C6-ArH). 13C NMR (75 MHz, CDCl3) δ
56.0, 110.2, 111.2, 119.0, 119.5, 123.2, 127.8, 130.4, 135.1, 144.5, 145.1, 149.3, 151.5, 188.8.
HRMS-TOF: m/z [M + H]+ 310.1191 (Calcd for C17H16N3O3: 310.1186).
3.4 General procedure for the synthesis of propynyloxy derivatives (5a-g)
A propargyl bromide (2.4 mmol) was added to a suspension of an appropiate phenol (2
mmol) and potassium carbonate (4 mmol) in acetone (15 mL). The suspension was heated
under reflux for 2 h. The reaction was allowed to cool and then concentrated under reduced
pressure. Water (30 mL) was added and extracted with EtOAc (3 × 30 mL). The organic
extracts were combined and washed with water (20 mL) and brine (20 mL). The organic layer
was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude
product was purified by column chromatography. 1H NMR spectra of (2-propynyloxy)benzene
(5a) (71), 1-methyl-4-(2-propynyloxy)benzene (5b) (72), 1-methyl-2-(2-propynyloxy)benzene
(5c) (73), methyl 2-(2-propynyloxy)benzoate (5d) (74), 4-(2-propynyloxy)benzaldehyde (5e)
(75), 4-methoxy-3-(2-propynyloxy)benzaldehyde (5f) (76), and 2-(2-propynyloxy)naphthalene
(5g) (77) were consistent with those reported in the literatures.
3.5 General procedure for the synthesis of chalcone-triazole derivatives (6a-g)

13
To a stirred solution of azidochalcone 4 (0.2 mmol) and alkyne 5 (0.22 mmol) in t-
BuOH:H2O (3:3 mL), CuSO45H2O (0.22 mmol) and sodium ascorbate (0.5 mmol) were added.
The reaction mixture was stirred at room temperature for 2 h and then concentrated under
reduced pressure. The residue was added water (10 mL) and extracted with dichloromethane
(3 × 20 mL). The combined organic phases were washed with water (20 mL), dried over
anhydrous sodium sulfate and evaporated to dryness. The crude product was purified using
silica gel column chromatography and eluted with methanol:dichloromethane (1:50) to give
compounds 6a-g.
3.5.1
(E)-3-(3,4-dimethoxyphenyl)-1-(4-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-
yl)phenyl)prop-2-en-1-one (6a)
Pale yellow solid. 78% yield. mp 173-174 oC. IR (UATR) cm-1: 1656, 1584, 1513. 1H
NMR (300 MHz, DMSO-d6) δ 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.27 (s, 2H, CH2O),
6.98 (t, J = 7.3 Hz, 1H, ArH), 7.04 (d, J = 8.4 Hz, 1H, ArH), 7.09 (d, J = 8.3 Hz, 2H, ArH),
7.33 (t, J = 7.9 Hz, 2H, ArH), 7.44 (d, J = 8.2 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.76 (d, J =
15.5 Hz, 1H, CH=CHCO), 7.90 (d, J = 15.5 Hz, 1H, CH=CHCO), 8.14 (d, J = 8.6 Hz, 2H,
13
ArH), 8.38 (d, J = 8.6 Hz, 2H, ArH), 9.14 (s, 1H, CHN). C NMR (75 MHz, DMSO-d6) δ
56.1, 56.3, 61.4, 111.5, 112.1, 115.2, 119.8, 120.4, 121.5, 123.5, 124.7, 127.9, 130.0, 130.8,
138.0, 139.8, 144.8, 145.6, 149.5, 152.0, 158.4, 188.3. HRMS-TOF: m/z [M + Na]+ 464.1581
(Calcd for C26H23N3NaO4: 464.1569).
3.5.2
yl)phenyl)prop-2-en-1-one (6b)
Pale yellow solid. 89% yield. mp 178-179 C. IR (UATR) cm-1: 1654, 1604, 1583,
(E)-3-(3,4-dimethoxyphenyl)-1-(4-(4-((p-tolyloxy)methyl)-1H-1,2,3-triazol-1-
o
1
1510. H NMR (300 MHz, DMSO-d6) δ 2.24 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 5.22 (s, 2H, CH2O), 6.97 (d, J = 8.4 Hz, 2H, ArH), 7.05 (d, J = 8.3 Hz, 1H, ArH), 7.12
(d, J = 8.5 Hz, 2H, ArH), 7.44 (d, J = 8.4 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.75 (d, J = 15.4
Hz, 1H, CH=CHCO), 7.90 (d, J = 15.5 Hz, 1H, CH=CHCO), 8.14 (d, J = 8.5 Hz, 2H, ArH),
8.38 (d, J = 8.6 Hz, 2H, ArH), 9.12 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-d6) δ 20.5, 56.1,
56.3, 61.5, 111.5, 112.1, 115.1, 119.8, 120.4, 123.4, 124.7, 127.9, 130.2, 130.4, 130.8, 138.0,
139.8, 144.9, 145.6, 149.5, 152.0, 156.3, 188.3. HRMS-TOF: m/z [M + Na]+ 478.1737 (Calcd
for C27H25N3NaO4: 478.1726).
3.5.3
(E)-3-(3,4-dimethoxyphenyl)-1-(4-(4-((o-tolyloxy)methyl)-1H-1,2,3-triazol-1-
yl)phenyl)prop-2-en-1-one (6c)

