Flavokawains B and C, melanogenesis inhibitors, isolated from the root of Piper methysticum and synthesis of analogs

Article abstract of DOI:10.1016/j.bmcl.2014.12.082

The ethanolic extract of the root of Piper methysticum was found to inhibit melanogenesis in MSH-activated B16 melanoma cells. Flavokawains B and C were isolated from this extract based on their anti-melanogenesis activity and found to inhibit melanogenesis with IC₅₀ values of 7.7 μM and 6.9 μM, respectively. Flavokawain analogs were synthesized through a Claisen-Schmidt condensation of their corresponding acetophenones and benzaldehydes and were evaluated in terms of their tyrosinase inhibitory and anti-melanogenesis activities. Compound 1b was the most potent of these with an IC₅₀ value of 2.3 μM in melanogenesis inhibition assays using MSH-activated B16 melanoma cells.

Full text of DOI:10.1016/j.bmcl.2014.12.082

Bioorganic & Medicinal Chemistry Letters 25 (2015) 799–802
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Flavokawains B and C, melanogenesis inhibitors, isolated
from the root of Piper methysticum and synthesis of analogs
Hye-Jin Jeong d,e, , Chang Seok Lee a, , Janggyoo Choi c, Yong Deog Hong a, Song Seok Shin a,
Jun Seong Park a, John Hwan Lee a, Seokyong Lee e, Kee Dong Yoon b, , Jaeyoung Ko a,
⇑
⇑
a Amorepacific R&D Unit, 314-1 Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 449-729, Republic of Korea
b College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon-si, Republic of Korea
c College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, Republic of Korea
d Development Group for Global New Drug Developing Human Resources (BK21 Plus Project), Republic of Korea
e School of Pharmacy, Sungkyunkwan University, Suwan, Gyeonggi-do 440-746, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The ethanolic extract of the root of Piper methysticum was found to inhibit melanogenesis in MSH-
Received 5 September 2014
Revised 22 December 2014
Accepted 26 December 2014
Available online 3 January 2015
activated B16 melanoma cells. Flavokawains B and C were isolated from this extract based on their
anti-melanogenesis activity and found to inhibit melanogenesis with IC50 values of 7.7 lM and 6.9 lM,
respectively. Flavokawain analogs were synthesized through a Claisen–Schmidt condensation of their
corresponding acetophenones and benzaldehydes and were evaluated in terms of their tyrosinase
inhibitory and anti-melanogenesis activities. Compound 1b was the most potent of these with an IC50
Keywords:
Piper methysticum
Chalcones
value of 2.3
l
M in melanogenesis inhibition assays using MSH-activated B16 melanoma cells.
Ó 2015 Elsevier Ltd. All rights reserved.
Flavokawain B and C
Melanogenesis
Melanin is a natural substance, synthesized by specialized cells
known as melanocytes, that gives color to hair, skin, eyes, and
other body features. The major function of melanin is to protect
against the damaging effects of ultraviolet radiation. However,
excessive synthesis and accumulation of melanin in the skin can
lead to a number of dermatologic disorders, including melasma,
solar lentigo, and age spot.1 Melanin biosynthesis can be reduced
by avoiding UV exposure, inhibiting melanocyte metabolism, and
regulating melanogenic enzymes or the uptake and distribution
of melanosomes in recipient melanocyte.2 While many melano-
genesis inhibitors have been reported and are used as cosmetic
additives, many other known compounds suffer from high toxicity,
low stability, poor skin penetration, and/or insufficient activity. For
example, consumer application of arbutin, kojic acid, and various
synthetic agents are limited by serious side effects in humans.3
Therefore, recent research on melanogenesis inhibitors has focused
on the use of natural products in the cosmetics industry.4
for ceremonial activities. Commercially, kava has been used in nat-
ural remedies for insomnia, anxiety, and menopausal symptoms.
However, the consumption of kava has recently been reported to
cause serious side effects such as hepatitis and acute liver failure.6
As a result, products containing kava extracts were banned in a
number of European countries, the USA, and Canada.7
During our investigation of melanogenesis inhibitors derived
from natural sources for use as constituents of cosmetic products
and depigmenting agents, we discovered that a crude ethanolic
extract of kava root exhibited as moderate inhibitory activity on
melanogenesis in MSH-stimulated B16 melanoma cells.
