Inhibition of human enterovirus 71 replication by pentacyclic triterpenes and their novel synthetic derivatives

Article abstract of DOI:10.1248/cpb.c14-00088

A large number of bioactive pentacyclic triterpenoids have been shown to have multiple biological activities. This study was conducted to evaluate the inhibitory activities of 6 newly synthesized and novel pentacyclic triterpenoids against enterovirus 71 (EV71). The parent compound, ursolic acid (UA), showed the greatest inhibitory activity against EV71, while oleanolic acid (OA), asiatic acid (AA), and synthetic derivatives of 18-β-glycyrrhetinic acid (GA) and OA also exhibited inhibitory effects, although to lesser extents. The results suggest these compounds show potential for further optimization as antiviral candidates for treatment of EV71 infections.

Full text of DOI:10.1248/cpb.c14-00088

764
Regular Article
Chem. Pharm. Bull. 62(8) 764–771 (2014)
Vol. 62, No. 8
Inhibition of Human Enterovirus 71 Replication by Pentacyclic
Triterpenes and Their Novel Synthetic Derivatives
,e
Chun-hui Zhao,a,b Jie Xu,a Ying-qiu Zhang,c,d Long-xuan Zhao,b and Bin Feng*
b
a School of Life Sciences, Liaoning Normal University; Dalian 116029, China: Liaoning Provincial Key Laboratory
c
of Biotechnology and Drug Discovery; Dalian 116029, China: Key Laboratory of Pathogenic Microbiology and
d
Immunology, Institute of Microbiology, Chinese Academy of Sciences; Beijing 100101, China: Institute of Cancer
e
Stem Cell, Dalian Medical University; and Department of Biotechnology, Dalian Medical University; Dalian 116044,
China.
Received January 26, 2014; accepted April 29, 2014
A large number of bioactive pentacyclic triterpenoids have been shown to have multiple biological activi-
ties. This study was conducted to evaluate the inhibitory activities of 6 newly synthesized and novel pentacy-
clic triterpenoids against enterovirus 71 (EV71). The parent compound, ursolic acid (UA), showed the great-
est inhibitory activity against EV71, while oleanolic acid (OA), asiatic acid (AA), and synthetic derivatives of
18-β-glycyrrhetinic acid (GA) and OA also exhibited inhibitory effects, although to lesser extents. The results
suggest these compounds show potential for further optimization as antiviral candidates for treatment of
EV71 infections.
Key words enterovirus 71; pentacyclic triterpenoid; inhibitory activity
Human enterovirus 71 (EV71) is a small, non-enveloped asiatic acid (AA), oleanolic acid (OA), and UA all contain
virus belonging to the family Picornaviridae. It has been hydroxyl groups and carboxylic groups (Fig. 1). While previ-
identified as the most frequent causative agent of hand, foot, ous reports have suggested that the activity of these pentacy-
and mouth disease (HFMD), and EV71 infection is usually clic triterpenoids is related to their basic triterpenoid skeletal
accompanied by severe neurological complications which can structure, the attached functional groups offer opportunities
cause high mortality, such as aseptic meningitis, brainstem for chemical modification and improvement of activity.15–17)
encephalitis, neurogenic pulmonary edema, and acute flaccid Optically active D-amino acids are important components in
paralysis.1) Children under 5 years old are particularly suscep- marketed drugs. Among these amino acids, D-phenylglycine
tible to the most severe forms of EV71 infection. EV71 was is used to synthesize drugs such as ampicillin, aspoxicillin,
initially isolated in 1969 from patients in California (U.S.A.) cefbuperazone, and cefpiramide. D-Phenylglycine, instead of
with central nervous system diseases,2) and has since been L-phenylglycine, was used as the building block in pharmaceu-
implicated in large worldwide outbreaks of HFMD associated ticals and developmental drugs to enhance enzymatic stability
with severe neurological complications, especially in the Asia- and improve pharmacodynamics and bioavailability.18,19) In
Pacific region,3,4) including Singapore, Malaysia, Japan, and the current study, functional groups attached to pentacyclic
Taiwan.5–8) Since 2008, large outbreaks of EV71 infection in triterpene molecules were modified to generate derivatives
mainland China have resulted in millions of cases and hun- while maintaining the pentacyclic triterpene skeletal structure.
