Synthesis and neuroprotective effects of the fluorine substituted salidroside analogues in the PC12 cell model exposed to hypoglycemia and serum limitation

Article abstract of DOI:10.1248/cpb.c13-00555

Salidroside (Sal) is a natural antioxidant extracted from the root of Rhodiola rosea L., a traditional Chinese medicinal plant, which elicits neuroprotective effects in the treatment of ischemic stroke. In an attempt to improve its neuroprotective effects, fluorine substituted Sal analogues were synthesized and their neuroprotective activities against the hypoglycemia and serum limitation induced cell injury in differentiated PC12 cells were evaluated. The target compounds displayed strong protective effects on the cell viability against the damage caused by hypoglycemia and serum limitation, especially for D1, which had a great potency superior to Sal and efficiently inhibited hypoglycemia and serum limitation induced cell nuclear morphologic changes and the increased apoptotic rate in a dose-dependent manner. These new findings may provide potentially important information for further development of Sal analogues and lay the basis for further studies on the cerebral ischemic stroke and neurodegenerative diseases for human clinical treatment.

Full text of DOI:10.1248/cpb.c13-00555

1192
Note
Chem. Pharm. Bull. 61(11) 1192–1196 (2013)
Vol. 61, No. 11
Synthesis and Neuroprotective Effects of the Fluorine Substituted
Salidroside Analogues in the PC12 Cell Model Exposed to
Hypoglycemia and Serum Limitation
,a
,c
Xiaoming Yang,a,# Wen Xu,a,# Weijia Zhao,b Yahong Zhao,a Yumin Yang,* and Yong Ling*
c
a JiangSu Key Laboratory of Neuroregeneration, Nantong University; Department of Pharmacy, School of Medical,
b
Nantong University; Nantong 226001, P.R. China: and School of Life Sciences, Nantong University; Nantong
226019, P.R. China.
Received July 14, 2013; accepted August 20, 2013
Salidroside (Sal) is a natural antioxidant extracted from the root of Rhodiola rosea L., a traditional
Chinese medicinal plant, which elicits neuroprotective effects in the treatment of ischemic stroke. In an at-
tempt to improve its neuroprotective effects, fluorine substituted Sal analogues were synthesized and their
neuroprotective activities against the hypoglycemia and serum limitation induced cell injury in differenti-
ated PC12 cells were evaluated. The target compounds displayed strong protective effects on the cell viabil-
ity against the damage caused by hypoglycemia and serum limitation, especially for D1, which had a great
potency superior to Sal and efficiently inhibited hypoglycemia and serum limitation induced cell nuclear
morphologic changes and the increased apoptotic rate in a dose-dependent manner. These new findings may
provide potentially important information for further development of Sal analogues and lay the basis for
further studies on the cerebral ischemic stroke and neurodegenerative diseases for human clinical treatment.
Key words salidroside (Sal) analogue; hypoglycemia serum limitation; neuroprotective effect; apoptosis
Focal cerebral ischemia or stroke remains has been a major natural Sal in R. roseaꢀ productionꢀ isꢀ lowꢀ (<1.99%), and its
cause of death and disability in clinical practice, especially in neuroprotectiveꢀ effectꢀ forꢀ treatmentꢀ cerebralꢀ ischemiaꢀ wasꢀ
the elderly population. Wide variety of cellular and molecular limited. Therefore, the development of novel Sal analogues
mechanisms involved in ischemic brain damage.1) Ischemia is withꢀ strongerꢀ neuroprotectiveꢀ effectsꢀ byꢀ inhibitingꢀ neuronalꢀ
characterizedꢀ byꢀ lackingꢀ theꢀ bloodꢀ flowꢀ toꢀ theꢀ brain,ꢀ whichꢀ apoptosisꢀ willꢀ beꢀ ofꢀ greatꢀ significance.ꢀ Ourꢀ previousꢀ studiesꢀ
resultsꢀ inꢀ insufficientꢀ supplyꢀ ofꢀ glucose,ꢀ oxygen,ꢀ serumꢀ andꢀ haveꢀ revealedꢀ thatꢀ Salꢀ analoguesꢀ exhibitedꢀ protectiveꢀ effectsꢀ
nutrient energy generation. Morphological manifestations of on hypoglycemia and serum limitation-induced cell apoptosis
programmed cell death in the ischemic brain and biochemical in cultured PC12 cells.11–13)
evidenceꢀhaveꢀrevealedꢀthatꢀapoptosisꢀwasꢀimplicatedꢀinꢀisch-
ToꢀdevelopꢀnewꢀSalꢀanaloguesꢀforꢀhumanꢀuse,ꢀweꢀwereꢀin-
emic stroke.2) Thus, the prevention and inhibition of neuronal spiredꢀ byꢀ theꢀ factꢀ thatꢀ introducingꢀ fluorineꢀ atomsꢀ intoꢀ drugsꢀ
apoptosis may be a reasonable choice for the treatment of per- can alter the rate and route of drug metabolism14) and stereo-
manent neurological damage for cerebral ischemia.
