Novel multifunctional ascorbic triazole derivatives for amyloidogenic pathway inhibition, anti-inflammation, and neuroprotection

molecules

Article

Novel Multifunctional Ascorbic Triazole Derivatives for

Amyloidogenic Pathway Inhibition, Anti-Inﬂammation,

and Neuroprotection

Jutamas Jiaranaikulwanitch ¹^,²^,* , Hataichanok Pandith , Sarin Tadtong

3

4

, Phanit Thammarat ,

2

Supat Jiranusornkul , Nattapong Chauthong , Supitcha Nilkosol and Opa Vajragupta ⁵

2

1

Innovation Center for Holistic Health, Nutraceuticals, and Cosmeceuticals, Faculty of Pharmacy,

Chiang Mai University, Chiang Mai 50200, Thailand

Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University,

2

Chiang Mai 50200, Thailand; phanit.thamma@cmu.ac.th (P.T.); supat.jira@cmu.ac.th (S.J.);

nattapong.chau@gmail.com (N.C.); supitchanilkosol@gmail.com (S.N.)

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand;

3

hataichanok.p@cmu.ac.th

Department of Pharmacognosy, Faculty of Pharmacy, Srinakharinwirot University,

4

Nakhonnayok 26120, Thailand; sarin@g.swu.ac.th

Ofﬁce of Research Affairs, Faculty of Pharmaceutical Sciences, Chulalongkorn University,

5

Bangkok 10330, Thailand; opa.v@chula.ac.th

*

Correspondence: jutamas.jia@cmu.ac.th; Tel.: +66-5394-4382

Abstract: Alzheimer’s disease (AD) is a common neurodegenerative disorder. The number of

patients with AD is projected to reach 152 million by 2050. Donepezil, rivastigmine, galantamine,

and memantine are the only four drugs currently approved by the United States Food and Drug

Administration for AD treatment. However, these drugs can only alleviate AD symptoms. Thus,

this research focuses on the discovery of novel lead compounds that possess multitarget regulation

of AD etiopathology relating to amyloid cascade. The ascorbic acid structure has been designated

as a core functional domain due to several characteristics, including antioxidant activities, amyloid

aggregation inhibition, and the ability to be transported to the brain and neurons. Multifunctional

ascorbic derivatives were synthesized by copper (I)-catalyzed azide–alkyne cycloaddition reaction

Citation: Jiaranaikulwanitch, J.;

Pandith, H.; Tadtong, S.; Thammarat,

P.; Jiranusornkul, S.; Chauthong, N.;

Nilkosol, S.; Vajragupta, O. Novel

Multifunctional Ascorbic Triazole

Derivatives for Amyloidogenic

Pathway Inhibition, Anti-Inflammation,

and Neuroprotection. Molecules 2021 ,

2

6 , 1562. https://doi.org/10.3390/

(

click chemistry). The in vitro and cell-based assays showed that compounds 2c and 5c exhibited

molecules26061562

prominent multifunctional activities as beta-secretase 1 inhibitors, amyloid aggregation inhibitors,

and antioxidant, neuroprotectant, and anti-inﬂammatory agents. Signiﬁcant changes in activities

promoting neuroprotection and anti-inﬂammation were observed at a considerably low concentration

at a nanomolar level. Moreover, an in silico study showed that compounds 2c and 5c were capable of

being permeated across the blood–brain barrier by sodium-dependent vitamin C transporter-2.

Academic Editor: Barbara Malawska

Received: 29 December 2020

Accepted: 9 March 2021

Published: 12 March 2021

Keywords: ascorbic derivatives; BACE1 inhibitor; amyloid aggregation inhibition; antioxidant;

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

neuroprotective; anti-inﬂammation

1. Introduction

Alzheimer’s disease (AD) is the main cause of dementia. It is a common neurodegen-

erative disorder, with signiﬁcant attributes relating to memory loss and cognitive function

impairments. It has negative impacts on patients’ thinking abilities, behaviors, and daily

routine activities. In 2020, over 50 million people have dementia, and the number of

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distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

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patients will reach 82 million in 2030 and 152 million in 2050 [

of several biological pathways that signiﬁcantly leads to the onset and progression of AD.

Considerable evidence has indicated that each form of amyloid- peptide aggregation

contributes to AD etiopathology. This includes the induction of toxic conditions to neurons

such as lipid peroxidation [ ], depolarization of synaptic membranes [ ], production of

1]. Amyloid cascade is one

β

2

4

.0/).

Molecules 2021, 26, 1562. https://doi.org/10.3390/molecules26061562

https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1562

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free radicals [

cytokines [

3

], induction of inﬂammatory response, and mediation of proinﬂammatory

4

]. Moreover, amyloid-β peptides can damage synapse-, neurite-, and neuron-

producing serotonin, epinephrine, glutamate, and acetylcholine neurotransmitters [5].

Beta-secretase 1 (BACE1) and gramma-secretase are important enzymes that cleave

the amyloid-

amyloid- peptides in neurons. Aggregations of these peptides set off the oligomerization

processes that produce amyloid- peptide oligomers. The oligomers then react with

the reactive metals in the brain; as a result, they produce neurotoxic reactive oxygen

species (ROS). Moreover, amyloid- peptide oligomers have been reported to induce

neuronal inﬂammation in the AD brain via the upregulation of inﬂammation-related genes,

cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [ ]. Altogether,

β precursor protein (APP), leading to the generation and accumulation of

β

6,7

increasing ROS and inﬂammation induce the activation of programmed cell death in brain

tissue, giving rise to the pathogenesis of AD [5].

Previously, the inhibitory mechanisms of amyloid-β peptide production and aggrega-

tion via BACE1 have been most intensively investigated. However, downstream processes

involving neuronal oxidative stress and inﬂammation are currently gaining attention and

have become strategies for the design and development of disease-modifying Alzheimer’s

drugs. Compounds with the capacities of both neuroprotection against neurotoxic ROS and

anti-inﬂammation have been deemed to, altogether, be beneﬁcial to promoting synergistic

effects to treat AD. As a result, they can increase the potencies of novel AD drug candi-

dates [6–8]. Additionally, during the past few decades, several single-targeted AD drugs

have failed in clinical trials, whereas multitargeted drugs or multitargeted-single-ligand

approaches have become more promising treatments. Diseases with etiological complexity,

like AD, could beneﬁt from this new paradigm shift. These novel treatments have also

recently attained more acceptance in AD research and clinical practices [9].

For drugs targeting the central nervous system, it is essential that AD drugs possess

the capacity to pass the blood–brain barrier (BBB). Prior studies have shown that ascorbic

acid crosses the BBB via interaction with sodium-dependent vitamin C transporter-2

(SVCT2) [10]. Additionally, its oxidized form, known as dehydroascorbic acid, permeates

the BBB via glucose transporters (GLUT) [10]. Ascorbic acid is a well-known potent

antioxidant. It also reduces amyloid oligomerization associated with a progression of AD

pathogenesis [11]. Therefore, ascorbic acid was selected as the functional core structure

in the design of the present study. This core domain was used as a skeletal structure for

subsequent chemical modiﬁcations. Despite the advantages mentioned earlier, ascorbic

acid has major pharmacological drawbacks such as low bioavailability [12]. Normally,

ascorbic acid predominantly exists in an anion form at neutral pH, causing a slow diffusion

rate across the plasma membrane. As the oral dose increases, percent bioavailability

is shown to be decreased, potentially due to saturated sodium-dependent vitamin C

transporter-1 (SVCT1) [13

organs relies on active transporters. Plasma ascorbic acid concentration is, therefore, higher

than that in tissue by 2.5-fold [12 15]. As a result, applying a higher daily dose may not

,14]. Moreover, a distribution from the bloodstream to targeted

,

be able to overcome this challenge. Moreover, a high intake increases risks of hemolysis

in patients with glucose 6 phosphate deﬁciency, paroxysmal nocturnal hemoglobinuria,

oxalate kidney stone development, and iron overload [16,17]. In addition, ascorbic acid can

act as a pro-oxidant due to a very good reducing ability. This behavior can reduce catalytic

metal, Cu²or Fe , to Cu and Fe and generate more ROS [18–20].

+

3+

+

2+

The present study aims to construct compounds exerting multitargeted functionality

to treat AD. The designs of the novel compounds emphasize the conjugation of the ascorbic

core structure to other functional motifs, namely, tryptoline and phenolic moieties, as

shown in Figure 1. Tryptoline was selected based on its well-known characteristics as

an inhibitor of several enzymes, including beta-secretase (BACE1), cholinesterase (ChE),

and monoamine oxidase (MAO) [21

well-recognized functioning against oxidative stress and

ties [24,25]. The functional motifs were linked to the ascorbic domain by a triazole ring to

–

23], while the phenolic motif was selected for its

β-amyloid aggregation activi-

Molecules 2021, 26, 1562

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generate ascorbic derivatives, which were then investigated for inhibition against BACE1,

amyloid aggregation, oxidative stress, and inﬂammation. In silico predictions of the bind-

ing capacities between the newly synthesized ascorbic derivatives to SVCT2, targeting BBB

permeation, were also evaluated.

Figure 1. Chemical structure of ascorbic acid and its conjugation via triazole linker with trypto-

line and phenolic moieties to create novel compounds with multitargeted functionality to treat

Alzheimer’s disease.

2. Results and Discussion

2.1. Synthesis

Six ascorbic derivatives (Figure 2) were designed as novel compounds, with multifunc-

tional activities impeding amyloid cascade toxicity pathways. In the ﬁrst series, ascorbic

acid was substituted at hydroxyl position C-6 to generate derivatives 2a 2c. In the sec-

ond series, ascorbic acid was substituted at hydroxyl position C-3 to generate derivatives

5c. The syntheses of these derivatives were carried out by the conjugating reactions of

–

5a–

the ascorbic acid core structure with either tryptoline or phenolic groups via the triazole

linker. The yields of these compounds ranged between 8.58% and 24.61%. The compounds’

yields (Table S1) and NMR spectrums (Figures S1–S12) are reported in the supplementary

information. The derivatives’ multifunctional modes of activities were determined in the

present study by in vitro chemical-based assays, cell-based assays, and in silico assays.

Figure 2. Structures of multifunctional novel ascorbic derivatives.

2.2. Biological Activity Assays

Ascorbic acid derivatives 2a–2c and 5a–5c were evaluated for biological activities,

including BACE1 inhibition, amyloid aggregation inhibition, and antioxidant, neuropro-

tection, and anti-inﬂammation activities. Table 1 shows the results for BACE1 inhibition,

amyloid aggregation inhibition, and antioxidant activities at a concentration of 400

Compounds 2c and 5c possessed moderate BACE1 inhibitory activities with %inhibition of

3.49 and 27.33, respectively. The BACE1 inhibitory activities of 2c and 5c were less than

µM.

