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S. Nakamura et al. / Tetrahedron 65 (2009) 2443–2450
3-12 (69 mg) was purified by HPLC [MeOH–H2O (85:15, v/v)] to
afford inoterpene A (1, 4.8 mg) and inoterpene B (2, 5.0 mg). Frac-
tion 6 (14.0 g) was subjected to reversed-phase silica gel CC [700 g,
MeOH–H2O (70:30/80:20/90:10, v/v)/MeOH] to afford nine
fractions {Fr. 6-1, Fr. 6-2, Fr. 6-3, Fr. 6-4, Fr. 6-5, Fr. 6-6 (1.440 g), Fr.
6-7 [7.232 g¼inotodiol (11)], Fr. 6-8, and Fr. 6-9}. Fraction 6-6
(580 mg) was purified by HPLC [MeOH–H2O (90:10, v/v)] to furnish
inoterpene F (6, 20 mg) and inotodiol (11, 116 mg). Fraction 8 (3.3 g)
was subjected to reversed-phase silica gel CC [165 g, MeOH–H2O
(60:40/70:30/80:20/90:10, v/v)/MeOH] to afford seven
fractions [Fr. 8-1, Fr. 8-2, Fr. 8-3, Fr. 8-4 (1.278 g), Fr. 8-5 (2.190 g), Fr.
8-6, and Fr. 8-7]. Fraction 8-4 (510 mg) was separated by HPLC
[MeOH–H2O (90:10, v/v)] to afford six fractions {Fr. 8-4-1, Fr. 8-4-2,
Fr. 8-4-3 (21 mg), Fr. 8-4-4, Fr. 8-4-5, and Fr. 8-4-6 [42 mg¼tra-
metenolic acid (10)]}. Fraction 8-4-3 (21 mg) was purified by HPLC
[MeOH–H2O (80:20, v/v)] to furnish inoterpene E (5, 2.6 mg).
Fraction 8-5 (200 mg) was purified by HPLC [MeOH–H2O (90:10,
1.26, 1.59, 1.59 (3H each, all s, H3-18, 30, 19, 29, 28, 26, 27), 1.03 (3H,
d, J¼6.1 Hz, H3-21), 3.47 (1H, m, H-3), 5.86 (1H, ddd like, J¼5.5, 8.6,
15.9 Hz, H-23), 5.99 (1H, d, J¼15.9 Hz, H-24); 13C NMR (pyridine-d5,
125 MHz) see Table 1; EIMS m/z 458 [M]þ; HREIMS m/z 458.3764
(calcd for C30H50O3 [M]þ, 458.3760).
3.3.4. Inoterpene D (4)
28
A white powder; [
a
]
þ66.0 (c 1.50, CHCl3); IR (KBr) nmax 3450,
D
2940 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 0.78, 0.81, 0.85, 0.98, 1.00,
1.68, 1.75 (3H each, all s, H3-18, 29, 30, 19, 28, 26, 27), 3.23 (1H, dd,
J¼4.4, 11.6 Hz, H-3), 3.72 (1H, dd, J¼10.7, 10.7 Hz, H-21a), 3.83 (1H,
m, H-22), 4.02 (1H, dd, J¼3.8, 10.7 Hz, H-21b), 5.22 (1H, m, H-24);
13C NMR (CDCl3, 125 MHz) see Table 1; EIMS m/z 458 [M]þ; HREIMS
m/z 458.3769 (calcd for C30H50O3 [M]þ, 458.3760).
3.3.5. Inoterpene E (5)
26
A white powder; [
a
]
þ30.8 (c 0.17, CHCl3); IR (KBr) nmax 3450,
v/v)] to furnish 3b,21-dihydroxylanosta-8,24-diene (8, 15 mg), tra-
D
2940 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 0.72, 0.80, 0.89, 0.97, 1.00,
metenolic acid (10, 35 mg), and inotodiol (11, 48 mg). Fraction 9
(3.4 g) was subjected to reversed-phase silica gel CC [170 g,
MeOH–H2O (70:30/80:20/90:10, v/v)/MeOH] to afford nine
fractions {Fr. 9-1, Fr. 9-2, Fr. 9-3, Fr. 9-4, Fr. 9-5, Fr. 9-6 (107 mg), Fr.