electronicꢀ factorsꢀ associatedꢀ withꢀ theꢀ fluorineꢀ atomꢀ canꢀ leadꢀ
2-(4-Hydroxyphenyl)ethyl β-d-Glucopyranoside Sali- to changes in the biological action of molecules in comparison
drosideꢀ (Sal),ꢀ 2-(4-hydroxyphenyl)ꢀethylꢀ β-D-glucopyranoside toꢀitsꢀanaloguesꢀsubstitutedꢀwithꢀhydroxyꢀgroupsꢀorꢀhydrogenꢀ
(Fig. 1A), belonging to the family Crassulaceae, is one of atoms.15)ꢀ Inꢀ addition,ꢀ theꢀ smallꢀ sizeꢀ ofꢀ theꢀ fluorineꢀ atom,ꢀ theꢀ
theꢀ activeꢀ ingredientsꢀ extractedꢀ fromꢀ theꢀ rootꢀ ofꢀ Rhodiola enhanced lipophilicity it imparts to the molecules and the
rosea L. R. rosea, mainly in the mountainous areas of a plant electronegativity of the atom often results in improved thera-
in high altitude areas in the Northeast Asia, has been used peutic drugs.16)ꢀ Therefore,ꢀ hydroxyꢀ groupꢀ orꢀ hydrogenꢀ atomsꢀ
as traditional Tibetan medicine for a long time. The plant ofꢀ Sal’sꢀ aromaticꢀ ringꢀ wereꢀ substitutedꢀ withꢀ fluorineꢀ atomsꢀ
alsoꢀ showsꢀ aꢀ rangeꢀ ofꢀ pharmacologicalꢀ properties,ꢀ includingꢀ soꢀ asꢀ toꢀ discoverꢀ andꢀ developꢀ ofꢀ newꢀ neuroprotectiveꢀ effectꢀ
antiaging,ꢀ antiinflammation,ꢀ antifatigue,ꢀ anticancer,ꢀ andꢀ car- agentsꢀ forꢀ humanꢀ use.ꢀ Theꢀ fluorineꢀ substitutedꢀ Salꢀ analoguesꢀ
dioprotection.3–8) Recently, more attention has been focused (D1,ꢀD2,ꢀseeꢀFig.ꢀ1B)ꢀwereꢀsynthesized,ꢀandꢀtheirꢀneuroprotec-
on the study of its neuroprotective effect.9,10)ꢀ However,ꢀ theꢀ tive activities and apoptosis effects in the cell injury induced
Fig.ꢀ 1.ꢀ (A)ꢀChemicalꢀStructureꢀofꢀSalidrosideꢀ(Sal,ꢀ2-(4-(Hydroxyphenꢀyl)ethylꢀβ-D-Glucopyranoside) and (B) Chemical Structures of D1 and D2
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
#ꢀTheseꢀauthorsꢀcontributedꢀequallyꢀtoꢀthisꢀwork.
© 2013 The Pharmaceutical Society of Japan
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀyangym@ntu.edu.cn;ꢀLyyy111@sina.com

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Reagentsꢀ andꢀ conditions:ꢀ (a)ꢀ n-BuLi/hexane,ꢀ ethyleneꢀ oxide,ꢀ ether,ꢀ −78°C, 2h; (b) tetra-O-acetyl-α-D-glucopyranosyl bromide or tetra-O-acetyl-α-D-galactopyranosyl
bromide, Ag2CO3, CH2Cl2,ꢀmolecularꢀsieveꢀpowder,ꢀrt,ꢀovernight;ꢀ(c)ꢀK2CO3, CH3OH,ꢀrt,ꢀ2ꢀh.
Chart 1. General Method for the Synthesis of D1 and D2
Fig. 2. Protective Effect of D1, D2 and Sal on Hypoglycemia and Serum Limitation Induced Apoptosis in PC12 Cells
PC12ꢀcellsꢀwereꢀpretreatedꢀwithꢀtestꢀcompoundsꢀonꢀdifferentꢀconcentrationsꢀ(20,ꢀ100,ꢀ300ꢀµg/mL)ꢀforꢀ24ꢀh,ꢀthenꢀexposedꢀtoꢀhypoglycemiaꢀandꢀserumꢀlimitationꢀforꢀanꢀ
additionalꢀ48ꢀhꢀwithꢀrespectiveꢀoriginalꢀconcentrationsꢀofꢀcompoundsꢀmaintained.ꢀViabilityꢀwasꢀcalculatedꢀasꢀtheꢀpercentageꢀofꢀlivingꢀcellsꢀinꢀtreatedꢀculturesꢀcomparedꢀ
to those in control cultures by using MTT assay. *p<0.05, versusꢀexposureꢀtoꢀhypoglycemiaꢀandꢀserumꢀlimitationꢀforꢀ48ꢀhꢀinꢀalone.ꢀ# p<0.05, versus the positive control
group Sal (300µg/mL).ꢀTheꢀdataꢀwereꢀpresentedꢀasꢀmean S.D.ꢀofꢀthreeꢀindependentꢀexperimentsꢀ(eachꢀinꢀtriplicate).
byꢀ hypoglycemiaꢀ andꢀ serumꢀ limitationꢀ wereꢀ investigatedꢀ andꢀ miaꢀ andꢀ serumꢀ limitationꢀ treatment,ꢀ weꢀ foundꢀ thatꢀ D1ꢀ andꢀ
comparedꢀwithꢀSal.