This Letter describes the inhibitory effects of flavokawain B (1a)
and flavokawain C (1d), isolated from Piper methysticum on
melanogenesis. We also investigated the structural requirements
for this inhibitory activity through the synthesis of new flavoka-
wain analogs and evaluation of their inhibitory activity on
melanogenesis.
Piper methysticum, or kava, of the family Piperaceae, grows as a
perennial shrub in the South Pacific islands.5 The root of kava is
commonly used by native Pacific islanders to prepare a beverage
Kava root (100 g), collected in Micronesia, was dried, ground,
and extracted with aqueous EtOH (80%) for 48 h. After filtering,
the brown extract solution was evaporated in vacuo to afford a
brown residue (8.5 g). The residue was then partitioned between
methanol and hexane. The hexane layer was evaporated in vacuo
to give a ‘hexane fraction’ (1.5 g). The MeOH-soluble layer was
dried and diluted with distilled water, and further partitioned by
⇑
Corresponding authors. Tel.: +82 31 280 5928; fax: +82 883 9289.
E-mail addresses: kdyoon@catholic.ac.kr (K.D. Yoon), jaeyoungko@amorepacific.
com (J. Ko).
These two authors contributed equally to the work.
http://dx.doi.org/10.1016/j.bmcl.2014.12.082
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

800
H.-J. Jeong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 799–802
successive solvent extractions with ethyl acetate and n-butanol.
Each fractions was evaporated in vacuo to yield an ‘ethyl acetate
fraction’ (7.7 g), an ‘n-butanol fraction’ (0.15 g), and an ‘H2O frac-
tion’ (0.4 g). The ‘hexane fraction’ was most effective in inhibiting
OH
O
H3CO
OCH3
R
melanogenesis in B16 melanoma cells (52% inhibition at 10 lg/
ml). This fraction was separated through a silica column to obtain
six fractions, hexane-1 to -6. Each of these was tested in terms of
the inhibition of melanogenesis. Fractions hexane-4 and hexane-
6 exhibited inhibitory effects on melanin production in B16 mela-
noma cells (Table 1).
1i
R = OCH3 flavokawain A (
)
H
flavokawain B (1a)
1d
OH flavokawain C (
)
Figure 1. Flavokawains isolated from Piper methysticum.
To identify compounds responsible for the observed inhibitory
activities, two major compounds were isolated from hexane-4,
and one compound from hexane-6, by silica column. These com-
pounds isolated from hexane-4 were identified as flavokawain A
and flavokawain B, and the compound isolated from hexane-6
was identified as flavokawain C by NMR and mass spectroscopic
analyze. Their physicochemical spectroscopic data of the isolated
compounds matched published values.8
Flavokawains are a class of chalcone found in kava plant.
Currently flavokawain A, B, and C were identified (Fig. 1).
For confirming the inhibitory effects of flavokawain A, B, and C
on melanogenesis, the production of melanin in MSH-activated
B16 melanoma cells was tested with a kojic acid as positive con-
trol. Flavokawain B and C inhibited the production of melanin in
A
solution of acetophenone (1.2 mmol) and aldehyde
(1.4 mmol) in methanol (3 ml) was added dropwise under nitrogen
to a stirred solution of KOH (26.7 mmol) in water (1.5 ml) that had
been cooled to 0 °C in an ice bath. The reaction mixture was held at
0 °C for 3 h and then at room temperature for 2 days. The mixture
was poured into ice-water, adjusted to pH 3–4 with 1 M HCl, and
then extracted with ethyl acetate. The organic layer was succes-
sively washed with water and saturated brine, and dried over
anhydrous Na2SO4. After concentration under reduced pressure,
the resultant solid was recrystallized from a solution hexane and
ethyl acetate.
Chalcones (1a–1n) were first evaluated for anti-melanogenic
activities in B16 melanoma cells at 10 ppm. Compounds exhibiting
with >50% inhibition 10 ppm were further evaluated other concen-
trations to determine IC50 values (Table 2). Cell viability assays of
all synthesized chalcones in Table 2 indicated that the observed
reduction in melanine production was not caused by cytotoxic
effects of these compounds. As shown in Table 2, most of the chal-
cones were potent inhibitors of melanogenesis.