2

Molecules 2021, 26, 1562

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BACE1 inhibitor I (%inhibition of 99.14 at a concentration of 100

µM). However, these two

compounds possessed inhibitory activity against BACE1 at least three times higher than

that of the ascorbic acid core structure (%inhibition of 7.84). The IC₅₀value of compounds

2c

and 5c were 725.70 and 593.10

µM, respectively. This suggests that the BACE1 inhibitory

activities of the core structure were improved by the conjugation to the tryptoline motif.

Table 1. Multifunctional in vitro activities of ascorbic derivatives at a concentration of 400 µM.

BACE1

Inhibition

±SEM)

6.80 ± 2.69

6.29 ± 0.98

23.49 ± 1.66 *

ND

Amyloid Aggregation

Antioxidant

%Inhibition

(±SEM)

66.54 ± 0.56 *

62.81 ± 2.62 *

94.67 ± 0.50

78.42 ± 0.93 *

77.37 ± 1.60 *

85.84 ± 2.43

94.85 ± 0.62

Compound

%

IC₅₀

(µM)

%Inhibition

(±SEM)

IC₅₀

(µM)

IC₅₀

(µM)

(

2

a

-

53.01 ± 0.60

50.99 ± 2.12 *

71.79 ± 1.42

34.53 ± 3.62 *

19.58 ± 1.83 *

68.26 ± 0.67

63.44 ± 0.87

318.60

318.80

92.33

-

206.50

155.70

72.26

91.15

103.10

62.89

21.28

2

b

c

725.70

5

a

-

5

b

c

ND

-

593.10

-

27.33 ± 1.41 *

136.00

146.10

Ascorbic acid

BACE1 inhibitor I

7.84 ± 1.75

9

9.14 ± 0.11 *

-

(

100 µM)

Curcumin

100 µM)

-

94.45 ± 1.15

-

(

ND indicates not detected. All data are presented as means

ascorbic acid of each group, based on paired t-test statistical analysis by using Microsoft Excel (* p ≤ 0.01).

±

SEM (n = 3). Asterisk denotes a signiﬁcant difference compared with the

All ascorbic derivatives exhibited the potential for amyloid aggregation inhibition

in the range of 19.58–71.79% and oxidative stress inhibition in the range of 62.81–94.67%.

These ﬁndings were expected due to the previously reported characteristics of the ascorbic

core structure. Despite lower inhibitory activities than curcumin against amyloid aggrega-

tion (94.45% inhibition at a concentration of 100

and 5c were able to hamper amyloid aggregation in a similar inhibitory magnitude to

ascorbic acid at a concentration of 400 M. Compounds 2c and 5c, containing the tryptoline

motif, were the best lead compounds for the inhibition of amyloid aggregation, with IC₅₀

µM), the newly synthesized compounds

2c

µ

values of 92.33 and 136.00

acid (IC₅₀value of 146.10

µ

M, respectively, which were stronger than that of ascorbic

µM). This suggests that the tryptoline moiety, conjugated to

the core structure, contributes to the rise of the amyloid aggregation inhibition activities

of compounds 2c and 5c. The substitution of the hydroxyl group at the C-6 position of

the ascorbic core structure led to higher antiamyloid aggregation activity than that of

substitution at the C-3 position. Additionally, the 3D structural model of compound 2c

showed that the distance between the aromatic end group and the ascorbic furanone ring

was 13.3 Å (Figure 3A). This distance is in good agreement with the previously reported

length of 13–14 Å between the aromatic nucleus of the amyloid aggregation inhibitor [26].

On the contrary, for compound 5c, the distance was 11.2 Å (Figure 3B), which was not

within the optimal range. The differences of this crucial distance might be the explanation

for the higher inhibitory activity of compound 2c against amyloid aggregation than that of

compound 5c.

Figure 3. Distance between the aromatic end group and the ascorbic furanone ring in the 3D structure

of ascorbic derivatives: (a) 2c (yellow) and (b) 5c (magenta).

Molecules 2021, 26, 1562

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Compounds 2c and 5c were the strongest antioxidants among the derivatives, with

IC₅₀values of 72.26 and 62.89 µM, respectively. However, they both exhibited lower antiox-

idant potential than ascorbic acid (IC₅₀value of 21.28

due to the structure s steric hindrance.

µM) by 3.0- to 3.4-fold, potentially

0

The results from the in vitro assays in the present study led to the conclusion that

the tryptoline moiety attributed to the multifunctional activities of compounds 2c and

5c. With regard to possession of BACE1 inhibition, amyloid aggregation inhibition, and

antioxidant activities, the novel compounds 2c and 5c could potentially produce the

mentioned synergistic effects for AD treatment.

Neuroprotective activities against oxidative stress of all the derivatives (2a

5c) and ascorbic acid were evaluated using cell viability assays of P19 embryonal carci-

noma cells. The P19 embryonal carcinoma cell is a pluripotent stem cell line that can be

differentiated into cholinergic neurons by all transretinoic acids [27 29]. The P19-derived

–2c and

5a–

–

neurons exhibit many characteristics of mature CNS neurons, containing neurotransmitter

acetylcholine [28], which can be used as a model to evaluate neurotoxicity and neuropro-

tective activity [29–33]. In the present study, neurotoxicity assays were performed prior

to neuroprotective activity assays, with test compounds at various concentrations of 1 to

4

1

0 nM to evaluate nontoxic concentrations. Cell viability was measured by the XTT assay

method, and 0.5% DMSO was used as a control. Ascorbic derivatives at 1 nM did not

exhibit cytotoxicity to P19-derived neurons (see supplementary information, Figure S13).

Conversely, percent cell viability was shown to be decreased by ascorbic acid. This could

partly be explained by ROS generation via an intracellular ascorbate-mediated production

of H O [34]. However, this effect depends on cell type and growth conditions [35]. Notice-

2

ably, the ascorbic derivatives exhibited higher percent cell viability than those in ascorbic

acid and 0.5% DMSO. This implies that a structural modiﬁcation of ascorbic acid might

help reduce pro-oxidation activity by potentially reducing reactivity or increasing steric

hindrance of the structure [36

intracellular ROS levels upon exposure to these derivatives.

The ascorbic derivatives (2a 2c and 5a 5c), at a nontoxic concentration (1 nM), were

,37]. However, further experiments are required to assess

–

selected to be further characterized for their neuroprotective effects against the toxic

conditions induced by serum deprivation. Serum is necessary for cell growth in cell culture

systems because it contains essential growth elements. Therefore, when cultured cells

are exposed to serum-deprivation conditions, this induces oxidative stress, leading to

apoptosis [38]; this can be used as an in vitro model to assess neuroprotective activity.

All ascorbic derivatives (2a–2c and 5a–5c) showed signiﬁcantly higher percentages of cell

survival than that of 0.5% DMSO in the toxic conditions, which was the absence of fetal

bovine serum (FBS; Figure 4; p < 0.05). Moreover, there were no statistical differences

in neuroprotective activities among compounds 2a, 2b, 5a–5c, and quercetin (positive

control; p > 0.05). However, because 2a and 5c (1 nM) showed signiﬁcantly higher viability

than 0.5% DMSO under a nontoxic condition (Figure S13), the observed neuroprotective

activities may be caused by this underlying effect. This effect could be partly due to

increased cell numbers or mitochondrial activities since the XTT assay is generally used

to measure cellular metabolic activities as the indicators of cell viability, cytotoxicity, and

proliferation [39

increase cell numbers [42

–

41]. Ascorbic acid and phenolic group have been previously shown to

45] and cellular ATP production [43 46]. Additionally, tryptoline

–

,

derivatives and phenolic derivatives were also able to promote neurite outgrowth [47],

which may be associated with increased mitochondria numbers, accumulated within the

neurite [48]. To understand the potential mechanisms of increased percent cell viability of

compounds 2a and 5c (1 nM) under a nontoxic condition, cell proliferation and intracellular

ATP levels should be further investigated.

The in vitro antioxidant activities of the synthesized compounds were in the micro-

molar range for the chemical-based assays. However, the neuroprotective effects against

neurotoxic ROS of these compounds in the cell-based assays were in the nanomolar range.

The differences in concentration magnitude might be partly explained by the experimental

Molecules 2021, 26, 1562

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conditions. There are several cellular defense mechanisms against ROS in the cell-based

study models, including mediation via enzymatic and nonenzymatic reactions. Therefore,

the effects of ascorbic acid and its derivatives against oxidative stress and cell apoptosis

have been determined by diverse markers at the cellular level. These include reduction of

malondialdehyde (MDA) [49,50], upregulation of the BCL2 gene (antiapoptotic regulator),

and downregulation of the TP53 gene (proapoptotic factor) [51].

Figure 4. Neuroprotective activities of ascorbic derivatives at a concentration of 1 nM on cultured P19-derived neurons

evaluated by serum-deprivation-induced oxidative stress. -MEM, supplemented with 10 M Ara-C, and 1% antibiotics–

antimycotic solution without FBS were used to induce the oxidative stress condition. Each bar is presented as mean

α

µ

±

SD (n = 3). Letter a denotes a signiﬁcant difference compared with the toxic condition (p < 0.05), and letter b denotes a

signiﬁcant difference compared with quercetin (1 nM) positive control (p < 0.05), based on one-way ANOVA using the

Origin Pro 9.0 program, Northampton, MA, USA.

Collectively, the results presented in this study prompted us to form conclusive as-

sumptions that compounds 2c and 5c, rather than other derivatives, had the best potential

for multifunctional BACE1 inhibition, amyloid aggregation inhibition, antioxidant, and neu-

roprotection activities. Thus, these two compounds were designated as the lead compounds

selected to be further investigated for anti-inﬂammatory activities at the cellular and molec-

ular levels. The ascorbic acid core structure and a well-known anti-inﬂammatory drug,

indomethacin, were also included in the studies for comparison to the lead compounds.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were appointed

as the inﬂammatory marker genes and study models for anti-inﬂammatory activities of the

lead compounds. These two genes were previously reported to be linked to AD. COX-2

expression is markedly elevated in the AD brain [6]. Regulation of the COX-2 gene has

also been correlated with clinical dementia in AD patients [52]. Additionally, the deﬁciency

of iNOS gene expression has been proven to substantially protect AD-like mice from

premature mortality, cerebral plaque formation, and increased β-amyloid levels. Therefore,

iNOS may play a crucial role in driving an initiation of β-amyloid deposition and disease

progression [7].

