7
76
Vol. 59, No. 6
light or by heating after spraying with 10% H SO in C H OH (v/v). The
1.06 (3H, s, H-12), 0.98 (3H, s, H-11); HR-ESI-MS (positive mode) m/z:
2
4
2
5
ꢀ
packing material for molecular sieve column chromatography was Sephadex
LH-20 (Pharmacia Co. U.S.A.). Low pressure liquid chromatography was
247.1307 [MꢀNa] (Calcd for C H O Na: 247.1310).
1
3
20
3
2
5
1
2a: [a]D ꢁ60.2° (cꢂ0.1, CHCl3); H-NMR (pyridine-d , 500 MHz) d:
5
®
carried out over a Merck Lichroprep Lobar -A Si 60 (240ꢅ10 mm) or a
7.30 (1H, d, Jꢂ16.0 Hz, H-7), 6.54 (1H, d, Jꢂ16.0 Hz, H-8), 4.31 (1H, dt,
Jꢂ12.3, 3.5 Hz, H-3), 2.28 (3H, s, H-10), 2.18 (1H, br t, Jꢂ12.3 Hz, H-2b),
1.54 (1H, dd, Jꢂ12.3, 3.0 Hz, H-2a), 1.51 (3H, s, H-13), 1.15 (3H, s, H-12),
®
Lichroprep Lobar -A RP-18 (240ꢅ10 mm) column with a FMI QSY-0
pump (ISCO).
ꢀ
Plant Materials Cardamine komarovii (NAKAI) plants (1.7 kg) were
collected at Goseong-gun in Gangwon-Do province, Korea in August 2008.
A voucher specimen of the plant (SKK-08-011) was deposited at the School
of Pharmacy in Sungkyunkwan University.
1.06 (3H, s, H-11); HR-ESI-MS (positive mode) m/z: 263.1254 [MꢀNa]
(Calcd for C H O Na: 263.1259).
1
3
20
4
2
5
3a: [a] ꢁ77.0° (cꢂ0.1, CHCl ); positive-ion EI-MS m/z: 265.15
[MꢀNa] ; H-NMR (CDCl , 500 MHz) d: 5.93 (1H, dd, Jꢂ15.5, 1.0 Hz, H-
D
3
ꢀ
1
3
Extraction and Isolation The half dried aerial parts of C. komarovii 7), 5.77 (1H, dd, Jꢂ15.5, 5.5 Hz, H-8), 4.42 (1H, dqd, Jꢂ6.5, 5.5, 1.0 Hz, H-
(
NAKAI) (1.7 kg) were extracted with 80% MeOH three times at room tem-
9), 4.03 (1H, d, Jꢂ3.0 Hz, H-4), 3.97 (1H, dt, Jꢂ12.2, 3.3 Hz, H-3), 1.65
(1H, br t, Jꢂ12.2 Hz, H-2b), 1.36 (3H, s, H-13), 1.34 (1H, m, H-2a), 1.30
perature. The resultant MeOH extracts (120 g) were suspended in distilled
water (800 mlꢅ3) and then successively partitioned with n-hexane, CHCl3, (3H, d, Jꢂ6.5 Hz, H-10), 1.12 (3H, s, H-12), 0.99 (3H, s, H-11); C-NMR
13
EtOAc and n-BuOH, yielding residues weighing 12 g, 13 g, 3 g and 26 g,
(CDCl , 125 MHz) d: 138.1 (C-8), 124.7 (C-7), 72.1 (C-3), 70.1 (C-6), 68.2
3
respectively. The CHCl soluble fraction (13 g) was chromatographed on a
(C-4), 68.0 (C-9), 65.9 (C-5), 39.6 (C-2), 34.2 (C-1), 29.3 (C-11), 24.4 (C-
3
silica gel column, eluting with a gradient solvent system of CHCl /MeOH
12), 23.7 (C-10), 16.8 (C-13); HR-ESI-MS (positive mode) m/z: 265.1412
3
ꢀ
(40 : 1—1 : 1) as the eluant to yield seven fraction (fr. PC1—PC7). Fraction
[MꢀNa] (Calcd for C H O Na: 265.1416).
