Synthesis and biological evaluation of analogs of methyl ursolate 3-o-chacotrioside as H5N1 viral entry inhibitors

Article abstract of DOI:10.1080/07328303.2012.687060

The earliest stage of influenza virus infection is the viral entry to the host cell. In our previous work, we discovered the first three small molecule H5N1 viral entry inhibitors 1-3. Here, based on saponin 3, methyl ursolate 3-O-chacotrioside, several analogs were synthesized and evaluated to understand the structure-activity relationships of this type of compound on the H5N1 viral entry inhibitory activity. The preliminary studies demonstrated that unlike saponins 1 and 2, it is possible to reduce the 3-O-chacotriosyl residue of compound 3 to a disaccharide without affecting the viral entry activities significantly. The results obtained will render new clues to the understanding of the antiviral profile for these types of compounds.

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Synthesis and Biological Evaluation
of Analogs of Methyl Ursolate 3-O-β-
Chacotrioside as H5N1 Viral Entry
Inhibitors
Sumei Ren a , Qing Chen b , Ning Ding a , Wei Zhang a , Yingxia Li a
Ying Guo b
&
a Department of Medicinal Chemistry, School of Pharmacy , Fudan
University , Shanghai , 201203 , China
b Department of Pharmacology, Institute of Materia Medica , Chinese
Academy of Medical Sciences and Peking Union Medical College ,
Beijing , 100050 , China
Published online: 31 Oct 2012.
To cite this article: Sumei Ren , Qing Chen , Ning Ding , Wei Zhang , Yingxia Li & Ying Guo
(2012) Synthesis and Biological Evaluation of Analogs of Methyl Ursolate 3-O-β-Chacotrioside
as H5N1 Viral Entry Inhibitors, Journal of Carbohydrate Chemistry, 31:8, 647-658, DOI:
10.1080/07328303.2012.687060
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Journal of Carbohydrate Chemistry, 31:647–658, 2012
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2012.687060
Synthesis and Biological
Evaluation of Analogs of
Methyl Ursolate
3-O-β-Chacotrioside as H5N1
Viral Entry Inhibitors
Sumei Ren,1 Qing Chen,2 Ning Ding,1 Wei Zhang,1 Yingxia Li,1
and Ying Guo2
1Department of Medicinal Chemistry, School of Pharmacy, Fudan University,
Shanghai 201203, China
2Department of Pharmacology, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China
The earliest stage of influenza virus infection is the viral entry to the host cell. In our
previous work, we discovered the first three small molecule H5N1 viral entry inhibitors
1–3. Here, based on saponin 3, methyl ursolate 3-O-β-chacotrioside, several analogs
were synthesized and evaluated to understand the structure-activity relationships of
this type of compound on the H5N1 viral entry inhibitory activity. The preliminary
studies demonstrated that unlike saponins 1 and 2, it is possible to reduce the 3-O-
chacotriosyl residue of compound 3 to a disaccharide without affecting the viral entry
activities significantly. The results obtained will render new clues to the understanding
of the antiviral profile for these types of compounds.
Keywords: Influenza; Entry inhibitors; H5N1; Structure-activity relationship
INTRODUCTION
Avian H5N1 influenza viruses pose a pandemic threat with continuously occur-
ring widespread infections of avian species, as well as sporadic human cases
with a mortality rate of approximately 60%.[1] The World Health Organization
Received March 19, 2012; accepted April 17, 2012.
Address correspondence to Ying Guo, Department of Pharmacology, Institute of Ma-
teria Medica, Chinese Academy of Medical Sciences and Peking Union Medical Col-
lege, Beijing 100050, China. E-mail: yingguo6@yahoo.com; or Yingxia Li, Department of
Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China.
E-mail: liyx417@fudan.edu.cn
647

S. Ren et al.
648
(WHO) and many individual nations have made tremendous efforts to fully un-
derstand the pandemic of the virus and develop effective therapies to control
the spread of the virus. However, only two kinds of agents, the M2 ion channel
blockers and the neuraminidase inhibitors, have been used to treat influenza.
