Synthesis and neuroprotective effects of novel chalcone-triazole hybrids

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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 Sooknual^a, Ratchanok Pingaew^a,*, Kamonrat Phopin^b,c,*, Waralee Ruankham^b,

Supaluk Prachayasittikul^d, Somsak Ruchirawat^e,f,g, Virapong Prachayasittikul^c

^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)

2

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

3

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-

5

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 H₂O₂concentration-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 H₂O₂was added to SH-SY5Y

cells for 24 h incubation, accompanied by compounds 6a and 6e pretreatment.

As shown in Table 1, treatment with 400ꢀµM H₂O₂significantly reduced cell viability

(63.94 ± 2.06%), in comparison with H₂O₂untreated cells, which indicated that H₂O₂model

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 H₂O₂treatment 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 Aβ_1-42

oligomer for 3 h. The Aβ_1-42oligomer was performed according to a previous procedure (49).

The confirming toxicity of the 10ꢁμM Aβ_1-42oligomer 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 Aβ_1-42oligomer-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 H₂O₂group (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.

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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 H₂O₂induced 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 H₂O₂group. 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 H₂O₂for a further 24 h. As shown in Fig. 4a, the cells treated with

H₂O₂significantly 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 H₂O₂was 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 H₂O₂group 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 H₂O₂evoked

ROS-induced neurotoxicity in in vitro model. After the neurons were exposed to 400ꢀµM of

H₂O₂for 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 H₂O₂exposure. Similarly, the fluorescence intensity was

strongly reduced in the cells exposed to 6a and 6e compared with the H₂O₂group (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 H₂O₂treatment 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.

9

Chalcone-O-alkylamine and -O-carbamate derivatives displayed good antioxidant activity,

inhibited Aβ_1-42aggregation, 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, H₂O₂treatment

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 H₂O₂were

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 H₂O₂alone.

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 Aβ_1-42against 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 Aβ_1-42and anticholinesterase

activities by 2-arylbenzofuran derivative bearing a substituted triazole (69). These results

11

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 F₂₅₄aluminum

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 IC₅₀values 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-cm²flasks 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 H₂O₂or 10 µM Aβ_1-42

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.

References

1.

derivatives as potent neuroprotective agents. Eur J Med Chem. 2015;95:313-23.

2. Zanforlin E, Zagotto G, Ribaudo G. The medicinal chemistry of natural and

Liu YG, Li X, Xiong DC, Yu B, Pu X, Ye XS. Synthetic phenylethanoid glycoside

semisynthetic compounds against Parkinson’s and Huntington’s diseases. ACS Chem

Neurosci. 2017;8(11):2356-68.

3.

Iqbal S, Shah FA, Naeem K, Nadeem H, Sarwar S, Ashraf Z, et al. Succinamide

derivatives ameliorate neuroinflammation and oxidative stress in scopolamine-induced

neurodegeneration. Biomolecules 2020;10(3):443.

4.

natural compounds. Pharmacol Rep 2017;69(5):851-60.

5. Bansal R, Singh R. Exploring the potential of natural and synthetic neuroprotective

Wąsik A, Antkiewicz Michaluk L. The mechanism of neuroprotective action of

steroids against neurodegenerative disorders: A literature review. Medicinal research reviews.

2018;38(4):1126-58.

6.

structure in medicinal chemistry. Chem Rev. 2017;117(12):7762-810.

7. Gomes MN, Muratov EN, Pereira M, Peixoto JC, Rosseto LP, Cravo PVL, et al.

Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z. Chalcone: A privileged

Chalcone derivatives: Promising starting points for drug design. Molecules (Basel,

Switzerland). 2017;22(8):1210.

8.

chemistry: A recent overview. Bioorg Med Chem. 2019;27(16):3511-31.

9. Dheer D, Singh V, Shankar R. Medicinal attributes of 1,2,3-triazoles: Current

developments. Bioorg Chem. 2017;71:30-54.

