Melanin synthesis inhibitors from Lespedeza cyrtobotrya

Article abstract of DOI:10.1021/np800535g

From the roots of Lespedeza cyrtobotrya, 45 flavonoids were isolated along with 20 new and 25 known compounds. Lipophilic flavonoids 2, 3, 7, 9,11, 28, 30, and 39 exhibited strong inhibitory activities on melanin synthesis in normal human epidermal melano

Full text of DOI:10.1021/np800535g

J. Nat. Prod. 2009, 72, 63–71
63
Melanin Synthesis Inhibitors from Lespedeza cyrtobotrya
Maya Mori-Hongo,†,‡ Hiromi Yamaguchi,† Tsutomu Warashina,§ and Toshio Miyase*,†
School of Pharmaceutical Sciences, UniVersity of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan, Cutaneous Drug Research
Department, POLA Chemical Industries, Inc., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan, and Instutute of EnVironmental
Sciences, UniVersity of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka, 422-8526, Japan
ReceiVed August 24, 2008
From the roots of Lespedeza cyrtobotrya, 45 flavonoids were isolated along with 20 new and 25 known compounds.
Lipophilic flavonoids 2, 3, 7, 9, 11, 28, 30, and 39 exhibited strong inhibitory activities on melanin synthesis in normal
human epidermal melanocytes.
We have reported chalcone and isoflavene derivatives from the
aerial parts of Lespedeza cyrtobotrya Miq. (Leguminosae),1,2 and
these isoflavene derivatives showed strong inhibitory activity on
melanin synthesis.3 In the course of further research for new
melanogenesis inhibitors from plants, 20 new and 25 known
flavonoids were isolated from the roots of this plant. The structures
of the new compounds were elucidated by NMR analysis, while
the absolute configurations were determined from the CD spectra.
The known compounds were identified as liquiritigenin (21),4
bavachin (22),5 isobavachin (23),6 abyssinone II (24),7 naringenin
(25),8 leachianone G (26),9 6-methylaromadendrin (27),10 isoba-
vachalcone (28),11 licoagrochalcone A (29),12 xanthoangerol (39),1
lespeol (31),1 genistein (32),1 6-methylgenistein (33),13 neobavai-
soflavone (34),14 uncinanone A (35),15 bavaisoflavanone (36),16
1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl-
)propanone (37),17 phaseolidin (38),18 erybraedin A (39),19 ery-
braedin C (40),20 lespeflorin G4 (41),21 phaseollin (42),18 gangetinin
(43),22 lespeflorin I2 (44),21 and lespeflorin J3 (45),21 by comparison
with reported NMR data. We measured inhibitory activities of these
compounds on the production of melanin in normal human
epidermal melanocytes and found that many compounds showed
inhibitory activities, with compounds 2, 3, 7, 9, 11, 28, 30, and 39
being more potent than hydroquinone, which is a major skin-
lightening drug.
(dd, J ) 10, 8 Hz), and a hydrogen-bonded hydroxyl proton at δ
13.50 (s). In the 13C NMR spectrum, an ether-linked methine carbon
signal was observed at δ 88.1.15,26 Sheng et al.27 reported the same
structure for artonin ZA from Artocarpus heterophyllus Lamk.
(Moraceae), having an oxygenated carbon signal at δ 77.1. This
carbon should be assigned to a carbon bearing a hydroxyl group.28
Lespecyrtin B2 (3) has a benzpyrene structure [δ 6.73 and 5.75
(each 1H, d, J ) 10 Hz)] and two tertiary methyl groups at δ 1.53
(6H, s). The 13C NMR spectrum of lespecrytin B3 (4) showed two
oxygenated carbon signals at δ 71.4 and 92.6. The former was
assigned to the γ-carbon and the latter to the ꢀ-carbon of a side
chain from the HMQC and HMBC spectra.29
Lespecrytin C1 (5) gave similar 1H NMR data to those of
lespecrytin B1 (2) except for a set of AA′BB′-type aliphatic proton
signals at δ 2.93 and 3.26 (each 2H, d, J ) 7.5 Hz), which were
characteristic of a dihydrochalcone.17
The 1H NMR spectrum of lespecrytin D1 (6) showed five
aliphatic proton signals at δ 2.75 (ddd, J ) 15.5, 5, 2.5 Hz), 2.95
(dd, J ) 15.5, 10 Hz), 3.47 (m), 3.95 (dd, J ) 10, 10 Hz), and
4.19 (ddd, J ) 10, 4, 2.5 Hz), which were characteristic of an
isoflavan.30 In the aromatic proton region, four singlet proton signals
were observed at δ 6.35, 6.47, 6.76, and 6.77. The aromatic proton
signal at δ 6.76 showed ROEs between the proton signals at δ 3.23
(2H, brd, J ) 7.5 Hz), 5.32 (1H, tsept, J ) 7.5, 1 Hz), and 2.95
(1H, dd, J ) 15.5, 10 Hz), due to the R-methylene protons, the
ꢀ-olefinic proton, and the H-4 proton signal, respectively. The
singlet proton signal at δ 6.77 showed ROEs between the proton
signals at δ 3.75 (3H, s), 2.75 (1H, ddd, J ) 15.5, 5, 2.5 Hz), 3.47
(1H, m), and 3.95 (1H, dd, J ) 10, 10 Hz), due to the methoxyl
protons, the H-4 proton, the H-3 proton, and the H-2 proton,
respectively. The absolute stereochemistry at C-3 was deduced as
R from the negative Cotton effect at 233 nm (-6648) and the
positive Cotton effect at 290 nm (+1583).31
Results and Discussion
1
The H NMR spectrum of lespecyrtin A1 (1) showed a typical
AB-type proton signal pattern at δ 4.53 and 5.03 (each 1H, d, J )
12 Hz), suggesting this compound to be a flavanonol.23 Also
observed were one set of isoprenyl proton signals, AA′BB′-type
proton signals at δ 6.91 and 7.46 (each 2H, d, J ) 8.5 Hz), and
AX-type proton signals at δ 6.69 and 7.59 (each 1H, d, J ) 8.5
Hz). The absolute stereochemistry was decided as 2R,3R from the
Lespecrytins E1 (7), E2 (8), E3 (9), E4 (10), E5 (11), E6 (12), and
E7 (13) were assigned with a pterocarpan skeleton from the
large coupling constant (12 Hz) between H-2 and H-3,24
a
laevoratory optical activity,25 and a positive Cotton effect at the
nfπ* absorption band in the CD spectrum25 of lespecyrtin A1.
