Two new cytotoxic nonsulfated pentasaccharide holostane (=20-hydroxylanostan-18-oic acid γ-lactone) glycosides from the sea cucumber Holothuria grisea

Article abstract of DOI:10.1002/hlca.200890158

Two new lanostane-type nonsulfated pentasaccharide triterpene glycosides, 17-dehydroxyholothurinoside A (1) and griseaside A (2), were isolated from the sea cucumber Holothuria grisea. Their structures were elucidated by spectroscopic methods, including 2D-NMR and MS experiments, as well as chemical evidence. Compounds 1 and 2 possess the same pentasaccharide moieties but differ slightly in their side chains of the holostane-type triterpene aglycone. The structures of the two new glycosides were established as (3β,12α)-22, 25-epoxy-3-{(O-β-D-glucopyranosyl-(1 → 4)-O-[O-3-O-methyl-β-D- glucopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-β-D-glucopyranosyl-(1 → 2)]-β-D-xylopyranosyl)-oxy}- 12,20-dihydroxylanost-9(11)-en-18-oic acid γ-lactone (1) and (3β,12α)-3-{(O-β-D-glucopyranosyl-(1 → 4)-O-[O-3-O-methyl- β-D-glucopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-β-D-glucopyranosyl-(1 → 2)]-β-D-xylopyranosyl)oxy}-12, 20,22-trihydroxylanost-9(11)-en-18-oic acid γ-lactone (2). The 17-dehydroxyholothurinoside A (1) and griseaside A (2) exhibited significant cytotoxicity against HL-60, BEL-7402, Molt-4, and A-549 cancer cell lines.

Full text of DOI:10.1002/hlca.200890158

Helvetica Chimica Acta – Vol. 91 (2008)
1453
Two New Cytotoxic Nonsulfated Pentasaccharide Holostane
(¼20-Hydroxylanostan-18-oic Acid g-Lactone) Glycosides from the Sea
Cucumber Holothuria grisea
by Guo-Quan Suna), Ling Li*a), Yang-Hua Yi*a), Wei-Hua Yuana), Bao-Shu Liua), Yi-Yi Wenga),
Shi-Long Zhanga), Peng Suna), and Zeng-Lei Wanga)
a) Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University,
325 Guohe Road, Shanghai 200433, P. R. China
(phone: þ 862165384988; fax: þ 862165483662; e-mail: yiyanghua@hotmail.com)
Two new lanostane-type nonsulfated pentasaccharide triterpene glycosides, 17-dehydroxyholothur-
inoside A (1) and griseaside A (2), were isolated from the sea cucumber Holothuria grisea. Their
structures were elucidated by spectroscopic methods, including 2D-NMR and MS experiments, as well as
chemical evidence. Compounds 1 and 2 possess the same pentasaccharide moieties but differ slightly in
their side chains of the holostane-type triterpene aglycone. The structures of the two new glycosides were
established as (3b,12a)-22,25-epoxy-3-{(O-b-d-glucopyranosyl-(1 ! 4)-O-[O-3-O-methyl-b-d-glucopyr-
anosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-6-deoxy-b-d-glucopyranosyl-(1 ! 2)]-b-d-xylopyranosyl)-
oxy}-12,20-dihydroxylanost-9(11)-en-18-oic acid g-lactone (1) and (3b,12a)-3-{(O-b-d-glucopyranosyl-
(1 ! 4)-O-[O-3-O-methyl-b-d-glucopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-6-deoxy-b-d-glu-
copyranosyl-(1 ! 2)]-b-d-xylopyranosyl)oxy}-12,20,22-trihydroxylanost-9(11)-en-18-oic acid g-lactone
(2). The 17-dehydroxyholothurinoside A (1) and griseaside A (2) exhibited significant cytotoxicity
against HL-60, BEL-7402, Molt-4, and A-549 cancer cell lines.
