Design and synthesis of π-extended resveratrol analogues and in vitro antioxidant and anti-inflammatory activity evaluation

Article abstract of DOI:10.3390/molecules26030646

The research on resveratrol (1) has been conducted intensively over a long time due to its proven antioxidant activity and disease-fighting capabilities. Many efforts have also been made to increase these biological effects. In the present study, six new extended aromatic resveratrol analogues containing naphthalene (2) and its bioisosteres quinoline (3 and 4), isoquinoline (5) quinoxaline (6) and quinazoline (7) scaffolds were designed and synthesized using an annulation strategy. The antioxidant and anti-inflammatory activities of these compounds were investigated. All compounds showed better antioxidant activity than resveratrol in ABTS assay. As for the anti-inflammatory test, 5 and 7 exhibited better activity than resveratrol. It is worth noting that nitrogen substitution on the extended aromatic resveratrol analogues has a significant impact on cell viability. Taking the antioxidant activities and NO inhibition activities into consideration, we conclude that isoquinoline analogue 5 may qualify for the further investigation of antioxidant and anti-inflammatory therapy. Furthermore, our study results suggest that in order to improve the biological activity of polyphenolic compounds, extended aromaticity and nitrogen substitution strategy could be a viable method for the design of future drug candidates.

Full text of DOI:10.3390/molecules26030646

molecules
Communication
Design and Synthesis of π-Extended Resveratrol Analogues and
In Vitro Antioxidant and Anti-Inflammatory Activity Evaluation
Ki Yoon Nam 1, Kongara Damodar 1 , Yeontaek Lee 2, Lee Seul Park 1, Ji Geun Gim 1, Jae Phil Park 2,
Seong Ho Jeon 2,* and Jeong Tae Lee 1,
*
1
Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 24252, Korea;
gugu4478@naver.com (K.Y.N.); kongaradamu@gmail.com (K.D.); hellobong3@nate.com (L.S.P.);
M17515@hallym.ac.kr (J.G.G.)
Department of Life Science and Multidisciplinary Genome Institute, Hallym University,
Chuncheon 24252, Korea; M18026@hallym.ac.kr (Y.L.); pjp8683@hallym.ac.kr (J.P.P.)
Correspondence: sjeon@hallym.ac.kr (S.H.J.); leo900516@gmail.com (J.T.L);
Tel.: +82-33-248-2096 (S.H.J.); +82-33-248-2071 (J.T.L.)
2
*
Abstract: The research on resveratrol (1) has been conducted intensively over a long time due to its
proven antioxidant activity and disease-fighting capabilities. Many efforts have also been made to
increase these biological effects. In the present study, six new extended aromatic resveratrol analogues
containing naphthalene (
2) and its bioisosteres quinoline (3 and 4), isoquinoline (5) quinoxaline (6)
and quinazoline ( ) scaffolds were designed and synthesized using an annulation strategy. The
7
antioxidant and anti-inflammatory activities of these compounds were investigated. All compounds
showed better antioxidant activity than resveratrol in ABTS assay. As for the anti-inflammatory test,
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
5
and 7 exhibited better activity than resveratrol. It is worth noting that nitrogen substitution on
ꢀꢁꢂꢃꢄꢅꢆ
the extended aromatic resveratrol analogues has a significant impact on cell viability. Taking the
antioxidant activities and NO inhibition activities into consideration, we conclude that isoquinoline
analogue 5 may qualify for the further investigation of antioxidant and anti-inflammatory therapy.
Furthermore, our study results suggest that in order to improve the biological activity of polyphenolic
compounds, extended aromaticity and nitrogen substitution strategy could be a viable method for
the design of future drug candidates.
