Synthesis and Antitumor Activity of a New Ergosterol Derivative

Article abstract of DOI:10.1007/s10600-016-1607-6

Ergosterol-3-O-(β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside) was efficiently synthesized and evaluated for its inhibitory activities against S180 cell lines compared with ergosterol. The structures of all synthesized compounds were fully characterized

Full text of DOI:10.1007/s10600-016-1607-6

DOI 10.1007/s10600-016-1607-6
Chemistry of Natural Compounds, Vol. 52, No. 2, March, 2016
SYNTHESIS AND ANTITUMOR ACTIVITY
OF A NEW ERGOSTEROL DERIVATIVE
1*
1
1
Zhen-Yuan Zhu, Zhen-qian Wang, Fei Liu,
1
2*
1
Xiao-Cui Liu, Li-Jing Chen, Xiao-Ran Ge,
An-Jun Liu, and Yong-Min Zhang
1
3
Ergosterol-3-O-(ꢀ-D-galactopyranosyl-(1ꢁ4)-ꢀ-D-glucopyranoside) was efficiently synthesized and
evaluated for its inhibitory activities against S180 cell lines compared with ergosterol. The structures of all
synthesized compounds were fully characterized by spectroscopic data (NMR, MS). The antitumor activity
of the new derivative was significantly enhanced compared with ergosterol.
Keywords: ergosterol, glycosylation, derivative, antitumor activity.
Saponins are natural compounds present in various higher plants that are usually found in roots, tubers, leaves,
blooms, or seeds. Saponins were classified into triterpenes and steroids based on the carbon skeleton. Their sugar parts are
mostly oligosaccharides, arranged either in linear or branched fashion, attached to hydroxyl groups through an acetal linkage.
Modern research has found that saponins have antitumor effects on many cancer cells. Several saponins inhibit tumor cell
growth by cell cycle arrest and apoptosis with IC values up to 0.2 mM. Meanwhile, saponins in combination with conventional
50
tumor treatment strategies have improved the success rate of therapeutics [1].
Ergosterol is one of the phytosterols that are widely distributed in fungal cultures and also the most important component
in membranes. It is involved in numerous biological functions such as membrane fluidity regulation, activity and distribution
of integral proteins, and control of the cellular cycle [2, 3]. Moreover, it posseses different pharmacological activities such as
antibacterial, antiviral, and anticancer [4].
Although a small quantity of ergosterol glucoside derivatives has been extracted from Cordyceps gunnii,
nevertheless, it was difficult to study it due to its low content and complicated composition. Chemical synthesis is one of the
prospective routes for obtaining homogeneous saponins, thus affording an opportunity to understand and apply this important
branch of natural products [5].
In our previous study [6], we successfully synthesized two new ergosterol glycosides, ergosterol-3-O-ꢀ-D-
glucopyranoside and ergosterol-3-O-ꢀ-D-galactopyranoside. The antitumor activities of these compounds have been investigated,
and the result indicate that both of them have significant cytotoxic and antitumor activity on the S180 cell line [7].
In order to further understand the effect of the sugar part of the ergosterol glycosides on the biological activity, we
selected D-lactose as the sugar part, then used benzoyl protection for the lactose hydroxyl groups; then we used the
trichloroacetimidate method [8] for the glycosylation procedure (Scheme 1). In this study, the glycosylation of the ergosterol
with the donor (compound 3) was achieved under the conditions described above, promoted by TMSOTf (trimethylsilyl
trifluoromethanesulfonate), to give compound 4 in 78.0% yield. The stereochemistry of the newly introduced glycosidic
linkage was determined to be ꢀ on the basis of the Glc H-1, H-2 coupling constant (J = 8.0 Hz) [9]. De-O-benzoylation of
1,2
compound 4 in methanol-dichloromethane quantitatively offered the target compound 5 in 81.0% yield (Scheme 2).
1) Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology,
Tianjin University of Science and Technology, 300457, Tianjin, P. R. China, e-mail: zhyanzhu@tust.edu.cn; 2) Key Laboratory
of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, 200090, P. R. China;
3) Universite Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moleculaire UMR CNRS 8232, 4 place Jussieu,
75005, Paris, France. Published in Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2016, pp. 222–224. Original article
submitted June 4, 2014.
252
0009-3130/16/5202-0252 02016 Springer Science+Business Media New York

