Facile synthesis and cytotoxicity of triterpenoid saponins bearing a unique disaccharide moiety: hederacolchiside A1 and its analogues

Article abstract of DOI:10.1016/j.carres.2007.12.014

An improved synthetic approach toward hederacolchiside A₁, an antitumor triterpenoid saponin bearing a unique disaccharide moiety, was established. This approach began from a partially protected intermediate and avoided tedious protection-deprotection manipulation. An abnormal ring conformation (¹C₄) of the center arabinose residue was found in the intermediate, which may account for the unusual regioselectivity between 3-OH and 4-OH of arabinose. Two analogues of hederacolchiside A₁ were then facilely prepared by this approach and exhibited significant cytotoxicity in preliminary in vitro assay.

Full text of DOI:10.1016/j.carres.2007.12.014

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 780–784
Note
Facile synthesis and cytotoxicity of triterpenoid saponins bearing a
unique disaccharide moiety: hederacolchiside A1 and its analogues
Mao-Cai Yan, Yang Liu, Wen-Xiang Lu, Hui Wang, Yu Sha and Mao-Sheng Cheng*
Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang 110016, PR China
Received 3 June 2007; received in revised form 6 December 2007; accepted 18 December 2007
Available online 25 December 2007
Abstract—An improved synthetic approach toward hederacolchiside A1, an antitumor triterpenoid saponin bearing a unique disac-
charide moiety, was established. This approach began from a partially protected intermediate and avoided tedious protection–
deprotection manipulation. An abnormal ring conformation (1C4) of the center arabinose residue was found in the intermediate,
which may account for the unusual regioselectivity between 3-OH and 4-OH of arabinose. Two analogues of hederacolchiside
A1 were then facilely prepared by this approach and exhibited significant cytotoxicity in preliminary in vitro assay.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Hederacolchiside A1; Triterpenoid saponin; Cytotoxicity; Regioselective glycosylation; 1C4 conformation; Arabinose
Hederacolchiside A1 (1) (Fig. 1), namely oleanolic acid
3-O-a-L-rhamnopyranosyl-(1?2)-[b-D-glucopyranosyl-
(1?4)]-a-L-arabinopyranoside, is an important triter-
penoid saponin that has prominent inhibitory activity
against many tumor cell lines.1 Its structure contains a
unique disaccharide substructure, a-L-rhamnopyran-
osyl-(1?2)-a-L-arabinopyranoside, which is generally
hypothesized to be a characteristic sugar sequence
strongly inducing the antitumor activity of oleanane-
type glycosides.1–3 Many triterpenoid saponins bearing
this disaccharide moiety commonly present significant
antitumor activity, such as 2–4.1,3,4 Moreover, as a nat-
ural derivative of 1, hederacolchiside A (5) exhibits
excellent in vivo antitumor activity against various solid
tumors in animal experiments.5 Therefore, much atten-
tion has been paid to the further study of this series of
saponins, including antitumor mechanism study, struc-
tural modification, and investigations into the struc-
ture–activity relationships.6–8
oleanolic acid.9 However, the synthesis of 1 had neces-
sary tedious protective group manipulation, and labori-
ous column chromatography was required for
purification of some intermediates. During the successive
work on the structure modification of these two sapo-
nins, we found a more convenient method to prepare 1
and its analogues.
Compound 69 (Scheme 1) is an important intermedi-
ate in the previous synthesis of 1. In general, the equato-
rial 3-OH of arabinose is considered to be more reactive
than the axial 4-OH. Thus, we attempted to glycosylate
COOH
OR2
O
O
R3
R1O
O
R1
H
H
-D-Glc
H
R2
-D-Glc
H
R3
H
H
H
OH
OH
In 2006, we reported the synthesis of hederacolchiside
A1 (1) and b-hederin (2) from commercially available
O
HO
HO
1
2
3
4
5
p-
β
OH
p- H
β
H
H
-D-Glcp-
β
*
Corresponding author. Tel.: +86 24 23986413; fax: +86 24
23995043; e-mail: mscheng@syphu.edu.cn
Figure 1.
0008-6215/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.12.014

M.-C. Yan et al. / Carbohydrate Research 343 (2008) 780–784
781
BzO
O
CCl3
NH
O
BzO
COOBn
COOBn
BzO
OBz
7
III
OH
HO
BzO
BzO
BzO
O
O
O
I
O
O
O
a
HO
O
BzO
6
O
8
O
II
BzO
O
BzO
BzO
BzO
OBz
OBz
˚
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 4 A MS, rt, 65%.
