Design, synthesis and biological evaluation of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.01.005

Since the influenza viruses can rapidly evolve, it is urgently required to develop novel anti-influenza agents possessing a novel mechanism of action. In our previous study, two pentacyclic triterpene derivatives (Q8 and Y3) have been found to have anti-influenza virus entry activities. Keeping the potential synergy of biological activity of pentacyclic triterpenes and l-ascorbic acid in mind, we synthesized a series of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives (18-26, 29-31, 35-40 and 42-43). Moreover, we evaluated these novel compounds for their anti-influenza activities against A/WSN/33 virus in MDCK cells. Among all evaluated compounds, the 2,3-O,O-dibenzyl-6-deoxy-l-ascorbic acid-betulinic acid conjugate (30) showed the most significant anti-influenza activity with an EC₅₀ of 8.7 μM, and no cytotoxic effects on MDCK cells were observed. Time-of-addition assay indicated that compound 30 acted at an early stage of the influenza life cycle. Further analyses revealed that influenza virus-induced hemagglutination of chicken red blood cells was inhibited by treatment of compound 30, and the interaction between the influenza hemagglutinin (HA) and compound 30 was determined by surface plasmon resonance (SPR) with a dissociation constant of K_D = 3.76 μM. Finally, silico docking studies indicated that compound 30 and its derivative 31 were able to occupy the binding pocket of HA for sialic acid receptor. Collectively, these results suggested that l-ascorbic acid-conjugated pentacyclic triterpenes were promising anti-influenza entry inhibitors, and HA protein associated with viral entry was a promising drug target.

Full text of DOI:10.1016/j.ejmech.2016.01.005

Accepted Manuscript
Design, synthesis and biological evaluation of novel L-ascorbic acid-conjugated
pentacyclic triterpene derivatives as potential influenza virus entry inhibitors
Han Wang, Renyang Xu, Yongying Shi, Longlong Si, Pingxuan Jiao, Zibo Fan, Xu
Han, Xingyu Wu, Xiaoshu Zhou, Fei Yu, Yongmin Zhang, Lihe Zhang, Demin Zhou,
Sulong Xiao
PII:
S0223-5234(16)30005-8
10.1016/j.ejmech.2016.01.005
EJMECH 8295
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 November 2015
Revised Date: 2 January 2016
Accepted Date: 6 January 2016
Please cite this article as: H. Wang, R. Xu, Y. Shi, L. Si, P. Jiao, Z. Fan, X. Han, X. Wu, X. Zhou, F. Yu,
Y. Zhang, L. Zhang, D. Zhou, S. Xiao, Design, synthesis and biological evaluation of novel L-ascorbic
acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.005.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel L-ascorbic acid-conjugated pentacyclic
triterpene derivatives as potential influenza virus entry inhibitors
Han Wanga,1, Renyang Xua,1, Yongying Shia, Longlong Sia, Pingxuan Jiaoa, Zibo Fana, Xu Hana,
Xingyu Wub, Xiaoshu Zhoua, Fei Yua,c, Yongmin Zhangd, Lihe Zhanga, Demin Zhoua,*, Sulong
Xiaoa,*
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
bInstitute of Systems Biomedicine, Department of Pathology, School of Basic Medical Sciences,
Beijing Key Laboratory of Tumor Systems Biology, Peking University, Beijing 100191, China
cMedical Faculty of Kunming University of Science and Technology, Kunming 650500, China
dInstitut Parisien de Chimie Moléculaire, CNRS UMR 8232, Université Pierre & Marie Curie-Paris
6, 4 place Jussieu, 75005 Paris, France
* Corresponding author. Tel.: 86-10-8280-5646; fax: 86-10-8280-2724.
