Cholesterol-derived novel anti-apoptotic agents on the structural basis of ginsenoside Rk1

Article abstract of DOI:10.1016/j.bmcl.2010.09.071

Design and synthesis of cholesterol-derived anti-apoptotic agents were described. The synthesized cholesterol analogs designed on the structural basis of ginsenoside Rk1 inhibited the undesirable apoptosis of human endothelial cells, which are induced by

Full text of DOI:10.1016/j.bmcl.2010.09.071

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7102–7105
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cholesterol-derived novel anti-apoptotic agents on the structural basis of
ginsenoside Rk1
Sujin Lee a, Sony Maharjan b, Kyeojin Kim a, Nam-Jung Kim a, Hyun-Jung Choi b, Young-Guen Kwon b,
,
⇑
,
Young-Ger Suh a, ,
⇑
a College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
b Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2010
Revised 13 September 2010
Accepted 14 September 2010
Available online 21 October 2010
Design and synthesis of cholesterol-derived anti-apoptotic agents were described. The synthesized cho-
lesterol analogs designed on the structural basis of ginsenoside Rk1 inhibited the undesirable apoptosis of
human endothelial cells, which are induced by a vascular injury. In particular, analogue 1 possessing 4,6-
di-O-acetyl-2,3-dideoxyhex-2-enopyran linked to hydroxyl group of cholesterol exhibited the most effec-
tive anti-apoptotic activities at both 5 and 10 lg/ml.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anti-apoptotic agents
Cholesterol
Ginsenoside Rk1
Homeostasis in tissues of multi organism can be achieved by
proper balance between cell proliferation and apoptosis.1 Many
pathological phenomenons such as cancer,2 autoimmunity,3 sep-
sis,4 and neuro degenerative diseases5 were observed at the col-
lapse of this equilibrium. Adequate maintenance of endothelial
cells constructing the inner cellular lining of blood vessels are in-
volved in vascular homeostasis for health.6 It has been known that
vascular endothelial cell (VEC) apoptosis can induce an alteration
in the integrity of vessels and the function of endothelium. This
programmed cell death may contribute to pathogenesis of a variety
of human diseases, such as atherosclerosis, allograft vasculopathy,
and congestive heart failure (CHF).7 Therefore, apoptosis inhibition
has been considered as a new approach for treatment of vascular
disorder. In addition, it has recently been revealed that ginsenoside
Rg3 isolated from the root of genus Panax (ginseng), which exerts
many pharmacological activities such as anti-tumor, anti-inflam-
matory, neuroprotection against the cerebral ischemia-induced in-
jury,8 has anti-apoptotic activity.9 Moreover, our efforts for
development of the potent anti-apoptotic agents have driven dis-
covery of ginsenoside Rk1, which exhibit potent anti-apoptotic
activity in human umbilical vein endothelial cell (HUVEC) lines.10
However, instability of Rk1 under acidic conditions and the extract
process-dependent composition of ginsenosides limited studies on
biological functions and underlying mechanisms of their biological
activities.11 Up to date, most researches on natural ginsenosides
has focused on their biological activities. Few studies on struc-
ture–activity relationship (SAR) with the synthetic ginsenoside
analogues was reported.12 Thus, we were interested in the anti-
apoptosis associated SAR of the ginsenoside Rk1 analogues.13 On
the basis of our preliminary studies that the glycosyl moiety at
C3 of Rk1 is crucial for the VEC apoptosis inhibiting effect, we
OH
HO
O
HO
O
HO
HO
HO
HO
H
O
O
ginsenoside Rk1
OH
carbohydrate
equivalent
O
⇑
cholesterol scaffold
Corresponding authors. Tel.: +82 2 880 7875; fax: +82 2 888 0649.
E-mail addresses: ygkwon@yonsei.ac.kr (Y.-G. Kwon), ygsuh@snu.ac.kr (Y.-G.
(protopanaxadiol equivalent)
Suh).
Figure 1. Cholesterol analogues based on ginsenoside Rk1.
These co-corresponding authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.071