14
Pale yellow solid. 87% yield. mp 159-160 oC. IR (UATR) cm-1: 1654, 1590, 1515. 1H
NMR (300 MHz, DMSO-d6) δ 2.16 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 5.26
(s, 2H, CH2O), 6.82-6.92 (m, 1H, ArH), 7.03 (d, J = 8.3 Hz, 1H, ArH), 7.13-7.20 (m, 3H, ArH),
7.42 (dd, J = 8.4, 1.7 Hz, 1H, ArH), 7.56 (d, J = 1.7 Hz, 1H, ArH), 7.75 (d, J = 15.5 Hz, 1H,
CH=CHCO), 7.88 (d, J = 15.5 Hz, 1H, CH=CHCO), 8.14 (d, J = 8.7 Hz, 2H, ArH), 8.36 (d, J
13
= 8.6 Hz, 2H, ArH), 9.10 (s, 1H, CHN). C NMR (75 MHz, DMSO-d6) δ 16.5, 56.1, 56.3,
61.7, 111.5, 112.1, 112.4, 119.9, 120.4, 121.3, 123.3, 124.6, 126.5, 127.4, 127.9, 130.8, 131.0,
138.0, 139.8, 145.1, 145.6, 149.5, 152.0, 156.6, 188.4. HRMS-TOF: m/z [M + Na]+ 478.1737
(Calcd for C27H25N3NaO4: 478.1724).
3.5.4
(E)-methyl2-((1-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-1H-1,2,3-
triazol-4-yl)methoxy)benzoate (6d)
Pale yellow solid. 73% yield.mp 163-164 oC. IR (UATR) cm-1: 1719, 1661, 1593, 1520.
1H NMR (300 MHz, DMSO-d6) δ 3.76 (s, 3H, COCH3), 3.82 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 5.35 (s, 2H, CH2O), 7.00-7.11 (m, 2H, ArH), 7.37-7.47 (m, 2H, ArH), 7.53-7.61 (m,
2H, ArH), 7.67 (dd, J = 7.7, 1.7 Hz, 1H, ArH), 7.77 (d, J = 15.5 Hz, 1H, CH=CHCO), 7.89 (d,
J = 15.5 Hz, 1H, CH=CHCO), 8.13 (d, J = 8.8 Hz, 2H, ArH), 8.38 (d, J = 8.8 Hz, 2H, ArH),
9.06 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-d6) δ 52.4, 56.1, 56.2, 62.6, 111.4, 112.1, 115.0,
119.8, 120.4, 121.4, 123.4, 124.7, 127.9, 130.8, 131.2, 134.0, 138.0, 139.8, 144.6, 145.7, 149.5,
152.0, 157.3, 166.5, 188.4. HRMS-TOF: m/z [M + Na]+ 522.1636 (Calcd for C28H25N3NaO6:
522.1632).
3.5.5
(E)-4-((1-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-1H-1,2,3-triazol-4-
yl)methoxy)benzaldehyde (6e)
o
Pale yellow solid. 82% yield. mp 149-150 C. IR (UATR) cm-1: 1688, 1655, 1602,
1581, 1509. 1H NMR (300 MHz, DMSO-d6) δ 3.82 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.41
(s, 2H, CH2O), 7.03 (d, J = 8.4 Hz, 1H, ArH), 7.29 (d, J = 8.7 Hz, 2H, ArH), 7.42 (dd, J = 8.4,
1.7 Hz, 1H, ArH), 7.56 (d, J = 1.7 Hz, 1H, ArH), 7.75 (d, J = 15.5 Hz, 1H, CH=CHCO), 7.88
(d, J = 15.5 Hz, 1H, CH=CHCO), 7.89 (d, J = 8.8 Hz, 2H, ArH), 8.13 (d, J = 8.7 Hz, 2H, ArH),
13
8.37 (d, J = 8.7 Hz, 2H, ArH), 9.14 (s, 1H, CHN), 9.89 (s, 1H, CHO). C NMR (75 MHz,
DMSO-d6) δ 56.1, 56.3, 61.8, 111.4, 112.1, 115.7, 119.8, 120.5, 123.8, 124.7, 127.9, 130.5,
130.8, 132.3, 138.1, 139.8, 144.1, 145.7, 149.5, 152.0, 163.3, 188.4, 191.9. HRMS-TOF: m/z
[M + Na]+ 492.1523 (Calcd for C27H23N3NaO5: 492.1523).

