Determination of the Absolute Configuration of Khellactone Esters from Peucedanum japonicum Roots

Article abstract of DOI:10.1021/acs.jnatprod.6b00947

Sixteen new angular dihydropyranocoumarins (1-16) and 24 known compounds were isolated from the roots of Peucedanum japonicum Thunb. The absolute configuration of diacylkhellactone was established by partial hydrolysis, the Mosher method, and X-ray crysta

Full text of DOI:10.1021/acs.jnatprod.6b00947

Article
pubs.acs.org/jnp
Determination of the Absolute Configuration of Khellactone Esters
from Peucedanum japonicum Roots
Min Jee Hong and Jinwoong Kim*
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul
08826, Republic of Korea
S
* Supporting Information
ABSTRACT: Sixteen new angular dihydropyranocoumarins (1−16) and 24 known
compounds were isolated from the roots of Peucedanum japonicum Thunb. The absolute
configuration of diacylkhellactone was established by partial hydrolysis, the Mosher method,
and X-ray crystallography. In addition, ECD spectroscopy was used to assign the absolute
configurations of several of the angular dihydropyranocoumarins. Enantiomers were detected
by RP-HPLC using MTPA esters while acyl migration of the substituents was observed in
cis-monoacylkhellactones.
Peucedanum japonicum Thunberg, a member of the Umbellifer-
ae family, is widely distributed in southern and eastern Asia. Its
roots have been used as a folk medicine for cold and neuralgic
diseases in Korea and Taiwan.1,2 Phytochemical studies have
revealed that the roots of this plant contain coumarins,3−7
chromones, polyacetylenes,8 inositols,9 and steroid glycosides.10
A literature survey showed that coumarins,11,12 flavonoids,
phenylpropanoid glycosides,13 phenol derivatives, C13 noriso-
prenoid glycosides, nucleosides, nucleobases, amino acids, and
benzofuran glycosides have been isolated from the leaves of P.
japonicum. In this study, 16 new angular dihydropyranocou-
marins (1−16) and 24 known angular dihydropyranocoumar-
ins (17−40) were isolated from the n-hexane and CHCl3
fractions of the MeOH extract of the roots of P. japonicum. In
the case of the diacylkhellactones, partial hydrolysis combined
with MTPA derivatization and X-ray crystallography were
applied to determine the configurations at C-3′ and C-4′. In
addition, ECD spectroscopy combined with TDDFT calcu-
lations was used to define the absolute configurations.
Compounds 2, 7, 9, 12, and 14 were assigned (3′S,4′S)
absolute configurations, while the enantiomers of these
compounds have been previously reported.14−17
RESULTS AND DISCUSSION
■
The air-dried roots of P. japonicum (30.0 kg) were extracted
with 100% MeOH under ultrasonication. After removing the
(2-methylbutyroyl)khellactone (7),15 3′-O-isovaleroyl-4′-O-an-
geloylkhellactone (9),16 3′-O-acetyl-4′-O-(2-methylbutyroyl)-
solvent under reduced pressure, the MeOH extract was
khellactone (hyuganin C) (12),17 and 4′-O-(2-
partitioned using n-hexane, CHCl3, EtOAc, and n-BuOH.
methylbutyroyl)khellactone (14),14 and 24 known compounds.
The n-hexane and CHCl3 fractions were subjected to repeated
chromatography to afford 16 new angular dihydropyranocou-
marins (1−16), including five new enantiomers 3′-O-acetyl-4′-
Received: October 19, 2016
O-senecioylkhellactone (isosamidin) (2),14 4′-O-angeloyl-3′-O-
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. 1H and 13C Spectroscopic Data of Compounds 1−2
The known compounds were identified as (3′S,4′S)-peujapo-
nisinol B (17),5 4′-O-senecioylkhellactone [(3′R,4′R)-peujapo-
nisinol B] (18),18,19 (3′S,4′S)-peujaponisinol A (19),5 3′-O-
senecioylkhellactone [(3′R,4′R)-peujaponisinol A] (20),18,19
4′-O-angeloyl-3′-O-senecioylkhellactone (21),20 3′,4′-di-O-sen-
ecioylkhellactone (22),21 peujaponisin (23),6 qianhucoumarin
B (24),22,23 3′-O-acetyl-4′-O-angeloylkhellactone (pteryxin)
(25),24 hyuganin D (26),20 corymbocoumarin (27),25 (−)-cis-
khellactone (28),20 qianhucoumarin C (29),23 3′-hydroxy-4′-
angeloyloxy-3′,4′-dihydroseselin (30),25 praeruptorin F (31),26
praeruptorin B (32),27 3′-angeloyloxy-4′-hydroxy-3′,4′-dihy-
droseselin (33),25 3′-O-angeloyl-4′-O-senecioylkhellactone
(calipteryxin) (34),27 3′-O-isovaleroyl-4′-O-senecioylkhellac-
tone (35),21 3′,4′-di-O-isovaleroylkhellactone (36),27 praer-
uptorin H (37),26 O-senecioyllomatin (38),28,29 O-isovaler-
oyllomatin (39),28,29 and qianhucoumarin D (40)30 (Figure S1,
Supporting Information).
