6-Hydroxy-benzofuran-3-(2H)-ones as Potential Anti-Inflammatory Agents: Synthesis and Inhibitory Activity of LPS-Stimulated ROS Production in RAW 264.7 Macrophage

Full text of DOI:10.1002/bkcs.12215

Communication
DOI: 10.1002/bkcs.12215
BULLETIN OF THE
A. Shrestha et al.
KOREAN CHEMICAL SOCIETY
6-Hydroxy-benzofuran-3-(2H)-ones as Potential Anti-Inflammatory
Agents: Synthesis and Inhibitory Activity of LPS-Stimulated ROS
Production in RAW 264.7 Macrophage
†
†
†
*
Aarajana Shrestha, Ritina Shrestha, Sumin Lee, Pil-Hoon Park, and Eung-Seok Lee
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea.
*E-mail: eslee@ynu.ac.kr
Received October 13, 2020, Accepted December 25, 2020, Published online January 22, 2021
Keywords: Aurone, 6-Hydroxy-benzofuran-3-(2H)-ones, Anti-inflammatory agents, Inhibition of ROS
production, RAW 264.7 macrophage
Reactive oxygen species (ROS) are oxygen containing
chemical reactive molecules, which are considered as a fac-
tor in the inflammatory responses induced by lipopolysac-
charide (LPS).1 LPSs are the exotoxins that activate
macrophages to liberate inflammatory cytokines including
interleukin-1 beta (IL-1), tumor necrosis factor (TNF), nitric
oxide (NO), and prostaglandin E2 (PGE2).2 The pathophysi-
ological processes of various inflammatory diseases are
mediated by these cytokines. Aurones are structural isomers
of flavones, which are biosynthesized from chalcones by
auresidin synthase.3 These possess not only anticancer,
antiparasitic, antimicrobial, antiviral properties but also anti-
inflammatory property.4 It is reported that the anti-inflamma-
tory activity of chalcone derivatives is accelerated by the
incorportaion of electron withdrawing groups like flourine,
chorine in the chalcone moiety.5 Previously our group also
reported significance of halogens on inhibitory effect of
LPS-stimulated ROS production in RAW 264.7 macro-
phages.6 It is further reported that the position and electronic
nature of the substituent on ring B of aurone scaffold affect
the potency, selectivity, and cytotoxicity of the compounds.7
The importance of hydroxyl group in benzofuran-3-(2H)-one
moiety is highlighted in the bioassay of naturally occurring
aurones such as aureusidin, sulfuretin, and maritimetin.8,9
Among chalcones, flavones and aurones, there have been
few studies on aurones and its antiinfammatory activity.
Figure 1. Rational design for target compounds (1–48).
(a)
(b)
Scheme 1. General synthetic representation for the synthesis of compounds 1–48. (a) Nonhydroxylated benzofuran-3(2H)-one (1–24); (b)
hydroxylated benzofuran-3(2H)-one (25–48). Reagents and conditions: (I) morpholine acetate, methanol, reflux 80 ꢀC, 3 h, (II) CH2Cl2,
Al2O3, 3–24 h, 25 ꢀC, (III) 50% aq. KOH, ethanol, 24 h, 25 ꢀC (IV) 50% aq. KOH, 2–3 h, 25ꢀC, (V) SOCl2, ethanol, 2 h, 25 ꢀC, (VI)
50% aq. KOH, ethanol, DMF, 4–6 h, 25 ꢀC.
†These authors contributed equally to this work.
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Table 1. Inhibitory activity against LPS-stimulated ROS production by compounds 1–24 in RAW 264.7 macrophages.