Lespecyrtins B1 (2), B2 (3), and B3 (4) were assigned with a
chalcone skeleton having a 1,4-disubstituted benzene ring. The 1H
NMR spectra of these chalcones showed a set of AX-type proton
signals in the aromatic proton region due to H-5′ and H-6′ and an
ether-ring side chain. Lespecyrtin B1 (2) showed a vinyl methyl
proton at δ 1.78 (brs), two exocyclic methylene protons at δ 4.94
(m) and 5.10 (m), a set of methylene protons at δ 3.03 (dd, J )
16, 8 Hz) and 3.37 (dd, J ) 16, 10 Hz), a methine proton at δ 5.33
1
characteristic H NMR spectroscopic data in the aliphatic proton
region, and in the aromatic proton region two singlet proton signals
due to H-1 and H-4 were observed. The former five compounds
also had one or two 2,2-dimethyl-2H-chromene rings. The 1H NMR
spectrum of lespecrytin E1 (7) showed two sets of 2,2-dimeth-
ylpyrene ring protons at δ 1.39, 1.41, 1.43, and 1.44 (each 3H, s)
and 5.54, 5.57, 6.32, and 6.51 (each 1H, d, J ) 10 Hz) and one set
of AX-type aromatic proton signals at δ 6.33 and 6.94 (each 1H,
d, J ) 8 Hz). The aromatic proton at δ 6.94 showed ROEs between
the proton signals at δ 3.48 (m), 3.59 (1H, dd, J ) 11, 11 Hz), and
4.21 (1H, dd, J ) 11, 5 Hz) due to H-6a, H-6ꢀ, and H-6R,
respectively. The 1H NMR spectrum of lespecrytin E2 (8) was
similar to that of lespecrytin E1 (7) except for one set of isoprenyl
proton signals. The HRFABMS showed a molecular ion peak at
* To whom correspondence should be addressed. Tel: +81-54-264-5625.
Fax: +81-54-264-5623. E-mail: miyase@ys7.u-shizuoka-ken.ac.jp.
† School of Pharmaceutical Sciences, University of Shizuoka.
‡ POLA Chemical Industries, Inc.
§ Instutute of Environmental Sciences, University of Shizuoka.
m/z 472.2258, suggesting the molecular formula C30H32O5. The 13
C
10.1021/np800535g CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/22/2008

64 Journal of Natural Products, 2009, Vol. 72, No. 1
Mori-Hongo et al.
NMR spectrum showed a specifically hindered R-methylene carbon
signal of an isoprenyl residue at δ 27.2 and ortho dioxygenated
aromatic carbon signals at δ 137.8 and 140.0.21 From these NMR
data, the structure of lespecrytin E2 was deduced as shown.
116.8), and C-9 (δ 154.1), and the R-methylene proton signal at δ
3.29 (2H, brd, J ) 7 Hz) was long-range coupled to C-9 (δ 154.1),
C-10 (δ 112.2), and C-10a (δ 157.7). The HRFABMS of lespecrytin
E6 (12) showed a molecular ion peak at m/z 410.2097, suggesting
1
1
Lespecrytins E3 (9) and E4 (10) gave similar H NMR spectra to
a molecular formula of C25H30O5. The H NMR spectrum was
that of lespecrytin E2 (8). In the H NMR spectrum of lespecrytin
similar to that of erybraedin C (40)20 except for one set of isoprenyl
proton signals. In the aliphatic proton region, isolated ethylene
proton signals at δ 1.77 and 2.70 (each 2H, m) and two singlet
methyl proton signals at δ 1.25 (6H, s) occurred. ROEs were
observed in the methylene ptoton signal at δ 2.70 and the methine
proton signal at δ 5.45 (1H, d, J ) 7 Hz) due to the R-methylene
proton and H-11a, on irradiation of the aromatic proton signal at δ
7.23 (1H, s). In the 13C NMR spectrum, an oxygenated carbon signal
was observed at δ 70.3, which was long-range coupled to the carbon
signals due to the methyl proton signal at δ 1.25 and the
R-methylene proton signal at δ 2.70.32 These NMR data showed
the structure of lespecrytin E6 to be 12. The 1H NMR spectrum of
lespecrytin E7 (13) showed a methoxyl proton signal at δ 3.80 (s),
four singlet aromatic proton signals at δ 6.22, 6.33, 7.01, and 7.15,
and ABX-type proton signals at δ 2.70 (1H, dd, J ) 16.5, 8.5 Hz),
2.99 (1H, dd, J ) 16.5, 5.5 Hz), and 3.77 (1H, dd, J ) 8.5, 5.5
Hz). The 13C NMR spectrum indicated the presence of a 2,2-
dimethyl-3-hydroxy-2H-chromane skeleton33,34 at δ 31.5, 69.8, and
78.1. ROEs were observed between the aromatic proton at δ 7.01
(1H, s) and two methylene protons at δ 3.57 (1H, dd, J ) 10, 10
1
E3 (9), two sets of isoprenyl proton signals were evident, and ROEs
were observed between the proton signal at δ 3.34 (1H, brd, J )
7.5 Hz) and an aromatic proton signal at δ 7.24 and between an
olefinic proton signal at δ 6.30 (1H, d, J ) 10 Hz) and one of the
methylene protons at δ 3.52 (1H, dd, J ) 11, 11 Hz) and an olefinic
proton signal at δ 5.66 (1H, d, J ) 10 Hz). The 1H NMR spectrum
of lespecyrtin E4 (10) exhibited two 2,2-dimethyl-2H-chromene
structures as well as a set of isoprenyl proton signals that was
specifically hindered as determined from the 13C NMR spectroscopic
data for the R-carbon (δ 23.9). ROEs were observed between an
aromatic proton at δ 7.18 (s) and an olefinic proton signal at δ
6.42 (1H, d, J ) 10 Hz) and a methine proton signal at δ 5.33
(1H, d, J ) 7 Hz), and between an olefinic proton signal at δ 6.51
(1H, d, J ) 10 Hz) and one methylene proton signal at δ 4.27 (1H,
dd, J ) 11.5, 5 Hz) and a methine proton signal at δ 3.60 (1H, m).
From these data, the structure of lespecyrtin E4 was determined to
be 10. Lespecrytin E5 (11) had one methyl group resonance at δ
2.17, which was attached to C-8. In the HMBC spectrum, this
methyl proton was long-range coupled to C-7 (δ 124.1), C-8 (δ

Melanin Synthesis Inhibitors from Lespedeza
Journal of Natural Products, 2009, Vol. 72, No. 1 65
Hz) and 4.23 (1H, dd, J ) 10, 10 Hz), a methine proton signal at
δ 3.55 (1H, m), and a methoxyl proton signal at δ 3.80. The absolute
stereochemistry of the ꢀ-carbon was determined to be R using
the Mosher ester method for its methyl ether derivative. The
absolute stereochemistries at C-6a and C-11a of lespecrytins E1-E7
were determined to be 6aR and 11aR from their levoratory optical
activities35 and positive Cotton effects at the region 260-310 nm
in their CD spectra.31
Lespecyrtins F1 (14) and F2 (15) showed a characteristic singlet
proton signal (H2-6) of a pterocarpene skelton near 5.5 ppm in their
1H NMR spectra.30 In the 1H NMR spectrum of 14, a set of ABX-
type aromatic proton signals at δ 6.44 (1H, d, J ) 2 Hz), 6.51
(1H, dd, J ) 8.5, 2 Hz), and 7.31 (1H, d, J ) 8.5 Hz) and a singlet
aromatic proton signal at δ 6.78 (1H, brs) were seen. The latter
showed ROEs between a methylene proton signal at δ 5.49 (2H,
s), which was assigned to H2-6, and a set of isoprenyl proton signals.