But concern has been raised by the isolation of viable mutant viruses that
are resistant to them.[2–5] Therefore, attempts should be made to develop new
strategies directed at unexplored targets such as the viral proteins hemagglu-
tinin (HA), the viral polymerase (and endonuclease), and the nonstructural
protein NS1.[6] To address the need for new antivirals against influenza, a
number of studies have been done to inhibit the interaction of HA with the
receptor.[7–10]
By screening a saponin library generated from semisynthesis with an
efficient HIV-based pseudotyping system, followed by preliminary structure-
activity relationship study, our group had discovered the first two classes of
saponins (compounds 1, 2, and 3, Fig. 1) as small molecule H5N1 viral entry
inhibitors.[11] Interestingly, these two classes of saponins differ not only in the
aglycone structure but also in the mechanism of action. The class of compounds
1 and 2 containing β-chacotrioside linked to the aglycone of chlorogenin is pos-
sible by the inhibitors of receptor binding disrupting the interaction of HA with
sialic acid sugars on the host cells. In contrast, compound 3, with a methyl ur-
solate aglycone, might be possible through interrupting the membrane fusion
of virus with the host cells (data not shown).
To further understand the mechanism of actions of these two classes of
saponins on the H5N1 viral entry and in order to search for potentially new
antiviral agents with high development potentials, extensive structure-activity
relationship (SAR) studies around compounds 1, 2, and 3 are highly demanded.
As part of this continuous work we would like to report here the design, syn-
thesis, and biological evaluations of several analogs of compound 3. Here, we
kept the aglycone of compound 3 intact and focused on the sugar chain. Cut-
ting down one or two sugar units from the chacotrioside resulted in saponins 4,
5, and 6, respectively. The aglycone, compound 7, was also synthesized (Fig. 2).
Figure 1: Three saponin inhibitors for H5N1 viral entry.

H5N1 Viral Entry Inhibitors
649
Figure 2: Saponins designed for SAR studies on the sugar chain.
RESULTS AND DISCUSSION
The synthetic route toward target compounds 4, 6, and 7 is depicted in
Scheme 1. Starting from ursolic acid 8, compound 7 was generated by protect-
ing the 28-CO2H with a methyl group. The free hydroxyl group of 7 was then
glycosylated with 2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl trichloroacetim-
idate 9[12] under the promotion of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) to afford 10. Removal of benzoyl groups in 10 furnished target
saponin 6.[11] Treatment of 3β-O-(D-glucopyranosyl)-ursolic-28-methyl ester
6 with benzyaldehyde dimethyl acetal catalyzed by camphor sulfonic acid
(CSA) at 60◦C in acetonitrile gave the 4,6-O-benzylidene-protected glycoside
11. The next step was to introduce an L-rhamnopyranosyl residue to the 2-
OH of 11. As we know, fully differentiating 2,3-diols of D-glucopyranoside is
still a challenge and usually needs extensive protecting group manipulations.
To rapidly access our designed target compound 4, we employed 1.0 equiv. of
2,3,4-tri-O-acetyl-L-rhamnopyranosyl trichloroacetimidate 12[13] as donor for
glycosylation with equal equivalents of acceptor 11, affording the disaccharide
glycosides 13 (37%) and 14 (63%) simultaneously. These two compounds can
be easily separated by silica gel column chromatography. Removal of the ben-
zylidene group in 13 by hydrogenolysis in the presence of Pd/C, followed by
removal of the benzoyl groups with MeONa in CH3OH/CH2Cl2, afforded tar-
get saponin 4.
The synthesis of compound 5 is shown in Scheme 2. The known trichloroac-
etamidate 12 and thioglycoside 15[11] were coupled under the promotion of
TMSOTf to afford disaccharide 16,[10] which was coupled with acceptor 7 pro-
moted by N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH)
in CH2Cl2 to give the intermediate 17. Final removal of the benzoyl groups
with NaOMe in CH3OH/CH2Cl2 obtained the target saponin 5.

S. Ren et al.
650
Scheme 1: Synthesis of compounds 4, 6, and 7.
Using the HIV-based particles pseudotyped with a highly pathogenic H5N1
HA model (A/Viet Nam/1203/2004), the influences of compounds 4–7 on HA-
mediated viral entry were evaluated, while VSVG/HIV was applied to evalu-
ate the specificity of H5N1 inhibitors. The results are summarized in Table 1.
