Synthesis of Clitocybin A, B and C and their Biological Evaluation for Antioxidant Activities

Article abstract of DOI:10.1002/bkcs.11825

Clitocybin A, isolated from the culture broth of mushroom Clitocybe aurantiaca, showed various biological activities such as cell death inhibition effects, anti-wrinkle effects, aortic smooth muscle cell proliferation inhibition effects. In an effort to further develop this promising compound toward a potent antioxidant, we have modified the structure of clitocybin A by changing the number of free phenolic hydrogen in isoindolinone moiety. In this paper, clitocybin A and its analogues, clitocybin B and C were synthesized and their antioxidant activities were evaluated. Clitocybin A possessing two phenolic hydrogens in isoindolinone moiety of clitocybin A analogues showed more potent radical scavenging activity than clitocybin B possessing one phenolic hydrogen. In inhibition of cellular DNA damage in H2O2treated cells, clitocybin C lacking free phenolic hydrogen in isoindolinone moiety showed more potent activity than clitocybin A possessing two phenolic hydrogens.Among the tested compounds, clitocybin A only showed dose-dependently HNE inhibitory activity with an IC50 value of 78.8 uM. The presence of free phenolic hydrogens inisoindolinone moiety of clitocybin A analogues plays an important role in antioxidant activities.

Full text of DOI:10.1002/bkcs.11825

Article
DOI: 10.1002/bkcs.11825
BULLETIN OF THE
S. Lee et al.
KOREAN CHEMICAL SOCIETY
Synthesis of Clitocybin A, B and C and their Biological Evaluation for
Antioxidant Activities
‡
§
†
†,
Sangku Lee,†, Jae Nyoung Kim, Jin Won Hyun, Ick-Dong Yoo, and Bo Yeon Kim
*
*
†Anticancer Agent Research Center, KRIBB, Cheongju 28116, Republic of Korea.
*E-mail: sangku@kribb.re.kr; bykim@kribb.re.kr
‡Department of Chemistry, Chonnam National University, Gwangju 61186, Republic of Korea
§School of Medicine, Cheju National University, Jeju 63243, Republic of Korea
Received May 8, 2019, Accepted June 7, 2019
Clitocybin A, isolated from the culture broth of mushroom Clitocybe aurantiaca, showed various biologi-
cal activities such as cell death inhibition effects, anti-wrinkle effects, aortic smooth muscle cell prolifera-
tion inhibition effects. In an effort to further develop this promising compound toward a potent
antioxidant, we have modified the structure of clitocybin A by changing the number of free phenolic
hydrogen in isoindolinone moiety. In this paper, clitocybin A and its analogues, clitocybin B and C were
synthesized and their antioxidant activities were evaluated. Clitocybin A possessing two phenolic hydro-
gens in isoindolinone moiety of clitocybin A analogues showed more potent radical scavenging activity
than clitocybin B possessing one phenolic hydrogen. In inhibition of cellular DNA damage in
H2O2treated cells, clitocybin C lacking free phenolic hydrogen in isoindolinone moiety showed more
potent activity than clitocybin A possessing two phenolic hydrogens.Among the tested compounds,
clitocybin A only showed dose-dependently HNE inhibitory activity with an IC50 value of 78.8 uM. The
presence of free phenolic hydrogens inisoindolinone moiety of clitocybin A analogues plays an important
role in antioxidant activities.
Keywords: Clitocybin, Antioxidant, Oxidative stress, Natural product
Introduction
superoxide, and DPPH radicals.4 Also, clitocybin A
showed various biological activities such as cell death inhi-
bition effects,6 anti-wrinkle effects,7 aortic smooth muscle
cell proliferation inhibition effects.8 In an effort to further
develop this promising compound toward a potent antioxi-
dant, we have modified the structure of clitocybin A by
changing the number of free phenolic hydrogen in iso-
indolinone moiety (Figure 1). In this paper, we described
the synthesis of clitocybin A and its analogues, clitocybin
B and C, and evaluated their free radical scavenging activi-
ties against ABTS, superoxide, and DPPH radicals, as well
as protective activities against cellular DNA damage
induced by oxidative stress and human neutrophil elastase
(HNE) inhibitory activity.