dreds of deaths in children.9) The EV71 capsid comprises four As a result of these modifications, the carboxylic group was
structural proteins: VP1, VP2, VP3, and VP4. Proteins VP1, converted to a methyl ester with retention of stereochemistry,
VP2, and VP3 are located at the surface of the viral capsid which may significantly influence its physical properties or
and exposed to immune pressures, whereas VP4 is inside the receptor interactions. D-Phenylglycine was introduced into
capsid.10) In recent years, studies on the molecular biology of the hydroxyl group at the C-2 position of AA, and at the C-3
EV71 have led to the development of new strategies for treat- positions of GA, OA, and UA. The dihydroxyl groups at C-3
ment and prevention of EV71 infection; however, there is still and C-23 of AA were protected with acetonide using 2,2-di-
no effective agent for treating EV71 infections.11) Therefore, it methoxy propane.15) The present report describes six novel
is essential to identify novel anti-EV71 agents as candidates synthetic pentacyclic triterpenoids and their anti-EV71 activi-
for further research and optimization.
ties, as compared to the activities of GA, AA, OA, and UA.
To date, ca. 20000 triterpenoids have been identified from
various parts of medicinal plants.12) While numerous triterpe-
noids have demonstrated antiviral activity,13) only the penta-
Results and Discussion
Synthesis of Pentacyclic Triterpenoid Derivatives Pen-
cyclic triterpenoid ursolic acid (UA) extracted from Ocimum tacyclic triterpenoids were used as lead compounds, and
basilicum has been reported to exhibit potent anti-EV activity structural modifications were made at positions C-3, C-30 of
(EC50 value of 0.5µg/mL and 50% cytotoxic concentration GA, C-2, C-3, C-23, C-28 of AA, and C-3 and C-28 of OA and
(CC50) value of 100.5µg/mL) in a human skin basal cell car- UA, respectively. Similar procedures were used to synthesize
cinoma cell line (BCC-1/KMC).14) Our knowledge concerning derivatives of GA, UA, and OA, and the synthetic pathways
the antiviral activity of natural and synthetic pentacyclic trit- are presented in Charts 1–3. The carboxylic groups (C-30 of
erpenoids against EV71 is quite limited. The naturally occur- GA, and C-28 of OA and UA) were subjected to methyl iodide
ring pentacyclic triterpenoids 18-β-glycyrrhetinic acid (GA), treatment in the presence of potassium carbonate in dimethyl
formamide to create methyl ester moieties with retention of
The authors declare no conflict of interest.
stereochemistry. The hydroxyl group at C-3 of these pentacy-
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: binfeng@dlmedu.edu.cn

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Fig. 1. Chemical Structures of Pentacyclic Triterpenoids
Reagents and conditions: a) CH3I, K2CO3, DMF; b) Fmoc-D-Phg-OH, EDCI, DMAP, CH2Cl2; c) Et2NH, CH2Cl2.
Chart 1. Synthesis of Glycyrrhetinic Acid Derivatives
clic triterpenoids was treated with Fmoc-D-phenylglycine in using the same synthetic pathways as used for other pentacy-
the presence of 4-(dimethylamino)pyridine in dichloromethane clic triterpenoids. All compounds were dissolved in dimethyl
to generate an intermediate, which in turn, was treated with sulfoxide (DMSO) prior to use in biological activity assays.
diethylamine in dichloromethane to obtain end products with
Assay of Anti-EV71 Activity The VP1 gene sequence is
good yield. AA was prepared and its derivatives were syn- specific to EV71 and is used to distinguish EV71 from other
thesized as previously described.20) As shown in Chart 4, the enteroviruses and in molecular epidemiologic studies.21) The
dihydroxyl groups at C-3 and C-23 of AA were treated with VP1 gene can be detected by using real-time polymerase
2,2-dimethoxypropane in the presence of p-toluenesulfonic chain reaction (PCR) with primers and probes targeting the
acid in dimethylformamide to generate acetonide. The car- VP1 region, and by Western blot analysis with an EV71
boxylic group at C-28 was converted to a methyl ester and the VP1 monoclonal antibody.22,23) To examine the effects GA,
hydroxyl group at C-2 was treated with Fmoc-D-phenylglycine AA, OA, UA, and their derivatives on EV71, virus-infected

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Reagents and conditions: a) (CH3)2C(OCH3)2, p-TsOH, DMF; b) CH3I, K2CO3, DMF; c) Fmoc-D-Phg-OH, EDCI, DMAP, CH2Cl2; d) Et2NH, CH2Cl2.
Chart 2. Synthesis of Asiatic Acid Derivatives19)
Reagents and conditions: a) CH3I, K2CO3, DMF; b) Fmoc-D-phg-OH, EDCI, DMAP; c) Et2NH, CH2Cl2.