Citation: Nam, K.Y.; Damodar, K.;
Lee, Y.; Park, L.S.; Gim, J.G.; Park, J.P.;
Jeon, S.H.; Lee, J.T. Design and
Synthesis of π-Extended Resveratrol
Analogues and In Vitro Antioxidant
and Anti-Inflammatory Activity
Evaluation. Molecules 2021, 26, 646.
https://doi.org/10.3390/molecules
26030646
Keywords: resveratrol; extended aromaticity; antioxidant; anti-inflammatory; cell viability
Academic Editor: Chang-Hee Lee
Received: 14 January 2021
Accepted: 21 January 2021
Published: 26 January 2021
1. Introduction
The oxygen element is essential for human life, but under certain circumstances, it also
has harmful effects. Free radicals are known as reactive oxygen species (ROS), generated
by the oxygen metabolism in the human body, and have many biochemical activities such
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iations.
as cell growth, proliferation, and survival at their physiological concentrations [1]. Never-
theless, excessive production of ROS and reactive nitrogen species (RNS) causes damage to
DNA, cellular proteins, carbohydrates, and membrane lipids and eventually cell injury and
death. The human body’s protective attempt against this damage is achieved by endoge-
nous antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase)
or nonenzymatic antioxidants (uric acid, bilirubin, lipoic acid, metallothioneins) [2]. The
antioxidant component can interact with ROS/RNS to stop the radical chain reactions, and
thereby delay or block cellular damage. When the balance between free radical generation
and antioxidant defenses is disturbed, the situation is called oxidative stress (OS) [3]. OS
causes various pathological conditions such as cancer, hypertension, neurological disorder,
and diabetes. When the endogenous antioxidant system fails to protect the organism
against ROS/RNS damage, exogenous antioxidants are needed as diet, nutritional sup-
plements or pharmaceutical products, which contain antioxidants as the principle active
compound [4].
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 646. https://doi.org/10.3390/molecules26030646
https://www.mdpi.com/journal/molecules

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Inflammation is a natural response of living tissue against injury or infection. Acti-
vated inflammatory pathway generates several inflammatory mediators such as cytokines
(IL-1β, IL6 and TNF-α, chemokines, free radicals, NO (nitric oxide) and eicosanoids
(prostaglandins)) in the affected tissues that eventually guide the restoration of the normal
tissue structure. However, their secretion for an extended period can lead to OS and chronic
inflammation-associated disorders. Excess ROS/RNS can trigger an intracellular signaling
cascade that augments pro-inflammatory gene expression [
NO magnitude by macrophages during the inflammatory response leads to undesirable
tissue destruction [ ]. Hence, inflammation and OS are closely related to pathophysiolog-
ical events [ ]. This mutual interdependence has a crucial role in the pathogenesis and
5]. Moreover, the upsurge of
6
7
management of various diseases, and we may expect beneficial effects from the antioxidant
and anti-inflammatory therapy.
Resveratrol (3,5,40-trihydroxy-trans-stilbene) (
1) (Figure 1) is a natural polyphenolic
stilbenoid produced in some plants as a phytoalexin. It is widely recognized as a nutraceu-
tical and therapeutic compound given its positive effects on human health. Promising
activities of
and cardioprotective properties [
1
include antioxidant, anti-aging, anticancer, anti-inflammatory, antidiabetic,
10]. Antioxidant activity of is due to its substantial
8–
1
scavenging property of ROS, RNS and other secondary organic radicals formed by the
reaction of ROS and RNS with biomolecules. It also acts as a useful metal ions chelator,
maintains the oxidation-reduction balance in a cell, and reduces the activity of the enzymes
which involve in ROS production. However, its potential application in some cancer studies
was ambiguous due to its poor bioavailability [11].
Figure 1. Structures of resveratrol (1) and its naphthalene (2), quinoline (3 and 4), isoquinoline (5) quinoxaline (6) and
quinazoline (7) analogues.