Compounds 1b–1d have monohydroxyl substituent at o-, m- or
p-position of the B-ring. o-Hydroxy substituted compound 1b was
the most potent inhibitor, with an IC50 value of 2.3
and para-substituted derivatives had IC50 value of 9.9 and 6.9
respectively. Among the monomethoxy substituents, the ortho-
and meta-substituted compounds 1g and 1h showed inhibitory
activities with IC50 values of 3.8 and 3.1 lM, respectively. However,
the para-methoxy-substituted compound 1i showed no inhibitory
activity. Flavokawain B (1a), which does not have a substituent on
its B-ring, showed a moderate inhibitory activity with an IC50 of
a dose-dependent manner with IC50 values of 7.7 and 6.9 lM,
respectively. However, flavokawain A exhibited no inhibitory
activity despite its structural similarities with flavokawain B and
C. These result indicated that the inhibitory activity of hexane-4
must result from the presence of flavokawain B, and that of
hexane-6 from the presence of flavokawain C. Cell viability assays
indicated that the observed reduction in melanine production was
not caused by cytotoxic effects of these compounds.9
Recently, several chalcones have been reported as potential
inhibitors of tyrosinase. The position of the hydroxyl group on
the chalcone rings was shown to be important for that activity.
In 2007, Jun reported 20,40,60-trihydroxy chalcones as a new class
of tyrosinase inhibitors. In particular, trihydroxy chalcones, which
have a 2,4-hydroxy resorcinol structure on their B-ring, were
lM. The meta-
lM,
potent inhibitors of mushroom tyrosinase with an IC50 = 1
l
M.10
Based on these Letters, we hypothesized that the inhibitory
activities of flavokawain B and C on melanogenesis result from
tyrosinase inhibition. However, the flavokawains did not inhibit
mushroom tyrosinase.11 This indicates that both flavokawain B
and C inhibit melanogenesis without inhibiting tyrosinase.
We hypothesized that the hydroxyl or methoxyl substituent on
the B ring of a chalcone derivative would be a key pharmacophore
for anti-melanogenesis.
To investigate the structure–activity relationship (SAR) of
chalcone B-ring, derivatives 1a–1n were synthesized through a
Claisen–Schmidt condensation of the corresponding acetophenon-
es and benzaldehydes with yields of 60–90%.
7.7 lM. To further investigate the effect of hydroxyl substituents,
dihydroxylated compounds 1e and 1f were evaluated in terms of
their melanogenesis inhibitory activity. The introduction of a
hydroxyl group at the o-position on the B-ring of flavokawain
C (1d) played an important role in its enhancing the activity
(1d ? 1f, IC50 6.9
m-hydroxy group decreased this activity (1d ? 1e, IC50
6.9 M ? 31.6 M). SAR studies revealed that the introduction of
lM ? 3.4 lM). However, an additional
l
l
p-methoxy functional groups on the B-ring (1i and 1j) leads to
no activity. In contrast, the introduction of o-hydroxy or methoxy
group on the B-ring has a considerable effect on their potency.
To evaluate the effect of o-substituent on B-ring, novel compounds
with o-alkyl group were synthesized and tested.12 Compared to fla-
The general procedure for these reactions was as follows.
Table 1
vokawain
increased activity (1a ? 1l, 1m, 1n: IC50 7.7
2.7 M). However, in the case of an o-dimethyl compound (1k),
the additional o-methyl group led to a decrease in activity
(1a ? 1k: IC50 7.7 M ? 9.9 M), likely due to steric hindrance
by the o-dimethyl group.
B
(1a), the introduction of alkyl group generally
Inhibitory effects of hexane-1–6 from Piper methysticum on melanogenesis in B16
melanoma cells
lM ? 3.0, 2.5,
l
Samples
Inhibition rate of melanogenesisa (%)
g/ml 10 g/ml
l
l
3
l
l
Hexane-1
Hexane-2
Hexane-3
Hexane-4
Hexane-5
Hexane-6
21 15
27 17
40 24
46 22
23 12
26 12
27 17
>99 10
24 10
>99 11
In conclusion, the ethanolic extract of the root of Piper methys-
ticum was found to inhibit melanogenesis in MSH-activated B16
melanoma cells. Flavokawains B and C were isolated from this
extract based on their anti-melanogenesis activity and found to
inhibit melanogenesis with IC50 values of 7.7 and 6.9
respectively. Flavokawain analogs were synthesized through a
26
4
45 15
lM,
a
Values are the of at least two measurements.