Chart 3. Synthesis of Oleanolic Acid Derivatives
human rhabdomyosarcoma (RD) cells were incubated with RT-PCR assays. RD cells treated with AA, OA, 3a, 3b, 4a, or
the selected individual compounds at a concentration of 10µg/ 4b showed partial suppression of VP protein expression. No
mL for 48h, and then harvested. The inhibitory effects of the significant decrease in VP1 protein levels was observed in RD
various compounds on EV71 replication were analyzed by cells treated with 1a, 2b, or 2c. GA showed slightly inhibition
reverse transcription (RT)-PCR and Western blot analysis. activity (90% of control). These results suggested that modifi-
RD cells incubated with compound 1b showed an ca. 35% cation of GA to form D-phenylglycine at the C-3 position, and
decrease in viral transcripts, while cells incubated with UA modification of OA at the C-2 position may increase antiviral
showed an ca. 45% reduction (Fig. 2A). However, there was activity, but antiviral activity would not be increased by in-
no significant decrease of EV71 viral RNA transcripts in the troduction of D-phenylglycine into the structural skeletons of
RD cells treated with the other compounds (Fig. 2A). West- UA and AA. These results also suggest that the inhibitory
ern blot studies were used to analyze EV71 viral proteins in effects of various compounds on EV71 infection might be
RD cells treated with various compounds (Fig. 2B). Results mediated through different mechanisms. Future studies using
showed that RD cells treated with 1b and UA, had decreased both of D-phenylglycine and L-phenylglycine to synthesize
levels of VP1 viral protein (ca. 80% and 90% reductions, new compounds would ascertain the structure–activity re-
respectively), which corresponded with results obtained from lationship of the two enantiomers. The cytotoxicity of these

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Reagents and conditions: a) CH3I, K2CO3, DMF; b) Fmoc-D-phg-OH, EDCI, DMAP; c) Et2NH, CH2Cl2.
Chart 4. Synthesis of Ursolic Acid Derivatives
compounds was evaluated against RD cells in vitro using ate (735mg, 5.32mmol) in dry dimethyl formamide (10mL)
the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- was added to methyl iodide (0.26mL, 4.25mmol) and stirred
2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine etho- at room temperature for 6h. The reaction mixture was then
sulfate (MTS/PMS) assay, and all compounds tested showed a diluted with ethyl acetate (50mL), and washed with water
low level of toxicity (CC50>50µg/mL).
(20mL×3) and saturated sodium chloride solution. The or-
In conclusion, data from the present study collectively sug- ganic layer was dried over anhydrous magnesium sulfate. The
gest that the pentacyclic triterpenoid compounds UA, OA, and solution was filtered, the remaining solvent was removed by
AA have inhibitory effects against EV71. Synthetic deriva- evaporation at reduced pressure, and the isolated material was
tives of GA and OA showed greater antiviral effects than the purified by silica gel chromatography using a gradient elu-
natural parent compounds, and lacked significant cytotoxic- tion of ethyl acetate–petrol ether (1:2, v/v) to afford a white
1
ity. Due to the lack of effective drugs for treatment of EV71 solid (875mg, 85%), mp 240.5–241.8°C; H-NMR (500MHz,
infections, these compounds should be further evaluated as CDCl3) δ: 5.65 (s, 1H, H-12), 3.68 (s, 3H, –OCH3), 3.24–3.21
antiviral agents. Future studies will be conducted to better de- (m, 1H, H-3), 2.79 (dt, J1=13.2Hz, J2=3.6Hz, 1H, H-18), 2.34
termine the accuracy of IC50 values obtained for the individual (s, 1H, H-9), 1.36, 1.15, 1.14, 1.12, 1.00 (s, each 3H, –CH3),
compounds and the effect of these compounds on key steps 0.80 (s, 6H, –CH3×2); 13C-NMR (125MHz, CDCl3) δ: 200.22,
involved in EV71 replication; i.e., translation of the internal 176.98, 169.22, 135.37, 128.70, 79.00, 62.08, 55.22, 52.03,
ribosomal entry site (IRES)-mediated viral polyprotein; the 48.67, 45.66, 44.31, 43.46, 41.39, 39.41, 38.03, 37.38, 33.06,
proteolytic activity of viral proteases 2A and/or 3C; and ac- 32.13, 31.43, 28.83, 28.63, 28.40, 27.63, 26.79, 26.73, 23.70,
tivity of viral 3D-RNA-dependent RNA polymerase (RdRp). 19.00, 17.81, 16.69, 15.89; IR νmax (KBr, cm−1): 3453, 2945,
Results of such studies will help to elucidate the molecular 1723, 1662; electrospray ionization (ESI)-MS: m/z 485.02
targets modulated by these compounds.
[M−H]−.