In the 1H NMR spectrum of 15, three singlet aromatic proton signals
at δ 6.45, 6.75, and 7.19 and two sets of isoprenyl proton signals
were observed. These NMR data led to the structures 14 and 15
for lespecyrtins F1 and F2, respectively.
6.36, 6.53, 6.59, 6.87, 7.12, and 7.19, and four sets of isoprenyl
proton signals and isolated methylene proton signals at δ 3.92 and
3.96 (each 1H, d, J ) 16.5 Hz), which showed long-range coupling
to C-2, C-3, and C-4a (upper moiety) and C-7, C-8, and C-9 (lower
moiety) in the HMBC spectrum. ROE experiments on irradiating
at δ 6.87 suggested that this proton could be assigned to H-7 of
the lower moiety, by showing ROEs at the proton signals at δ 3.92,
3.44, 3.48, 3.96, 6.53, and 7.12. Irradiating at δ 7.12 showed ROEs
at δ 3.29 (2H, brd, J ) 7.5 Hz) and 5.36 (1H, tsept, J ) 7.5, 1
Hz), and irradiating at δ 7.19 showed ROEs at δ 3.25 (2H, brd, J
) 7.5 Hz) and 5.30 (1H, tsept, J ) 7.5, 1 Hz). From these data,
the structure of lespecyrtin J1 was assigned as 17 like lespedezols
B2 and B3, which were isolated from Lespedeza homoloba Nakai.37
Lespecyrtin H2 (18) showed similar NMR data to those of
lespeflorin J4,36 but lacked a methoxyl signal. The NMR spectra
of lespecyrtins H3 and H4 showed three sets of isoprenyl proton
signals and a dimethylbenzopyrene structure. In a ROE experiment
on lespecyrtin H3, irradiation at δ 7.33 showed ROEs at δ 6.37 (R
proton) and δ 7.41 (1H, brs). In a ROE experiment of lespecyrtin
H4, on irradiating at δ 7.27 (1H, s), a ROE occurred at δ 5.30 (1H,
tsept, J ) 7.5, 1 Hz), and on irradiation at δ 7.18 (1H, s) ROEs
occurred at δ 3.27 (2H, brd, J ) 7.5 Hz) and 5.35 (1H, t sept, J )
7.5, 1 Hz). In the HMBC spectrum of lespecyrtin H4, long-range
couplings were observed between H-R at δ 6.84 (1H, d, J ) 10
Hz) and the carbon signals at δ 107.0, 139.3, and 144.3, which
were assigned to C-8, C-9, and C-8a of the upper moiety,
respectively, from the upfield shifted oxygenated aromatic carbon
Lespecyrtin G1 (16) was assumed to have a coumestan skeleton
from the characteristic carbon signal at δ 158.8.30 The H NMR
1
spectrum of lespecyrtin G1 showed a set of ABX-type aromatic
proton signals at δ 6.91 (1H, d, J ) 2 Hz), 6.96 (1H, dd, J ) 8.5,
2 Hz), and 7.80 (1H, d, J ) 8.5 Hz) and a set of isoprenyl proton
signals. These data led to the assignment of structure 16 for
lespecyrtin G1.
Lespecyrtins H1 (17), H2 (18), H3 (19), and H4 (20) were assumed
signals. The absolute stereochemistry of the pterocarpan skeleton
of these compounds was deduced as 6aR, 11aR from their levoratory
optical activities.35
to be dimeric flavonoids composed of a pterocarpan and a
2-arylbenzofuran derivative, from their molecular formulas and 13
C
NMR spectra like lespeflorins J1-J4, which were isolated from
Lespedeza floribunda Bunge.21 The 1H NMR spectra of lespecyrtin
H1 (17) showed a characteristic spin system of a pterocarpan at δ
3.48 (1H, dd, J ) 10.5, 10.5 Hz), 4.12 (1H, m), 3.40 (1H, m), and
5.39 (1H, d, J ) 7 Hz), six singlet aromatic proton signals at δ
To evaluate the inhibitory activities on melanin synthesis of the
isolated flavonoids, we measured 50% inhibition concentration
(IC50) values of melanin synthesis in normal human epidermal
melanocytes (NHEM).37 As a result, 37 compounds showed
inhibitory activities among 45 compounds from this plant (Table

66 Journal of Natural Products, 2009, Vol. 72, No. 1
Mori-Hongo et al.
Table 1. NMR Spectroscopic Data (400 MHz) for Compounds
1 and 6
Some flavonoid derivatives from plants, such as quercetin and
artocarpanone, were demonstrated previously to have inhibitory
activities against mushroom tyrosinase, the rate-limiting enzyme
of melanin synthesis.38 We are now studying the inhibitory
mechanisms of these active compounds.
1a
δH (J in Hz)
5.03 (d, 12)
6a
position
δC
δH (J in Hz)
δC
2
84.8 3.95 (dd, 10, 10)
4.19 (ddd, 10, 4, 2.5)
73.9 3.47 (m)
193.6 2.75 (ddd, 15.5, 5, 2.5)
2.95 (dd, 15.5, 11)
113.3
70.5
Experimental Section
3
4
4.53 (d, 12)
33.0
31.3
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-1000 digital polarimeter. UV spectra were measured
in methanol on a JASCO V-630 spectrophotometer. Circular dichroism
spectra were measured on JASCO J-20A spectrometer. 1H (400 MHz)
and 13C (100 MHz) NMR spectra were recorded on a JEOL JNM R-400
FT-NMR spectrometer, and chemical shifts are given as δ values with
TMS as an internal standard at 35 °C. Inverse-detected heteronuclear
correlations were measured using HMQC (optimized for 1JC-H ) 145
Hz) and HMBC (optimized for nJC-H ) 8 Hz) pulse sequences with a
pulse field gradient. HRFABMS data were obtained on a JEOL JMS
700 mass spectrometer in the positive mode using a m-nitrobenzyl
alcohol matrix. Preparative HPLC was performed on a JASCO 800
instrument.
4a
5
6
7
8
8a
1′
2′
3′
4′
5′
6′
110.3
130.9
121.0
154.6
103.5
154.0
118.0
150.2
104.3
146.9
142.0
113.1
7.59 (d, 8.5)
6.69 (d, 8.5)
126.6 6.76 (s)
111.0
162.8
116.6 6.30 (s)
162.1
129.7
130.1
115.8 6.47 (s)
158.6
115.8
130.1 6.77 (s)
7.46 (d, 8.5)
6.91 (d, 8.5)
6.91 (d, 8.5)
7.46 (d, 8.5)
Plant Material. The roots of Lespedeza crytobotrya were harvested
from our medicinal plant garden in April 2007. The plant was
authenticated by Prof. Akira Ueno, University of Shizuoka, and a
voucher specimen (200704023) has been deposited at the Herbarium
of the University of Shizuoka.
side chain
at 6
R
3.23 (brd, 7.5)
5.32 (tsept, 7.5, 1)
28.4
124.6
131.6
25.9
ꢀ
γ
Extraction and Isolation. Air-dried roots (4.0 kg) of L. cyrtobotrya
were extracted with methanol (10 L) under reflux for 3 h twice. The
methanol extract was concentrated under reduced pressure to give a
brown residue (345 g). The residue was suspended in hot water (2 L)
and extracted with ether continuously for 3 h. The ether layer was
concentrated under reduced pressure to give a brown residue (85 g).
