Synthesis and antitumor activity of two natural N-acetylglucosamine-bearing triterpenoid saponins: Lotoidoside D and E

Article abstract of DOI:10.1016/j.bmcl.2006.05.086

Two natural triterpenoid saponins bearing N-acetylglucosamine, lotoidoside D and lotoidoside E, which had been available only from Glinus lotoides growing in Egyptian desert, were facilely synthesized from readily available oleanolic acid. Preliminary pha

Full text of DOI:10.1016/j.bmcl.2006.05.086

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4200–4204
Synthesis and antitumor activity of two natural
N-acetylglucosamine-bearing triterpenoid saponins:
Lotoidoside D and E
Mao-Cai Yan,a Yang Liu,a Hong Chen,b Ying Ke,b Qing-Chun Xua and
Mao-Sheng Chenga,*
aKey Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang 110016, PR China
bStaff Room of Pharmacognosy, Medical College of Chinese People’s Armed Police Force, Tianjin 300162, PR China
Received 12 March 2006; revised 8 May 2006; accepted 29 May 2006
Available online 12 June 2006
Abstract—Two natural triterpenoid saponins bearing N-acetylglucosamine, lotoidoside D and lotoidoside E, which had been avail-
able only from Glinus lotoides growing in Egyptian desert, were facilely synthesized from readily available oleanolic acid. Prelimin-
ary pharmacological research showed their antitumor activity against HeLa cell.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Triterpenoid saponins are a large family of natural
products with great variety of structures and bioactivi-
ties.1 Some of them have N-acetylglucosamine in the
carbohydrate moiety, namely N-acetylglucosamine-
bearing triterpenoid saponins. These rarely reported
natural products have been drawing more and more
attention of phytochemists and pharmacologists in re-
cent years. Quite a number of these saponins were found
to have remarkable cytotoxicity or antiproliferative
activities.2 For instance, lotoidoside D (1) and lotoido-
side E (2) (Fig. 1) are two N-acetylglucosamine-bearing
triterpenoid saponins isolated from the roots of Glinus
lotoides (a medical plant growing in Egyptian desert)
by Hamed et al.3 in 2005. These triterpenoid saponins
Figure 1. Structures of lotoidosides D (1) and E (2).
show prominent antiproliferative activity against several
murine and human cell lines.3 For example, IC50 values
of 1 and 2 against murine fibrosarcoma cell line WEHI-
the nature turns out to be a great obstacle for further
pharmacological research. Considering the rapid dis-
164 are 0.36 and 0.03 lM, respectively.
covery of N-acetylglucosamine-bearing triterpenoid
saponins with attractive bioactivities in recent years,
it is valuable to develop a convenient way to prepare
this kind of saponins to promote further studies, such
as thorough pharmacological research and further
structure–activity relationship (SAR) investigation.
Herein, we report the chemical synthesis of 1 and 2
from commercially available oleanolic acid (3). The
preliminary antitumor activity of these synthetic sapo-
nins was then investigated.
However, 1 and 2 are only available from the tedious
isolation procedure from the roots of Glinus lotoides
in low isolated yields (only 25 mg of 1 and 20 mg of
2 from 800 g roots).3 The scarceness of 1 and 2 in
Keywords: Lotoidoside D; Lotoidoside E; Triterpenoid saponin; Gly-
cosylation; Antitumor.
*
Corresponding author. Tel.: +86 24 23986413; fax: +86 24
23995043; e-mail: mscheng@syphu.edu.cn
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.086

M.-C. Yan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4200–4204
4201
Stepwise glycosylation was adopted to construct the
oligosaccharide moiety because it is a preferable meth-
od of preparing glycoside analogues for SAR research
by altering monosaccharide donors.4 It is known that
ester linkage at C-28 carboxyl of pentacyclic triterpe-
noid is extremely resistant to basic hydrolysis due to
its high steric hindrance.5 Therefore, it is difficult to
deprotect general alkyl protective groups from C-28
alkyl ester. Based on our experience of triterpenoid
saponin synthesis,6 benzyl was adopted as a perma-
nent protective group for it can be removed conve-
niently through catalytic hydrogenolysis.7 Especially
in this work, the benzyl ester would be cleaved togeth-
er with benzylidene acetal or benzyl ether in carbohy-
drate moiety during catalytic hydrogenolysis, which
would make the synthetic route more concise and
efficient.