D2ꢀ exhibitedꢀ significantꢀ protectiveꢀ effects,ꢀ whichꢀ increasedꢀ
cell viability to 81.32 2.31% and 70.27 2.68%, respectively,
whichꢀ superiorꢀ toꢀ Salꢀ groupꢀ (65.62 3.87%) at the same con-
Results and Discussion
Chemistry The synthetic route of Sal’s analogue centration of 300µg/mL.ꢀ Especially,ꢀ compoundꢀ D1ꢀ exhibitedꢀ
(D1,ꢀ D2)ꢀ wasꢀ outlinedꢀ inꢀ Chartꢀ 1.ꢀ Theꢀ startingꢀ materialꢀ theꢀ mostꢀ significantꢀ protectiveꢀ effect,ꢀ whichꢀ increasedꢀ cellꢀ
1-bromo-2,3,4,5,6-pentafluorobenzeneꢀ (1)ꢀ wasꢀ convertedꢀ toꢀ viability to 81.32 2.31%, 68.39 1.56% and 47.92 1.31% at
2-(pentafluorophenyl)ꢀethanolꢀ (2)ꢀ byꢀ treatmentꢀ withꢀ n-BuLi/ the different concentration of 20, 100, 300µg/mL,ꢀ andꢀ wereꢀ
hexaneꢀandꢀethyleneꢀoxideꢀatꢀ−78°C in 76% yield. Compound significantlyꢀhigherꢀthanꢀSalꢀandꢀD1ꢀgroupsꢀrespectively.ꢀOneꢀ
2ꢀwasꢀtreatedꢀwithꢀtetra-O-acetyl-α-D-glucopyranosyl bromide plausibleꢀexplanationꢀwasꢀthatꢀtheꢀglucoseꢀfragmentꢀofꢀD1ꢀmayꢀ
or tetra-O-acetyl-α-D-galactopyranosyl bromide in the pres- contributeꢀ toꢀ itsꢀ bioactivitiesꢀ superiorꢀ toꢀ D2ꢀ withꢀ galactoseꢀ
enceꢀ ofꢀ AgCO3 to give intermediate 3a or 3b,ꢀ whichꢀ wereꢀ fragment. Furthermore, the protective activities of D1 and D2
deacetylatedꢀ withꢀ K2CO3 to yield the targeted compound D1 withꢀfluorineꢀatomsꢀsubstitutedꢀaromaticꢀringꢀwereꢀalsoꢀbetterꢀ
orꢀD2.ꢀTheꢀstructureꢀofꢀD1ꢀandꢀD2ꢀwereꢀconfirmedꢀbyꢀofꢀMS,ꢀ thanꢀ thatꢀ ofꢀ phenethylꢀ analoguesꢀ withoutꢀ phenolicꢀ hydroxylꢀ
1
IR, H-NMR spectra and elemental analysis (EA).
whichꢀ hadꢀ beenꢀ researchedꢀ byꢀ ourꢀ group.11) Therefore, these
Biological Evaluation The neuroprotective effects of resultsꢀ suggestedꢀ thatꢀ theꢀ fluorineꢀ substitutedꢀ Salꢀ analoguesꢀ
targetꢀcompoundsꢀD1ꢀandꢀD2ꢀwereꢀinvestigatedꢀonꢀpreventingꢀ displayed neuroprotective activities in the cell injury induced
decreased cell viability in PC12 cells induced by hypoglyce- byꢀ hypoglycemiaꢀ andꢀ serumꢀ limitation.ꢀ D1ꢀ wasꢀ furtherꢀ re-
mia and serum limitation using Sal as positive controls. The searchedꢀbyꢀHoechstꢀ33342ꢀstainingꢀandꢀflowꢀcytometryꢀwithꢀ
results of these studies are summarized in Fig. 2. Hypoglyce- annexinꢀV/PIꢀstaining.
miaꢀ andꢀ serumꢀ limitationꢀ ledꢀ toꢀ significantꢀ reductionꢀ ofꢀ cellꢀ
Apoptosis after cerebral ischemia is one of the major path-
viability to 38.12 0.87% for 48h, compared to control. When waysꢀthatꢀleadꢀtoꢀtheꢀprocessꢀofꢀcellꢀdeath.17) Therefore, mor-
cellsꢀ pretreatedꢀ withꢀ D1,ꢀ D2ꢀ andꢀ Salꢀ underꢀ differentꢀ concen- phologicalꢀ examinationsꢀ usingꢀ Hoechstꢀ 33342ꢀ stainingꢀ wereꢀ
tration (20, 100, 300µg/mL)ꢀ forꢀ 24ꢀhꢀ followedꢀ byꢀ hypoglyce- performed to determine the anti-apoptotic effects of the target

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Vol. 61, No. 11
Fig.ꢀ 3.ꢀ (A)ꢀ Representativeꢀ Fluorescenceꢀ Imagesꢀ ofꢀ Hoechstꢀ 33342ꢀ Stainingꢀ Showingꢀ Morphologicalꢀ Apoptosisꢀ forꢀ Culturedꢀ PC12ꢀ Cellsꢀ inꢀ Controlꢀ
Groupꢀ (a),ꢀ Hypoglycemiaꢀ andꢀ Serumꢀ Limitationꢀ Aloneꢀ Groupꢀ (b),ꢀ Pretreatmentꢀ withꢀ Salꢀ (300ꢀµg/mL)ꢀ Plusꢀ Exposureꢀ toꢀ Hypoglycemiaꢀ andꢀ Serumꢀ
Limitationꢀ(c),ꢀandꢀPretreatmentꢀwithꢀD1ꢀ(300ꢀµg/mL)ꢀPlusꢀExposureꢀtoꢀHypoglycemiaꢀandꢀSerumꢀLimitationꢀ(d),ꢀRespectively
Scale bar for (a–d), 10µm.ꢀ(B)ꢀTheꢀnumberꢀpercentageꢀofꢀapoptoticꢀcellsꢀinꢀtheꢀtotalꢀcellꢀpopulationꢀisꢀshownꢀforꢀsixꢀdifferentꢀtreatmentsꢀofꢀPC12ꢀcells.ꢀ*ꢀp<0.01 and
**p<0.05 vs. hypoglycemia and serum limitation alone. # p<0.01 vs. Sal (300µg/mL) group.
compoundꢀ D1.ꢀ Accordingꢀ toꢀ Fig.ꢀ 3A,ꢀ thereꢀ wasꢀ onlyꢀ 3.1
1.3%ꢀofꢀtheꢀcellsꢀinꢀtheꢀcontrolꢀgroupꢀthatꢀunderwentꢀapopto-
sisꢀorꢀcellꢀdeath,ꢀwhereasꢀhypoglycemiaꢀandꢀserumꢀlimitationꢀ
induced apoptotic/necrotic cells to 18.9 ꢀ5.2%.ꢀHowever,ꢀtreat-
ment of Sal (100, 300µg/mL)ꢀsignificantlyꢀdecreasedꢀhypogly-
cemia and serum limitation induced apoptotic/necrotic cells
to 10.8 1.9% and 8.2 2.0%; simultaneously treatment of D1
presentꢀnewꢀfindingsꢀmayꢀprovideꢀpotentiallyꢀimportantꢀinfor-
mation for further development of Sal analogues and lay the
basis for further studies on the cerebral ischemic stroke and
neurodegenerative diseases for human clinical treatment.