The ether extract was chromatographed on a silica gel (Fiji Silysia PSQ-
100B, 800 g) column using mixture of hexane-ethyl acetate (9:1-5:
5) as solvents to give 33 fractions (Frs. 1-33). Fr. 3 (584 mg) was
subjected to semipreparative HPLC [column, Cosmosil 5C18-AR-II 2
× 25 cm; solvent, H2O-CH3CN (20:80), UV 280 nm] to give 7 (27
mg). Fr. 4 (105 mg) was subjected to semipreparative HPLC [column,
Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN (15:85), UV 280
nm] to give 43 (2.1 mg). Fr. 5 (101 mg) was subjected to semiprepara-
tive HPLC [column, Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN
(15:85), UV 280 nm] to give 10 (1.8 mg) and 8 (2.3 mg). Fr. 6 (221
mg) was subjected to semipreparative HPLC [column, Inertsil ODS-3,
3 × 50 cm; solvent, H2O-CH3CN (15:85), UV 280 nm] to give 11
(3.1 mg). Fr. 12 (731 mg) was subjected to semipreparative HPLC
δ
1.70 (brs)
1.70 (brs)
ε
17.8
at 8
R
3.30 (m)
5.21 (tsept, 7.5, 1) 122.9
131.9
1.61 (brs)
1.58 (brs)
22.7
ꢀ
γ
δ
25.9
17.9
ε
OMe
3.75 (s)
57.4
a Measured in acetone-d6.
6), and none affected cell viability (data not shown). In particular,
compounds 2, 3, 7, 9, 11, 28, 30, and 39, which have more lipophilic
structures than the other compounds, showed strong inhibitory
activities. The IC50 values of these compounds were lower than 2
µM, while that of hydroquinone, a positive control, was 2.2 µM.
Thus, these compounds were more potent in cell culture than
hydroquinone, which is widely used as a skin lightening agent.
Table 2. NMR Spectroscopic Data (400 MHz) for Compounds 2-5
2a
3a
4a
δH (J in Hz)
5a
δH (J in Hz)
position
1′
2′
3′
4′
5′
6′
CdO
δH (J in Hz)
δC
δH (J in Hz)
δC
δC
δC
115.0
161.5
113.3
166.9
101.8
131.9
192.1
118.3
144.0
127.9
130.5
116.0
158.0
116.0
130.5
121.8
157.7
110.1
154.8
109.2
132.1
189.3
126.3
141.7
128.7
130.8
116.8
160.2
116.8
130.8
115.5
168.1
114.6
162.1
102.3
133.1
193.1
118.7
145.1
127.7
131.7
116.7
160.9
116.7
131.7
115.1
161.0
113.6
167.6
102.4
132.7
205.3
30.4
6.44 (d, 9)
7.79 (d, 9)
6.54 (d, 8.5)
7.52 (d, 8.5)
6.40 (d, 8.5)
8.09 (d, 8.5)
6.39 (d, 9)
7.83 (d, 9)
R
7.44 (d, 17)
7.82 (d, 17)
7.57 (d, 15.5)
7.63 (d, 15.5)
7.78 (d, 15.5)
7.83 (d, 15.5)
2.93 (dd, 7.5, 7.5)
3.26 (dd, 7.5, 7.5)
ꢀ
40.6
1
133.8
130.2
116.0
156.5
116.0
130.2
2
3
4
5
7.55 (d, 8.5)
6.88 (d, 9)
7.58 (d, 8.5)
6.93 (d, 8.5)
7.74 (d, 8.5)
6.93 (d, 8.5)
7.10 (d, 8.5)
6.75 (d, 8.5)
6.88 (d, 9)
7.55 (d, 8.5)
6.93 (d, 8.5)
7.58 (d, 8.5)
6.93 (d, 8.5)
7.74 (d, 8.5)
6.75 (d, 8.5)
7.10 (d, 8.5)
6
side chain
at 3′
R
3.03 (dd, 16, 8)
3.37 (dd, 16, 10)
5.33 (dd, 10, 8)
31.1
6.73 (d, 10)
5.75 (d, 10)
117.6
3.14 (dd, 15.5, 9.5)
3.18 (dd, 15.5, 8)
4.81 (dd, 9.5, 8)
27.6
2.93 (dd, 15.5, 7.5)
3.34 (dd, 15.5, 10)
5.38 (dd, 10, 7.5)
31.6
ꢀ
γ
δ
ε
88.1
143.3
17.0
129.3
77.7
20.8
20.8
92.6
71.4
25.4
25.9
88.5
144.8
17.1
1.78 (brs)
4.94 (m)
5.10 (m)
1.53 (s)
1.53 (s)
1.29 (s)
1.24 (s)
1.77 (brs)
4.92 (m)
5.08 (m)
112.5
112.4
a Measured in acetone-d6.

Melanin Synthesis Inhibitors from Lespedeza
Journal of Natural Products, 2009, Vol. 72, No. 1 67
Table 3. NMR Spectroscopic Data (400 MHz) for Compounds 7-13
7a
8b
9a
10a
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
4a
6
6
6a
11a
7.14 (s)
128.5
116.3
154.6
104.7
156.5
66.6
7.17 (s)
129.8
117.5
155.4
104.9
157.3
66.6
7.24 (s)
132.1
122.0
155.6
103.9
154.9
66.1
7.18 (s)
129.7
116.9
155.3
104.9
157.3
66.8
6.37 (s)
6.29 (s)
6.41 (s)
6.28 (s)
3.59 (dd, 11, 11)
4.21 (dd, 11, 5)
3.48 (m)
3.38 (dd, 11, 11)
4.28 (dd, 11, 5)
3.52 (ddd, 11, 6.5, 5)
5.31 (d, 6.5)
3.52 (dd, 11, 11.5)
4.19 (dd, 11, 3)
3.49 (m)
3.45 (dd, 11.5, 11.5)
4.27 (dd, 11.5, 5)
3.60 (m)
39.8
78.9
39.9
78.8
38.8
77.5
39.5
78.3
5.45 (d, 7)
5.31 (d, 6)
5.33 (d, 7)
11b
10b
7
8
9
10
10a
112.3
119.1
123.8
108.6
153.8
106.2
155.5
113.6
118.4
126.0
137.8
140.0
116.9
148.4
112.6
112.6
115.1
133.1
143.0
111.6
151.8
114.0
113.1
116.6
134.7
144.4
112.2
152.7
6.94 (d, 8)
6.33 (d, 8)
Me
OMe
side chain at 2
R
6.32 (d, 10)
5.54 (d, 10)
121.7
129.1
76.7
28.0
28.3
6.42 (d, 10)
5.67 (d, 10)
122.4
129.9
77.3
27.9
28.4
3.34 (brd, 7.5)
5.34 (tsept., 7.5, 1)
29.2
120.9
134.7
25.8
6.42 (d, 10)
5.66 (d, 10)
122.4
129.9
77.2
27.6
28.3
ꢀ
γ
δ
1.43 (s)
1.44 (s)
1.40 (s)
1.42 (s)
1.79 (brs)
1.79 (brs)
1.45 (s)
1.45 (s)
ε
17.9
at 7
R′
ꢀ′
3.35 (brd, 7.5)
