1
264 Journal of Natural Products, 2007, Vol. 70, No. 8
Zhou et al.
Figure 1. Key HMBC correlations of 1.
and 13C NMR spectra of 2 showed two anomeric protons at δ
H
4
.95 (d, J ) 7.8 Hz) and 4.96 (d, J ) 7.0 Hz) and two anomeric
Figure 2. Key HMBC correlations of 3.
C
carbons at δ 102.1 and 105.4, suggesting that there were only
two sugar units in 2. The differences in the H and C NMR spectra
of 1 and 2 showed that 2 lacked the xylose unit found in 1. J-
correlations were observed between the arabinose H-1 (δ
and the glucose C-6 (δ 69.5) as well as the glucose H-1 (δ
and the aglycone C-3 (δ
findings indicated that the terminal arabinose was linked at C-6 of
the inner glucose and the sugar chain was attached to C-3 of the
aglycone. The carbon and proton signals of 2 were fully assigned
1
13
showing that the terminal xylose and rhamnose were linked at the
C-2 and C-4 positions of the inner glucose. J-correlations between
3
3
H
4.96)
4.95)
H-1 (δ
H-1 (δ
H
5.02) of xylose and C-2 (δ
6.38) of rhamnose and C-4 (δ
C
81.5) of glucose and between
76.7) of glucose in the
C
H
H
C
C
76.8) in the HMBC spectrum. These
HMBC spectrum also supported the above observations. In the
HMBC spectrum, long-range correlation between the glucose H-1
(δ 4.82) and the aglycone C-3 (δ 75.8) indicated that the sugar
H C
chain was attached to C-3 of the aglycone (see Figure 2). On the
basis of the observed data, the structure of 3 was established as
1
1
by H- H COSY, TOCSY, HSQC, and HMBC experiments. In
conclusion, the structure of 2 was elucidated as (25R)-3â-hydroxy-
(
25S)-5â-spirostan-3â-hydroxy-3-O-â-xylopyranosyl(1f2)-[R-rham-
5
R-spirostan-12-one-3-O-R-arabinopyranosyl(1f6)-â-glucopyrano-
side.
Filiasparoside C (3) was obtained as a white, amorphous solid.
Its molecular formula was determined as C44 16 on the basis
nopyranosyl(1f4)]-â-glucopyranoside.
Filiasparoside D (4), obtained as a white, amorphous solid, has
the molecular formula C38
H
62
O
12 from HRESIMS measurement
H
72
O
-
1
13
(m/z 709.4167, [M - H] ). Comparison of the H NMR and
C
+
of HRESIMS data (m/z 879.4722, [M + Na] ). The IR spectrum
of 3 exhibited absorptions for hydroxyl groups (3442, 1449 cm
and strong absorption bands at 984, 914, 890, and 865 cm
characteristic of a spirostane-type steroidal sapogenin. However,
NMR spectroscopic data showed that 4 and 3 possess the same
aglycone. Two anomeric proton signals at δ 4.86 (d, J ) 7.8 Hz)
and 4.98 (d, J ) 7.4 Hz) and two anomeric carbon signals at δ
-
1
)
H
-
1
C
1
13
1
02.9 and 105.3 were observed in the H NMR and C NMR
-
1
unlike 1, the weaker intensity of the band at 890 than 914 cm
spectra, respectively, suggesting that the sugar moiety of 4 consists
7
1
showed that 3 belonged to the 25S series of spirostanes. The H
NMR spectrum of 3 showed four methyl proton signals at δ 0.78
s, Me-18), 1.06 (s, Me-19), 1.04 (d, J ) 7.0 Hz, Me-27), and 1.12
d, J ) 6.6 Hz, Me-21) and two typical methylene proton signals
3.34 (d, J ) 11.3 Hz, 26-Ha) and 4.04 (26-Hb) in the
spirostanol. Acid hydrolysis of 3 yielded the aglycone (3a) that
13
of two units. The C NMR data for the sugar moieties of 4 agreed
H
well with those of 2, so these two compounds should contain the
(
(
3
same sugar moieties. As for 2, J-correlations [arabinose H-1 (δ
H
4.98) with glucose C-6 (δ
C
69.6); glucose H-1 (δ
H
4.86) with
at δ
H
aglycone C-3 (δ
C
74.3)] in the HMBC spectrum of 4 indicated that
the terminal arabinose was linked at C-6 of the inner glucose and
the sugar chain was attached to C-3 of the aglycone. All carbon
and proton signals of 4 were fully assigned by H- H COSY,
TOCSY, HSQC, and HMBC experiments. From the above data,
the structure of 4 was elucidated as (25S)-5â-spirostan-3â-hydroxy-
3-O-R-arabinopyranosyl(1f6)-â-glucopyranoside.