Previous studies of
1 have evidenced that the presence of three hydroxy groups in 3, 5
0
and 4 positions, presence of aromatic rings and a trans-double bond between the aromatic
rings, are vital for its antioxidant activity [12,13]. Analogues of 1 bearing electron-donating
groups on the benzene rings displayed increased its antioxidant activity [14]. The free
radical scavenging property undoubtedly depends on the resonance stabilization of the
resulting phenoxy radicals. Hence, an extension of π-conjugation could lead to the better
stabilization of the phenoxy radical, which can result in lowering the phenolic O–H bond
dissociation energy, thereby increased antioxidant activity can be obtained [15]. Zhou et al.
applied this concept in designing analogues of
between two benzene rings and observed increased antioxidant activity compared to
1 [16]. Another promising approach to achieve the extended -conjugation would be
extended aromaticity strategy. It is also apparent that -delocalization is maximized in
1 by introducing additional double bonds
π
π

Molecules 2021, 26, 646
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cyclic
π-systems compared to acyclic ones because the former systems contain extended
π-surfaces [17]. We believe that no report has been observed in the literature based on the
π-system elongation of 1 through aromaticity extension in a systematic way.
According to a recent report, heterocyclic replacements for benzene can have a sig-
nificant impact on ADME (absorption, distribution, metabolism, and excretion) -related
parameters [18]. Many biologically active natural products such as alkaloids, niacin, and
vitamin B6 contain nitrogen. These two facts inspired us to investigate the effect of nitrogen
incorporation in extended aromatic compounds on biological activity. In this context, we
designed and synthesized new analogues of
4’-hydroxy group-containing benzene ring is fused with benzene (naphthalene analogue
and its bioisosteres pyridine (quinoline and isoquinoline analogues and ), pyrazine
(quinoxaline analogue ) and pyrimidine (quinazoline analogue ) scaffolds (Figure 1), and
1 with extended aromaticity in which the
2)
3,
4
5
6
7
investigated their trends in their antioxidant and anti-inflammatory activity with respect to
the extended aromaticity as well as the substitution with nitrogen atoms.
2. Results and Discussion
2.1. Chemistry
Synthesis of resveratrol analogues
hydroxyl groups of 3,5-dihydroxybenzaldehyde
bromide (BnBr) in the presence of K2CO3 afforded bis(Bn) ether
transformed to the corresponding phosphonate ester 10 after reduction, bromination and
Arbuzov reactions on the aldehyde group of , successively [19]. Naphthalene aldehyde
2
–
7
commenced with the protection of phenolic
(Scheme 1). Treatment of with benzyl
which was further
8
8
9
9
13, which was used for the resveratrol analogue synthesis, was obtained from 6-hydroxy-
2-naphthoic acid 11. Treatment of 11 with BnBr/K2CO3 resulted in compound 12, which
was subjected to reduction and oxidation synthetic sequence to provide the aldehyde 13
in high yields. For the synthesis of quinoline-3-carbaldehyde moiety 16, we employed
Vilsmeier–Haack reaction. 3-Benzyloxyacetanilide 14 efficiently underwent Vilsmeier–
Haack cyclization [20] to afford 2-chloro-3-formyl quinoline derivative 15 and subsequent
hydrodechlorination of 15 was realized to yield 16 using Pd(PPh3)4/Et3N/HCO2 H system
◦
in DMF at 110 C. Next, a metal-free, mild and efficient method was employed for the
synthesis of 2-methylquinoline moiety 18 by condensation of 4-(benzyloxy)aniline 17 with
ethyl vinyl ether in the presence of catalytic amount of iodine [21]. To achieve isoquino-
line moiety 23, Heck reaction was performed on 5-(benzyloxy)-2-bromobenzaldehyde
20 which in turn obtained from 2-bromo-5-hydroxybenzaldehyde 19. Treatment of 20
with methyl ester of N-acetyl dehydroalanine using Pd(OAc)2/P(o-tol)3/TEA system in
DMSO underwent Heck reaction, whereupon a spontaneous intramolecular transesterifi-
cation resulted in the isoquinoline-3-carboxylate derivative 21. Subsequent reduction to
the corresponding alcohol 22 and oxidation with IBX gave the isoquinoline aldehyde 23
.