5.27 (tsept., 7.5, 1)
27.2
124.0
131.7
25.8
6.30 (d, 10)
5.66 (d, 10)
118.9
131.8
76.5
27.8
27.8
6.51 (d, 10)
5.79 (d, 10)
119.9
133.3
76.6
27.3
27.5
γ′
δ′
ε′
1.70 (d, 1)
1.80 (brs)
1.44 (s)
1.45 (s)
1.45 (s)
1.45 (s)
18.0
at 10
R′′
ꢀ′′
γ′′
δ′′
ε′′
6.51 (d, 10)
5.57 (d, 10)
116.8
129.6
76.1
27.8
27.9
6.44 (d, 10)
5.65 (d, 10)
117.5
129.9
77.2
27.8
28.2
3.30 (m)
5.29 (tsept., 7.5, 1)
23.3
122.0
131.9
25.8
3.26 (brd, 7.5)
5.27 (tsept., 7.5, 1)
23.9
123.4
131.4
25.9
1.39 (s)
1.41 (s)
1.40 (s)
1.42 (s)
1.69 (brs)
1.79 (brs)
1.63 (brs)
1.74 (brs)
17.7
17.9
11b
12b
13a
13aa
13ba
position
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
δ
H (J in Hz)
δH (J in Hz)
1
2
3
4
4a
6
6
6a
11a
11b
10b
7
8
9
7.16 (s)
129.7
116.9
155.2
104.9
157.5
67.2
7.23 (s)
132.8
124.4
157.0
103.7
155.6
67.2
7.15 (s)
132.9
115.2
155.0
104.7
155.9
67.2
7.19 (s)
7.16 (s)
6.38 (s)
6.23 (s)
6.35 (s)
6.22 (s)
6.40 (s)
3.63 (dd, 11, 11)
4.25 (dd, 11, 4.5)
3.55 (m)
3.55 (dd, 10, 10)
4.20 (dd, 10, 4)
3.53 (m)
3.57 (dd, 10, 10)
4.23 (dd, 10, 6)
3.55 (m)
3.66 (dd, 11, 11)
4.24 (dd, 11, 5)
3.55 (m)
3.61 (dd, 11, 11)
4.23 (dd, 11, 5)
3.53 (m)
41.2
78.4
41.1
79.1
41.2
78.8
5.42 (d, 7)
5.45 (d, 7)
5.42 (d, 7)
5.47 (d, 7)
5.45 (d, 7)
114.5
118.7
124.1
116.9
154.1
112.2
157.7
16.5
112.0
119.1
122.6
108.0
159.9
113.0
156.7
114.0
117.8
110.4
142.6
148.4
98.6
6.89 (s)
2.17 (s)
6.94 (d, 8)
6.38 (d, 8)
7.01 (s)
6.33 (s)
3.80 (s)
6.83 (s)
6.50 (s)
6.81 (s)
6.49 (s)
10
10a
Me
OMe
OMe
side chain
at 2
R
155.1
57.4
3.83 (s)
3.85 (s)
3.82 (s)
3.84 (s)
6.41 (d, 10)
5.65 (d, 10)
122.4
2.70 (m)
1.77 (m)
25.2
2.70 (dd, 16.5, 8.5)
2.99 (dd, 16.5, 5.5)
3.77 (dd, 8.5, 5.5)
31.5
2.90 (dd, 17, 7)
3.23 (dd, 17, 5)
5.14 (dd, 7, 5)
2.76 (dd, 16.5, 8)
3.22 (dd, 16.5, 5)
5.14 (dd, 8, 5)
ꢀ
129.8
77.1
28.4
28.2
45.0
70.3
29.2
29.4
69.8
78.1
20.6
26.1
γ
δ
1.38 (s)
1.41 (s)
1.25 (s)
1.25 (s)
1.23 (s)
1.34 (s)
1.21 (s)
1.30 (s)
1.24 (s)
1.36 (s)
ε
at 7
R′
ꢀ′
γ′
δ′
ε′
at 10
R′′
ꢀ′′
γ′′
δ′′
ε′′
3.29 (brd, 7.5)
5.23 (tsept., 7.5, 1)
23.8
123.5
131.8
25.9
3.27 (brd, 7.5)
5.26 (tsept., 7.5, 1)
23.5
123.6
131.3
25.9
1.61 (brs)
1.73 (brs)
1.62 (brs)
1.74 (brs)
17.9
17.9
a Measured in CDCl3. b Measured in acetone-d6.
[column, Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN (40:60-30:
70) linear gradient, UV 280 nm] to give 42 (1.8 mg), 5 (16 mg), 41
(50 mg), 9 (18 mg), and 31 (123 mg), and fr. 12-1 (35 mg). Fr. 12-1
was subjected to semipreparative HPLC [column, Develosil C30-UG-
5, 2 × 25 cm; solvent, H2O-CH3CN (35:65), UV 280 nm] to give 39
(9.3 mg) and 37 (14 mg). A 70 mg portion of fr. 15 (1.161 g) was
subjected to semipreparative HPLC [column, Capcell Pak ODS, 2 ×
25 cm; solvent, H2O-CH3CN (32.5:67.5), UV 280 nm] to give 2 (12

68 Journal of Natural Products, 2009, Vol. 72, No. 1
Mori-Hongo et al.
Table 4. NMR Spectroscopic Data (400 MHz) for Compounds 14-16
14a
15a
16b
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
4a
6
6a
11a
11b
10b
7
8
9
10
10a
7.31 (d, 8.5)
6.51 (dd, 8.5, 2)
121.5
109.3
159.4
104.5
155.9
66.0
106.8
147.3
110.0
117.8
101.6
142.9
142.2
113.0
149.6
7.19 (s)
121.9
121.3
156.5
104.2
153.8
65.9
106.7
147.6
109.9
117.9
101.5
142.9
142.1
112.9
149.6
7.80 (d, 8.5)
6.96 (dd, 8.5, 2)
122.3
113.4
160.9
104.5
154.4
158.8
102.4
157.9
103.1
113.7
102.0
144.0
143.0
112.5
148.0
6.44 (d, 2)
5.49 (s)
6.45 (s)
5.43 (s)
6.91 (d, 2)
6.78 (s)
6.75 (s)
7.17 (s)
side chain
at 2
R
3.30 (brd, 7.5)
5.38 (m)
28.1
123.7
132.6
25.9
ꢀ
γ
δ
1.77 (brs)
1.74 (brs)
ε
17.9
at 10
R′
ꢀ′
γ′
δ′
ε′
3.65 (brd, 7)
5.43 (tsept., 7, 1)
23.8
122.9
132.1
25.9
3.64 (brd, 7.5)
5.38 (m)
23.8
122.9
132.0
25.9
3.59 (brd, 7.5)
5.35 (brt, 7.5)
22.8
121.6
131.5
25.5
1.68 (brs)
1.89 (brs)
1.69 (brs)
1.92 (brs)
1.67 (brs)
1.87 (brs)
17.9
17.9
17.7
a Measured in acetone-d6. b Measured in DMSO-d6.
mg). Then, 50 mg of fr. 17 (564 mg) was subjected to preparative
HPLC [column, TSKgel ODS-80TS, 5.5 60 cm; solvent,
UV 280 nm] to give 18 (77 mg), 17 (39 mg), 45 (36 mg), 20 (11 mg),
and fr. 27-1 (266 mg). Fr. 27-1 was subjected to semipreparative HPLC
[column, Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN (60:40),
UV 280 nm] to give 21 (3.9 mg), 27 (3.1 mg), 1 (5.7 mg), 13 (6.2
mg), 26 (12 mg), 3 (3.3 mg), 23 (1.0 mg), 4 (6.5 mg), 35 (40 mg), 34
(6.8 mg), 12 (2.5 mg), and 16 (25 mg).