The cell-based studies were designed to proﬁle differential expressions of COX-2

and iNOS genes in comparison with 0.001% DMSO control by using lipopolysaccharide

(LPS)-activated RAW 264.7 macrophages. The effects of ascorbic acid, 2c, and 5c on the

viability of RAW 264.7 cells were determined by MTT assays. The results suggest that

all compounds, at concentrations from 0.001 to 100 nM, did not exhibit cytotoxicity to

RAW 264.7 after 24-h exposure. Cell viabilities were signiﬁcantly higher than 80% in all

experiments (n = 3, p-value < 0.05; see supplementary information, Figure S14).

The expression levels of the two inﬂammatory marker genes were measured by two-

step reverse transcription-quantitative PCR (RT-qPCR), with GAPDH as the reference

gene. RAW 264.7 cells were pretreated with either ascorbic acid, 2c, or 5c at two different

Molecules 2021, 26, 1562

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concentrations (10 and 100 nM) for one hour prior to induction of the inﬂammatory process

by LPS. Figure 5 shows that ascorbic acid, 2c, and 5c were able to downregulate the

expression of both COX-2 and iNOS genes compared to 0.001% DMSO control (n = 3,

p-value < 0.05). Additionally, the magnitudes of the downregulation of both genes by 2c

and 5c were notably stronger than that of indomethacin (50 µM) by about 1.5- to 2-fold.

Figure 5. Effects of ascorbic acid, compound 2c, and compound 5c at 10 and 100 nM, on mRNA expression levels of

COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) genes in LPS inﬂammatory-activated RAW 264.7

macrophages. Each bar is presented as mean

means between the groups (p-value < 0.05) based on Tukey’s HSD one-way ANOVA using PAST version 3.14, Oslo, Norway

±

SD (n = 3). Different letters (a, b, c, d, and e) indicate signiﬁcant differences of

.

The gene expression studies enabled us to effectively assess a pattern of the anti-

inﬂammatory capacities of ascorbic acid, 2c, and 5c at the molecular level. The expression

of COX-2 decreased between 30% to 70%, whereas iNOS expression was suppressed by

about 50% to 70% by ascorbic acid, 2c, and 5c. The ability to inhibit COX-2 gene expression

was ranked from high to low, as follows: 2c

and 100 nM), indomethacin (100 M); 2c (10 nM), and indomethacin (50

the capacity for iNOS inhibition was ranked from high to low, as follows: 5c

ascorbic acid (100 nM); 2c (100 nM), 5c 100 nM), ascorbic acid (10 nM), indomethacin

100 µM), 2c (10 nM), and indomethacin (50 µM). This leads to an initial conclusion that

the underlying molecular mechanisms of neuroprotective activities of the lead compounds

and 5c involve anti-inﬂammatory activities mediated through the inhibitions of both

(

100 nM), 5c (10 and 100 nM), ascorbic acid (10

M). Additionally,

10 nM),

µ

(

2c

COX-2 and iNOS gene expressions. The present study also reafﬁrms previous studies that

have reported the modulation activities of ascorbic acid to downregulate the expressions of

COX-2 and iNOS [53

,54]. However, this study is the ﬁrst to report the inhibitions of COX-2

and iNOS gene expression by ascorbyl tryptoline derivatives.

2.3. In Silico Study of Sodium-Dependent Vitamin C Transporter2 (SVCT2) Binding

Ascorbic acid was selected as a core structure in the present study not only for its

multifunctional activities but also its ability to pass through the BBB by binding to an active

transporter. Sodium-dependent vitamin C transporter 2 (SVCT2), which is ubiquitously

expressed in various brain tissues, including the cortex, cerebellar stem cells, neurons,

and neuroblastoma cells, is a well-known transporter of ascorbic acid into the brain [55].

The conjugation of ascorbic acid to several drugs such as nipecotic, kynurenic, and di-

clophenamic acids and ibuprofen has demonstrated competitive binding with the SVCT2

receptor and increased drug penetration into the brain over nonconjugated drugs [56,57].

Thus, 2c and 5c were studied for their binding afﬁnity with the SVCT2 receptor using

molecular docking.

Docking parameters used in the present study were validated by the redocking of

ascorbic acid. The docking pose of ascorbic acid was able to bind with the SVCT2 receptor

(pdb code: 4RP9) [58], similar to the crystal pose, with the refRMS value of 0.93 Å at the

binding free energy of 7.59 kcal/mol. Docking pose and crystal pose both had hydrogen

−

Molecules 2021, 26, 1562

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bond interaction with the same amino acids, including Thr86, Tyr87, Gln139, His135,

His194, Gln195, and Asp314. Residues having the van der Waals interaction with ascorbic

acid around the active site were Thr86, Tyr87, His135, Ile136, Gln139, His194, Gln195,

Asp314, Cys315, Ala316, Ile358, Phe362, and Met410. The binding modes of ascorbic acid

and the SVCT2 transporter are shown in Figure 6.

Figure 6. Ascorbic binding modes with sodium-dependent vitamin C transporter-2 (SVCT2) (pdb code: 4RP9). (

Superimposition of ascorbic acid crystal pose (yellow) and dock pose (cyan); ( ) van der Waals interaction of the residues

around the active sites, with ascorbic acid dock pose.

a)

b

In silico prediction by a docking study revealed that the designed compounds, 2c and

c, were able to bind with the SVCT2 receptor in a similar binding mode as ascorbic acid.

5

Compounds 2c and 5c exhibited binding free energy values of

−

10.36 and −11.97 kcal/mol

,

respectively, which were lower than that of ascorbic acid. This might indicate that com-

pounds 2c and 5c were able to form the complex to the transporter with more stable

conformations than that of ascorbic acid.

Compound 5c was able to bind with SVCT2, with a binding energy of -10.36 kcal/mol,

and the binding forces were comprised of van der Waals and H-bond interactions of the

residues around the active sites. Several amino acids exhibited involvement in the binding

interactions at the active sites, including Phe52, Leu55, Gln56, Ser59, Gly60, Thr63, Lys67,

Thr86, Tyr87, His135, Ile136, Gln139, His194, Gln195, Asp314, Cys315, Ala316, Ile358,

Phe362, and Met410. Compound 5c formed two hydrogen bonds with Gln56 and Thr63

residues of the transporter (Figure 7a). Nevertheless, compound 2c showed a binding

energy of -11.97 kcal/mol to SVCT2, suggesting a greater binding afﬁnity than that of

5

c

despite the similar manner of binding interactions. The explanation might be that

compound 2c interacts differently with SVCT2 at the residue positions of Lys67 and Tyr87

Figure 7b).

Interestingly, compounds 2c and 5c are bound to the transporter at the ascorbic

(

position via the tryptoline moiety instead of the ascorbic acid core structure. However,

both 2c and 5c were able to bind with the transporter at the amino acid residues, similar

to the ascorbic acid binding site (Thr86, Tyr87, His135, Ile136, Gln139, His194, Gln195,

Asp314, Cys315, Ala316, Ile358, Phe362, and Met410), a cocrystallized ligand. Thus, the

computational analysis presented in this study indicates that both 2c and 5c utilize sodium-

dependent vitamin C transporter 2 (SVCT2) to facilitate permeability across the BBB into

the brain.

3. Conclusions

The most potent lead compounds in the present study are 2c and 5c. Both novel com-

pounds showed promising multifunctional activities against amyloid cascade, including

BACE1 inhibition, amyloid aggregation inhibition, antioxidant activity, neuroprotective

activity, and anti-inﬂammatory activity. Chemical-based assays showed that BACE1 in-

hibition, amyloid aggregation inhibition, and antioxidant activity were exerted in the

micromolar range for both 2c and 5c. However, the neuroprotective and anti-inﬂammatory

Molecules 2021, 26, 1562

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activities, determined by cell-based assays, were present at the nanomolar level for both

compounds. The imbalance of activities on different biological targets indicates that 2c

and 5c should be used for anti-inﬂammation and neuroprotection; however, the lower

potency of BACE1 inhibition and anti-amyloid aggregation might help by increasing the

synergistic activity of the compounds. Moreover, the in silico study predicted that 2c and

5c could potentially pass through the BBB into the brain via interaction with the transporter

SVCT2. Compounds 2c and 5c are potential novel candidates to be further investigated

in preclinical studies. However, brain transport of these newly synthesized compounds

should be further conﬁrmed in animal studies. Additionally, the activities of these com-

pounds are still required to be improved. The target site of actions, particularly in brain

cells, and functional potency are essential to investigate. Regarding the low bioavailability

of ascorbic acid, compounds 2c and 5c are also required to be further assessed for their

pharmacokinetic proﬁles. The molecular structures of 2c and 5c could also be further modi-

ﬁed and synthesized as prodrugs to enhance and extend their capacities to treat AD beyond

their structural breakdown, as a result of drug metabolism, which may produce active

metabolites such as ascorbic acid (a potent amyloid aggregation inhibitor and antioxidant

and anti-inﬂammatory agent) and tryptoline (BACE1, ChE, and MAO inhibitors [21–23]).

In conclusion, the novel compounds 2c and 5c are potential candidates in the discovery

and development of new multitargeted AD drug designs.

Figure 7. Binding modes of ascorbic derivatives and vitamin C transporter 2 (pdb code: 4RP9).

(

a) Binding position of 5c (magenta) compared with ascorbic acid (cyan), and (

b) binding position of

2

c (yellow) compared with ascorbic acid (cyan).

4. Materials and Methods

4

.1. General

1

Chemical reagents were purchased from Aldrich, Chem-Impex, or TCI chemical. H-

NMR and ¹³C-NMR spectra were acquired on Bruker Avance 300 or 400 MHz instruments.

Mass spectra were recorded on LCMS Bruker MicroTof. IR spectra were recorded on Nicolet

FTIR 550. BACE1 enzyme and BACE1 substrate were purchased from Sino Biological ,

®

Beijing, China and Calbiochem , San Diego, CA, USA, respectively. Amyloid-

β

(1–42) from

®

Anaspec , Fremont, CA, USA was used in ThT assays. Neuroprotective assay statistics

were determined by ANOVA, calculated with Origin Pro 9.0 program, Northampton, MA,

USA. Statistical analysis of cell viability and gene expression data for the anti-inﬂammatory

activity study was conducted by Tukey’s HSD one-way ANOVA using PAST version 3.14,

Oslo, Norway. Molecular dockings were performed using AutoDockTools-1.5.6, San Diego,

CA, USA. All compounds were generated and optimized with ChemDraw Ultra 13.0 and

ChemBio3D Ultra 13.0, Waltham, MA, USA. Microsoft Excel 2013, Redmond, WA, USA

and GraphPad Prism version 5.02, San Diego, CA, USA were used in the generation of

graph visualization and the calculation of IC₅₀values.

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4

.2. Synthesis

Two series of ascorbic derivatives were synthesized from ascorbic alkyne,

reacting with azides by a copper (I)-catalyzed azide–alkyne cycloaddition reaction.