Bozorov K, Zhao J, Aisa HA. 1,2,3-Triazole-containing hybrids as leads in medicinal

19

10.

significance of chalcone scaffold: A review. Curr Med Chem. 2012;19(2):209-25.

11. Daniela Ilieva B, Iva Todorova T. Trends in utilization of the pharmacological

potential of chalcones. Curr Clin Pharmacol. 2010;5(1):1-29.

12. Zhang X, Rakesh KP, Bukhari SNA, Balakrishna M, Manukumar HM, Qin HL.

Sahu NK, Balbhadra SS, Choudhary J, Kohli DV. Exploring pharmacological

Multi-targetable chalcone analogs to treat deadly Alzheimer’s disease: Current view and

upcoming advice. Bioorg Chem. 2018;80:86-93.

13.

De Freitas Silva M, Pruccoli L, Morroni F, Sita G, Seghetti F, Viegas Jr C, et al. The

Keap1/Nrf2-ARE pathway as a pharmacological target for chalcones. Molecules (Basel,

Switzerland). 2018;23(7).

14.

Bai P, Wang K, Zhang P, Shi J, Cheng X, Zhang Q, et al. Development of chalcone-

O-alkylamine derivatives as multifunctional agents against Alzheimer's disease. Eur J Med

Chem. 2019;183:111737.

15.

Sang Z, Wang K, Zhang P, Shi J, Liu W, Tan Z. Design, synthesis, in-silico and

biological evaluation of novel chalcone derivatives as multi-function agents for the treatment

of Alzheimer's disease. Eur J Med Chem. 2019;180:238-52.

16.

Sang Z, Wang K, Shi J, Liu W, Tan Z. Design, synthesis, in-silico and biological

evaluation of novel chalcone-O-carbamate derivatives as multifunctional agents for the

treatment of Alzheimer's disease. Eur J Med Chem. 2019;178:726-39.

17.

Wu JZ, Cheng CC, Shen LL, Wang ZK, Wu SB, Li WL, et al. Synthetic chalcones

with potent antioxidant ability on H₂O₂-induced apoptosis in PC12 cells. Int J Mol Sci

2014;15(10):18525-39.

18.

Jiwrajka M, Phillips A, Butler M, Rossi M, Pocock JM. The plant-derived chalcone

2,2',5'-trihydroxychalcone provides neuroprotection against toll-like receptor 4 triggered

inflammation in microglia. Oxid Med Cell Longev. 2016;2016:6301712.

19.

Cong L, Dong X, Wang Y, Deng Y, Li B, Dai R. On the role of synthesized

hydroxylated chalcones as dual functional amyloid-β aggregation and ferroptosis inhibitors

for potential treatment of Alzheimer's disease. Eur J Med Chem. 2019;166:11-21.

20.

Xiao G, Li Y, Qiang X, Xu R, Zheng Y, Cao Z, et al. Design, synthesis, and

biological evaluation of 4′-aminochalcone-rivastigmine hybrids as multifunctional agents for

the treatment of Alzheimer's disease. Bioorg Med Chem. 2017;25(3):1030-41.

20

21.

Jeon KH, Lee E, Jun KY, Eom JE, Kwak SY, Na Y, et al. Neuroprotective effect of

synthetic chalcone derivatives as competitive dual inhibitors against μ-calpain and cathepsin

B through the downregulation of tau phosphorylation and insoluble Aβ peptide formation.

Eur J Med Chem. 2016;121:433-44.

22.

Liu Hr, Huang Xq, Lou Dh, Liu Xj, Liu Wk, Wang Qa. Synthesis and

acetylcholinesterase inhibitory activity of Mannich base derivatives flavokawain B. Bioorg

Med Chem Lett. 2014;24(19):4749-53.

23.

Kang L, Gao XH, Liu HR, Men X, Wu HN, Cui PW, et al. Structure-activity

relationship investigation of coumarin-chalcone hybrids with diverse side-chains as

acetylcholinesterase and butyrylcholinesterase inhibitors. Mol Divers. 2018;22(4):893-906.