Introduction. – More than hundred triterpene glycosides found in holothurians
have been described to date, and the majority of them are lanostane-type triterpenes
with an 18,20-lactone moiety and a sugar chain linked to C(3) of the aglycone [1]. The
carbohydrate chain consists mostly of disaccharides, tetrasaccharides, and hexasac-
charides. These glycosides are the predominant secondary metabolites of holothurians
and responsible for their general toxicity and have been reported to have a wide
spectrum of biological activities, including cytotoxic, antifungal, hemolytic, and
immunomodulatory effects [2][3]. Holothuria grisea Selenka (family Holothuridae)
is an abundant sea cucumber distributed from Florida south through the Bahamas to
Brazil. Although previous studies of this sea cucumber have yielded a series of
triterpene glycosides [4], knowledge on the constituents of Holothuria grisea remains
fragmentary. Herein, we report the isolation and structure elucidation of two new
constituents, 17-dehydroxyholothurinoside A (1) and griseaside A (2), as well as their
potent cytotoxicities against various human tumor cell lines.
Results and Discussion. – The EtOH extract of H. grisea was partitioned between
H2O and BuOH. The BuOH extract was subjected to repeated chromatographic
purification. Finally, reversed-phase HPLC on Zobax SBC-18 afforded compounds 1
and 2. DEPT and 13C-NMR Spectral data of compounds 1 and 2 (Table 1) exhibited
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

1454
Helvetica Chimica Acta – Vol. 91 (2008)
that the carbohydrate moieties of these new glycosides were identical to each other and
coincident with that of the earlier known holothurinoside A [5]. The relative
configurations at all stereogenic C-atoms of the oligosaccharide chain were established
by NOESY experiments, as shown in the Figure. Glycosides 1 and 2 were also treated
with 2m CF3COOH and gave d-quinovose (¼6-deoxy-d-glucose; Qui), d-xylose
(Xyl), 3-O-methyl-d-glucose (MeGlc), and d-glucose (Glc) in the ratio 1:1:1:2,
together with a mixture of triterpene aglycones. The monosaccharides were identified
by GC/MS in the form of the peracetates of the corresponding alditols [6].
Figure. 1H,1H-NOESY of the carbohydrate moieties of 1 and 2

Helvetica Chimica Acta – Vol. 91 (2008)
1455
Table 1. 1H- and 13C-NMR Data and Selected HMBC Correlations of the Carbohydrate Moieties of 1 and
2. d in ppm, J in Hz.
d(H)
d(C)
1H,13C-HMBC
Xyl1(1 ! C(3))
HꢀC(1)
4.78 (d, J ¼ 7.2)
105.5
83.7
76.0
78.1
64.4
C(3)
HꢀC(2)
4.08 – 4.11 (m)
C(1)(Qui2), C(1)(Xyl1)
C(2)(Xyl1)
HꢀC(3)
4.29 – 4.33 (m)
4.07 – 4.10 (m)
3.68 – 3.72 (m), 4.43 – 4.46 (m)
HꢀC(4)
C(1)(Glc5)
C(1,3)(Xyl1)
CH2(5)
Qui2(1 ! C(2)(Xyl1))
HꢀC(1)
5.20 (d, J ¼ 7.2)
4.18 – 4.20 (m)
4.10 – 4.12 (m)
3.70 – 3.73 (m)
3.83 – 3.86 (m)
1.82 (d, J ¼ 7.3)
105.7
76.1
76.6
87.6
71.9
18.4
C(2)(Xyl1)
C(4)(Qui2)
C(1)(Qui2)
C(1,6)(Glc3)
C(1,6)(Qui2)
C(4,5)(Qui2)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
