June 2011
745
small pieces and percolated with 70% EtOH for 5 d at room temperature, 8
times. The 70% EtOH extract was evaporated to dryness under reduced pres-
sure and then partitioned between H2O and EtOAc and then BuOH (778 g).
The EtOAc extract was added to 90% aqueous MeOH and extracted with
hexane to give hexane (100 g) and 90% MeOH (106 g) extracts. The 90%
MeOH extract (106 g) was fractionated by column chromatography over sil-
ica gel with CH2Cl2/MeOH (gradient) to yield 50 subfractions (Fr. 90M01—
90M50). Fraction 90M32 (5.6 g) was further purified on a silica gel column
with EtOAc saturated with water to yield 45 subfractions (Fr. 90M32-01—
90M32-45). Fraction 90M32-28 (500 mg) was chromatographed on a silica
gel column with MeOH/H2O (10 : 1) to yield compounds 1 (2 mg) and 2
(50 mg) from 90M32-28-15. Fraction 90M32-40 (2 g) was chromatographed
on a silica gel column with EtOAc saturated with water/MeOH (95 : 5) to
afford subfraction 90M-32-40-45 (800 mg), which was further purified on an
RP-18 column with 50% MeOH to yield compound 3 (100 mg).
Glutinolic Acid (1): Amorphous white powder. [a]D27 ꢁ9.0° (cꢂ0.5,
MeOH); UV lmax (MeOH) nm (log e): 202 (3.47); IR (KBr) nmax cmꢀ1
:
3357 (OH), 1700 (CꢂO), 1670 (CHꢂCH), 1456, 1251, 1150, 1033, 1004,
860; EI-MS m/z (rel. int., %): 502 [MꢀH2O]ꢁ (6), 484 [Mꢀ2H2O]ꢁ (3), 469
[Mꢀ(3H2OꢁCH3)]ꢁ (2), 466 [Mꢀ3H2O]ꢁ (2), 453 [Mꢀ(2H2OꢁCH2OH)]ꢁ
(4), 440 [Mꢀ(H2Oꢁ2CH2OH)]ꢁ (7), 278 [D/E ring (a)ꢀH2O]ꢁ (66), 263
[aꢀ(H2OꢁCH3)]ꢁ (33), 224 [A/B ring (b)]ꢁ (11), 223 [bꢀH]ꢁ (19), 217
[aꢀ(H2OꢁCOOHꢁH)]ꢁ (22), 206 [bꢀH2O]ꢁ (58), 175 [bꢀ(H2Oꢁ
CH2OH)]ꢁ (100), 146 (32), 133 (44), 119 (43), 105 (49), 81 (38); (HR)-(ꢀ)-
FAB-MS m/z: 519.3354. Calcd for C30H47O7: 519.3322; HR-EI-MS m/z:
502.3299. Calcd for C30H46O6 [MꢀH2O]ꢁ: 502.3294; (ꢀ)-FAB-MS m/z:
519 [MꢀH]ꢀ; 1H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz,
CD3OD) data: Table 1.
Aeginetic Acid 5-O-b-D-Quinovoside (2): Amorphous white powder.
[a]D23 ꢀ80.1° (cꢂ1.0, MeOH); UV lmax (MeOH) nm (log e): 260 (4.47);
IR (KBr) nmax cmꢀ1: 3398 (OH), 1686 (CꢂO), 1610 (CHꢂCH), 1375, 1245,
1168, 1067, 1004 (glycosidic C–O), 866; HR-FAB-MS m/z: 437.2166.
Calcd for C21H34O8Na: 437.2151; FAB-MS m/z: 437 [MꢁNa]ꢁ, 419
1
[(MꢁNa)ꢀH2O]ꢁ; H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz,
CD3OD) data: Table 2.
Aeginetoyl Ajugol 5ꢃ-O-b-D-Quinovoside (3): Amorphous white powder.