a
and 16
1
2
16
δH (J in
δH (J in
Hz)
δH (J in
Hz)
position
δC
δC
δC
Hz)
2
3
159.7
159.8
160.1
113.3 6.20, d
(9.5)
113.3 6.19, d
(9.5)
113.1 6.21, d
(9.4)
4
5
6
143.1 7.57, d
(9.5)
143.1 7.56, d
(9,5)
143.3 7.58, d
(9.4)
129.2 7.33, d
(8.6)
129.1 7.32, d
(8.6)
129.1 7.32, d
(8.6)
114.4 6.78, d
(8.6)
114.4 6.77, d
(8.6)
114.5 6.79, d
(8.6)
7
156.6
107.5
154.0
112.4
77.4
156.7
107.5
154.0
112.5
77.4
157.0
8
107.6
9
154.2
10
2′
3′
112.6
Absolute Configuration Determination of the Diac-
ylkhellactones by Partial Hydrolysis Combined with the
Mosher Method and X-ray Crystallography. Compound 1
was isolated as colorless needles. The molecular formula was
determined as C23H28O7 based on the m/z 417.1912 ion [M +
79.3
70.3 5.30, d
(4.8)
70.6 5.29, d
(4.8)
73.7 3.93, br s
4′
60.4 6.53, d
(4.8)
59.6 6.56, d
(4.8)
67.8 6.08, d
(3.4)
1
H]+ in HRCIMS. In the H NMR spectrum, 1 showed signals
5′
6′
25.5 1.38, s
22.0 1.43, s
25.3 1.40, s
22.3 1.44, s
3′-ester
24.9 1.46, s
21.0 1.37, s
attributable to an α-pyrone moiety at δH 6.20 (1H, d, J = 9.5
Hz, H-3) and 7.57 (1H, d, J = 9.5 Hz, H-4), and an o-
disubstituted benzene moiety at δH 7.33 (1H, d, J = 8.6 Hz, H-
5) and 6.78 (1H, d, J = 8.6 Hz, H-6), typical patterns of a
disubstituted coumarin. A pair of methine doublets [δH 5.30
(1H, d, J = 4.8 Hz, H-3′) and 6.53 (1H, d, J = 4.8 Hz, H-4′)]
and geminal dimethyls [δH 1.38 and 1.43 (each 3H, s, H3-5′,
H3-6′)] revealed the presence of an angular dihydropyran unit
(Table 1). The signals at δH 2.55 (1H, sep, J = 7.0 Hz, H-2″),
1.18, and 1.17 (each 3H, d, J = 7.0 Hz, H3-3″, H3-4″) were due
to an isobutyroyl group with 2-methylbutyroyl signals
resonating at δH 2.37 (1H, sext, J = 7.0 Hz, H-2‴), 1.71, 1.44
(each 1H, m, H-3‴), 0.91 (3H, t, J = 7.4 Hz, H3-4‴), and 1.19
(3H, d, J = 7.0 Hz, H3-5‴). The isobutyroyl (C-1″) and 2-
methylbutyroyl (C-1‴) groups were correlated to H-3′ (δH
5.30) and H-4′ (δH 6.53), respectively, as indicated by the MS
fragmentation (base peak, M+ − OMeBu) and confirmed by
HMBC data. The relative configurations of the C-3′ and C-4′
stereogenic carbons were considered to be cis based on the
coupling constant (4.8 Hz) and NOESY correlation between
H-3′ and H-4′.27,31
1
2
175.7
169.9
33.9 2.55, sep
(7.0)
20.7 2.07, s
3
4
19.1 1.18, d
(7.0)
18.5 1.17, d
(7.0)
4′-ester
1
2
175.4
165.2
167.3
41.3 2.37, sxt
(7.0)
115.0 5.62, s
115.0 5.68, s
3
26.5 1.71, m
1.44, m
158.2
159.6
4
5
11.6 0.91, t
(7.4)
20.4 2.21, s
27.5 1.87, s
20.6 2.21, s
27.6 1.89, s
16.4 1.19, d
(7.0)
a
Data were obtained at 400 MHz in CDCl3.
The 2D structure of the angular dihydropyranocoumarin
moiety was readily defined by 1D and 2D NMR data. The
Mosher’s method was selected to define the absolute
configurations at C-3′ and C-4′. In the case of the
diacyldihydropyranocoumarins, partial alkaline hydrolysis was
required to release a hydroxy group prior to the MTPA (α-
methoxy-α-trifluoromethylphenylacetyl) esterification.
Upon alkaline hydrolysis, 1 gave a mixture of three products
(1a−c), which were isolated by HPLC (Figure 1). Compounds
1a and 1b showed epimerization at C-4′ as previously
reported.27 The partially hydrolyzed product (1a) was found
to be trans-configured based on the J3′,4′ value of 3.9 Hz (Table
S1, Supporting Information).27,31 The fully hydrolyzed
products were identified as (+)-trans-khellactone (1b) and
(−)-cis-khellactone (1c) by NMR data analysis and specific
rotations (Table S1, Supporting Information).5 The absolute
configuration of 1a was defined using Mosher’s reagents to
yield the diastereomeric (S)- and (R)-MTPA esters (1aa and
1ab). In the (S)-MTPA ester of 1a, the protons in the C-3′, C-
Figure 1. Partial and total alkaline hydrolysis of 1.
4′, C-5′, C-6′, and C-6 portion were shielded, and those in the
C-3 and C-4 portion were deshielded (vice versa in the (R)-
MTPA ester) (Figure 2). Thus, the absolute configuration of
compound 1 was assigned as (3′S,4′S). The (3′S,4′S) absolute
B
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Figure 2. Δδ (δS − δR) values obtained from the MTPA esters of
compound 16 and partial hydrolysis product 1a.
configuration was confirmed by single-crystal X-ray diffraction
analysis (Figure 3).
Figure 4. X-ray crystallographic structure of 2 (ORTEP drawing).
agreement with the experimental ECD spectra (Figures S3 and
S5, Supporting Information).
Compounds 3−15 displayed similar patterns to compounds
1 and 2 in their experimental ECD spectra (Figure S2,
Supporting Information). Therefore, the absolute configura-
tions of compounds 3−15 were defined as (3′S,4′S). If the
khellactone ester showed negative ECD Cotton effects at ca.
220 and 320 nm, a NOESY correlation between H-3′ and H-4′,
3
and a J3′,4′ value of 4.6−5.0 Hz, the absolute configuration
could be identified as (3′S,4′S), regardless of the nature of the
C-3′ and C-4′ substituents.
Structure Determination of trans-Monoacylkhellac-
tone. Compound 16 (tR = 37.8 min) was isolated along with
E1 (enantiomeric mixture of 17 and 18, tR = 36.6 min) and E2
(enantiomeric mixture of 19 and 20, tR = 39.6 min) by RP-
HPLC [CH3CN−H2O (55:45, 5 mL/min)] from fraction H39.
The molecular composition of compound 16, C19H20O6, was
determined by HRCIMS data (m/z 345.1335 [M + H]+). The
1H and 13C NMR spectra of 16 were similar to those of its
diastereomer, cis-4′-O-senecioylkhellactone (peujaponisinol B,
17)5 except for the protons and carbons of the dihydropyran
ring, especially at C-4′ [16: δH 6.08 (1H, d, J = 3.4 Hz, H-4′),
δC 67.8 (C-4′); 17: δH 6.43 (1H, d, J = 4.8 Hz, H-4′), δC 63.0
(C-4′)] (Tables 1 and S3 and S5, Supporting Information).