Glc3(1 ! C(4)(Qui2))
HꢀC(1)
5.03 (d, J ¼ 7.6)
103.5
73.9
88.3
70.1
78.4
62.4
C(4)(Qui2)
C(1,3)(Glc3)
HꢀC(2)
3.98 – 3.99 (m)
HꢀC(3)
4.28 – 4.32 (m)
4.07 – 4.11 (m)
4.33 – 4.36 (m)
4.24 – 4.28 (m), 4.50 – 4.54 (m)
C(1)(MeGlc4), C(1)(Glc3)
C(3)(Glc3)
HꢀC(4)
HꢀC(5)
C(1)(Glc3)
CH2(6)
MeGlc4(1 ! C(3)(Glc3))
HꢀC(1)
5.35 (d, J ¼ 7.8)
4.05 – 4.07 (m)
3.76 – 3.79 (m)
4.20 – 4.24 (m)
4.02 – 4.05 (m)
4.31 – 4.33 (m), 4.52 – 4.55 (m)
3.92 (s)
105.8
75.3
88.2
70.8
78.5
62.4
61.0
C(3)(Glc3)
HꢀC(2)
C(1)(MeGlc4)
C(4)(MeGlc4), MeGlc4
C(5)(MeGlc4)
C(4)(MeGlc4)
C(4)(MeGlc4)
C(3)(MeGlc4)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
MeOꢀC(3)
Glc5(1 ! C(4)(Xyl1))
HꢀC(1)
5.09 (d, J ¼ 7.4)
105.2
74.5
79.0
71.8
77.5
62.7
C(4)(Xyl1)
C(1)(Glc5)
C(2)(Glc5)
C(5)(Glc5)
C(4,6)(Glc5)
C(4)(Glc5)
HꢀC(2)
4.10 – 4.12 (m)
HꢀC(3)
3.90 – 3.93 (m)
4.26 – 4.29 (m)
4.28 – 4.31 (m)
4.37 – 4.39 (m), 4.60 – 4.63 (m)
HꢀC(4)
HꢀC(5)
CH2(6)
The presence of five monosaccharide units in 1 and 2 was confirmed by the 13C-NMR signals of the
anomeric centers at d(C) 105.5, 105.7, 103.5, 105.8, and 105.2, correlated by HMQC with the
corresponding anomeric H-atoms at d(H) 4.78 (d, J ¼ 7.2 Hz), 5.20 (d, J ¼ 7.2 Hz), 5.03 (d, J ¼ 7.6 Hz ) ,
5.35 (d, J ¼ 7.8 Hz), and 5.09 ppm (d, J ¼ 7.4 Hz). The large vicinal coupling constants of each anomeric
H-atom indicated trans-diaxial orientations with respect to their coupling partners (b-configurations) [7].
A DQCOSY experiment allowed the sequential assignment of most of the resonances for each sugar
moiety, starting from the easily distinguished signals due to the anomeric H-atoms. Complete
assignments were achieved by a combination of HMQC, HMBC, DQCOSY, and TOCSY results. The
HMQC experiment allowed us to correlate all sugar 1H- and 13C-NMR resonances. The data from these
experiments (Table 1) indicated that the sugar residues were all in their pyranose form, and the d-
configuration for the carbohydrate units was assumed also according to those most often encountered

1456
Helvetica Chimica Acta – Vol. 91 (2008)
among the sea-cucumber glycosides [8][9]. The interglycosidic linkages were deduced by analysis of the
chemical shifts d(C) for C(2) of Xyl1 (83.7), for C(4) of Xyl1 (78.1), for C(4) of Qui2 (87.6), and for C(3)
of Glc3 (88.3), which were shifted downfield relative to the resonances expected for the corresponding
methyl glycopyranosides [10]. The structure of the carbohydrate chain was corroborated by an HMBC
experiment: a cross-peak between C(3) of the aglycone and HꢀC(1) of Xyl1 indicated that this sugar
residue was connected to C(3) of the aglycone. Similarly, the location of the interglycoside linkage in the
oligosaccharide chain was confirmed on the basis of the cross-peaks HꢀC(1)(Qui2)/C(2)(Xyl1),
HꢀC(1)(Glc3)/C(4)(Qui2), HꢀC(1)(MeGlc4)/C(3)(Glc3), and HꢀC(1)(Glc5)/C(4)(Xyl1). Therefore,
the sugar sequence was elucidated as Glc-(1 ! 4)-[MeGlc-(1 ! 3)-Glc-(1 ! 4)-Qui-(1 ! 2)]-Xyl.
