Oploxynes A and B, polyacetylenes from the stems of Oplopanax elatus

Article abstract of DOI:10.1021/np900628j

Two new polyacetylenes, oploxynes A (1) and B (2), and the known oplopandiol (3) and falcarindiol (4) were isolated from the stem of Oplopanax elatus. The structures of compounds 1 and 2 were determined to be 9,10-epoxyheptadeca-4,6-diyne-3,8-diol and 10-methoxyheptadeca-4,6-diyne-3,8,9- triol, respectively, on the basis of their UV, MS, and NMR data. The absolute configurations of these compounds were determined using the modified Mosher's method and acetonide formation. Oploxyne A (1), oplopandiol (3), and falcarindiol (4) inhibited the formation of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in lipopolysaccharide (LPS)-induced murine macrophage RAW 267.7 cells.

Full text of DOI:10.1021/np900628j

J. Nat. Prod. 2010, 73, 801–805
801
Oploxynes A and B, Polyacetylenes from the Stems of Oplopanax elatus
Min Cheol Yang,†,§ Hak Cheol Kwon,†,§ Young-Joo Kim,† Kang Ro Lee,‡ and Hyun Ok Yang*,†
Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 210-340, Republic of Korea, and
Natural Products Laboratory, College of Pharmacy, Sungkyunkwan UniVersity, Suwon 440-746, Republic of Korea
ReceiVed October 6, 2009
Two new polyacetylenes, oploxynes A (1) and B (2), and the known oplopandiol (3) and falcarindiol (4) were isolated
from the stem of Oplopanax elatus. The structures of compounds 1 and 2 were determined to be 9,10-epoxyheptadeca-
4,6-diyne-3,8-diol and 10-methoxyheptadeca-4,6-diyne-3,8,9-triol, respectively, on the basis of their UV, MS, and NMR
data. The absolute configurations of these compounds were determined using the modified Mosher’s method and acetonide
formation. Oploxyne A (1), oplopandiol (3), and falcarindiol (4) inhibited the formation of nitric oxide (NO) and
prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 267.7 cells.
Plants of the genus Oplopanax (Araliaceae) contain various
bioactive secondary metabolites, such as lignans,1 saponins,2,3
sesquiterpenes,4,5 polyacetylenes,6,7 and anthraquinones.8 In par-
ticular, O. horridus, commonly known as Devil’s Club, is used for
the treatment of diabetes, rheumatism, tuberculosis, colds, head-
aches, and lung hemorrhages. Polyacetylenes are major constituents
of Devil’s Club, and these compounds exhibit significant antimy-
cobacterial and antifungal activity.7
Oplopanax elatus NAKAI has been used in Korean and Chinese
traditional medicine for anti-inflammatory and analgesic purposes.9,10
Saponins,2,3 an anthraquinone,8 an antipsoriasis lignin,1 and the
antibacterial essential oil11,12 have been identified from this plant.
However, neither the active principles in the traditional medicine
nor the polyacetylenes in this plant have been investigated. Because
of the ethnopharmacological prescription of this plant for inflam-
mation, we examined the inhibitors produced in the stem on the
formation of nitric oxide (NO) and prostaglandin E2 (PGE2) in
lipopolysaccharide (LPS)-induced murine macrophage RAW 267.7
cells. During this survey, we isolated two new diynes, oploxynes
A (1) and B (2), together with two known diynes, oplopandiol (3)7
and falcarindiol (4),13 from the CH2Cl2 extract of the stem of O.
elatus.
The polyacetylenes, including conjugated diynes, possess a
variety of biological activities, particularly antitumor, anti-inflam-
matory, antimicrobial, and antiviral activities. For example, pen-
tadeca-(8Z,13Z)-dien-11-yn-2-one (from the roots of Echinacea
pallida) exhibited anticancer activity against MIA PaCa-2 and
COLO320 cells.14 (3Z,5E,11E)-Tridecatriene-7,9-diynyl-1-O-(E)-
ferulate (from the rhizomes of Atractylodes lancea) showed strong
inhibition of 5-LOX and COX-1 activities.15 Octadeca-1,9-diene-
4,6-diyne-3,8,18-triol and 18-acetoxyoctadeca-1,9-diene-4,6-diyne-
3,8-diol (from Angelica gigas) downregulated expression of the
inducible form of nitric oxide synthase.16 13(E),17-Octadecadiene-
9,11-diynoic acid (from Mitrephora celebica) demonstrated sig-
nificant activity against methicillin-resistant Staphylococcus aureus
and Mycobacterium smegmatis.17 R-Terthienyl was extremely toxic
against murine cytomegalovirus (CMV) and Sindbis virus.18
However, the anti-inflammatory effects of acyclic, conjugated
diynes lacking double bonds have not been evaluated.
were extracted using CH2Cl2 at room temperature. The extract (25.0
g) was subjected to a diversity of chromatographic purification steps
to afford oploxynes A (1) and B (2), together with two known
polyacetylenes, oplopandiol (3) and falcarindiol (4).