Brieﬂy, the hydroxyl group of ascorbic acid at carbon position 6 was substituted with

the propargyl group to yield the ascorbic alkynes . Additionally, ascorbic alkynes were

synthesized in three steps. Firstly, the hydroxyl group, at carbon position 5 and 6, were

protected with the acetonide group to generate compound . Secondly, the hydroxyl group

of compound , at carbon position 3, was substituted with the propargyl group to yield

compound . Finally, the acetonide protecting group of compound was cleaved in an

. The complete routes of synthesis are shown in

2

and 5,

a–c

1

2

5

3

4

acidic condition to yield ascorbic alkynes

Scheme 1.

5

Scheme 1. The synthesis route of ascorbic derivatives. (a) K CO , propargyl bromide, DMSO/THF (9:8), rt, 2 h. (b) N R,

2

3

5

% mol CuSO , 20% mol NaAsc, t-BuOH/EtOH/H O (2:2:1), rt, 2 h. (c) 2,2 dimethoxypropane, p-toluenesulfonyl chloride,

4 2

rt, 24 h. (d) K CO , propargyl bromide, DMSO/THF (9:8), rt, 2 h. (e) 12 N HCl, THF, rt, 2.5 h. (f) N R, 5% mol CuSO , 20%

2

3

4

mol NaAsc, t-BuOH/H O (4:2) or t-BuOH/EtOH/H O (4:4:2), rt, 2 h.

2

4

.2.1. (R)-3,4-Dihydroxy-5-((S)-1-hydroxy-2-(prop-2-yn-1-yloxy)ethyl)furan-2(5H)-one; 2

Ascorbic alkyne was synthesized according to the procedure of Wimalasena and

Mahindaratne [59]. A mixture of ascorbic acid (2.0000 g, 9.34 mmol) and 0.5 equivalent of

K CO (0.6452 g, 4.67 mmol) in DMSO/THF (9:8) was stirred at room temperature for 20

2

3

min. Propargyl bromide (0.83 mL, 9.34 mmol) in the same solvent was added dropwise to

the reaction by dropping funnel. The reaction was vigorously stirred at room temperature

under nitrogen for 2 h. The reaction was diluted with water (30 mL) and extracted with

ethyl acetate (3

×

30 mL). The organic layer was washed with brine and dried over Na SO .

2 4

The solvent was removed under reduced pressure, and the obtained residue was puriﬁed

by column chromatography (Hex/EtOAc 2:3) to yield of yellow viscous oil (0.5832 g, 29%);

FTIR (ATR, cm ): 3425 (O-H, st), 3279 (O-H, st), 2983, 2957, 2894 (aliphatic C-H, st), 2123

−

1

(

C

≡

C, st), 1771 (C=O, st), 1695 (C=C, st), 1326, 1210, 1127 (C-O, st); H-NMR (300 MHz,

DMSO-d ):

δ

2.47 (1H, m), 2.66 (1H, dd, J = 2.70, 16.45 Hz), 2.95 (1H, t, J = 2.68 Hz), 3.84

6

(

1H, dd, J = 4.22, 9.40 Hz), 4.12 (1H, dd, J = 6.18, 9.41 Hz), 4.27 (1H, m), 4.52 (1H, s), 5.60

+

1H, d, J = 4.14 Hz), 5.96 (1H, s), 6.96 (1H, s); HRMS (ESI) m/z calcd for [M] 214.04774,

+

Found 237.03855 [M+Na] .

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4

.2.2. (R)-5-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)-3,4-dihydroxyfuran-2(5H)-one; 3

Ascorbic acid (20.0000 g, 113.56 mmol) was dissolved in 80 mL of acetone. Then, 2,2

dimethoxypropane (13.91 mL, 113.56 mmol) and p-toluenesulfonyl chloride (43.3000 g,

27.118 mmol) was added to the reaction and stirred at room temperature for 24 h. After the

2

reaction was completed, the reaction mixture was ﬁltered and the precipitate was washed

◦

with cooled acetone to yield of white powder (16.1737 g, 65.88%); m.p. 218–220 C; FTIR

−

1

(

ATR, cm ): 3236 (O-H, st), 2957, 2923, 2853 (aliphatic C-H, st), 1748 (C=O, st), 1658 (C=C,

st), 1330, 1137, 1063 (C-O, st); H-NMR (300 MHz, DMSO-d ): 1.24 (6H, s), 3.88 (1H, dd,

1

6

J = 6.32, 8.40 Hz), 4.10 (1H, dd, J = 7.05, 8.36 Hz), 4.26 (1H, td, J= 2.92, 6.48 Hz), 4.71 (1H, d,

+

J = 2.91 Hz), 8.49 (1H, s), 11.31 (1H, s); HRMS (ESI) m/z calcd for [M] 216.06339, Found

+

2

39.05475 [M+Na] .

4

2

.2.3. (R)-5-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)-3-hydroxy-4-(prop-2-yn-1-yloxy)furan-

(5H)-one; 4

Compound

daratne. A mixture of compound

0.6392 g, 4.63 mmol) in DMSO/THF (9:8) was stirred at room temperature for 20 min.

4

was synthesized according to the procedure of Wimalasena and Mahin-

3

(2.0000 g, 9.25 mmol) and 0.5 equivalent of K CO

2

3

(

Propargyl bromide (0.80 mL, 9.25 mmol) in the same solvent was added dropwise to the

reaction by dropping funnel. The reaction was vigorously stirred at room temperature

under nitrogen for 2 h. The reaction was diluted with water (30 mL) and extracted with

ethyl acetate (3

×

30 mL). The organic layer was washed with brine and dried over Na SO .

2 4

The solvent was removed under reduced pressure and the obtained residue was puriﬁed by

column chromatography (Hex/EtOAc 8:2) to yield of yellow viscous oil (1.2025 g, 51.13%);

FTIR (ATR, cm ): 3285 (O-H, st), 2987, 2937, 2897 (aliphatic C-H, st), 2130 (C

−

1

≡

C, st),

1

1764 (C=O, st), 1695 (C=C, st), 1373, 1319, 1213, 1143, 1060 (C-O, st); H-NMR (300 MHz,

DMSO-d ): 1.26 (6H, s), 3.72 (1H, t, J = 2.42 Hz), 3.86 (1H, dd, J = 6.04, 8.44 Hz), 4.10 (1H,

6

dd, J = 7.09, 8.38 Hz), 4.23 (1H, m), 4.84 (1H, d, J = 2.81 Hz), 5.04 (1H, m), 9.27 (1H, s);

+

HRMS (ESI) m/z calcd for [M] 254.07904, Found 256.26494 [M+2H] , 277.06949 [M+Na] .

4

.2.4. (R)-5-((S)-1,2-Dihydroxyethyl)-3-hydroxy-4-(prop-2-ynyloxy)furan-2(5H)-one; 5

Compound was synthesized by deprotection of the acetonide group in an acidic

condition. The pH of the compound solution (0.5923 g, 2.33 mmol) in THF was adjusted

to pH 1 by 1 mL of 12 N HCl. The resulting mixture was stirred at room temperature for

.5 h. After reaction was completed, the reaction mixture was diluted with water (30 mL)

and extracted with ethyl acetate (3 30 mL). The organic layer was washed with brine

and dried over Na SO . The solvent was removed under reduced pressure to obtained

5

4

2

×

2

4

yellow oil (0.4298 g, 86.14%). This residue was puriﬁed by column chromatography

−

1

(

Hex/EtOAc/MeOH 4:6:0.1) for identiﬁcation; FTIR (ATR, cm ): 3360, 3290 (O-H, st),

2

923, 2853 (aliphatic C-H, st), 2123 (C

≡

C, st), 1759 (C=O, st), 1696 (C=C, st), 1327, 1156,

1

1064 (C-O, st); H-NMR (300 MHz, DMSO-d ): 3.45 (2H, m), 3.65 (1H, t, J = 7.25 Hz), 3.70

6

(

5

1H, t, J = 2.40 Hz), 4.79 (1H, d, J = 1.25 Hz), 4.97 (1H, dd, J = 15.5, 32.44 Hz), 4.97 (1H, br),

+

.11 (1H, dd, J = 15.53, 2.41 Hz), 9.07 (1H, s); HRMS (ESI) m/z calcd for [M] 214.04774,

+

Found 215.05676 [M H] .

4.2.5. 4-(Azidomethyl)phenol; a

4

-Hydroxybenzyl alcohol (2.5000 g, 20.00 mmol) and potassium ﬂuoride (1.3922 g,

◦

2

4.00 mmol) were dissolved in acetic acid (5 mL). The reaction mixture was stirred at 80 C

for 1 h. After reaction was completed, water (30 mL) was added to the reaction mixture and

extracted with ethyl acetate (3 30 mL). The organic phase was washed with saturated

NaHCO , brine, dried over Na SO , and concentrated under reduced pressure. The

×

3

2

4

obtained residue was puriﬁed by column chromatography using dichloromethane/ethyl

acetate (1:1) to give a 63.13% yield of the intermediate. This intermediate and NaN (2 eq)

3

were dissolved in dried DMF under nitrogen. Boron triﬂuoride etherate (3 eq) was added

to the reaction and stirred at room temperature, with light protection, for 3 h. After reaction

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was completed, dichloromethane (30 mL) was added to the reaction and washed with

saturated NaHCO , brine, dried over Na SO , and concentrated under reduced pressure.

3

2

4

The product was puriﬁed by column chromatography (Hex/EtOAc 9:1) to yield of dark

−

1

liquid (0.4815 g, 16.15%); FTIR (ATR, cm ): 3354 (O-H, st), 3059 (aromatic C-H, st), 2926,

2

854 (aliphatic C-H, st), 2097 (N=N=N, st), 1515 (C=C, st), 1264 (C-O, st), 1240 (C-N, st);

1

H-NMR (300 MHz, DMSO-d₆):

Hz), 9.57 (1H, s). HRMS (ESI) m/z calcd for [M] 149.05891, Found 107.05055 [M-42] .

δ 4.29 (2H, s), 6.77 (2H, d, J = 8.52 Hz), 7.18 (2H, d, J = 8.52

+

4.2.6. 4-(Azidomethyl)-2-methoxyphenol; b

4-Hydroxy-3-methoxybenzyl alcohol (3.0000 g, 19.46 mmol) and potassium ﬂuoride

(

1.3547 g, 23.35 mmol) were dissolved in acetic acid (5 mL). The reaction mixture was stirred

◦

at 80 C for 1 h. After reaction was completed, water (30 mL) was added to the reaction

mixture and extracted with ethyl acetate (3

×

30 mL). The organic phase was washed with

4

saturated NaHCO , brine, dried over Na SO , and concentrated under reduced pressure.