The 3′,4′-trans relative configurations were characterized based
on the J value (3.4 Hz) and the absence of a NOESY
correlation between H-3′ and H-4′.
Figure 3. X-ray crystallographic structure of 1 (ORTEP drawing).
Compound 2 was obtained as colorless needles. The
molecular formula was identified as C19H20O6 by HRESIMS
1
data. The H NMR spectrum of 2 was similar to that of 1
except for the substituents at C-3′ and C-4′ (Table 1). The
linkage between the khellactone, acetyl (C-1″), and senecioyl
(C-1‴) moieties was confirmed by the HMBC cross-peaks
from H-3′ (δH 5.29) to C-1″ (δC 169.9) and from H-4′ (δH
6.56) to C-1‴ (δC 165.2). The 3′,4′-cis relative configuration
3
was identified based on the J3′,4′ value of 4.8 Hz and NOESY
interactions.
The Mosher method could not be applied to compound 2,
because the ester functionalities at both C-3′ and C-4′ were
hydrolyzed under alkaline conditions. The (3′S,4′S) absolute
configuration of compound 2 was defined by single-crystal X-
ray diffraction analysis (Figure 4).
The 3′,4′-trans relative configuration of 16 was determined
1
via H NMR data. The absolute configuration was determined
1
by the Mosher method using the free 3′-OH group. The H
NMR chemical shift differences of the MTPA esters (16a and
16b) [H-3 (−0.02 ppm), H-4 (−0.02 ppm), H-4′ (−0.12
ppm), H-6 (+0.00 ppm), H-5′ (+0.09 ppm), H-6′ (+0.15
ppm), and H-3′ (+0.03 ppm)] indicated a (3′S) configuration
(Figure 2). Therefore, the C-4′ absolute configuration had to
be R based on the 3′,4′-trans relative configuration. Thus, the
structure of compound 16 was assigned as (3′S,4′R)-4′-O-
senecioylkhellactone. Interestingly, a structure identical to
compound 16 was reported but without substantial spectro-
scopic evidence (Table S6, Supporting Information).32
Assignment of the Absolute Configuration of the
Khellactone Esters by ECD Spectroscopy. The structures
of compounds 3−15 were similar to 1 except for the 3′ and 4′
1
substituents that were confirmed by H and 13C NMR data
(Tables S2−S5, Supporting Information). The locations of the
C-3′ and C-4′ substituents were determined by the HMBC
interactions from H-3′ to C-1″ and from H-4′ to C-1‴. The
3′,4′-cis relative configuration was confirmed by the NOESY
interactions and J values (4.6−5.0 Hz).
The configuration of 1 was assigned as (3′S,4′S) based on
the MTPA experiment and X-ray diffraction analysis, while the
(3′S,4′S) configuration of 2 was defined via X-ray data only. In
addition, the calculated ECD spectra of 1 and 2 were in good
Separation of the Senecioylkhellactone Enantiomers.
Before the presence of enantiomers was recognized, E1
(enantiomeric mixture of compounds 17 and 18) was esterified
by the Mosher method to determine its absolute configuration.
After the MTPA reaction of E1 with (R)-(−)-MTPA-Cl, major
C
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and minor peaks (17a and 18a, respectively) were isolated by
HPLC (Figure S10, Supporting Information). MTPA esters
with (S)-(+)-MTPA-Cl gave 17b and 18b as the major and
minor constituents, respectively. Based on the Mosher method,
the absolute configurations of the major (17) and minor (18)
compounds were assigned as (3′S,4′S) and (3′R,4′R),
respectively (Figure S11, Supporting Information).
observed between 17 and 19 and between 18 and 20 due to
transesterification involving the hydroxy and senecioyl moieties
(Figures S16 and S17, Supporting Information). Equilibrium
was established through mutual acyl migration between C-3′
and C-4′ approximately 4 days after purification. The migration
of the C-3′ acyl moiety to C-4′ was slightly dominant compared
to the reverse migration. Positional isomers, such as
compounds 13 and 14, 17 and 19, 18 and 20, 24 and 29,
and 30 and 33, were isolated in this experiment.
MS Fragmentation of the Khellactone Esters. For
compound 1, the location of the isobutyroyl (C-3′) and 2-
methylbutyroyl (C-4′) groups was deduced by the diagnostic
fragment at m/z 315 (base peak, M+ − OMeBu) in the CI mass
spectrum. In addition, the fragment ion of 2 was detected at m/
z 287 (M+ − OSen), and that of 16 (4′-O-senecioylkhellac-
tone) was detected at m/z 245 (base peak, M+ − OSen) (Table
S7, Supporting Information).
The MS fragment peaks of khellactone esters without a C-4′
substituent were detected as major peaks, which were more
dominant than those from esters without a 3′-ester moiety. The
ion resulting from fragmentation of the 3′-substituent was small
or undetectable, depending on the ionization method. The
determination of the position of substituents by MS
fragmentation analysis corresponded with the HMBC analysis.
Based on these results, the positions of the C-3′ and C-4′
substituents could be determined by MS fragmentation.
E2, the enantiomeric mixture of compounds 19 and 20, was
also derivatized by Mosher reagent (Figure S12, Supporting
Information). The absolute configurations of the major (19)
and minor (20) compounds were determined as (3′S,4′S) and
(3′R,4′R), respectively (Figure S13, Supporting Information).