Compound 1 was obtained as an optically active colorless oil
([R]D +123.4). The molecular formula was assigned as C17H26O3
by HRFABMS (obsd [M + Na]+ at m/z 301.1775; calcd 301.1780).
The IR spectrum of 1 displayed absorption bands at 3384, 2253,
and 2146 cm-1, indicating the presence of hydroxy and triple-bond
functionalities, while UV absorption bands at 231, 243, and 257
nm suggested a diyne moiety.19 In addition, the 13C NMR signals
at δC 81.0, 68.7, 70.3, and 77.4 were attributable to a conjugated
diyne.20,21 The characteristic features of the 1H NMR spectrum of
1 included the presence of four oxymethine protons at δH 4.40 (1H,
Results and Discussion
The stems of O. elatus were collected in Gangwon Province
(Korea) in September 2006. The air-dried stems (1.0 kg) of O. elatus
* To whom correspondence should be addressed. Tel: 82-33-650-3501.
Fax: 82-33-650-3529. E-mail: hoyang@kist.re.kr.
† Korea Institute of Science and Technology.
‡ Sungkyunkwan University.
§ These authors contributed equally to this work.
10.1021/np900628j  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/13/2010

802 Journal of Natural Products, 2010, Vol. 73, No. 5
Yang et al.
br dt, J ) 6.0, 6.0 Hz), 4.36 (1H, br dd, J ) 7.5, 3.5 Hz), 3.16
(1H, dd, J ) 7.5, 4.0 Hz), and 3.07 (1H, br ddd, J ) 7.0, 5.5, 4.0
Hz) and two exchangeable protons at δH 2.19 (1H, br d, J ) 6.0
1
Hz) and 2.50 (1H, br d, J ) 3.5 Hz). The H-1H COSY NMR
spectrum showed that these two exchangeable protons correlated
with protons at δH 4.40 and 4.36. Also, correlations between δH
1
4.36, 3.16, and 3.07 were observed in the H-1H COSY NMR
spectrum of 1. The HSQC spectrum showed that the four oxyme-
thine proton signals correlated with 13C NMR signals at δC 64.0,
60.7, 58.0, and 58.1, respectively. In addition, the 1H NMR spectrum
of 1 displayed signals attributable to two methyl protons [δH 1.03
(3H, t, J ) 7.5 Hz) and 0.89 (3H, t, J ) 7.0 Hz)] and seven aliphatic
methylenes [δH 1.76, 1.64, 1.51, 1.35 (all are 2H, m), and 1.29
(6H, m)]. The 13C NMR and HSQC spectra of 1 allowed assignment
1
of all protons to their respective carbons. The H and 13C NMR
spectra of 1 were similar to those of a known heptadeca-1-ene-
9,10-epoxy-4,6-diyne-3,8-diol.20,21 The key difference between 1
and heptadeca-1-ene-9,10-epoxy-4,6-diyne-3,8-diol was the pres-
ence of a methyl group [δH 1.03 and δC 9.3 (C-1)] in 1, instead of
the terminal olefinic methylene group in heptadeca-1-ene-9,10-
epoxy-4,6-diyne-3,8-diol.
The relative configuration of the epoxide ring in 1 was assigned
by the analysis of coupling constants in its 1H NMR spectrum and
1H-1H NOE correlations. H-8 and H-9 showed consistent NOESY
correlations with H2-11 and H-10, respectively, while there was
no NOESY correlation between H-8 and H-10 in the 2D NOESY
spectrum of 1. These NOE data and the coupling constant (4.0 Hz)
between H-9 and H-10 supported the cis configuration for the
epoxide.22
The absolute configurations of C-3, C-8, C-9, and C-10 of
compound 1 were assigned on the basis of chiral derivatization.
Treatment of 1 with (R)-(-)-R-methoxy-R-(trifluoromethyl)phe-
nylacetyl chloride [(R)-MTPA-Cl] and DMAP in pyridine yielded
a mixture of 3,8-bis-(S)-Mosher ester (5a) and 3,9-bis-(S)-Mosher
ester (5b) of 10-chloroheptadeca-4,6-diyne-3,8,9-triol. Similar treat-
ment of 1 with (S)-(+)-MTPA-Cl afforded a mixture of 3,8-bis-
(R)-Mosher ester (6a) and 3,9-bis-(R)-Mosher ester (6b) of 10-
chloroheptadeca-4,6-diyne-3,8,9-triol. Analysis of the 1H NMR
23
chemical shift differences (∆δS-R
)
(Figure 1) of the mixtures
allowed the assignment of the absolute configuration of C-3, C-8,
and C-9 of 1 as S, R, and R, respectively. The absolute configuration
of C-10 was thus assigned as S.