Methyl 3β-O-(N-Fluorenylmethoxycarbonyl)-phenylglycyl-
11-oxo-olean-12-en-30-oate (1a): A solution of compound
(500mg, 1.03mmol), Fmoc-D-phenylglycine (578.7mg,
and N,N-dimethylaminopyridine (DMAP)
Experimental
Materials and Methods Melting points (mp, uncor-
rected) were determined in glass capillaries using an X-5 1.55mmol),
1
melting point apparatus (Beijing Tech Instrument Co., Ltd., (189.3mg, 1.55mmol) in methylene chloride (5mL) was added
China). Nuclear magnetic resonance (NMR) experiments to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
(1H-NMR and 13C-NMR) were conducted using a Bruker (197.1mg, 1.55mmol) at 0°C and stirred for 3h at room
Avance 500MHz spectrometer (Bruker, Karlsruhe, Germany). temperature. The reaction mixture was then diluted with
Infrared radiation (IR) data were analyzed using a Shimadzu methylene chloride and washed with water and saturated
IRPrestige-21 FT-IR spectrophotometer (Shimadzu, Kyoto, sodium chloride solution (10mL). The solution was filtered,
Japan). Mass spectra were recorded on a Micromass QTof-mi- the remaining solvent was removed by evaporation at reduced
cro™ spectrometer (Micromass, Manchester, U.K.). Elemental pressure, and the isolated material was purified by silica gel
analyses were performed using a Elementar Analysensysteme chromatography using a gradient elution of ethyl acetate–pe-
GmbH (Elementar, Hanau, Germany). All reagents and chemi- troleum ether (1:5, v/v) to afford a white solid (347.2mg,
cals were of analytical grade or chemically pure.
40.9%), mp 125.4–126.0°C; 1H-NMR (500MHz, CDCl3) δ:
Chemical Synthesis. Synthesis of GA Derivatives 7.68 (d, J=7.4Hz, 2H, fluorenyl H-4′, H-5′), 7.51 (d, J=4.0Hz,
Methyl 3β-Hydroxyl-11-oxo-olean-12-en-30-oate (1): A solu- 2H, fluorenyl H-1′, H-8′), 7.32 (d, J=7.0Hz, 2H, fluorenyl H-3′,
tion of GA (1g, 2.125mmol) and anhydrous potassium carbon- H-6′), 7.23 (d, J=6.5Hz, 2H, fluorenyl H-2′, H-7′), 7.34–7.21

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Vol. 62, No. 8
Fig. 2. Inhibition of Enterovirus 71 (EV71) Replication by Various Compounds
(A) Levels of the EV71 RNA transcripts were determined using real-time PCR. Results represent the mean percentage of EV71 RNA transcripts S.D. from three inde-
pendent experiments. The ANOVA for comparison of EV71 RNA transcript levels in untreated cells and cells treated with 10 µg/mL UA or 1b showed p<0.05. (B) West-
ern blot analysis was performed using specific monoclonal antibodies against VP1 structural proteins of EV71. β-Actin was used as an internal control with anti-β-actin
monoclonal antibodies. Data represent results obtained from three independent experiments. The difference among different treatments was assessed by one-way ANOVA
followed by post-hoc Tukey’s multiple comparison test (*p<0.05, **p<0.001 versus the control group with DMSO).
(m, 5H, Ph-H), 5.96 (d, J=7.1Hz, 1H, Ph-CH), 5.58 (s, 1H, 55.05, 51.78, 48.45, 47.21, 45.40, 44.08, 43.24, 41.15, 38.73,
H-12), 5.88 (d, J=6.9Hz, 1H, –NH–), 5.20–5.16 (m, 1H, H-2), 38.25, 38.11, 37.78, 36.92, 32.69, 31.87, 31.18, 29.72, 28.54,
4.52–4.42 (m, 2H, –CH2O–), 4.19 (t, J=7.0Hz, 1H, fluorenyl 28.34, 26.46, 23.70, 23.37, 22.68, 19.20, 18.70, 17.38, 17.24,
H-9′), 3.53 (s, 3H, –OCH3), 2.72 (dt, J1=14.0Hz, J2=3.9Hz, 16.65, 16.35, 14.42, 14.12; IR νmax (KBr, cm−1): 3439, 2954,
1H, H-18), 2.25 (s, 1H, H-9), 1.07, 0.89, 0.85, 0.80,0.79 (s, 2890, 1710, 1532, 1438, 1369, 1206, 1156, 796, 710; ESI-MS:
each 3H, –CH3), 0.71 (s, 6H, –CH3×2); 13C-NMR (125MHz, m/z 839.83 [M−H]−. Anal. Calcd for C54H65NO7: C, 77.20; H,
CDCl3) δ: 199.92, 176.93, 169.26, 143.91, 128.89, 128.84, 7.80; N, 1.67. Found: C, 77.13; H, 7.75; N, 1.66.