E-mail address: deminzhou@bjmu.edu.cn (D. Zhou); slxiao@bjmu.edu.cn (S. Xiao)
1 These authors contributed equally to this work.
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ABSTRACT:
Since the influenza viruses can rapidly evolve, it is urgently required to develop novel
anti-influenza agents possessing a novel mechanism of action. In our previous study, two
pentacyclic triterpene derivatives (Q8 and Y3) have been found to have anti-influenza virus entry
activities. Keeping the potential synergy of biological activity of pentacyclic triterpenes and
L-ascorbic acid in mind, we synthesized a series of novel L-ascorbic acid-conjugated pentacyclic
triterpene derivatives (18-26, 29-31, 35-40 and 42-43). Moreover, we evaluated these novel
compounds for their anti-influenza activities against A/WSN/33 virus in MDCK cells. Among all
evaluated compounds, the 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid-betulinic acid conjugate (30)
showed the most significant anti-influenza activity with an EC50 of 8.7 ꢀM, and no cytotoxic
effects on MDCK cells were observed. Time-of-addition assay indicated that compound 30 acted
at an early stage of the influenza life cycle. Further analyses revealed that influenza virus-induced
hemagglutination of chicken red blood cells was inhibited by treatment of compound 30, and the
interaction between the influenza hemagglutinin (HA) and compound 30 was determined by
surface plasmon resonance (SPR) with a dissociation constant of KD = 3.76 ꢀM. Finally, silico
docking studies indicated that compound 30 and its derivative 31 were able to occupy the binding
pocket of HA for sialic acid receptor. Collectively, these results suggested that L-ascorbic
acid-conjugated pentacyclic triterpenes were promising anti-influenza entry inhibitors, and HA
protein associated with viral entry was a promising drug target.
Keywords: Influenza virus, pentacyclic triterpene, L-ascorbic acid, entry inhibitor, hemagglutinin,
sialic acid receptor
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1. Introduction
Influenza viruses cause substantial morbidity and mortality through routine seasonal spread
and seasonal epidemics. Currently, anti-influenza therapies include universal influenza vaccines
[1], broadly-neutralizing therapeutic antibodies [2] and small-molecule inhibitors [3]. However,
the high mutation rate of the RNA genome of the influenza virus, combined with assortment of its
multiple genomic segments, promotes antigenic diversity and new subtypes, allowing the virus to
avoid vaccines and become resistant to antiviral drugs [4]. For example, the 2008-2009 H1N1
strain of influenza exhibits ∼100% resistance against Tamiflu (oseltamivir) [5]. Therefore, there
is a continuous and urgent need for prevention or treatment strategies that can cover a broad range
of viral strains and subtypes. As the first step to prevent virus infection, a favorable target for
drug discovery is to inhibit influenza virus entry, resulting in an efficient blockage of virus
propagation [6-7]. It would be an effective approach of anti-influenza, and it could prevent the
infection of influenza as vaccination since many viruses share similar entry routes. Moreover, it
also might be a type of broad-spectrum anti-influenza inhibitor, and the resistance is less likely to
occur [8]. One possible strategy is to target the sialic acid (also named N-acetylneuraminic acid,
Neu5Ac) receptor-binding hemagglutinin (HA) glycoprotein to disrupt the interactions between
the viral particles and host cell proteins [9]. HA is the major surface envelope protein of influenza
viruses A and B, and it carries essential functions in the viral life cycle. The entry of influenza
virus is mediated by attachment through HA binding to sialic acid receptors on the host
membrane and then internalization of viral particles into the late endosome [10]. A large portion
of the sialic acid receptor-binding sites is highly conserved for the recognition of the common
ligands. The critical role of HA in influenza entry renders it an attractive target for the
development of prophylactic and therapeutic anti-influenza virus drugs that prevent viral infection
at the first stage [11-12].
Pentacyclic triterpenes are secondary plant metabolites and biosynthetically derived from the
cyclization of squalene [13]. It is generally believed that their physiological functions enhance the
immunity of host plants and increase plant resistance to pathogens [14]. Many of these naturally
occurring compounds exhibit a variety of antiviral activities, such as anti-HIV [15], anti-HCV
[16], anti-influenza [17], anti-HSV [18] and so on. In our previous studies [19-24], we have also
found that echinocystic acid (EA, Fig. 1), an oleanane-type triterpene, displays substantial
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inhibitory activity on HCV entry with one derivative showing EC50 at nanomolar level. In view of
significant inhibitory effects of pentacyclic triterpene on broad antiviral spectrum, we sought to
learn whether such triterpenes were also active against influenza viruses. Recently, we found that
Q8, an EA−galactose conjugate, and it analog Y3 (Fig. 1) are the most potent entry inhibitors
against influenza A/WSN/33 virus with EC50 values of 5.0 and 14.2 ꢀM, respectively [25]. The
molecular basis of their activity is potentially due to their high affinity to HA protein, thus leading
to the blockage of HA-SA receptor interaction and the attachment of influenza viruses to host
cells.