Compound 2 was isolated as an optically active, colorless oil
([R]D +11.7) and gave the molecular formula C18H31O4, based on
HRCIMS data (obsd [M + Na]+ at m/z 311.2222). The IR and UV
spectra of 2 were similar to those of 1, suggesting the presence of
hydroxy and conjugated diyne functionalities. As with compound
1, comprehensive analysis of the 2D NMR (1H-1H COSY, HSQC,
HMBC, and NOESY) data of 2 allowed assignment of proton and
carbon signals. The 1H NMR spectrum of 2, including HMBC and
1H-1H COSY data, showed signals attributable to three exchange-
able hydroxy groups (δH 1.85, 2.68, and 3.41) attached to C-3 (δC
64.3), C-9 (δC 73.1), and C-8 (δC 65.7), and a methoxy group (δH
3.44) attached to C-10 (δC 81.7). Furthermore, the C-9 and C-10
signals were observed at δC 73.1 and 81.7, respectively, while the
chemical shifts of C-9 and C-10 in the 13C NMR spectrum of 1
were δC 58.0 and 58.1, respectively. These data indicated that
compound 2 contains hydroxy and methoxy functionalities at C-9
and C-10, respectively, in place of the epoxide in 1. The chemical
shifts of C-8, C-9, and C-10 were similar to those of the reported
10-methoxyheptadec-16-ene-4,6-diyne-8,9-diol, ciryneol B.22 The
C-8, C-9, and C-10 resonances in ciryneol B were observed at δC
65.3, 73.5, and 81.3.22 However, the chemical shifts of C-8-C-10
carbons in heptadec-16-ene-4,6-diyne-8,9,10-triol were at δC 65.5,
75.0, and 71.3, respectively.
Figure 1. ∆δS-R values for (A) the bis-Mosher esters 5a/6a, (B)
the bis-Mosher esters 5b/6b, (C) the tris-Mosher esters 8/9, (D)
the bis-Mosher esters 10/11, (E) the bis-Mosher esters 12a/13a,
and (F) the bis-Mosher esters 12b/13b, in pyridine-d5.
chemical transformation. Treatment of 2 with 2,2-dimethoxypropane
and pyridinium p-toluenesulfonate in acetone afforded the acetonide
7,24 which resulted from the acetal formation involving the C-8
and C-9 hydroxy groups. The NOE correlation between H-8 (δH
4.71) and H-9 (δH 4.05) in the NOESY spectra of 7 revealed a
2,3-syn-isopropylidene moiety. The acetonide methyl groups showed
chemical shifts at δH 1.57 and 1.37, which further supported the
assignment of a syn relationship between H-8 and H-9.25 The
relative configuration of C-10 in 2 was deduced from the 1H
coupling constants and the NOESY data of compound 7 (Figure
2). The coupling constant between H-9 and H-10 of 7 was 8.5 Hz
The relative configurations of C-3, C-8, C-9, and C-10 of
oploxyne B (2) were again assigned by chiral derivatization and

Polyacetylenes from Oplopanax elatus
Journal of Natural Products, 2010, Vol. 73, No. 5 803
allowed us to assign S configuration at both C-3 and C-8, which
confirmed the previously described configurations of oplopandiol.7
Although the chemical and physical properties of falcarindiol
(4) have been described,7,28,29 NMR and optical rotation data related
to the absolute configuration at C-3 and C-8 are inconclusive.
Review of reported physical data of (3S,8S)- and (3R,8S)-falcar-
indiols revealed that it is hard to distinguish the isomers by
comparison of specific rotation and NMR data (Tables S1, S2, and
S3). This uncertainty of the stereochemical assignments for
falcarindiol (4) prompted us to examine CD spectra and prepare
its Mosher esters. The CD spectra of both 3 and 4 exhibited positive
Cotton effects at 202 nm (Figures S21 and S28). These data
suggested that the absolute configuration of chiral carbons flanked
by an acetylene and an olefin of 4 is identical with those of 3.
