Synthesis and biological evaluation of two salidroside analogues in the PC12 cell model exposed to hypoglycemia and serum limitation

Article abstract of DOI:10.1248/cpb.59.1045

Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has displayed a broad spectrum of pharmacological properties. In this paper, two analogues were prepared with the glucosamine and N-acet

Full text of DOI:10.1248/cpb.59.1045

August 2011
Note
Chem. Pharm. Bull. 59(8) 1045—1047 (2011)
1045
Synthesis and Biological Evaluation of Two Salidroside Analogues in the
PC12 Cell Model Exposed to Hypoglycemia and Serum Limitation
a
b
b
a
,b
Yibing GUO,a,b,c Xiaohong LI, Yahong ZHAO, Yongxing SI, Hui ZHU, and Yumin YANG
*
a Surgical Comprehensive Laboratory of Affiliated Hospital, Nantong University; b Jiangsu Key Laboratory of
Neuroregeneration, Nantong University; and c Medical School, Nantong University; No. 19, Qixiu Road, Nantong, Jiangsu
226001, P. R. China. Received March 1, 2011; accepted May 22, 2011; published online May 30, 2011
Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal
plant, and has displayed a broad spectrum of pharmacological properties. In this paper, two analogues were pre-
pared with the glucosamine and N-acetylglucosamine as glycosyl donor, 2-(4-hydroxyphenyl)ethanol as glycosyl
acceptor. The effects of them over PC12 cell model exposed to hypoglycemia and serum limitation were assessed
with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Flow Cytometry and Western
blot analysis.
Key words salidroside; analogues; N-acetylglucosamine; hypoglycemia; PC12 cell
Salidroside (Fig. 1), a traditional Chinese medicine, which zinc chloride and 4,4ꢂ-dimethoxytriphenylmethyl chloride as
may be synthesized by glucosylation of 2-(4-hydroxy- catalysts. Compound 1 can readily be prepared in a single
phenyl)ethanol, showed a range of pharmacological proper- step from N-acetylglucosamine by reaction with acetyl chlo-
ties, such as resisting anoxia,1) anti-radiation and anti- ride.13) The designed target compound 5 was obtained from 3
fatigue,2,3) postponing ageing,4) preventing cardiovascular by direct deacetylation with CH3ONa/CH3OH and catalytic
disease5) and anti-tumor.6) It has been reported that the hydrogenation by refluxing with 5% Pd/C and HCOONH4 in
species of glycosyl donor could affect activities of glyco- anhydrous methanol. Compound 6 was obtained from 5 by
sides, for example, the antioxidant activity increases in the refluxing with 30% KOH/ethanol (m/v) (Chart 1). Com-
1
order glucoseꢀmannoseꢁfructose.7)
pounds 5 and 6 were beta configuration identified from H-
N-Acetylglucosamine (GlcNAc) can be obtained by either NMR studies.
chemical or enzymatic hydrolysis of chitin and chitosan.8)
According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
Both natural and synthetic glucosamine-containing com- tetrazolium bromide (MTT) assay, after the PC12 cells were
pounds have been reported to have a range of biological ac- exposed to hypoglycemia and serum limitation for 24 h, cell
tivities.9—12) N-Acetylglucosamine has been shown, there- viabilities were significantly decreases, as compared to con-
fore, to be an available starting material for physiologically trol group. And further showed that pretreatment with com-
or pharmacologically important products.
pounds 5, 6 and salidroside at different concentrations (80,
The purpose of this study was to synthesize two salidro- 160, 320 mg/ml) significantly attenuated the cell viability loss
side analogues with the glucosamine and N-acetylglu- evoked by hypoglycemia and serum limitation, and the atten-
cosamine as glycosyl donor, 2-(4-hydroxyphenyl)ethanol as uating effect on the survival of cultured PC12 cells displayed
glycosyl acceptor, and to investigate their activities in the a dose dependent pattern (Fig. 2).
PC12 cell model exposed to hypoglycemia and serum limita-
tion, as compared with salidroside.
The change in the percentage of apoptotic cells were also
analyzed by Hoechst 33342 staining (Fig. 3). In comparison
The synthesis of the compound 3 was completed, starting to control group, exposure to hypoglycemia and serum limi-
from 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyra- tation alone produced significantly apoptosis. Pre-incubation
nosyl chloride 1 and 2-(4-(benzyloxy)phenyl)ethanol 2. The with 80, 160, and 320 mg/ml of compounds 5, 6 and salidro-
reaction was conducted in dry dichloromethane for 3 h with side, the apoptotic percentages were significantly lower than
that produced by exposure to hypoglycemia and serum limi-
tation alone.
The quantitative comparison indicated that the percentage
of early apoptotic PC12 cells induced by exposure to hypo-
glycemia and serum limitation alone was significantly larger
Fig. 1. The Chemical Structure of Salidroside
than in control group, and also significantly larger than that
Reagents: a) ZnCl2, DMT·Cl, CH2Cl2; b) NaOMe, MeOH, rt, 3 h; c) HCOONH4, 5% Pd/C, CH3OH, reflux; d) 30% KOH, EtOH.
Chart 1. Synthetic Route for Compounds 5 and 6
∗ To whom correspondence should be addressed. e-mail: yangym@ntu.edu.cn
© 2011 Pharmaceutical Society of Japan