4
6 with donor 710 to prepare 3-O-a-L-rhamnopyranosyl-
(1?2)-[b-D-glucopyranosyl-(1?3)]-a-L-arabinopyrano-
syl oleanolic acid (3), which is also a natural triterpenoid
saponin with high cytotoxicity.2 The glycosylation yield
was 65%; however, NMR spectra showed that the
glucose moiety had been introduced to 4I-OH, and no
by-product with (1?3) linkage was found.
to the normal C1 conformation after deprotection and
yielded the final products, b-hederin and hederacolchi-
side A1, correctly.
Afterward, benzyl group and benzoyl groups of 80
were removed by catalytic hydrogenolysis and ester
interchange in turn to give 1 at 89% yield. The C1 con-
formation of arabinose was found in the product (J1–2
4
=
Similar unusual regioselectivity in glycosylation of 3-
OH and 4-OH of arabinose has been reported previ-
ously11 and the reason was suggested to be the reduced
activity of 3-OH by the bulky group at 2-OH. However,
5.9). However, some differences were found between the
1H NMR spectra of 1 and those reported previously9,15
(Table 1).
1
Thereupon, H NMR data of the original synthetic
1
the small H NMR coupling constant of arabinose in 6
sample9 were measured again at 600 MHz. Surprisingly,
the new spectra of the original sample agreed with that
of the new product, but differed from data determined
previously.9 Nevertheless, other physical properties
and analytical data (such as 13C NMR spectra) were
in good agreement.9,15,16 Chemical shift (d) of carbo-
hydrate protons of saponins is sensitive to various fac-
tors (such as temperature and residual water in NMR
solvent) and sometimes show considerable instability,
while 13C chemical shifts are much less variable.
Although isolation and structure characterization of 1
have been reported repeatedly,4,15–18 1H NMR data
were rarely presented in the literatures except Ref. 15.
Interestingly, Lu et al. isolated 1 from rhizome of
Anemone raddeana and we found that our new 1H
NMR spectra were in agreement with these data.19,20
In succession, analogues of hederacolchiside A1 were
facilely prepared through this route (Scheme 2). Trichlo-
roacetimidates 9 and 10 were used in the regioselective
glycosylation of 60 according to the same procedure.
After deprotection with catalytic hydrogenolysis and
ester interchange, two analogues 13 and 14 were
furnished in moderate yields (24% and 47% from 60,
respectively).
(J1–2 = 1.3) and 8 (J1–2 = 3.5, J2–3 = 4.8) indicated that
1
the arabinose ring may adopt the unusual C4 confor-
4
mation rather than the C1 form. Signal peaks of H-2I,
3I, and 4I in 6 were overlapped, such that the coupling
constants cannot be calculated; nevertheless, the 3I-ben-
zoate of 6 (that is, intermediate 22 in Ref. 9) showed
small J values (J1–2 = 3.8, J2–3 = 2.8, J3–4 = 5.8) in
600 MHz 1H NMR spectra, suggesting a 1C4 conforma-
tion of arabinose. This unusual chair form had been ob-
served on arabinose, xylose, and rhamnose during
glycosylation,12–14 especially when bulky protective
groups are present in two adjacent positions; however,
they usually return to normal chair forms (4C1 for arab-
1
inose and xylose, while C4 for rhamnose) after depro-
tection. Therefore, structures of 6 and 8 should be 60
and 80, respectively (Scheme 2). In this case, 4I-OH of
60 was in the equatorial position and should have a high-
er reactivity than 3I-OH in the glycosylation.
Thereupon, we checked the 1H NMR spectra of inter-
mediates in our previous work9 and found that the arab-
4
inose kept the normal C1 form in the first several steps
1
and did not convert to C4 until the isopropylidene was
1
removed. Apparently, this C4 form allows two bulky
groups (aglycon and benzoylated rhamnose) at 1I-OH
and 2I-OH an antiperiplanar conformation, which is
more favorable than the synclinal conformation in the
4C1 form. It is conceivable that the arabinose ring has
been inclined to convert into the 1C4 form since the
installation of rhamnose, while the presence of isopro-
pylidene prevented this conversion. That is, arabinose
of intermediates 17–25 (except sugar donor 23) in Ref.
A preliminary in vitro pharmacology assay was then
performed to evaluate the cytotoxicity of these saponins
against HeLa cell using a standard MTT assay. Syn-
thetic b-hederin (2) was used as a reference compound.