Free radicals are reactive chemical species to cause damage
to biomolecules by abstracting hydrogens. Reactive oxygen
species (ROS) such as hydroxyl radical and superoxide
anion are known to cause oxidative damage to proteins,
DNA alteration, membrane lipid peroxidation, and enzyme
inactivation.1 This oxidative stress generated by ROS,
induces human diseases including inflammatory disease,
cardiovascular disease, cancer, and aging.2 Antioxidants are
well recognized to prevent cells from ROS-induced oxida-
tive stress. Most of natural antioxidants are obtained from
plants including herbs, fruits, vegetables, and tea as com-
pounds of flavonoids or polyphenols. Also, mushrooms
have been known to include significant biological potent
compounds.3 In the course of searching an antioxidant from
various mushrooms, we isolated a new isoindolinone com-
pound, named clitocybin A,4 from the culture broth of
mushroom Clitocybe aurantiaca.5 Clitocybin A exhibited
strong free radical scavenging activity against ABTS,
Experimental
General. All reactions were performed under a nitrogen
atmosphere in oven-dried glassware, unless otherwise
noted. Reactions were monitored by thin-layer chromatog-
raphy (TLC) using Merck silica gel 60 F254 plates. TLC
plates were visualized with a combination of UV light, p-
anisaldehyde or aqueous ceric ammonium molybdate solu-
tion stain and subsequent charring on a hot-plate. Column
chromatography was conducted with indicated solvents
using Merck Silica Gel (40–63 μm). Yields refer to
Figure 1. Structure of Clitocybin A, B, and C.
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chromatographically and spectroscopically pure compounds
unless otherwise stated. Melting points are uncorrected
The Superoxide Anion Scavenging Activity. Each well
of a 96-well plate including of the following reagents
(100 μL): NBT (nitroblue tetrazolium, 0.04 mM), potas-
sium phosphate buffer (50 mM, pH 7.8), EDTA (1 mM),
xanthine (0.18 mM), xanthine oxidase (250 mU/mL), and
the test compounds at various concentrations was incubated
1
unless otherwise noted. H and 13C NMR analyses were
performed using a Varian-300 or 400 spectrometer (Palo
1
Alto, CA, USA) at ambient temperature. H and 13C NMR
chemical shifts are reported as δ values relative to the tetra-
methylsilane (TMS) signal at 0.00 ppm. High resolution
mass spectra were obtained at Korea Basic Science Institute
(KBCI, Daejeon, Korea) on the Synapt G2-HDMS mass
spectrometer (Waters, Manchester, UK), which was oper-
ated on the MassLynx 4.1 software.
ꢀ
for 30 min at 37 C in the dark. The xanthine oxidase cata-
lyzes the oxidation of xanthine to superoxide and uric acid,
and the superoxide reduces NBT to blue formazan. Then,
the reduction of NBT into blue formazan was recorded
(absorbance) by a microplate reader at 560 nm.
The ABTS Radical Scavenging Activity. ABTS was dis-
solved in H2O to form a solution of 7 mM concentration.
The ABTS•+ cation radical was obtained by the reaction of
the ABTS stock solution with potassium persulfate
(2.45 mM) and followed by standing the mixture in the
dark for 12 h. After addition of the ABTS radical cation
solution (0.1 mL) to the test compounds (5 μL) in ethanol,
the absorbance was recorded at 734 nm in an ELISA reader
after mixing for up to 6 min.
Synthesis of Clitocybin C. To a solution of methyl
2-formyl-3,5-dimethoxybenzoate (2.24 g, 10.0 mmol) in
methanol (40 mL) was added 4-aminophenol (1.1 g,
10.0 mmol) at room temperature, and the mixture was
stirred for 1 h. Then, NaBH4 (0.75 g, 20.0 mmol) was
added and the reaction mixture was stirred for 18 h. The
mixture was concentrated in reduced pressure, and the resi-
due was diluted with H2O (40 mL) and adjusted to be
acidic by adding a small amount of acetic acid. The formed
solid was filtered through a glass filter and the collected
solid was washed with water and hot EtOH successively to
The Scavenging Effect on the DPPH Radicals.