(Figure 1)
L-ascorbic acid (vitamin C, Fig. 2), which is usually mentioned as an essential nutrient for
humans and a reducing agent to scavenge free radicals, has been also shown to have antiviral
activity against a broad spectrum of RNA and DNA viruses in vitro [26-27]. Some derivatives of
L-ascorbic acid, such as 1,2,4-triazole and imidazole [28], 2-O-phosphate [29], 2-O-glycoside [30]
and nucleoside derivatives [31-33], have been found to have antiviral activities. In vivo, oral and
intravenous administration of L-ascorbic acid has been proved to have clinical improvements in
patients with influenza virus [34], hepatitis C virus [35], herpes simplex virus [36] and
Epstein-Barr virus infection [37]. In addition, L-ascorbic acid is a weak acid (pKa = 4.2) with
multi-hydroxyl side chain structurally related to sialic acid, which is the primary receptor for
influenza A virus during the attachment of viruses to the host cells. In the development of
neuraminidase (NA) inhibitors, many analogs of sialic acid (Neu5Ac), such as
2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and the 4-guanidino analog of
Neu5Ac2en (zanamivir), are found to inhibit NA with a potency similar or more stronger than
sialic acid (Fig. 2)[38-41]. Therefore, L-ascorbic acid has been considered as a useful synthetic
precursor to many molecules of potential biological utility [42-44].
(Figure 2)
To continue our studies towards the development of novel antiviral inhibitors [19-24], we
prepared new types of molecules containing pentacyclic triterpene (EA, oleanolic acid (OA),
ursolic acid (UA) and betulinic acid (BA)) moieties connected to L-ascorbic acid via triazole and
amide linker in the present study. In addition, we report the synthesis, NMR, anti-influenza
activity and docking studies in this paper.
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2. Results and discussion
2.1 Chemistry
There are four hydroxy groups in the L-ascorbic acid molecule, and two of them are enol
hydroxy groups. Many investigations have pointed out that the two hydroxy groups at C-2 and
C-3 play particularly important roles to maintain its inherent biological activity in vivo [45]. On
the contrary, the 6-hydroxy group of L-ascorbic acid has been proved to be unimportant in
transport and function of this vitamin [46-48]. Because of these reports, we also focused on the
preparation of C-6-O-modified L-ascorbic acid derivatives.
(Scheme 1)
Scheme 1 outlines that the 6-deoxy-6-azide-2,3-O,O-dialkyl-L-ascorbic acid (9 and 10) and
6-deoxy-6-amino-2,3-O,O-dibenzyl-L-ascorbic acid (11), key intermediates for coupling with
pentacyclic triterpene (OA, EA, UA and BA), were synthesized from the starting L-ascorbic acid.
Briefly, the treatment of L-ascorbic acid with acetyl chloride in acetone was performed to afford
the 5,6-ketal of L-ascorbic acid (2) according to the procedure described by Spickenreither and
Tahir with minor modifications [47-48]. Benzylation or methylation of the C-2 and C-3 hydroxy
groups of the lactone ring in 2 was accomplished using K2CO3 and methyl iodide or benzyl
bromide in DMF to provide 3 and 4, respectively. Deblocking of the 5,6-O,O-protected derivative
of L-ascorbic acid 3 or 4 with 2N HCl in THF-CH3OH solution gave 2,3-O,O-dialkyl-L-ascorbic
acid (5 and 6). Tosylation of the C-6 hydroxy group in 5 or 6 using TsCl in the presence of
triethylamine provided 6-O-tosyl-2,3-O,O-dialkyl-L-ascorbic acid (7 and 8). This was followed
by nucleophilic substitution with sodium azide in DMF to provide the key intermediates 9 and 10.