Experimental
MeltingꢀpointsꢀwereꢀdeterminedꢀonꢀaꢀMel-TEMPꢀIIꢀmeltingꢀ
(100, 300µg/mL)ꢀ significantlyꢀ decreasedꢀ hypoglycemiaꢀ andꢀ pointꢀapparatusꢀandꢀuncorrected.ꢀOpticalꢀrotationsꢀwereꢀmea-
serum limitation induced apoptotic/necrotic cells to 8.4 2.1% suredꢀwithꢀaꢀJASCOꢀP-1020ꢀpolarimeterꢀ(cellꢀlength:ꢀ100ꢀmm).ꢀ
and 3.8 1.9% (Fig. 3B). Interesting, treatment of D1 (300µg/ Infraredꢀ(IR)ꢀspectraꢀ(KBr)ꢀwereꢀrecordedꢀonꢀaꢀNicoletꢀImpactꢀ
mL)ꢀ significantlyꢀ wasꢀ betterꢀ protectiveꢀ effectꢀ thanꢀ Salꢀ groupꢀ 410ꢀ instrumentꢀ (KBrꢀ pellet).ꢀ 1H-NMRꢀ spectraꢀ wereꢀ recordedꢀ
withꢀtheꢀsameꢀdose.
withꢀaꢀBrukerꢀAvanceꢀ300ꢀMHzꢀspectrometerꢀatꢀ300ꢀK,ꢀusingꢀ
To further determine the anti-apoptotic effects of the target tetramethylsilane (TMS) as an internal standard. MS spectra
compoundꢀ D1,ꢀ flowꢀ cytometryꢀ withꢀ annexinꢀ V/PIꢀ stainingꢀ wereꢀ recordedꢀ onꢀ aꢀ Shimadzuꢀ GC-MSꢀ 2010ꢀ (electronꢀ ioniza-
provided further evidence that Sal pretreatment prevented tion (EI)) or a Mariner Mass Spectrum (electrospray ioniza-
hypoglycemia and serum limitation induced apoptosis in tionꢀ (ESI)).ꢀ Elementꢀ analysisꢀ wasꢀ performedꢀ onꢀ anꢀ Eagerꢀ
culturedꢀ PC12ꢀ cells.ꢀ Theꢀ percentagesꢀ ofꢀ apoptoticꢀ cellsꢀ wereꢀ 300ꢀ instrument.ꢀ Allꢀ compoundsꢀ wereꢀ routinelyꢀ checkedꢀ byꢀ
1
determinedꢀbyꢀflowꢀcytometryꢀanalysisꢀinꢀFig.ꢀ4A.ꢀTheꢀquan- TLC and H-NMR. TLCs and preparative thin-layer chroma-
titative comparison indicated that the percentages of early and tographyꢀ wereꢀ performedꢀ onꢀ silicaꢀ gelꢀ GF/UVꢀ 254,ꢀ andꢀ theꢀ
lateꢀapoptoticꢀrateꢀinꢀPC12ꢀcellsꢀinducedꢀbyꢀexposureꢀtoꢀhypo- chromatogramsꢀwereꢀconductedꢀonꢀsilicaꢀgelꢀ(200–300ꢀmesh)ꢀ
glycemiaꢀandꢀserumꢀlimitationꢀaloneꢀwereꢀ29.63%ꢀandꢀ15.16%ꢀ andꢀvisualizedꢀunderꢀUVꢀlightꢀatꢀ254ꢀandꢀ365ꢀnm.ꢀAllꢀsolventsꢀ
respectively.ꢀ Whileꢀ PC12ꢀ cellsꢀ wereꢀ pretreatedꢀ withꢀ 300ꢀµg/ wereꢀ reagentꢀ gradeꢀ and,ꢀ whenꢀ necessary,ꢀ wereꢀ purifiedꢀ andꢀ
mL of D1 prior to hypoglycemia and serum limitation, early dried by standards methods. Solutions after reactions and
stageꢀandꢀlateꢀstageꢀofꢀapoptosisꢀwereꢀsurprisinglyꢀprevented,ꢀ extractionsꢀ wereꢀ concentratedꢀ usingꢀ aꢀ rotaryꢀ evaporatorꢀ op-
andꢀ theꢀ apoptoticꢀ rateꢀ significantlyꢀ decreasedꢀ toꢀ 12.08%ꢀ andꢀ erating at a reduced pressure of ca.ꢀ20ꢀTorr.ꢀOrganicꢀsolutionsꢀ
6.61%,ꢀ whichꢀ wereꢀ betterꢀ thanꢀ thatꢀ inducedꢀ byꢀ pretreatmentꢀ wereꢀ driedꢀ overꢀ anhydrousꢀ sodiumꢀ sulfate.ꢀ Compoundꢀ 1ꢀ wasꢀ
withꢀ Salꢀ atꢀ theꢀ sameꢀ concentrationꢀ (Fig.ꢀ 4B).ꢀ Theꢀ aboveꢀ re- commercially available, and tetra-acetyl glucosyl bromide and
sults revealed that the target compound D1 could prevented tetra-acetylꢀgalactosylꢀbromideꢀwereꢀpreparedꢀaccordingꢀtoꢀtheꢀ
the increased apoptotic rate and nuclear morphologic changes literature.18,19)
induced by hypoglycemia and serum limitation.