As shown in Table 2, 14 exhibited a considerable cyto-
toxicity against HeLa cells, while the activity of 13 was a
bit lower (IC50 > 10 lM). However, compared to the
leading compounds 1 and 2, the cytotoxicity was
remarkably reduced after structural modification. This
1
9 should be in the C4 form. Fortunately, it returned

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M.-C. Yan et al. / Carbohydrate Research 343 (2008) 780–784
7 R = R1
9 R = R2
COOBn
10 R = R3
NH
COOBn
b, c
CCl3
RO
COOH
O
O
OH
O
a
OH
OR
HO
O
HO
O
O
6'
8' R = R1
11 R = R2
12 R = R3
RO
1 R = R4
13 R = R5
14 R = R6
O
O
O
O
O
BzO
BzO
O
HO
BzO
BzO
HO
OBz
OH
OBz
OBz
BzO
BzO
BzO
O
O
O
R1
=
=
R2
R5
=
=
R3 =
BzO
BzO
OBz
BzO
OBz
OBz
OH
HO
HO
HO
HO
O
O
R4
R6 =
O
HO
OH
OH
HO
OH
˚
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, 4 A MS, rt; (b) 10% Pd–C, H2, EtOAc, reflux; (c) NaOMe, CH2Cl2–MeOH, rt.
Table 1. 1H NMR and 13C NMR data of 1 (carbohydrate moiety)
Position
13Ca
1H NMRa
1H NMRb
d (ppm)
d (ppm)
J (Hz)
d (ppm)
J (Hz)
1I
105.0
76.4
74.2c
79.8
64.7
4.77, d
5.9
4.76, d
6.2
2I
4.50–4.47, m
4.29–4.25, m
4.29–4.25, m
4.40, dd
3.81, br d
6.13, br s
4.71, t
4.60–4.57, m
4.29–4.25, m
4.62–4.59, m
1.63, d
5.12, d
4.01, dd
4.17, dd
4.22, t
3.90–3.87, m
4.50–4.47, m
4.36, dd
3.22, dd
5.47, br s
3.29, dd
4.67–4.60, m
4.30–4.16, m
4.30–4.16, m
4.41–4.34, m
3.80, d
3I
4I
5I-a
5I-b
1II
12.2, 4.0
11.0
11.3
101.8
72.4
72.5
74.1c
69.9
18.5
106.5
75.5
78.6
71.3
78.9
62.6
6.20, s
2II
1.6
4.75–4.73, m
4.67–4.60, m
4.41–4.34, m
4.67–4.60, m
1.64, d
3II
4II
5II
6II
6.1
7.9
8.8, 8.1
9.0, 8.9
9.1
6.2
7.8
7.9
1III
2III
3III
4III
5III
6III-a
6III-b
3
5.14, d
4.03, t
4.30–4.16, m
4.30–4.16, m
3.91, m
4.53–4.48, m
4.53–4.48, m
3.22, dd
11.7, 4.8
11.7, 4.1
88.7
122.6
42.0
11.5, 3.8
10.2
12
18
5.46, br s
3.27, br d
13.7, 3.8
a Synthetic product of the present work. Data were determined at 600 MHz in pyridine-d5.
b Spectra of Ref. 9.
c Assignments may be interchangeable.
result provided further support for the hypothesis that
this unique disaccharide contributes essentially to the
cytotoxicity of this class of triterpenoid saponins, and
on the other hand, substituents on the arabinose residue
may influence the bioactivity significantly. Conse-
quently, structural modification of this class of triter-
penoid saponin using b-hederin as a core structure is
expected to provide novel compounds with higher anti-
tumor activity.
In conclusion, an improved synthesis of hederacolchi-
side A1 has been achieved from intermediate 60 through
three steps at a yield of 58%, while seven steps were
Table 2. IC50 values of synthetic triterpenoid saponins against HeLa
cell
Compound
1
2
13
14
IC50 (lM)
0.69
0.80
12.80
4.86

M.-C. Yan et al. / Carbohydrate Research 343 (2008) 780–784
783
needed to convert 60 to the target molecule in our former
approach. This newly developed approach avoids
tedious protection–deprotection operation. From this
approach, two analogues of hederacolchiside A1 were
facilely prepared in moderate yields, and exhibited sig-
nificant cytotoxicity in preliminary in vitro study. More-
over, 1C4 conformation of arabinose was found when its
1-OH and 2-OH were occupied by some bulky groups,
which may be meaningful in the synthesis of complex
arabinosides.