15
3.5.6
(E)-3-((1-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-1H-1,2,3-triazol-4-
yl)methoxy)-4-methoxybenzaldehyde (6f)
o
Pale yellow solid. 83% yield. mp 151-152 C. IR (UATR) cm-1: 1683, 1658, 1593,
1511. 1H NMR (300 MHz, DMSO-d6) δ 3.83 (s, 3H, OCH3), 3.87 (s, 6H, 2 × OCH3), 5.34 (s,
2H, CH2O), 7.04 (d, J = 8.4 Hz, 1H, ArH), 7.22 (d, J = 8.3 Hz, 1H, ArH), 7.43 (d, J = 7.3 Hz,
1H, ArH), 7.57 (s, 1H, ArH), 7.61 (d, J = 8.3 Hz, 1H, ArH), 7.67 (s, 1H, ArH), 7.74 (d, J =
15.4 Hz, 1H, CH=CHCO), 7.89 (d, J = 15.4 Hz, 1H, CH=CHCO), 8.14 (d, J = 8.6 Hz, 2H,
13
ArH), 8.37 (d, J = 8.6 Hz, 2H, ArH), 9.12 (s, 1H, CHN), 9.87 (s, 1H, CHO). C NMR (75
MHz, DMSO-d6) δ 56.1, 56.3, 56.4, 62.1, 111.5, 112.1, 112.2, 112.4, 119.9, 120.4, 123.8,
124.6, 126.9, 127.9, 130.1, 130.8, 138.1, 139.8, 144.3, 145.6, 148.3, 149.5, 152.0, 155.0, 188.4,
191.8. HRMS-TOF: m/z [M + Na]+ 522.1640 (Calcd for C28H25N3NaO6: 522.1641).
3.5.7
(E)-3-(3,4-dimethoxyphenyl)-1-(4-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-
triazol-1-yl)phenyl)prop-2-en-1-one (6g)
Pale yellow solid. 89% yield. mp 190-191 oC. IR (UATR) cm-1: 1654, 1601, 1512. 1H
NMR (300 MHz, DMSO-d6) δ 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.40 (s, 2H, CH2O),
7.05 (d, J = 8.4 Hz, 1H, ArH), 7.25 (dd, J = 8.9, 2.4 Hz, 1H, ArH), 7.34-7.60 (m, 5H, ArH),
7.75 (d, J = 15.5 Hz, 1H, CH=CHCO), 7.80-7.95 (m, 4H, ArH and CH=CHCO), 8.15 (d, J =
13
8.7 Hz, 2H, ArH), 8.39 (d, J = 8.6 Hz, 2H, ArH), 9.20 (s, 1H, CHN). C NMR (75 MHz,
DMSO-d6) δ 56.1, 56.3, 61.6, 107.8, 111.5, 112.1, 119.1, 119.8, 120.4, 123.6, 124.3, 124.7,
127.0, 127.3, 127.9, 128.0, 129.2, 129.9, 130.8, 134.7, 138.0, 139.9, 144.7, 145.6, 149.5, 152.0,
156.3, 188.3. HRMS-TOF: m/z [M + Na]+ 514.1737 (Calcd for C30H25N3NaO4: 514.1720).
3.6 Biological activity
3.6.1 Chemical and reagents
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7′-
dichlorofluorescein diacetate (DCFDA) were obtained from Molecular Probes (Eugene, OR,
USA). Annexin V & Dead Cell Assay Kit and Immobilon ECL Ultra Western HRP Substrate
were purchased from Merck Millipore (Darmstadt, Germany). Protease inhibitor cocktail
were obtained from Calbiochem (Cambridge, MA, USA). RIPA and primary antibodies
including anti-BAX, BCL-2, SIRT1, SIRT2, SIRT3, FOXO3a, SOD2, and β-actin as well as
secondary antibodies were supplied from Cell Signaling Technology (Beverly, MA, USA).