Compounds 4 and 5, each bearing a disaccharide sugar chain, showed good
inhibitory activity. The position of the terminal L-rhamnosyl residue on the in-
terglucose has no important effect on the activity (IC50, 22.6 vs. 22.1 μM)). This
result is very interesting because during the SAR studies around compounds
1 and 2 we found that removing any of the two L-rhamnosyl residues from

H5N1 Viral Entry Inhibitors
651
Scheme 2: Synthesis of compound 5.
the trisaccharide chain led to total inactivation.[11] The finding is also attrac-
tive since analogs with reduced sugar chains are easier to access and modify.
However, we failed to further reduce the sugar chain from compounds 4 and
5, since compound 6 with a monosaccharide residue displayed strong cytotoxi-
city under the testing concentrations. Not surprisingly, the aglycon itself (i.e.,
methyl ursolate 7) did not show viral entry inhibition at the concentration of
50 μM.
Table 1: Effect of compounds on HA mediated viral entry.
HA(Viet)a/HIV
IC50 (µM)
VSVGb/HIV
Conc. (30 µM)
Compounds
3
4
5
6
7
16.6
22.6
99.6%c
106.1%
22.1
91.1%
d
d
>50
103%
aHA(Viet), HA, H5N1 (A/Viet Nam/1203/2004).
bVSVG, vesicular stomatitis virus glycoprotein.
cThe percentages in the table presented the infectivity compared to the same amount solvent
as 100%.
dCytotoxicity was observed at the final concentration of 10 µM.

S. Ren et al.
652
CONCLUSION
In this work, based on our previously discovered small molecule inhibitor
saponin 3, several analogs were synthesized and evaluated as inhibitors of
H5N1 viral entry. The results demonstrated that unlike the class of saponins
1 and 2, it’s possible to reduce the 3-O-chacotriosyl residue of compound 3 to a
disaccharide without affecting the viral entry activities significantly. This work
offered more lead compounds for the following studies on the action mecha-
nisms and will render new clues to the understanding of the antiviral profile
for these types of compounds.
EXPERIMENTAL PROTOCOLS
General Methods
All chemical reagents were used as supplied unless indicated. Solvents
used in organic reactions were distilled under an inert atmosphere. Unless
otherwise noted, all reactions were carried out at rt and were performed un-
˚
der a positive pressure of argon. Crushed 4 A molecular sieves were acti-
vated by thorough flame-drying and cooled in vacuo prior to use. Flash col-
umn chromatography was performed on silica gel (200–300 mesh, Qingdao,
China). Amberlite 15 (H+ form) was used where acidic ion-exchange resin
is indicated. Analytical thin layer chromatography (TLC) was performed on
glass plates precoated with a 0.25-mm thickness of silica gel. 1H NMR and 13
C
NMR spectra were taken on a Jeol JNM-ECP 600 or a Bruker Avance III 400
1
spectrometer at rt. Chemical shifts of the H NMR spectra are expressed in
ppm relative to the solvent residual signal 7.26 in CDCl3 or to tetramethyl-
silane (δ = 0.00). Chemical shifts of the 13C NMR spectra are expressed in
ppm relative to the solvent signal 77.00 in CDCl3 or to tetramethylsilane (δ =
0.00) unless otherwise noted. COSY, HMQC, and HMBC were routinely used
1
to definitively assign the signals of H NMR and 13C NMR spectra. Electro-
spray ionization (ESI) mass spectra were recorded on a Global Q-TOF mass
spectrometer.