Compd
R
ROS Inhibition (IC50, μM)a Toxicity Compd
R
ROS Inhibition (IC50, μM)a Toxicity
1
2
3
4
5
6
7
8
2-F
3-F
4-F
>10
>10
>10
>10
>10
>10
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
13
14
15
16
17
18
19
20
21
22
23
24
2-Cl
3-Cl
4-Cl
>10
>10
9.55 Æ 1.05
>10
>10
>10
>10
>10
>10
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
2-CF3
3-CF3
4-CF3
2-OCF3 >10
3-OCF3 >10
4-OCF3 >10
2-Br
3-Br
4-Br
2-pyridyl
3-pyridyl
4-pyridyl
2-furanyl
3-furanyl
2-thiophenyl
3-thiophenyl
5-chloro furanyl
9
10
11
12
>10
>10
>10
>10
>10
5-chloro thiophenyl >10
NT: nontoxic.
a Each data represent mean Æ SEM from at least three different experiments.
Therefore, in this study, we aim to synthesize total
48 halogenated/arylated and hydroxylated/nonhydroxylated
2-arylidene-1-benzofuranone analogues as aurone derivatives
in order to evaluate inhibition of LPS-stimulated ROS pro-
duction in RAW 264.7 macrophages. The structure–activity
relationship (SAR) study was performed based on presence
or absence of 6-hydroxyl group in 1-benzofuranone along
with the position of ortho-, meta-, or para-halogen (F−,
CF3 , OCF3 , Cl−, Br−) groups in the phenyl ring of aurone
and pyridyl, furanyl, thiophenyl, 5-chlorofuranyl, and 5-
−
−
Table 2. Inhibitory activity against LPS-stimulated ROS production by compounds 25–48 in RAW 264.7 macrophages.
Compd
R
ROS Inhibition (IC50, μM)a Toxicity Compd
R
ROS Inhibition (IC50, μM)a Toxicity
25
26
27
28
29
30
31
32
33
34
35
36
2-F
3-F
4-F
>10
>10
>10
>10
10.06 Æ 3.19
>10
NT
NT
NT
Toxicb
NT
NT
NT
NT
Toxicb
NT
37
38
39
40
41
42
43
44
45
46
47
48
2-Cl
3-Cl
4-Cl
>10
>10
>10
>10
>10
6.41 Æ 1.25
>10
>10
>10
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
2-CF3
3-CF3
4-CF3
2-OCF3 6.80 Æ 2.51
3-OCF3 >10
2-pyridyl
3-pyridyl
4-pyridyl
2-furanyl
3-furanyl
2-thiophenyl
3-thiophenyl
5-chloro furanyl
4-OCF3 >10
2-Br
3-Br
4-Br
>10
>10
>10
6.25 Æ 1.2
>10
NT
NT
5-chloro thiophenyl >10
NT: Non-toxic.
a Each data represents mean Æ SEM from at least three different experiments.
b Less than 90% cell viability at 10 μM; Malvidin was used as a positive control.
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Figure 2. Structure of the prepared compounds; nonhydroxylated (series A, 1–24) and hydroxylated (series B, 25–48) benzofuranone derivatives.
1
chlorothiophenyl moieties of 2-arylidene-1-benzofuranone
derivatives of aurone (Figure 1).
compounds was characterized by H NMR, 13C NMR, and
LC–MS (Supporting Information).
Based on previously reported synthetic method,10 com-
pounds 1–48 were prepared using either base catalyzed
(50% KOH or activated basic aluminium oxide) or acid
catalyzed (thionyl chloride or morpholine acetate) Claisen–
Schmidt condensation reaction (Scheme 1). Specially, com-
pound 37 having ortho-chloride group on B-ring was pre-
pared using thionyl chloride, while compounds 16–18, and
40 containing ortho-, meta-, or para-pyridyl group on B-
ring were prepared using morpholine acetate. Remaining
compounds were synthesized by using potassium hydroxide
or aluminium oxide as a base (Supporting Information).
