525
dd, J = 10.7, 6.1 Hz, H-26b), 2.47 (1H, d, J = 10.1 Hz,
H-17), 1.57 (3H, s, CH3-21), 1.02 (3H, s, CH3-19), 0.94
(3H, d, J = 6.7 Hz, CH3-27) and 0.65 (3H, s, CH3-18);
(protons on galactopyranose) δ 5.29 (1H, d, J = 3.7 Hz,
H-4′), 4.93 (1H, dd, J = 10.1, 3.7 Hz, H-3′), 4.32 (1H, d,
J = 7.6 Hz, H-1′), 3.98 (1H, dd, J = 10.1, 7.6 Hz, H-2′),
3.82 (1H, dd, J = 7.3, 7.3 Hz, H-5′) and 4.05–4.15 (H-6′a
and H-6′b); (protons on glucopyranose) δ 5.13 (1H, dd, J
= 9.8, 9.5 Hz, H-3′′), 4.98 (1H, dd, J = 9.8, 9.8 Hz, H-4′′),
4.83 (1H, dd, J = 9.5, 8.2 Hz, H-2′′), 4.73 (1H, d, J = 8.2
Hz, H-1′′) and 4.05–4.15 (H-6′′a and H-6′′b), 3.73 (1H,
ddd, J = 9.8, 5.2, 3.7 Hz, H-5′′) and 2.15, 2.09, 2.08, 2.06,
2.05, 2.04, 2.01, 1.99 and 1.98 (each 3H, Ac); 13C-NMR
(CDCl3) δ 171.1, 170.5, 170.5, 170.3, 170.1, 170.1,
169.8, 169.3 and 169.2 (each CO), 151.3 (C-22), 103.7
(C-20), 100.8 (C-1′), 100.4 (C-1′′), 84.2 (C-16), 77.0
(C-2), 74.0 (C-2′), 73.7 (C-3′), 72.7 (C-3′′), 71.7 (C-5′′),
71.4 (C-2′′), 70.5 (C-5′), 70.0 (C-3), 69.1 (C-26), 68.7
(C-4′′), 67.3 (C-4′), 64.2 (C-17), 62.5 (C-6′′), 61.2 (C-6′),
54.6 (C-14), 45.3 (C-13), 41.0 (C-9), 39.6 (C-12), 36.7
(C-10), 35.1 (C-8), 35.0 (C-5), 33.9 (C-15), 30.6 (C-1),
30.6 (C-24), 30.1 (C-4), 26.6 (C-6), 25.6 (C-7), 23.5
(C-19), 23.1 (C-23), 21.0 (C-11), 21.2 (C-25), 20.9–20.3
(COCH3 × 9), 16.6 (C-27), 14.0 (C-18) and 11.5 (C-21);
Anal. calcd. for C57H82O23: C, 60.31; H, 7.28. Found: C,
60.01; H, 7.30.
J = 8.9, 7.6 Hz, H-2′′),.4.01 (1H, dd, J = 6.4, 6.1 Hz,
H-5′) and 3.85 (1H, ddd, J = 9.5, 6.1, 3.4 Hz, H-5′′);
13C-NMR spectrum of 20 was listed in table III; Anal.
calcd. for C39H64O13.H2O: C, 61.72; H, 8.77. Found; C,
61.66; H, 8.81. FABMS of 21 m/z 763 [M + Na]+; signals
of CH3-18, CH3-19, CH3-21, CH3-27, H-20, H-26a and
H-26b in the 1H-NMR spectra (pyridine-d5) were listed in
table I; other assignable signals were as follows: δ 5.23
(1H, d, J = 7.6 Hz, H-1′′), 4.89 (1H, d, J = 7.6 Hz, H-1′),
4.62 (1H, dd, J = 9.5, 7.6 Hz, H-2′), 4.61 (1H, ddd, J =
7.3, 7.3, 3.4 Hz, H-16), 4.58 (1H, d, J = 4.0 Hz, H-4′),
4.53 (1H, m, H-16), 4.31 (1H, br. s, H-3), 4.35–4.47 (4H,
H-6′a, 6′b, 6′′a and 6′′b), 4.27 (1H, dd, J = 9.5, 9.2 Hz,
H-4′′), 4.24 (1H, dd, J = 9.5, 4.0 Hz, H-3′), 4.14 (1H, dd,
J = 9.2, 8.9 Hz, H-3′′), 4.03 (1H, dd, J = 8.9, 7.6 Hz,
H-2′′), 4.00 (1H, dd, J = 6.1, 6.1 Hz, H-5′) and 3.81 (1H,
ddd, J = 9.5, 5.5, 4.0 Hz, H-5′′); 13C-NMR spectrum of
21 was listed in table III;Anal. calcd. for C39H64O13.H2O:
C, 61.72; H, 8.77. Found: C, 61.33; H, 8.65.