4.12 (m, 1H, H-5I-a), 4.08 (m, 1H, H-4I), 3.89 (m, 1H,
H-3I), 3.84 (dd, 1H, J 3.6, 4.8, H-2I), 3.69 (br d, 1H, J
9.7, H-5I-b), 3.10 (dd, 1H, J 11.6, 3.8, H-3), 2.91 (dd,
1H, J 13.8, 3.2, H-18), 1.29 (d, 3H, J 6.1, H-6II), 1.11,
1.03, 0.92, 0.90, 0.86, 0.81, 0.60 (s, 7 ꢀ 3H, CH3); 13C
NMR (CDCl3): d 177.4 (C-28), 166.1, 165.8, 165.7,
165.5, 165.4, 165.3, 165.1 (7 ꢀ PhCO), 143.6 (C-13),
136.4, 133.5–133.1, 129.9–127.9 (aromatic C), 122.5
(C-12), 102.5 (C-1I), 101.9 (C-1III), 97.8 (C-1II), 90.1
(C-3), 76.2 (C-4I), 76.0 (C-2I), 72.8 (C-3III), 72.3
(C-5III), 72.0 (C-2II), 71.7 (C-4II), 70.7 (C-3I, C-2III),
69.8 (C-3II), 69.6 (C-4III), 67.0 (C-5II), 65.9 (PhCH2),
63.0 (C-6III), 60.8 (C-5I), 55.6, 47.6, 46.7, 45.9, 41.6,
41.4, 39.3, 39.1, 38.6, 36.7, 33.8, 33.1, 32.7, 32.4, 30.7,
28.2, 27.6, 25.9, 25.8, 23.6, 23.4, 23.0, 18.2 (C-6II),
17.5, 16.9, 16.5, 15.3; ESI-MS m/z: 1738.7 ([M+Na]+);
1714.8 ([MꢁH]ꢁ).
1. Experimental
1.1. Chemical synthesis
1.1.1. General methods. Commercial reagents were
used without further purification. Boiling range of
petroleum ether was 60–90 °C. Analytical TLC was per-
formed using TLC plates pre-coated with silica HF254
and visualized by UV radiation (254 nm) or I2 fuming.
Preparative column chromatography was performed
with silica gel (200–300 mesh). Melting points were
1.1.3. Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1?2)-
[b-D-glucopyranosyl-(1?4)]-a-L-arabinopyranoside (1).
A suspension of 80 (90 mg, 0.0524 mmol) and 10%
Pd–C (40 mg) in EtOAc (8 mL) was refluxed and bub-
bled up with H2 (20 mL/min). When TLC (2:1, petro-
leum–EtOAc) showed that the reaction had completed,
Pd–C was removed through filtration and the filtrate
was concentrated to dryness. The resulted amorphous
solid was dissolved in dry CH2Cl2–MeOH (1:2,
24 mL), to which a newly prepared NaOMe/MeOH
(1.0 mol/L, 1.20 mL) was added. The solution was stir-
red at rt for 2 h and then neutralized with Dowex H+
resin to pH 7 and filtered. The filtrate was concentrated
and subjected to a flash column chromatography
(CHCl3–MeOH–H2O 7:3:1, organic layer) to give 1
(42 mg, 89%) as a white powder. Mp 249–251 °C, lit.9
251–253 °C, lit.16 250–260 °C; [a]D +0.8 (c 0.33, MeOH),
lit.16 0 (c 2.97, MeOH); Rf = 0.24 (CHCl3–MeOH–H2O
¨
detected with BUCHI Melting Point B-540. Optical
rotations were measured at the sodium D-line at room
temperature with a Perkin–Elmer 241 MC polarimeter.
NMR spectra were recorded on Avance AV 600 MHz
spectrometer. J values were given in hertz. ESI mass
spectra were obtained on a Waters Quattro Micro mass
spectrometer. HRMS was detected on High resolution
ESI-FTICR mass spectrometry (Ion spec 7.0T).