Among two series (A and B) (Figure 2), series A contains
halogenated or arylated aurones (1–24) without hydroxyl
moiety while series B (25–48) contains halogenated or
arylated aurones with hydroxyl group at 6-position of
benzofuranone moiety. The structure of these synthesized
Compounds 1–48 were assessed for their inhibitory
activity against LPS-stimulated ROS production in RAW
Figure 3. Compounds exhibiting LPS stimulated ROS inhibition
in RAW 264.7 macrophage.
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264.7
macrophages
compared
to
Malvidin
In conclusion, we synthesized 48 benzofuranone deriva-
tives and tested for LPS-stimulated ROS production in
RAW 264.7 macrophages. Compounds 31, 42 and 46 with
hydroxy at 6-position of benzofuranone exhibited potent
inhibitory activity (IC50 = 6.25–6.80 μM) against LPS-stimu-
lated ROS production in RAW 364.7 macrophages as com-
pared to reference compound. These results showed
importance of hydroxyl group along with ortho-
trifluoromethoxyl, para-pyridyl, and para-thiophenyl moie-
ties at phenyl ring of aurone derivatives. These findings
would provide useful information for the group of
researchers working on the development of anti-inflamma-
tory agents.
(IC50 = 9.0 Æ 0.8 μM), positive control and data are pres-
ented in Tables 1 and 2.11
From series A, compounds 1–24 bearing no hydroxyl
group at 6-position of ring A exhibited no toxicity against
RAW 264.7 macrophages. Among 24 aurone derivatives
only aurone 15 bearing chloro group at para- position of
B-ring showed comparable suppression of ROS production
(IC50 = 9.55 μM) in RAW 264.7 macrophage to positive
control, Malvidin. Therefore, except compound 15, non-
hydroxylated aurones are found to exhibit insignificant
inhibitory activity against LPS-stimulated ROS production
in RAW 264.7 macrophages.
As shown in Table 2, most of the C-6 hydroxy-substituted
compounds (series B, 25–48) demonstrated weak inhibition
of ROS production in RAW 264.7 macrophages. Com-
pounds possessing ortho-trifluoromethoxyphenyl group (31),
para-pyridyl group (42) and para-thiophenyl group (46)
exhibited better LPS-stimulated ROS production inhibitory
activity than malvidin with IC50 = 6.80, 6.41, and 6.25 μM,
respectively. Changing the position of trifluoromethoxy
group from ortho- to para-position in B-ring of 6-hydroxyl-
ated aurone results in compound 33 which exhibited higher
toxicity and no inhibitory activity. Similar to compound 33,
compound 28 possessing ortho-trifluoromethylphenyl moiety
also showed toxicity while all other prepared compounds are
confirmed to be nontoxic.
Previously our group reported that among the eight com-
pounds (13–15, 23, 24, 37–39), compound 24 showed sig-
nificant inhibition of TNF-α induced ROS production in
HT-29 cell.10 Surprisingly, the same eight compounds did
not exhibit any inhibitory effect on LPS-stimulated ROS
production in RAW 264.7 macrophages which may be due
to different target and mechanism.
As far as SAR study is concerned, insignificant inhibi-
tory activity (IC50 > 10 μM) demonstrated by most of the
compounds resulted in difficulty to withdraw a concrete
SAR. However, it is interesting to note that the presence of
6-hydroxybenzofuran-3(2H)-one pharmacophore in three
compounds (31, 42, and 46) has direct impact on their
higher inhibitory activity against ROS production stimu-
lated by LPS in RAW 264.7 macrophages. This indicates
the importance of hydroxyl group at 6-position of
benzofuranone to improve the ROS inhibitory activity. In
addition, the significant inhibitory results shown by the
these compounds suggested the presence of ortho-
trifluoromethoxyl, para-pyridyl, and para-thiophenyl moie-
ties at phenyl ring of aurone derivatives is advantageous for
the inhibition of ROS production as shown in Figure 3.
Acknowledgments. This work was supported by the
National Research Foundation of Korea (NRF) grant funded
by the Korea government (MSIT) NRF-2017R1A2B2003944.
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
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