3

2

The product was puriﬁed by column chromatography using dichloromethane to give a

3.10% yield of the intermediate. This intermediate and NaN (4 eq) were dissolved in

7

3

dried DMF under nitrogen. Boron triﬂuoride etherate (6 eq) was added to the reaction and

stirred at room temperature with light protection for 3 h. After reaction was completed,

dichloromethane (30 mL) was added to the reaction and washed with saturated NaHCO₃,

brine, dried over Na SO , and concentrated under reduced pressure, and the obtained

2

4

residue was puriﬁed by column chromatography using hexane: ethyl acetate (9:1) to yield

−

1

of yellow liquid (0.4709 g, 13.51%); FTIR (ATR, cm ): 3511, 3423 (O-H, st), 3011 (aromatic

C-H, st), 2936, 2844 (aliphatic C-H, st), 2096 (N=N=N, st), 1514 (C=C, st), 1272, 1237 (C-O,

1

st), 1154 (C-N, st); H-NMR (300 MHz, DMSO-d ):

δ

3.77 (3H, s), 4.30 (2H, s), 6.78 (2H, d,

6

+

J = 1.07 Hz), 6.94 (1H, s), 9.12 (1H, s). HRMS (ESI) m/z calcd for [M] 179.06948, Found

+

1

37.06065 [M-42] .

4.2.7. 4-(Azidomethyl)-2-methoxyphenol; c

Azide compound was synthesized, following the previous method of tryptoline

c

azide, with some modiﬁcation of deprotection reagent [60]. Brieﬂy, the carboxylic group of

tryptoline-3-carboxylic acid was ﬁrstly activated by esteriﬁcation to obtain In-c1, which

was further reduced to yield In-c2. Then, the hydroxyl group of In-c2 was replaced with

nosyl, Ns, a good leaving group for azide substitution. Finally, the nosyl group at amine,

Ns, was removed by p-toluenethiol in the basic condition to yield (3-azidomethyl)tryptoline

c, as shown in Scheme 2.

Scheme 2. The synthesis route of (3-azidomethyl)tryptoline,

c. (a) MeOH, H SO , reﬂux, 18 h. (b) NaBH , MeOH/THF

2 4 4

◦

(

1:1), reﬂux, 18 h. (c) NsCl, TEA, CH Cl , rt, 4 h. (d) NaN , DMF, 70 C, 6 h. (e) p-toluenethiol, DMF, 50 C, 2 h.

2

3

4.2.8. Methyl 2,3,4,9-tetrahydro-1H-pyrido [3,4-b]indole-3-carboxylate; In-c1

A mixture of tryptoline-3-carboxylic acid (5.00 g, 23.10 mmol) and 3 mL of sulfuric

acid in 30 mL of methanol was heated at reﬂux for 18 h. Then, 30 mL of water was added

Molecules 2021, 26, 1562

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to the reaction mixture, and the organic solvent was removed under reduced pressure.

After pH was adjusted to pH 8 with saturated sodium bicarbonate solution, the aqueous

phase was extracted with ethyl acetate (30 mL

sodium sulfate, concentrated, and puriﬁed by crystallization with ethyl ether and methanol

×

2). The organic solution was dried over

(

1

10:2) to yield compound In-c1. Product was a light brown crystal (5.2997 g, 99.64%); m.p.

66–168 C; FTIR (ATR) (cm ): 3388, 3299 (N-H, st), 3056 (aromatic C-H, st), 2950, 2845

◦ −1

(

aliphatic C-H, st) 1732 (C=O, st), 1452, 1436 (aromatic C=C, st), 1331 (C-N, st), 1269 (C-O,

1

st), 1201 (C-N, st), 741 (aromatic C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 10.76

6

(1H, s), 7.39 (1H, d, J = 7.6 Hz), 7.28 (1H, d, J = 7.9 Hz), 7.03 (1H, t, J = 6.9 Hz), 6.95 (1H, t,

J = 6.8 Hz), 4.02 (2H, q, J = 16.0 Hz), 3.85 (1H, dd, J = 8.9, 4.9 Hz), 3.70 (3H, s), 3.45 (1H, s),

2.98 (1H, dd, J = 15.0, 4.8 Hz), 2.78 (1H, dd, J = 15.1, 8.9 Hz).

4.2.9. (2,3,4,9-Tetrahydro-1H-pyrido [3,4-b]indol-3-yl)methanol; In-c2

Compound In-c1 (5.2997 g, 23.02 mmol), in 30 mL of dried methanol and dried

tetrahydrofuran (1:1), was added slowly to a stirred solution of sodium borohydride

3.0475 g, 50.56 mmol) at room temperature. The mixture was heated to boiling under

reﬂux for 18 h. The solution was evaporated under reduced pressure to remove organic

solvent. Then, 30 mL water was added and extracted with ethyl acetate (30 mL 2). The

organic solution was dried, concentrated, and puriﬁed by column chromatography on

silica gel (CH Cl /EtOAc/MeOH/NH OH 5:4.5:0.5:0.01) to yield compound In-c2. The

(

×

2

4

◦

−1

product was white powder (2.7148 g, 58.32%); m.p. 194–196 C; FTIR (ATR) (cm ): 3395

indole N-H, st), 3279 (N-H, st), 3200 (O-H, st), 3050 (aromatic C-H, st), 2885, 2841 (aliphatic

(

C-H, st), 1450 (aromatic C=C, st), 1326 (C-N, st), 1215 (C-O, st), 1023 (C-N, st) 743 (aromatic

1

C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 10.65 (1H, s), 7.33 (1H, d, J = 7.5 Hz), 7.25

6

(1H, d, J = 7.8 Hz), 6.99 (1H, t, J = 6.8 Hz), 6.92 (1H, t, J = 6.8 Hz), 4.69 (1H, t, J = 5.4 Hz),

3

.91 (1H, s), 3.59–3.41 (1H, m), 2.85 (1H, dd, J = 10.2, 5.4 Hz), 2.62 (1H, dd, J = 14.9, 4.0 Hz),

2.37–2.22 (2H, m).

4

.2.10. (S)-3-(Azidomethyl)-2-(4-nitrophenylsulfonyl)-2,3,4,9-tetrahydro-1H

–

pyrido[3,4-b]indole; In-c4

A mixture of compound In-c2 (2.0654 g, 10.21 mmol) and triethylamine (2.25 mL,

3

0.64 mmol) in 30 mL of dried dichloromethane was added with 4-nitrobenzenesulfonyl

◦

chloride (5.6580 g, 25.53 mmol) in dried dichloromethane at 0 C. The reaction mixture

was adjusted to room temperature and stirred for 4 h. Water (30 mL) was added to the

reaction. Organic phase was washed with 1N HCl, saturated NaHCO and brine. The

3

organic solution was dried and concentrated to yield compound In-c3 intermediate. This

intermediate was redissolved in dried DMF. NaN (3.3194 g, 51.06 mmol) was added to

3

◦

the solution and stirred at 70 C for 6 h. After reaction was completed, 30 mL of water

was added to the reaction mixture. The resulting solution was extracted with ethyl acetate

(

30 mL × 2). The organic phase was washed with saturated NaHCO and brine. The

3

resulting solution was dried, concentrated, and puriﬁed by column chromatography on

silica gel with CHCl to yield a yellow powder of compound In-c4 (3.2969 g, 78.28%); m.p.

3

◦

−1

2

05–207 C; FTIR (ATR) (cm ): 3389 (indole N-H, st), 3018 (aromatic C-H, st), 2927, 2868

(

aliphatic C-H, st), 2101 (N=N=N, st), 1667 (N-H, bending), 1529 (N=O, st), 1454 (aromatic

C=C, st), 1349 (N=O, st), 1347 (S=O, st), 1167 (S=O, st), 1094 (C-N, st), 854 (nitroaromatic

1

C-N, st), 760 (aromatic C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 10.84 (1H, s,

6

J = 8.1 Hz), 8.29 (2H, d, J = 8.9 Hz), 8.10 (2H, d, J = 8.9 Hz), 7.28 (2H, t, J = 6.9 Hz), 7.03 (1H,

t, J = 7.5 Hz), 6.91 (1H, t, J = 7.4 Hz), 4.87 (1H, d, J = 17.4 Hz), 4.69–4.59 (1H, m), 4.46 (1H, d,

J = 17.5 Hz), 3.50 (1H, dd, J = 13.0, 9.7 Hz), 3.39 (1H, dd, J = 13.0, 5.4 Hz), 2.70–2.57 (2H, m).

4.2.11. (S)-3-(Azidomethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole; c

A mixture of compound In-c4 (3.2969 g, 7.99 mmol), potassium carbonate (3.3143 g,

2

5

3.98 mmol), and p-toluenethiol (4.9643 g, 39.97 mmol) in 5 mL of DMF was stirred at

◦

0 C for 2 h. After the reaction was completed, 15 mL of saturated aqueous NH Cl was

4

added. The mixture solution was extracted with ethyl acetate (30

×

2 mL). The organic

Molecules 2021, 26, 1562

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phase was washed with water, saturated NaHCO and brine. The resulting solution was

3

dried, concentrated, and puriﬁed by column chromatography on silica gel (CHCl /MeOH

3

1

4

0:0.2). A yellow powder product of (3-azidomethyl)tryptoline, c, was obtained (0.7484 g,

1.19%); m.p. 179–181 C; FTIR (ATR) (cm ): 3396 (indole N-H, st), 3161 (N-H, st), 3056

◦

−1

(

1

aromatic C-H, st), 2923, 2842 (aliphatic C-H, st), 2100 (N=N=N, st), 1450 (aromatic C=C, st),

1

267 (C-N, st), 742 (aromatic C-H, bending); HNMR (300 MHz, DMSO-d ):

δ

10.70 (1H, s,

6

H9), 7.34 (1H, d, J = 7.57 Hz, H5), 7.26 (1H, d, J = 7.82 Hz, H8), 7.00 (1H, t, J = 6.83 Hz, H7),

.93 (1H, t, J = 6.79 Hz, H6), 3.91 (2H, s, H1), 3.44 (2H, dd, J = 2.51, 6.19 Hz, H10), 3.03 (1H,

m, H3), 2.68 (1H, dd, J = 4.11, 14.93 Hz, H4b), 2.36 (1H, dd, J = 9.92, 14.96 Hz, H4a); HRMS

6

+

(

ESI) m/z calcd for [M] 227.1171, Found 228.12485 [M+H] , 144.08179 [M-84] .