Synthesis of D1 and D2ꢀ ꢀ2-(Pentafluorophenyl)eꢀ thanolꢀ(2):ꢀ
Inꢀ conclusion,ꢀ twoꢀ fluorineꢀ substitutedꢀ Salꢀ analoguesꢀ D1ꢀ 1-Bromo-2,3,4,5,6-pentafluorobenzeneꢀ(1,ꢀ23.0ꢀg,ꢀ93ꢀmmol)ꢀwasꢀ
andꢀ D2ꢀ wereꢀ synthesized,ꢀ andꢀ theirꢀ neuroprotectiveꢀ activitiesꢀ dissolved in ether (30mL) and cooled to −78°C. To this solu-
against the hypoglycemia and serum limitation-induced injury tion, n-BuLiꢀ (1.6ꢀmol/Lꢀ solutionꢀ inꢀ hexanes,ꢀ 60ꢀmL,ꢀ 96ꢀmmol)ꢀ
ofꢀ differentiatedꢀ PC12ꢀ cellsꢀ wereꢀ studied.ꢀ Bothꢀ D1ꢀ andꢀ D2ꢀ wasꢀslowlyꢀadded.ꢀAfterꢀ1ꢀh,ꢀethyleneꢀoxideꢀinꢀtolueneꢀsolutionꢀ
displayed strong protective effects on the cell viability against wasꢀaddedꢀtoꢀtheꢀreactionꢀmixture,ꢀandꢀtheꢀflaskꢀwasꢀremovedꢀ
the damage caused by hypoglycemia and serum limitation, fromꢀtheꢀcoolingꢀbathꢀandꢀtheꢀmixtureꢀwasꢀstirredꢀatꢀrtꢀforꢀ1ꢀh.ꢀ
Clꢀ solutionꢀ (100ꢀmL)ꢀ wasꢀ added,ꢀ
especiallyꢀ forꢀ D1,ꢀ whichꢀ hadꢀ aꢀ greatꢀ potencyꢀ superiorꢀ toꢀ Salꢀ After saturated aqueous NH4
theꢀ productꢀ wasꢀ extractedꢀ withꢀ diethylꢀ etherꢀ (2×300mL).
Theꢀ combinedꢀ organicꢀ layersꢀ wereꢀ driedꢀ overꢀ magnesiumꢀ
sulfate and evaporated in vacuo.ꢀTheꢀresultantꢀyellowꢀresidueꢀ
wasꢀ purifiedꢀ byꢀ rapidꢀ columnꢀ chromatographyꢀ (AcOEt–PE=
andꢀ efficientlyꢀ preventedꢀ theꢀ increasedꢀ apoptoticꢀ rateꢀ andꢀ
nuclear morphologic changes induced by hypoglycemia and
serum limitation. Although a detailed molecular mechanism
that links all these events should be further investigated, the

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Fig.ꢀ 4.ꢀ ProtectiveꢀEffectꢀofꢀD1ꢀonꢀExposureꢀtoꢀHypoglycemiaꢀandꢀSerumꢀLimitation-InducedꢀApoptosisꢀinꢀPC12ꢀCells
(A)ꢀPC12ꢀcellsꢀinꢀcontrolꢀgroup,ꢀhypoglycemiaꢀandꢀserumꢀlimitationꢀaloneꢀgroup,ꢀpretreatmentꢀwithꢀSalꢀ(300ꢀµg/mL) or D1 (300µg/mL)ꢀplusꢀexposureꢀtoꢀhypoglycemiaꢀ
andꢀserumꢀlimitationꢀgroupꢀwereꢀstainedꢀwithꢀFITC-AnnexinꢀVꢀandꢀPI,ꢀfollowedꢀbyꢀflowꢀcytometryꢀanalysis.ꢀ(B)ꢀQuantitativeꢀanalysisꢀofꢀtheꢀpercentageꢀofꢀapoptoticꢀandꢀ
necroticꢀcellsꢀinꢀtotalꢀcellꢀpopulationꢀwereꢀshownꢀafterꢀdifferentꢀtreatments.ꢀDataꢀareꢀexpressedꢀasꢀmeans S.D. of the percentages of apoptotic cells from three indepen-
dentꢀexperiments.ꢀ*ꢀp<0.01 vs.ꢀexposureꢀtoꢀhypoglycemiaꢀandꢀserumꢀlimitationꢀalone.ꢀ# p<0.01 vs. Sal (300µg/mL) group.
1ꢀ:ꢀ10–1ꢀ:ꢀ5,ꢀv/v)ꢀtoꢀaffordꢀcolorlessꢀoilꢀ(15.0ꢀg,ꢀyield:ꢀ75%).