128.54, 128.51, 125.12, 82.70, 77.29, 77.03, 76.78, 67.15, 61.67,
Methyl 3β-O-Phenylglycyl-11-oxo-olean-12-en-30-oate (1b):

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A solution of compound 1a (133mg, 0.16mmol) in dry methyl- washed with water (15mL×3) and saturated sodium chloride
ene chloride (2mL) was added to diethylamine (3.32mL, solution (30mL). The organic layer was dried over anhydrous
32.24mmol) and stirred at room temperature for 2h. The sol- magnesium sulfate. The residue was filtered, the remaining
vent was evaporated at reduced pressure, and the residue was solvent was removed by evaporation at reduced pressure, and
purified by silica gel chromatography using a gradient elution the isolated material was purified by silica gel chromatog-
of ethyl acetate–petroleum ether (1:5, v/v) to afford a white raphy with a gradient elution of ethyl acetate–petrol ether
1
solid (83.2mg, 85%), mp 226.8–227.1°C; H-NMR (500MHz, (1:10, v/v) and the obtained white solid was recrystallized
CDCl3) δ: 7.32–7.21 (m, 5H, Ph-H), 5.58 (s, 1H, H-12), 4.53 from methanol, providing 3a (410mg, 58.1%) as colorless
1
(s, 2H, –NH2), 4.43 (dd, J1=11.8Hz, J2=4.8Hz, 1H, H-3), solid; mp 117.7–118.6°C; H-NMR (500MHz, CDCl3) δ: 7.76
4.22 (t, J=6.7Hz, 1H, Ph-CH), 3.61(s, 3H, –OCH3), 2.72 (dt, (d, 2H, J=7.5Hz, fluorenyl H-4, H-5), 7.58 (d, 2H, J=6.2Hz,
J1=14.0Hz, J2=3.9Hz, 1H, H-18), 2.25 (s, 1H, H-9), 1.27, 1.07, fluorenyl H-1, H-8), 7.30–7.41 (m, 9H, fluorenyl H-2, H-7, H-6,
1.06, 1.03, 0.80, 0.58, 0.38 (s, each 3H); 13C-NMR (125MHz, H-3, 5×Ph-H), 5.99 (dd, J=6.8Hz, 1H, CH–NH–CO), 5.32
CDCl3) δ: 199.98, 176.93, 169.21, 130.90, 128.86, 128.64, (dd, 1H, J=7.1Hz, CH–NH–CO), 5.25 (t, 1H, J=3.5Hz, H-12),
128.53, 127.95, 127.02, 81.72, 65.57, 61.69, 59.09, 54.99, 51.77, 4.47–4.58 (m, 1H, H-3), 4.39 (m, 2H, –CHCH2OCONH–),
48.44, 45.39, 44.07, 43.22, 41.13, 38.74, 38.23, 37.77, 36.91, 4.25 (m, 1H, fluorenyl H-9), 3.60 (s, 3H, OCH3), 2.85 (dd, 1H,
32.69, 31.86, 31.17, 30.61, 28.53, 28.33, 27.47, 26.49, 26.45, J=14Hz, H-18), 1.08, 0.95, 0.93, 0.87, 0.75, 0.67, 0.48 (s, each
23.68, 23.35, 19.20, 18.69, 17.24, 16.34, 13.73; IR νmax (KBr, 3H); 13C-NMR (125MHz, CDCl3) δ: 178.28, 143.86, 128.85,
cm−1): 3448, 3380, 2967, 2824, 1724, 1653, 1465, 1384, 1256, 127.71, 127.21, 127.09, 119.99, 82.99, 55.26, 51.52, 47.21, 45.91,
1167, 769, 712; ESI-MS: m/z 617.44 [M−H]−. Anal. Calcd for 41.68, 39.31, 38.07, 37.90, 37.77, 36.91, 33.90, 33.12, 32.59,
C39H55NO5: C, 75.81; H, 8.97; N, 2.27. Found: C, 75.66; H, 30.72, 27.71, 25.91, 23.66, 23.43, 23.10, 18.06, 16.85, 16.35,
8.85; N, 2.16.
