Synthesis and protective effects of aralkyl alcoholic 2-acetamido-2-deoxy- β-d-pyranosides on hypoglycemia and serum limitation induced apoptosis in PC12 cell

Article abstract of DOI:10.1016/j.bmc.2011.07.031

Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological activities, especially neuroprotection. In an at

Full text of DOI:10.1016/j.bmc.2011.07.031

Bioorganic & Medicinal Chemistry 19 (2011) 5577–5584
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Bioorganic & Medicinal Chemistry
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Synthesis and protective effects of aralkyl alcoholic
2-acetamido-2-deoxy-b-D-pyranosides on hypoglycemia and serum
limitation induced apoptosis in PC12 cell
Ying Meng a, , Yibing Guo a,b, , Yong Ling c, Yahong Zhao a, Qi Zhang a, Xinyang Zhou a, Fei Ding a,
Yumin Yang a,
⇑
a Jiangsu Key Laboratory of Neuroregeneration, Nantong University, No. 19, Qixiu Road, Nantong, Jiangsu 226001, PR China
b Surgical Comprehensive Laboratory of Affiliated Hospital, Nantong University, No. 20, Xisi Road, Nantong, Jiangsu 226001, PR China
c Medical School, Nantong University, NO. 19, Qixiu Road, Nantong, Jiangsu 226001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidro-
side, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological
activities, especially neuroprotection. In an attempt to improve neuroprotective effects of new salidroside
Received 21 June 2011
Revised 18 July 2011
Accepted 19 July 2011
Available online 24 July 2011
analogs for ischemic stroke, a series of novel aralkyl alcoholic 2-acetamido-2-deoxy-b-D-pyranosides
were synthesized and their protective activities against the hypoglycemia and serum limitation induced
cell death in rat pheochromocytoma cells (PC12 cells) were studied. Most compounds showed strong
neuroprotective effects, especially for 4g and 4h, which exhibited a great potency superior to salidroside.
MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed
that pretreatment with 4g and 4h attenuated cell viability loss and apoptotic cell death in cultured PC12
cells. Caspase-3 colorimetric assay and Rhodamine 123 staining revealed the changes in expression levels
of caspase-3 and mitochondrial membrane potential in PC12 cells on exposure to hypoglycemia and
serum limitation with and without 4g and 4h pretreatment, respectively. All the results suggested that
4g and 4h protects the PC12 cells against hypoglycemia and serum limitation induced apoptosis possibly
by modulation of apoptosis-related gene expression and restoration of the mitochondrial membrane
potential. Therefore, these novel findings may provided a new framework for the design of new aralkyl
Keywords:
Salidroside
N-Acetylglucosamine
PC12 cell
Hypoglycemia and serum limitation
Apoptosis
Cerebral ischemic
alcoholic 2-acetamido-2-deoxy-b-
D-pyranosides as neuroprotective agents for treating cerebral ischemic
stroke and neurodegenerative diseases.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
stroke and neuroprotective agents could attenuate lots of the clin-
ical problems of ischemic stroke, the development of new neuro-
protective agents that inhibit neuronal apoptosis induced by
cerebral ischemia will be of great significance.8
Stroke is one of the leading causes of death and permanent dis-
ability in adults1 Most strokes (80%) are ischemic and the disease
leads loss of blood flow in a specific cerebral region, which upsets
the critical balance between the demand and supply of glucose,
oxygen, serum, and nutrient that are indispensable for the energy
generation.2 Morphological manifestations of programmed cell
death in the ischemic brain and biochemical evidence have
revealed that apoptosis was implicated in neurological diseases
including ischemic stroke.3,4 In general, cerebral ischemia could
induce neuronal apoptosis, which is a complex process with multi-
ple mechanisms, such as the release of proapoptotic proteins or
stimulation of caspase-3, mitochondrial dysfunction and so on.5–7
Given that there is no effective chemotherapy for cerebral ischemic
Salidroside (Fig. 1), one of the most potent compounds in
Rhodiola rosea L. was well known as an adaptogen in traditional
Chinesemedicineand has been shownto have diversepharmacolog-
ical activities, including anti-oxidation,9 anti-aging,10 anti-fatigue11
and anti-carcinogenic.12 Many recent reports and reviews have also
highlighted that salidroside exhibit potent neuroprotective activ-
ity.13,14 Our previous studies have revealed that salidroside exhib-
ited protective effects on hypoglycemia and serum limitation
induced cell apoptosis in cultured PC12 cells.15 It supposed that sal-
idroside probably possess an ability to protect neurons from ische-
mic damage.
Both natural and synthetic glucosamine-containing compounds
have been reported to possess neuroprotective action,16,17 immuno-
modulatory activity, anticoagulation18 and were used to treat
clinically kidney disorders19 and heart disease.20 N-acetylglucosa-
⇑
Corresponding author. Tel./fax: +86 513 85511585.
E-mail address: yangym@ntu.edu.cn (Y. Yang).
These authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.031

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Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
OH
firmed by IR, MS, 1H NMR, and elemental analysis. All the gluco-
O
sidic bonds of compounds were identified as b configurations by
the coupling constants (J) of the protons between C1 and C2 in
their 1H NMR spectra. The proton at C1 gave the signal as a doublet
at d 4.39–4.54 ppm (J = 8.4–8.5 Hz).