Synthesis of 3β-O-(4,6-O-benzylidene-β-D-Glucopyranosyl)-
Ursolic-28-Methyl Ester (11)
Compound 6 (438.9 mg, 0.69 mmol) was dissolved in dried CH3CN
(50 mL) containing PhCH(OCH3)2 (0.208 mL, 1.39 mmol) and CSA (29.0 mg,
0.13 mmol). The reaction mixture was stirred for 2 h at rt, then neutralized
with Et3N and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (CH2Cl2/CH3OH gradient elution) to
give crude 11 as a white solid (323.0 mg, 65%): Rf = 0.27 (1:6, EtOAc/petroleum

H5N1 Viral Entry Inhibitors
653
ether); 1H NMR (CDCl3, 400 MHz): δ 7.51–7.48 (m, 2H), 7.39–7.27 (m, 3H), 5.53
(s, 1H), 5.25 (s, 1H), 4.47 (d, 1H, J = 7.6 Hz), 4.31 (dd, 1H, J = 10.4, 4.9 Hz),
3.85–3.77 (m, 2H), 3.61 (s, 3H), 3.57–3.54 (m, 2H), 3.48–3.44 (m, 1H), 3.21 (dd,
1H, J = 11.6, 4.6 Hz), 2.81 (s, 1H), 2.51 (s, 1H), 2.23 (d, 1H, J = 11.3 Hz),
1.07, 1.03, 0.70 (each s, each 3H); 13C NMR (CDCl3, 100 MHz): δ 178.1, 140.0,
138.1, 129.3, 128.3, 126.3, 125.5, 105.2, 101.9, 90.0, 80.5, 75.2, 73.1, 68.8, 66.3,
55.5, 52.8, 51.5, 48.1, 47.6, 42.0, 39.5, 39.2, 38.8, 38.6, 36.7, 36.6, 32.9, 30.6,
29.7, 28.3, 28.0, 25.9, 24.2, 23.6, 23.3, 21.2, 18.1, 17.0, 16.9, 16.7, 15.4; MALDI-
HRMS calcd for C44H64NaO8 [M+Na]+ 743.4493, found 743.4622.
Synthesis of 3β-O-[2-O-(2,3,4-Tri-O-benzoyl-α-L-rhamnopyran-
osyl)-4,6-O- benzylidene-β-D-glucopyranosyl]-ursolic-28-
methyl ester (13) and 3β-O-[4-O- (2,3,4-tri-O-benzoyl-α-L-
rhamnopyranosyl)-4,6-O-benzylidene-β-D-Glucopyranosyl]
-Ursolic-28-Methyl Ester (14)
A mixture of 11 (100.0 mg, 0.14 mmol), 12 (103.0 mg, 0.17 mmol), and
˚
powdered 4 A molecular sieves in dried CH2Cl2 (15 mL) was stirred at rt
for 30 min and then cooled to 0◦C. TMSOTf (4.8 μL, 27.6 μmol) was added
slowly. The reaction was stirred for 1 h at 0◦C. After completion of the re-
action (monitored by TLC), the reaction was quenched with Et3N, and then
filtered. The filtrate was concentrated and purified by silica gel column chro-
matography (EtOAc/petroleum ether, 1:10–1:7) to give 13 (61.0 mg, 37%) and
1
14 (104.0 mg, 63%). Compound 13: white solid; Rf = 0.24 (CH2Cl2); H NMR
(CDCl3, 400 MHz) for 13: δ 8.09 (d, 2H, J = 7.3 Hz), 7.97 (d, 2H, J = 7.3, Hz),
7.82 (d, 2H, J = 7.3 Hz), 7.60–7.21 (m, 14H), 5.90 (dd, 1H, J = 10.1, 3.1 Hz),
5.83 (s, 1H),, 5.77 (s, 1H), 5.69 (t, 1H, J = 10.1 Hz), 5.54 (s, 1H), 4.67 (d, 1H, J
= 7.3 Hz), 4.58–4.55 (m, 1H), 4.34 (dd, 1H, J = 4.6 Hz), 4.08 (t, 1H, J = 8.0 Hz),
3.85 (t, 1H, J = 8.2, 7.9 Hz), 3.78 (t, 1H, J = 10.1 Hz), 3.61 (s, 3H), 3.56 (t, 1H,
J = 9.5, 9.2 Hz), 3.49–3.42 (m, 1H), 3.26 (dd, 1H), 3.12 (brs, 1H), 2.24 (d, 1H, J
= 11.0 Hz), 1.09, 0.75 (each s, each 3H); 13C NMR (CDCl3, 100 MHz): δ 178.1,
165.7, 165.6, 165.6, 138.2, 137.0, 133.4, 133.3, 133.0, 129.9, 129.7, 129.5, 129.4,
129.3, 129.2, 128.5, 128.4, 128.2, 126.2, 125.6, 114.1, 104.6, 101.8, 97.5, 89.9,
80.7, 77.7, 77.2, 75.0, 71.8, 70.6, 69.8, 68.8, 67.1, 65.7, 55.9, 52.9, 51.5, 48.1,
47.6, 42.0, 39.5, 39.2, 39.0, 39.0, 38.9, 36.7, 36.6, 33.0, 31.9, 30.6, 29.7, 29.4,
28.0, 26.3, 24.2, 23.6, 23.3, 22.7, 21.2, 18.1, 17.5, 17.0, 16.9, 16.6, 15.5, 14.1;
MALDI-HRMS calcd for C71H86NaO15 [M+Na]+ 1201.5859, found 1201.6154.