1,4-Difluoro-2-nitrobenzene 24 was used as a precursor to obtain the quinoxaline moiety
29 [22]. Nucleophilic displacement reaction of 24 with L-alanine ethyl ester hydrochloride
furnished 25, which subsequently underwent cyclization to give quinoxalone 26. When
26 was subjected to Vilsmeier–Haack condition, quinoxaline moiety 27 was formed which
finally transformed to the desired moiety 29 by hydrodechlorination and nucleophilic
displacement reaction synthetic sequences, respectively. The requisite quinazoline moiety
30 was constructed by copper-catalyzed cascade coupling of 20 with acetamidine HCl [23].
With all the key fragments secured, their linkage to construct analogues of
then executed (Scheme 2). At first, we tried the Wittig–Horner reaction between 13 and
10 using NaH in DMF, and the resulting benzyl protected naphthalene based analogue
31 was obtained in high yield. In a similar manner, quinoline and isoquinoline analogues
32 and 33 were also acquired. Next, compounds 34
36 were built up through C(sp3)–H
functionalization of methyl azaarenes 18 29 and 30 with the aldehyde , respectively [24].
Notably, excellent stereoselectivity (based on 1H-NMR) was achieved in both conditions.
Finally, benzyl (Bn) group deprotection of 31 35 using 1.0 M BBr3 (in CH2Cl2 ) smoothly
furnished the desired target compounds , respectively. Unfortunately, very poor yield
1 was
1
–
,
9
–
2–6

Molecules 2021, 26, 646
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was observed for quinazoline-based analogue
7
even after extended reaction time. To
solve this problem, we employed AlCl3/N,N-dimethylaniline system [25]. This reagent
system afforded the product in 3 h, albeit in low yield. All the structures of the final
products
were settled from their spectral data (1H and 13C NMR and MS) (see the
7
2–7
Supplementary Materials).
Scheme 1. Reagents and conditions: (a) BnBr, K2CO3 , acetone, reflux, 5 h, 78%; (b), (c) CH2Cl2 , rt,
◦
6 h; 93%; (d) P(OEt)3, xylene, reflux, 10 h, 99% (over 3 steps); (e) BnBr, K2CO3 , DMF, 60 C, 2 h, 77%;
(f) LiAlH4 (2.0 M in THF), THF, rt, 2 h; (g) IBX, DMSO, rt, 1 h, 94% (over 2 steps); (h) POCl3, DMF,
90 ◦C, 2 h, 78%; (i) TEA, HCO2H, Pd(PPh3)4, DMF, 110 ◦C, 2 h, 74%; (j) Iodine, toluene, 80 ◦C, 2 h,
◦
49%; (k) BnBr, K2CO3 , DMF, rt, 2 h, 90%; (l) TEA, P(o-tol)3, Pd(OAc)2, DMSO, 110 C, 18 h, 56%; (m)
1.0 M LiAlH4 (in THF), THF, rt, 1 h, 61%; (n) IBX, DMSO, rt, 1 h, 66%; (o) TEA, pyridine, reflux, 17 h,
93%; (p) (i) SnCl2
·
2H2O, EtOH, H2 O, reflux, 1 h; (ii) DDQ, acetonitrile, 0.5 h, rt, 18 h, quantitative,
(over 2 steps); (q) POCl3, N, N-dimethylaniline, reflux, 1 h, 86%; (r) TEA, HCOOH, Pd(PPh3)4, DMF,
◦
◦
110 C, 2 h, 83% (s) BnOH, 60% NaH (in mineral oil), DMF, 100 C, 2 h, 83%; (t) L-proline, Cs2CO3 ,
◦
CuI(I), DMF, 110 C, 18 h, 27%.

Molecules 2021, 26, 646
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Scheme 2. Reagents and conditions: (a) 10, 60% NaH (in mineral oil), DMF, rt, 2 h, 82–89%; (b) 1.0 M BBr3 (in CH2Cl2),
◦
CH2 Cl2 , 0 C, 2 h, 61–87%; (c) 9, Fe(OAc)2, TFA, toluene, reflux, 24 h, 56% (34); 64% (35); (d) 9, t-BuOK, DMF, rt, 4 h, 39%
(36); (e) AlCl3, N,N-dimethylaniline, CH2Cl2 , rt, 3 h, 20% (7).