The 17-dehydroxyholothurinoside A (1), a colorless amorphous powder, tested
positive in both the Liebermann – Burchard and Molish tests. The molecular formula
was established as C60H96O28 by the pseudomolecular ion [M þ Na]þ at m/z 1287.5991 in
the HR-ESI-MS (positive-ion mode). The IR spectrum of 1 showed the presence of a
g-lactone moiety (1762), olefinic C¼C bonds (1635), and strong, broad absorptions
due to sugar OH functions (3376 cmꢀ1). The NMR data of the aglycon part of 1
(Table 2) were very close to those of the known holothurinoside A [5]. But the signals
of C(16) and C(17) were shifted upfield (d 23.9 and 47.9), correspondingly, due to the
absence of an OH group at C(17). The absence of OHꢀC(17) was also confirmed by
the upfield shift of the signal of C(20) (d 83.8) [10]. On the basis of the 13C-NMR
spectrum of the aglycon part of 1, it was concluded that this aglycon was identical to
that of holothurin B3 from the holothurian H. polii [11]. This structure was confirmed
by extensive NMR spectroscopy (1H- and 13C-NMR, HMQC, HMBC, DQCOSY,
TOCSY, and NOESY). In further confirmation of the carbohydrate-chain structure,
the ESI-MS of 1 exhibited a pseudomolecular ion peak at m/z 1287 ([M þ Na]þ) in the
positive-ion mode, and a pseudomolecular ion peak at m/z 1263 ([M ꢀ H]ꢀ) as well as
peaks resulting from the loss of sugar moieties from the [M ꢀ H]ꢀ ion at m/z 1087
([M ꢀ H ꢀ MeGlc]ꢀ), 925 ([1087 ꢀ Glc]ꢀ), 763 ([925 ꢀ Glc]ꢀ), and 617 ([763 ꢀ Qui]ꢀ)
in the negative-ion mode. All these data including results of the 2D-NMR experiments
indicate that 17-dehydroxyholothurinoside A (1) is (3b,12a)-22,25-epoxy-3-{(O-b-d-
glucopyranosyl-(1 ! 4)-O-[O-3-O-methyl-b-d-glucopyranosyl-(1 ! 3)-O-b-d-glucopy-
ranosyl-(1 ! 4)-6-deoxy-b-d-glucopyranosyl-(1 ! 2)]-b-d-xylopyranosyl)oxy}-12,20-di-
hydroxylanost-9(11)-en-18-oic acid g-lactone.
Griseaside A (2), a colorless amorphous powder, tested positive in both the
Liebermann – Burchard and Molish tests. The molecular formula was established as
C60H98O28 by the pseudomolecular ion [M þ Na]þ at m/z 1289.6139 in the HR-ESI-MS
(pos.). 13C-NMR and DEPT spectra of 2 (Table 3) showed that the signals of C(1) to
C(18) are coincident with those of the aglycon part of 17-dehydroxyholothurinoside A
(1). The 13C-NMR spectrum of the side chain of the aglycon moiety of 2 was identical
to that of the tetraoside holothurin A1 [4]. The configuration at C(22) was not
established.
The presence of an OH group at C(12) of 2 was indicated by the characteristic signal at d 68.5. The
Me(21) (d(H) 1.90) exhibited a NOESY correlation with HbꢀC(12) (d(H) 4.72 (J ¼ 6.1)), which
indicated the a-configuration of the OH group at C(12) [12] (Table 3). On the other hand, the signals of
the side chain differed from those of 1 by the absence of the signals at d(C) 80.4 and 81.4, characteristic
for a 22,25-epoxy group. Moreover, the signal of C(22) was observed at d(C) 75.2, which indicated the
presence of an OH group at this position of the side chain of griseaside A (2).

Helvetica Chimica Acta – Vol. 91 (2008)
1457
Table 2. 1H- and 13C-NMR Data and Selected HMBC and NOESY Correlations of the Aglycon Moieties
of 1. d in ppm, J in Hz.