The synthetic route of 2 is shown in Schemes 1 and 2.
Oleanolic acid (3) was first converted to its benzyl ester
4 by treating 3 with BnBr and K2CO3 in aqueous THF
in high yield (98%).8 Next, a straightforward prepara-
tion of intermediate 5 using readily available 2-acetami-
no-2-deoxyglucose donor 69 was investigated. However,
little desired product was obtained while the corre-
sponding oxazoline derivative became the major prod-
uct. Therefore, 2-phthalimido-2-deoxyglucose donor
7
10 was employed to be coupled with 4 under the promo-
tion of TMSOTf to give 8 in a high yield (96%), which
was then treated with hot ethylenediamine/butanol and
Ac2O/pyridine afterward to afford 5. The overall yield
for 3 steps from 4 to 5 was 84%.
Then, the O-acetyl groups of 5 were removed by
NaOMe/MeOH to give 10, and benzylidene was
˚
Scheme 1. Synthesis of intermediate 5. Reagents and conditions: (a) BnBr, K2CO3, THF–H2O, rt, 98%; (b) TMSOTf, 4 A MS, CH2Cl2, 0 ꢁC to rt,
˚
4%; (c) TMSOTf, 4 A MS, CH2Cl2, 0 ꢁC to rt, 96%; (d) H2NCH2CH2NH2, n-BuOH, 90 ꢁC; (e) Ac2O, pyridine, rt, 88% for two steps.
Scheme 2. Synthesis of lotoidoside E (2). Reagents and conditions: (a) NaOMe, CH2Cl2–MeOH, rt; (b) PhCH(OMe)2, CSA, DMF, 70 ꢁC, 87% for
two steps; (c) TMSOTf, CH2Cl2, 0 ꢁC to rt, 69%; (d) H2, Pd–C (10%), HOAc–THF, 40 ꢁC; (e) NaOMe, CH2Cl2–MeOH, rt, 82% for two steps.

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M.-C. Yan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4200–4204
introduced to protect 40,60-OH by the reaction of 10
with PhCH(OMe)2 and DL-camphorsulfonic acid
(CSA) in dry DMF (Scheme 2). 3-OH of 4,6-O-benzyli-
dene-2-acetamido-2-deoxyglucopyranosides have been
found to be unreactive sugar acceptors in glycosyla-
tion.11 In current study, no reaction product was detect-
ed from TLC (petroleum ether/EtOAc = 4:1) when a
catalytic amount of TMSOTf (0.05–0.20 equiv) was
used in the glycosylation of 11 with 12. Furthermore,
adding excess galactose donor 1212 (1.60 equiv) and
TMSOTf (1.20 equiv) gave a yield of merely 22%.
Therefore, the ‘inverse procedure,’13 which was devel-
oped by Schmidt et al. originally to prevent sensitive su-
gar donors from decomposing and later was employed
in glycosylation of unreactive acceptors, was applied
here and worked well to give the desired product 13 in
a yield of 69%. Catalytic hydrogenolysis of 13 was per-
formed to remove the benzyl and benzylidene cleanly.
Finally, the resulted 14 was treated with NaOMe in
CH2Cl2–MeOH to give the target product 2. The overall
yield from oleanolic acid was 41%.
condition Et3SiH–BF3ÆEt2O15 was applied to 13 to give
benzyl ether 15 in a yield of 79%. Coupling of 15 and
1616 afforded fully protected saponin 17 in a yield of
66%. Deprotection of 17 through catalytic hydrogenoly-
sis and ester interchange gave the final product 1. The
overall yield from oleanolic acid to 1 was 19%. The ana-
lytical data of synthetic 1 and 2 were identical with those
reported for the natural products.3,17
The in vitro antiproliferative activity of these triterpe-
noid saponins against HeLa tumor cell was investigated.