1
3
22
4
PC3 (1.0 g) was isolated by sephadex LH-20 chromatography (80% MeOH)
and was purified with a silica gel preparatory HPLC with a solvent system
Preparation of the (R)- and (S)-MTPA-Cl Ester Derivatives of 1a, 2a
and 3a by a Convenient Mosher Ester A solution of 1a (0.7 mg), 2a
(0.7 mg) and 3a (1.0 mg) in CH Cl (1.0 ml) was treated with (R)-MTPA
of CHCl : MeOH (25 : 1) to yield 11 (5 mg). The n-BuOH soluble fraction
3
2
2
(
26 g) was chromatographed on a diaion HP-20 column, eluting with a
(5 ml) in the presence of 1-ethyl-3-(3-dimethoxylaminopropyl)-carbodiimide
hydrochloride (EDC·HCl, 1.5 mg) and 4-methylaminopyridine (4-DMAP,
0.7 mg), and the mixture was stirred at room temperature for 7 h. After cool-
ing, the reaction mixture was poured into water and the whole reaction mix-
ture was extracted with EtOAc. The EtOAc extract was successively washed
gradient solvent system consisting of 100% water and 100% MeOH. This
yielded two subfractions. Fraction B (48 g) was isolated using a silica gel
column, eluting with a solvent system of CHCl /MeOH/water (10 : 4 : 0.5).
According to TLC analysis, fourteen crude fractions (fr. PB1—PB14) were
collected. Fr. B3 (0.2 g) purified using a Lobar -A RP-18 (240ꢅ10 mm)
3
®
with 5% aqueous HCl, saturated aqueous NaHCO , and brine, then dried
3
column (40% MeOH), and purified further by preparative reverse-phase
over MgSO powder and filtered. Removal of the solvent from the filtrate
4
HPLC, using a solvent system of 45% MeOH and 25% MeCN, yielded 4 under reduced pressure furnished a residue, which was separated by HPLC
®
(
28 mg) and 8 (14 mg). Fr. B4 (0.5 g) purified by a Lobar -B RP-18 (310ꢅ [MeOH : H Oꢂ45 : 55] to give 1b (0.4 mg), 2b (0.4 mg) and 3b (0.5 mg),
2
25 mm) column (30% MeOH), and purified further by a preparative reverse- respectively. Using a similar procedure, (S)-MTPA esters 1c (0.4 mg), 2c
phase HPLC, using a solvent system of 30% MeOH and 20% MeCN,
(0.4 mg) and 3c (0.6 mg) obtained from 1a, 2a, and 3a.
yielded 1 (13 mg), 2 (7 mg) and 5 (19 mg). Fr. PB5 (0.3 g) purified by a
Lobar -A RP-18 (240ꢅ10 mm) column (35% MeOH), and purified further
by preparative reverse-phase HPLC, using a solvent system of 15% MeCN,
1b: (500 MHz, pyridine-d ) d: 7.457—7.334 (1H, m, H-7), 6.258 (1H, d,
5
®
Jꢂ16.5 Hz, H-8), 5.449 (dt, Jꢂ12.3, 3.5 Hz, H-3), 4.458 (1H, d, Jꢂ3.5 Hz,
H-4), 2.410 (1H, d, Jꢂ12.3 Hz, H-2b), 2.293 (3H, s, H-10), 1.960 (3H, s, H-
yielded 6 (5 mg), 7 (10 mg) and 9 (15 mg). Fr. B7 (0.3 g), which was purified
13), 1.842 (1H, dd, Jꢂ12.3, 3.5 Hz, H-2a), 1.139 (3H, s, H-12), 1.023 (3H,
®
ꢀ
by a Lobar -B RP-18 (310ꢅ25 mm) column (30% MeOH), and purified s, H-11); HR-ESI-MS (positive mode) m/z: 463.1702 [MꢀNa] (Calcd for
further by preparative normal-phase HPLC, using a solvent system of CHCl3/
C H O F Na: 463.1708).
23 27 5 3
MeOH (7 : 1, 3 : 1) yielded 3 (12 mg) and 10 (9 mg).