Position
d(H)
d(C) 1H,1H-NOESY
1H,13C-HMBC
CH2(1)
CH2(2)
HꢀC(3)
1.53 – 1.57, 1.92 – 1.98 (2m)
2.19 – 2.23, 1.98 – 2.03 (2m)
3.24 (dd, J ¼ 4.0, 11.6)
36.7
27.3 Me(19)
88.8 HꢀC(1)(Xyl1),
C(1)(Xyl1), C(4)
HꢀC(5), Me(28)
C(4)
40.2
HꢀC(5)
CH2(6)
CH2(7)
HꢀC(8)
C(9)
1.09 (d, J ¼ 6.9)
53.0 HꢀC(3), Me(28)
C(6), C(19), C(28)
C(9), C(11), C(14)
C(8), C(10), C(12), C(13)
1.62 – 1.66, 1.81 – 1.85 (2m)
1.52 – 1.54, 1.85 – 1.89 (2m)
3.50 – 3.53 (m)
21.4
29.0
40.3 CH2(15), Me(19)
153.4
39.8
C(10)
HꢀC(11) 5.82 (d, J ¼ 5.1)
HꢀC(12) 4.65 (d, J ¼ 4.0)
C(13)
116.3 HꢀC(12), Me(19)
68.1 HꢀC(11), HꢀC(12) C(9), C(11), C(14), C(18)
64.0
C(14)
46.4
CH2(15)
CH2(16)
1.51 – 1.55, 1.74 – 1.77 (2m)
2.14 – 2.19, 2.43 – 2.48 (2m)
37.5
23.9 Me(30)
HꢀC(17) 3.46 – 3.50 (m)
47.9 Me(21), HꢀC(24)
C(12), C(13), C(18), C(21)
C(1), C(5), C(9), C(10)
C(18)
177.7
Me(19)
1.50 (s)
22.7 CH2(2), HꢀC(8),
HꢀC(11), Me(29)
83.8
C(20)
Me(21)
1.69 (s)
21.0 HꢀC(12), HꢀC(17) C(17), C(20), C(22)
HꢀC(22) 4.30 – 4.33 (m)
80.4
C(17), C(20), C(21)
CH2(23)
CH2(24)
C(25)
1.97 – 2.03 (m)
2.09 – 2.23, 1.70 – 1.75 (2m)
27.3
C(20), C(24), C(25)
38.8 HꢀC(17)
C(22), C(23), C(26), C(27)
81.4
Me(26)
Me(27)
Me(29)
Me(28)
Me(30)
1.34 (s)
1.30 (s)
1.19 (s)
1.35 (s)
1.38 (s)
28.8
27.5
C(24), C(25), C(27)
C(24), C(25), C(26)
C(3), C(4), C(5), C(28)
C(3), C(4), C(5), C(29)
C(8), C(13), C(14), C(15)
16.9 Me(19)
28.3 HꢀC(3), HꢀC(5)
22.3 HꢀC(16)
The ESI-MS of 2 exhibited a pseudomolecular ion peak at m/z 1289 ([M þ Na]þ) in
the positive-ion mode, and pseudomolecular ion peaks at m/z 1265 ([M ꢀ H]ꢀ) as well
as peaks resulting from the loss of sugar moieties from the [M ꢀ H]ꢀ ion at m/z 1089
([M ꢀ H ꢀ MeGlc]ꢀ), 927 ([1089 ꢀ Glc]ꢀ), 765 ([927 ꢀ Glc]ꢀ), and 619 ([765 ꢀ Qui]ꢀ)
in the negative-ion mode. All these data including results of the 2D-NMR experiments
indicate that griseaside A (2) is (3b,12a)-3-{(O-b-d-glucopyranosyl-(1 ! 4)-O-[O-3-O-
methyl-b-d-glucopyranosyl-(1 ! 3)-O-b-d-glucopyranosyl-(1 ! 4)-6-deoxy-b-d-gluco-
pyranosyl-(1 ! 2)]-b-d-xylopyranosyl)oxy}-12,20,22-trihydroxylanost-9(11)-en-18-dio-
ic acid g-lactone.
Glycosides 1 and 2 were tested for in vitro cytotoxicity against four human tumor
cell lines (A549, HL-60, BEL-7402, and Molt-4) by using the sulforhodamine B (SRB)
method [13]; 10-hydroxycamptothecine (HCP) was used as reference compound (IC50

1458
Helvetica Chimica Acta – Vol. 91 (2008)
Table 3. 1H- and 13C-NMR Data and Selected HMBC and NOESY Correlations of the Aglycon Moieties
of 2. d in ppm, J in Hz.