24.

Rampa A, Bartolini M, Pruccoli L, Naldi M, Iriepa I, Moraleda I, et al. Exploiting the

chalcone scaffold to develop multifunctional agents for Alzheimer’s disease. Molecules

(Basel, Switzerland). 2018;23(8).

25.

Burmaoglu S, Yilmaz AO, Polat MF, Kaya R, Gulcin İ, Algul O. Synthesis and

biological evaluation of novel tris-chalcones as potent carbonic anhydrase,

acetylcholinesterase, butyrylcholinesterase and α-glycosidase inhibitors. Bioorg Chem.

2019;85:191-7.

26.

Jung JC, Jang S, Lee Y, Min D, Lim E, Jung H, et al. Efficient synthesis and

neuroprotective effect of substituted 1,3-diphenyl-2-propen-1-ones. J Med Chem.

2008;51(13):4054-8.

27.

Kühnl J, Roggenkamp D, Gehrke SA, Stäb F, Wenck H, Kolbe L, et al. Licochalcone

A activates Nrf2 in vitro and contributes to licorice extract-induced lowered cutaneous

oxidative stress in vivo. Exp Dermatol. 2015;24(1):42-7.

28.

Kim SS, Lim J, Bang Y, Gal J, Lee SU, Cho YC, et al. Licochalcone E activates

Nrf2/antioxidant response element signaling pathway in both neuronal and microglial cells:

therapeutic relevance to neurodegenerative disease. J Nutr Biochem. 2012;23(10):1314-23.

29.

Yao J, Zhang B, Ge C, Peng S, Fang J. Xanthohumol, a polyphenol chalcone present

in hops, activating Nrf2 enzymes to confer protection against oxidative damage in PC12

cells. J Agric Food Chem. 2015;63(5):1521-31.

30.

Xu Z, Zhao SJ, Liu Y. 1,2,3-Triazole-containing hybrids as potential anticancer

agents: Current developments, action mechanisms and structure-activity relationships. Eur J

Med Chem. 2019;183:111700.

21

31.

Jones MR, Dyrager C, Hoarau M, Korshavn KJ, Lim MH, Ramamoorthy A, et al.

Multifunctional quinoline-triazole derivatives as potential modulators of amyloid-β peptide

aggregation. J Inorg Biochem. 2016;158:131-8.

32.

Yazdani M, Edraki N, Badri R, Khoshneviszadeh M, Iraji A, Firuzi O. Multi-target

inhibitors against Alzheimer disease derived from 3-hydrazinyl 1,2,4-triazine scaffold

containing pendant phenoxy methyl-1,2,3-triazole: Design, synthesis and biological

evaluation. Bioorg Chem. 2019;84:363-71.

33.

Rastegari A, Nadri H, Mahdavi M, Moradi A, Mirfazli SS, Edraki N, et al. Design,

synthesis and anti-Alzheimer’s activity of novel 1,2,3-triazole-chromenone carboxamide

derivatives. Bioorg Chem. 2019;83:391-401.

34.

Jalili Baleh L, Nadri H, Forootanfar H, Samzadeh Kermani A, Küçükkılınç TT,

Ayazgok B, et al. Novel 3-phenylcoumarin–lipoic acid conjugates as multi-functional agents

for potential treatment of Alzheimer's disease. Bioorg Chem. 2018;79:223-34.

35.

Najafi Z, Mahdavi M, Saeedi M, Karimpour Razkenari E, Edraki N, Sharifzadeh M,

et al. Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer’s compounds:

In vitro and in vivo biological evaluation and docking study. Bioorg Chem 2019;83:303-16.

36.

chalcones: the odyssey continues. Expert Opin Drug Discov. 2019;14(3):249-88.