1
Analysis of H NMR chemical shift differences between the (S)-
(12a) and (R)-(13a) Mosher esters revealed that the absolute
configurations of C-3 and C-8 are both S. However, small proton
signals, which were readily distinguishable from the proton signals
of 12a or 13a by an approximately 30% smaller integral, were also
observed in the 1H NMR spectra of the Mosher esters (Figures S56
and S57). The ∆δS-R values calculated from these resonances were
positive for H2-1 and H-2 and negative for H-8 (Figure 1). These
data indicate that the Mosher esters of 4 were composed of a
mixture of (3S,8S)- and (3R,8S)-isomers in a ca. 3.3:1 ratio.
However, it was not possible to determine whether the (3R,8S)-
isomer was a naturally occurring metabolite or an artifact of the
isolation or the reaction procedure.
Because O. elatus is commonly used for the treatment of
inflammation in Korean traditional medicine, the potential anti-
inflammatory properties of compounds 1-4 were investigated by
using RAW264.7 macrophage cells, which can produce NO and
PGE2 upon stimulation with LPS (Gram-negative bacterial li-
popolysaccharide). Compounds 1, 3, and 4 showed inhibition of
NO and PGE2 production with an IC50 range of 1.28 to 3.08 µM
(Table S4 and Table 2). Compound 4 showed significant inhibitory
effects on NO and PGE2 production with IC50 values of 1.28 and
1.54 µM, respectively, while 4 was cytotoxic to the tested cells
with an IC50 value of 3.77 µM. However compounds 1, 2, and 3
showed no cytotoxicity against the tested cells. Furthermore, both
the production of NO and the quantity of PGE2 secreted decreased
to an approximately basal level in RAW264.7 cells exposed to 8
µM of 1 or 3 for 24 h (Table 2 and Table S4).
Figure 2. Key NOE correlations in the NOESY spectra of the
acetonide 7 in CDCl3.
and suggested an anti configuration.26 The NOESY correlations
were in support of this assignment. In the 2D NOESY spectrum of
7, H-9 and H-10 showed consistent correlations with H2-11 and
H-8, respectively. However, there was no apparent correlation
between H-9 and H-10 in its 1D NOE spectrum (Figure 2). The
coupling constant and NOE data indicated that H-9 and H-10 in 7
were in the anti configuration. The correlation between H-8 and
H2-11 was also observed in the 2D NOESY spectrum of 7. These
NOESY data, in combination with the large 3JH-9,H-10 value, allowed
us to assign the relative configurations at C-9 and C-10 in 7 as S*
and R*.
The CD spectra of both 1 and 2 showed negative Cotton effects
at 202 nm (Figures S4 and S14). These data suggested that the
absolute configurations of carbinol carbons adjacent to triple bonds
of 2 were identical with those of 1. The absolute configurations of
C-3, C-8, and C-9 of 2 were determined by application of the
modified Mosher method.27 Separate treatment of 2 in pyridine with
(R)-MTPA-Cl and (S)-MTPA-Cl yielded the tris-(S)- (8) and tris-
1
(R)-Mosher esters (9), respectively. Analysis of the H chemical
shift differences (∆δS-R) between 8 and 9 (Figure 1) revealed
negative ∆δS-R values for H-1 and H-2 of the C-3 ester, negative
values for H-10-H-12, and a positive value for H-8 of the C-9
ester. Furthermore, a negative ∆δS-R value for H-9 of the C-8 ester
was observed, while the H-3 signals showed a positive difference.
These data allowed the absolute configurations at C-3, C-8, and
C-9 of 2 to be assigned as S, R, and R, respectively.23,27 The
absolute configuration of C-10 was determined to be R on the basis
of the relative configuration of C-9 and C-8 in compound 7.
The NMR data of compound 3 were in accordance with published
data.7 Those authors assigned H-9 and C-9 at δH 5.6 (1H, ddt, J )
10.8, 7.3, 1.5 Hz) and δC 134.6, respectively, while the resonances
at δH 5.5 (1H, ddt, J ) 10.8, 8.3, 1.5 Hz) and δC 127.8 were
assigned to H-10 and C-10, respectively. However, analysis of the
2D NMR data of 3 allowed us to reassign H-9 and H-10 at δH 5.47
(1H, ddt, J ) 11.0, 8.5, 1.0 Hz, H-9) and 5.55 (1H, dtd, J ) 11.0,
8.0, 1.0 Hz, H-10). The chemical shifts of C-9 and C-10 should
accordingly be changed to δC 127.8 and 134.0, respectively.