4

.2.12. (R)-3,4-Dihydroxy-5-((S)-1-hydroxy-2-((1-(4-hydroxybenzyl)-1H-1,2,3-triazol-4-

yl)metho-xy)ethyl)furan-2(5H)-one; 2a

A mixture of compound (0.1806 g, 0.843 mmol), 4-(azidomethyl)phenol, a, (0.1509 g,

1

.012 mmol), 5% mol CuSO , and 20% mol sodium ascorbate in 8 mL of t-BuOH/EtOH/H O

4

2

(

2:2:1) was stirred at room temperature for 2 h. After reaction was completed, water (10 mL)

was added to the reaction mixture. The aqueous solution was extracted with ethyl acetate

20 mL). The organic solution was washed with brine, dried, concentrated, and puriﬁed

by column chromatography (Hex/EtOAc/MeOH 1:4:0.1). A yellow viscous of compound

(3

×

−

1

2a was obtained (0.0263 g, 8.58%); FTIR (ATR) (cm ): 3358 (O-H, st), 3194 (aromatic C-H,

st), 2955, 2851 (aliphatic C-H, st), 1659, 1632 (C=O, st), 1468 (aromatic C=C, st), 1411 (C-N,

1

st), 1237 (C-O, st), 722 (aromatic C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 3.40 (2H,

6

m, H6), 3.62 (1H, d, J = 6.27 Hz, H5), 4.75 (1H, d, J = 0.74 Hz, H4), 4.82 (1H, s, OH), 5.01

(

[

1H, d, J = 5.92 Hz, OH), 5.47 (4H, m, H7, H10), 6.74 (2H, d, J = 8.43 Hz, H13, H15), 7.18

1

3

2H, d, J = 8.48 Hz, H12, H16), 8.18 (1H, s, H9), 9.00 (1H, s, OH), 9.56 (1H, s, OH); C-NMR

300 MHz, DMSO-d₆): 53.07 (C10), 62.19 (C5), 64.03 (C7), 68.86 (C6), 74.96 (C4), 115.90

δ

C13, C15), 120.29 (C2), 124.84 (C9), 126.50 (C11), 130.13 (C12, C16), 142.90 (C8), 150.45

+

C14), 157.85 (C3), 170.80 (C1); HRMS (ESI) m/z calcd for [M] 363.10665, Found 386.09639

+

M+Na] , 364.11406 [M+H] .

4

.2.13. (R)-3,4-Dihydroxy-5-((S)-1-hydroxy-2-((1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-

triazol-4-yl)methoxy)ethyl)furan-2(5H)-one; 2b

A mixture of compound (0.1403 g, 0.655 mmol), 4-(azidomethyl)-2-methoxyphenol,

2

b, (0.1409 g, 0.786 mmol), 5% mol CuSO , and 20% mol sodium ascorbate in 8 mL of

4

t-BuOH/EtOH/H O (2:2:1) was stirred at room temperature for 2 h. After reaction was

2

completed, water (10 mL) was added to the reaction mixture. The aqueous solution was

extracted with ethyl acetate (3

dried, concentrated and puriﬁed by column chromatography (Hex/EtOAc/MeOH 1:4:0.1).

×

20 mL). The organic solution was washed with brine,

−

1

A yellow viscous of compound 2b was obtained (0.0317 g, 12.30%); FTIR (ATR) (cm ):

360 (O-H, st), 3195 (aromatic C-H, st), 2923, 2852 (aliphatic C-H, st), 1660, 1632 (C=O, st),

3

1

467 (aromatic C=C, st), 1410 (C-N, st), 1264 (C-O, st), 1132 (C-N, st), 737, 704 (aromatic

1

C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 3.41 (2H, m, H6), 3.61 (1H, dd, J = 7.38,

6

1

3.74 Hz, H5), 3.74 (3H, s, CH ), 4.75 (1H, s, H4), 4.82 (1H, t, J = 5.66 Hz, OH), 5.01 (1H,

3

d, J = 6.15 Hz, OH), 5.47 (4H, m, H7, H10), 6.74 (2H, d, J = 8.29 Hz, H12, H16), 6.98 (H, d,

13

J = 4.35 Hz, H15), 8.20 (1H, s, H9), 9.01 (1H, s, OH), 9.13 (1H, s, OH); C-NMR (300 MHz,

DMSO-d₆): 53.39 (CH ), 56.08 (C10), 62.18 (C5), 64.02 (C7), 68.87 (C6), 74.97 (C4), 113.01

δ

3

(

C12), 115.98 (C15), 120.29 (C2), 121.47 (C16), 124.88 (C9), 126.90 (C11), 142.89 (C8), 147.09

+

C14), 148.08 (C13), 150.44 (C3), 170.79 (C1); HRMS (ESI) m/z calcd for [M] 393.11721,

+

Found 416.10439 [M+Na] , 394.12280 [M+H] .

4

.2.14. (R)-3,4-Dihydroxy-5-((S)-1-hydroxy-2-((1-((2,3,4,9-tetrahydro-1H-pyrido[3,4-

b]indol-3-yl)-methyl)-1H-1,2,3-triazol-4-yl)methoxy)ethyl)furan-2(5H)-one; 2c

A mixture of compound (0.2693 g, 1.257 mmol), 3-(azidomethyl)-2,3,4,9-tetrahydro-

2

1

H-pyrido[3,4-b]indole, c, (0.1858 g, 0.817 mmol), 5% mol CuSO , and 20% mol sodium

4

ascorbate in 8 mL of t-BuOH/EtOH/H O (2:2:1) was stirred at room temperature for

2

Molecules 2021, 26, 1562

15 of 22

2

h. After reaction was completed, water (10 mL) was added to the reaction mixture.

The aqueous solution was extracted with ethyl acetate (3

was washed with brine, dried, concentrated, and puriﬁed by column chromatography

EtOAc/EtOH 7:3). A light brown powder of compound 2c was obtained (0.1361 g, 24.52%);

×

20 mL). The organic solution

(

◦

−1

m.p. 172–174 C; FTIR (ATR) (cm ): 3360 (O-H, st), 3192 (aromatic C-H, st), 2922, 2851

(

aliphatic C-H, st), 1659, 1632 (C=O, st), 1467 (aromatic C=C, st), 1409 (C-N, st), 1264 (C-O,

1

st), 738, 703 (aromatic C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 2.42 (1H, dd,

6

J = 9.84, 14.86 Hz, H12a) 2.67 (1H, dd, J = 3.96, 14.98 Hz, H12b), 3.42 (3H, m, H6, H11), 3.65

1H, m, H5), 3.91 (2H, q, J = 16.57 Hz, H10), 4.55 (2H, m, H21), 4.78 (1H, d, 0.93 Hz, H4),

.84 (1H, s, OH), 5.03 (1H, s, OH), 5.48 (1H, d, J = 12.16 Hz, H7a), 5.57 (1H, d, J = 12.18

(

4

Hz, H7b), 6.93 (1H, t, J = 7.28 Hz, H16), 7.01 (1H, t, J = 7.48 Hz, H17), 7.27 (1H, d, J = 7.84

Hz, H18), 7.34 (1H, d, J = 7.51 Hz, H15), 8.27 (1H, s, H9), 9.08 (1H, s, OH), 10.72 (1H, s,

NH-indole); ¹³C-NMR (300 MHz, DMSO-d₆):

δ

25.70 (C12), 42.24 (C21), 53.85 (C11), 54.21

(

C10), 62.20 (C5), 64.09 (C7), 68.88 (C6), 74.99 (C4), 106.28 (C13), 111.33 (C18), 117.59 (C2),

1

4

18.74 (C15), 120.37 (C16), 120.89 (C17), 125.92 (C14), 127.39 (C9), 134.08 (C20), 136.23 (C19),

+

42.44 (C8), 150.48 (C3), 170.84 (C1); HRMS (ESI) m/z calcd for [M] 441.16483, Found

+

42.17209 [M+H] .

4

.2.15. (R)-5-((S)-1,2-Dihydroxyethyl)-3-hydroxy-4-((1-(4-hydroxybenzyl)-1H-1,2,3-

triazol-4-yl)-methoxy)furan-2(5H)-one; 5a

A mixture of compound

5 (0.4298 g, 2.007 mmol), 4-(azidomethyl)phenol, a, (0.4490 g,

3

.010 mmol), 5% mol CuSO , and 20% mol sodium ascorbate in 6 mL of t-BuOH/H O

4

2

(

4:2) was stirred at room temperature for 2 h. After reaction was completed, water (10 mL

was added to the reaction mixture. The aqueous solution was extracted with ethyl acetate

3 × 20 mL). The organic solution was washed with brine, dried, concentrated, and puriﬁed

by column chromatography (Hex/EtOAc/EtOH 1:4:0.1). A yellow semisolid of compound

)

(

−

1

5a was obtained (0.0999 g, 13.70%); FTIR (ATR) (cm ): 3358 (O-H, st), 3197 (aromatic C-H,

st), 2921, 2851 (aliphatic C-H, st), 1659, 1632 (C=O, st), 1468 (aromatic C=C, st), 1410 (C-N,

1

st), 1264 (C-O, st), 738, 704 (aromatic C-H, bending); H-NMR (300 MHz, DMSO-d ):

δ 3.43

6

(1H, m, H6), 3.61 (1H, t, J = 7.09 Hz, H5), 4.75 (1H, s, H4), 4.83 (1H, t, J = 5.58 Hz, OH),

5

.02 (1H, d, J = 6.11 Hz, OH), 5.41 (1H, d, J = 12.16 Hz, H7a), 5.46 (2H, s, H10), 5.52 (1H, d,

J = 12.14 Hz, H7b), 6.74 (2H, d, J = 8.41 Hz, H13, H15), 7.18 (2H, d, J = 8.45 Hz, H12, H16),

8

.18 (1H, s, H9), 9.01 (1H, s, OH), 9.56 (1H, s, OH); ¹³C-NMR (300 MHz, DMSO-d₆):

δ

53.07

(

C10), 62.18 (C7), 64.02 (C6), 68.86 (C5), 74.97 (C4), 115.90 (C13, C15), 120.29 (C2), 124.84

C9), 126.50 (C11), 130.13 (C12, C16), 142.90 (C3), 150.45 (C8), 157.84 (C14), 170.80 (C1);

+

HRMS (ESI) m/z calcd for [M] 363.10665, Found 364.11522 [M+H] , 386.09827 [M+Na] .