×
H2O-CH3CN (60:40), UV 280 nm] to give 40 (18 mg). Next, 50 mg
of fr. 20 (371 mg) was subjected to semipreparative HPLC [column,
Cosmosil 5C18-AR-II 2 × 25 cm; solvent, H2O-CH3CN (57.5:42.5),
UV 280 nm] to give 38 (24 mg). Fr. 23 (4.05 g) was subjected to
preparative HPLC [column, TSKgel ODS-80TS, 5.5 × 60 cm; solvent,
H2O-CH3CN (60:40-40:60) linear gradient, UV 280 nm] to give 25
(15 mg), 36 (65 mg), 24 (16 mg), 29 (108 mg), and 28 (834 mg), and
frs. 23-1-23-2. A 30 mg portion of fr. 23-1 (211 mg) was subjected
to semipreparative HPLC [column, Develosil C30-UG-5, 2 × 25 cm;
solvent, H2O-CH3CN (25:75), UV 280 nm] to give 30 (20 mg). Fr.
23-2 (46 mg) was subjected to semipreparative HPLC [column,
Develosil C30-UG-5, 2 × 25 cm; solvent, H2O-CH3CN (20:80), UV
280 nm] to give 19 (15 mg). Fr. 24 (3.27 g) was subjected to
semipreparative HPLC [column, Cosmosil 5C18-AR-II 2 × 25 cm;
solvent, H2O-CH3CN (20:80), UV 280 nm] to give 32 (316 mg), 23
(47 mg), and 14 (229 mg), and frs. 24-1-24-5. Fr. 24-1 (17 mg) was
subjected to semipreparative HPLC [column, Develosil C30-UG-5, 2
× 25 cm; solvent, H2O-CH3CN (35:65), UV 280 nm] to give 32 (1.2
mg) and 33 (1.6 mg). Fr. 24-2 (37 mg) was subjected to semipreparative
Lespecyrtin A1 (1): colorless, amorphous solid; [R]2D3 -30.6 (c 0.73,
MeOH); UV (MeOH) λmax (log ꢀ) 285 (4.08) nm; CD (MeOH) λmax
1
nm ([θ]) 261 (+4082), 305 (+9070), 332 (+12 698); H NMR and
13C NMR, Table 1; HRFABMS m/z 341.1417 (calcd for C20H20O5 +
H, 341.1389).
Lespecyrtin B1 (2): colorless, amorphous solid; [R]2D3 +34.1 (c 1.48,
1
MeOH); UV (MeOH) λmax (log ꢀ) 370 (4.47) nm; H NMR and 13C
NMR, Table 2; HRFABMS m/z 323.1320 (calcd for C20H18O4 + H,
323.1284).
Lespecyrtin B2 (3): colorless, amorphous solid; UV (MeOH) λmax
(log ꢀ) 226 (4.05), 284 (3.89), 347 (3.96) nm; 1H NMR and 13C NMR,
Table 2; HRFABMS m/z 323.1311 (calcd for C20H18O4 + H, 323.1284).
Lespecyrtin B3 (4): colorless, amorphous solid; [R]2D3 -11.7 (c 0.75,
1
MeOH); UV (MeOH) λmax (log ꢀ) 369 (4.35) nm; H NMR and 13C
NMR, Table 2; HRFABMS m/z 341.1393 (calcd for C20H20O5 + H,
341.1389).
HPLC [column, Develosil C30-UG-5,
2
×
25 cm; solvent,
H2O-CH3CN (52.5:47.5), UV 280 nm] to give 6 (10 mg). Fr. 24-3
(14 mg) was subjected to semipreparative HPLC [column, Develosil
C30-UG-5, 2 × 25 cm; solvent, H2O-CH3CN (52.5:47.5), UV 280
nm] to give 22 (4.5 mg). Fr. 24-4 (43 mg) was subjected to
semipreparative HPLC [column, Develosil C30-UG-5, 2 × 25 cm;
solvent, H2O-CH3CN (40:60), UV 280 nm] to give 28 (33 mg). Fr.
24-5 (162 mg) was subjected to semipreparative HPLC [column, Inertsil
ODS-3, 3 × 50 cm; solvent, H2O-CH3CN (40:60-30:70) linear
gradient, UV 280 nm] to give 15 (36 mg) and 44 (4.3 mg). A 100 mg
sample of fr. 25 (651 mg) was chromatographed on a Sephadex LH-
20 column (2.5 × 100 cm) using methanol as solvent to give fr. 25-1
(24 mg). Fr. 25-1 was subjected to semipreparative HPLC [column,
Develosil C30-UG-5, 2 × 25 cm; solvent, H2O-CH3CN (20:80), UV
280 nm] to give 45 (6.3 mg). Fr. 26 (594 mg) was subjected to
semipreparative HPLC [column, Inertsil ODS-3, 3 × 50 cm; solvent,
H2O-CH3CN (15:85), UV 280 nm] to give fr. 26-1 (176 mg). Fr. 26-1
was subjected to semipreparative HPLC [column, Develosil C30-UG-
5, 2 × 25 cm; solvent, H2O-CH3CN (15:85), UV 280 nm] to give 45
(36 mg). Fr. 27 (209 mg) was subjected to semipreparative HPLC
[column, Inertsil ODS-3, 3 × 50 cm; solvent, H2O-CH3CN (15:85),
Lespecyrtin C1 (5): colorless, amorphous solid; [R]2D3 +59.4 (c 1.08,
MeOH); UV (MeOH) λmax (log ꢀ) 220 sh (4.44), 241 sh (4.05), 288
(4.27) nm; 1H NMR and 13C NMR, Table 2; HRFABMS m/z 325.1457
(calcd for C20H20O4 + H, 325.1440).
Lespecyrtin D1 (6): colorless, amorphous solid; [R]2D3 -42.8 (c 1.02,
MeOH); UV (MeOH) λmax (log ꢀ) 220 sh (4.24), 292 (3.93) nm; CD
1
(MeOH) λmax nm ([θ]) 233 (-6648), 257 (+1108), 290 (-1583); H
NMR and 13C NMR, Table 1; HRFABMS m/z 356.1609 (calcd for
C21H24O5, 356.1624).