Clitocybin A, B, and C at various concentrations were
added to 95 μL DPPH ethanol solution (150 μM) and the
mixture was incubated for 20 min at room temperature, and
then the absorbance was recorded by an ELISA reader at
517 nm.
1
afford 2.6 g (91%) of clitocybin C: H NMR (300 MHz,
DMSO‑d6) δ 9.38 (s, 1H), 7.65 (s, 1H), 7.62 (s, 1H),
6.82–6.76 (m, 4H), 4.73 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H);
13C NMR (75 MHz, DMSO‑d6) δ 165.9, 161.4, 154.9,
154.2, 134.7, 131.1, 121.5, 121.1, 115.2, 102.5, 97.8, 55.7,
55.6, 48.3; HRMS (ESI) m/z 286.1083 [(M + H)+, calcd for
C16H16NO4 286.1079].
Comet Assay. The cell used in comet assay was Chinese
hamster lung fibroblast cell (V79-4). Briefly, the cell pellets
were mixed with low melting point agarose (0.5%). Triple
layered slides were incubated with lysis buffer (10 mM Tris
pH 10, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100,
1% sodium lauroyl sarcosinate was added fresh to this solu-
tion by stirring for at least 15 min) for 1 h. After 15 min
unwinding and 40 min electrophoresis the slides were neu-
tralized with 0.4 M Tris (pH 7.5). Then, the slides were sta-
ined with ethidium bromide and were observed under
fluorescent microscope and image analysis at 515–560 nm.
The percentage of total fluorescence in the tail and the tail
length of 50 cells per slide were measured.
Synthesis of Clitocybin A and B. A mixture of clitocybin
C (2.0 g, 7.0 mmol) and 48% aqueous HBr (10 mL) in
AcOH (20 mL) was heated to reflux for 16 h. The reaction
mixture was cooled to room temperature, poured into ice-
water, and extracted with ethyl acetate. The organic layer
was washed with H2O, dried over MgSO4, and concen-
trated under reduced pressure. The residue was purified by
silica gel column chromatography (10:1 CH2Cl2-CH3OH)
1
to give 1.5 g (85%) of clitocybin A: H NMR (300 MHz,
DMSO‑d6) δ 9.98 (s, 1H), 9.61 (s, 1H), 9.36 (s, 1H), 7.61
(dd, J = 6.6, 1.8 Hz, 2H), 6.79 (dd, J = 6.6, 1.8 Hz, 2H),
6.56 (d, J = 1.8 Hz, 1H), 6.49 (d, J = 1.8 Hz, 1H), 4.65 (s,
2H); 13C NMR (75 MHz, DMSO‑d6) δ 166.4, 158.9,
154.1, 153.0, 134.9, 131.3, 121.5, 117.8, 115.2, 105.9,
100.1, 48.5; HRMS (ESI) m/z 258.0757 [(M + H)+, calcd
for C14H12NO4 258.0766]. Refluxing the mixture of
clitocybin C and 48% aqueous HBr in AcOH for 4 h pro-
vided unreacted clitocybin C (0.54 g, 30%) along with
0.76 g (40%) of clitocybin B: 1H NMR (300 MHz,
DMSO‑d6) δ 9.85 (s, 1H), 9.36 (s, 1H), 7.62(d, J = 9.3 Hz,
2H), 6.78 (d, J = 9.3 Hz, 2H), 6.67 (d, J = 1.8 Hz, 1H),
6.63 (d, J = 1.8 Hz, 1H), 4.70 (s, 2H), 3.84 (s, 3H); 13C
NMR (75 MHz, DMSO‑d6) δ 166.0, 159.3, 154.9, 154.1,
134.7, 131.2, 121.5, 119.0, 115.2, 102.5, 100.5, 55.4, 48.3;
HRMS (ESI) m/z 272.0927 [(M + H)+, calcd for
C15H14NO4 272.0923].