Compound 14: white solid; Rf = 0.17 (CH2Cl2); 1H NMR (CDCl3, 400 MHz) for
14: δ 8.08 (d, 2H, J = 8.3 Hz), 7.82 (d, 4H, J = 6.6 Hz), 7.60 (t, 1H, J = 7.0 Hz),
7.54–7.41 (m, 6H), 7.35 (t, 2H, J = 7.9, 7.6 Hz), 7.28–7.21 (m, 5H), 5.83 (dd,
1H, J = 10.1, 3.7 Hz), 5.73 (s, 1H), 5.64 (s, 1H), 5.58 (t, 1H, J = 10.1 Hz), 5.53
(s, 1H), 5.25 (s, 1H), 4.53 (dd, 1H, J = 9.9, 6.1 Hz), 4.47 (d, 1H, J = 7.6 Hz),

S. Ren et al.
654
4.36 (dd, 1H, J = 10.7, 5.2 Hz), 4.01 (t, 1H, J = 9.5, 8.9 Hz), 3.86 (t, 1H, J
= 10.4, 10.1 Hz), 3.79–3.71 (m, 2H), 3.61 (s, 3H), 3.54–3.46 (m, 1H), 3.21 (dd,
1H, J = 11.3, 4.6 Hz), 2.60 (d, 1H, J = 3.1 Hz), 2.23 (d, 1H, J = 11.6 Hz),
1.07, 0.74 (each s, each 3H); 13C NMR (CDCl3, 100 MHz): δ 178.0, 165.6, 138.1,
137.2, 133.4, 133.2, 133.0, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.5,
128.3, 128.2, 128.1, 126.1, 125.5, 105.6, 101.7, 97.6, 90.2, 79.0, 77.2, 76.4, 76.2,
71.8, 70.8, 70.1, 68.9, 66.8, 66.2, 60.4, 55.5, 52.9, 51.5, 48.1, 47.6, 42.0, 39.5,
39.0, 38.8, 38.6, 36.7, 36.6, 32.9, 30.6, 29.7, 28.3, 28.0, 25.9, 24.2, 23.6, 23.3,
21.2, 18.1, 17.0, 16.9, 16.7, 15.4, 14.2; MALDI-HRMS calcd for C71H86NaO15
[M+Na]+ 1201.5859, found 1201.6154.