HO
HO
O
OH
OH
Figure 1. Chemical structure of salidroside (p-hydroxyphenethyl-b-
D
-glucoside).
2.2. Effects of target compounds 4a–h, 5i–k against hypogly-
cemia and serum limitation induced cytotoxicity in
differentiated PC12 cells
mine has been shown, therefore, to be an available starting material
for physiologically or pharmacologically important products. In
prior studies, we described the synthesis and preliminary biological
evaluationof some salidroside analogs and found that different agly-
cons of aralkyl alcohol influenced their activities.21 In an ongoing
study on the discovery and development of new anti-ischemic
agents, we hypothesized that novel type of aralkyl alcoholic 2-acet-
The aim of the research was to assess the neuroprotective
effects of aralkyl alcoholic 2-acetamido-2-deoxy-b-D-pyranosides
on hypoglycemia and serum limitation-induced cell injury to par-
tially model the pathological process of cerebral ischemia.2 PC12
cells were chosen for in vitro studies because they constitute a
widely used neuronal model system.24 First, we investigated the
effects of target compounds on preventing decreased cell viability
in PC12 cells induced by hypoglycemia and serum limitation. As
the results shown in Fig. 3, hypoglycemia and serum limitation
led to significant reduction of cell viability to 45.08 3.45%, com-
pared to control. When cells pretreated with different concentra-
amido-2-deoxy-b-D-pyranosides could have neuroprotective effects
on the PC12 cells against the damage caused by hypoglycemia and
serum limitation and inhibit hypoglycemia and serum limitation
induced cell apoptosis. Therefore, a total of 11 target compounds
(4a–h, 5i–k) were designed and synthesized by coupling the
N-acetylglucosamine with various aralkyl alcohols. Their in vitro
neuroprotective activities in the cell injury induced by hypoglyce-
mia and serum limitation, the inhibition of caspase-3 like protease
activity, and the preservation of mitochondrial membrane potential
were evaluated. Herein, the synthesis and preliminary biological
evaluation of these compounds were reported.
tions (8, 40, 200 lM) of compounds 4a–h and 5i–k for 24 h
followed by hypoglycemia and serum limitation treatment, most
compounds showed protective effects. Especially, compounds 4g
and 4h exhibited the most significant protective effects at the con-
centration of 40 lM, which increased cell viability to 86.62 3.99%
and 80.41 3.99%, respectively, whereas 4d, 4e, 5i, and 5k restored
cell survival to 60.65 2.34%, 65.90 2.45%, 65.36 3.21%, and
62.38 3.45%, which was approximate to salidroside group
2. Results and discussion
2.1. Synthesis of aralkyl alcoholic 2-acetamido-2-deoxy-b-D-
pyranosides
(65.62 3.87%). At the same time, 4c, 4f, and 5j (40
l
M) were
found to be moderately active, 4a and 4b (40
l
M) didn0t exert
obvious protective effects.
The synthesis route for aralkyl alcoholic b-
ransides 4 and 5 was shown in Fig. 2. 2-acetamido-3, 4, 6-tri
-O-acetyl-2-deoxy- -glucopyranosyl chloride 2 was prepared
from 2-acetamido-2-deoxy-
D-N-acetylglucopy-
Compounds 4g and 4h which superior to salidroside were fur-
ther researched by Hoechst 33342 staining, flow cytometry with
annexin V/PI staining, caspase-3 colorimetric assay and Rhodamine
123 staining.
a-D
D
-glucose by esterification using a
well-known procedure.22 The intermediates 3a–k were obtained
in moderate yields at room temperature by the glycosidation of
compound 2 in dry dichloromethane with different substituted
2.3. Anti-apoptotic effects of the compounds 4g and 4h by
Hoechst 33342 staining and flow cytometry with annexin V/PI
staining
aromatic alcohol using zinc chloride as
a
catalyst and
4,40-dimethoxytrityl chloride(DMT-Cl) as a cocatalyst.23 Further,
3a–k were deacetylated with CH3ONa/CH3OH and afforded the
Apoptosis after cerebral ischemia is one of the major pathways
that leads to the process of cell death.25 Therefore, we investigated
the anti-apoptotic effects of the compounds 4g and 4h by Hoechst
33342 staining assay. In the control group, the nuclei of PC12 cells
were round and homogeneously stained. After hypoglycemia and
corresponding aralkyl alcoholic b-D-N-acetylglucopyransides
4a–k with high yields. Finally, 4i–k on catalytic hydrogenation,
using ammonium formate and 5% Pd/C in anhydrous methanol,
gave 5i–k. The structures of the compounds 4a–h, 5i–k were con-
OH
OAc
OAc
O
O
R
O
a
b
R
AcO
AcO
HO
HO
AcO
AcO
+
HO
O
n
n
OH
NHAc
NHAc
Cl
NHAc
2
1
3a-3k
OH
c
4a: R=H
4b: R=H
4c: R=3-OH
4d: R=4-OH
n=1
n=2
n=1
n=1
4e: R=3-OMe, 4-OH n=1
O
R
HO
HO
4f: R=3,5-OMe
4g: R=4-OMe
4h: R=4-OMe
n=1
n=2
n=3
O
n
NHAc
4a-4h,4i-4k
OH
O
OH
O
R
d
R
HO
HO
HO
HO
O
O
n
n
NHAc
5i-5k
NHAc
4i-4k
4i: R=4-OBn
4j: R=4-OBn
n=2 5i: R=4-OH
n=3 5j: R=4-OH
n=2
n=3
4k:R=3-OMe,4-OBn n=3 5k: R=3-OMe,4-OH n=3
Figure 2. Synthetic route for aralkyl alcoholic 2-acetamido-2-deoxy-b-D-pyranosides. Reagents and conditions: (a) CH3COCl; (b) ZnCl2, DMT-Cl, CH2Cl2; (c)CH3ONa, CH3OH;
(d) HCOONH4, 5% Pd/C, CH3OH, reflux.

Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
5579
with 4g, 4h or salidroside significantly attenuated these increase
of caspase-3 activity induced by hypoglycemia and serum limita-
tion, causing the decrease in caspase-3 activity back to 1.55, 1.69,
or 1.97-fold, compared to control. The obtained data demonstrated
that the two compounds suppressed hypoglycemia and serum lim-
itation induced apoptosis in PC12 cells.
2.5. Restoration of mitochondrial membrane potential in PC12
cells
Increasing evidence suggests that mitochondrial dysfunction
participates in the induction of apoptosis and even be central to
the apoptotic pathway. Indeed, depolarization of the transmem-
brane potential, release of apoptogenic factors and loss of oxidative
phosphorylation could be induced by opening the mitochondrial
permeability transition pore.27 The loss of mitochondrial mem-
brane potential may be a consequence of the apoptotic-signaling
pathway.28 Therefore, we investigated the changes of the mito-
chondrial membrane potential (MMP) by Rhodamine fluorescence.
After exposure to hypoglycemia and serum limitation for 24 h
alone, the MMP of PC12 cells was significantly decreased to
42.12% compared with that of control. However, the MMP decrease
were markedly recovered after being pretreated with 4g, 4h or sal-
Figure 3. Protective effect of aralkyl alcoholic b-D-N-acetylglucopyransides on
hypoglycemia and serum limitation induced apoptosis in PC12 cells. PC12 cells
were pretreated with test compounds or salidroside on different concentrations (8,
40, 200 lM) for 24 h, then exposed to hypoglycemia and serum limitation for an
idroside (40
(Fig. 6).
lM), up to 82.86%, 79.05% or 71.43%, respectively.
additional 24 h with respective original concentrations of compounds maintained.
Viability was calculated as the percentage of living cells in treated cultures
compared to those in control cultures by using MTT assay. ⁄P <0.05 versus exposure
to hypoglycemia and serum limitation in alone. The data were presented as
mean SD of three independent experiments (each in triplicate).
2.6. Preliminary analysis of structure activity relationship
From structure activity relationship (SAR) studies, the protec-
tive effects of target compounds were influenced by the molecular
structure of the chemicals. It was indicated that the neuroprotec-
serum limitation insult for 24 h, 13.93% of cultured cells showed
typical characteristics of apoptosis, including the condensation of
chromatin, the shrinkage of nuclear and the appearance of a few
tive activities of the aralkyl alcoholic 2-acetamido-2-deoxy-b-D-
pyranosides against hypoglycemia and serum limitation-induced
cell injury were markedly influenced by the aromatic substituents.
Among the synthesized compounds, 4g and 4h containing para-
methoxy group of phenyl ring generally showed stronger protec-
tive activities than their analogs 5i and 5j with hydroxy at the
same position. Also, protective activities of 4g and 4h were higher
than those compounds with methoxy group at the meta-position
such as 4e, 4f, 5k. The enhancement of activity might be related
to the increment of hydrophobicity, which means that compounds
with higher lipophilicity would be easier to penetrate cell mem-
brances.29 From the data of 4c and 4d, it seemed that para-hydroxy
of phenyl ring might be favorable for protective efficacy in compar-
ison with meta-hydroxy. There was unapparent correlation be-
tween the neuroprotective activities and the length of alkyl chain
connecting benzene ring with N-acetylglucosamine. Compound 5i
containing alkyl chain with two carbons exhibited better activity
than that of one or three carbons. The space length of the alkyl
chain of two carbons may be appropriate for small molecules bind-
ing with correlative acceptors. In addition, compounds 4a and 4b
without aromatic substituents displayed poor protective effects
and this may be oxygen atom of substituent on the aromatic ring
could form a hydrogen bond with protein. Additional study is nec-
essary to elucidate their mechanism and structure activity
relationships.
apoptotic bodies. However, under pretreatments of 40 lM 4g or
4h, the apoptotic percentages were significantly reduced to 5.90%
or 7.12%, respectively (Fig. 4A and B). These apoptotic percentages
were lower than that of salidroside (8.31%).
To provide further evidence that 4g and 4h pretreatment can
prevent hypoglycemia and serum limitation induced apoptosis in
cultured PC12 cells, flow cytometer analysis with annexin V and
PI double-staining was investigated. We found that a significant
induction of PC12 cells apoptosis by hypoglycemia and serum lim-
itation, increasing early and late apoptotic percent, and the total
apoptotic rate reached to about 41.94%. While PC12 cells were
pre-cultured with 40 lM of 4g, 4h or salidroside prior to hypogly-
cemia and serum limitation, early stage of apoptosis were pre-
vented, and the apoptotic rate decreased to 11.21%, 15.51% and
17.56% respectively. The percentage of late stage of apoptotic cells,
however, displayed no significant difference. (Fig. 4C). Therefore,
we believed that 4g and 4h prevented the increased apoptosis rate
and nuclear morphologic changes induced by hypoglycemia and
serum limitation. In addition, the result revealed that the protec-
tive effects of 4g and 4h pretreatment manifested itself mainly in
an inhibition of early cell apoptosis.