OBz
OH
OBz
OH
BzO
BzO
HO
HO
O
O
a, b, c
O
O
O
O
OR
OH
BzO
HO
OBz
OH
OBz
OH
lactose
1 - 3
3:R = C(NH)-C(Cl)
1: R = Bz
2: R = H
3
a. BzCl, Py, 0°C to r.t., 16 h; b. THF, methylamine, r.t., 2h; c. CH Cl , DBU, CCl CN r.t., 3 h
2
2
3
Scheme 1
28
21
18
26
23
25
27
17
19
5
11
13
1
10
15
H
H
3
OR
1
7
O
O
6''
3
+
1''
H
H
5''
3''
OR
1
OR
1
O
HO
O
R O
R O
1
1
1'
5'
ergosterol
4, 5
4: R = Bz
OR
1
R O
1
1
5:R = H
1
Scheme 2
The antitumor activity of the new derivative in vitro was evaluated using the MTT assay [10].
The new glycoside was tested for its anticancer activity against S108 cell lines by the MTT-based assay in vitro. The
data showed that the antitumor activity of the new derivative was higher than that of ergosterol. In 48 h, the new derivative
displayed the best inhibition rate of 93.4% at the concentration of 200 ꢂg/mL (Fig. 1). We found that the antitumor activity of
this new derivative was higher than that of ergosterol-3-O-(ꢀ-D-galactopyranoside) in 24 h compared with our previous study,
and the antitumor activity of the new derivative was higher than that of ergosterol-3-O-(ꢀ-D-glucopyranoside) in 48 and 72 h
[6]. Thus, the sugar part of the ergosterol glycosides plays an important role in the improvement of its biological activity, so
further study is currently being carried out in our laboratory.
b
a
c
100
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
12.5
25
50
100
200
12.5
25
50
100
g/mL
200
12.5
25
50
100
g/mL
200
Concentration,
ꢀ
Concentration,
ꢀ
g/mL
Concentration,
ꢀ
- Compound
5
- Ergosterol
Fig. 1. Growth inhibition activities of compound 5 and ergosterol on S180 cell lines at 24 h (a), 48 h (b), and 72 h (c).
EXPERIMENTAL
Ergosterol-3-O-(2,3,4,6-tetra-O-benzoyl-ꢀ-D-galactopyranosyl-(1ꢁ4)-2,3,6-tri-O-benzoyl-ꢀ-D-glucopyranoside)
(4). To a stirred solution of 2,3,4,6-tetra-O-benzoyl-ꢀ-D-galactopyranoyl-(1ꢁ4)-2,3,6-tri-O-benzoyl-ꢀ-D-glucopyranosyl,
ꢃꢄꢄ
trichloroacetimidate (3, 2.84 g, 2.34 mmol) in 200 mL of dry CH Cl 4A molecular sieves (1.5 g) was added, followed by
2
2
ergosterol (0.90 g, 2.27 mmol) as a solid powder. The mixture was stirred for 0.5 h at 0ꢃC, then TMSOTf (0.17 mL, 1.0 mmol)
was added under Ar protection. The mixture was continuously stirred at 0ꢃC for 1 h and room temperature for another 0.5 h,
253