15.30; IR νmax (KBr, cm−1): 3340, 2922, 1730, 1577, 1472;
Synthesis of Asiatic Acid Derivatives AA deriva- ESI-MS: m/z 825.82 [M−H]−. Anal. Calcd for C54H67NO6: C,
tives (compounds 2 and 2a–c, structures shown in Chart 2) 78.51; H, 8.17; N, 1.70. Found: C, 78.41; H, 8.03; N, 1.59.
were synthesized as previously described.19) Anal. Calcd for
Methyl 3β-O-Phenylglycylurs-12-ene-28-oate (3b): A solu-
C57H71NO8 (2b): C, 76.22; H, 7.97; N, 1.56. Found: C, 76.08; tion of compound 3a (200mg, 0.24mmol) in dry methylene
H, 7.86; N, 1.43. Anal. Calcd for C42H61NO6 (2c): C, 74.63; H, chloride (5mL) was added to diethylamine (5mL, 49.5mmol)
9.10; N, 2.07. Found: C, 74.48; H, 8.98; N, 1.94.
Synthesis of Oleanolic Acid Derivatives Methyl evaporated at reduced pressure and the residue was purified
3β-Hydroxyolean-12-ene-28β-oate (3): solution of OA by silica gel chromatography using a gradient elution of ethyl
and stirred at room temperature for 2h. The solvent was
A
(5g, 11mmol) and anhydrous potassium carbonate (3.78g, acetate–petrol ether (1:2, v/v) and the obtained white solid
27.35mmol) in dry dimethyl formamide (50mL) was added to was recrystallized from ethyl ether, providing 3b as colorless
1
methyl iodide (1.37mL, 21.89mmol) and stirred at room tem- solid; (88mg, 60.0%), mp 195.6–196.9°C; H-NMR (500MHz,
perature for 6h. The reaction mixture was then diluted with CDCl3) δ: 7.39–7.27 (m, 5H, 5×Ph-H), 5.25 (t, J=3.6Hz, 1H,
ethyl acetate (300mL) and washed with water (100mL×3) H-12), 4.60 (d, J=13.9Hz, 1H, NH2CHOCO), 4.52–4.45 (m,
and saturated NaCl solution (50mL). The organic layer was 1H, H-3), 3.60 (s, 3H, –OCH3), 2.85 (dd, J=13.9, 4.0Hz, 1H,
dried over anhydrous magnesium sulfate. The solution was 4.2Hz, H-18), 1.12, 0.98, 0.89, 0.87, 0.70, 0.63, 0.44 (s, each
filtered, the remaining solvent was removed by evaporation 3H); 13C-NMR (125MHz, CDCl3) δ: 178.29, 143.85, 128.63,
at reduced pressure, and the isolated material was purified 129.90, 126.96, 126.69, 122.28, 81.96, 59.21, 55.27, 51.51,
by silica gel chromatography using a gradient elution of 47.56, 46.76, 41.68, 39.31, 38.10, 37.98, 37.89, 37.76, 36.91,
ethyl acetate–petrol ether (1:5, v/v) to afford a white solid 36.91, 33.90, 30.71, 29.73, 28.12, 27.71, 25.91, 23.66, 23.43,
1
(5g, 97%), mp 197.0–198.0°C; H-NMR (500MHz, CDCl3) δ: 23.10, 18.08, 16.85, 16.66, 16.37, 15.30; IR νmax (KBr, cm−1):
5.27 (t, J=3.6Hz, 1H, H-12), 3.61 (s, 3H, –OCH3), 3.20 (dd, 3444, 2935, 1729, 1530, 1460; ESI-MS: m/z 635.39 [M−H]−.
J=10.4, 5.2Hz, 1H, H-3), 2.85 (dd, J=14.0, 4.0Hz, 1H, H-18), Anal. Calcd for C39H57NO4: C, 77.58; H, 9.51; N, 2.32. Found:
1.06, 1.00, 0.92, 0.78, 0.73 (s, each 3H); 13C-NMR (125MHz, C, 77.46; H, 9.45; N, 2.24.
CDCl3) δ: 178.25, 143.76, 122.36, 78.96, 77.29, 77.03, 76.78,
Synthesis of Ursolic Acid and Its Derivatives Methyl
55.24, 51.49, 47.63, 46.71, 45.88, 41.63, 41.29, 39.27, 38.74, 3β-Hydroxyurs-12-ene-28β-oate (4): Compound 4 was synthe-
38.45, 37.03, 33.86, 33.10, 32.67, 32.37, 30.67, 28.10, 27.70, sized as a white solid (4.9g, 95%) using UA (5g, 11mmol) as
27.19, 25.93, 23.63, 23.39, 23.07, 18.33, 16.82, 15.57, 15.29; IR a starting material. The procedure was the same as that used
νmax (KBr, cm−1): 3448, 2948, 1728, 1426; ESI-MS: m/z 471.28 for synthesis of compound 3 from OA, mp 171.0–172.5°C;
[M−H]−.