HeLa cell in RPMI1640 medium with 10% fetal bovine
serum was plated on 96-well microtiter plates
(4.0 · 104 cells per well), and allowed to adhere at
37 ꢁC with 5% CO2 for 4 h. Then the test compound
was added, and the cells were incubated at 37 ꢁC with
5% CO2 for 72 h. The cell viability was assessed through
standard MTT assay.18
As shown in Table 1, 1 displayed prominent inhibition
activity to HeLa cell even at very low concentrations.
The inhibition at 10 nM was 34.44%. On the other hand,
saponin 2, which was more potent than 1 in the previous
research,3 showed relatively lower inhibition against
HeLa cell. Despite the sensitivity difference between
these cell lines, it may be indicated that 1 and 2 possibly
have different inhibition mechanisms against various tu-
mor cells. Extensive antitumor screening and intensive
mechanism investigation are demanded in this area to
The key intermediate 13 was also utilized for the facile
synthesis of 1 (Scheme 3). Regioselective cleavage of
4,6-O-benzylidene acetal of carbohydrate derivatives is
proved to be an outstanding strategy to furnish 4-OH-
6-benzyl ether or 6-OH-4-benzyl ether.14 Considering
the compatibility to the glycosidic linkages and various
protecting groups on the substrate, mild reducing
˚
˚
Scheme 3. Synthesis of lotoidoside D (1). Reagents and conditions: (a) Et3SiH, BF3ÆEt2O, 4 A MS, CH2Cl2, 0 ꢁC, 79%; (b) TMSOTf, 4 A MS,
CH2Cl2, 0 ꢁC to rt, 66%; (c) H2, Pd–C (10%), HOAc–THF, reflux; (d) NaOMe, CH2Cl2–MeOH, rt, 74% for two steps.
Table 1. Antitumor activity of synthetic saponins against HeLa tumor cell line
Saponin
Inhibition rate at different concentration (%)
IC50 (lM)
10ꢀ5 M
10ꢀ6 M
10ꢀ7 M
10ꢀ8 M
1
2
56.43
42.02
35.33
10.04
32.84
1.71
34.44
0.79
2.74
>10

M.-C. Yan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4200–4204
4203
5.06 (dd, 2H, J = 18.6, 12.5), 5.02 (t, 1H, J = 9.6), 4.68
(d, 1H, J = 8.3, H-10), 4.24–4.21 (m, 1H), 4.09 (dd, 1H,
J = 12.0, 2.6), 3.86 (dd, 1H, J = 14.6, 8.3), 3.69–3.66 (m,
1H), 3.07 (dd, 1H, J = 11.3, 4.6), 2.84 (dd, 1H, J = 13.1,
3.4), 2.07 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.92 (s, 3H),
1.11 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.87
(s, 3H), 0.77 (s, 3H), 0.59 (s, 3H); ESI-MS m/z: 898.6
(M+Na)+. Compound 11: [a]D +13.2 (c 1.30, acetone);
1H NMR (600 MHz, CDCl3) d 7.50–7.30 (m, 10H, Ar-
H), 5.78 (d, 1H, J = 6.5), 5.53 (s, 1H), 5.28 (br s, 1H),
5.07 (dd, 1H, J = 30.2, 12.5), 4.80 (d, 1H, J = 8.2, H-10),
4.29 (dd, 1H, J = 10.4, 4.9), 4.19 (t, 1H, J = 9.4), 3.78 (t,
1H, J = 10.3), 3.74 (br s, 1H), 3.53 (t, 1H, J = 9.3), 3.46–