Because enantiomeric mixtures E1 and E2 were discovered
during the separation of the MTPA reaction product,
separation of the enantiomer was performed on a CHIRALPAK
IC column (4.6 mm × 250 mm, 5 μm). (3′S,4′S)-
Peujaponisinol B (17) and (3′R,4′R)-peujaponisinol B (18)
(ratio = 2.44:1) were isolated from E1 (Figure S14, Supporting
Information), and (3′S,4′S)-peujaponisinol A (19) and
(3′R,4′R)-peujaponisinol A (20) (ratio = 2.70:1) were isolated
from E2 (Figure S15, Supporting Information). The absolute
configurations of the enantiomers were confirmed by their
opposite specific rotations, mirror-image related ECD patterns,
and similar NMR spectra (Tables S3 and S5, Supporting
Information). It is difficult to find appropriate columns that
have good resolution to resolve enantiomers (Figures S11 and
S12, Supporting Information). Therefore, HPLC analysis after
MTPA derivatization is a feasible alternative to detect the
presence of enantiomers. In addition, the ratios of the major
and minor MTPA reaction products and enantioseparation
products were similar.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-2000 polarimeter at 20 °C using a sodium
lamp (589 nm). UV spectra were recorded using a PerkinElmer
Lambda 25 UV/vis spectrophotometer. ECD and UV spectra were
measured on an Applied Photophysics Chirascan-plus CD spectrom-
eter. IR spectra were acquired using a JASCO FT/IR-4200
ECD Spectroscopy of the Senecioylkhellactone
Enantiomers. The monoacylkhellactones 16−18 have the
same 2D structures. The ECD curves of 16 and 18 showed
mirror-image related Cotton effects compared to that of 17.
The calculated ECD spectra of 16−18 (Figure 5) showed
1
spectrophotometer. The H and 13C NMR, COSY, HSQC, HMBC,
and NOESY spectra were obtained in CDCl3 on Bruker AVANCE
digital 400 or 500 spectrometers, a JEOL JNM-ECA 600 spectrometer,
or a Bruker AVANCE III HD 800 spectrometer with a 5 mm CPTCI
cryoprobe. Solvent peaks were used as internal standards. High-
resolution and low-resolution CIMS were conducted on a JEOL JMS
700 spectrometer. High-resolution and low-resolution ESIMS data
were obtained using an AB SCIEX Q-TOF 5600 mass spectrometer or
an Agilent Technologies 6130 Quadrupole LC/MS spectrometer with
an Agilent Technologies 1260 Infinity LC system and an INNO C18
column (4.6 × 150 mm, S-5 μm, 12 nm). Column chromatography
(CC) was performed with Merck silica gel 60 (40−63 μm, 230−400
mesh). TLC was conducted using Merck silica gel 60 F254-precoated
TLC plates and Merck RP-18 F254s-precoated TLC plates. Spots were
visualized by spraying with anisaldehyde−H2SO4 followed by heating
at 120 °C. The MPLC system was equipped with Combi Flash
Companion (Teledyne Isco Inc., Lincoln, NE, USA) and a Reveleris
C18 Reversed-Phase 120 g Cartridge (Grace & Co., Columbia, MD,
USA). Fisher HPLC-grade solvents were used for the MeOH−H2O
solution. The HPLC system was composed of a Gilson 321 pump,
Gilson UV/vis 151 detector (detection at 254 or 327 nm), and a
Hydrosphere C18 column (20 × 250 mm, S-5 μm, 12 nm, YMC Co.,
Ltd., Kyoto, Japan), INNO C18 column (20 × 250 mm, S-5 μm, 12
nm, Young Jin Biochrom Co., Ltd., Seoul, South Korea), YMC
J’sphere ODS H80 column (10 × 250 mm, S-4 μm, 8 nm), INNO C18
column (10 × 250 mm, S-5 μm, 12 nm), or CHIRALPAK IC column
(4.6 × 250 mm, 5 μm, Daicel Corporation, Osaka, Japan). Fisher
HPLC-grade solvents were used for the CH3CN−H2O and MeOH
system. Pyridine-d5 (Cambridge Isotope Laboratories, Inc.), (R)-(−)-
and (S)-(+)-MTPA-Cl, and DMAP (4-dimethylaminopyridine)
(Sigma-Aldrich Co., St. Louis, MO, USA) were used to prepare the
Mosher esters.
Figure 5. Experimental ECD spectra of 16−18 in MeOH.
similar patterns to the experimental ECD spectra (Figures S18,
S20, and S22, Supporting Information). The (4′R)-khellactone
ester without a 3′-O-acyl substituent displayed a mirror-image
related ECD pattern with the (3′S,4′S)-cis-khellactone ester.
Acyl Migration of the cis-Monoacylkhellactones.
Compounds 17−20 were separated from fraction H39. After
their isolation, interchange of the substituents in solution was
D
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Plant Material. P. japonicum roots were collected at Taean-gun,
Chungcheongnam-do, Korea, in November 2012. Plant specimens
were deposited at the herbarium of Seoul National University in Korea
(ref: SNUPH 2012−02/KOR) after identification by Prof. Je Hyun
Lee, College of Oriental Medicine, Dongguk University.
Extraction and Isolation. Dried roots of P. japonicum (30.0 kg)
were extracted with MeOH (3 × 45.5 L, 200 min each) at room
temperature in an ultrasonicator and filtered. After condensing the
filtrate under reduced pressure, the MeOH extract (2.7 kg, yield: 8.9%)
was partitioned into n-hexane (454.2 g, yield: 16.9%), CHCl3 (87.9 g,
yield: 3.3%), EtOAc (19.9 g, yield: 0.7%), n-BuOH (97.5 g, yield:
3.6%), and residual aqueous fractions (1.6 kg, yield: 59.6%).
(3′S,4′S)-3′-O-Isobutyroyl-4′-O-(2S-methylbutyroyl)khellactone
(1). Colorless needles; [α]20 + 1 (c 0.1, MeOH); UV (MeOH) λmax
D
(log ε) 220 (3.69), 255 (3.10), 321 (3.64) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 224 (−3.73), 244 (+0.92), 256 (+0.73), 323
1
(−0.87) nm; H and 13C NMR data, see Supporting Information;
CIMS m/z 417 [M + H]+; HRCIMS m/z 417.1912 [M + H]+ (calcd
for C23H29O7, 417.1913).
(3′S,4′S)-3′-O-Acetyl-4′-O-senecioylkhellactone (2). Colorless nee-
dles; mp 104−106 °C; [α]20 − 6 (c 1, MeOH); UV (MeOH) λmax
D
(log ε) 218 (4.28), 255 (3.50), 323 (3.87) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 226 (−17.00), 245 (+2.86), 255 (+2.46), 319
1
(−2.44) nm; H and 13C NMR data, see Supporting Information;
HRESIMS m/z 409.1258 [M + Na]+ (calcd for C21H22O7Na,
409.1258).