9-7 [2.225 g¼trametenolic acid (10)], Fr. 9-8, and Fr. 9-9}. Fraction
9-6 (107 mg) was purified by HPLC [MeOH–H2O (90:10, v/v)] to
furnish trametenolic acid (10, 9.9 mg). Fraction 11 (5.2 g) was
subjected to reversed-phase silica gel CC [260 g, MeOH–H2O
(10:90/20:80/40:60/60:40/80:20, v/v)/MeOH] to afford 19
fractions {Fr. 11-1, Fr. 11-2, Fr. 11-3, Fr. 11-4, Fr. 11-5, Fr. 11-6, Fr. 11-7,
Fr. 11-8, Fr. 11-9, Fr. 11-10, Fr. 11-11, Fr. 11-12, Fr. 11-13, Fr. 11-14, Fr.
11-15, Fr. 11-16 (1.960 g), Fr. 11-17 (415 mg), Fr. 11-18, and Fr. 11-19}.
Fraction 11-16 (240 mg) was purified by HPLC [MeOH–H2O (90:10,
v/v)] to furnish inoterpene D (4, 5.1 mg) and trametenolic acid (10,
21 mg). Fraction 11-17 (415 mg) was purified by HPLC [MeOH–H2O
(90:10, v/v)] to furnish inotodiol (11, 5.1 mg). Fraction 12 (18.9 g)
was subjected to reversed-phase silica gel CC [950 g, MeOH–H2O
(20:80/40:60/60:40/80:20, v/v)/MeOH] to afford 16 frac-
tions {Fr. 12-1, Fr. 12-2, Fr. 12-3, Fr. 12-4, Fr. 12-5, Fr. 12-6, Fr. 12-7, Fr.
12-8, Fr. 12-9, Fr. 12-10, Fr. 12-11, Fr. 12-12 (493 mg), Fr. 12-13
(164 mg), Fr.12-14, Fr.12-15, and Fr.12-16}. Fraction 12-12 (493 mg)
was purified by HPLC [MeOH–H2O (85:15, v/v)] to furnish ino-
terpene D (4, 19 mg). Fraction 12-13 (164 mg) was purified by HPLC
[MeOH–H2O (85:15, v/v)] to furnish inoterpene A (1, 3.2 mg) and
inoterpene B (2, 3.7 mg). The known compounds were identified by
1.32, 1.33 (3H each, all s, H3-18, 29, 30, 19, 28, 26, 27), 3.24 (1H, dd,
J¼4.1, 11.7 Hz, H-3), 3.64 (1H, dd, J¼6.2, 11.7 Hz, H-21a), 3.78 (1H,
dd, J¼2.8, 11.7 Hz, H-21b), 5.59 (1H, d, J¼15.1 Hz, H-24), 5.81 (1H,
ddd like, J¼7.9, 7.9, 15.1 Hz, H-23); 13C NMR (CDCl3, 125 MHz) see
Table 1; EIMS m/z 474 [M]þ; HREIMS m/z 474.3711 (calcd for
C30H50O4 [M]þ, 474.3709).
3.3.6. Inoterpene F (6)
27
A white powder; [
a
]
þ38.8 (c 0.85, CHCl3); IR (KBr) nmax 3450,
D
2950, 1655 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 0.78, 0.81, 0.89, 0.98,
1.00, 1.81 (3H each, all s, H3-18, 29, 30, 19, 28, 27), 3.23 (1H, dd,
J¼4.4,11.6 Hz, H-3), 3.97 (1H, d like, J¼ca. 5 Hz, H-21), 4.84, 5.00 (1H
each, both s-like, H2-26); 13C NMR (CDCl3, 125 MHz) see Table 1;
EIMS m/z 440 [M]þ; HREIMS m/z 440.3663 (calcd for C30H48O2
[M]þ, 440.3654).