2.2. Antioxidant Activity
ABTS (2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assay
was used for the antioxidant estimation. Though many methods are available, this appears
to be the most popular because of its operational simplicity, short experimental time,
and the employment of the inexpensive spectrophotometer [26]. In vitro ABTS radical
scavenging activities of
illustrates that all the analogues
with better IC50 values (43~23% better activity, 3.14
2.81 0.034, 3.10 0.953 and 3.54 0.714 M, respectively) than the standard/positive
control (4.88 0.35 M). We believe that the high ABTS radical scavenging activity of
was due to the enhanced resonance stabilization of the resulting phenoxy radicals.
Compared to , all the analogues showed bicyclic aromatic moieties instead of a
benzene ring, and this feature facilitated the extended and planar conjugation by which
the stronger capability to stabilize the radical species via delocalization of the -electrons
can be achieved. The lower the IC50 value of a compound, the higher its antioxidant activity.
Compound , which contains an isoquinoline moiety, expressed the best activity among
all analogues. The nitrogen in the Y position (such as compounds and in Scheme 2)
increased the antioxidant activity, and the nitrogen in the Z position (such as compounds
and in Scheme 2) decreased the antioxidant activity. However, there appears to be no clear
1
and its analogues
showed excellent ABTS free radical scavenging activity
0.445, 3.57 0.496, 3.61 0.253,
2–7 are summarized in Table 1. The table
2–7
±
±
±
±
±
±
µ
1
±
µ
2–7
1
2–7
π
π
5
5
7
4
6
correlation between antioxidant activity and the number of nitrogen atom substitutions in
the ABTS assay results. It seems that the position of nitrogen substitution is more important
than the number of nitrogen substitution on the activity.
Table 1. In vitro ABTS radical scavenging activities of ascorbic acid, resveratrol
1
and analogues 2–7.
Compound
IC50 (µM)
19.82 ± 0.934
4.88 ± 0.350
3.14 ± 0.445
3.57 ± 0.496
Compound
IC50 (µM)
3.61 ± 0.253
2.81 ± 0.034
3.10 ± 0.953
3.54 ± 0.714
Ascorbic acid
4
5
6
7
1
2
3
The results are reported as mean value ± SEM for n = 3.

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2.3. Anti-Iinflammatory Effects on Macrophage
In the pathogenesis of inflammatory-related disorders, NO is recognized as one of the
important mediators. Hence, its inhibition is considered as a beneficial approach for the
prevention of inflammation [27]. Herein, to understand the anti-inflammatory properties
of the resveratrol analogues, we studied their inhibitory effects against NO production in
LPS-treated RAW 264.7 cells. Although it is not possible to measure NO quantitatively in
a biological environment due to its rapid oxidation to NO2 (nitrite), short physiological
half-life and presence of other free radicals, NO secretion activity in cells is indirectly
determined by Griess assay counting its stable product NO2 levels [28]. The inhibitory
effects of
analogues
treated cells (see Supplementary Materials, Figure S2). The median effective doses (ED50)
for inhibiting the production of NO by were calculated on the basis of NO2 released
into the culture media and were observed in the range of 13.08–56.88 M. The activities
(26.89 1.210),
(ED50 = 38.81 M) and
. Notably, isoquinoline analogue exerted
the highest reduction in NO production. Next, an assessment of cellular metabolic activity
was conducted by MTT assay to prove that inhibitory effects of on LPS-stimulated NO
secretion was not associated with their cytotoxic effect. Cell viability was not affected up to
50 M except for . We also applied in vitro efficacy index (iEI) paradigm [29], the ratio of
1–
7
on NO production were collected in Table 2. Pretreatment with
1 and its
2–7 showed some reduction in the NO production dose-dependently in LPS-
1–7
µ
of
while the activities of
(ED50 = 56.88
5
(ED50 = 13.08
µ
M) and
7
(ED50 = 21.67
M),
µ
M) were superior to that of
1
±
2
(ED50 = 29.50
µ
3
(ED50 = 44.60 M),
µ
4
µ
6
µM) were inferior to that of
1
5
1–7
µ
2
toxicity (LD50)/anti-inflammatory potency (ED50), to confirm that the anti-inflammatory
activity is in the viable cells only. All nitrogen substituted extended resveratrol analogues
3–7
(iEI = 2.11~4.84) are more potent than their non-nitrogenated bioisostere
2 (iEI = 0.98);
especially, compounds and showed better iEI values than even the parent compound 1.