2-(2,3,4,5,6-Pentafluorophenyl)ꢀethylꢀ β-D-Glucopyranoside
2-(Pentafluorophenyl)ꢀethylꢀ 2,3,4,6-Tetra-O-acetyl-β-D-glu- (D1):ꢀ Toꢀ 3aꢀ (1.14ꢀg,ꢀ 2.1ꢀmmol)ꢀ inꢀ MeOHꢀ (20ꢀmL)ꢀ wasꢀ addedꢀ
copyranoside (3a):ꢀ Toꢀ CH2Cl2 (10mL) solution of 2 (0.76g, K2CO3 (0.31g, 2.3mmol) to pH=ꢀ8–10ꢀ andꢀ theꢀ mixtureꢀ wasꢀ
3.6ꢀmmol)ꢀ wereꢀ addedꢀ Ag2CO3 (2.00g, 7.2mmol) and mo- allowedꢀ toꢀ stirꢀ forꢀ 2ꢀhꢀ atꢀ roomꢀ temperature.ꢀ Theꢀ solutionꢀ
lecularꢀ sieveꢀ powderꢀ (1.2ꢀg)ꢀ byꢀ nitrogenꢀ protection,ꢀ andꢀ theꢀ wasꢀ neutralizedꢀ withꢀ Amberliteꢀ IRA-120ꢀ (H+) resin to pH
mixtureꢀ wasꢀ stirredꢀ awayꢀ fromꢀ lightꢀ forꢀ 0.5ꢀhꢀ atꢀ roomꢀ tem- 7,ꢀ thenꢀ filtratedꢀ andꢀ concentratedꢀ in vacuo, and the residue
perature, then tetra-O-acetyl-α-D-glucopyranosyl bromide wasꢀ purifiedꢀ byꢀ rapidꢀ columnꢀ chromatographyꢀ (MeOH–
(2.95ꢀg,ꢀ7.2ꢀmmol)ꢀwasꢀaddedꢀquickly,ꢀandꢀcontinuouslyꢀstirredꢀ CHCl3=1ꢀ:ꢀ10–1ꢀ:ꢀ5,ꢀ v/v)ꢀ toꢀ affordꢀ whiteꢀ solidꢀ D1;ꢀ yield:ꢀ 85%;ꢀ
overnight.ꢀ Theꢀ precipitateꢀ wasꢀ filtratedꢀ andꢀ theꢀ filtrateꢀ wasꢀ mp:ꢀ 134–136°C;ꢀ [α]D26.9 −18.5 (c=0.15,ꢀ MeOH).ꢀ IRꢀ (KBr,ꢀ ν,
concentrated in vacuo,ꢀ andꢀ theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ rapidꢀ cm−1):ꢀ3406,ꢀ2940,ꢀ1507,ꢀ1120,ꢀ1094,ꢀ1037.ꢀ1H-NMR (CD3OD,ꢀ
columnꢀ chromatographyꢀ (PE–EtOAc=ꢀ1ꢀ:ꢀ2–1ꢀ:ꢀ3,ꢀ v/v)ꢀ toꢀ affordꢀ 300ꢀMHz):ꢀ δ (ppm) 3.06 (t, J=6.9Hz, 2H), 3.13 (t, J=8.4Hz,
3aꢀasꢀcolorlessꢀviscousꢀliquid;ꢀyield:ꢀ75%.
1H), 3.23–3.29 (m, 3H), 3.63 (t, J=5.7Hz, 1H), 3.74–3.86 (m,
2-(Pentafluorophenyl)ꢀethylꢀ 2,3,4,6-Tetra-O-acetyl-β-D-ga- 2H), 4.06 (t, J=6.9Hz, 1H), 4.45 (d, J=8.4Hz, 1H). MS (ESI)
lactopyranoside (3b):ꢀ Compoundꢀ 3bꢀ wasꢀ synthesizedꢀ fromꢀ 2 m/z=375ꢀ [M+H]+; Anal. Calcd for C14H15F5O6:ꢀ C,ꢀ 44.93;ꢀ H,ꢀ
(0.76g, 3.6mmol) and tetra-O-acetyl-α-D-galactopyranosyl bro- 4.04.ꢀFound:ꢀC,ꢀ44.99;ꢀH,ꢀ4.06.
mide (2.95g, 7.2mmol) according to the synthetic procedure
of 3a in yield 72%, colorless viscous liquid.
2-(2,3,4,5,6-Pentafluorophenyl)ꢀethylꢀ β-D-Galactopyranoside
(D2):ꢀ Compoundꢀ D2ꢀ wasꢀ synthesizedꢀ fromꢀ 3b (1.14g,

1196
Vol. 61, No. 11
2.1ꢀmmol)ꢀ andꢀ K2CO3 (0.31g, 2.3mmol) according to the (25°C) in the dark, and added 400µL of 1X Binding Buffer to
syntheticꢀ procedureꢀ ofꢀ D1ꢀ inꢀ yieldꢀ 78%,ꢀ whiteꢀ solid.ꢀ mpꢀ eachꢀ tube.ꢀ Cellsꢀ wereꢀ analyzedꢀ byꢀ flowꢀ cytometryꢀ withinꢀ 1ꢀhꢀ
110–113°C;ꢀ [α]D26.9 −8.2 (c=0.11,ꢀ MeOH).ꢀ IRꢀ (KBr,ꢀ ν, cm−1):ꢀ (BD, CA).
1
3385, 2935, 1506, 1121, 1085. H-NMR (CD3OD,ꢀ300ꢀMHz):ꢀδ
Statistical Analysisꢀ ꢀAllꢀ Dataꢀ wereꢀ expressedꢀ asꢀ means
(ppm) 3.06 (t, J=7.0Hz, 2H), 3.43–3.50 (m, 3H), 3.65–3.70 (m, S.E.M.ꢀ Statisticalꢀ differencesꢀ betweenꢀ groupsꢀ wereꢀ analyzedꢀ
2H), 3.73–3.81 (m, 2H), 4.01–4.09 (t, J=6.9Hz, 1H), 4.40 (d, byꢀ onewayꢀ analysisꢀ ofꢀ varianceꢀ withꢀ subsequentꢀ Turkey’sꢀ
J=8.4Hz, 1H). MS (ESI) m/z=375ꢀ [M+H]+; Anal. Calcd for tests. In all cases, p<0.05ꢀwasꢀconsideredꢀstatisticallyꢀsignifi-
C14H15F5O6:ꢀC,ꢀ44.93;ꢀH,ꢀ4.04.ꢀFound:ꢀC,ꢀ44.89;ꢀH,ꢀ4.04.
Cell Treatment PC-12 cells, obtained from the American
TypeꢀCultureꢀCollectionꢀ(Manassas,ꢀVA,ꢀU.S.A.),ꢀwereꢀplatedꢀ
cant.