S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7102–7105
7103
1
2
4
6
R= Ac
R= Bz
R= H
RO
RO
f
g
R= Me
O
O
a or b
d
b or c
OR
O
RO
RO
O
O
HO
O
cholesterol
e
10
3
5
7
R= Bz
R= H
f
g
h
R= Me
OR
O
RO
OR
O
RO
8
R= Bz
R= H
O
O
f
9
11
Scheme 1. Reagents and conditions: (a) 3,4,6- tri-O-acetyl-
glucan, CAN, CH3CN; (d) DHP, PPTS, CH2Cl2, 68%; (e) 2,3,4,6-tetrabenzoyl-
0 °C, 32%; (h) H2, Pd/C, EtOAc, 98%.
D
-glucan, BF3ꢀOEt2, CH2Cl2, 43%; (b) 3,4,6-tri-O-benzoyl-
D
-glucan, TsOH, CH2Cl2, 26%; (c) 3,4,6-tri-O-methoxy-
D-
D
-gluconosyl bromide, AgOTf, CH2Cl2, ꢁ20 °C, 45%; (f) NaOMe, MeOH/CH2Cl2, 60%; (g) MeI, NaH, THF,
attempted to develop novel and structurally simplified VEC apop-
tosis inhibitors by introducing the carbohydrate equivalents. In
addition, we selected cholesterol scaffold to substitute for the pro-
topanaxadiol backbone of Rk1 in terms of structural similarity and
ready accessibility. Taking together these, we have conducted de-
sign and synthesis of a series of cholesterol analogues. We also
investigated their inhibitory effects on the serum deprivation-
induced apoptosis of human endothelial cells (HECs). We herein
report our successful results.
As an equivalent of the glycosyl unit, cyclic ether (hydropyran),
acyclic ether (alkyl ether) and aryl moieties were introduced to C3
alcohol of the cholesterol scaffold (Fig. 1). Our synthesis com-
menced with an introduction of dihydro- or tetrahydropyran moi-
eties to C3 alcohol of cholesterol. Dihydropyran analogue 1 was
prepared by reaction of 3,4,6-tri-O-acetyl-D-glucal with cholesterol
in the presence of boron trifluoride. Treatment of tri-O-benzoyl
glucal with cholesterol in acidic media yielded a mixture of 2
and 3, which could be separated by column chromatography.
Removal of benzoyl group of 2 and 3 by using NaOMe afforded 4
and 5, which could be further transformed into 6 and 7 by methyl-
ation (NaH, iodomethane), respectively. Analogue 9 was prepared
by reaction of cholesterol with 2,3,4,6-tetrabenzoyl-D-glucopyran-
osyl bromide in the presence of silver triflate,14 followed by
HO
a or b
HO
a or b
RO
O
12 R=
13 R=
14 R=
15 R=
O
c
OH
RO
16 R= benzyl
O
17 R= naphthalenyl
18 R= CONH
2
Scheme 2. Reagents and conditions: (a) iPr2NEt, CH2Cl2, MEMCl, 48% for 12; (b)
NaH, THF/DMF (1:1), allyl bromide, reflux, 36% for 9; 2S-(+)-glycidol tosylate, 45%
for 15; (c) Grubbs’ 2nd catalyst, 1,4-butene diol, CH2Cl2, reflux, 30%.
Scheme 3. Reagents and conditions: (a) NaH, TBAI, aryl bromide, THF, reflux, 30%;
(b) Cl3CCONCO, CHCl3, 31%.