Synthesis of 3β-O-[2-O-(α-L-rhamnopyranosyl)-β-D-glucopy-
ranosyl]-Ursolic- 28-Methyl Ester (4)
A mixture of 13 (81.0 mg, 0.068 mmol) and 10% Pd–C (30.0 mg) in
CH3OH/CH2Cl2 (15 mL, v:v = 1:1) was stirred under 1 atm of H2 for 5 h. The
reaction mixture was then filtered, and the filtrate was concentrated to dry-
ness to give a white solid. The solid was dissolved in CH3OH/CH2Cl2 (8 mL,
v:v = 1:1), and then NaOMe was added until pH = 9. After stirring at 35◦C
for 8 h, the solution was neutralized with ion-exchange resin (H+) and then
filtered and concentrated. The residue was purified by silica gel column chro-
matography (CH2Cl2/MeOH, 10:1) to afford 4 as a white solid (33.4 mg, 62%):
1
Rf = 0.13 (CH2Cl2/MeOH, 4:1); H NMR (DMSO-d6, 400 MHz): δ 5.23 (s, 1H),
5.12 (brs, 1H), 4.96 (brs, 1H), 4.62 (brs, 1H), 4.55 (s, 1H), 4.45 (brs, 1H), 4.40
(brs, 1H), 4.23 (d, 1H, J = 7.4 Hz), 3.82–3.78 (m, 1H), 3.66 (s, 1H), 3.61 (d,
1H, J = 11.7 Hz), 3.49 (s, 3H), 3.46–3.44 (m, 1H), 3.28–3.16 (m, 4H), 3.04–3.01
(m, 3H), 2.12 (d, 1H, J = 8.6 Hz), 1.03 (d, 3H, J = 9.0 Hz), 1.02, 0.92, 0.89,
0.84, 0.72, 0.64 (each s, each 3H), 0.79 (d, 3H, J = 5.9 Hz); 13C NMR (DMSO-
d6, 100 MHz): δ 177.4, 138.4, 125.3, 104.3, 100.2, 88.3, 78.6, 77.0, 76.7, 72.5,
70.9, 70.8, 70.8, 68.4, 61.5, 55.7, 52.9, 51.8, 47.8, 47.4, 42.0, 39.0, 38.9, 38.7,
36.6, 33.0, 31.7, 30.4, 29.4, 29.1, 27.9, 27.8, 26.2, 24.2, 23.7, 23.3, 22.5, 21.4,
18.3, 18.1, 17.4, 17.0, 16.7, 15.7, 14.4; MALDI-HRMS calcd for C43H70NaO12
[M+Na]+ 801.4759, found 801.4999.
Synthesis of 2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→4)-2,
3,6-tri-O-benzoyl- 1-(4-tolyl) thio-β-D-glucopyranoside (16)
A mixture of 12 (700.0 mg, 1.17 mmol), 15 (871.0 mg, 1.40 mmol), and
˚
powdered 4 A molecular sieves in dried CH2Cl2 (50 mL) was stirred at rt for
30 min and then cooled to 0◦C. TMSOTf (20 μL, 117 μmol) was added slowly.
The reaction was stirred for 1 h at 0◦C. After completion of the reaction (mon-
itored by TLC), the reaction was quenched with Et3N and then filtered. The
filtrate was concentrated and purified by silica gel column chromatography

H5N1 Viral Entry Inhibitors
655
(EtOAc/petroleum ether, 1:10) to give crude 16 as a white solid (1.1 g, 90%): Rf
= 0.54 (1:3, EtOAc/petroleum ether); 1H NMR (CDCl3, 400 MHz): δ 8.10 (d, 2H,
J = 6.8 Hz), 7.94 (d, 6H, J = 7.2 Hz), 7.86–7.81 (m, 4H), 7.61–7.33 (m, 17H),
7.27–7.24 (m, 3H), 6.89 (d, 2H, J = 7.6 Hz), 5.85 (t, 1H, J = 9.6, 9.2 Hz), 5.66
(dd, 1H, J = 10.4, 3.2 Hz), 5.55–5.53 (m, 1H), 5.49 (t, 1H, J = 10.0 Hz), 5.20
(d, 1H, J = 1.2 Hz), 5.14 (dd, 1H, J = 12.4, 1.6 Hz), 4.93 (d, 1H, J = 10.0 Hz),
4.65 (dd, 1H, J = 12.4, 4.0 Hz), 4.21 (t, 1H, J = 9.6, 9.2 Hz), 4.07 (dd, 1H,
J = 9.6, 2.4 Hz), 3.97–3.93 (m, 1H), 2.24 (s, 3H), 0.74 (d, 3H, J = 6.0 Hz); 13
C
NMR (CDCl3, 125 MHz): δ 165.9, 165.6, 165.5, 165.1, 139.4, 134.9, 134.7, 134.6,
134.5, 134.3, 133.4, 133.2, 133.1, 133.0, 129.9, 129.8, 129.7, 129.5, 129.4, 128.4,
128.3, 128.2, 98.8, 85.7, 75.1, 71.3, 71.1, 70.9, 69.5, 67.8, 62.5,60.3, 21.1, 21.0,
17.1, 14.2; ESIMS: calcd for [M+Na]+ m/z 1079.3; found: 1079.1.