Lespecyrtin E1 (7): colorless, amorphous solid; [R]2D3 -59.9 (c 0.46,
MeOH); UV (MeOH) λmax (log ꢀ) 230 (4.84), 279 (4.23), 308 (4.01),
1
319 (4.02) nm; CD (MeOH) λmax nm ([θ]) 318 (+18977); H NMR
and 13C NMR, Table 3; HRFABMS m/z 388.1668 (calcd for C25H24O4,
388.1675).
Lespecyrtin E2 (8): colorless, amorphous solid; [R]2D3 -108.2 (c 0.30,
MeOH); UV (MeOH) λmax (log ꢀ) 227 (4.62), 283 (4.09), 319 (3.88)
nm; CD (MeOH) λmax nm ([θ]) 270 (+3148), 303 (+3779), 320
(+5247); 1H NMR and 13C NMR, Table 3; HRFABMS m/z 472.2258
(calcd for C30H32O5, 472.2251).

Melanin Synthesis Inhibitors from Lespedeza
Journal of Natural Products, 2009, Vol. 72, No. 1 69
Table 5. NMR Spectroscopic Data (400 MHz) for Compounds 17-20
17a
18a
19a
20a
δH (J in Hz)
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δC
upper moiety
2
3
4
149.9
118.8
26.3
153.9
120.3
196.4
153.4
120.3
196.3
155.1
121.9
196.0
3.92 (d, 16.5)
3.96 (d, 16.5)
4a
5
6
7
8
8a
1′
2′
3′
4′
5′
6′
114.8
103.1
142.2
142.1
112.0
148.8
110.6
154.2
103.6
157.3
120.9
131.8
116.4
103.1
143.2
142.8
112.2
148.3
111.2
156.9
103.6
160.9
108.6
131.7
116.8
103.1
143.3
142.9
112.2
148.3
112.0
156.7
103.6
156.4
114.9
128.2
116.3
105.5
144.5
139.3
107.0
144.3
110.4
155.4
103.8
158.2
121.1
131.7
6.53 (s)
6.93 (s)
6.93 (s)
6.98 (s)
6.59 (s)
7.12 (s)
6.37 (d, 2.5)
6.35 (s)
7.33 (s)
6.40 (s)
7.27 (s)
6.51 (dd, 8.5, 2.5)
7.49 (d, 8.5)
isoprenyl at 8
R
3.61 (brd, 7.5)
5.44 (tsept., 7.5, 1)
23.8
124.0
132.4
25.8
3.67 (brd, 7.5)
5.48 (overlapped)
23.8
122.7
132.0
25.9
3.67 (brd, 7.5)
5.48 (overlapped)
23.8
122.9
132.5
25.9
6.84 (d, 10)
5.90 (d, 10)
116.6
132.0
77.8
27.6
27.6
ꢀ
γ
δ
1.74 (brs)b
1.83 (brs)
1.66 (brs)i
1.87 (brs)
1.74 (brs)
1.88 (brs)
1.50 (s)
1.50 (s)
ε
17.7
18.0
18.0
at 5′
R′
3.29 (brd, 7.5)
5.36 (tsept., 7.5, 1)
28.1
123.7d
132.0
25.9
6.39 (d, 10)
5.62 (d, 10)
122.1
129.0
77.7
28.7
28.7
3.25 (brd, 7.5)
5.30 (tsept., 7.5, 1)
28.3
123.8
132.5m
17.8n
25.9
ꢀ′
γ′
δ′
1.73 (brs)b
1.75 (brs)c
1.39 (s)
1.44 (s)
1.69 (brs)l
1.65 (brs)l
ε′
17.8
lower moiety
1
2
3
4
4a
6
6
6a
7.19 (s)
132.4
122.8
156.8
103.9
155.5
67.3
7.19 (s)
6.36 (s)
132.3
123.8
157.2
103.8
155.6
66.8
7.19 (s)
132.3
123.1
157.1
104.3
155.6
66.9
7.18 (s)
123.1
132.2
157.2
103.7
155.7
66.8
6.36 (s)
6.24 (s)
6.37 (s)
3.48 (dd, 10.5, 10.5)
4.12 (m)
3.44 (m)
3.25 (dd, 11, 11)
3.90 (dd, 11, 5)
3.40 (m)
3.25 (dd, 10.5, 10.5)
3.96 (dd, 10.5, 5.5)
3.41 (m)
3.20 (dd, 11, 11)
3.87 (dd, 11, 5)
3.35 (m)
41.2
78.8
39.9
80.3
40.0
80.3
40.0
80.4
11a
5.39 (d, 7)
5.48 (d, 7)
5.46 (d, 7)
5.46 (d, 7)
11b
10b
7
8
9
10
10a
113.0
118.9
123.4
122.0
154.3
112.0
158.2
111.6
119.3
127.9
115.2
164.5
111.9
164.4
112.0
119.3
127.9
115.3
164.4
111.6
164.4
111.9
119.3
127.9
115.0
164.6
111.6
164.5
6.87 (s)
7.42 (s)
7.41 (s)
7.39 (s)
isoprenyl at 2
R′′
3.25 (brd, 7.5)
5.30 (tsept., 7.5, 1)
28.4
123.2d
131.8
25.9
3.29 (brd, 7.5)
5.35 (tsept., 7.5, 1)
28.4
122.9
132.3j
25.9
3.29 (brd, 7.5)
5.35 (tsept., 7.5, 1)
28.4
123.8
132.2
25.9
3.27 (brd, 7.5)
5.35 (tsept., 7.5, 1)
28.4
123.8
132.4m
25.9
ꢀ′′
γ′′
δ′′
1.63 (brs)b
1.63 (brs)c
1.78 (brs)i
1.70 (brs)i
1.67 (brs)
1.75 (brs)
1.74 (brs)l
1.78 (brs)
ε′′
18.0
17.8k
17.9
17.8n
at 10
R′′′
ꢀ′′′
γ′′′
δ′′′
ε′′′
3.29 (brd, 7.5)
5.24 (tsept., 7.5, 1)
23.8
123.4d
131.8
25.9
3.27 (brd, 7.5)
5.26 (tsept., 7.5, 1)
22.8
122.9
132.2j
25.9
3.28 (brd, 7.5)
5.26 (tsept., 7.5, 1)
22.9
122.7
132.0
25.9
3.29 (brd, 7.5)
5.26 (tsept., 7.5, 1)
22.9
122.7
132.6
25.9
1.63 (brs)c
1.74 (brs)c
1.73 (brs)i
1.74 (brs)i
13.44 (s)
1.67 (brs)
1.78 (brs)
13.35 (s)
1.70 (brs)l
1.73 (brs)l
13.40 (s)
17.9
17.9k
17.9
17.9n
OH at 9
a Measured in acetone-d6.b-nAssignments are interchangeable in each column.
Lespecyrtin E3 (9): colorless, amorphous solid; [R]2D3 -191.1 (c 0.67,
MeOH); UV (MeOH) λmax (log ꢀ) 225 sh (4.55), 276 (4.27), 285 (4.28),
342 (3.73) nm; CD (MeOH) λmax nm ([θ]) 236 (-68 501), 270
(+17 916), 288 (-24 239), 345 (-2347); 1H NMR and 13C NMR, Table
3; HRFABMS m/z 474.2405 (calcd for C30H34O5, 474.2407).