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S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7102–7105
debenzoylation. Reaction of cholesterol with 3,4-dihydro-2H-pyr-
an in the presence of pyridinium p-toluene sulfonate in CH2Cl2
afforded 10, which was readily converted to 11 by Pd/C catalyzed
hydrogenation (Scheme 1).
Next, we synthesized a series of cholesterol analogues linked
with the acyclic, or alkyl ether moieties. Analogue 12 was synthe-
sized by reaction with MEMCl in the presence of Hunig’s base. Ally-
lation of cholesterol by treatment with sodium hydride and allyl
bromide in refluxing THF/DMF provided allyl ether 13. Cross
metathesis of 13 with 1,4-butene diol by employing Grubbs’ 2nd
generation catalyst afforded 14. Reaction of cholesterol with so-
dium hydride and then with (2S)-glycidol tosylate afforded 15
(Scheme 2).
To confirm the spatial influence of glycosyl residue, aryl moiety
as a carbohydrate equivalent was also introduced to the cholesterol
scaffold. Analogues 16 and 17 were prepared by O-benzylation and
O-naphthylation of cholesterol under reaction conditions of sodium
hydride and TBAI in refluxing THF. Treatment of cholesterol with
trichloroacetyl isocyanate gave carbamate 18 (Scheme 3).
Inhibitory effect of the cholesterol analogues were investi-
gated on the growth factor deprivation-induced HUVEC cell
death using the MTT colorimetric method at concentration of
5 lg/ml and 10 lg/ml for 24 h and 48 h, respectively. The result
of MTT assay is summarized in Figure 2. It is noticeable that the
acetyl dihydropyran derivative 1 was more potent than ginseno-
side Rk1 known as one of the potent anti-apoptotic agent. In
contrast, analogues 5 and 6 possessing methoxy or hydroxy
dihydropyran exhibited less potent activity compared to Rk1.
This result revealed that the acyl group of the carbohydrate
equivalents is important to manifest the inhibitory effect of the
cholesterol analogues. The tetrahydropyran analogue 10 also
exhibited comparable activity to that of Rk1 after 24 h treat-
ment. However it was slightly less potent than Rk1 after 48 h
treatment. Analogues 4, 7, and 9 possessing the substituted pyr-
an moiety did not show any significant inhibitory activity. Ana-
logues, 12, 13, 14, and 17 possessing the acyclic carbohydrate
equivalents exhibited moderate inhibitory activities at the con-
centration of 5 lg/ml. It is also noteworthy that the tetrahydro-
pyran analogue 10 was more potent than the dihydrocholesteryl
tetrahydropyran analog 11.
For further investigation of the cell protective effect of ana-
logues 1 and 10, their anti-apoptotic effect on cell viability in hu-
man retina endothelial cells (HRECs) was examined. In addition,
their dose-dependent activities were confirmed by the cell survival
counting assay, which was performed at 0.1 lg/ml to 10 lg/ml,
respectively. The concentration-dependent cell viabilities by both
analogues 1 and 10 are well revealed in Figure 3.
The anti-apoptotic effects of analogues 1 and 10 were further
confirmed by their inhibitory effects on nuclear DNA condensa-
tion and fragmentation in VECs, which were analyzed by
Figure 2. Effects of the cholesterol analogs on apoptosis of HUVECs. (A) 5 lg/ml for
24 h treatment, (B) 5 lg/ml, 48 h, (C) 10 lg/ml 24 h, (D) 10 lg/ml, 48 h treatment.
0 h, the viability of the cells cultured in the medium without any derivatives. DM,
the viability of the cells cultured in the medium containing DMSO used as a vehicle
control. R, the viability treated with Rk1.
Figure 3. Inhibitory effects by analogs 1, and 10 on apoptosis of HRECs. (A) Treatment with analog 1 at sequential concentration from 0.1 to 10
with analog 10 at sequential concentration from 0.1 to 10 g/ml for 48 h.
lg/ml for 48 h. (B) Treatment
l

S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7102–7105
7105
with DAPI revealed increased cell survivals by 1 or 10 after 48 h of
serum deprivation. TUNEL assay specifically detecting DNA frag-
mentation also revealed that analogues 1 and 10 suppressed the
apoptotic cell death induced by serum deprivation in a dose-
dependent manner (Figs. 4 and 5).
In conclusion, we identified a series of potent anti-apoptotic
agents derived from easily accessible cholesterol. In particular,
analogues 1 and 10 possessing the cholesterol scaffold and the cyc-
lic carbohydrate equivalents exhibited excellent cell survival activ-
ities, which are equipotent to that of ginsenoside Rk1. In addition,
these analogues exhibited dose-dependent cell viability on HREC
line. The excellent human endothelial cell protective activities of
our novel Rk1-based anti-apoptotic agents envisage its valuable
application for treatment of vascular disorders. Currently, system-
atic studies on their therapeutic applications including in vivo test
of the potent cholesterol analogs as well as their mechanistic as-
pects are in good progress.
Acknowledgement
This work is supported by a grant from the Korea Health 21 R&D
Project, Ministry of Health Welfare & Family Affairs (A085136),
Korea.
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