1c: (500 MHz, pyridine-d ) d: 7.420—7.334 (1H, m, H-7), 6.261 (1H, d,
5
2
5
Komaroveside A (1): Colorless gum. [a]D ꢁ36.8° (cꢂ0.25, MeOH); UV Jꢂ16.5 Hz, H-8), 5.470 (dt, Jꢂ12.3, 3.5 Hz, H-3), 4.612 (1H, d, Jꢂ3.5 Hz,
ꢁ
1
(
2
MeOH) lmax (log e) 227 (4.0), 279 (3.9) nm; IR (KBr) n
cm : 3382, H-4), 2.327 (1H, d, Jꢂ12.3 Hz, H-2b), 2.303 (3H, s, H-10), 1.993 (3H, s, H-
max
1
924, 1660, 1608, 1425, 1365, 1076; H-NMR (CD OD, 500 MHz): see
13), 1.717 (1H, dd, Jꢂ12.3, 3.5 Hz, H-2a), 1.100 (3H, s, H-12), 0.954 (3H,
3
1
3
ꢀ
Table 1; C-NMR (CD OD, 125 MHz): see Table 1; HR-ESI-MS (positive
s, H-11); HR-ESI-MS (positive mode) m/z: 463.1689 [MꢀNa] (Calcd for
3
ꢀ
mode) m/z: 409.1831 [MꢀNa] (Calcd for C H O Na: 409.1838).
Komaroveside B (2): Colorless gum. [a]D ꢁ65.4° (cꢂ0.1, MeOH); UV
MeOH) l
C H O F Na: 463.1708).
1
9
30
8
23 27
5 3
2
5
2b: (500 MHz, pyridine-d ) d: 7.304 (1H, Jꢂ16.0 Hz, H-7), 6.556 (1H, d,
5
ꢁ
1
(
1
1
[
(log e) 229 (4.1) nm; IR (KBr) nmax cm : 3383, 2948, 1658,
Jꢂ16.0 Hz, H-8), 5.705 (dt, Jꢂ12.3, 3.5 Hz, H-3), 4.547 (1H, d, Jꢂ3.5 Hz,
max
1
13
450, 1370; H-NMR (CD OD, 500 MHz): see Table 1; C-NMR (CD OD, H-4), 2.439 (1H, d, Jꢂ12.3 Hz, H-2b), 2.329 (3H, s, H-10), 1.567 (1H, dd,
3
3
25 MHz): see Table 1; HR-ESI-MS (positive mode) m/z: 425.1781
Jꢂ12.3, 3.0 Hz, H-2a), 1.507 (3H, s, H-13), 1.233 (3H, s, H-12), 1.167 (3H,
ꢀ
ꢀ
MꢀNa] (Calcd for C H O Na: 425.1788).
s, H-11) HR-ESI-MS (positive mode) m/z: 479.4398 [MꢀNa] (Calcd for
19
30
9
2
5
Komaroveside C (3): Colorless gum. [a] ꢁ61.4° (cꢂ0.1, MeOH); IR C H O F Na: 479.4410).
D
23 27
6 3
ꢁ1
1
(
(
KBr) nmax cm : 3382, 2966, 1739, 1627, 1450, 137; positive; H-NMR
2c: (500 MHz, pyridine-d ) d: 7.291 (1H, d, Jꢂ16.0 Hz, H-7), 6.535 (1H,
5
1
3
CD OD, 500 MHz): see Table 1; C-NMR (CD OD, 125 MHz): see Table
d, Jꢂ16.0 Hz, H-8), 5.730 (dt, Jꢂ12.3, 3.5 Hz, H-3), 4.723 (1H, d, Jꢂ3.5
Hz, H-4), 2.334 (1H, d, Jꢂ12.3 Hz, H-2b), 2.311 (3H, s, H-10), 1.565 (1H,
3
3
ꢀ
1; HR-ESI-MS (positive mode) m/z: 427.1936 [MꢀNa] (Calcd for
C H O Na:427.1944)
dd, Jꢂ12.3, 3.0 Hz, H-2a), 1.550 (3H, s, H-13), 1.155 (3H, s, H-12), 1.107
19
32
9
ꢀ
Enzymatic Hydrolysis of 1, 2 and 3 Compound 1 (3.0 mg) with 1 ml of (3H, s, H-11); HR-ESI-MS (positive mode) m/z: 479.4401 [MꢀNa] (Calcd
H O and 3 mg of b-glucosidase (Emulsin) was shaken for 7 d at 37 °C. The
for C H O F Na: 479.4410).