[a]D22 ꢀ120.0° (cꢂ1.0, MeOH); UV lmax (MeOH) nm (log e): 269 (4.73);
IR (KBr) nmax cmꢀ1: 3409 (OH), 1700 (CꢂO), 1651, 1612 (CHꢂCH), 1237,
1157, 1070, 1005 (glycosidic C–O); HR-FAB-MS m/z: 767.3441. Calcd for
C36H56O16Na: 767.3466; FAB-MS m/z: 767 [MꢁNa]ꢁ, 745 [MꢁH]ꢁ, 621
[(MꢁNa)ꢀ146]ꢁ, 603 [(MꢁNa)ꢀ146ꢀH2O]ꢁ, 437 [(MꢁH)ꢀ146ꢀ162]ꢁ,
419 [(MꢁH)ꢀ146ꢀ162ꢀH2O]ꢁ, 401 [(MꢁH)ꢀ146ꢀ162ꢀ(2ꢅH2O)]ꢁ,
397 [C20H33O6C≡Oꢁ], 379 [(C20H33O6C≡Oꢁ)ꢀH2O]ꢁ, 251 [(C20H33O6C≡
Oꢁ)ꢀ146]ꢁ, 233 [(C20H33O6C≡Oꢁ)ꢀ146ꢀH2O]ꢁ; 1H-NMR (400 MHz,
CD3OD) and 13C-NMR (100 MHz, CD3OD) data: Table 2.
Fig. 3. Important HMBC Correlations for 3
Acid Hydrolysis of 2 Compound 2 (30 mg) was refluxed with 5% HCl
in 60% aqueous dioxane (5 ml) for 1 h. The reaction mixture was concen-
trated, added to crushed ice, and extracted with CHCl3. The CHCl3 extract
was purified on a silica gel column with CHCl3/MeOH/H2O (7 : 1.5 : 0.5)
solution to afford aeginetic acid, which was crystallized from MeOH to give
fine needles. Aeginetic acid: mp 204—205°C, [a]D27 ꢀ63.9° (cꢂ1.0, MeOH);
1H-NMR (400 MHz, CD3OD) d: 0.79 (3H, s, 1b-CH3), 1.06 (3H, s, 5-CH3),
1.20 (3H, s, 1a-CH3), 2.30 (3H, br s, 9-CH3), 5.78 (1H, br s, H-10), 6.41
(1H, d, Jꢂ16.0 Hz, H-8), 6.69 (1H, d, Jꢂ16.0 Hz, H-7); EI-MS m/z (rel. int.,
%): 268 [M]ꢁ (4.3), 250 [MꢀH2O]ꢁ (3.9), 207 (37.5), 181 (10.2), 127
(63.3), 109 (100), 95 (57.8), 69 (53.1). The aqueous layer was neutralized
with Ag2CO3, filtered and then concentrated to dryness in vacuo to give a
residue that was subjected to RP-18 column chromatography with 40%
aqueous MeOH to yield pure quinovose ([a]D27 ꢁ48.3° (cꢂ1.0, H2O), Rf
0.51 with BuOH/HOAc/H2O (4 : 1 : 2) on cellulose plate. The dried sugar
(1 mg) was treated with pyridine (0.1 ml), and then the solution was added to
a pyridine solution (0.1 ml) of L-cysteine methyl ester hydrochloride (2 mg)
and warmed at 60 °C for 1 h. The solvent was evaporated under a N2 stream
and dried in vacuo. The residue was trimethylsilylated with TMS-HT
(0.1 ml) at 60 °C for 30 min. After the addition of hexane and water, the
hexane layer was removed and checked by GC. The retention times (tR) of
the peaks were 4.493 and 5.053 min. The tR of the peaks of the authentic
sample were 4.490 and 5.048 min (D-quinovose).