5.1.1.14. Acid hydrolysis of 20 and 21
Solutions of 20 and 21 in 2 N HCl in dioxane/H2O
were heated at 80 °C for 16 h. After-treatment of both
solutions in the same manner as hydrolysis of 3, 2 was
obtained quantitatively.
5.1.1.15. Ring-closure reaction of 19
5.1.1.13. Ring-closure reaction of 18
A solution of 19 (0.6 g, 0.53 mmol) in 5% KOH in
EtOH/H2O (1:1, 5 mL) was allowed to stand overnight at
room temperature. After-treatment of the solution in the
same manner as the ring-closure reaction of 18 gave
compounds 4 (15 mg, 3.8%), 22 (166 mg, 41.5%), 23
(45 mg, 11.3%) and 24 (8 mg, 2.0%). Compound 4 was
identified with marcogenin diglycoside by comparison of
HPLC and 1H-NMR spectrum. FABMS of 22 m/z 779 [M
+ Na]+; signals of CH3-18, CH3-19, CH3-21, CH3-27,
H-20, H-26a and H-26b in the 1H-NMR specrum
(pyridine-d5) were listed in table I. Other assignable
signals were as follows: δ 5.27 (1H, d, J = 7.6 Hz, H-1′′),
4.99 (1H, d, J = 7.6 Hz, H-1′), 4.71 (1H, dd, J = 8.9, 7.6
Hz, H-2′), 4.51 (1H, d, J = 3.1 Hz, H-4′), 4.53–4.43 (5H,
H-16, 6′a, 6′b, 6′′a and 6′′b), 4.38 (1H, br. s, H-3), 4.27
(2H, H-3′ and 4′′), 4.19 (1H, dd, J = 8.9, 8.9 Hz, H-3′′),
4.11 (1H, dd, J = 8.9, 7.6 Hz, H-2′′), 4.09 (1H, m, H-5′)
and 3.85 (1H, m, H-5′′); 13C-NMR spectrum were listed
in table III; Anal. calcd. for C39H64O14.H2O: C, 60.45; H,
8.58. Found: C, 60.19; H, 8.47. FABMS of 23 m/z 779 [M
+ Na]+; signals of CH3-18, CH3-19, CH3-21, CH3-27,
H-20, H-26a and H-26b in the 1H-NMR specrum
(pyridine-d5) were listed in table I; other assignable
signals of 23 were as follows; δ 5.29 (1H, d, J = 7.6 Hz,
H-1′′), 5.03 (1H, d, J = 7.6 Hz, H-1′), 4.73 (1H, dd, J =
A solution of 18 (5 g, 4.64 mmol) in 5% KOH in
EtOH/H2O (1:1, 20 mL) was allowed to stand overnight
at room temperature. The reaction mixture was acidified
with acetic acid (pH 4.0), then neutralized with pyridine.
The mixture was evaporated to give a residue that was
subjected to column chromatography (CHCl3/MeOH/
H2O, 65:35:10, lower layer), followed by application of
preparative HPLC (3:7 H2O/MeOH), to give compound 3
(fine needles, m.p. more than 300 °C after recrystalliza-
tion from acetone/ethanol, 95 mg, 2.8%), 20 (white
needles, m.p. more than 300 °C after recrystallization
from acetone/ethanol, 1.6 g, 46.5%), and 21 (white foam,
690 mg, 20.1%). Compound 3 was identified as timosa-
ponin A-III by HPLC and 1H-NMR spectrum. FABMS of
20 m/z 763 [M + Na]+; signals of CH3-18, CH3-19,
CH3-21, CH3-27, H-20, H-26a and H-26b in the 1H-NMR
spectra (pyridine-d5) were listed in table I. Other assign-
able signals were as follows: δ 5.25 (1H, d, J = 7.6 Hz,
H-1′′), 4.90 (1H, d, J = 7.6 Hz, H-1′), 4.64 (1H, dd, J =
9.5, 7.6 Hz, H-2′), 4.54 (1H, d, J = 3.1 Hz, H-4′), 4.53
(1H, ddd, J = 7.3, 7.3, 5.5 Hz, H-16), 4.35–4.55 (4H,
H-6′a, 6′b, 6′′a and 6′′b), 4.32 (1H, br. s, H-3), 4.29 (1H,
dd, J = 9.5, 9.2 Hz, H-4′′), 4.26 (1H, dd, J = 9.5, 3.1 Hz,
H-3′), 4.17 (1H, dd, J = 9.2, 8.9 Hz, H-3′′), 4.06 (1H, dd,