1H-NMR (500MHz, CDCl3) δ: 5.25 (t, J=3.6Hz, 1H, H-12),
Methyl 3β- O -(N-Fluorenonemethyoxycarbonyl)- 3.60 (s, 3H, –OCH3), 3.20 (dd, J=10.4, 5.2Hz, 1H, H-3), 2.22
phenylglycylurs-12-ene-28β-oate (3a): A solution of compound (d, J=11.2Hz, 1H, H-18), 1.06, 1.00, 0.93, 0.92, 0.85, 0.78, 0.73
3 (400mg, 0.86mmol) and Fmoc-D-phenylglycine (382mg, (s, each 3H); 13C-NMR (125MHz, CDCl3) δ: 178.08, 138.17,
1.02mmol) in dry methylene chloride (7mL) was added to 125.59, 79.05, 77.29, 77.04, 76.78, 55.25, 52.91, 51.46, 48.11,
4-dimethylaminopyridine (124mg, 1.02mmol) and stirred. A 47.59, 42.02, 39.51, 39.07, 38.89, 38.77, 38.64, 37.00, 36.66,
solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 33.00, 30.68, 28.16, 28.05, 27.25, 24.25, 23.63, 23.32, 21.20,
hydrochloride (197mg, 1.02mmol in dry dichloromethane 18.34, 17.05, 16.93, 15.64, 15.45; IR νmax (KBr, cm−1): 3452,
(3mL)) was added drop-wise to the reaction mixture at 0°C 2923, 1722, 1458; ESI-MS: m/z 471.13 [M−H]−.
and the mixture was stirred for 5min at 0°C, followed by
Methyl 3β- O -(N-Fluorenonemethyoxycarbonyl)-
additional stirring for 5h at room temperature. The reaction phenylglycylurs-12-ene-28β-oate (4a): Compound 4a was
mixture was then diluted with methylene chloride (50mL) and synthesized as a colorless solid (510mg, 57.7%) from com-

770
Vol. 62, No. 8
pound 4 (500mg, 1.07mmol). The procedure was the same TBST. Primary antibody specific for EV71 VP1 protein was
as that used for synthesis of compound 3a from compound obtained from Millipore (Billerica, MA, U.S.A.) and β-actin
1
3, mp 115.6–116.5°C; H-NMR (500MHz, CDCl3) δ: 7.76 (d, was obtained from Santa Cruz Biotechnology (Santa Cruz,
J=7.5Hz, 2H, fluorenyl H-4, H-5), 7.58 (d, J=6.2Hz, 2H, CA, U.S.A.). The membrane was then incubated with goat
fluorenyl H-1, H-8), 7.41–7.30 (m, 9H, fluorenyl H-2, H-3, anti-mouse immunoglobulin G conjugated with horseradish
H-6, H-7, 5×Ph-H), 5.99 (d, J=6.8Hz, 1H, –CH–NH–CO), peroxidase (1:2000; GE Healthcare, U.S.A.) as a secondary
5.32 (d, J=7.05Hz, 1H, –CH–NH–CO), 5.25 (t, J=3.5Hz, antibody at RT for 1h. The immuno-reactive bands were de-
1H, H-12), 4.57–4.48 (m, 1H, H-3), 4.39–4.38 (m, 2H, tected by an enhanced chemiluminescence detection system
–CHCH2OCONH–), 4.22–4.20 (m, 1H, fluorenyl H-9), 3.60 (s, (GE Healthcare).
3H, –OCH3), 2.22 (d, J=11.2Hz, 1H, H-18), 1.08, 0.95, 0.93,
Real-Time Quantitative PCR RD cells were treated as
0.87, 0.75, 0.67, 0.48 (s, each 3H); 13C-NMR (125MHz, CDCl3) for Western blot analysis. Cellular RNA was extracted from
δ: 178.04, 143.84, 128.85, 127.71, 127.21, 127.09, 119.99, 83.00, cells using TRIzol reagent (Life Technologies, Grand Island,
55.26, 52.91, 41.44, 48.13, 42.03, 39.08, 38.91, 37.89, 37.75, NY, U.S.A.). RNA was reverse-transcribed into cDNA using
36.85, 36.67, 32.89, 30.68, 28.04, 24.25, 23.69, 23.60, 23.33, a reverse transcription kit (Promega, Sunnyvale, CA, U.S.A.)