2.4. Inhibition of caspase-3-like protease activity in PC12 cells
Caspases are cysteine dependent enzymes that play important
role in the induction, transduction and amplification of intracellu-
lar apoptotic signals. Among them, caspase-3 is a frequently
activated death protease, catalyzing the specific cleavage of many
key cellular proteins.26 Therefore, we measured the effects of
3. Conclusion
In summary, we synthesized a series of novel aralkyl alcoholic
2-acetamido-2-deoxy-b-D-pyranosides and studied their protective
activities against the hypoglycemia and serum limitation-induced
cell death in differentiated PC12 cells. Most target compounds dis-
played strong protective effects on the cell viability against the
damage caused by hypoglycemia and serum limitation, especially
4g and 4h (40 lM) on the activity of caspase-3 by colorimetric as-
say. As shown in Fig. 5, after exposure to hypoglycemia and serum
limitation for 24 h, the activity of caspase-3 was enhanced by
4.2-fold, as compared to control group. However, pretreatment

5580
Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
Figure 4. Protective effect of 4 g and 4 h on hypoglycemia and serum limitation-induced apoptosis in PC12 cells. (A) Representative fluorescence micrographs of Hoechst
33342 staining, which showed morphological apoptosis for PC12 cells in control (a), exposure to hypoglycemia and serum limitation alone (b), 40 M 4 g pretreatment plus
hypoglycemia and serum limitation (c), 40 M 4 h pretreatment plus hypoglycemia and serum limitation (d), and 40 M salidroside pretreatment plus hypoglycemia and
l
l
l
serum limitation (e), respectively. Original magnification ꢀ600. (B) The percentage of nuclear condensation in total cell population was counted after different treatments of
PC12 cells. ⁄P <0.01 versus exposure to hypoglycemia and serum limitation alone. (C) Flow cytometry with annexin V/PI staining for PC12 cells in the above five treatments
and the percentage of apoptotic and necrotic cells in total cell population were shown after different treatments. ⁄P <0.01 versus exposure to hypoglycemia and serum
limitation alone. The data were expressed as mean SD of three independent experiments (each in triplicate).
for 4g and 4h, which had a great potency superior to salidroside and
efficiently inhibited hypoglycemia and serum limitation induced
cell apoptosis through the inhibition of caspase-3 activation and
restoration of mitochondrial membrane potential. Although a
detailed molecular mechanism that links all these events should
be further investigated, the present findings provided a preliminary
pharmacological basis to exploit preventive or therapeutic strate-
gies for stroke and other neurological insults.
4. Experiment
Melting points were determined on an X-6 melting point appa-
ratus and were uncorrected. 1H NMR spectra were recorded using
TMS as an internal standard on a Bruker AC 400 instrument at
500 MHz or 300 MHz with D2O or CD3OD as solvent. IR was
recorded on AVATAR-370 FT-IR Thermo Nicolet with KBr disk. Ele-
mental analysis(C, N and H) were performed on Elementar VarioEL

Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
5581
(D2O, 500 MHz) d (ppm): 7.38 (2H, t, J = 7.55 Hz, Ar-H), 7.29 (3H,
d, J = 7.35 Hz, Ar - H), 4.47 (1H, d, J = 8.5 Hz, H-10), 4.19 (1H, dd,
J = 10.0 and J = 5.5 Hz, OCH2), 3.92 (1H, d, J = 12.05 Hz, H-60a),
3.84 (1H, dd, J = 8.65 and J = 5.1 Hz, OCH2), 3.75 (1H, d,
J = 12.05 Hz, H-60b), 3.62–3.40 (4H, m, H-20, H-30, H-40, H-50),
2.93–2.83 (2H, m, ArCH2), 1.83 (3H, s, NAc); Anal. Calcd for
C16H23NO6: C, 59.06; H, 7.13; N, 4.31. Found: C, 59.03; H, 7.20; N,
4.25.
4.1.1.3. 1-(3-Hydroxyphenyl)methyl-2-acetamido-2-deoxy-b-
D
-
pyranoside (4c). White solid; Yield: 90%; mp 166–168 °C; [
a
]
D
ꢁ48.4 (c 0.09, CH3OH); IR (KBr,
m
, cmꢁ1): 3336, 2935, 2841, 1635,
1591, 1459, 1376, 1251, 1153, 1038, 883, 761; MS: m/z 328
[M+H]+; 1H NMR (D2O, 300 MHz) d (ppm): 7.33 (1H, t, J = 7.77 Hz,
Ar-H), 6.94–6.87 (3H, m, Ar-H), 4.88 (1H, d, J = 12.25 Hz, ArCH2),
4.62 (1H, d, J = 12.3 Hz, ArCH2), 4.53 (1H, d, J = 8.4 Hz, H-10),
3.84–3.45 (6H, m, H-20,H-30,H-40, H-50, H-60), 2.0 (3H, s, NAc); Anal.
Calcd for C15H21NO7: C, 55.04; H, 6.47; N, 4.28. Found: C, 55.13; H,
6.55; N, 4.26.