A part of the n-hexane fraction (406.0 g) was subjected to silica
column chromatography (CC, 34 × 15.3 cm) using gradient mixtures
of n-hexane-EtOAc (95:5 → 0:100) to afford 51 fractions (H1−H51).
Fraction H32 (5.3 g) was separated using RP-MPLC (MeOH−H2O,
75:25, 20 mL/min) to yield 16 subfractions, H32−1 to H-32−16.
H32−4 was subjected to RP-HPLC (CH3CN−H2O, 75:25, 5 mL/
min) to obtain compound 15 (0.8 mg, tR = 26.9 min). H32−5 was
divided by RP-HPLC (CH3CN−H2O, 75:25, 5 mL/min) to obtain
seven subfractions (H32−5−1 to H32−5−7). H32−5−5 was applied
to chiral-selective HPLC (1 mL/min) using MeOH to furnish 1 (15.1
mg, tR = 6.3 min), 3 (24.0 mg, tR = 6.0 min), 6 (4.7 mg, tR = 5.5 min),
and a subfraction (H32-5-5-4). H32-5-5-4 was chromatographed using
chiral-selective HPLC (1 mL/min) with MeOH to give 8 (0.6 mg, tR =
6.6 min). H32-6 was separated by RP-HPLC (CH3CN−H2O, 75:25, 5
mL/min) to give compound 9 (2.2 mg, tR = 41.6 min). Fraction H34
(45.2 g) was chromatographed using RP-MPLC (MeOH-H2O, 70:30,
40 mL/min) to give 13 subfractions, H34−1 to H-34−13. H34−7 was
separated by RP-HPLC (CH3CN−H2O, 75:25, 7 mL/min) to acquire
compound 7 (11.1 mg, tR = 37.2 min) and subfractions (H34-7-3, 4).
Compound 5 (2.1 mg, tR = 45.8 min) was obtained from H34-7-4
by RP-HPLC (7 mL/min), using a mixture of CH3CN−H2O (70:30).
Subfraction H34-7-3 was subjected to HPLC (1 mL/min) using a
chiral-selective column with MeOH to provide compound 4 (3.5 mg,
tR = 5.5 min). Fraction H35 (14.7 g) was applied to silica CC and
eluted using a mixture of n-hexane−EtOAc−MeOH (60:4:1) followed
by RP-HPLC (CH3CN−H2O, 80:20, 5 mL/min) to obtain
subfractions (H35-11-1, 2). Compound 2 (2.8 mg, tR = 27.4 min)
was isolated from H35-11-1 by RP-HPLC using a mixture of
CH3CN−H2O (65:35, 7 mL/min), and 12 (109.6 mg, tR = 24.5
min) was isolated from H35-11-2 by eluting with CH3CN−H2O
(80:20, 5 mL/min). Fraction H37 (5.8 g) was subjected to RP-MPLC
using a MeOH−H2O (55:45, 15 mL/min) elution system followed by
RP-HPLC (CH3CN−H2O, 55:45, 5 mL/min) to yield subfractions
H37-9-1 and H37-9-4. Chiral-selective HPLC was performed with
MeOH to obtain compounds 13 (6.7 mg, tR = 5.4 min) and 14 (21.7
mg, tR = 4.7 min) from H37-9-4. Fraction H39 (1.7 g) was purified by
RP-HPLC using a CH3CN−H2O (55:45, 5 mL/min) elution system
to give compounds 16 (2.5 mg, tR = 37.8 min) and 40 (11.4 mg, tR =
34.6 min) and the enantiomeric mixtures E1 (8.1 mg, tR = 36.6 min)
and E2 (7.2 mg, tR = 39.6 min). The CHIRALPAK IC column
(MeOH, 1 mL/min) was used to yield compounds 17 (1.0 mg, tR =
4.5 min) and 18 (0.5 mg, tR = 5.9 min) from E1 and compounds 19
(0.8 mg, tR = 6.3 min) and 20 (0.5 mg, tR = 5.3 min) from E2.
Fractions H41−43 (3.7 g) were applied to MPLC over ODS and
eluted with MeOH−H2O (55:45, 10 mL/min) to give ten subfractions
H41-43-1 to H41-43-10. H41-43-7 was chromatographed by RP-
HPLC (5 mL/min) using an isocratic solvent system of CH3CN−H2O
(45:55) to afford 10 (3.6 mg, tR = 50.9 min).
(3′S,4′S)-4′-O-Isobutyroyl-3′-O-(2-methylbutyroyl)khellactone
(3). Colorless needles; [α]20D + 52 (c 0.1, MeOH); UV (MeOH) λmax
(log ε) 219 (4.36), 256 (3.84), 323 (4.23) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 225 (−19.43), 245 (+3.31), 256 (+2.63), 323
(−4.16) nm; IR νmax 2974, 2938, 2875, 1745, 1608, 1491, 1220, 1147,
772 cm−1; 1H and 13C NMR data, see Supporting Information; CIMS
m/z 417 [M + H]+; HRCIMS m/z:417.1910 ([M + H]+, calcd for
C23H29O7:417.1913).
(3′S,4′S)-3′-O-(2-Methylbutyroyl)-4′-O-senecioylkhellactone (4).
White amorphous powder; [α]20 −20 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 218 (4.50), 256 (3.85), 323 (4.17) nm; ECD
(c 50 μM, MeOH) λmax (Δε) 225 (−29.78), 246 (+4.94), 256 (+4.21),
326 (−5.38) nm; IR νmax 2976, 2938, 2877, 1741, 1608, 1491, 1221,
1
1144, 1074, 772 cm−1; for H and 13C NMR data, see Supporting
Information; CIMS m/z:429 [M + H]+; HRCIMS m/z:429.1909 [M +
H]+ (calcd for C24H29O7, 429.1913).
(3′S,4′S)-4′-O-(2-Methylbutyroyl)-3′-O-senecioylkhellactone (5).
White amorphous powder; [α]20 + 6 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 219 (4.55), 256 (3.86), 323 (4.23) nm; ECD
(c 50 μM, MeOH) λmax (Δε) 225 (−17.32), 246 (+3.05), 256 (+2.14),
323 (−4.93) nm; IR νmax 2976, 2934, 1747, 1608, 1220, 1146, 772
cm−1; 1H and 13C NMR data, see Supporting Information; CIMS m/z
429 [M + H]+; HRCIMS m/z 429.1908 [M + H]+ (calcd for
C24H29O7, 429.1913).