1.1.2. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl-(1?2)-[2,3,4,6-tetra-O-benzoyl-b-D-glu-
copyranosyl-(1?4)]-a-L-arabinopyranoside (80). A sus-
pension of 60 (133 mg, 0.117 mmol), 7 (92 mg, 0.124
1
mmol), and powdered 4 A molecular sieves (400 mg) in
7:3:1, organic layer); H NMR: see Table 1; 13C NMR
˚
dry CH2Cl2 (4 mL) was stirred for 30 min at rt. A solu-
tion of TMSOTf in dry CH2Cl2 (5% v/v, 95 lL,
0.024 mmol) was added and the mixture was stirred
for 40 min before Et3N (0.10 mL) was added to quench
the reaction. The mixture was then filtered and the fil-
trate was concentrated and purified by a flash column
chromatography (3:1, petroleum ether–EtOAc) to afford
80 (131 mg, 65%) as a white foam. [a]D +56.7 (c 0.61,
CH2Cl2); Rf = 0.38 (2:1, petroleum ether–EtOAc); 1H
NMR (CDCl3): d 8.09–7.81 (m, 14H), 7.62 (m, 1H),
7.53–7.48 (m, 5H), 7.43–7.37 (m, 6H), 7.36–7.31 (m,
6H), 7.30–7.24 (m, 8H) (aromatic H), 5.94 (t, 1H, J
9.6, H-3III), 5.79 (dd, 1H, J 10.1, 2.5, H-3II), 5.71 (dd,
1H, J 9.7, 9.6, H-4III), 5.65–5.61 (m, 2H, H-2II, H-4II),
5.59 (m, 1H, H-2III), 5.29 (br s, 1H, H-12), 5.20 (br s,
1H, H-1II), 5.13 (d, 1H, J 7.8, H-1III), 5.07 (dd, 2H, J
33.4, 12.5, PhCH2), 4.71 (dd, 1H, J 12.1, 2.4, H-6III-a),
4.65 (d, 1H, J 3.5, H-1I), 4.52 (dd, 1H, J 12.0, 4.7,
H-6III-b), 4.32 (m, 1H, H-5II), 4.25 (m, 1H, H-5III),
(pyridine-d5): aglycon moiety d 180.2 (C-28), 144.9
(C-13), 122.6 (C-12), 88.7 (C-3), 56.0 (C-5), 48.0 (C-9),
46.7 (C-17), 46.5 (C-19), 42.2 (C-14), 42.0 (C-18), 39.7
(C-4), 39.5 (C-8), 38.9 (C-1), 37.1 (C-10), 34.2 (C-21),
33.3 (C-29), 33.2, 33.1 (C-7, C-22), 31.0 (C-20), 28.3
(C-15), 28.1 (C-23), 26.7 (C-2), 26.2 (C-27), 23.8
(C-30), 23.7 (C-11, C-16), 18.7 (C-6), 17.4 (C-26), 17.1
(C-24), 15.6 (C-25), carbohydrate moiety: see Table 1;
HRMS m/z: calcd for [C47H76NaO16]+ ([M+Na]+)
919.5026, found: 919.5030.
1.1.4. Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1?2)-
[a-L-rhamnopyranosyl-(1?4)]-a-L-arabinopyranoside (13).
C. ompound 13 was prepared from 60 by the same proce-
dure as for 1. Yield: 24%; white powder, [a]D ꢁ15.4 (c
0.44, MeOH); Rf = 0.33 (CHCl3–MeOH–H2O 7:3:1,
1
organic layer); H NMR (pyridine-d5): d 5.97 (s, 1H,
H-1II), 5.72 (s, 1H, H-1III), 5.43 (br s, 1H, H-12), 4.87
(d, 1H, J 4.3, H-1I), 4.70 (br s, 1H), 4.59 (br d, 1H, J

784
M.-C. Yan et al. / Carbohydrate Research 343 (2008) 780–784
9.4), 4.55–4.50 (m, 2H), 4.49–4.45 (m, 2H), 4.42 (dd, 1H,
J 11.6, 4.6), 4.37–4.31 (m, 3H), 4.28 (t, 1H, J 9.2), 4.23
(t, 1H, J 9.2), 3.84 (br d, 1H, J 11.2), 3.26 (br d, 1H, J
12.8, H-18), 3.18 (dd, 1H, J 11.9, 3.4, H-3), 1.64 (d,
3H, J 5.8, H-6II), 1.58 (d, 3H, J 5.9, H-6III), 1.26, 1.13,
1.02, 0.97, 0.95, 0.92, 0.78 (s, 7 ꢀ 3H, CH3); 13C NMR
(pyridine-d5): d 180.2 (C-28), 144.8 (C-13), 122.5
(C-12), 104.9 (C-1I), 102.6 (C-1III), 102.0 (C-1II), 89.0
(C-3), 76.4 (C-2I), 75.0 (C-4I), 74.1 (C-3I, C-4II), 72.6
(C-2II, C-3II), 72.4, 72.2, 72.1 (C-2III, C-3III, C-4III),
70.3, 70.2 (C-5II, C-5III), 63.0 (C-5I), 55.9 (C-5), 39.8
(C-4), 39.5 (C-8), 38.8 (C-1), 37.0 (C-10), 34.3 (C-21),
33.3 (C-29), 33.2, 33.2 (C-7, C-22), 31.0 (C-20), 28.3
(C-15), 28.1 (C-23), 26.5 (C-2), 26.2 (C-27), 23.8, 23.8,
23.7 (C-30, C-11, C-16), 18.7 (C-6II), 18.6 (C-6III), 18.6
(C-6), 17.4 (C-26), 16.9 (C-24), 15.5 (C-25); HRMS
m/z: calcd for [C47H76ClO15]ꢁ ([M+Cl]ꢁ) 915.4878,
found: 915.4881.