13
was identified as sarsasapogenin by comparison of its C NMR
data with those in the literature.8
1
1
+
Fragment ions at m/z 725 [M + H - 132] , 579 [M + H - 132
+
+
-
146] , and 417 [M + H - 132 - 146 - 162] in the ESIMS
1
spectrum suggested that there were three sugar units in 3. The H
NMR spectrum displayed three anomeric protons at δ 4.82 (d, J
7.2 Hz), 5.02 (d, J ) 7.5 Hz), and 6.38 (s), with corresponding
H
Compounds 5 and 6 were identified as aspafiliosides A and B
)
1
13
2
by comparing their H NMR and C NMR data with reported data.
carbon signals at δ
C
101.8, 105.6, and 101.5, respectively, confirm-
All isolates were evaluated for in Vitro cytotoxicity against human
lung carcinoma (A549) and breast adenocarcinoma (MCF-7) cell
lines. The results are listed in Table 5. Compounds 1-6 showed
varying degrees of cytotoxic activity against the two cell lines,
except that 5 had no effect on the MCF-7 cell line (EC50 >20 µg/
mL). The most potent compound, 3, exhibited significant cytotox-
icity against A549 and MCF-7 cell lines with EC50 values of 2.3
and 3.0 µg/mL, respectively. Compound 4 was as potent against
the A549 cell line (EC50 2.4 µg/mL) but less potent against the
MCF-7 cell line (EC50 10.3 µg/mL).
ing that the sugar moiety of 3 consists of three units. Complete
acid hydrolysis of 3 yielded glucose, xylose, and rhamnose,
1
identified by TLC and GC analysis. H NMR coupling constants
3
(
J
1,2 > 7 Hz) for anomeric protons indicated that the anomeric
carbon configurations were â for the xylose and glucose moieties.
An R-configuration for rhamnose was deduced from the C-5 signal
of rhamnose at δ 69.4. The combined use of H- H COSY,
TOCSY, HSQC, and HMBC experiments allowed the sequential
assignments of all resonances for each monosaccharide. Starting
9
1
1
C
from the anomeric proton signal at δ
at δ 4.20 (1 H, m), 4.22 (1 H, m), 4.25 (1 H, m), 3.78 (1 H, m),
.40 (1 H, dd, J ) 3.1, 12.4 Hz), and 4.50 (1 H, dd, J ) 3.7, 12.4
Hz) were assigned to the glucose H-2, H-3, H-4, H-5, and H -6,
respectively, on the basis of H- H COSY analysis. The carbon
signals at δ 101.8, 81.5, 77.5, 76.7, 76.1, and 61.6 were assigned
H
4.82, the proton resonances
Because compounds 3-6 have the same aglycone, differences
in their cytotoxic activities likely are related to the numbers,
identities, and linkages of the sugar moieties. Compound 3 is the
only compound with an attached rhamnose unit or with a sugar at
C-2 of the inner glucose. The aglycone unit also affects activity,
as compounds 2 and 4 have the same sugar units and linkage
positions, but the cytotoxic activity of 2 is weaker than that of 4.
The aglycones differ in the stereochemistries at C-5 and C-25 and
in the presence of a carbonyl at C-12. This latter difference has
been associated previously with decreased cytotoxicity, as discussed
H
4
2
1
1
C
to the glucose C-1, C-2, C-3, C-4, C-5, and C-6, respectively, from
the HSQC analysis. The proton and carbon signals of xylose and
rhamnose were fully assigned by the same method.
The glucose C-2 and C-4 resonances were downfield shifted to
-glucose,10
in the literature.
11
δ
C
81.5 and 76.7 in 3 from δ
C
74.2 and 70.8 in 1-OCH
3