Experimental Section
General Experimental Procedures. Optical rotations were measured
using a 35 polarimeter (PerkinElmer Co., USA). CD spectra were
measured on a JASCO J-715 polarimeter (JASCO Co., Japan). UV
spectra were recorded on a Lambda 35 UV/vis spectrophotometer
(PerkinElmer Co., USA), and FT-IR spectra were recorded on a Bruker
IFS-66/S instrument (Bruker Co., Germany). The NMR spectra were
obtained on a Varian Unity Plus 500 MHz NMR system (Varian Co.,
USA). Low-resolution ESIMS data were obtained either on an Agilent
1100 HPLC/MS system (Agilent Co., USA) at Kangnung-Wonju
National University or on a Varian 320-MS (Varian Co., USA) mass
spectrometer. The HRFABMS and HRCIMS were measured on JMS-
AX505WA (JEOL Co., Japan) and JMS-600W (JEOL Co., Japan) mass
spectrometers at the National Center for Inter-University Facilities of
Seoul National University, Seoul, Korea. The analytical HPLC data
were obtained on a Waters analytical HPLC system that included a
1525 binary HPLC pump, a Waters 717 plus autosampler, and a Waters
2996 PDA (Waters Co., USA). Preparative HPLC was performed using
a Gilson 321 HPLC system (Gilson Co., France), with a UV detector
(254 nm) and either a Luna C18(2) (10 × 250 mm, 10 µm and 21 ×
250 mm, 15 µm, Phenomenex Co., USA), a Luna C8(2) (10 × 250
mm, 10 µm, Phenomenex Co., USA), a Luna silica (10 × 250 mm, 10
µm, Phenomenex Co., USA), or a Waters Delta Pak C18 (30 × 300
mm, 15 µm, Waters Co., Tokyo, Japan) column. Open column
chromatography was performed using silica gel (particle size 70-230
mesh, Silicycle Co., Canada). TLC was performed on silica gel 60 F254
and RP-18 F254s (Merck Co., Germany).
The absolute configurations of C-3 and C-4 in oplopandiol (3)
were defined by the analysis of ∆δS-R values for the two fluorine
signals in the 19F NMR spectra of the (S)- and (R)-MTPA esters.
Because the assignment of absolute configuration using only 19F
NMR data may not always be feasible, we prepared MTPA esters
1
of 3 in order to obtain H NMR data for the (S)-MTPA (10) and
(R)-MTPA (11) esters. The modified Mosher ester analysis resulted
in negative ∆δS-R values for H3-1, H2-2, H-9, H-10, and H2-11,
while a positive value was obtained for H-3 (Figure 1). These data

804 Journal of Natural Products, 2010, Vol. 73, No. 5
Yang et al.
Table 1. NMR Data of Compounds 1 and 2
1
2
b
b
position
δH mult (J (Hz))a
1.03 t (7.5)
1.76 m
4.40 br dt (6.0, 6.0)
δC
δH mult (J (Hz))a
δC
1
2
3
4
9.3 CH3
30.6 CH2
64.0 CH
81.0 C
1.04 t (7.5)
1.77 m
4.40 dt (6.0, 6.0)
9.5 CH3
30.8 CH2
64.3 CH
80.7 C
5
68.7 C
69.0 C
6
70.3 C
70.7 C
7
77.4 C
77.8 C
8
9
4.36 br dd (7.5, 3.5)
3.16 dd (7.5, 4.0)
3.07 br ddd (7.0, 5.5, 4.0)
1.64 m
1.51 m
1.35 m
1.29 m
1.29 m
1.29 m
60.7 CH
58.0 CH
58.1 CH
27.5 CH2
26.5 CH2
29.4 CH2
29.2 CH2
31.8 CH2
22.6 CH2
14.1 CH3
4.53 dd (9.0, 4.5)
3.66 ddd (8.5, 4.5, 3.5)
3.61 ddd (7.5, 5.0, 3.5)
1.64 m
65.7 CH
73.1 CH
81.7 CH
29.4c CH2
25.2 CH2
30.0 CH2
29.3c CH2
32.0 CH2
22.9 CH2
14.3 CH3
10
11
12
13
14
15
16
17
3-OH
8-OH
9-OH
OCH3
1.31 m
1.31 m
1.30 m
1.31 m
0.90 t (7.0)
1.85 d (6.0)
3.41 d (9.0)
2.68 d (8.5)
3.44 s
0.89 t (7.0)
2.19 br d (6.0)
2.50 br d (3.5)
57.5 CH3
a 500 MHz, CDCl3 (7.27 ppm). b 125 MHz, CDCl3 (77.0 ppm). c Exchangeable.