CH3), 1.13 (3H, s, 5-CH3), 1.18 (3H, s, 1a-CH3), 1.16 (1H, overlap, H-2a),
1.21 (3H, d, Jꢂ6.2 Hz, Qui 6-CH3), 1.28 (1H, m, H-3a), 1.59—1.73 (3H, m,
H-2b, 4), 2.11 (1H, qt, Jꢂ3.4, 13.4 Hz, H-3b), 2.29 (3H, d, Jꢂ0.9 Hz, 9-
CH3), 3.00 (1H, t, Jꢂ9.0 Hz, Qui H-5), 3.20 (1H, dd, Jꢂ7.1, 8.9 Hz, Qui H-
2), 3.22 (1H, t, Jꢂ9.3 Hz, Qui H-4), 3.25 (1H, t, Jꢂ8.8 Hz, Qui H-3), 3.68
(3H, s, COOCH3), 4.41 (1H, d, Jꢂ7.5 Hz, Qui H-1), 5.78 (1H, br s, H-10),
6.36 (1H, d, Jꢂ16.2 Hz, H-8), 6.83 (1H, d, Jꢂ16.2 Hz, H-7); 13C-NMR
(75.5 MHz, CD3OD) d: 39.9 (C-1), 37.1 (C-2), 18.7 (C-3), 32.9 (C-4), 83.6
(C-5), 80.4 (C-6), 141.4 (C-7), 133.5 (C-8), 154.6 (C-9), 118.2 (C-10), 169.4
(C-11), 51.4 (COOCH3), 25.9 (1a-CH3), 27.6 (1b-CH3), 21.9 (5-CH3), 14.4
(9-CH3), 98.0 (Qui C-1), 75.8 (Qui C-2), 78.8 (Qui C-3), 72.6 (Qui C-4),
77.1 (Qui C-5), 18.4 (Qui C-6); FAB-MS m/z: 451 [MꢁNa]ꢁ, 429 [MꢁH]ꢁ,
283 [(MꢁH)ꢀ146]ꢁ, 265 [(MꢁH)ꢀ146ꢀH2O]ꢁ. The aqueous layer was
neutralized with 10% HCl and then chromatographed on an MCI gel column
with distilled water to afford ajugol as an amorphous powder. [a]D27 ꢀ103.8°
(cꢂ0.5, MeOH); UV lmax (MeOH) nm (log e): 201 (3.63); IR (KBr) nmax
cmꢀ1: 3376 (OH), 1658 (CHꢂCH), 1077, 1005 (glycosidic C–O), 969, 946,
1
749; H-NMR (500 MHz, CD3OD) d: 1.31 (3H, s, 10-CH3), 1.78 (1H, dd,
Jꢂ4.5, 13.4 Hz, H-7a), 2.03 (1H, dd, Jꢂ5.6, 13.4 Hz, H-7b), 2.54 (1H, br d,
Jꢂ9.5 Hz, H-9), 2.72 (1H, br d, Jꢂ9.4 Hz, H-5), 3.19 (1H, t, Jꢂ8.9 Hz, H-
2ꢆ), 3.26 (1H, t, Jꢂ9.6 Hz, H-4ꢆ), 3.36 (1H, t, Jꢂ9.8 Hz, H-3ꢆ), 3.65 (1H, dd,
Jꢂ5.5, 11.8 Hz, H-6ꢆa), 3.88 (1H, br d, Jꢂ11.8 Hz, H-6ꢆb), 3.91 (1H, m, H-
6), 4.63 (1H, d, Jꢂ7.9 Hz, H-1ꢆ), 4.90 (overlap with HDO, H-4), 5.45 (1H,
br s, H-1), 6.15 (1H, br d, Jꢂ6.1 Hz, H-3); 13C-NMR (125 MHz, CD3OD) d:
25.2 (C-10), 41.3 (C-5), 50.0 (C-7), 51.8 (C-9), 62.9 (C-6ꢆ), 71.7 (C-4ꢆ),
74.8 (C-2ꢆ), 77.8 (C-3ꢆ), 78.0 (C-5ꢆ), 78.2 (C-6), 79.4 (C-8), 93.7 (C-1), 99.4
(C-1ꢆ), 105.9 (C-4), 140.4 (C-3); FAB-MS m/z: 371 [MꢁNa]ꢁ, 349
[MꢁH]ꢁ.
Alkaline Hydrolysis of 3 A solution of 3 (12 mg) in 2% NaOH in
MeOH (1 ml) was kept at room temperature for 3 h. The reaction mixture
was added to water (5 ml) and extracted with CH2Cl2 (5 ml). The CH2Cl2
layer was concentrated and crystallized from MeOH to yield methyl aegine-
tate 5-O-b-D-quinovoside (5 mg) as an amorphous powder. Methyl aegine-
tate 5-O-b-D-quinovoside: 1H-NMR (300 MHz, CD3OD) d: 0.80 (3H, s, 1b-