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Vol. 59, No. 8
B-cell lymphoma-2 (Bcl-2) antibody, primary monoclonal anti-Bax anti-
body, and primary monoclonal anti-b-actin antibody were purchased from
Sigma. AnnexinV-fluorescein isothiocyanate (FITC) ApoptosisDetection Kit
was purchased from BenderMedsystens. IRDye 800 Conjugated Affinity
Purified Goat Anti-Mouse immunoglobulin G (IgG) was purchased from
KPL (USA). Slidroside was purchased from Aladdin.
induced by pretreatment with 320 mg/ml compounds 5, 6 and
salidroside followed by exposure to hypoglycemia and serum
limitation. The percentage of necrotic cells, however, dis-
played no significant difference among the different groups
(Fig. 4).
General Procedure for Compounds 5 and 6 2-Acetamido-3,4,6-tri-O-
acetyl-2-deoxy-a-D-glucopyranosyl chloride 1 (1.0 g, 2.7 mmol) and 2-(4-
(benzyloxy)phenyl)ethanol 2 (500 mg, 2.3 mmol) were added to a suspen-
sion of zinc chloride (280 mg, 2 mmol) and 4,4ꢂ-dimethoxytriphenylmethyl
(710 mg, 2 mmol) in a dry dichloromethane. The mixture was stirred at
room temperature for 5 h until reaction was complete as shown by TLC, then
20 ml dichloromethane was added, and the mixture was washed with satu-
rated sodium bicarbonate. Solvent was removed in vacuo and the residue
was purified by column chromatography to yield the glycoside 3 0.91 g
(72.2%).
Western blot analysis showed that compounds 5 and 6
against hypoglycemia and serum limitation-induced cell
apoptosis in the PC12 cells partly attributed to their evoked
modulation of apoptosis-related gene expression, which was
in accordance with salidroside14) (Fig. 5).
Experimental
General Commercial reagents were used without further purification
1
unless otherwise stated. Melting points were uncorrected. H-NMR spectra
were recorded on a Bruker AC 400 instrument at 300 MHz, with tetra-
methylsilane (TMS, d 0.00) as the internal standard and were run in D2O,
J values were expressed in Hz. Elemental analysis (C, N and H) were per-
formed on ElementarVarioEL III analyzer (German). Flash chromatography
was performed on silica gel (200—300 mesh). Primary monoclonal anti-
To a solution of compound 3 (1.6 mmol) in MeOH (25 ml) was added
NaOMe (40 mg), and the reaction mixture was stirred at room temperature
for 3 h. After that diluted with MeOH and acidified with Amberlite IRA-120
(Hꢃ) resin to pHꢄ7. The reaction mixture was filtered and evaporated to
dryness under reduced pressure afford 4 (0.69 g, 86.9%) as white solid.
A mixture of 4 (600 mg, 1.4 mmol), 5% Pd/C (210 mg) and ammonium
formate (441 mg, 7 mmol) in MeOH (10 ml) was stirred at refluxing for 6 h.
Then filtered, concentrated and purified by column chromatography using
8 : 1 CHCl3–MeOH to give white solid 430 mg (90%) of 5, mp 176—178 °C,
Rfꢄ0.22 (MeOH : CHCl3ꢄ1 : 5). 1H-NMR (D2O) d: 7.17 (2H, d, Jꢄ8.07
Hz), 6.87 (2H, d, Jꢄ7.86 Hz), 4.46 (1H, d, Jꢄ8.07 Hz), 4.12—4.17 (1H, m),
3.94 (1H, d, Jꢄ12.3 Hz), 3,73—3.76 (2H, m), 3.61 (1H, t, Jꢄ9.8, 8.58 Hz),
3.43—3.48 (3H, m), 2.77—2.82 (2H, m), 1.81 (3H, s). IR (KBr) cmꢅ1
:
3345, 2928, 2842, 1655, 1520, 1451, 1382, 1274, 1128, 1082, 1006, 825.
Anal. Calcd for C16H23NO7: C, 56.30; H, 6.79; N, 4.10. Found: C, 56.36; H,
6.83; N, 4.03.
Compound 5 (375 mg, 1.1 mmol) was added to 15 ml 30% KOH/EtOH
(m/v) in 50 ml flask, refluxing for 10 h, extracted with dichloromethane, con-
centrated and purified by column chromatography using 1 : 5 MeOH–CHCl3
to give 290 mg (88%) of 6, mp 151—153 °C, Rfꢄ0.34 (MeOH : CHCl3ꢄ
Fig. 2. Protective Effect of Compounds 5, 6 and Salidroside on Hypo-
glycemia and Serum Limitation Induced Cytotoxicity in PC12 Cells
The incubation in the high-glucose medium during the whole treatment period
served as control group, and the treatment only with hypoglycemia and serum limita-
tion for 24 h served as hypoglycemia and serum limitation alone group. The cell viabili-
ties were expressed as the percent (%) of the control value by using MTT assay. The
data were expressed as meansꢆS.D. of three independent experiments (nꢄ3). ∗ pꢀ0.01
vs. hypoglycemia and serum limitation alone group.
Fig. 4. Flow Cytometry with AnnexinV/PI Staining for Cultured PC12
Cells in Control Group, Hypoglycemia and Serum Limitation Alone Group,
Pretreatment with 320 mg/ml Compounds 5, 6 and Salidroside Plus Expo-
sure to Hypoglycemia and Serum Limitation
The percentage of viable, early apoptotic, and necrotic cells in the total cell popula-
tion was shown for the above five different treatments. The data were expressed as
meansꢆS.D. of three independent experiments (nꢄ3). ∗ pꢀ0.01 vs. hypoglycemia and
serum limitation alone group.
Fig. 3. The Percentage of Apoptotic Cells in the Total Cell Population Is
Shown for Different Treatments of PC12 Cell the Same as in Fig. 2
∗ pꢀ0.01 vs. hypoglycemia and serum limitation alone group.
Fig. 5. Western Blotting Image for (a) Compound 5, (b) Compound 6