Preparation of (S)- and (R)-MTPA Esters (5a/b, 6a/b, 8, 9, 10,
11, 12a/b, and 13a/b).23 Oploxyne A (1) (1 mg) was dissolved in 1
mL of pyridine, and DMAP (1 mg) and (R)-MTPA-Cl (10 µL) were
added. The mixture was stirred for 3 h at room temperature, and then
two drops of H2O were added. The solution was concentrated under
reduced pressure, and the residue was subjected to silica gel chroma-
tography (normal phase Sep-Pak (1 g)) using n-hexane-EtOAc (10:1)
to afford a mixture (0.5 mg) of two chlorinated bis-(S)-Mosher esters,
5a and 5b. In a similar fashion, a solution of 1 in pyridine was treated
with DMAP (1 mg) and (S)-MTPA-Cl (10 µL) to afford a mixture
(0.5 mg) of two chlorinated bis-(R)-Mosher esters, 6a and 6b.
In a similar manner, separate treatment of 2 in pyridine with (R)-
MTPA-Cl and (S)-MTPA-Cl yielded tris-(S)- (8) and tris-(R)- (9)
Mosher esters, respectively. Each of these compounds was purified by
reversed-phase HPLC using a Lunar C8 (2) column (250 × 10 mm, 10
µm) with a CH3CN-H2O gradient solvent system (10 to 100% for 30
min, 4 mL/min).
Table 2. IC50 Values of NO, PGE2, and Cytotoxicity
NO inhibition
PGE2 inhibition
cytotoxicity
compound
(IC50, µM)
(IC50, µM)
(IC50, µM)
1
2
3
4
1.98 ( 0.28
>40
2.72 ( 0.10
1.28 ( 0.14
3.08 ( 0.37
>40
2.99 ( 0.12
1.54 ( 0.01
>40
>40
>40
3.77
Plant Material. The stems of O. elatus were collected in Gangwon
Province (Korea) in September 2006. The botanical identification was
made by Prof. Kyu Song Lee at Kangnung-Wonju National University.
A voucher specimen (KIST NP-2006-10-001) was deposited at the
herbarium, KIST Gangneung Institute.
Extraction and Isolation. The air-dried stems (1.0 kg) of O. elatus
were extracted with CH2Cl2 (3 × 10 L) at room temperature. The
CH2Cl2 extract (25.0 g) was subjected to silica gel (300 g) column
chromatography, eluting with a step gradient of n-hexane-CH2Cl2
(10:1, 5:1, and 2:1) and CH2Cl2-MeOH (20:1, 10:1, 5:1, 2:1, and 1:1)
to afford eight fractions, R1-R8 (each 1 L). The R3 fraction (2.95 g),
which eluted with n-hexane-CH2Cl2 (2:1), was then fractionated by
preparative HPLC using a Delta Pak C18 column (300 × 30 mm, 15
µm) and a CH3CN-H2O gradient solvent system (60 to 100% for 60
min, 15 mL/min) to obtain nine subfractions, R31-R39. Subfraction
R36 (453 mg) was further purified by repeated HPLC using a silica
gel column (Luna, silica, 250 × 10 mm, 10 µm) with n-hexane-EtOAc
(3:1) isocratic solvent (4 mL/min) to afford compound 3 (300 mg).
Subfraction R35 (498 mg) was purified by repeated reversed-phase (RP)
HPLC using a Luna C18 (2) column (250 × 10 mm, 10 µm) and 75%
MeOH (4 mL/min) to afford compound 4 (100 mg), as well as two
subfractions, R351 and R352. Compound 1 (23 mg) was obtained from
subfraction R351 (25 mg) by C18 RP HPLC in the same manner as
was used to purify compound 4. Fraction R33 (9.5 mg) was purified
by repeated HPLC with a Luna C18 (2) column (250 × 10 mm, 10
µm) with 75% MeOH, followed by a Luna silica column (250 × 10
mm, 10 µm) with n-hexane-EtOAc (2:1) to afford compound 2 (1.5
mg).
Compound 3 (6.0 mg) was divided into two portions, and each was
dissolved in 700 µL of 99.5% pyridine-d5 in a 5 mm NMR tube. DMAP
(1 mg) was added to both NMR tubes. (R)-MTPA-Cl (10 µL) and (S)-
MTPA-Cl (10 µL) were then added into each NMR tube, respectively.
After 1 h, the 1H NMR spectra for (S)-Mosher ester 10 and (R)-Mosher
ester 11 were recorded (see Supporting Information for NMR data).
Compound 4 (3 mg) was divided into two portions, and each was
treated with pyridine-d5, DMAP, (R)-MTPA-Cl, and (S)-MTPA-Cl in
1
separate 5 mm NMR tubes as above. The H NMR spectra for the
(S)-Mosher ester 12a/b and (R)-Mosher ester 13a/b were recorded (see
Supporting Information for NMR data).