Acknowledgments Weꢀ gratefullyꢀ acknowledgeꢀ theꢀ fi-
andꢀ maintainedꢀ inꢀ high-glucoseꢀ Dulbecco’sꢀ modifiedꢀ Eagle’sꢀ nancial support by the Natural Science Foundation of China
mediumꢀ(DMEM)ꢀsupplementedꢀwithꢀ5%ꢀfetalꢀbovineꢀserum,ꢀ (GrantꢀNos.ꢀ21242005ꢀandꢀ81171457),ꢀBiotechnologyꢀandꢀNewꢀ
10%ꢀ horseꢀ serum,ꢀ 100ꢀU/mLꢀ streptomycin,ꢀ andꢀ 100ꢀU/mLꢀ Medicine Projects of Nantong City (AS2011016), Nature Sci-
penicillinꢀinꢀaꢀhumidifiedꢀatmosphereꢀofꢀ5%ꢀCO2 and 95% air enceꢀ Foundationꢀ ofꢀ Nantongꢀ Universityꢀ (Grantꢀ Nos.ꢀ 10Z074ꢀ
atꢀ 37°C.ꢀ Theꢀ cellsꢀ wereꢀ differentiatedꢀ byꢀ treatingꢀ withꢀ nerveꢀ and 12ZY035), and also thank a project funded by the Priority
growthꢀfactorꢀ(NGF)ꢀ(50ꢀng/mL)ꢀeveryꢀotherꢀdayꢀforꢀ6ꢀd.
Academic Programs Development of Jiangsu Higher Educa-
PC12ꢀcellsꢀwereꢀpre-incubatedꢀwithꢀD1,ꢀD2ꢀorꢀSalꢀforꢀ24ꢀh,ꢀ tion Institutions (PAPD).
thenꢀ wereꢀ exposedꢀ toꢀ hypoglycemiaꢀ andꢀ serumꢀ limitation,ꢀ
withꢀ theꢀ glucose-freeꢀ DMEMꢀ supplementedꢀ includingꢀ 1%ꢀ
fetalꢀbovineꢀserum,ꢀ1%ꢀhorseꢀserum,ꢀ100ꢀU/mLꢀstreptomycin,ꢀ
andꢀ 100ꢀU/mLꢀ penicillin,ꢀ forꢀ anꢀ additionalꢀ 48ꢀhꢀ withꢀ originalꢀ
concentrations.ꢀ Atꢀ theꢀ sameꢀ time,ꢀ treatmentꢀ onlyꢀ withꢀ cul-
ture medium containing high glucose/enough serum or only
exposureꢀ toꢀ hypoglycemiaꢀ andꢀ serumꢀ limitationꢀ (butꢀ withoutꢀ
pretreatment)ꢀwasꢀconsideredꢀcontrolꢀgroupꢀorꢀnegativeꢀgroup.ꢀ
Atꢀtheꢀendꢀofꢀcellꢀtreatments,ꢀweꢀcarriedꢀoutꢀtheꢀfollowingꢀdif-
ferent tests.
References
ꢀ 1)ꢀ Wongꢀ P.ꢀ C.,ꢀ Pardoꢀ C.ꢀ A.,ꢀ Borcheltꢀ D.ꢀ R.,ꢀ Leeꢀ M.ꢀ K.,ꢀ Copelandꢀ N.ꢀ
G., Jenkins N. A., Sisodia S. S., Cleveland D. W., Price D. L., Neu-
ron, 14, 1105–1116 (1995).
2) Stadelmann C., Lassmann H., Cell Tissue Res., 301, 19–31 (2000).
3) Díaz Lanza A. M., Abad Martínez M. J., Fernández Matellano L.,
Recuero Carretero C., Villaescusa Castillo L., Silván Sen A. M.,
Bermejo Benito P., Planta Med., 67, 219–223 (2001).
ꢀ 4)ꢀ Iaremiĭꢀ I.ꢀ N.,ꢀ Grigor’evaꢀ N.ꢀ F.,ꢀ Eksp. Klin. Farmakol., 65, 57–59
(2002).
Cell Viability Assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-di-
phenyltetrazoliumꢀ bromideꢀ (MTT)ꢀ assayꢀ wasꢀ usedꢀ toꢀ detectꢀ
cellꢀ viabilityꢀ asꢀ describedꢀ previously.ꢀ MTTꢀ wasꢀ dissolvedꢀ inꢀ
DMEMꢀ mediumꢀ andꢀ addedꢀ theꢀ cellꢀ samplesꢀ toꢀ makeꢀ finalꢀ
concentrationꢀ ofꢀ 0.5ꢀmg/mLꢀ atꢀ 37°Cꢀ forꢀ 4ꢀh.ꢀ Afterward,ꢀ me-
diumꢀwasꢀaddedꢀtoꢀaꢀsolutionꢀofꢀ20%ꢀsodiumꢀdodecylꢀsulfideꢀ
(SDS) to dissolve formazan generated. The absorbance (optical
densityꢀ (OD))ꢀ valuesꢀ wereꢀ measuredꢀ byꢀ spectrophotometryꢀ
atꢀ 570ꢀnmꢀ withꢀ anꢀ EIX-800ꢀ Microelisaꢀ readerꢀ (Bio-Tekꢀ Inc.,ꢀ
U.S.A.).
ꢀ 5)ꢀ Kanupriyaꢀ Prasadaꢀ D.,ꢀ Saiꢀ R.ꢀ M.,ꢀ Kumarꢀ R.,ꢀ Sawhneyꢀ R.ꢀ C.,ꢀ
SharmaꢀS.ꢀK.,ꢀIlavazhaganꢀG.,ꢀKumarꢀD.,ꢀBanerjeeꢀP.ꢀK.,ꢀMol. Cell.
Biochem., 275, 1–6 (2005).
6) Ming D. S., Hillhouse B. J., Guns E. S., Eberding A., Xie S., Vima-
lanathanꢀS.,ꢀTowersꢀG.ꢀH.,ꢀPhytother. Res., 19, 740–743 (2005).