August 2011
1047
1 : 3), 1H-NMR (D2O) d: 8.46 (1H, s), 7.24 (2H, d, Jꢄ8.07 Hz), 6.90 (2H, d,
Jꢄ8.07 Hz), 4.70 (1H, d, Jꢄ8.9 Hz), 4.12—4.17 (1H, m), 3.90—3.94 (2H,
m), 3.72—3.76 (1H, m), 3.61—3.67 (1H, m), 3.45—3.47 (2H, m), 3.01 (1H,
d, Jꢄ9.27 Hz), 2.89—2.95 (2H, m). IR (KBr) cmꢅ1: 3404, 2917, 2840,
1639, 1517, 1460, 1095, 1066, 713, 627. Anal. Calcd for C14H21NO6: C,
56.18; H, 7.07; N, 4.68. Found: C, 56.22; H, 7.12; N, 4.60.
control.
Statistical Analysis All measurements were repeated 3 times, data were
expressed as meansꢆS.D. Statistical differences among groups were ana-
lyzed by one-way analysis of variance (ANOVA) tests, and differences were
considered significant if pꢀ0.05.
Acknowledgments The financial supports of Hi-Tech Research and De-
velopment Program of China (863 Program, Grant No.2006AA02A128),
Nature Science Foundation of China (Grant No.30970713), Basic Research
Program of Jiangsu Province (Grant No. BK2009518) and the Priority Aca-
demic Program Development of Jiangsu Higher Education Institutions are
gratefully acknowledged.
Cell Culture Rat PC-12 cells, obtained from the American Type Cul-
ture Collection (Manassas, VA, U.S.A.), were plated and maintained in high-
glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% horse serum, 5% fetal bovine serum (FBS), 100 U/ml penicillin, and
100 U/ml streptomycin in 5% CO2/air at 37 °C. After being pretreated with
80, 160, or 320 mg/ml 5, 6 and salidroside for 24 h, the cells were subjected
to hypoglycemia and serum limitation by replacing the culture medium with
the glucose-free DMEM supplement with 1% horse serum and 1% FBS,
100 U/ml penicillin, and 100 U/ml streptomycin, and in the presence of 5, 6
and salidroside at original concentrations for another 24 h incubation.15) At
the end of incubation, the MTT solution was added and further incubated for
4 h at 37 °C followed by the addition of dimethyl sulfoxide (DMSO) to dis-
solve the resulting formazan. The absorbance (OD) values were measured at
570 nm with Thermo Multiskan MK3 (U.S.A.).
Detection of Apoptosis On the detection of apoptosis, PC12 cells were
fixed in 4.0% paraformaldehyde, then stained with Hoechst 33342 at 37 °C,
followed by observation under a fluorescence microscope (Olympus, Japan).
In order to quantify the apoptotic process, cells with fragmented or con-
densed DNA and normal DNA were counted, data were expressed as the
ratio of apoptotic cells to total cells.
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