Bis-(S)-MTPA esters (5a and 5b). 5a: 1H NMR (CDCl3, 500 MHz,)
δ 0.89 (3H, t, J ) 7.0 Hz, H3-17), 0.94 (3H, t, J ) 7.5 Hz, H3-1), 1.26
(8H, m, H2-13-16), 1.36 (2H, m, H2-12), 1.65 (1H, m, H2-11a), 1.79
(1H, m, H2-11b), 1.86 (2H, m, H2-2), 2.23 (1H, d, J ) 9.0 Hz, 9-OH),
3.57-3.63 (6H, s, OCH3), 3.60 (1H, m,a H-10), 3.87 (1H, br dd, J )
9.0, 9.0 Hz, H-9), 5.56 (1H, br t, J ) 6.0 Hz, H-3), 5.62 (1H, d, J )
9.0 Hz, H-8), 7.42 (5H, m, aromatic H), 7.53 (3H, m, aromatic H),
a
7.62 (2H, m, aromatic H). The multiplicity could not be interpreted
because of peak overlap. 5b: 1H NMR (CDCl3, 500 MHz,) δ 0.89 (3H,
t, J ) 7.0 Hz, H3-17), 0.94 (3H, t, J ) 7.5 Hz, H3-1), 1.26 (8H, m,
H2-13-16), 1.36 (2H, m, H2-12), 1.51 (1H, m, H2-11a), 1.59 (1H, m,
H2-11b), 1.86 (2H, m, H2-2), 2.49 (1H, d, J ) 6.5 Hz, 8-OH), 3.57-3.63
(6H, s, OCH3), 4.85 (1H, dd, J ) 7.0, 6.5 Hz, H-8), 4.28 (1H, br ddd,
J ) 8.5, 5.0, 3.0 Hz, H-10), 5.35 (1H, dd, J ) 7.0, 3.0 Hz, H-9), 5.56
(1H, br t, J ) 6.0 Hz, H2-3), 7.42 (5H, m, aromatic H), 7.53 (3H, m,
aromatic H), 7.62 (2H, m, aromatic H); ESIMS m/z 769 [M + Na]+,
771 [M + 2 + Na]+.
Oploxyne A (1): colorless oil; [R]25 +123.4 (c 0.4, MeOH); UV
D
(MeOH) λmax (log ε) 257 (2.34), 243 (2.55), 231 (2.56) nm; CD (MeOH)
λmax (∆ε) 202 (-7.28) nm; IR (neat) νmax 3384 (OH), 2253 (Ct C),
1
2146 (Ct C) cm-1; H and 13C NMR data, see Table 1; HRFABMS
m/z 301.1775 [M + Na]+ (calcd for C17H26O3Na, 301.1780).
Oploxyne B (2): colorless oil; [R]25 +11.7 (c 0.06, MeOH); UV
D
(MeOH) λmax (log ε) 257 (2.42), 243 (2.60), 231 (2.62) nm; CD (MeOH)
λmax (∆ε) 202 (-7.64) nm; IR (neat) νmax 3383 (OH), 2257 (Ct C),
2149 (Ct C) cm-1; 1H and 13C NMR data, see Table 1; HRCIMS m/z
311.2222 [M + Na]+ (calcd for C18H31O4Na, 311.2222).
Bis-(R)-MTPA esters (6a and 6b). 6a: 1H NMR (CDCl3, 500 MHz,)
δ 0.89 (3H, t, J ) 7.0 Hz, H3-17), 1.05 (3H, t, J ) 7.5 Hz, H3-1), 1.27
(8H, m, H2-13-16), 1.44 (2H, m, H2-12), 1.79 (1H, m, H2-11a), 1.89

Polyacetylenes from Oplopanax elatus
Journal of Natural Products, 2010, Vol. 73, No. 5 805
(1H, m, H2-11b), 1.91 (2H, m, H2-2), 2.30 (1H, d, J ) 8.5 Hz, 9-OH),
3.55-3.57 (6H, s, OCH3), 3.95 (1H, ddd, J ) 8.5, 8.5, 2.0 Hz, H-9),
4.00 (1H, br ddd, J ) 8.5, 5.0, 2.0 Hz, H-10), 5.53 (1H, t, J ) 6.5 Hz,
H-3), 5.56 (1H, d, J ) 8.0 Hz, H-8), 7.43 (5H, m, aromatic H), 7.52
(3H, m, aromatic H), 7.62 (2H, m, aromatic H). 6b: 1H NMR (CDCl3,
500 MHz,) δ 0.89 (3H, t, J ) 7.0 Hz, H3-17), 1.05 (3H, t, J ) 7.5 Hz,
H3-1), 1.27 (8H, m, H2-13-16), 1.44 (2H, m, H2-12), 1.68 (1H, m,
H2-11a), 1.71 (1H, m, H2-11b), 1.91 (2H, m, H-2), 2.33 (1H, d, J )
6.5 Hz, 8-OH), 3.55-3.57 (6H, s, OCH3), 4.33 (1H, br ddd, J ) 8.5,
5.5, 3.0 Hz, H-10), 4.78 (1H, dd, J ) 7.0, 6.5 Hz, H-8), 5.38 (1H, dd,
J ) 7.0, 3.0 Hz, H-9), 5.53 (1H, t, J ) 6.5 Hz, H-3), 7.43 (5H, m,
aromatic H), 7.52 (3H, m, aromatic H), 7.62 (2H, m, aromatic H);
ESIMS m/z 769 [M + Na]+, 771 [M + 2 + Na]+.