37. Singh G, Arora A, Kalra P, Maurya IK, Ruizc CE, Estebanc MA, et al. A strategic

Rani A, Anand A, Kumar K, Kumar V. Recent developments in biological aspects of

approach to the synthesis of ferrocene appended chalcone linked triazole allied

organosilatranes: Antibacterial, antifungal, antiparasitic and antioxidant studies. Bioorg Med

Chem. 2019;27(1):188-95.

38.

Singh G, Arora A, Mangat SS, Rani S, Kaur H, Goyal K, et al. Design, synthesis and

biological evaluation of chalconyl blended triazole allied organosilatranes as giardicidal and

trichomonacidal agents. Eur J Med Chem. 2016;108:287-300.

39.

chalcones: A mini review. Eur J Med Chem. 2014;85:758-77.

40. Yadav P, Lal K, Kumar A, Guru SK, Jaglan S, Bhushan S. Green synthesis and

anticancer potential of chalcone linked-1,2,3-triazoles. Eur J Med Chem. 2017;126:944-53.

41. Lal K, Yadav P, Kumar A, Kumar A, Paul AK. Design, synthesis, characterization,

Singh P, Anand A, Kumar V. Recent developments in biological activities of

antimicrobial evaluation and molecular modeling studies of some dehydroacetic acid-

chalcone-1,2,3-triazole hybrids. Bioorg Chem. 2018;77:236-44.

22

42.

Pingaew R, Saekee A, Mandi P, Nantasenamat C, Prachayasittikul S, Ruchirawat S, et

al. Synthesis, biological evaluation and molecular docking of novel chalcone–coumarin

hybrids as anticancer and antimalarial agents. Eur J Med Chem. 2014;85:65-76.

43.

oxidative stress in neuronal cells. RAS. 2015;3:40-5.

44. Konyalioglu S, Armagan G, Yalcin A, Atalayin C, Dagci T. Effects of resveratrol on

Othman SB, Yabe T. Use of hydrogen peroxide and peroxyl radicals to induce

hydrogen peroxide-induced oxidative stress in embryonic neural stem cells. Neural Regen

Res. 2013;8(6):485-95.

45.

induced hydrogen peroxide toxicity in neuronal cells. Neuroreport. 2012;23(17):989-94.

46. Cline EN, Bicca MA, Viola KL, Klein WL. The amyloid-beta oligomer hypothesis:

Beginning of the third decade. J Alzheimers Dis. 2018;64(s1):S567-S610.

47. Cieslik M, Czapski GA, Strosznajder JB. The molecular mechanism of amyloid

Peritore CS, Ho A, Yamamoto BK, Schaus SE. Resveratrol attenuates L-DOPA-

beta42 peptide toxicity: The role of sphingosine kinase-1 and mitochondrial sirtuins. PLoS

One. 2015;10(9):e0137193.

48.

neurodegenerative diseases. Oxid Med Cell Longev. 2014;2014:270291.

49. Zhang LL, Sui HJ, Liang B, Wang HM, Qu WH, Yu SX, et al. Atorvastatin prevents

Perez de la Cruz V, Korrapati SV, Pedraza-Chaverri J. Redox status and aging link in

amyloid-beta peptide oligomer-induced synaptotoxicity and memory dysfunction in rats

through a p38 MAPK-dependent pathway. Acta Pharmacol Sin. 2014;35(6):716-26.

50.

neuroprotection mediated by a targeted antioxidant. Sci Rep. 2014;4:4983.

51. Martinou JC, Youle RJ. Mitochondria in apoptosis: BCL-2 family members and

mitochondrial dynamics. Dev Cell. 2011;21(1):92-101.

52. Brunelle JK, Letai A. Control of mitochondrial apoptosis by the BCL-2 family. J Cell

Sci. 2009;122(Pt 4):437-41.

Giordano CR, Terlecky LJ, Bollig-Fischer A, Walton PA, Terlecky SR. Amyloid-beta

53.

Perelman A, Wachtel C, Cohen M, Haupt S, Shapiro H, Tzur A. JC-1: alternative

excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death

Dis. 2012;3(11):e430-e.

54.