4

.2.16. (R)-5-((S)-1,2-Dihydroxyethyl)-3-hydroxy-4-((1-(4-hydroxy-3-methoxybenzyl)-1H-

1

,2,3-triazol-4-yl)methoxy)furan-2(5H)-one; 5b

A mixture of compound (0.2319 g, 1.083 mmol), 4-(azidomethyl)-2-methoxyphenol,

5

b, (0.2910 g, 1.624 mmol), 5% mol CuSO , and 20% mol sodium ascorbate in 6 mL of

4

t-BuOH/H O (4:2) was stirred at room temperature for 2 h. After reaction was completed,

2

water (10 mL) was added to the reaction mixture. The aqueous solution was extracted with

ethyl acetate (3

×

20 mL). The organic solution was washed with brine, dried, concentrated,

and puriﬁed by column chromatography (Hex/EtOAc/MeOH 1:4:0.1). A light yellow

−

1

semisolid of compound 5b was obtained (0.1048 g, 24.61%); FTIR (ATR) (cm ): 3358

O-H, st), 3195 (aromatic C-H, st), 2922, 2851 (aliphatic C-H, st), 1659, 1632 (C=O, st),

(

1

468 (aromatic C=C, st), 1410 (C-N, st), 1264 (C-O, st), 737, 704 (aromatic C-H, bending);

1

H-NMR (300 MHz, DMSO-d₆):

δ 3.40 (2H, m, H6), 3.61 (1H, dd, J = 7.01, 13.35 Hz, H5),

3

.74 (3H, s, CH3), 4.75 (1H, d, J = 1.20 Hz, H4), 4.82 (1H, t, J = 5.64 Hz, OH), 5.01 (1H, d,

J = 6.14 Hz, OH), 5.41 (1H, d, J = 12.18 Hz, H7a), 5.46 (2H, s, H10), 5.53 (1H, d, J = 12.19

Hz, H7b), 6.74 (2H, d, J = 7.66 Hz, H12, H16), 6.97 (1H, s, H15), 8.20 (1H, s, H9), 9.01 (1H, s,

OH), 9.13 (1H, s, OH); ¹³C-NMR (300 MHz, DMSO-d₆):

δ 53.39 (CH ), 56.08 (C10), 62.18

3

(

C7), 64.02 (C6), 68.87 (C5), 74.96 (C4), 113.02 (C12), 115.98 (C2), 120.29 (C15), 212.47 (C16),

Molecules 2021, 26, 1562

16 of 22

1

24.88 (C9), 126.90 (C11), 142.89 (C3), 147.09 (C8), 148.08 (C14), 150.43 (C13), 170.79 (C1);

+

HRMS (ESI) m/z calcd for [M] 393.11721, Found 394.12523 [M+H] , 416.10913 [M+Na] .

4

.2.17. (R)-5-((S)-1,2-Dihydroxyethyl)-3-hydroxy-4-((1-((2,3,4,9-tetrahydro-1H-pyrido[3,4-

b]indol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)furan-2(5H)-one; 5c

A mixture of compound (0.5439 g, 2.540 mmol), 3-(azidomethyl)-2,3,4,9-tetrahydro-

5

1

H-pyrido[3,4-b]indole, c, (0.3607 g, 1.587 mmol), 5% mol CuSO , and 20% mol sodium

4

ascorbate in 8 mL of t-BuOH/EtOH/H O (4:4:2) was stirred at room temperature for 2 h.

2

After reaction was completed, alcohol was evaporated out and water (10 mL) was added

to the reaction mixture. The aqueous solution was extracted with ethyl acetate (3 × 30 mL).

The organic solution was washed with brine, dried, concentrated, and puriﬁed by column

chromatography (EtOAc/EtOH 9:1). A brown powder of compound 5c was obtained

◦ −1

0.1493 g, 13.32%); m.p. 144–146 C; FTIR (ATR) (cm ): 3358 (O-H, st), 3195 (aromatic

(

C-H, st), 2921, 2851 (aliphatic C-H, st), 1741, 1658, 1632 (C=O, st), 1468 (aromatic C=C, st),

1

410 (C-N, st), 1220, 1197 (C-O, st), 738, 703 (aromatic C-H, bending); H-NMR (300 MHz

,

DMSO-d₆):

δ 2.54 (1H, m, H12a), 2.77 (1H, d, J = 12.11 Hz, H12b), 3.40 (2H, dd, J = 7.08

,

1

4.72 Hz, H6), 3.65 (2H, d, J = 6.89 Hz, H5, H11), 4.03 (3H, m, H10, OH), 4.62 (3H, d,

J = 5.89 Hz, H21, OH), 4.78 (1H, d, J = 1.01 Hz, H4), 5.48 (1H, d, J = 12.19 Hz, H7a), 5.58 (1H,

d, J = 12.19 Hz, H7b), 5.76 (1H, s, OH), 6.95 (1H, t, J = 7.07 Hz, H17), 7.03 (1H, t, J = 7.45 Hz

H17), 7.29 (1H, d, J = 7.91 Hz, H18), 7.36 (1H, d, J = 7.60 Hz, H15), 8.32 (1H, d, J = 15.52 Hz,

H9), 10.80 (1H, s, NH-indole); ¹³C-NMR (300 MHz, DMSO-d₆):

24.98 (C12), 31.15 (C21),

2.03 (C11), 53.77 (C10), 62.19 (C7), 64.04 (C6), 68.90 (C5), 74.99 (C4), 105.97 (C13), 111.45

,

δ

4

(

4

C18), 117.73 (C2), 118.93 (C15), 120.40 (C16), 121.18 (C17), 126.06 (C14), 127.13 (C9), 132.51

+

C20), 136.34 (C19), 142.57 (C3), 150.45 (C8), 170.82 (C1); HRMS (ESI) m/z calcd for [M]

+

41.16483, Found 442.17604 [M+H] , 464.15840 [M+Na] .

4.3. Biological Activity Assays

4.3.1. β-Secretase (BACE1) Inhibition Assay

All ascorbic derivatives (2a

uated for -secretase inhibitory activities by using the FRET assay. Brieﬂy, the reaction

composing 30 L of 0.01 unit/ L BACE1 enzyme stock solution, 20 L of 5% DMSO of

test compound, 30 L of 125 -secretase

–2c and 5a–5c), at a concentration of 400 µM, were eval-

β

µ

L of sodium acetate buffer (pH 4.5), and 20

µ

µM β

®

◦

substrate IV (Calbiochem , San Diego, CA, USA) was incubated at 37 C for 30 min.

Fluorescence was then measured at E

x

= 380 nm and E

m

= 510 nm using a SpectraMax

™

®

Gemini EM , San Jose, CA, USA [26

,61].

β-secretase inhibitor I (Anaspec , Fremont, CA,

USA) was used as a positive control, at a concentration of 100 µM. The experiments were

performed in triplicate. For the compounds exerting inhibitory activities more than 20%,

an IC50 value was further determined.

4.3.2. Amyloid Aggregation Inhibition Assay

The ascorbic derivatives ware evaluated for activities of amyloid

β

aggregation in-

L of

L of test compound were

hibition at a concentration of 400

µM. Brieﬂy, in each well of transparent plate, 9 µ

2

5 µM working solution of amyloid-

β

(1-42) peptide and 1

µ

◦

gently mixed and then incubated in the dark at 37 C for 48 h. After the incubation, 200 µL

®

of 5

µ

M ThT (Sigma , Saint Louis, MO, USA) in 50 mM Tris buffer (pH 7.4) was added

to each well. Fluorescence was then measured at E

x

= 446 nm and E

m

= 480 nm using a

™

SpectraMax Gemini EM , San Jose, CA, USA [26

,62]. Curcumin was used as a positive

control, at a concentration of 100 µM.

4.3.3. Antioxidant Activity Assay

The ascorbic derivatives were evaluated for antioxidant activities by DPPH assay at a

concentration of 400

in 80% methanol), 10

µ

M. The reaction was a mixture of 70

µL of DPPH solution (500 µM

µ

L of 80% methanol, and 20 L of test compound. The reaction was

µ

Molecules 2021, 26, 1562

17 of 22

incubated at room temperature in the dark for 30 min. The absorbance was then measured

™

at 517 nm using Inﬁnite 200 Pro (Tecan) [63]. Ascorbic acid was used as a positive control.

4.3.4. Neuroprotective Activity Assay

Cell Culture Preparation

P19 cells were grown in alpha minimal essential medium (α-MEM) supplemented

with 7.5% newborn calf serum (NCS), 2.5% fetal bovine serum (FBS), and 1% antibiotics–

◦

antimycotic solution in a 5% CO humidiﬁed atmosphere at 37 C. Cells in monolayer

2

cultures were maintained in exponential growth by subculturing every two days [27].

Differentiation of P19 Cells into P19-Derived Neurons

Exponentially grown cultures were trypsinized and dissociated into single cells. P19

6

cells (2

1

×

10 cells/mL) were then suspended in 10 mL

α

-MEM supplemented with 5% FBS,

% antibiotics–antimycotic solution, and 0.5

µM all transretinoic acid (RA) and seeded onto

a 100-mm bacteriological culture dish. The cells formed large aggregates in suspension.

After four days of RA treatment, the aggregates were dissociated by 5-mL glass measuring

pipette and replated on poly-L-lysine-precoated multiwell plates (the multiwell plates

were coated with 50

under UV light for 30 min) at 7

µ

g/mL poly-L-lysine, dissolved in PBS overnight, and sterilized

4

×

10 cells/mL (150

µL/well in 96-well plate) in

α

-MEM

supplemented with 10% FBS and 1% antibiotics–antimycotic solution and incubated for

4 h. Cytosine-1- -D-arabinoside or Ara-C (10 M) was added at day 1 after plating,

2

β

µ

and the medium was changed every two to three days. The differentiated neuronal cells,

P19-derived neurons, were used after day 14 of the differentiation process [27–29].

Viability Assay

The assay was carried out on P19-derived neurons cultured in a 96-well plate. After

1

4 days of the differentiation process, the

Ara-C, and 1% antibiotics–antimycotic solution, was removed and DMSO solutions of the

sample, diluted with -MEM supplemented with 10% FBS and 1% antibiotics–antimycotic

solution in the presence of 10 M Ara-C, were added to produce the concentrations of

0,000, 1000, 100, 10, and 1 nM. A concentration of DMSO was added to the cultures at

α-MEM, supplemented with 10% FBS, 10 µM

α

µ

1

0

.5% v/v. The

α

-MEM, supplemented with 10% FBS, 10

µ

M Ara-C, and 1% antibiotics–

◦

antimycotic solution, was added to the control wells. Cells were incubated for 18 h at 37 C.

Then, 150 µL of medium was removed, and 50

◦

µL of XTT solution (1 mg/mL XTT in 60 C

◦

α

-MEM + 25 µM PMS) was added to the cells. After being incubated at 37 C for four hours,

1

00

determined by using a microplate reader at 450 nm. Data were expressed as mean ± SEM

n = 3, each n was run in triplicate), with the medium as a control, representing 100% cell

µL of PBS (phosphate buffer saline solution), pH 7.4, was added. The OD value was

(

viability. The samples that enhanced the survival of cultured neurons more than the control

were further investigated for their neuroprotective ability [64].