2
23 27 6 3
H O solution was then extracted with CHCl three times, and the CHCl
3b: (500 MHz, pyridine-d ) d: 6.202 (1H, d, Jꢂ15.8, H-7), 6.083 (1H, dd,
2
3
3
5
extract was evaporated in vacuo. The CHCl extract (2.0 mg) was purified
Jꢂ15.8, 5.5 Hz, H-8), 5.851 (1H, m, H-9), 5.675 (1H, m, H-3), 4.486 (1H, d,
3
using reverse-phase HPLC (50% MeOH) to yield the aglycone 1a as a color- Jꢂ3.5 Hz, H-4), 2.355 (1H, br t, Jꢂ12.8 Hz, H-2b), 1.484 (3H, s, H-13),
2
5
1
less gum [a] ꢁ60.5° (cꢂ0.1, CHCl ), H-NMR (pyridine-d , 500 MHz).
1.499 (1H, dd, Jꢂ12.8, 3.5 Hz, H-2a), 1.391 (3H, d, Jꢂ6.5 Hz, H-10), 1.118
(3H, s, H-12), 1.089 (3H, s, H-11); HR-ESI-MS (positive mode) m/z:
D
3
5
Compound 2 (3.0 mg) was treated by the same method. The CHCl extract
2.0 mg) was purified using reverse-phase HPLC (40% MeOH) to yield the
aglycone 2a as a colorless gum [a] ꢁ60.2° (cꢂ0.1, CHCl3), H-NMR
pyridine-d , 500 MHz). Compound 3 (3.0 mg) was treated by the same
3
ꢀ
(
697.2198 [MꢀNa] (Calcd for C H O F Na: 697.2212).
3
3
36
8 6
2
5
1
3c: (500 MHz, pyridine-d ) d: 6.287 (1H, d, Jꢂ15.8, H-7), 6.195 (1H, dd,
D
5
(
Jꢂ15.8, 5.5 Hz, H-8), 5.879 (1H, m, H-9), 5.791 (1H, m, H-3), 4.689 (1H, d,
Jꢂ3.5, H-4), 2.273 (1H, br t, Jꢂ12.8 Hz, H-2b), 1.554 (3H, s, H-13), 1.474
5
method. The CHCl extract (1.0 mg) was purified using reverse-phase HPLC
3
2
5
(
(
40% MeOH) to yield the aglycone 3a as a colorless gum [a] ꢁ77.0° (1H, dd, Jꢂ12.8, 3.5 Hz, H-2a), 1.265 (3H, d, Jꢂ6.5 Hz, H-10), 1.125 (3H,
D
1
cꢂ0.1, CHCl ), H-NMR (pyridine-d , 500 MHz). The sugar in the distilled s, H-12), 1.069 (3H, s, H-11); HR-ESI-MS (positive mode) m/z: 697.2215
3
5
ꢀ
water layer was identified as D-glucose by co-TLC (EtOAc : MeOH : H Oꢂ
[MꢀNa] (Calcd for C H O F Na: 697.2212).
2
33 36 8 6
9
: 3 : 1, Rf value: 0.2, 1a: 1.5 mg, 2a: 1.5 mg, 3a: 2.0 mg) with a D-glucose
Cytotoxicity Assay A sulforhodamin B bioassay (SRB) was used to
standard (Aldrich Co., U.S.A.).
determine the cytotoxicity of the compounds. The cytotoxic activity of each
25
1
1
a: [a] ꢁ60.5° (cꢂ0.1, CHCl3); H-NMR (pyridine-d , 500 MHz) d: compound against four cultured human tumor cells was examined in vitro at
D
5
7
4
1
.31 (1H, d, Jꢂ16.4 Hz, H-7), 6.25 (1H, d, Jꢂ16.4, H-8), 4.23 (1H, br s, H- the Korea Research Institute of Chemical Technology. The tumor cell lines
), 4.10 (1H, dt, Jꢂ12.3, 3.0 Hz, H-3), 2.25 (3H, s, H-10), 2.19 (1H, br t, Jꢂ were A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer
2.3 Hz, H-2b), 1.99 (3H, s, H-13), 1.71 (1H, dd, Jꢂ12.3, 2.3 Hz, H-2a),
17)
cells), SK-MEL-2 (skin melanoma) and HCT15 (colon cancer cells).