HNE Inhibitory Activity. About 60 μL of the sample
solution [the sample compounds in DMSO (500 μL) were
diluted with pH 7.5 Tris–HCl buffer to provide the sample
solution],
125 μL
of
substrated
solution
[N-
Scheme 1. Synthesis of Clitocybin A, B, and C
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Synthesis and Antioxidant Activities of Clitocybin A, B and C
KOREAN CHEMICAL SOCIETY
Table 2. Protective effect of clitocybin A, B, and C on DNA
damage induced by H2O2.
methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (1.4 mM)
was dissolved in Tris–HCl buffer], and 390 μL of pH 7.5
Tris–HCl buffer solution were combined and vortexed.
Then, 25 μL of enzyme solution (EC 3.4.21.37, approx.
0.18 U elastase of human neutrophils) was added, the
Group
DNA tail (%)c
Control
5.7 ꢁ 0.7
61.1 ꢁ 2.4
7.0 ꢁ 0.8
6.5 ꢁ 0.8
5.1 ꢁ 0.6
35.4 ꢁ 2.6
38.3 ꢁ 2.6
32.3 ꢁ 2.3
16.2 ꢁ 1.3
H2O2
ꢀ
resulting mixture was incubated at 37 C for 1 h. Then, the
Clitocybin Aa
mixture was quenched by adding 500 μL soybean trypsin
inhibitor [0.2 mg/mL, Tris–HCl buffer] and absorbance
was promptly recorded at 405 nm. As a positive control,
epigallocatechin gallate (EGCG) was utilized.
Clitocybin Ba
Clitocybin Ca
Clitocybin Aa+ H2O2
Clitocybin Ba+ H2O2
Clitocybin Ca+ H2O2
N-acetylcysteineb + H2O2
Results and Discussion
a The concentration of clitocybin A, B, and C were at 10 μg/mL.
b The concentration of N-acetylcysteine was at 2 mM.
c Results percentaged as the mean (n = 3) ꢁ SE.
Chemistry. The synthesis of clitocybin A and its ana-
logues was achieved through the route in Scheme 1. First,
clitocybin C was synthesized by reaction of methyl
2-formyl-3,5-dimethoxybenzoate with 4-aminophenol, and
subsequent reduction of the resulting enamine to the amine
and in situ cyclization to yield clitocybin C. Clitocybin A
was synthesized in 85% yield by demethylation of
clitocybin C with 48% HBr in refluxing acetic acid for
18 h. Clitocybin C on refluxing for 4 h with 48% HBr in
acetic acid afforded clitocybin B in 40% yield along with
unreacted clitocybin C in 30% yield.
phenolic hydrogen. But, clitocybin A, B, and C exhibited
no activity against the DPPH radical.
Comet Assay. Inhibition of clitocybin A, B, and C against
damage of cellular DNA induced by H2O2 treatment was
conducted by the comet assay, which evaluate oxidative
DNA damage.12 The cells used in comet assay were Chi-
nese hamster lung fibroblast cells (V79-4). Concentration
of hydrogen peroxide was adjusted to 1 mM. The cell pel-
lets were referred to electrophoresis and stained with
ethidium bromide, and then monitored utilizing a fluores-
cence microscope and image analysis. The percentage of
total fluorescence in the tail and the tail length of 50 cells
per slide were observed. The comet assay exhibited that the
exposure of the cells to H2O2 resulted in increasing their
tail length to 61.1, whereas their treatment with
clitocybin A, B, and C decreased their tail length to 35.4,
38.3, and 32.3 as shown in Table 2. This data suggested
that clitocybin A, B, and C had a protective effect against
damage of cellular DNA induced by oxidative stress, and
Antioxidant Activity.