(3′S,4′S)-3′-O-Isobutyroyl-4′-O-isovaleroylkhellactone (6). White
amorphous powder; [α]20D −9 (c 0.1, MeOH); UV (MeOH) λmax (log
ε) 218 (4.09), 255 (3.49), 323 (4.05) nm; ECD (c 50 μM, MeOH)
λmax (Δε) 224 (−8.79), 245 (+2.53), 255 (+1.94), 323 (−1.80) nm;
1H and 13C NMR data, see Supporting Information; CIMS m/z 417
[M + H]+; HRCIMS m/z 417.1917 [M + H]+ (calcd for C23H29O7,
417.1913).
(3′S,4′S)-4′-O-Angeloyl-3′-O-(2-methylbutyroyl)khellactone (7).
White amorphous powder; [α]20 −59 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 219 (4.33), 255 (3.62), 323 (4.12) nm; ECD
(c 50 μM, MeOH) λmax (Δε) 226 (−7.15), 246 (+1.36), 254 (+1.24),
318 (−1.25) nm; 1H and 13C NMR data, see Supporting Information;
HRESIMS m/z 451.1729 [M + Na]+ (calcd for C24H28O7Na,
451.1727).
(3′S,4′S)-3′-O-Butyroyl-4′-O-(2-methylbutyroyl)khellactone (8).
Colorless needles; [α]20 + 7 (c 0.1, MeOH); UV (MeOH) λmax
D
(log ε) 219 (3.78), 255 (3.23), 323 (3.75) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 224 (−4.58), 243 (+1.03), 257 (+0.90), 326
1
(−1.04) nm; H and 13C NMR data, see Supporting Information;
HRESIMS m/z 439.1719 [M + Na]+ (calcd for C23H28O7Na,
439.1727).
(3′S,4′S)-4′-O-Angeloyl-3′-O-isovaleroylkhellactone (9). White
amorphous powder; [α]20 −40 (c 0.1, MeOH); UV (MeOH) λmax
The CHCl3 fraction was divided by CC (64 × 15.3 cm) over silica
gel with mixtures of n-hexane−EtOAc (85:15 → 0:100) as a gradient
solvent system to produce 36 fractions (C1−C36). Fraction C20−22
(4.8 g) was subjected to MPLC (20 mL/min) over ODS using
MeOH−H2O (50:50) as the eluent to give 16 subfractions (C20-22-1
to C20-22-16). C20-22-12 was separated by RP-HPLC (CH3CN−
H2O, 45:55, 5 mL/min) to give seven subfractions (C20-22-12-1 to
C20-22-12-7). C20-22-12-7 was purified by semipreparative RP-HPLC
(CH3CN−H2O, 45:55, 2 mL/min) to obtain compound 11 (0.8 mg,
tR = 31.5 min).
D
(log ε) 219 (4.32), 255 (3.62), 323 (4.01) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 225 (−19.19), 246 (+2.61), 256 (+2.66), 326
1
(−2.57) nm; H and 13C NMR data, see Supporting Information;
HRESIMS m/z 451.1724 [M + Na]+ (calcd for C24H28O7Na,
451.1727).
(3′S,4′S)-3′-O-Acetyl-4′-O-(3-hydroxyisovaleroyl)khellactone
(10). White amorphous powder; [α]20 + 23 (c 0.3, CHCl3); UV
D
(MeOH) λmax (log ε) 219 (4.27), 256 (3.78), 322 (4.19) nm; ECD (c
50 μM, MeOH) λmax (Δε) 224 (−7.73), 245 (+2.32), 255 (+1.72),
E
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322 (−1.43) nm; 1H and 13C NMR data, see Supporting Information;
CIMS m/z 405 [M + H]+; HRCIMS m/z 405.1548 [M + H]+ (calcd
for C21H25O8, 405.1549).
7.1 min, 0.2 mg, 0.7 μmol, yield: 8.9%), and 1c (tR = 7.6 min, 1.0 mg,
2.9 μmol, yield: 37.2%) were purified by RP-HPLC using a mixture of
CH3CN−H2O (75:25, 2 mL/min, 327 nm). The structures of 1a, 1b,
and 1c were confirmed by 1H NMR (CDCl3, 600 MHz) spectroscopy,
see Supporting Information. In addition, (S)-(+)-2-methylbutyric acid
was isolated (tR = 8.6 min, 210 nm) using HPLC (CH3CN−H2O, 5:95
→ 95:5, 1 mL/min) with a BDS Hypersil C18 column (4.6 × 150 mm,
S-5 μm, Thermo Scientific, Waltham, MA, USA).
Preparation of the MTPA Esters of 1a. Compound 1a (0.5 mg,
1.4 μmol) was resuspended in pyridine-d5 (800 μL), and (R)-
(−)-MTPA-Cl (10 μL, 0.05 mmol) and DMAP were added under a
stream of nitrogen gas. The reaction mixture was incubated at room
temperature overnight, dried, and dissolved in CHCl3 (50 μL). Silica
gel TLC using n-hexane and EtOAc (2:1) was conducted to monitor
progress of the reaction. Using an INNO C18 column (10 × 250 mm,
S-5 μm, 12 nm), the resulting (S)-MTPA ester 1aa (tR = 13.9 min, 0.4
mg, 0.7 μmol, yield: 52.7%) was isolated (2 mL/min) with CH3CN−
H2O (90:10). In the same manner, (S)-(+)-MTPA-Cl (10 μL, 0.05
mmol) was added to 1a (0.5 mg, 1.5 μmol) to obtain the (R)-MTPA
ester 1ab (tR = 13.1 min, 0.3 mg, 0.5 μmol, yield: 37.1%).
Preparation of the MTPA Esters of 16. Compound 16 (0.4 mg,
1.2 μmol) was dissolved in deuterated pyridine (600 μL), and mixed
with (R)-(−)-MTPA-Cl (10 μL, 0.05 mmol) under a stream of
nitrogen gas. The resulting mixture was kept at room temperature
overnight, evaporated, dissolved in CHCl3 (50 μL), and subjected to
silica capillary CC (7 × 0.5 cm) using a mixture of n-hexane-EtOAc
(5:1). Fifty drops were collected per vial, and the (S)-MTPA ester (0.2
mg, 0.3 μmol, yield: 27.0%) was obtained from the 15th−21st vials.