then neutralized by Et N. The yellow solution was diluted with CH Cl and filtered to remove the molecular sieves. The
3
2
2
organic layer was concentrated under reduced pressure to give a yellow syrup, which was purified by column chromatography
(EtOAc–petroleum ether, 1:4, v/v) to give compound 4 (2.57 g, 1.77 mmol, 78.0%) as a white powder with R 0.61
f
25
(EtOAc–petroleum ether, 1:4, v/v); [ꢅ] +20ꢃ (c 0.1, CH Cl ).
D
2
2
1
H NMR (400 MHz, CDCl , , ppm, J/Hz): 7.10–8.03 (35H, m, ArH), 5.84 (1H, d, J = 5.6, H-6), 5.63 (1H, d, J = 8.0,
3
H-1ꢆꢆ), 5.78–5.69 (3H, m, H-7, 22, 23), 5.47 (1H, dd, J = 3.1, H-4ꢆ), 5.43 (1H, t, J = 9.5, H-3ꢆꢆ), 5.36 (1H, dd, J = 10.1, 8.2, H-2ꢆ),
5.21 (1H, dd, J = 9.5, 8.0, H-2ꢆꢆ), 5.11 (1H, dd, J = 10.1, 3.1, H-3ꢆ), 4.79 (1H, d, J = 8.1, H-1ꢆ), 4.63 (1H, dd, J = 12.3, 2.1,
H-6ꢆꢆ), 4.38–4.27 (3H, m, H-6ꢆꢆ, H-6aꢆ, 6bꢆ), 4.11–4.04 (2H, m, H-5ꢆ, 4ꢆꢆ), 3.96–3.87 (1H, m, H-5ꢆꢆ), 3.82–3.74 (1H, m, H-3),
1.55 (3H, d, J = 6.4, H-21), 1.47 (3H, d, J = 3.4, H-27), 1.35 (3H, d, J = 8.9, H-25), 1.08 (3H, s, H-19), 0.96 (3H, t, J = 6.1,
13
H-28), 0.89 (3H, s, H-18). C NMR (100 MHz, CDCl , , ppm): 165.84, 165.81, 165.59, 165.45, 165.41, 165.24, 165.18
3
(C=O, Bz), 158.53 (C-8), 147.49 (C-5), 140.63 (C-23), 135.51 (C-22), 135.37–123.01 (CH, Ar), 117.95 (C-6), 99.69 (C-1ꢆꢆ),
99.24 (C-1ꢆ), 86.97 (C-7), 77.95 (C-5ꢆꢆ), 76.75 (C-4ꢆꢆ), 72.55 (C-2ꢆꢆ), 71.11 (C-2ꢆ), 70.99 (C-5ꢆ), 69.26 (C-4ꢆ), 68.71 (C-3ꢆꢆ),
67.12 (C-3ꢆ), 63.07, 62.69 (C-6ꢆ, 6ꢆꢆ), 57.62 (C-3), 56.63 (C-17), 55.23 (C-14), 48.52 (C-9), 48.41 (C-13), 46.31 (C-24), 43.49
(C-20), 41.58 (C-12), 40.08 (C-4), 39.43 (C-1), 35.82 (C-25), 34.69 (C-2), 30.81 (C-10), 29.92 (C-16), 28.22 (C-15), 24.89
(C-26), 23.84 (C-11), 20.98 (C-21), 20.88 (C-27), 20.64 (C-28), 20.25 (C-19), 20.07 (C-18), 19.14 (C-9). HR-ESI-MS m/z
+
1471.6234, [M + Na] (calcd for C H O Na, 1471.6182).
89 92 18
Ergosterol-3-O-(ꢀ-D-galactopyranosyl-(1ꢁ4)-ꢀ-D-glucopyranoside) (5). Compound 4 (2.04 g, 1.41 mmol) was
dissolved in MeOH–CH Cl (1:1, 80 mL), and then 172 mg NaOMe was added. After stirring at room temperature for 3.5 h,
2
2
+
the solution was neutralized by ion-exchange resin (H ), and then filtered and concentrated. The white residue was purified
by column chromatography (CH Cl –MeOH, 8:1ꢁ5:1) to give 5 as a white solid (0.82 g, 1.14 mmol, 81.0%) with R 0.28
2
2
f
25
1
(CH Cl –MeOH, 5:1, v/v) [ꢅ] –15ꢃ (c 0.1, CH Cl ). H NMR (400 MHz, DMSO-d , , ppm, J/Hz): 5.71 (1H, d, J = 5.7, H-6),
2
2
D
2
2
6
5.68–5.42 (3H, m, H-7, 22, 23), 5.35 (1H, d, J = 8.0, H-1ꢆꢆ), 5.26 (1H, dd, J = 3.2, H-4ꢆ), 5.24 (1H, t, J = 9.2, H-3ꢆꢆ), 5.11 (1H,