Position
d(H)
d(C) 1H,1H-NOESY
1H,13C-HMBC
C(3)
CH2(1)
CH2(2)
HꢀC(3)
1.54 – 1.58, 1.95 – 1.97 (2m)
2.23 – 2.27 (m)
3.20 – 3.23 (m)
36.6
27.3 Me(19)
88.9 HꢀC(1)(Xyl1),
C(1)(Xyl1), C(4)
HꢀC(5), Me(28)
C(4)
40.2
HꢀC(5)
CH2(6)
CH2(7)
HꢀC(8)
C(9)
1.09 (d, J ¼ 5.8)
1.89 – 1.93 (m)
1.78 – 1.82 (m)
3.53 – 3.57 (m)
53.0 HꢀC(3), Me(28)
C(6), C(19), C(28)
C(9), C(11), C(14)
C(8), C(12), C(13)
21.4
29.1
40.4 CH2(15), Me(19)
153.3
39.8
C(10)
HꢀC(11) 5.81 (d, J ¼ 4.6)
HꢀC(12) 4.72 (d, J ¼ 6.1)
C(13)
116.4 HꢀC(12), Me(19)
68.5 HꢀC(11), HꢀC(12) C(9), C(11), C(14), C(18)
64.2
C(14)
46.5
CH2(15)
CH2(16)
1.58 – 1.62, 1.77 – 1.79 (2m)
2.41 – 2.45, 2.97 – 3.03 (2m)
37.5
23.9 Me(30)
HꢀC(17) 3.66 – 3.71 (m)
47.9 Me(21), HꢀC(24)
C(13), C(14), C(18)
C(18)
177.9
Me(19)
1.52 (s)
22.7 CH2(2), HꢀC(8),
HꢀC(11), Me(29)
85.8
C(1), C(5), C(9), C(10)
C(20)
Me(21)
1.90 (s)
21.0 HꢀC(12), HꢀC(17) C(17), C(20), C(22)
HꢀC(22) 4.05 – 4.08 (m)
75.2 HꢀC(23)
31.0 HꢀC(22)
36.5 HꢀC(17)
28.7
C(17), C(20), C(21)
CH2(23)
CH2(24)
1.84 – 1.88, 1.96 – 1.99 (2m)
1.40 – 1.43, 1.87 – 1.89 (2m)
C(20), C(24), C(25)
C(22), C(23), C(26), C(27)
HꢀC(25) 1.61 – 1.63 (m)
Me(26)
Me(27)
Me(29)
Me(28)
Me(30)
0.98 (s)
1.33 (s)
1.19 (s)
1.47 (s)
1.43 (s)
23.1
C(24), C(25), C(27)
22.8
C(24), C(25), C(26)
16.9 Me(19)
28.3 HꢀC(3), HꢀC(5)
22.3 HꢀC(16)
C(3), C(4), C(5), C(28)
C(3), C(4), C(5), C(29)
C(8), C(13), C(14), C(15)
values in Table 4). The results showed that 1 and 2 exhibited significant cytotoxicity
against the four cell lines with IC50 values in the range of 0.245 – 1.114 mm. Comparison
of the activities of 1, 2, and the positive control showed that 1 and 2 are more active
than HCP on these four cell lines. Based on these initially promising results, 17-
dehydroxyholothurinoside A (1) and griseaside A (2) deserve further study as potent
antitumor agents.
W.-H. Y. thanks the ꢁ863ꢂ Hi-tech Research and Development Program of China (Grant No.
2006AA09Z417), and the Shanghai Leading Academic Discipline Project (Grant No. B906) for generous
financial support.

Helvetica Chimica Acta – Vol. 91 (2008)
1459
Table 4. IC50 Values of Compounds 1 and 2 against Human Tumor Cell Lines in vitro
IC50 [mm]a)
A-549
BEL-7402
HL-60
Molt-4
1
2
0.886 ꢂ 0.042
1.074 ꢂ 0.053
2.335 ꢂ 0.088
0.973 ꢂ 0.038
1.114 ꢂ 0.062
2.610 ꢂ 0.133
0.245 ꢂ 0.006
0.427 ꢂ 0.009
1.978 ꢂ 0.083
0.340 ꢂ 0.009
0.521 ꢂ 0.014
2.252 ꢂ 0.083
HCPb)
a) IC50 Values are means from independent experiments (mean ꢂ s.d.). b) Positive control.