Subsequent reduction of the azide group in 10 was carried out through the treatment with Ph3P in
THF/H2O (1:1) to yield intermediate 11.
(Scheme 2)
Compounds 15-17 and 28 were prepared from OA, EA, UA and BA, respectively, as
previously described [19, 21-22]. Coupling of them with key intermediate 9 and 10 via click
reaction in THF/H2O (1:1) in the presence of a catalytic amount of copper sulfate and sodium
ascorbate yielded pentacyclic triterpene derivatives of 2,3-O,O-dialkyl-L-ascorbic acid (18-21, 23,
25 and 29-30). Debenzylation of 21, 23, 25 and 30 was accomplished by hydrogenolysis, yielding
22, 24, 26 and 31, respectively (Scheme 2). While the hydrogenation of the double bond 20 (29)
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of compound 30 did not occur under this condition (300 kPa), whereas a higher pressure, such as
800 kPa, could ensure the reaction to be fully completed [52]. Activation of the carboxyl group of
pentacyclic
triterpene
with
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) gave the stable intermediates 32-34 and 41, and then condensation with
intermediate 11 in the presence of EDC thus yielded 35, 37, 39 and 42, followed by
hydrogenolysis with 10% Pd/C to afford 36, 38, 40 and 43, respectively (Scheme 3). The
structures of all L-ascorbic acid-triterpene conjugates were characterized by 1H NMR, 13C NMR
and ESI-HRMS.
(Scheme 3)
2.2 Biological assays
2.2.1 Anti-influenza A/WSN/33 virus activity
As part of our biological profiling, we evaluated the newly synthesized pentacyclic
triterpene-L-ascorbic acid conjugates (18-26, 29-31, 35-40 and 42-43) against the influenza
A/WSN/33 (H1N1) virus that was propagated in MDCK cells by the CellTiter-Glo assay and the
cytopathic effect (CPE) reduction assay [50]. Moreover, we employed the CellTiter-Glo
screening, an assay used to monitor cell proliferation, to screen and exclude compounds with
significant toxicity toward MDCK cells. The CPE screening, an assay to determine the damage to
host cells during virus invasion, was used to screen and identify compounds with a reduction of
the CPE on influenza A/WSN/33 virus. Except compounds 21, 23, 25, 30, 31 and 42-43
significantly reduced the viral CPE in the CellTiter-Glo assay, all the other compounds had
cytotoxicity at the same concentration (Table 1 and 2). Compound 30 exhibited the most potent
anti-influenza virus activity with an EC50 of 8.7 µM (Figure 3A), while no cytotoxicity against
uninfected MDCK cells was observed at a concentration of 200 µM. The reduction of CPE was
confirmed by direct microscopic observation, which detected far less CPE than that in the DMSO
control (Figure 3B).
(Table 1)
(Table 2)
(Figure 3)
2.2.2 Structure-activity relationship
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Further analysis of the structure-activity relationship of the L-ascorbic acid-triterpene
conjugates revealed a clear co-relationship as potential anti-influenza virus agents. Analogues
exhibited a variety of anti-influenza virus activities dependent on the substituent groups of
L-ascorbic acid, the linker and triterpene skeleton.
Table 1 shows that substituents at C-2 and C-3 hydroxy groups of L-ascorbic acid exerted
important effects on the antiviral potency. The synthetic analogs showed high activities when they
had a common structural feature, the 2,3-O,O-dibenzyl substitution of L-ascorbic acid. These
results also confirmed that all conjugates (compounds 21, 23, 25, 35, 37 and 39) with such
structural property showed high inhibitory rate of anti-influenza virus at a dose of 50 µM no
matter the triterpene skeleton was OA, EA, UA or BA, or the linker moiety was 1,2,3-triazole or
amide bond. However, the removal of benzyl groups of C-2 and C-3 hydroxyl groups
(compounds 22, 24, 26, 36, 38 and 40) resulted in the loss of anti-influenza activity. This result
might be caused by the reduction of hydrophobicity, which decreased the affinity of L-ascorbic
acid-triterpene conjugates to the protein targets. Moreover, most L-ascorbic acid-BA conjugates
(compounds 30-31 and 42-43) exhibited substantial effects against influenza A/WSN/33 virus
activity, except for compound 29 which possessed cytoxicity against uninfected DMCK cells.