H.-J. Jeong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 799–802
801
Table 2
Tyrosinase inhibitory and anti-melanogenic activities of 20-hydroxy, 40,60-dimethoxychalcones with 2-hydroxyl substituents on B-ring of chalcones on melanogenesis in B16
melanoma cells
OH
A
O
R1
R2
R3
B
H3CO
R4
OCH3
Compounds
R1
R2
R3
R4
Tyrosinase inhibition (IC50
l
M)
Melanogenesis inhibition (IC50 lM)
Kojic acid12
Flavokawain B (1a)
—
H
OH
H
H
H
OH
OCH3
H
—
H
H
OH
H
OH
H
H
OCH3
H
H
—
H
H
H
OH
OH
OH
H
—
H
H
H
H
H
H
H
H
H
H
30
—
—
—
—
—
—
—
—
—
—
—
—
—
—
>500
7.7
2.3
9.9
6.9
31.6
3.4
3.8
3.1
—
1b
1c17
Flavokawain C (1d)
1e10
1f17
1g13
1h14
H
Flavokawain A (1i)
H
OCH3
OCH3
H
H
H
1j15
OCH3
CH3
CH3
CH2CH3
CH(CH3)2
—
1k17
1l16
H
H
H
H
CH3
9.9
3.0
2.5
2.7
H
H
H
1m17
1n17
H
were seeded into 24-well plates and the test compounds/fractions were added
in triplicate. The medium was charged daily and after 4 d of culture, the cells
Claisen–Schmidt condensation of their corresponding acetophe-
nones and benzaldehydes and were evaluated in terms of their
tyrosinase inhibitory and anti-melanogenesis activities. All of the
isolated flavokawain analogs were inactive in mushroom
tyrosinase assays. In particular, flavokawain derivatives were over
20–200 times stronger inhibitors than kojic acid. Compound 1b
were lysed with 1 ml of 1 N NaOH. Then 200
transferred into 96-well plates. The relative melanin content was measured at
400 nm with microplate reader (Molecular Devices). Cell viability was
ll of each crude cell extract was
a
determined using a modified crystal violet assay. After removing the medium
from each well, the cells were washed with PBS and stained with 0.1% crystal
violet in 10% ethanol for 5 min at room temperature. The cells were then rinsed
four times with distilled water, and crystal violet retained by adherent cells
was extracted with 95% ethanol at room temperature for 10 min. Crystal violet
absorption was measured at 590 nm (Molecular Devices Co., Sunnyvale, CA,
USA).
was the most potent of these with an IC50 value of 2.3 lM in mela-
nogenesis inhibition assays using MSH-activated B16 melanoma
cells. Based on structure–activity analysis, we concluded that an
o-hydroxyl or alkyl group on the B-ring is a pharmacophore for
increased inhibitory activity. In contrast, an m-methoxyl group
on B-ring is a pharmacophore for decreased activity. Further
studies on the mechanism of action of flavokawain analogs in
melanogenesis are underway.
10. (a) Nerya, O.; Vaya, J.; Musa, R.; Izrael, S.; Ben-Arie, R.; Tamir, S. J. Agric. Food
Chem. 2003, 51, 1201; (b) Nerya, O.; Musa, R.; Khatib, S.; Tamir, S.; Vaya, J.
Phytochemistry 2004, 65, 1389; (c) Khatib, S.; Nerya, O.; Musa, R.; Shmuel, M.;
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11. Measurement of mushroom tyrosinase activity: Mushroom tyrosinase,
and -DOPA were purchased from Sigma Chemical. The reaction mixture for
mushroom tyrosinase activity consisted of 150 l of 0.1 M phosphate buffer
(pH 6.5), 3 l of sample solution, 8 l of mushroom tyrosinase (2100 unit/ml,
0.05 M phosphate buffer at pH 6.5), and 36 l of 1.5 mM -tyrosine or -DOPA.
L-tyrosine,
L
l
Acknowledgments
l
l
l
L
L
Tyrosinase activity was determined by reading the optical density at 490 nm
on a microplate reader (Bio-Rad 3550, Richnmond, CA, USA) after incubation
for 20 min at 37 °C. The inhibitory activity of the sample was expressed as the
concentration that inhibits 50% of the enzyme activity (IC50).
This study was partially supported by the Research Fund of the
Catholic University of Korea (2011).