Synthesis of 3β-O-[4-O-(2,3,4-Tri-O-benzoyl-α-L-rhamnopyran-
osyl)-2,3,6-tri-O- benzoyl-β-D-glucopyranosyl]-Ursolic-28-
Methyl Ester (17)
A mixture of 16 (300.0 mg, 0.28 mmol), 7 (111.0 mg, 0.24 mmol), and
˚
powdered 4 A molecular sieves in dried CH2Cl2 (25 mL) was stirred at rt for
30 min and then cooled to 0◦C. NIS (80.0 mg, 0.35 mmol) and TfOH (8.4 μL,
94 μmol) were added. The reaction was stirred for 2 h. After completion of
the reaction (monitored by TLC), the reaction mixture was filtered and con-
centrated. Then the residue was diluted with CH2Cl2 (100 mL) and washed
with aqueous Na2S2O3 (50 mL), satd aq NaHCO3 (50 mL), and brine (2 ×
50 mL). The organic layer was dried over Na2SO4 and concentrated under re-
duced pressure. The residue was purified by silica gel column chromatography
(EtOAc/petroleum ether, 1:10) to give crude 17 as a white amorphous solid
1
(150.0 mg, 45%): Rf = 0.27 (1:6, EtOAc/petroleum ether); H NMR (CDCl3,
400 MHz): δ 8.10 (d, 2H, J = 7.2 Hz), 7.97 (t, 4H, J = 7.2, 6.8 Hz), 7.91 (d,
2H, J = 6.8 Hz), 7.89 (d, 2H, J = 6.8 Hz), 7.83 (d, 2H, J = 7.2 Hz), 7.57–7.33
(m, 14H), 7.30–7.24 (m, 4H), 5.85 (t, 1H, J = 9.6, 9.2 Hz), 5.69 (dd, 1H, J =
10.0, 3.2 Hz), 5.57–5.55 (m, 1H), 5.51 (t, 1H, J = 10.0 Hz), 5.44 (t, 1H, J = 8.0,
7.6 Hz), 5.25 (t, 1H, J = 3.6, 3.2 Hz), 5.20 (d, 1H, J = 1.2 Hz), 4.97 (dd, 1H, J =
12.0, 2.0 Hz), 4.80 (d, 1H, J = 7.6 Hz), 4.73 (dd, 1H, J = 12.0, 6.0 Hz), 4.23 (t,
1H, J = 9.6, 9.2 Hz), 4.06–4.02 (m, 1H), 3.98 (dd, 1H, J = 9.6, 6.0 Hz), 3.59 (s,
3H), 3.05 (dd, 1H, J = 11.6, 4.4Hz), 2.23 (d, 1H, J = 11.2 Hz), 1.04, 0.97, 0.80,
0.57 (each s, each 3H), 0.76 (d, 3H, J = 6.0 Hz), 0.68 (s, 6H); 13C NMR (CDCl3,
100 MHz): δ 138.0, 133.5, 133.3, 133.2, 133.1, 133.0, 130.0, 129.9, 129.7, 129.4,
129.3, 129.2, 129.1, 128.5, 128.4, 128.3, 128.3, 125.6, 103.1, 98.7, 90.3, 73.9,
73.3, 72.6, 71.3, 71.0, 69.5, 67.7, 62.7, 55.4, 53.4, 52.9, 51.5, 48.1, 47.5, 41.9,
39.4, 39.1, 38.9, 38.7, 38.4, 36.6, 36.5, 32.9, 31.9, 30.7, 29.7, 29.7, 29.4, 28.0,

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27.6, 25.7, 24.2, 23.6, 23.2, 22.7, 21.2, 18.0, 17.0, 16.8, 16.1, 15.3, 14.1; ESIMS:
calcd for [M+K]+ m/z 1441.6; found: 1442.1.