Experimental Part
General. Column chromatography (CC): silica gel (200 – 300 mesh, 10 – 40 mm; Yantai, P. R. China)
and Lobar Lichroprep RP-18 (40 – 63 mm; Merck). TLC: precoated silica gel GF254 plates (10 – 40 mm;
Yantai), detection by spraying with 10% aq. H2SO4 soln. followed by heating. HPLC: Agilent 1100 system
equipped with a refractive-index detector; Zorbax-300-SB-C18 column (250 ꢁ 9.4 mm i.d., 5 mm). M.p.:
XT5-XMTapparatus; uncorrected. Optical rotations: Perkin-Elmer 341 polarimeter. IR Spectra: Bruker
Vector-22 IR spectrometer; KBr pellets; in cmꢀ1 1H- and 13C-NMR Spectra: Varian Inova-600
.
spectrometer, at 600 and 150 MHz; d in ppm, J in Hz; assignments supported by 1H,1H-COSY, HMQC,
HMBC, and NOESY experiments. ESI- and HR-ESI-MS: Waters Q-TOF-Micromass spectrometer. GC/
MS: Finnigan Voyager apparatus with a DB-5 column (0.25 mm ꢁ 30 m i.d.); in m/z. Cell lines: A-549 and
Molt-4: ATCC; BL-60 and BEL-7402: Institute of Biochemistry and Cell Biology, SIBS, CAS.
Animal Material. Specimens of H. grisea were collected from Guangzhou (Guangdong Province) in
the South China Sea in November 2005. The organisms were identified by Professor Yu-Lin Liao
(Institute of Oceanology, Chinese Academy of Science, P. R. China). A voucher specimen was deposited
with the Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University,
Shanghai, China, under the registration code number HG-2005-11.
Extraction and Isolation. The sea cucumbers (2.5 kg, dry weight) were crumbled and extracted with
65% aq. EtOH soln. (5 ꢁ 5 l) for 2 h each. The combined extract was concentrated, and the residue was
dissolved in H2O (4 l) and then partitioned with BuOH (6 ꢁ 4 l). The BuOH fraction (46 g) was subjected
to CC (silica gel, CHCl3/MeOH/H2O 9 :1:1 ! 6.5 :3.5 :1 (lower phase)): Fractions A – I. Fr. I (7.9 g)
mainly contained triterpene glycosides and was subjected to LPLC (Lobar column (Lichroprep RP-18,
40 – 63 mm; Merck), MeOH/H2O 45 :55 ! 80 :20): Fr. I1 and I2. Fr. I1 (960 mg) was further purified by
repeated semi-prep. HPLC (Zorbax-300-SB-C18; 67% aq. MeOH soln., 2 ml/min): pure 1 (860 mg, tR
28.3 min). Fr. I2 was further purified by HPLC (Zorbax-300-SB-C18; 59% aq. MeOH soln., 2 ml/min):
pure 2 (15 mg, tR 27.2 min).
17-Dehydroxyholothurinoside A (1): Colorless amorphous powder. M.p. 225 – 2278. [a]2D0 ¼ ꢀ11.2
(c ¼ 0.785, MeOH). IR (KBr): 3376 (OH), 1762 (C¼O), 1635 (C¼C). 1H- and 13C-NMR: Tables 1 and
2. ESI-MS (pos.): 1287 ([M þ Na]þ). ESI-MS (neg.): 1263 ([M ꢀ H]ꢀ), 1087 ([M ꢀ H ꢀ MeGlc]ꢀ), 925
([1087 ꢀ Glc]ꢀ), 763 ([925 ꢀ Glc]ꢀ), 617 ([763 ꢀ Qui]ꢀ). HR-ESI-MS (pos.): 1287.5991 ([M þ Na]þ,
C60H96O28Naþ; calc. 1287.5986).