12. Kim, K. D.; Song, M. H.; Yum, E. K.; Jeon, O. S.; Ju, Y. W.; Chang, M. S. Bull. Kor.
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DMSO-d6): 13.52 (s, 1H), 9.68 (s, 1H), 7.69 (d, J = 15.4 Hz, 1H), 7.55 (d,
J = 15.4 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 7.5 Hz, 1H), 7.07 (s, 1H), 6.83
(m, 1H), 6.15 (d, J = 1.7 Hz, 1H), 6.12 (d, J = 1.7 Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H).
13C NMR (125 MHz, DMSO-d6) 192.3, 165.8, 165.7, 162.0, 157.8, 142.6, 136.1,
130.2, 127.3, 119.8, 117.8, 114.4, 106.3, 93.9, 91.2, 56.3, 55.8; HRMS m/z
[MꢁH]ꢁ 299.0942, calcd for C17H15O5 299.0919, mp 158–161 °C, compound 1f,
yellowish solid, 1H NMR (500 MHz, DMSO-d6): 13.88 (s, 1H), 10.23 (s, 1H), 9.99
(s, 1H), 7.90 (d, J = 15.6 Hz, 1H), 7.71 (d, J = 15.6 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H),
6.37 (d, J = 2.3 Hz, 1H), 6.32 (dd, J = 8.6, 2.3 Hz, 1H), 6.13 (d, J = 2.3 Hz, 1H), 6.09
(d, J = 2.4 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 3H). 13C NMR (125 MHz, DMSO-d6)
192.6, 165.6, 165.1, 161.8, 161.4, 159.2, 139.7, 130.6, 122.7, 113.6, 108.2, 106.3,
102.6, 93.9, 91.0, 56.1, 55.6; HRMS m/z [MꢁH]ꢁ 315.0898, calcd for C17H15O6
315.0868, compound 1k, yellowish solid, 1H NMR (500 MHz, CDCl3): 7.95 (d,
J = 16.0 Hz, 1H), 7.52 (d, J = 16.0 Hz, 1H), 7.11 (m, 3H), 6.11 (d, J = 2.4 Hz), 6.05
(d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 2.42 (s, 6H). 13C NMR (125 MHz,
CDCl3) 192.7, 168.5, 166.4, 162.6, 140.8, 137.2, 135.2, 132.9, 106.5, 93.9, 91.3,
55.9, 55.7, 21.4; HRMS m/z [M+H]+ 313.1444, calcd for C19H21O4 312.1440, mp
81–84 °C, compound 1m, yellowish solid, 1H NMR (500 MHz, CDCl3): 8.12 (d,
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number were measured in melan-a melanocytes. The cells (2 ꢀ 105 cells/ml)

802
H.-J. Jeong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 799–802
J = 15.4 Hz, 1H), 7.82 (d, J = 15.4 Hz, 1H), 7.65–7.67 (m, 1H), 7.32–7.35 (m, 1H),
1H), 7.62 (dd, J = 7.7, 1.2 Hz, 1H), 7.44–7.32 (m, 2H), 7.23 (m, 1H), 6.12 (d,
J = 2,4 Hz, 1H), 5.96 (d, J = 2.4 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.44 (hept,
J = 6.8 Hz, 1H), 1.29 (d, J = 6.9 Hz, 6H) 13C NMR (125 MHz, CDCl3) 192.8, 168.5,
166.4, 162.7, 148.5, 140.1, 133.7, 130.1, 129.3, 126.9, 126.1, 125.6, 106.4, 93.9,
91.4, 55.9, 55.7, 29.3, 23.8; HRMS m/z [M+H]+ 327.1601, calcd for C20H23O4
326.1596 mp 92–94 °C.
7.23–7.28 (m, 2H), 6.12 (d, J = 2.4 Hz, 1H), 5.97 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H),
3.84 (s, 3H), 2.85 (dd, J = 7.3, 15.1 Hz, 2H), 1.27 (t, J = 7.3 Hz, 3H). 13C NMR
(125 MHz, CDCl3) 192.7, 168.6, 166.5, 162.8, 144.6, 140.0, 134.1, 130.1, 129.4,
128.8, 126.8, 126.4, 106.5, 93.9, 91.4, 55.9, 55.7, 26.6, 16.0; HRMS m/z [M+H]+
313.1446, calcd for C19H21O4 313.1440, mp 96–99 °C compound 1n, yellowish
solid, 1H NMR (500 MHz, CDCl3): 8.22 (d, J = 15.3 Hz, 1H), 7.79 (d, J = 15.3 Hz,