Figure 5. Effects of 4 g and 4 h on activities of caspase-3 induced by hypoglycemia
and serum limitation in PC12 cells. PC12 cells were pre-incubated with 4 g, 4 h and
salidroside (40
lM) respectively for 24 h then exposed to hypoglycemia and serum
limitation for 24 h. The activity of caspase
3
in PC12 was measured using
colorimetric assay and expressed as the percentage of the activity of control cells.
The values presented were mean SD. ⁄P <0.05 versus exposure to hypoglycemia
and serum limitation alone group.
4.1.1.4. 1-(4-Hydroxyphenyl)methyl-2-acetamido-2-deoxy-b-
pyranoside (4d). White solid; Yield: 98%; mp 169–172 °C; [
D
-
a
]
D
ꢁ37.0 (c 0.14, CH3OH); IR (KBr,
m
, cmꢁ1): 3322, 2947, 2869, 1667,
1547, 1449, 1375, 1261, 1151, 1033, 820; MS: m/z 328 [M+H]+;
1H NMR (D2O, 300 MHz) d (ppm): 7.27 (2H, d, J = 8.28 Hz, Ar-H),
6.93 (2H, d, J = 8.4 Hz, Ar- H), 4.89 (1H, d, J = 12.21 Hz,
ArCH2), 4.59 (1H, d, J = 11.9 Hz, ArCH2), 4.52 (1H, d, J = 8.46 Hz,
H-10), 3.95 (1H, d J = 12.09 Hz, H-60a), 3.76 (1H, d, J = 12.42 Hz, H-
60b), 3.70–3.45 (4H, m, H-20, H-30, H-40, H-50), 1.91 (3H, s, NAc);
Anal. Calcd for C15H21NO7: C, 55.04; H, 6.47; N, 4.28. Found: C,
55.04; H, 6.55; N, 4.29.
III analyzer (German). Optical rotations were measured with a
JASCO P-1020 polarimeter (cell length, 100 mm). Flash chromatog-
raphy was performed on Silica gel (200–300 mesh). Commercial
reagents were used without further purification unless otherwise
stated. Solutions after reactions and extractions were concentrated
using a rotary evaporator operating at a reduced pressure of ca.
20 Torr.
4.1. Synthesis
4.1.1.5. 1-(3-Methoxy-4-hydroxyphenyl)methyl-2-acetamido-2-
deoxy-b-
174 °C; [
D
-pyranoside (4e). White solid; Yield: 91%; mp 170–
, cmꢁ1): 3266, 2962,
4.1.1. General procedure for preparation of compounds 4a–k
Compound 2 (2.1 g, 5.7 mmol) and substituted aromatic alcohol
(3.8 mmol) were added to a suspension of zinc chloride (0.51 g,
3.8 mmol) and 4,40-dimethoxytrityl chloride (1.28 g, 3.8 mmol) in
dry dichloromethane (15 ml), then stirred overnight at room tem-
perature. After diluting with dichloromethane, the mixture was
washed with saturated aqueous sodium hydrogen carbonate. The
solvent was removed in vacuum and the residue was purified by
column chromatography (petroleum ether–EtOAc 2:1, v/v) to yield
the glycosides 3a–k (54–71%). To compounds 3a–k (3.8 mmol) in
MeOH (15 ml) was added NaOMe (0.2 g, 3.8 mmol) and the
mixture was allowed to stir for 2 h at room temperature. On
completion of the reaction as indicated by TLC (methanol/chloro-
form 1/5–1/3, v/v), the solution was neutralized with Amberlite
IRA-120 (H+) resin to pH 7, then filtered and evaporated to dryness
under reduced pressure afford 4a–k.
a]
ꢁ34.6 (c 0.10, CH3OH); IR (KBr,
m
D
2849, 1655, 1558, 1457, 1353, 1268, 1161, 1058, 801,628; MS:
m/z 358 [M+H]+; 1H NMR (D2O, 500 MHz) d (ppm): 7.03 (1H, s,
Ar-H), 6.94 (1H, d, J = 7.95 Hz, Ar-H), 6.89 (1H, d, J = 7.95 Hz, Ar-
H), 4.89 (1H, d, J = 11.50 Hz, ArCH2), 4.68 (1H, d, J = 11.43 Hz,
ArCH2), 4.53 (1H, d, J = 8.4 Hz, H-10), 3.90 (1H, d, J = 12.25 Hz, H-
60a), 3.89 (3H, s, OCH3), 3.77 (1H, d, J = 9.35 Hz, H-60b), 3.67–3.45
(4H, m, H-20, H-30, H-40, H-50), 1.89 (3H, s, NAc); Anal. Calcd for
C16H23NO8: C, 53.78; H, 6.49; N, 3.92. Found: C, 53.76; H, 6.58;
N, 3.90.