5% CO2 for 4 d. 100 lL MTT (1.0 mg/mL in nutrient
medium) was added and cells continued to incubate
for a further 4 h. Then, 150 lL DMSO was added to
each well and the absorbance of samples was measured
at 490 nm. The IC50 values were calculated according to
Logit method after getting the inhibitory rate.
Acknowledgments
This work was supported by National Natural Science
Foundation of China (Nos. 20472054 and 30772641).
We also thank Professor Hong Chen in Medical College
of Chinese People’s Armed Police Force for pharma-
cology evaluation.
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2004; Chem. Abstr. 2004, 140, 139489u.
1.1.5. Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1?2)-
[a-L-arabinopyranosyl-(1?4)]-a-L-arabinopyranoside (14).
Compound 14 was prepared from 60 by the same proce-
dure as for 1. Yield: 47%; white powder, [a]D 0.0 (c 0.31,
MeOH); Rf = 0.30 (CHCl3–MeOH–H2O 7:3:1, organic
layer); 1H NMR (pyridine-d5): d 6.12 (s, 1H, H-1II),
5.43 (br s, 1H, H-12), 4.88 (d, 1H, J 7.5, H-1I), 4.76
(d, 1H, J 6.1, H-1III), 4.69 (m, 1H), 4.63–4.58 (m, 2H),
4.46–4.42 (m, 3H), 4.27 (t, 1H, J 9.4), 4.25–4.22 (m,
4H), 4.06 (dd, 1H, J 9.1, 3.2), 3.82 (br d, 1H, J 10.8),
3.66 (br d, 1H, J 10.9), 3.26 (dd, 1H, J 13.3, 3.8,
H-18), 3.20 (dd, 1H, J 11.8, 4.1, H-3), 1.60 (d, 3H, J
6.1, H-6II), 1.25, 1.15, 1.07, 0.97, 0.94, 0.92, 0.79 (s,
7 ꢀ 3H, CH3); 13C NMR (pyridine-d5): d 180.2 (C-28),
144.8 (C-13), 122.6 (C-12), 107.0 (C-1III), 105.0 (C-1I),
101.8 (C-1II), 88.8 (C-3), 79.3 (C-4I), 76.4 (C-2I), 74.9
(C-3I, C-4II), 74.1, 74.1 (C-2III, C-3III), 72.9 (C-4III),
72.6, 72.4 (C-2II, C-3II), 69.9 (C-5II), 67.5 (C-5III), 64.6
(C-5I), 56.0 (C-5), 48.1 (C-9), 46.7 (C-17), 46.5 (C-19),
42.2 (C-14), 42.0 (C-18), 39.8 (C-4), 39.6 (C-8), 38.9
(C-1), 37.1 (C-10), 34.3 (C-21), 33.3 (C-29), 33.2, 33.2
(C-7, C-22), 31.0 (C-20), 28.4 (C-15), 28.1 (C-23), 26.7
(C-2), 26.2 (C-27), 23.8, 23.8, 23.7 (C-11, C-16, C-30),
18.7 (C-6II), 18.6 (C-6), 17.4 (C-26), 17.1 (C-24), 15.6
(C-25); HRMS m/z: calcd for [C46H74NaO15]+
([M+Na]+) 889.4920, found: 889.4924.
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Cytotoxicity of test compounds against HeLa cell line
was evaluated by the MTT method in vitro. Cells were
plated at a density of 4000 cells per well into 96-well
plate and incubated at 37 °C with 5% CO2 for 24 h.
The cells were then treated with increasing concentra-
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