dd, J = 10.4, 8.0, H-2ꢆ), 5.05 (1H, dd, J = 9.2, 8.4, H-2ꢆꢆ), 4.96 (1H, dd, J = 10.4, 3.2, H-3ꢆ), 4.57 (1H, d, J = 8.0, H-1ꢆ), 4.47
(1H, dd, J = 12.5, 2.0, H-6ꢆꢆ), 4.16–4.05 (3H, m, H-6ꢆꢆ, H-6aꢆ, 6bꢆ), 3.89–3.82 (2H, m, H-5ꢆ, 4ꢆꢆ), 3.77–3.74 (1H, m, H-5ꢆꢆ),
3.71–3.63 (1H, m, H-3), 1.25 (3H, d, J = 6.7, H-21), 1.21 (3H, d, J = 3.5, H-27), 0.95 (3H, d, J = 8.6, H-25), 0.91 (3H, s, H-19),
13
0.86 (3H, t, J = 6.4, H-28), 0.73 (3H, s, H-18). C NMR (100 MHz, DMSO-d , , ppm): 158.93 (C-8), 147.29 (C-5), 134.83
6
(C-23), 130.57 (C-22), 118.35 (C-6), 117.62 (C-7), 102.99 (C-1ꢆꢆ), 102.47 (C-1ꢆ), 77.71 (C-2ꢆ), 76.65 (C-4ꢆꢆ), 75.85 (C-2ꢆꢆ),
74.48 (C-5ꢆꢆ), 74.13 (C-5ꢆ), 73.42 (C-3ꢆ), 71.58 (C-3ꢆꢆ), 71.56 (C-4ꢆ), 67.62 (C-3), 62.50, 62.19 (C-6ꢆ, 6ꢆꢆ), 56.53 (C-17), 55.23
(C-14), 49.52 (C-9), 48.41 (C-13), 46.31 (C-24), 41.59 (C-20), 40.38 (C-12), 35.78 (C-4), 34.47 (C-1), 30.98 (C-25), 29.69
(C-2), 28.76 (C-10), 27.92 (C-16), 25.32 (C-15), 24.69 (C-26), 23.84 (C-11), 22.68 (C-21), 20.98 (C-27), 20.64 (C-28), 20.26
+
(C-19), 19.10 (C-18), 17.14 (C-9). HR-ESI-MS m/z 743.4386 [M + Na] (calcd for C H O Na, 743.4347).
40 64 11
Antitumor Activity. Growth inhibition against S180 from the laboratory of Tianjin University of Science and
Technology caused by compound 5 and ergosterol was studied to compare their antitumor activities. The inhibitory effects at
24 h (a) and 48 h (b) were assessed by the colorimetric-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays
after measuring the number of viable cells that survived through treatment with 25, 50, 100, 200, and 400 mg/mL of tested
compounds for 1–3 days.
The cell lines were maintained in a medium consisting of 10% RPMI 1640/FCS at 37ꢃC in an atmosphere of 5% CO .
2
6
Malignant cell lines, 10 cells/mL in 96-well round bottom plates (Costar Corporation, Cambridge, MA, USA), were cultured
with various concentrations at 24 (a) and 48 h (b) in a 5% CO -air humidified atmosphere at 37ꢃC for 20, 44, and 68 h, then
2
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. MTT uptake was measured
after 4 h of incubation using a scintillation counter. The inhibitory effect of each compound on cell proliferation was calculated
using the following equation:
Inhibition persent (%) = (Control group (cpm) – Experimental group (cpm)/Control group (cpm)) ꢇ 100.
ACKNOWLEDGMENT
This work was financially supported by the Foundation of Tianjin University of Science and Technology (No. 20120106),
the Foundation of Tianjin Educational Committee (No. 20090604), and National Spark Program of China (2012GA610005).
254

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