3.3.7. Preparation of the (S)- and (R)-MTPA esters (1a and 1b)
from 1
A solution of 1 (1.9 mg, 0.004 mmol) in pyridine (1 mL) was
treated with (ꢀ)-MTPACl (0.015 mL, 0.08 mmol), and the mixture
was stirred at rt for 6 h. The reaction mixture was poured into water
(1 mL) and the whole was extracted with EtOAc (6 mL). The EtOAc
extract was washed with brine, then dried over Na2SO4 powder and
filtered. Removal of the solvent from the filtrate under reduced
pressure furnished a residue, which was purified by reversed-phase
silica gel CC [MeOH–H2O (60:40, v/v)/MeOH] to give (S)-MTPA
ester derivative (1a, 2.8 mg, 76%). Through a similar procedure, the
(R)-MTPA ester derivative (1b, 2.0 mg, 75%) was obtained from 1
(1.4 mg, 0.003 mmol) using (þ)-MTPACl.
comparison of their physical data ([
reported values.
a
]D, 1H NMR, 13C NMR, MS) with
3.3.1. Inoterpene A (1)
23
A white powder; [
a
]
þ141.0 (c 0.21, CHCl3); IR (KBr) nmax 3450,
D
2945 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 0.69, 0.81, 0.88, 0.99, 1.01,
1.17, 1.22 (3H each, all s, H3-18, 29, 30, 19, 28, 26, 27), 0.92 (3H, d,
J¼6.1 Hz, H3-21), 3.24 (1H, dd, J¼4.3, 11.6 Hz, H-3), 3.29 (1H, m,
H-24); 13C NMR (CDCl3, 125 MHz) see Table 1; EIMS m/z 460 [M]þ;
HREIMS m/z 460.3909 (calcd for C30H52O3 [M]þ, 460.3916).
3.3.7.1. (S)-MTPA ester derivative (1a). A white powder; 1H NMR
(CDCl3, 600 MHz) d 0.64, 0.84, 0.86, 0.93, 0.98 (3H each, all s, H3-18,
29, 30, 28, 19), 0.84 (3H, d, J¼6.0 Hz, H3-21), 1.17, 1.23 [3H each, all s,
H3-26, 27 (interchangeable)], 1.33, 1.76 (1H each, both m, H2-1),
4.72 (1H, dd, J¼4.8, 11.7 Hz, H-3), 4.95 (1H, m, H-24); positive-ion
FABMS m/z 915 [MþNa]þ; HRFABMS m/z 915.4616 (calcd for
C50H66O7F6Na [MþNa]þ, 915.4610).
3.3.2. Inoterpene B (2)
22
A white powder; [
a
]
þ132.1 (c 0.28, CHCl3); IR (KBr) nmax
D
3450, 2945 cmꢀ1
;
1H NMR (CDCl3, 500 MHz)
d 0.70, 0.81, 0.88,
0.98, 1.00, 1.17, 1.22 (3H each, all s, H3-18, 29, 30, 19, 28, 26, 27),
0.91 (3H, d, J¼6.1 Hz, H3-21), 3.24 (1H, dd, J¼4.3, 11.6 Hz, H-3),
3.35 (1H, m, H-24); 13C NMR (CDCl3, 125 MHz) see Table 1; EIMS
m/z 460 [M]þ; HREIMS m/z 460.3912 (calcd for C30H52O3 [M]þ,
460.3916).
3.3.7.2. (R)-MTPA ester derivative (1b). A white powder; 1H NMR
(CDCl3, 600 MHz) d 0.67, 0.83, 0.84, 0.87, 1.02 (3H each, all s, H3-18,
29, 28, 30, 19), 0.90 (3H, d, J¼6.0 Hz, H3-21), 1.15, 1.18 [3H each, all s,
H3-26, 27 (interchangeable)], 1.35, 1.79 (1H each, both m, H2-1),
4.74 (1H, dd, J¼2.8, 12.0 Hz, H-3), 4.95 (1H, m, H-24); positive-ion
FABMS m/z 915 [MþNa]þ; HRFABMS m/z 915.4617 (calcd for
C50H66O7F6Na [MþNa]þ, 915.4610).
3.3.3. Inoterpene C (3)
23
A white powder; [
a
]
þ36.1 (c 2.10, MeOH); IR (KBr) nmax 3450,
D
2945 cmꢀ1
;
1H NMR (pyridine-d5, 500 MHz)
d 0.78, 0.97, 1.09, 1.10,