3.43 (m, 2H), 3.13 (dd, 1H, J = 11.6, 4.3), 2.90 (dd, 1H,
J = 13.6, 3.8), 2.01 (s, 3H), 1.11 (s, 3H), 0.96 (s, 3H), 0.92
(s, 3H), 0.90 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H), 0.60 (s,
3H); ESI-HRMS m/z 860.5069 (M+Na)+ (C52H71NNaO8
requires 860.5072). Compound 13: [a]D +45.2 (c 0.30,
acetone); 1H NMR (600 MHz, CDCl3) d 8.06–7.20 (m,
30H, Ar-H), 5.88 (d, 1H, J = 3.3), 5.72 (dd, 1H, J = 10.2,
8.1), 5.55 (s, 1H), 5.53–5.51 (m, 2H), 5.28 (br s, 1H), 5.18
(d, 1H, J = 8.1, H-10), 5.06 (dd, 2H, J = 31.2, 12.5), 4.98
(d, 1H, J = 8.1, H-100), 4.76 (t, 1H, J = 9.0), 4.31–4.28 (m,
3H), 3.89 (dd, 1H, J = 7.3, 6.3), 3.78 (t, 1H, J = 10.3),
3.73 (t, 1H, J = 9.1), 3.57 (dd, 1H, J = 9.8, 5.0), 3.05 (dd,
2H, J = 11.2, 4.1), 2.89 (dd, 1H, J = 13.7, 3.6), 1.60 (s,
3H), 1.09 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H),
0.80 (s, 3H), 0.72 (s, 3H), 0.58 (s, 3H); ESI-HRMS m/z
elucidate the mechanism and develop more desirable
bioactive agents.
In summary, lotoidosides D (1) and E (2) were efficiently
synthesized from oleanolic acid in high yields. This syn-
thesis may provide a convenient way for the preparation
of other N-acetylglucosamine-bearing triterpenoid sapo-
nins. Preliminary biological evaluation was performed
and showed significant antitumor activity. Further bio-
logical studies and SAR research of their derivatives
are in progress and will be reported in due course.
References and notes
1. Hostettmann, K.; Marston, A. Saponins; Cambridge
University Press: Cambridge, 1995, Chapter 5.
2. For some examples reported in 2005, see: (a) Liang, H.;
Tong, W. Y.; Zhao, Y. Y.; Cui, J. R.; Tu, G. Z. Bioorg.
Med. Chem. Lett. 2005, 15, 4493; (b) Zou, K.; Cui, J. R.;
Ran, F. X.; Wang, B.; Zhao, Y. Y.; Zhang, R. Y.; Zheng,
J. H. Chin. J. Org. Chem. 2005, 25, 654; (c) Krief, S.;
Thoison, O.; Sevenet, T.; Wrangham, R. W.; Lavaud, C.
J. Nat. Prod. 2005, 68, 897; (d) Zou, K.; Tong, W. Y.;
Liang, H.; Cui, J. R.; Tu, G. Z.; Zhao, Y. Y.; Zhang, R. Y.
Carbohydr. Res. 2005, 340, 1329.
3. Hamed, A. I.; Piacente, S.; Autore, G.; Marzocco, S.;
Pizza, C.; Oleszek, W. Planta Med. 2005, 71, 554.
4. Yu, B.; Hui, Y. In Glycochemistry: Principles, Synthesis
and Applications; Wang, P. G., Bertozzi, C. R., Eds.;
Marcel Dekker: New York, 2001; pp 167–174.
5. Ohtani, K.; Mizutani, K.; Kasai, R.; Tanaka, O. Tetra-
hedron Lett. 1984, 25, 4537.
6. Cheng, M. S.; Yan, M. C.; Liu, Y.; Zheng, L. G.; Liu, J.
Carbohydr. Res. 2006, 341, 60.
7. Double bond between C-12 and C-13 of oleanolic acid is
inert to catalytic hydrogenation. See: Winterstein, A.;
Stein, G. Z. physiol. Chem. 1931, 202, 222.