Cenini G, Lloret A, Cascella R. Oxidative stress in neurodegenerative diseases: From

a mitochondrial point of view. Oxid Med Cell Longev. 2019;2019:2105607.

23

55.

Gay NH, Suwanjang W, Ruankham W, Songtawee N, Wongchitrat P,

Prachayasittikul V, et al. Butein, isoliquiritigenin, and scopoletin attenuate neurodegeneration

via antioxidant enzymes and SIRT1/ADAM10 signaling pathway. RSC Advances.

2020;10(28):16593-606.

56.

Van Hau T, Ruankham W, Suwanjang W, Songtawee N, Wongchitrat P, Pingaew R,

et al. Repurposing of nitroxolinedrug for the prevention of neurodegeneration. Chem Res

Toxicol. 2019;32(11):2182-91.

57.

Ruankham W, Suwanjang W, Wongchitrat P, Prachayasittikul V, Prachayasittikul S,

Phopin K. Sesamin and sesamol attenuate H₂O₂-induced oxidative stress on human neuronal

cells via the SIRT1-SIRT3-FOXO3a signaling pathway. Nutr Neurosci. 2019:1-12.

58.

Gay NH, Phopin K, Suwanjang W, Songtawee N, Ruankham W, Wongchitrat P, et al.

Neuroprotective effects of phenolic and carboxylic acids on oxidative stress-induced toxicity

in human neuroblastoma SH-SY5Y cells. Neurochem Res. 2018;43(3):619-36.

59.

Jin H, Kanthasamy A, Ghosh A, Anantharam V, Kalyanaraman B, Kanthasamy AG.

Mitochondria-targeted antioxidants for treatment of Parkinson's disease: Preclinical and

clinical outcomes. Biochim Biophys Acta. 2014;1842(8):1282-94.

60.

Radic Biol Med. 2013;62:4-12.

61. Ren Z, He H, Zuo Z, Xu Z, Wei Z, Deng J. The role of different SIRT1-mediated

signaling pathways in toxic injury. Cell Mol Biol Lett. 2019;24(1):36.

62. Xu J, Jackson CW, Khoury N, Escobar I, Perez-Pinzon MA. Brain SIRT1 mediates

metabolic homeostasis and neuroprotection. Front Endocrinol 2018;9(702).

63. Salvatori I, Valle C, Ferri A, Carrì MT. SIRT3 and mitochondrial metabolism in

neurodegenerative diseases. Neurochem Int 2017;109:184-92.

64. Shih J, Donmez G. Mitochondrial sirtuins as therapeutic targets for age-related

disorders. Genes & Cancer. 2013;4(3-4):91-6.

65. Maiese K. Forkhead transcription factors: Formulating a FOXO target for cognitive

loss. Curr Neurovasc Res. 2017;14(4):415-20.

66. Gomes BAQ, Silva JPB, Romeiro CFR, dos Santos SM, Rodrigues CA, Gonçalves

Flynn JM, Melov S. SOD2 in mitochondrial dysfunction and neurodegeneration. Free

PR, et al. Neuroprotective mechanisms of resveratrol in Alzheimer’s disease: Role of SIRT1.

Oxid Med Cell Longev. 2018;2018:8152373.

24

67.

Gay N, Phopin K, Suwanjang W, Ruankham W, Wongchitrat P, Prachayasittikul S, et

al. Attenuation of oxidative stress-induced neuronal cell death by Hydnophytum formicarum

Jack. Asian Pac J Trop Med. 2018;11(7):415-22.

68.

Jones MR, Mathieu E, Dyrager C, Faissner S, Vaillancourt Z, Korshavn KJ, et al.

Multi-target-directed phenol–triazole ligands as therapeutic agents for Alzheimer's disease.

Chem Sci. 2017;8(8):5636-43.

69.

Montanari S, Mahmoud AM, Pruccoli L, Rabbito A, Naldi M, Petralla S, et al.