1H NMR spectra for 10-13, and the previously reported optical rotation
values and NMR data for falcarindiol. This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
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Tris-(S)-MTPA ester (8): 1H NMR (CDCl3, 500 MHz) δ 0.89 (3H,
t, J ) 7.0 Hz, H3-17), 0.94 (3H, t, J ) 7.5 Hz, H3-1), 1.26 (8H, m,
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Tris-(R)-MTPA ester (9): 1H NMR (CDCl3, 500 MHz) δ 0.89 (3H,
t, J ) 7.0 Hz, H3-17), 1.04 (3H, t, J ) 7.5 Hz, H3-1), 1.26 (8H, m,
H2-13-16), 1.36 (1H, m, H2-12), 1.55 (1H, m, H2-11), 1.90 (2H, m,
H2-2), 3.30, 3.40, 3.54, and 3.55 (each 3H, s, OCH3), 3.40 (1H, m, H-10),
5.45 (1H, dd, J ) 6.0, 4.5 Hz, H-9), 5.52 (1H, t, J ) 6.5 Hz, H-3), 5.82
(1H, d, J ) 4.5 Hz, H-8), 7.36-7.54 (15H, m, aromatic H); ESIMS m/z
981 [M + Na]+.
Formation of Acetonide 7.24 Oploxyne B (2) (0.3 mg) was dissolved
in 2,2-dimethoxypropane (1 mL) and MeOH (0.2 mL), and pyridinium
p-toluenesulfonate (2 mg) was added. The reaction was allowed to stir
for 6 h in an ice bath. The reaction mixture was quenched with 1%
aqueous NaHCO3, and the aqueous phase was extracted three times
with CH2Cl2. The CH2Cl2 was removed under reduced pressure, and
the residue was purified by silica gel column chromatography using
CH2Cl2-MeOH (10:1) to provide the acetonide 7 (0.2 mg).
1
Acetonide 7: H NMR (CDCl3, 500 MHz) δ 0.89 (3H, t, J ) 6.5
Hz, H3-17), 1.04 (3H, t, J ) 7.5 Hz, H3-1), 1.31 (10H, m, H2-13-17),
1.37 (3H, s, acetonide Me), 1.57 (3H, s, acetonide Me), 1.44 (2H, m,
H2-11), 1.52 (2H, m, H2-12), 1.78 (2H, m, H2-2), 1.80 (1H, d, J ) 6.0
Hz, 3-OH), 3.50 (1H, br ddd, J ) 9.0, 8.5, 1.5 Hz, H-10), 3.51 (3H, s,
OCH3), 4.05 (1H, dd, J ) 8.5, 5.5 Hz, H-9), 4.41 (1H, td, J ) 6.0, 6.0
Hz, H-3), 4.71 (1H, d, J ) 5.5 Hz, H-8); ESIMS m/z 373 [M + Na]+.
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ethylenediamine], and incubated at room temperature for 5-10 min.
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was used to generate a standard reference curve of nitrite concentration.
The control was exposed to phenol red-free medium containing 0.5%
(v/v) DMSO. These experiments were done in triplicate wells.
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endogenous arachidonic acid was measured using the PGE2 Parameter
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Acknowledgment. This study was supported by the Korea Institute
of Science and Technology institutional program, grant numbers
2Z03100 and 2Z03270. We thank the Seoul National University
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use of the HRMS, and the Center for Scientific Instruments at
Kangnung-Wonju National University for use of the LC/MS and the
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Supporting Information Available: Physical and spectral data sets
(1D and 2D NMR, ESIMS, UV/vis, CD, IR, HRMS, etc.) for
compounds 1-4, 1H NMR, 1H-1H COSY, and ESIMS data for 5-9,
NP900628J