4.1.1.6. 1-(3, 5-Dimethoxyphenyl)methyl-2-acetamido-2-deoxy-
b-D
-pyranoside (4f). White solid; Yield: 95%; mp 220–223 °C;
[a
]
D
ꢁ31.4 (c 0.11, CH3OH); IR (KBr,
m
, cmꢁ1): 3345, 2950, 2836,
1628, 1545, 1473, 1375, 1212, 1164, 1056, 830; MS: m/z 372
[M+H]+; 1H NMR (D2O, 500 MHz) d (ppm): 6.62 (2H, s, Ar-H),
6.60 (1H, s, Ar-H), 4.84 (1H, d, J = 12 Hz, ArCH2), 4.62 (1H, d,
J = 10 Hz, ArCH2), 4.54 (1H, d, J = 8.5 Hz, H-10), 3.95 (1H, d,
J = 12.5 Hz, H-60a), 3.85 (6H, s, 2ꢀ OCH3), 3.77 (1H, d, J = 10 Hz,
H-60b), 3.70–3.47 (4H, m, H-20, H-30, H-40, H-50), 1.93 (3H, s,
NAc); Anal. Calcd for C17H25NO8: C, 54.98; H, 6.79; N, 3.77. Found:
C, 54.93; H, 6.83; N, 3.75.
4.1.1.1. Benzyl-2-acetamido-2-deoxy-b-
solid; Yield: 96%; mp 194–196 °C; [ ]D: ꢁ39.0 (c 0.16, CH3OH); IR
(KBr,
, cmꢁ1): 3347, 2941, 2834, 1649, 1543, 1431, 1379, 1231,
D-pyranoside (4a). White
a
m
1124, 1057, 726; MS: m/z 312 [M+H]+; 1H NMR (D2O, 300 MHz) d
(ppm): 7.46–7.37 (5H, m, Ar-H), 4.89 (1H, d, J = 12 Hz, ArCH2),
4.68 (1H, d, J = 12 Hz, ArCH2) 4.54 (1H, d, J = 8.4 Hz, H-10), 3.95
(1H, d, J = 12 Hz, H-60a), 3.76 (1H, d, J = 12.5 Hz, H-60b), 3.70–3.46
(4H, m, H-20,H-30,H-40, H-50), 1.94 (3H, s, NAc); Anal. Calcd for
4.1.1.7.
2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-b-
D
-
pyranoside (4g). White solid; Yield: 93%; mp 190–191 °C; [
a
]
D
, cmꢁ1): 3469, 2934, 2853, 1654,
C15H21NO6: C, 57.87; H, 6.80; N, 4.50. Found: C, 57.82; H, 6.87;
ꢁ21.1 (c 0.15, CH3OH); IR (KBr,
m
1571, 1485, 1379, 1253, 1161, 1031, 825; MS: m/z 356 [M+H]+;
1H NMR (D2O, 500 MHz) d (ppm): 7.24 (2H, d, J = 8.5 Hz, Ar-H),
6.98 (2H, d, J = 8.5 Hz, Ar- H), 4.46 (1H, d, J = 8.5 Hz, H-10), 4.17
(1H, dd, J = 10.0 and J = 5.0 Hz, OCH2), 3.93 (1H, d, J = 12 Hz, H-
60a), 3.84 (3H, s, OCH3), 3.82–3.73 (2H, m, OCH2, H-60b), 3.62–
3.42 (4H, m, H-20, H-30, H-40, H-50), 2.88–2.80 (2H, m, ArCH2),
N, 4.40.
4.1.1.2. Phenethyl-2-acetamido-2-deoxy-b-
D
-pyranoside (4b).
]D: ꢁ22.0 (c 0.18,
, cmꢁ1): 3337, 2935, 2842, 1658, 1541, 1420,
1370, 1242, 1125, 1055, 710; MS: m/z 326 [M+H]+; 1H NMR
White solid; Yield: 95%; mp 207–208 °C; [
CH3OH); IR (KBr,
a
m

5582
Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
Figure 6. Effects of 4g and 4h on decrease of MMP induced by hypoglycemia and serum limitation in PC12 cells. (A) Representative fluorescent micrographs of Rhodamine
123 staining for comparing the mitochondrial membrane potential of PC12 cells in control (a), exposure to hypoglycemia and serum limitation alone (b), 40 M 4 g
pretreatment plus hypoglycemia and serum limitation (c), 40 M 4 h pretreatment plus hypoglycemia and serum limitation (d), and 40 M salidroside plus hypoglycemia
l
l
l
and serum limitation (e), respectively. Original magnification ꢀ400. (B) the percentage of fluorescent intensity of Rhodamine 123 in treated cultures compared to those in
control cultures was also shown. ⁄P <0.05 versus exposure to hypoglycemia and serum limitation alone. The data were expressed as mean SD of three independent
experiments (each in triplicate).
1.82 (3H, s, NAc); Anal. Calcd for C17H25NO7: C, 57.45; H, 7.09; N,
3.94. Found: C, 57.41; H, 7.15; N, 3.89.
4.1.1.10. 3-(4-Benzyloxyphenyl)propyl-2-acetamido-2-deoxy-b-
-pyranoside (4j). White solid; Yield: 95%. The product could be
used in subsequent reactions without further purification.