16
All chemical reagents used in this experiment were analytical grade from Sigma (St. Louis,
MO, USA).
3.6.2 Cytotoxicity assay
Green Fluorescent Protein (GFP) detection method was conducted to determine
cytotoxicity (78). The GFP-expressing Vero cell line was produced in-house by transfection of
African green monkey kidney cell line (Vero, ATCC CCL-81) with pEGFP-N1 plasmid
(Clontech). Minimal essential medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.8 mg/mL geneticin, and 1.5 g/L
sodium bicarbonate were utilized to culture the cell line. 45 μL of cell suspension at 3.3 × 104
cells/mL was added into to 384-well plates with 5 μL of chalcone-triazole derivatives or
ellipticine formerly diluted in 0.5% DMSO followed by 37 oC incubation for 4 days with 5%
CO2. SpectraMax M5 microplate reader (Molecular Devices, USA) was used to detect
fluorescence signals with excitation and emission wavelengths of 485 and 535 nm,
respectively. Fluorescence signal at day 4 was subtracted with background fluorescence at day
0. The values of IC50 values were obtained from dose-response curves with 6 concentrations of
3-fold serially diluted samples using the SOFTMax Pro software (Molecular device). 0.5%
DMSO and ellipticine were negative and positive controls, respectively.
3.6.3 Cell viability by MTT assay
SH-SY5Y cell line (human neuroblastoma) obtained from American Type Culture
Collection (ATCC) (Manassas, VA, USA) was utilized in this work. Dulbecco’s Modified
Eagle Medium (DMEM), 1% penicillin-streptomycin, and 10% heat-inactivated fetal bovine
serum (FBS) from Gibco BRL (Gaithersburg, MD, USA) were used to culture SH-SY5Y
cells in 75-cm2 flasks at 37°C with 5% CO2. Next, for testing the effect of all compounds on
cell viability, the cells were grown in 96-well plates for 24 h until reaching 80% confluence,
then various concentrations of chalcone-triazole derivatives or resveratrol (0.1-100 µM) were
added in the plates for 3 h, followed by incubation with 400 µM H2O2 or 10 µM Aβ1-42
oligomer. Subsequently, 5 mg/mL of MTT was included in all wells followed by incubation
for 3 h at 37 °C. Upon MTT removal, an extraction buffer (0.04 N HCl in isopropanol) was
applied to each well. The resulting purple formazan was measured by microplate reader
(BioTek Instruments, VT, USA) at 570 nm.
3.6.4 Cell apoptosis by Flow cytometry

17
For evaluation of cell apoptosis, 6-well plates were used for seeding SH-SY5Y cells,
and 1 µM of chalcone-triazole derivatives or resveratrol was added in the wells for 3 h,
followed by 24 h incubation with 400 µM H2O2. Following the incubation, the cells were
harvested and determined apoptosis profiles using Annexin V & Dead Cell Assay Kit.
Muse™ cell analyzer (Merck Millipore, USA) was utilized for analyzing the percentages of
living, apoptotic, and dead cells.
3.6.5 Mitochondrial membrane potential by rhodamine staining
Mitochondrial membrane potential was determined by rhodamine 123. After the
treatment, cells were washed twice with PBS, followed by incubation with 10 µM rhodamine
123 at 37°C for 30 min. Emission spectra at 488ꢀnm and excitation spectra at 525ꢀnm were
measured by microplate reader and imaged by fluorescence microscope.
3.6.6 Intracellular ROS levels by DCFDA assay
The cells were treated as described above and stained with 10 µM DCFDA for 30 min
in the dark. The fluorescence signals within the cells were measured using microplate reader
at excitation and emission spectra of 495 nm and 527 nm, respectively and visualized by
fluorescence microscope.
3.6.7 Western blot analysis
Following pretreatment with chalcone-triazole derivatives or resveratrol, SH-SY5Y
cells were lysed in the RIPA lysis buffer. Based on the protein concentration measured by the
Bradford assay, 20ꢀμg of samples were run on sodium dodecyl sulphate-polyacrylamide gels
and electrotransferred to polyvinylidene difluoride (PVDF) membranes. After blocking with
5% skimmed milk in Tris-buffer containing 0.1% Tween-20 (TBST) for 2 h, the primary
antibodies (SIRT1, SIRT2, SIRT3, FOXO3a, BAX, BCL-2, and SOD2) were applied for
overnight incubation at 4°C. Next, the blots were added with horseradish peroxidase-
conjugated secondary antibodies incubated at room temperature for an hour, then the
membrane was developed using ECL.The protein levels of all bands were determined using
ChemiDoc™ MP Imaging System and Image Lab Software (Bio-Rad Laboratories, Inc,
Hercules, CA, USA) and normalized to β-actin.
3.6.8 Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 software (GraphPad
Software Inc.) and one-way ANOVA followed by a Tukey-Kramer post-test was employed
for comparisons among three or more groups. Data were expressed as mean ± S.E.M. values