Griseaside A (2): Colorless amorphous powder. M.p. 230 – 2328. [a]2D0 ¼ ꢀ10 (c ¼ 0.10, MeOH). IR
(KBr): 3445 (OH), 1764 (C¼O), 1632 (C¼C). 1H- and 13C-NMR: Tables 1 and 3. ESI-MS (pos.): 1289
([M þ Na]þ). ESI-MS (neg.): 1265 ([M ꢀ H]ꢀ), 1089 ([M ꢀ H ꢀ MeGlc]ꢀ), 927 ([1089 ꢀ Glc]ꢀ), 765
([927 ꢀ Glc]ꢀ), 619 ([765 ꢀ Qui]ꢀ). HR-ESI-MS (pos.): 1289.6139 ([M þ Na]þ, C60H98O28Naþ; calc.
1289.6142).
Acid Hydrolysis. The glycoside 1 or 2 (1 mg) was heated in 2m aq. CF3COOH (1 ml) at 1208 for 2 h.
The mixture was concentrated and the residue partitioned between CH2Cl2 and H2O. The aq. phase was
evaporated to furnish a monosaccharide mixture. Pyridine (1 ml) and NH2OH · HCl (2 mg) were added
to the dried residue, and the mixture was heated at 908 for 30 min. Then, Ac2O (0.8 ml) was added, and
heating at 908 was continued for 1 h. The resulting soln. was concentrated, and the formed aldononitrile

1460
Helvetica Chimica Acta – Vol. 91 (2008)
peracetates were analyzed by GC/MS by using authentic reference samples for identification. The
derivatives of d-xylose (Xyl), d-quinovose (Qui), d-glucose (Glc), and 3-O-methyl-d-glucose (MeGlc)
were detected with the retention times (tR) 5.53, 5.44, 6.76, and 6.57 min, resp. Glycoside 1 and 2 gave
peaks of the derivatives of d-Xyl, d-Qui, d-Glc, and MeGlc in a ratio of 1:1:2 :1 [9].
REFERENCES
[1] V. A. Stonik, G. B. Elyakov, in ꢁBioorganic Marine Chemistyꢂ, Ed. P. J. Scheuer, Springer-Verlag,
Berlin, 1988, p. 43 – 88.
[2] H. Chludil, C. C. Muniain, A. M. Seldes, M. S. Maier, J. Nat. Prod. 2002, 65, 860.
[3] M. S. Maier, A. J. Roccatagliata, A. Kuriss, H. Cludil, A. M. Seldes, C. A. Pujol, E. B. Damonte, J.
Nat. Prod. 2001, 64, 732.
[4] G. K. Oleinikova, T. A. Kuznetsova, N. S. Ivanova, A. I. Kalinovskii, N. V. Rovnykh, G. B. Elyakov,
Chem. Nat. Compd. 1983, 18, 501.
[5] J. Rodriguez, R. Castro, R. Riguera, Tetrahedron 1991, 47, 4753.
[6] H. F. Tang, Y. H. Yi, L. Li, P. Sun, S. Q. Zhang, Y. P. Zhao, Planta Med. 2005, 71, 458.
[7] F. S. Shashkov, O. S. Chizhov, Bioorg. Khim. 1976, 2, 437.
[8] V. A. Stonik, V. I. Kalinin, S. A. Avilov, J. Nat. Toxins 1999, 8, 235.
[9] S. A. Avilov, V. I. Kalinin, A. V. Smirnov, Biochem. Syst. Ecol. 2004, 32, 715.
[10] E. Breitmaier, W. Voelter, ꢁCarbon-13 NMR Spectroscopyꢂ, VCH, Weinheim, 1987.
[11] A. S. Silchenko, V. A. Stonik, S. A. Avilov, V. I. Kalinin, A. I. Kalinovsky, A. M. Zaharenko, A. V.
Smirnov, E. Mollo, G. Cimino, J. Nat. Prod. 2005, 68, 564.
[12] I. Kitagawa, T. Nishino, T. Matsuno, H. Akutsu, Y. Kyogoku, Tetrahedron Lett. 1978, 11, 985.
[13] P. Skehan, R. Storeng, D. Scudiero, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney,
M. R. Boyd, J. Natl. Cancer Inst. 1990, 82, 1107.
Received February19, 2008