Synthesis of 3β-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyrano-
syl]-Ursolic-28-Methyl Ester (5)
Compound 17 (105.0 mg, 0.075 mmol) was dissolved in CH3OH/CH2Cl2
(10 mL, v:v = 1:1), and then NaOMe was added until pH = 9. After stirring
at 35◦C for 8 h, the solution was neutralized with ion-exchange resin (H+)
and then filtered and concentrated. The residue was purified by silica gel col-
umn chromatography (CH2Cl2/MeOH, 10:1–4:1) to afford 5 as a white solid
(53.0 mg, 91%): Rf = 0.29 (CH2Cl2/MeOH, 4:1); 1H NMR (CD3OD, 400 MHz): δ
5.23 (t, 1H, J = 3.5, 3.1 Hz), 4.85 (d, 1H, J = 1.6 Hz), 4.33 (d, 1H, J = 7.8 Hz),
3.98–3.94 (m, 1H), 3.83–3.77 (m, 2H), 3.67–3.60 (m, 2H), 3.59 (s, 3H), 3.52 (t,
1H, J = 9.4, 9.0 Hz), 3.45–3.37 (m, 2H), 3.22 (t, 1H, J = 8.6, 8.2 Hz), 3.17 (dd,
1H, J = 11.7, 9.3 Hz), 2.22 (d, 1H, J = 11.3 Hz), 1.26 (d, 3H, J = 5.9 Hz), 1.11,
1.05, 0.84, 0.79 (each s, each 3H), 0.96 (s, 6H), 0.88 (d, 3H, J = 6.3 Hz); 13C
NMR (CD3OD, 100 MHz): δ 177.1, 138.1, 128.5, 105.4, 100.7, 88.2, 75.5, 74.4,
72.1, 70.9, 70.8, 68.8, 60.4, 55.1, 52.6, 51.5, 47.6, 47.1, 41.7, 38.9, 38.6, 38.5,
36.4, 32.7, 30.1, 29.2, 28.9, 27.8, 25.7, 23.9, 23.5, 23.0, 21.2, 17.9, 17.2, 16.8,
16.7, 15.4; MALDI-HRMS calcd for C43H70NaO12 [M+Na]+ 801.4759, found
801.5149.
Plasmids and Cell Lines
A codon-optimized HA gene is from A/Viet Nam/1203/2004 (H5N1) and
was cloned into pcDNA3. NA A/PR/8/34 influenza virus (H1N1) in the vector
pEF6/V5-His-TOPO was kindly provided by John C. Olsen (University of North
Carolina, Chapel Hill).
A549 and 293T cells were grown in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and
100 μg/mL streptomycin and 100 units/mL penicillin (GIBCO) at 37◦C, 5%
CO2.
Production of HA/HIV Pseudovirions
Human embryonic kidney 293T cells were transiently cotransfected with
8 μg hemagglutinin envelope expression plasmid with 5 μg NA and 10 μg
Env-deficient HIV vector (pNL4–3-Luc-R−E−) in 100-mm plates by a standard
Ca3(PO4)2 protocol. Sixteen hours posttransfection, cells were washed by PBS
without Ca2+ and Mg2+, and then 10 mL fresh medium was added into each
plate. Forty-eight hours posttransfection, the supernatants were collected and

H5N1 Viral Entry Inhibitors
657
filtered through a 0.45-micron pore size filter (Nalgene) and the pseudovirions
were directly used for infection.
Infection Assay of HA/HIV Pseudovirions
To investigate the effect of the test compounds, A549 cells were seeded
into 24-well plates at 50,000 cells per well and tested compounds were added
to each well incubating for 15 min followed by adding HA/HIV or VSVG/HIV
pseudovirions to each well and incubating at 37◦C. Cells were lysed in 50 μL of
cell culture lysis reagent (Promega) 48 h postinfection. The luciferase activity
was measured with a luciferase assay kit (Promega) and an FB15 luminometer
(Berthold detection system) according to the supplier’s protocols.
ACKNOWLEDGMENTS
This work was supported by National Natural Science Foundation of China
(81072525 and 30701035), the central level, scientific research institutes for
basic R & D special fund business (2010ZD03 and 2011ZD06) and Shanghai
outstanding academic leaders program (11XD1400200). Sumei Ren and Qing
Chen contributed equally to this work.
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