NATuRAl PRODuCT RESEARCH
3
the β-configurations of the oxygenated substituents at C-1 and C-3. Moreover, the configura-
tions of C-23 and C-24 were characterised as S by the NOESY correlations between H-23 and
H-20, between H-23 and H2–21/H2–27, between H-24 and H2–27 (Mimaki & Watanabe 2008; Hayes
et al. 2009). Comparison of the 1H and 13C NMR spectroscopic data of the aglycone moiety of 1
with those of bethoside A (Hayes et al. 2009), along with the above analysis, the structure of the
aglycone of 1 was elucidated as (23S,24S)-1β,3β,21,23,24-pentahydroxy-spirosta-5,25(27)-diene.
For the sugar moiety, the five anomeric protons at δH 6.46 (1H, br s), 5.14 (1H, d, J = 7.0 Hz), 5.13
(1H, d, J = 7.0 Hz), 4.91 (1H, d, J = 7.0 Hz) and 4.67 (1H, d, J = 7.5 Hz) showed correlations with
the anomeric carbon resonances at δC 100.7, 105.9, 106.8, 106.6 and 100.5 in the HSQC spec-
trum, respectively. And two Me groups were observed at δH 1.36 (3H, d, J = 5.5 Hz), 1.50 (3H, d,
J = 6.0 Hz) in the 1H NMR spectrum, and δC 18.3, δC 17.4 in the 13C NMR spectrum, respectively,
which implied two of them were 6-deoxyhexose units. The Me group at δH 2.00 (3H, s) and δC
20.9 and C=O signal at δC 170.7 were assignable to one acetyl group. Acid hydrolysis of 1 with
1 M HCl in dioxane–H2O (1:1) followed by TlC analysis showed the presence of arabinose (Ara),
rhamnose (Rha), xylose (Xyl), fucose (Fuc) and glucose (Glc). One glycosyl group attached to C-1
position of the aglycone was established from the HMBC correlations of signals at δH 4.67 (H-1 of
Ara) with δC 83.7 (C-1 of the aglycone), H-1 (δH 6.46) of Rha with C-2 (δC 72.6) of Ara, H-1 (δH 4.91)
of Xyl with C-3 (δC 85.0) of Ara, which was also supported by the NOESY correlations of signals at
H-1 (δH 3.79) of aglycone with H-1 (δH 4.67) of Ara, H-2 (δH 4.59) of Ara with H-1 (δH 6.46) of Rha,
H-3 (δH 4.07) of Ara with H-1 (δH 4.91) of Xyl. The other sugar chain located at C-24 (δC 82.2) of 1
was deduced by a downfield shift of 8.1 ppm of C-24 (δC 74.1) compared with clintonioside B
(Mimaki & Watanabe 2008), and the HMBC correlations between H-24 (δH 4.75) of the aglycone
and C-1 (δC 105.9) of Fuc, and between C-4 (δC 83.2) of Fuc to H-1 (δH 5.13) of Glc proved the
linkage of the sugars and the aglycone, which was further supported by the NOESY cross-peaks
between H-24 (δH 4.75) of aglycone and H-1 (δH 5.14) of Fuc, between H-4 (δH 4.04) of Fuc and
H-1 (δH 5.13) of Glc. Full assignments of 1 were achieved by a comprehensive analysis of DEPT,
COSY, HSQC, NOESY and HMBC spectra. On the basis of the above evidence, the structure of the
new spirostanol glycoside 1 was fully determined to be (23S,24S)-21-hydroxymethyl-24-{[O-β-
d-glucopyranosyl-(1→4)-β-d-fucopyranosyl]oxy}-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl
O-(4-O-acetyl-α-l-rhamnopyranosyl)-(1→2)-O-[β- -xylopyranosyl-(1→3)]-α-l-arabinopyranoside.
d
Compound 2, a white amorphous solid, exhibited the ion peak at m/z 1193.5226
[M − H]− in the HRESIMS data, corresponding to the molecular formula C55H85O28, which
displayed the lack of C2H3O compared with 1. The IR absorption at 3424 cm−1 revealed
1
the presence of hydroxyl groups. Complete assignments of the H and 13C NMR signals
of 2 were accomplished by a combined analysis of DEPT, COSY, HSQC, NOESY and HMBC
1
spectra. A detailed comparison of the H, 13C NMR chemical shifts of 1 and 2, revealed
that they shared the same skeleton and same glycosidic positions at C-1 and C-24, except
for the disappearance of the carbon signal at δC 170.7 and δC 20.9 in the 13C NMR spec-
1
trum and the proton signal of δH 2.00 (3H, s) in the H NMR spectrum of compound 2.
Meanwhile, the chemical shift of Me signal of Rha at δH 1.36 (3H, d, J = 5.5 Hz) moved down-
field to δH 1.66 (3H, d, J = 6.0 Hz), these data suggested the acetyl linked at C-4 of Rha was
absent in compound 2. Acid hydrolysis of 2 with 1 M HCl in dioxane-H2O (1:1) gave Ara,
Rha, Xyl, Fuc and Glc. Thus, 2 was deduced to be (23S,24S)-21-hydroxymethyl-24-{[O-β-d-
glucopyranosyl-(1→4)-β-
d-fucopyranosyl]oxy}-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl
O-(α-l-rhamnopy-ranosyl)-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-α-l-arabinopyranoside.