8. In our first practice, the previous benzylation condition
(BnBr, Bu4NI, Et3N, and THF) (see Ref. 6) was utilized
but give unstable yield. Therefore another benzylation
condition using BnBr and K2CO3 in aqueous THF was
investigated and proved to give a high yield steadily.
9. Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 11,
1826.
10. Kretzschmar, G.; Stahl, W. Tetrahedron 1998, 54, 6341.
11. Jankowska, M.; Madaj, J. Carbohydr. Res. 2005, 340,
2048.
1438.6655
(M+Na)+
(C86H97NNaO17
requires
1438.6649). Compound 15: [a]D +84.8 (c 1.20, acetone);
1H NMR (600 MHz, CDCl3) d 8.09–7.22 (m, 30H, Ar-
H), 5.96 (d, 1H, J = 3.4), 5.81 (dd, 1H, J = 10.4, 8.1),
5.67 (dd, 1H, J = 10.4, 3.4), 5.29 (br s, 1H), 5.26 (d, 1H,
J = 6.7, NH), 5.06 (dd, 2H, J = 31.8, 12.6), 5.01 (d, 1H,
J = 8.7, H-10), 4.90 (d, 1H, J = 8.0, H-100), 4.63 (d, 1H,
J = 8.0), 4.61 (dd, 2H, J = 31.4, 7.9) 4.60–4.58 (m, 1H),
4.46–4.43 (m, 2H), 3.85 (d, 1H, J = 10.6), 3.64 (dd, 1H,
J = 10.7, 6.0), 3.55–3.52 (m, 2H), 3.07 (dd, 1H, J = 11.8,
4.4), 2.90 (dd, 1H, J = 10.6, 2.3), 2.89–2.86 (m, 1H), 1.34
(s, 3H), 1.09 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.85 (s,
3H), 0.79 (s, 3H), 0.71 (s, 3H), 0.58 (s, 3H); HRMS m/z
1418.7001 (M+H)+ (C86H100NO17 requires 1418.6986).
Compound 17: [a]D +31.4 (c 0.35, CHCl3); 1H NMR
(600 MHz, CDCl3) d 8.10–7.24 (m, 30H, Ar-H), 6.11 (d,
1H, J = 9.3), 6.04 (d, 1H, J = 3.2), 5.81 (dd, 1H, J = 10.6,
8.1), 5.70 (dd, 1H, J = 10.6, 3.3), 5.34 (d, 1H, J = 8.0, H-
100), 5.30 (br s, 1H), 5.19 (t, 1H, J = 9.6), 5.11 (dd, 2H,
J = 25.2, 12.6), 5.05 (t, 1H, J = 9.6), 4.93 (t, 1H, J = 9.4),
4.65 (dd, 1H, J = 11.0, 6.0), 4.59 (d, 1H, J = 11.0, H-1000),
4.50 (dd, 2H, J = 20.6, 8.8), 4.47–4.46 (m, 1H), 4.44 (t,
1H, J = 6.4), 4.31 (d, 1H, J = 5.3, H-10), 4.25–4.22 (m,
2H), 4.18 (br s, 1H), 4.11 (m, 1H), 4.04 (br d, 1H,
J = 12.2), 3.76–3.73 (m, 2H), 3.64 (dd, 1H, J = 12.7, 8.9),
3.43 (br d, 1H, J = 10.0), 2.93 (dd, 1H, J = 13.7, 2.8),
2.78 (dd, 1H, J = 11.7, 3.8, H-3), 2.06 (s, 3H), 2.05 (s,
3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.11 (s, 3H),
0.94 (s, 3H), 0.92 (s, 3H), 0.79 (s, 3H), 0.79 (s, 3H), 0.63
(s, 3H), 0.61 (s, 3H); ESI-HRMS m/z 1770.7772
(M+Na)+ (C100H117NNaO26 requires 1770.7756). Com-
pound 1: [a]D +22.1 (c 0.50, MeOH); 1H NMR
(600 MHz, MeOH-d4) d 5.29 (t, 1H, J = 3.3), 4.96–4.94
(m, 1H), 4.67 (d, 1H, J = 7.6, H-1000), 4.57 (d, 1H, J = 7.8,
H-100), 4.51 (d, 1H, J = 7.5, H-10), 4.15 (t, 1H, J = 9.2),
4.00 (t, 1H, J = 11.4), 3.98 (dd, 1H, J = 8.7, 7.4), 3.93–
3.90 (m, 2H), 3.87 (d, 1H, J = 2.8), 3.85–3.83 (dd, 1H,
J = 11.8, 2.6), 3.77–3.75 (dd, 1H, J = 11.4, 4.5), 3.73 (dd,
1H, J = 11.7, 4.3), 3.65 (dd, 1H, J = 9.0, 7.9), 3.59–3.57
(m, 1H), 3.52 (dd, 1H, J = 9.0, 3.3), 3.47–3.45 (m, 1H),
3.42–3.41 (m, 1H), 3.39 (dd, 1H, J = 9.3, 7.6), 3.37–3.35
12. Rio, S.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res. 1991,
219, 71.