The progress of the reaction was monitored by NP-TLC using a
mixture of n-hexane and EtOAc (3:1). To obtain the (R)-MTPA ester
(0.1 mg, 0.2 μmol, yield: 11.7%), (S)-(+)-MTPA-Cl (10 μL, 0.05
mmol) was added to 16 (0.5 mg, 0.002 mmol), and subsequent
processes were performed in the same way as above.
X-ray Crystallographic Analysis of 1 and 2. The absolute
configurations of 1 and 2 were determined using data collected on a
SuperNova, Dual, Cu at zero, AtlasS2 diffractometer with Cu Kα
radiation (λ = 1.54184). With Olex2, the structure was solved by direct
methods using the ShelXT structure solution program and was refined
by least-squares minimization using the ShelXL refinement package.
Crystallographic data for 1 and 2 have been deposited at the
Cambridge Crystallographic Data Centre (1: CCDC 1488681, 2:
CCDC 1474283). Copies of the data can be obtained free of charge by
application to the Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (fax: + 44-(0)1223−336033 or e-mail: deposit@ccdc.cam.ac.
uk).
Crystal Data of 1. Colorless crystal, C23H28O7, M = 416.45,
monoclinic, crystal size 0.3 × 0.101 × 0.031 mm3, space group P21
(no. 4), a = 24.3633(4) Å, b = 9.12727(13) Å, c = 31.0898(6) Å, β =
109.128(2)°, V = 6531.8(2) Å3, Z = 12, T = 100(80) K, μ (Cu Kα) =
0.774 mm−1, Dcalc = 1.270 g/cm3, Reflections collected: 39478 (7.274°
≤ 2Θ ≤ 153.672°). Independent reflections: 20639 (Rint = 0.0243,
Rsigma = 0.0335). The goodness of fit on F2 = 1.031. The final R1 values
were 0.0417 (I > 2σ (I)) and 0.0464 (all data). The final wR2 values
were 0.1069 (I > 2σ (I)) and 0.1107 (all data). Flack parameter =
0.08(6).
Crystal Data of 2. Colorless crystal, C21H22O7, M = 386.38,
monoclinic, crystal size 0.2 × 0.059 × 0.024 mm3, space group P21
(no. 4), a = 9.68785(12) Å, b = 6.62844(9) Å, c = 15.2024(2) Å, β =
103.8888(13)°, V = 947.69(2) Å3, Z = 2, T = 100.0(2) K, μ (Cu Kα) =
0.851 mm−1, Dcalc = 1.354 g/cm3, Reflections collected: 19091 (9.404°
≤ 2Θ ≤ 152.988°). Independent reflections: 3883 (Rint = 0.0347,
Rsigma = 0.0220). The goodness of fit on F2 = 1.062. The final R1 values
were 0.0276 (I > 2σ (I)) and 0.0287 (all data). The final wR2 values
were 0.0705 (I > 2σ (I)) and 0.0715 (all data). Flack parameter =
−0.03(6).
(3′S,4′S)-3′-O-Acetyl-4′-O-(3-hydroxy-2-methylbutyroyl)-
khellactone (11). White amorphous powder; [α]20 − 3 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 219 (4.06), 257 (3.56), 323 (4.00)
nm; ECD (c 50 μM, MeOH) λmax (Δε) 224 (−8.32), 244 (+1.98), 255
1
(+1.52), 321 (−1.44) nm; H and 13C NMR data, see Supporting
Information; CIMS m/z 404 [M]+; HRCIMS m/z 404.1466 [M]+
(calcd for C21H24O8, 404.1471).
(3′S,4′S)-3′-O-Acetyl-4′-O-(2-methylbutyroyl)khellactone (12).
White amorphous powder; [α]20 + 5 (c 2, CHCl3); UV (MeOH)
D
λmax (log ε) 220 (3.37), 255 (2.71), 325 (3.23) nm; ECD (c 50 μM,
MeOH) λmax (Δε) 224 (−11.8), 244 (+3.16), 256 (+2.24), 324
1
(−3.03) nm; H and 13C NMR data, see Supporting Information;
HRESIMS m/z 411.1400 [M + Na]+ (calcd for C21H24O7Na,
411.1414).
(3′S,4′S)-3′-O-(2-Methylbutyroyl)khellactone (13). White amor-
phous powder; [α]20 − 3 (c 1, MeOH); UV (MeOH) λmax (log ε)
D
219 (4.18), 256 (3.56), 326 (4.20) nm; ECD (c 50 μM, MeOH) λmax
1
(Δε) 224 (−6.05), 235 (+1.58), 258 (+0.67), 328 (−2.16) nm; H
and 13C NMR data, see Supporting Information; HRESIMS m/z
369.1309 [M + Na]+ (calcd for C19H22O6Na, 369.1309).
(3′S,4′S)-4′-O-(2-Methylbutyroyl)khellactone (14). White amor-
phous powder; [α]20 − 73 (c 1, MeOH); UV (MeOH) λmax (log ε)
D
220 (4.22), 257 (3.65), 323 (4.25) nm; ECD (c 50 μM, MeOH) λmax
1
(Δε) 225 (−4.15), 243 (+0.68), 257 (+0.53), 318 (−2.01) nm; H
and 13C NMR data, see Supporting Information; HRESIMS m/z
369.1312 [M + Na]+ (calcd for C19H22O6Na, 369.1309).
(3′S,4′S)-4′-O-Methyl-3′-O-(2-methylbutyroyl)khellactone (15).
White amorphous powder; [α]20 + 4 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 220 (3.66), 255 (3.11), 323 (3.39) nm; ECD
(c 50 μM, MeOH) λmax (Δε) 223 (−1.16), 248 (+0.15), 259 (+0.13),
329 (−0.33) nm; 1H and 13C NMR data, see Supporting Information;
HRESIMS m/z 383.1452 [M + Na]+ (calcd for C20H24O6Na,
383.1465).