3–
5
7
This result leads us to conclude that nitrogen substitution of extended aromatic resveratrol
conferred a significant advantage over non-substituted extended aromatic resveratrol in
terms of cytotoxicity. On the whole, compounds
compared to any other compounds. When we combine the results from the antioxidant
activity and anti-inflammatory activity, we may conclude that isoquinoline analogue had
5 and 7 have much-improved iEI values
5
a strong correlation between antioxidant and anti-inflammatory effects (Tables 1 and 2)
and could be the lead compound for both antioxidant and anti-inflammatory agents.
Table 2. Inhibitory effects of resveratrol 1 and its analogues 2–7 on NO production.
NO/MTT Assay
Cell Viability
LD50 (µM)
iEI
Compound
ED50 (µM)
(LD50/ED50)
1
2
3
4
5
6
7
26.89 ± 1.210
29.50 ± 0.168
44.60 ± 7.273
38.81 ± 1.878
13.08 ± 0.690
56.88 ± 1.613
21.67 ± 1.101
81.08 ± 9.258
28.82 ± 0.767
167.12 ± 13.652
123.47 ± 6.388
63.30 ± 1.139
120.46 ± 4.237
92.58 ± 8.745
3.02
0.98
3.75
3.18
4.84
2.11
4.27
The results are reported as the mean value ± SEM for n = 3.
We have further investigated whether resveratrol and its analogues can reduce the
inflammatory activity of macrophages. For this purpose, we examined the downregulation
of pro-inflammatory cytokine gene expression of LPS-stimulated RAW 264.7 cells by potent
inhibitors of NO production (
and IL-6 were significantly reduced by treatment of resveratrol and its analogues. On the
other hand, the level of TNF- did not change upon treatment. Treatment with 50 M of
1, 2, 5, and 7) (Figure 2). As expected, the levels of IL-1β
α
µ

Molecules 2021, 26, 646
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analogue
2
was highly toxic to the cells. Since most of the RNA was degraded by cell death,
gene amplification by PCR did not occur.
Figure 2. Inhibitory effects of
cells. RAW 264.7 cells were treated with 1
amounts (12.5~50 M) of compounds. Gene expression levels were analyzed by endpoint reverse
1–
7
on pro-inflammatory cytokine production of activated macrophage
µg/mL LPS for 24 h in the presence of the indicated
µ
transcriptase-polymerase chain reaction and 1.5% agarose gel electrophoresis. GAPDH was used as
a control.
3. Materials and Methods
All starting materials, chemical and biological reagents, and solvents were purchased
from commercial sources and used without any additional purification. Solvents used for re-
actions were purchased as either anhydrous grade products or freshly distilled using proper
1
dehydrating agents. H NMR and 13C NMR spectra were recorded at room temperature on
a Varian Mercury TM 300 MHz FT-NMR (Varian, CA, USA) and JNM-ECZ400S 400 MHz
FT-NMR (JEOL Ltd., Tokyo, Japan). Chloroform-d/acetone-d6/methanol-d4/dimethyl
sulfoxide-d6 were used as NMR solvents. Chemical shifts (δ) were reported in parts per
million (ppm) downfield relative to tetramethylsilane (TMS). The following pattern was
1
used for H NMR data:
δ value (multiplicity (singlet = s), (doublet = d), (triplet = t),
(quartet = q), (doublet of doublet = dd), and (multiplet = m), number of protons, coupling
constants (J) quoted in Hz). Mass spectra analyses were taken on a JMS-700 (JEOL Ltd.)
spectrometer at the central laboratory of Kangwon National University. Melting points
were uncorrected and observed on a MEL-TEMP II (Triad Scientific, Manasquan, NJ, USA)
open capillary melting points apparatus. All reactions were monitored through thin-layer
chromatography utilizing Merck silica gel plates 60 F254 and spots visualization was ac-
complished by UV light absorption and/or p-anisaldehyde/phosphomolybdic acid stain.