The antioxidant activity of
clitocybin A and its analogues, clitocybin B and C was
investigated by measurement of their free radical scaveng-
ing effects utilizing three different assays, ABTS radical
cation decolorization assay,9 DPPH radical scavenging
assay,10 and the superoxide radical anion scavenging activ-
ity assay.11 As a result, clitocybin A and B showed potent
superoxide radical scavenging activity with an IC50 value
of 10.3 and 11.2 μM, and exhibited a significant ABTS rad-
ical cation scavenging effect with an IC50 value of 6.4 and
7.2 μM, which were similar to those of well-known antioxi-
dant ferulic acid, used as a positive control (Table 1).
Clitocybin A possessing two phenolic hydrogens in iso-
indolinone moiety of clitocybin A showed more potent rad-
ical scavenging activity than clitocybin B possessing one
clitocybin
C
lacking free phenolic hydrogen in
HNE Inhibitory Activity
100
80
60
40
20
0
Clitocybin A
Clitocybin C
Clitocybin B
EGCG
Table 1. Free radical scavenging activities of clitocybin A-C
(IC50 μM).a
DPPH
ABTS
Superoxide
radicald
Compound
radicalb
radicalc
Clitocybin A
Clitocybin B
Clitocybin C
Ferulic acid
> 100
> 100
> 100
> 100
6.4 ꢁ 0.2
7.2 ꢁ 0.3
10.1 ꢁ 2.4
8.6 ꢁ 0.2
10.3 ꢁ 1.8
11.2 ꢁ 0.3
59.4 ꢁ 2.4
48.4 ꢁ 2.6
a In all three assays, results percentaged as the mean (n = 3) ꢁ SD.
b 2,2-Diphenyl-1-picrylhydrazyl.
1
3
10
30
100
c 2,20-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid).
d Xanthine/xanthine oxidase.
Concentration (uM)
Figure 2. HNE inhibitory activity of clitocybin A, B, and C.
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Synthesis and Antioxidant Activities of Clitocybin A, B and C
KOREAN CHEMICAL SOCIETY
isoindolinone moiety showed more potent activity than
Acknowledgments. This research was supported by a
grant from Korea Research Institute of Bioscience and Bio-
technology Research Initiative Program.
clitocybin
A possessing two phenolic hydrogens in
inhibiting cellular DNA damage in H2O2 treated cells.
However, in evaluation of free radical scavenging activities
against superoxide and ABTS, clitocybin A exhibited more
potent radical scavenging activity than clitocybin C as
shown in Table 1.
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HNE Inhibitory Activity. Elastic fibers play a crucial role
in the elasticity and resilience of skin, even though they
compose of only 1–2% of its dry weight.13 Human neutro-
phil elastase (HNE), a serine protease mainly sited in the
azurophil granules of polymorphonuclear leukocytes, has
wide substrate specificity. HNE is able to hydrolyze elastin,
an insoluble elastic fibrous protein, as well as connective
tissue proteins including fibronectin, cartilage, and colla-
gen.14 Elastase activity increases significantly with age,
which results in lowering skin elasticity.15 Therefore, inhi-
bition of the elastase activity can also be used as a method
of protecting against skin aging.16 The HNE inhibitory
activity of clitocybin A, B, and C were evaluated using a
reported procedure.17 Clitocybins
A dose-dependently
inhibited HNE activity with an IC50 value of 78.8 μM,
which was less potent than the positive control, EGCG
(12.9 μM) (Figure 2).
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Conclusion
In conclusion, clitocybin A and its analogues by modify-
ing free phenolic hydrogens in isoindolinone moiety were
prepared in an efficient route, and their antioxidant activi-
ties were evaluated. Clitocybin A, B, and C exhibited
potent free radical scavenging activities against ABTS and
superoxide, and clitocybin A possessing two phenolic
hydrogens showed the most potent free radical scavenging
activity. In evaluation of protective activities against cellu-
lar DNA damage induced by oxidative stress, clitocybin C
lacking free phenolic hydrogen showed stronger activity
than clitocybin B and clitocybin A. In evaluation of
protecting against skin aging, clitocybin A showed dose-
dependently HNE inhibitory activity. The presence of free
phenolic hydrogens in isoindolinone moiety of clitocybin
A analogues plays an important role in antioxidant
activities.
Bull. Korean Chem. Soc. 2019
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