Neuroprotective Assay

The assay was carried out with P19-derived neurons cultured in a 96-well plate by

using the serum-deprivation method. DMSO solutions of the samples, diluted with P19SM

plus 10 µM Ara-C and α-MEM supplemented with 10 µM Ara-C and 1% antibiotics–

antimycotic solution without FBS, were added to give the ﬁnal concentration of 1 nM,

which enhanced survival of cultured neurons more than control. A concentration of DMSO

was added to the cultures at 0.5%. P19SM plus 10

µM Ara-C was added into control wells.

The -MEM, supplemented with 10 M Ara-C and 1% antibiotics–antimycotic solution

α

µ

without FBS, was used to make an oxidative stress condition. The cells were incubated

◦

for 18 h at 37 C. Cell viability was assayed by the XTT reduction method. Data were

expressed as mean

±

SEM (n = 3, each n was run in triplicate), with the medium as a

control, representing 100% cell viability [30]. Quercetin, at concentration of 1 nM, was used

as a positive control [31].

Molecules 2021, 26, 1562

18 of 22

4.3.5. Anti-Inﬂammatory Activity Assay

Indomethacin, a well-known anti-inﬂammatory drug, prepared at 50 and 100 mM in

DMSO and 0.001% DMSO, was used as the experimental control in both the cell viability

assay and the anti-inﬂammatory gene expression study. The compounds of interest, which

are ascorbic acid (AA) and ascorbic derivatives (2c and 5c), were prepared as a 10-fold

serial dilution in DMSO, from 0.001, 0.01, 0.1, 1, 10 and 100

lines, indomethacin, ascorbic acid, and ascorbic derivatives were mixed with the cell media,

DMEM (Dulbecco’s modiﬁed Eagle medium), at a ratio of 1:999 l to produce 50 and

00 M of indomethacin solutions and 0.001, 0.01, 0.1, 1, 10, and 100 nM of ascorbic acid

µM. Prior to testing with cell

µ

1

µ

solutions and ascorbic derivative (2c and 5c) solutions.

Cell Culture Preparation

Cell lines, RAW 264.7 (murine) macrophages (ATCC, Manassas, VA, USA), were cul-

tured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/mL),

streptomycin (100

maintained in an incubator at 37 C, 5% CO , and 90% relative humidity.

µ

g/mL), and amphotericin B (0.25

µ

g/mL). The cell culture was then

◦

2

Determination of Cell Viability

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction as-

say was used to determine in vitro cell viability. RAW 264.7 starting cells were seeded in

5

◦

9

5

6-well plates at a density of 5

×

10 cells/100 µL per well and then incubated at 37 C,

% CO , and 90% relative humidity. Cell lines were monitored to ensure their proper

2

growth and the health of the cells in order to reach 80% conﬂuence within 24 h. DMEM

was immediately removed from each well prior to tests with either controls or compounds

of interest. To determine cytotoxicity, 100

00 M indomethacin, 0.1% PBS, or the proposed compounds at different concentrations

0.001–100 nM), prepared in DMEM, were separately loaded in each well, and each ex-

periment was carried out in ﬁve replicates. After cell lines were preincubated with the

testing samples for 24 h, the cell media was replaced with 50 L MTT solution (1 mg/mL

in PBS). The MTT reaction was incubated for four hours; then, 200 L DMSO was added to

µL of either 0.001% DMSO, 50 µM indomethacin,

1

µ

(

µ

each reaction and kept in the dark at room temperature for 10 min. The amount of MTT

formazan product formed was determined by measuring absorbance by using a microplate

reader at 570 nm. The % cell viability was calculated according to the following equation,

with 0.1% PBS as a reference control:

%

viability = (O.D. 570 sample/O.D. 570 control) × 100

Determination of mRNA Expression Levels of COX-2 and iNOS Genes

6

RAW 264.7 starting cells were seeded in six-well plates at a density of 3 × 10 cells/2 mL

◦

per well and then incubated at 37 C, 5% CO , and 90% relative humidity. Cell lines that

2

reached 80% conﬂuence within 48 h were selected for further gene expression study. DMEM

was replaced with the test samples, which were prepared as follows: 0.001% DMSO, 50 and

1

(

00

2c and 5c). The experiment was conducted in triplicate. After the cell lines were treated

with the testing samples for one hour, 2 g/mL LPS (lipopolysaccharide; Sigma-Aldrich,

µM indomethacin, 10 and 100 nM ascorbic acid, and 10 and 100 nM ascorbic derivatives

µ

St. Louis, MO, USA) was added to the cell lines and then maintained in an incubator for

another 24 h to induce the cell inﬂammatory process.

To study the effects of ascorbic acid and ascorbic derivatives (2c and 5c) on the

expressions of the inﬂammatory marker genes, COX-2 and iNOS, total RNA was isolated

™

from the cell lines by a PureLink RNA Mini Kit (Thermo Fisher Scientiﬁc Inc., Waltham,

MA, USA). Total RNA quality and quantity were examined by NanoDrop. An equivalent

of 2000 ng total RNA from each sample was then reverse-transcribed to produce ﬁrst-

strand complementary DNA (cDNA) using a Tetro cDNA Synthesis Kit (Bioline USA Inc.,

Memphis, TN, USA), according to the manufacturer’s instruction. Brieﬂy, the total RNA

Molecules 2021, 26, 1562

19 of 22

template was mixed with 4

µL Rnase inhibitor, and 1

water was then added to a mixture to adjust the volume to a ﬁnal 20-

µ

L 5X RT buffer, 1

µ

L oligo dT₍₁₈₎, 1

µ

L dNTP mix (10 µM),

1

µ

L reverse transcriptase. Diethylpyrocarbonate (DEPC)-treated

µl reaction. An

incubation process to produce ﬁrst strand cDNA was carried out in a thermal cycler

◦

(

3

SensoQuest Labcycler). Primer annealing and strand extending occurred at 45 C for

◦

0 min and a reverse transcriptase was ﬁnally heat-inactivated at 85 C for 5 min. The

corresponding cDNA products were then used as a template in a quantitative polymerase

chain reaction (qPCR) to study COX-2 and iNOS gene expressions, with GAPDH as a

reference gene.

0

The primer sequences for qPCR quantiﬁcations were as follows: COX-2: 5 -cccccacagt

0

caaagacact-3 (forward), 5 -gagtccatgttccaggagga-3 (reverse); iNOS: 5 -gtcttgcaagctgatggtc-

(forward), 5 -catgatggtcacattctgc-3 (reverse), and GAPDH: 5 -caggagcgagaccccactaacat-3

0

3

(

0

forward), 5 -gtcagatccacgacggacacatt-3 (reverse). These oligonucleotides were synthesized

by Invitrogen. qPCR analysis was performed according to the instructions of a SensiFAST^™

SYBR No-ROX Kit (Bioline USA Inc., USA). Then, 20 L qPCR reaction was prepared

by mixing a 1- L cDNA template with 0.8 L forward primer (10 mM), 0.8 L reverse

primer (10 mM), 10

µ

µL SYBR, and 7.4

µ

L DI water. Gene expression was measured in a

™

9

6-well plate using a BIO-RAD iCycler IQ 5 detection system. qPCR reaction was initially

◦

heated to 95 C 10 min, and then underwent 40 cycles of 95 C 15 s and 60 C 1 min.

The modulations of indomethacin, ascorbic acid, and ascorbic derivatives (2c and 5c) to

the expression of COX-2 and iNOS were compared against 0.001% DMSO control. Each

−

delta delta Ct

corresponding expression was computed as a relative fold change using the 2

method [65], normalized to the GAPDH gene.

4.3.6. In Silico Study of Sodium-Dependent Vitamin C Transporter 2 (SVCT2) Binding

The 3D ligands of the synthesized compounds, 2a–2c and 5a–5c, from ChemBio3D

were geometry optimized using the Guassian 09w program with the B3LYP/6-31G(d,p)

method. Subsequently, the aromatic bonds and rotatable bonds of each molecule were

identiﬁed. All hydrogen atoms and Gasteiger’s charges were assigned through the usage

of AutoDockTools-1.5.6. These conformations were then saved in pdbqt ﬁle format.

The crystallographic model of the bacterial vitamin c transporter (pdb code: 4RP9) [24

]

was used in the present study since structural information of human SVCTs is not available.

All cocrystallized ligands, metal ions, and water molecules were removed from the structure

through Discovery Studio 2017R2. All hydrogen atoms and Gasteiger’s charges were

assigned using AutoDockTool-1.5.6. Then, this pretreated protein was saved in pdb ﬁle

format. A grid box was generated from AutoGrid 4.0, presenting AutoDockTool-1.5.6 as

a graphical interface, which is deﬁned as a coordinate grid center. The size of the grid

3

box was set as 60

×

60

×

60 Å , with grid spacing of 0.375 Å. All docking simulations

were performed utilizing the AutoDock 4.0 program. Total binding energies of 100 docked

conformers, generated from 300 population sizes, were calculated based on Lamarkian

genetic algorithms (LGA).

Supplementary Materials: Table S1: The percent yields and structures of synthesized compounds,

Figures S1–S12: NMR spectrums (300 MHz) of synthesized compounds, Figure S13: Viability assay

4

by XTT of ascorbic derivatives at concentration of 1 to 10 nM; Figure S14: Effects of ascorbic acid

(A), 2c (B), and 5c (C) on the viability of RAW 264.7.

Author Contributions: Conceptualization, J.J. and O.V.; methodology, J.J. and O.V.; Synthesis, J.J.,

N.C. and S.N.; in vitro study and data analysis, J.J., N.C., and S.N.; cell base assay and data analysis,

H.P., S.T., and P.T.; in silico study, S.J.; writing—original draft preparation, J.J. and P.T.; writing—

review and editing, J.J., P.T., and O.V.; visualization, J.J.; project administration, J.J.; funding acquisi-

tion, J.J. and O.V. All authors have read and agreed to the published version of the manuscript.

Funding: This research was mostly funded by The Thailand Research Fund (Grant Number MRG

6

080112) and partially supported by Chiang Mai University.

Institutional Review Board Statement: Not applicable.

Molecules 2021, 26, 1562

20 of 22

Informed Consent Statement: Not applicable.

Data Availability Statement: Data sharing not applicable.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Sample Avalability: The sample of compounds are not available.

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Article Doi

Novel multifunctional ascorbic triazole derivatives for amyloidogenic pathway inhibition, anti-inflammation, and neuroprotection

DOI: 10.3390/molecules26061562

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Article abstract of DOI:10.3390/molecules26061562

Full text of DOI:10.3390/molecules26061562

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