Silica gel 60 (230–400 mesh (40–63
µm), Merck, Darmstadt, Germany) was employed for
chromatographic purification.
Antioxidant activity was tested in the dark room by ABTS assay [30]. UV-1800
(Shimadzu Corporation, Kyoto, Japan) was used to measure UV absorption. Hellma’s
104.600-QC Quartz cell was used as a UV cell. IC50 values were obtained using OriginPro
8.0 software (OriginLab Corporation, Northampton, MA, USA).
Anti-inflammatory activity was measured as the effect of compounds on inhibiting
NO production versus cell viability [31]. Their inhibitory effect was also tested for pro-
inflammatory cytokine gene expression.
Detailed synthetic methods and characterization of compounds and assay methods
are included in Supplementary Materials.
4. Conclusions
Six new extended aromatic resveratrol analogues containing naphthalene (
its bioisosteres quinoline ( and ), isoquinoline ( ) quinoxaline ( ) and quinazoline (
scaffolds were designed using an annulation strategy and investigated for their in vitro
2) and
3
4
5
6
7)

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ABTS radical scavenging activity. All compounds exhibited enhanced antioxidant activ-
ity in the ABTS test (IC50 = 2.81~3.61
dose-dependent inhibition of NO production in LPS-treated RAW 264.7 cells. Analogues
(ED50 = 13.08 M; iEI = 4.84) and (ED50 = 21.67 M; iEI = 4.27) furnished superior activity
than (ED50 = 26.89 M; iEI = 3.02). The inhibition potency was further evidenced by
the downregulation of the pro-inflammatory cytokine (IL-1 and IL-6) gene expression
of activated macrophage cells. Though further research is required to explore the exact
mechanisms of -induced regulation of inflammatory signaling pathway and antioxidant
activity, our study indicates that isoquinoline analogue may show therapeutic potential
µM) compared with 1. Besides, 2–7 also showed
5
µ
7
µ
1
µ
β
5
5
in both antioxidant and anti-inflammatory therapy. Our research verified that extended
aromaticity renders better antioxidant activity. In addition to extended aromaticity, nitro-
gen substitution gives better cell viability regardless of location and number of nitrogen
atoms. Therefore, it suggests that in order to improve the biological activity of resveratrol
analogues, aromaticity extension and nitrogen substitution could be a viable method for
the design of future drug candidates. We are currently working on the substitution of
heteroatoms other than nitrogen in the extended aromatic resveratrol.
Supplementary Materials: The following are available online. All the synthetic procedures of
compounds
effect of compounds
1H & 13C NMR spectra of compounds 2–7, IR spectra and UV-Vis spectra.
2
–
7, Figure S1: ABTS assay of compounds
1–7 (statistical analysis), Figure S2: Inhibitory
1–
7
on NO production., Figure S3: Cell viability (MTT) assay of compounds 1–7,
Author Contributions: Conceptualization: J.T.L.; methodology: K.Y.N., Y.L., L.S.P., J.G.G., J.P.P.;
investigation: K.Y.N., Y.L., L.S.P., J.G.G., J.P.P.; resources: K.Y.N. and Y.L.; data curation: K.Y.N., K.D.,
Y.L., L.S.P., J.G.G. and J.P.P.; writing—original draft preparation: K.Y.N., K.D. and Y.L.; writing—
review and editing: K.D., J.T.L. and S.H.J.; supervision: J.T.L. and S.H.J.; project administration: J.T.L.
and S.H.J.; funding acquisition: J.T.L. and S.H.J. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grant
funded by the Korea government (MOE) (2017R1D1A1A02018831 to J.T. Lee) and (MIST)
(No. 2020R1A2C101017311, S.H. Jeon).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 2–7 are available from the authors.
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