(3′S,4′R)-4′-O-Senecioylkhellactone (16). White amorphous pow-
der; [α]20D + 81 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 219 (4.33),
257 (3.69), 324 (3.99) nm; ECD (c 50 μM, MeOH) λma1x (Δε) 226
(+18.91), 247 (−3.39), 256 (−2.86), 324 (+2.57) nm; H and 13C
NMR data, see Supporting Information; CIMS m/z 345 [M + H]+;
HRCIMS m/z 345.1335 [M + H]+ (calcd for C19H21O6, 345.1338).
(3′S,4′S)-4′-O-Senecioylkhellactone (17). White amorphous pow-
der; [α]20 − 52 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220
D
(4.31), 257 (3.53), 326 (3.99) nm; ECD (c 50 μM, MeOH) λmax (Δε)
226 (−6.38), 247 (+0.44), 255 (+0.33), 324 (−1.75) nm; 1H and 13
C
NMR data, see Supporting Information; ESIMS m/z 367 [M + Na]+.
(3′R,4′R)-4′-O-Senecioylkhellactone (18). White amorphous pow-
der; [α]20D + 39 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (4.14),
257 (3.38), 326 (3.83) nm; ECD (c 50 μM, MeOH) λma1x (Δε) 226
(+11.74), 248 (−2.48), 257 (−2.29), 326 (+2.31) nm; H and 13C
NMR data, see Supporting Information; HRESIMS m/z 367.1144 [M
+ Na]+ (calcd for C19H20O6Na, 367.1152).
(3′S,4′S)-3′-O-Senecioylkhellactone (19). White amorphous pow-
der; [α]20D + 24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.32),
256 (3.45), 326 (4.00) nm; ECD (c 50 μM, MeOH) λm1ax (Δε) 226
(−4.31), 246 (+1.25), 257 (+1.63), 323 (−1.25) nm; H and 13C
NMR data, see Supporting Information; ESIMS m/z 367 [M + Na]+.
(3′R,4′R)-3′-O-Senecioylkhellactone (20). White amorphous pow-
der; [α]20D − 2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (4.13),
255 (3.28), 326 (3.81) nm; ECD (c 50 μM, MeOH) λm1ax (Δε) 224
(+0.78), 233 (−1.06), 257 (−0.36), 333 (+0.53) nm; H and 13C
NMR data, see Supporting Information; ESIMS m/z 367 [M + Na]+.
Partial and Total Alkaline Hydrolysis of 1. A solution of 1 (3.2
mg, 7.7 μmol) in 1,4-dioxane (0.5 mL) containing 0.5 M KOH (0.5
mL) was stirred at room temperature for 2 min. The progress of the
reaction was monitored by NP-TLC using a mixture of n-hexane and
EtOAc (2:1). The reaction mixture was acidified with 5% H2SO4
(∼120 μL), extracted with CHCl3 (1 mL), and evaporated. Reaction
products 1a (tR = 11.0 min, 0.2 mg, 0.7 μmol, yield: 9.4%), 1b (tR =
ECD Calculations. Conformational searches were performed using
the MMFF94s force field in Conflex 7 with a search limit of 10.0 kcal/
mol. Conformers used for geometry optimization were selected by a
lower energy level up to 90% population. Turbomole was used for the
ground-state geometry optimization with the def-SV(P) basis set for all
F
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atoms, B3LYP functional, and density functional theory (DFT). The
calculated ECD data were obtained using TDDFT at the B3LYP/def-
SV(P) level with the COSMO model in MeOH. The ECD spectra
were simulated using Gaussian functions overlapping each transition,
where σ is the width of the band at 1/e height and ΔEi and Ri are the
excitation energies and rotatory strengths for transition i, respectively.
The value of σ was 0.10 eV and R was Rlen in this work.
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A
2
2
1
1
2πσ
ΔE R e[−(E−ΔE ) /(2σ) ]
(11) Jang, K.-C.; Kim, S.-C.; Song, E.-Y.; Um, Y.-C.; Kim, S. C.; Lee,
Y.-J. Acta Hortic. 2008, 765, 49−53.
i
Δϵ(E) =
∑
i
i
2.297 × 10−39
i
(12) Hisamoto, M.; Kikuzaki, H.; Ohigashi, H.; Nakatani, N. J. Agric.
Food Chem. 2003, 51, 5255−5261.
ASSOCIATED CONTENT
* Supporting Information
(13) Hisamoto, M.; Kikuzaki, H.; Nakatani, N. J. Agric. Food Chem.
2004, 52, 445−450.
■
S
(14) Chen, Y.-C.; Chen, P.-Y.; Wu, C.-C.; Tsai, I.-L.; Chen, I.-S.
Yaowu Shipin Fenxi 2008, 16, 15−25.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00947.
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X-ray crystallographic data for 1 (CIF)
X-ray crystallographic data for 2 (CIF)
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Data including 1H (1−20, and 1a−1c), 13C (1−20), and
2D (1−16) NMR, experimental ECD (1−27, and 29−
40), conformational analysis (1−2, and 16−18),
calculated ECD (1−2, and 16−18), UV (1−2, and
16−18), MS (1−20), 1H NMR of MTPA esters (1a, and
16−20), experimental ECD of MTPA esters (17−20),
HPLC chromatograms of MTPA esters (17−20),
enantioseparation (17−20), acyl migration (17−20),
isolation of compounds 21−39, preparation of MTPA
esters of 17−20, comparison of 3′α-hydroxy-4′β-
senecioyloxy-3′,4′-dihydroseselin and 16, structures of
compounds 21−40, and HPLC chromatograms of E1
and E2 on CHIRALPAK IA-IF (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: jwkim@snu.ac.kr (J. Kim). Tel: +82-2-880-7853. Fax:
+82-2-888-0649.
ORCID
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Molecules 2012, 17, 4236−4251.
Jinwoong Kim: 0000-0001-9579-738X
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was supported by the Brain Korea 21 Plus program
in 2017. We would like to thank Prof. Sang Beom Han (Chung-
Ang University, Seoul, Republic of Korea) for analytical
experiments on the enantiomeric mixtures using chiral columns.
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DOI: 10.1021/acs.jnatprod.6b00947
J. Nat. Prod. XXXX, XXX, XXX−XXX