21.19, 18.05, 17.07, 16.92, 15.44; IR νmax (KBr, cm−1): 3348, following the manufacturer’s instructions. Quantification of
2931, 1722, 1564, 1452; ESI-MS: m/z 826.31 [M−H]−. Anal. mRNA expression was performed using an Applied Biosys-
Calcd for C54H67NO6: C, 78.51; H, 8.17; N, 1.70. Found: C, tems 7500 Fast Real-Time PCR System (Applied Biosystems,
78.38; H, 8.05; N, 1.58.
U.S.A.). Thermal cycling was performed at 95°C for 10min,
Methyl 3β-O-Phenylglycylurs-12-ene-28β-oate (4b): Com- followed by 40 cycles of 95°C for 10s and 60°C for 1min.
pound 4b was obtained as a colorless solid (78mg, 53.8%) Specific primers for the EV71 vp1 gene were 5′-AGTATG
from 4a (200mg, 0.24mmol). The procedure was the same as ATTGAGACTCGGTG-3′ (forward) and 5′-GCGACAAAA
that used for synthesis of compound 3b from compound 3a, GTGAACTCTGC-3′ (reverse). β-Actin was used to normal-
1
mp 54.0–55.0°C; H-NMR (500MHz, CDCl3) δ: 7.40–7.25 (m, ize levels of gene expression. Primers for the β-actin gene
5H, 5×Ph-H), 5.25 (t, J=3.5Hz, 1H, H-12), 4.6 (d, J=13.5Hz, were 5′-ACCCACACTGTGCCCATCTACGA-3′ (forward)
1H, H2NCHOCO), 4.45–4.52 (m, 1H, H-3), 3.60 (s, 3H, and 5′-GCCGTGGTGGTGAAGCTGTAGCC-3′ (reverse).
–OCH3), 2.24 (d, J=11.2Hz, 1H, H-18), 1.08, 0.95, 0.93, 0.87,
0.75, 0.67, 0.48 (s, each 3H); 13C-NMR (125MHz, CDCl3) δ:
176.07, 138.20, 128.64, 127.94, 126.94, 126.68, 125.44, 81.98,
Each reaction was repeated three times. Expression levels of
mRNAs were analyzed by 2−ΔΔCt
Cytotoxicity Analysis RD cells were seeded into 96-well
.
59.11, 55.21, 52.88, 51.44, 48.09, 47.44, 41.99, 39.48, 39.04, plates at a concentration of 5000 cells per well and incubated
38.87, 38.22, 37.85, 36.81, 36.64, 32.85, 30.65, 29.71, 28.00, for 24h at 37°C with 5% CO2. The incubation medium was
27.47, 24.22, 23.65, 23.57, 23.30, 21.19, 18.03, 17.06, 16.89, then replaced with fresh medium and a 2-fold serial dilution
16.41, 15.42; IR νmax (KBr, cm−1): 3447, 2937, 1731, 1581, 1436; of test compound was added to each well. After incubation for
ESI-MS: m/z 635.51 [M−H]−. Anal. Calcd for C39H57NO4: C, 48h, the culture medium was replaced with 100µL DMEM
77.58; H, 9.51; N, 2.32. Found: C, 77.43; H, 9.42; N, 2.23.
medium containing 20µL MTS/PMS (Promega). After incuba-
Biological Activity. Cells and Viruses Human rhabdo- tion at 37°C for 3h, the absorbance was measured at 490nm
myosarcoma cells (RD cells, ATCC accession no. CCL-136) using a microplate reader. Survival rates of treated cells were
were maintained in Dulbecco’s modified Eagle’s medium determined with the following equation: cell viability=(OD490
(DMEM) supplemented with 10% fetal bovine serum (FBS), of treated cells−OD490 of blank)/(OD490 of control cells−OD490
100U/mL penicillin and 0.1mg/mL streptomycin at 37°C with of blank). Data are expressed as means S.D. of triplicate de-
5% CO2. RD cells were used in propagation of enterovirus 71 terminations.
(BrCr-Ts strain GenBank: AB 204853.1).
Western Blot Analysis RD cells were inoculated into
Acknowledgments This work was jointly supported
12-well plates containing complete medium at a concentration by the National Natural Science Foundation of China (No.
of 2×105 cells per well and cultured for 24h. The pentacyclic 21102067, No. 81071248, No. 81371676). The authors thank Dr.
triterpenes and their derivatives were added into each well Huaiyi Yang, Key Laboratory of Pathogenic Microbiology and
at a final concentration of 0.01µg·µL−1, respectively. Wells Immunology, Institute of Microbiology, Chinese Academy of
containing cells and control wells received equal amounts of Sciences for his help in the experiments.
DMSO. Six hours later, the RD cells were infected with 10µL
of EV71 (tissue culture infective dose (TCID50)=1×109), fol-
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