18
from three independent experiments. A p-value less than 0.05 was set as statistically
significant.
Acknowledgments
This project is supported by the research grants from Srinakharinwirot University (grant
no. 085/2563). We are indebted to Chulabhorn Research Institute for recording mass spectra.
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Table legend
Table1 Cell survival rate and cytotoxicity of chalcone-triazole derivativesa,b
% Viability
Without H2O2
Cytotoxicity
Compound
(IC50, µg/mL)d
With H2O2
63.94 ± 2.06*
87.82 ± 5.08#
57.66 ± 3.95
71.97 ± 2.12
75.87 ± 8.78
87.16 ± 1.99#
61.42 ± 1.12
65.02 ± 2.22
86.08 ± 4.53#
NDe
Untreated
100.0 ± 0
100.3 ± 1.09
108.0 ± 6.51
96.86 ± 1.97
102.9 ± 4.05
98.36 ± 6.41
91.09 ± 3.83
94.34 ± 4.90
97.94 ± 0.25
NDe
-
6a
> 50
6b
> 50
6c
> 50
6d
> 50
6e
6f
> 50
> 50
6g
> 50
Resveratrolc
Ellipticinec
NDe
0.724
a Significance: *푃 < 0.05 vs. the untreated cells; #푃 < 0.05 vs. the H2O2 group.
b The data are expressed as mean ± S.E.M.
c Resveratrol and ellipticine were used as the reference compounds.
d Cytotoxicity against Vero cells: IC50 > 50 μg/mL is defined as low cytotoxic effect.
e ND = not determined.

26
Figure legends
O
O
O
HO
O
OH
HO
O
OH
Chalcone
Licochalcone E
Licochalcone A
OH
O
OH
HO
HO
OH
O
OH
Resveratrol
Xanthohumol
Fig. 1 Chemical structures of chalcone, licochalcones (A and E), xanthohumol and resveratrol

27
O
O
O
O
O
O
O
(i)
H
+
NH2
NH2
2
1
3
(ii)
O
O
OR
O
O
O
O
5a-g
(iii)
N
N
N3
OR
6a-g
4
N
Reagents and conditions:
(i) 40% KOH, EtOH, rt
(ii) NaNO2, HCl/CH3COOH, 0 oC, 15 min then NaN3, rt, 30 min
.
(iii) alkyne 5, CuSO4•5H2O, sodium ascorbate, t-BuOH/H2O, rt
O
O
c
a
e
b
d
g
R =
O
H
H
O
f
O
Scheme 1 synthesis of chalcone-triazole derivatives

28
Fig. 2 Chalcone-triazole derivatives (6a and 6e) alleviate the cytotoxic effect in the neuronal
cells. (a) The SH-SY5Y cells were incubated with 1 µM of 6a, 6e, or resveratrol for 3 h
followed by 400 µM H2O2 for 24 h, and evaluated by MTT assay. (b) Morphological alteration
was observed under a light microscope (20X magnification). Scale bar = 200 μm. (c) The cells
were preincubated with compounds followed by 10 µM Aβ1-42 oligomer for 3 h, and evaluated
by MTT assay. The data are expressed as mean ± S.E.M. *푃 < 0.05 compared with the control
cells; #푃 < 0.05 compared with the H2O2 or Aβ1-42 oligomer groups.

29
Fig. 3 Chalcone-triazole derivatives (6a and 6e) inhibit H2O2-induced apoptosis in SH-SY5Y
cells. (a) Apoptotic profiles were determined by annexin V/7-AAD. (b) Flow cytometric plots
represented the percentages of live, early apoptotic, late apoptotic, or necrotic cells. The data
are expressed as mean ± S.E.M. *푃 < 0.05 compared with the control; #푃 < 0.05 compared with
the H2O2 group.