D
4.1.1.8. 3-(4-Methoxyphenyl)propyl-2-acetamido-2-deoxy-b-
pyranoside (4h). White solid; Yield: 93%; mp 201–203 °C; [
ꢁ10.9 (c 0.13, CH3OH); IR (KBr,
1552, 1514, 1482, 1383, 1243, 1168, 1051, 820; MS: m/z 370
[M+H]+; 1H NMR (CD3OD, 300 MHz)
(ppm): 7.09 (2H, d,
D
-
a]
D
4.1.1.11.
ido-2-deoxy-b-D
3-(3-methoxy-4-benzyloxyphenyl)propyl-2-acetam-
-pyranoside (4k). White solid; Yield: 91%. The
m
, cmꢁ1): 3464, 2928, 2866, 1653,
product could be used in subsequent reactions without further
purification.
d
J = 8.7 Hz, Ar-H), 6.81 (2H, d, J = 7.2 Hz, Ar-H), 4.39 (1H, d,
J = 8.4 Hz, H-10), 3.91–3.84 (2H, m, OCH2, H-60a), 3.75 ((3H, s,
OCH3), 3.71–3.62 (2H, m, OCH2, H-60b), 3.50–3.41 (4H, m, H-20,
H-30, H-40, H-50), 2.6 (2H, t, J = 6.9 Hz, ArCH2), 1.99 (3H, s, NAc),
1.8 (2H, t, J = 6.9 Hz, BnCH2); Anal. Calcd for C18H27NO7: C, 58.52;
H, 7.37; N, 3.79. Found: C, 58.65; H, 7.32; N, 3.81.
4.1.2. General procedure for preparation of compounds 5i–k
To a stirred suspension of 4i–k (2 mmol) in methanol (10 mL),
5% Pd/C (90 mg) and ammonium formate (650 mg, 10 mmol) were
added, then the mixture was heated to reflux for 6 h. After reaction
the mixture was filtered and concentrated, then resorted to column
chromatography using 8:1 CHCl3–MeOH to afford compounds 5i–k.
4.1.1.9. 2-(4-Benzyloxyphenyl)ethyl-2-acetamido-2-deoxy-b-D-
pyranoside (4i). White solid; Yield: 93%. The product could be
4.1.2.1.
2-(4-Hydroxyphenyl)ethyl-2-acetamido-2-deoxy-b-
D
-
used in subsequent reactions without further purification.
pyranoside (5i). White solid; Yield: 95%; mp 175–178 °C; [a]
D

Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
5583
ꢁ31.4 (c 0.18, CH3OH); IR (KBr,
m
, cmꢁ1): 3346, 2928, 2845, 1658,
4.5. Hoechst 33342 staining
1519, 1452, 1384, 1274, 1129, 1081, 1007,825; MS: m/z 342
[M+H]+; 1H NMR (D2O, 500 MHz) d (ppm): 7.16 (2H, d, J = 7.5 Hz,
Ar-H), 6.86 (2H, d, J = 8.0 Hz, Ar-H), 4.45 (1H, d, J = 8.5 Hz, H-10),
4.16 (1H, d, J = 9.0 Hz, OCH2), 3.92 (1H, d, J = 12.5 Hz, H-60a),
3.76–3.73 (2H, m, OCH2, H-60a), 3.61–3.42 (4H, m, H-20, H-30, H-
40, H-50), 2.86–2.78 (2H, m, ArCH2), 1.81 (3H, s, NAc); Anal. Calcd
for C16H23NO7: C, 56.30; H, 6.79; N, 4.10; Found: C, 56.20; H,
6.85; N, 4.11.
At the end of cell treatments, the PC12 cells were fixed with
4.0% paraformaldehyde for 20 min, washed with 0.15 M NaCl for
3 times then stained with 5 lg/ml Hoechst 33342 dye at 37 °C
for 10 min. Morphologic changes in apoptotic nuclei were ob-
served under a DMR fluorescence microscope (Leica Microsys-
tems, Wetzlar, Germany) with ultraviolet illumination. The dye
was excited at 340 nm, and emission was filtered with
a
510 nm barrier filter. In order to quantify the apoptotic process,
cells with fragmented or condensed DNA and normal DNA were
respectively counted. Data were expressed as the ratio of apopto-
tic cells to total cells.
4.1.2.2. 3-(4-Hydroxyphenyl)propyl-2-acetamido-2-deoxy-b-
pyranoside (5j). White solid; [
ꢁ15.7 (c 0.17, CH3OH); IR
(KBr,
, cmꢁ1): 3308, 2934, 2866, 1615, 1557, 1517, 1449, 1370,
D-
a
]
D
m
1248, 1166, 1072, 820; MS: m/z 356 [M+H]+; 1H NMR (D2O,
300 MHz) d (ppm): 7.16 (2H, d, J = 8.4 Hz, Ar - H), 6.87 (2H, d,
J = 8.3 Hz, Ar-H), 4.5 (2H, d, J = 8.5 Hz, H-10), 3.94–3.85 (2H, m,
OCH2, H-60a), 3.78–3.68 (2H, m, OCH2, H-60b), 3.60–3.44 (4H, m,
H-20, H-30, H-40, H-50), 2.59 (2H, t, J = 7 Hz, ArCH2), 2.04 (3H, s,
NAc), 1.83 (2H, t, J = 6.66 Hz, BnCH2); Anal. Calcd for C17H25NO7:
C, 57.45; H, 7.09; N, 3.94. Found: C, 57.44; H, 7.20; N, 3.99.