7) Yu S., Liu M., Gu X., Ding F., Cell. Mol. Neurobiol., 28, 1067–1078
(2008).
8) Zhang J., Liu A., Hou R., Zhang J., Jia X., Jiang W., Chen J., Eur. J.
Pharmacol., 607, 6–14 (2009).
ꢀ 9)ꢀ Chenꢀ X.,ꢀ Zhangꢀ Q.,ꢀ Chengꢀ Q.,ꢀ Dingꢀ F.,ꢀ Mol. Cell. Biochem., 332,
85–93 (2009).
Hoechst 33342 Staining Assaysꢀ ꢀTheꢀ PC12ꢀ cellsꢀ wereꢀ
fixedꢀ inꢀ 4.0%ꢀ paraformaldehydeꢀ forꢀ 20ꢀmin,ꢀ andꢀ thenꢀ stainedꢀ
withꢀ5ꢀµg/mLꢀHoechstꢀ33342ꢀdyeꢀatꢀ37°Cꢀforꢀ10ꢀmin,ꢀfollowedꢀ
byꢀobservationꢀunderꢀaꢀDMRꢀfluorescenceꢀmicroscopeꢀ(Leicaꢀ
Microsystems,ꢀ Wetzlar,ꢀ Germany)ꢀ withꢀ ultravioletꢀ illumina-
tion.ꢀTheꢀdyeꢀwasꢀexcitedꢀatꢀ340ꢀnm,ꢀandꢀemissionꢀwasꢀfilteredꢀ
withꢀaꢀ510ꢀnmꢀbarrierꢀfilter.ꢀInꢀorderꢀtoꢀquantityꢀtheꢀapoptoticꢀ
process,ꢀcellsꢀwithꢀfragmentedꢀorꢀcondensedꢀDNAꢀandꢀnormalꢀ
DNAꢀwereꢀcounted,ꢀrespectively.ꢀTheꢀdataꢀwereꢀexpressedꢀasꢀ
a percent of control value and mean ꢀS.E.M.ꢀofꢀthreeꢀexperi-
ments.
10)ꢀ Shiꢀ T.ꢀ Y.,ꢀ Fengꢀ S.ꢀ F.,ꢀ Xingꢀ J.ꢀ H.,ꢀ Wuꢀ Y.ꢀ M.,ꢀ Liꢀ X.ꢀ Q.,ꢀ Zhangꢀ N.,ꢀ
Tian Z., Liu S. B., Zhao M. G., Neurotox. Res., 21, 358–367 (2012).
11) Guo Y., Zhao Y., Zheng C., Meng Y., Yang Y., Chem. Pharm. Bull.,
58, 1627–1629 (2010).
12) Guo Y., Li X., Zhao Y., Si Y., Zhu H., Yang Y., Chem. Pharm. Bull.,
59, 1045–1047 (2011).
13)ꢀ Mengꢀ Y.,ꢀ Guoꢀ Y.,ꢀ Lingꢀ Y.,ꢀ Zhaoꢀ Y.,ꢀ Zhangꢀ Q.,ꢀ Zhouꢀ X.,ꢀ Dingꢀ F.,ꢀ
Yang Y., Bioorg. Med. Chem., 19, 5577–5584 (2011).
14)ꢀ ParkꢀB.ꢀK.,ꢀKitteringhamꢀN.ꢀR.,ꢀO’neill P. M., Annu. Rev. Pharma-
col., 41, 443–470 (2001).
15)ꢀ O’Hagan D., Rzepa H. S., Chem. Commun. (Camb.), 7, 645–652
(1997).
Flow Cytometry with Annexin V/PI Staining Apo-
ptotic/necroticꢀ cellsꢀ wereꢀ measuredꢀ byꢀ flowꢀ cytometryꢀ asꢀ
described previously.
hedꢀcellsꢀtwiceꢀwithꢀcoldꢀPBSꢀandꢀthenꢀresuspendꢀcellsꢀinꢀ1Xꢀ
Binding Buffer at a concentration of 1×106 cells/mL per assay.
Transfer 100µL of the solution (1×105 cells) to a 5mL culture
tube, 5µLꢀ ofꢀ fluoresceinꢀ isothiocyanateꢀ (FITC)-conjugatedꢀ
Annexinꢀ Vꢀ andꢀ 5ꢀµLꢀ propidiumꢀ iodideꢀ (PI)ꢀ wereꢀ added,ꢀ thenꢀ
Gentlyꢀ vortexedꢀ theꢀ cellsꢀ andꢀ incubatedꢀ forꢀ 15ꢀminꢀ atꢀ RTꢀ
16)ꢀ KirkꢀK.ꢀL.,ꢀFillerꢀR.,ꢀ“BiomedicalꢀFrontiersꢀofꢀFluorineꢀChemistry,ꢀ
Symposiumꢀ Series,”ꢀ ed.ꢀ byꢀ Ojimaꢀ I.,ꢀ McCarthyꢀ J.ꢀ R.,ꢀ Welchꢀ T.,ꢀ
American Chemical Society, Washington, D.C., 1996, pp. 1–24.
17) Mattson M. P., Duan W., Pedersen W. A., Culmsee C., Apoptosis, 6,
69–81 (2001).
18) Horton D., Wolfrom M. L., J. Org. Chem., 27, 1794–1800 (1962).
19)ꢀ BárczaiꢀMartosꢀM.,ꢀKörösyꢀF.,ꢀNature (London), 165, 369 (1950).
20) Behbahani H., Rickle A., Concha H., Ankarcrona M., Winblad B.,
CowburnꢀR.ꢀF.,ꢀJ. Neurosci. Res., 82, 432–441 (2005).
20)ꢀ Theꢀ PC12ꢀ cellsꢀ wereꢀ harvested,ꢀ wasꢀ