Lespecyrtin E6 (12): colorless, amorphous solid; [R]2D3 -110.3 (c
0.37, MeOH); UV (MeOH) λmax (log ꢀ) 290 (3.86), 363 (3.53) nm;
1
CD (MeOH) λmax nm ([θ]) 239 (-17 320), 290 (+29 626); H NMR
and 13C NMR, Table 3; HRFABMS m/z 410.20974 (calcd for C25H30O5,
410.2094).
Lespecyrtin E4 (10): colorless, amorphous solid; [R]2D3 -84.0 (c 0.21,
MeOH); UV (MeOH) λmax (log ꢀ) 228 (4.50), 273 (4.08), 307 (3.89)
Lespecyrtin E7 (13): colorless, amorphous solid; [R]2D3 -359.6 (c
0.50, MeOH); UV (MeOH) λmax (log ꢀ) 295 (4.19) nm; CD (MeOH)
1
nm; CD (MeOH) λmax nm ([θ]) 270 (+5457), 315 (+1049); H NMR
1
λmax nm ([θ]) 236 (-106 930), 282 (-77 318), 296 (+190 829); H
and 13C NMR, Table 3; HRFABMS m/z 472.2225 (calcd for C30H32O5,
472.2251).
NMR and 13C NMR, Table 3; HRFABMS m/z 370.1423 (calcd for
C21H22O6, 370.1417).
Lespecyrtin E5 (11): colorless, amorphous solid; [R]2D3 -93.9 (c 0.35,
MeOH); UV (MeOH) λmax (log ꢀ) 226 (4.64), 274 (4.06), 306 (3.88),
319 (3.85) nm; CD (MeOH) λmax nm ([θ]) 279 (+7186), 304 (+10 779),
315 (+7725); 1H NMR and 13C NMR, Table 3; HRFABMS m/z
404.1992 (calcd for C26H28O4, 404.1988).
Lespecyrtin F1 (14): colorless, amorphous solid; UV (MeOH) λmax
1
(log ꢀ) 220 sh (4.46), 241 (4.06), 288 (4.29) nm; H NMR and 13C
NMR, Table 4; HRFABMS m/z 338.1173 (calcd for C20H18O5,
338.1154).

70 Journal of Natural Products, 2009, Vol. 72, No. 1
Mori-Hongo et al.
M254 calcium-free medium supplemented with human melanocyte
growth supplement (HMGS), 50 µg/mL streptomycin, and 50 U/mL
penicillin. NHEM were seeded in a 24-well culture plate. After
incubation for 24 h, the medium was exchanged for that containing
the sample compound dissolved in DMSO and 0.25 µCi 2-[2-14C]-
thiouracil (Amersham, Freiburg, Germany). After incubation for 72 h,
the cells were washed twice with phosphate-buffered saline and lysed
with trichloroacetic acid (TCA). On centrifugation, the pellets were
washed twice with 10% TCA. The pellets were mixed with scintillation
fluid and the incorporated radioactivity was determined with a liquid-
scintillation counter (LSC6100; Aloka, Tokyo, Japan). Cell viability
was examined by a colorimetric assay using a WST-8 cell-counting
kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s
protocol. The IC50 value of melanin synthesis was determined using
SAS software version 9.1.3 (SAS Institute Inc., Cary, NC) within the
range of the concentration where cell viability remains unaffected.
Table 6. Melanin Synthesis Inhibitory Effects (IC50 µM) of
Isolated Flavonoids
compound
flavanone
IC50 (µM)
compound
pterocarpan
IC50 (µM)
21
22
23
24
25
26
14.7
7
18.0
8
3.7
0.40
4.5
1.5
3.4
16.0
8.7
2.0
5.5
3.2
7.5
4.6
a
9
2.0
10
11
12
13
38
39
40
41
42
43
73.5
a
flavanonol
isoflavone
a
a
1
27
32
33
34
29.2
a
9.4
isoflavanone
chalcone
pterocarpen
coumestan
dimer
Supporting Information Available: Structures of known com-
pounds and ROE and HMBC data. This material is available free of
charge via the Internet at http://pubs.acs.org.
a
35
36
5.4
38.4
14
15
15.9
a
2
1.3
1.0
9.1
1.4
3.2
0.98
2.4
16
44
3
25.7
References and Notes
4
28
29
30
31
17
18
19
20
20
8.9
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a
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6.2
37.7
dihydrochalcone
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37
3.0
2.0
hydroquinone
arbutin
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103
6
2.1
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a Inhibitory activity not shown within the range of the concentration
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Lespecyrtin F2 (15): colorless, amorphous solid; UV (MeOH) λmax
1
(log ꢀ) 211 (4.54), 254 (4.12), 308 (3.79), 354 (4.18) nm; H NMR
and 13C NMR, Table 4; HRFABMS m/z 406.1808 (calcd for C25H26O5,
406.1781).
Lespecyrtin G1 (16): colorless, amorphous solid; UV (MeOH) λmax
1
(log ꢀ) 247 (4.04), 342 (4.11) nm; H NMR and 13C NMR, Table 4;
HRFABMS m/z 353.1030 (calcd for C20H16O6 + H, 353.1025).
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CD (MeOH) λmax nm ([θ]) 233 (-55 886), 290 (+35 483); H NMR
and 13C NMR, Table 5; HRFABMS m/z 798.3726 (calcd for C50H54O9,
798.3762).
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Lespecyrtin H2 (18): colorless, amorphous solid; [R]2D3 -62.9 (c
0.75, MeOH); UV (MeOH) λmax (log ꢀ) 309 (4.33) nm; CD (MeOH)
λmax nm ([θ]) 235 (-31 426), 270 (-13 232), 329 (-12 239), 393
1
(+860); H NMR and 13C NMR, Table 5; HRFABMS m/z 745.3022
(calcd for C45H44O10 + H, 745.3014).
Lespecyrtin H3 (19): colorless, amorphous solid; [R]2D3 -48.3 (c
1.65, MeOH); UV (MeOH) λmax (log ꢀ) 264 (4.44), 294 (4.35), 310
(4.36) nm; CD (MeOH) λmax nm ([θ]) 235 (-36 015), 258 (+45 019),
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285 (+7203), 326 (-5867), 394 (+28 812); H NMR and 13C NMR,
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Lespecyrtin H4 (20): colorless, amorphous solid; [R]2D3 -80.8 (c
1.10, MeOH); UV (MeOH) λmax (log ꢀ) 278 (4.40), 348 (4.26) nm;
CD (MeOH) λmax nm ([θ]) 275 (-27 732), 302 (-13 686), 342
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(each ca. 0.5 mg) in pyridine (30 µL) was added (+)-MTPA chloride
or (-)-MTPA chloride (3 µL), and the solution was left overnight at
room temperature. 3-[(Dimethylamino)prolyl]amine (3 µL) was added
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1
mg) and the (+)-MTPA ester (13b, 0.3 mg). H NMR: Table 3.
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Melanin Synthesis Inhibitors from Lespedeza
Journal of Natural Products, 2009, Vol. 72, No. 1 71
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NP800535G