4.6. Flow cytometry with annexin V/PI staining
The PC12 cells were harvested by centrifugation after treatment
as described above (treated with either 4g, 4h, or salidroside plus
hypoglycemia and serum limitation). The pellets resuspended in
1 ꢀ binding buffer (10 mM HEPES, 140 mmM NaCl, 2.5 mM CaCl2)
at a concentration of 1 ꢀ 106 cell/ml. In addition to a 100
ll aliquot
4.1.2.3. 3-(3-Methoxy-4-hydroxyphenyl)propyl-2-acetamido-2-
of the cell suspension, 5 ll of FITC-conjugated annexin V (PharM-
deoxy-b-
CH3OH); IR (KBr,
D
-pyranoside (5k). White solid; [
a
]
D
ꢁ15.6 (c 0.17,
ingen, San Diego, CA) and 5 ll of 50 lg/ml propidium iodide (PI)
m
, cmꢁ1): 3367, 2935, 2865, 1651, 1516, 1452,
were added. After 15 min incubation in the dark at room tempera-
ture, cells were analyzed for annexin V binding within 1 h with a
flow cytometer (BD FACScalibur, BD Bioscience, San Jose, CA).
Apoptotic and necrotic cells were quantitated by annexin V bind-
ing and PI uptake.
1375, 1273, 1155, 1034, 810, 641; MS: m/z 385 [M+H]+; 1H NMR
(D2O, 300 MHz): 6.94 (1H, s, Ar-H), 6.89 (1H, d, J = 8.27 Hz, Ar-H),
6.80 (1H, d, J = 8.07 Hz, Ar-H), 4.51 (1H, d, J = 8.5 Hz, H-10), 3.95–
3.90 (2H, m, OCH2, H-60a), 3.87 (3H, s, OCH3), 3.82–3.60 (2H, m,
OCH2, H-60b), 3.57–3.46 (4H, m, H-20, H-30, H-40, H-50), 2.60 (2H,
t, J = 7.35 Hz, ArCH2), 2.03 (3H, s, NAc), 1.85 (2H, t, J = 7.2 Hz,
BnCH2); Calcd for C18H27NO8: C, 56.09; H, 7.06; N, 3.63. Found: C,
56.18; H, 7.09; N, 3.57.
4.7. Caspase-3 colorimetric assay
Caspase-3 activity was determined using the caspase-3 activity
kit, which was based on the ability of caspase-3 to change acetyl-
Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into the yellow
4.2. Cell culture
formazan product, p-nitroaniline (pNA). According to the manufac-
turer’s instruction, treated cells were lysed, then the supernatant
was centrifuged at 12,000 g for 10 min, and protein concentrations
were determined by Bradford protein assay. After incubating the
PC12 cells, obtained from the American Type Culture Collection
(Manassas, VA), were plated and maintained in high-glucose
DMEM supplemented with 10% horse serum, 5% fetal bovine ser-
um, 100 U/ml penicillin, and 100 U/ml streptomycin in a humidi-
fied atmosphere of 5% CO2 at 37 °C. The cells were differentiated
by treating with nerve growth factor (NGF) (50 ng/ ml) every other
day for 6 days.
mixture composed of 10
ll of cell lysate, 80
ll of reaction buffer
and 10 l of 2 mM Ac-DEV
l
D-pNA in 96-well microtiter plates at
37 °C for 4 h, caspase-3 activity was quantified in the samples with
an EIX-800 Microelisa reader (Bio-Tek Inc., USA) at an absorbance
of 405 nm. The caspase activities were expressed as percentage
compared to control.
4.3. Exposure target compounds (4a–h, 5i–k) to differentiated
PC12 cells
After being pretreated with 8, 40, and 200
and synthetic aralkyl alcoholic 2-acetamido-2-deoxy-b-
l
mol/L salidroside
4.8. Measurement of mitochondrial membrane potential
D
-pyrano-
side for 24 h, respectively, the cells were subjected to hypoglyce-
mia and serum limitation by changing the culture medium with
the glucose-free DMEM supplemented with 1% horse serum and
1% fetal bovine serum, in the presence of compounds at original
concentrations for another 24 h incubation. In the aforementioned
process, treatment only with culture medium containing high glu-
cose/enough serum was considered control group, meanwhile,
only exposure to hypoglycemia and serum limitation as hypoglyce-
mia and serum limitation alone group.
At the end of cell treatments, the fluorescent dye Rhodamine
123 was added to the PC12 cells to make a final concentration of
2 lM. After 30 min culture at 37 °C, the cells were fixed with
4.0% paraformaldehyde for 20 min, and washed twice with PBS,
followed by visualization under a TCS SP2 confocal microscope
(Leica Microsystems, Wetzlar, Germany). The signal of Rhoda-
mine 123 was excited by 488 nm laser light and emission was
captured at 530 nm. Each field of cells was photographed (mag-
nification, 400) for calculation of the relative fluorescence
intensity.
4.4. Cell viability assay
4.9. Statistical analysis
At the end of cell treatments, MTT solution was added to the cell
samples to make final concentration of 0.5 mg/ml, then incubate at
37 °C for 4 h followed by the addition of 20% sodium dodecyl sul-
fide (SDS) to dissolve the resulting formazan. The absorbance
(OD) values were measured by spectrophotometry at 570 nm with
an EIX-800 Microelisa reader (Bio-Tek Inc., USA).
Data were expressed as means SD (n = 3, where n represents
the number of independent experiments). Statistical differences
between groups were analyzed by one-way ANOVA tests. Differ-
ences were considered significant at P <0.05.

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Y. Meng et al. / Bioorg. Med. Chem. 19 (2011) 5577–5584
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Science Foundation of China (Grant no.30970713), Basic Research
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Academic Program Development of Jiangsu Higher Education Insti-
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