Discovery of novel benzofuran-based compounds with neuroprotective and

immunomodulatory properties for Alzheimer's disease treatment. Eur J Med Chem.

2019;178:243-58.

70.

Adarsh N, Shanmugasundaram M, Ramaiah D. Efficient reaction based colorimetric

probe for sensitive detection, quantification, and on-site analysis of nitriteions in natural

water resources. Anal Chem. 2013;85(21):10008-12.

71.

Guantai EM, Ncokazi K, Egan TJ, Gut J, Rosenthal PJ, Smith PJ, et al. Design,

synthesis and in vitro antimalarial evaluation of triazole-linked chalcone and dienone hybrid

compounds. Bioorg Med Chem. 2010;18(23):8243-56.

72.

Efe C, Lykakis IN, Stratakis M. Gold nanoparticles supported on TiO₂ catalyse the

cycloisomerisation/oxidative dimerisation of aryl propargyl ethers. ChemComm

2011;47(2):803-5.

73.

Achard T, Lepronier A, Gimbert Y, Clavier H, Giordano L, Tenaglia A, et al. A regio-

and diastereoselective platinum-catalyzed tandem [2+1]/[3+2]ꢁcycloaddition sequence.

Angew Chem Int Ed. 2011;50(15):3552-6.

74.

Lykakis IN, Efe C, Gryparis C, Stratakis M. Ph3PAuNTf2 as a superior catalyst for

the selective synthesis of 2H-chromenes: Application to the concise synthesis of benzopyran

natural products. Eur J Org Chem. 2011;2011(12):2334-8.

75.

Hans RH, Guantai EM, Lategan C, Smith PJ, Wan B, Franzblau SG, et al. Synthesis,

antimalarial and antitubercular activity of acetylenic chalcones. Bioorg Med Chem Lett.

2010;20(3):942-4.

76.

Zammit SC, Cox AJ, Gow RM, Zhang Y, Gilbert RE, Krum H, et al. Evaluation and

optimization of antifibrotic activity of cinnamoyl anthranilates. Bioorg Med Chem Lett.

2009;19(24):7003-6.

25

77.

Tsuzuki Y, Nguyen TKN, Garud DR, Kuberan B, Koketsu M. 4-Deoxy-4-fluoro-

xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis. Bioorg Med Chem Lett.

2010;20(24):7269-73.

78.

Hunt L, Jordan M, De Jesus M, Wurm FM. GFP-expressing mammalian cells for fast,

sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnol Bioeng.

1999;65(2):201-5.

Table legend

Table1 Cell survival rate and cytotoxicity of chalcone-triazole derivatives^a,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^#

ND^e

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

ND^e

-

6a

> 50

6b

> 50

6c

> 50

6d

> 50

6e

6f

> 50

6g

> 50

Resveratrol^c

Ellipticine^c

ND^e

0.724

^aSignificance: ^*푃 < 0.05 vs. the untreated cells; ^#푃 < 0.05 vs. the H₂O₂group.

^bThe data are expressed as mean ± S.E.M.

^cResveratrol and ellipticine were used as the reference compounds.

^dCytotoxicity against Vero cells: IC₅₀> 50 μg/mL is defined as low cytotoxic effect.

^eND = not determined.

26

Figure legends

O

HO

O

OH

HO

O

OH

Chalcone

Licochalcone E

Licochalcone A

OH

O

OH

HO

OH

O

OH

Resveratrol

Xanthohumol

Fig. 1 Chemical structures of chalcone, licochalcones (A and E), xanthohumol and resveratrol

27

O

(i)

H

+

NH₂

2

1

3

(ii)

O

OR

O

5a-g

(iii)

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

c

a

e

b

d

g

R =

O

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 H₂O₂for 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 Aβ_1-42oligomer 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 H₂O₂or Aβ_1-42oligomer 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 H₂O₂group.

Article Doi

Synthesis and neuroprotective effects of novel chalcone-triazole hybrids

DOI: 10.1016/j.bioorg.2020.104384

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

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