13. ‘Inverse procedure’ here means mixing the sugar acceptor
and promoter (herein TMSOTf) before the sugar donor is
added, which is different to common glycosylation proce-
dure (adding the promoter to the mixture of sugar
acceptor and donor). See: Schmidt, R. R.; Toepfer, A.
Tetrahedron Lett. 1991, 32, 3353.
14. Garegg, P. J. In Preparative Carbohydrate Chemistry;
Hanessian, S., Ed.; Marcel Dekker: New York, 1997; pp
53–67.
15. Debenham, S. D.; Toone, E. J. Tetrahedron: Asymmetry
2000, 11, 385.
16. Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl.
1980, 19, 731.
17. Selected data for products and key intermediates: Com-
pound 5: [a]D +24.8 (c 0.62, acetone); 1H NMR
(300 MHz, CDCl3) d 7.35–7.30 (m, 5H, Ar-H), 5.52 (br
t, J = 8.3, NH), 5.31 (br s, 1H), 5.25 (t, 1H, J = 10.2),

4204
M.-C. Yan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4200–4204
(m, 2H), 3.15 (dd, 1H, J = 11.5, 4.2), 2.90 (dd, 1H,
1H, J = 8.6), 3.61–3.58 (m, 2H), 3.51 (dd, 1H, J = 8.5,
3.0), 3.47 (t, 1H, J = 9.0), 3.35–3.34 (m, 1H, H-50), 3.17
(dd, 1H, J = 11.6, 4.2), 2.89 (dd, 1H, J = 13.7, 3.7,), 2.00
(s, 3H), 1.21 (s, 3H), 1.02 (s, 3H), 1.01 (s, 3H), 0.99 (s,
3H), 0.96 (s, 3H), 0.86 (s, 3H), 0.82 (s, 3H); ESI-HRMS
m/z 844.4824 (M+Na)+ (C44H71NNaO13 requires
844.4818).
J = 13.8, 4.0), 2.02 (s, 3H), 1.20 (s, 3H), 1.02 (s, 3H),
1.00 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.86 (s, 3H), 0.81
(s, 3H); ESI-HRMS m/z 1006.5339 (M+Na)+
(C50H81NNaO18 requires1006.5346). Compound 2: [a]D
1
+18.6 (c 0.50, MeOH); H NMR (600 MHz, MeOH-d4) d
5.29 (br s, 1H), 4.55 (d, 1H, J = 8.1, H-10), 4.33 (d, 1H,
J = 7.6, H-100), 3.91 (dd, 1H, J = 11.4, 1.4), 3.85 (d, 1H,
J = 3.0), 3.81–3.79 (m, 2H), 3.76–3.74 (m, 2H), 3.72 (t,
18. Green, L. M.; Reade, J. L.; Ware, C. F. J. Immunol.
Methods 1984, 70, 257.