CPE reduction assay revealed that compound 30 exerted the most potent anti-influenza virus
activity with an EC50 of 8.7 ꢀM (Fig. 3A), which was in the same order as that found for the
reference anti-influenza drug oseltamivir (EC50 = 12.5 ꢀM), and no cytotoxicity effects were
observed on MDCK cells. In addition, pentacyclic triterpene derivatives of
2,3-O,O-dimethyl-L-ascorbic acid derivatives (compounds 18-20 and 29) showed obviously
cytotoxicity, which was also consistent with our previously studies that per-O-methylated
cyclodextrin-triterpene conjugates also exhibit some degree of cytotoxicity at 5 ꢀM and strong
cytotoxicity at 50 ꢀM in MDCK and 293T cell lines [22]. Clearly, the CH3O groups at C-2 and
C-3 of L-ascorbic acid are an unfavorable group in the scaffold of triterpene-L-ascorbic acid
conjugates for antiviral effects. Furthermore, the CPE assay indicated that compound 35, an
analogue of 21 in which the 1,2,3-trizole linker was replaced by an amide bond, had potency
almost identical to that of 21, and similar results were also found for other analogues (23 vs. 37,
25 vs. 39, 30 vs. 42 and 31 vs. 43), indicating the compatibility of 1,2,3-trizole and amide linkers
for anti-influenza A/WSN/33 virus.
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2.2.3 Compound 30 inactivates virions and prohibits influenza virus entry
In order to characterize at which stage(s) of virus life cycle was affected by L-ascorbic
acid-triterpene conjugates, we first sought to determine if they acted on viruses or on host cells. A
time-of-addition experiment using 30 as a representative was carried out [49]. We administrated
compound 30 at various time points (0, 2, 4, 6, 8 h) post infection. The reduction of NP protein
level at the interval 0-10 h (covering the whole life cycle) and 0-2 h (covering the entire viral
entry process) indicated that 30 was only effective at the attachment or the fusion of virus with
host cell (Figure 4A). There was no inhibitory effect for the remaining steps, such as viral genome
replication/translation or virion assembly/release. These results suggested that 30 acted in the
early stage of the influenza virus replication cycle, including entry and/or an early replication
phase.
Then three experiments were conducted in parallel, including the co-treatment, the
pre-treatment of cells and the pre-treatment of virus, in order to clarify whether the host cell or the
influenza virus was targeted. In the co-treatment assay, compound 30 at a concentration of 50 ꢀM
exerted an inhibition rate of 75%, while the pre-treatment of virus led to an inhibition rate of 87%
(Figure 4B). We concluded that compound 30 targeted the influenza virus particles but not host
cells, which was consistent with our previous study [25].
2.2.4 Inhibition of HA by compound 30
Since compound 30 could inhibit the early step of the influenza A virus life cycle by acting
on the virus and but not on the cells, we investigated the effect of it on the activity of the
influenza A virus surface proteins HA [51]. It is well known that influenza virus HA plays an
essential role in virus infection. The attachment mediated by HA initiates the fusion of the viral
envelope with the host membrane to release the viral genome into the target cells. Influenza A
virus HA can bind to sialic acid on the surface of RBCs, causing agglutination. Therefore, we
performed a hemagglutination inhibition (HI) assay to examine whether compound 30 blocked
the ability of virus particles to bind to cell receptors. Influenza A virus A/WSN/33 was pretreated
with compound 30 for 1 h before the addition of chicken erythrocytes, and a further incubation
was conducted at RT for 1 h. Compound 30 inhibited the binding of the influenza A virus
A/WSN/33 to chicken erythrocytes, showing similar capability as anti-HA antibody. The
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inhibitory effect was in a concentration-dependent manner, indicating that compound 30, as
anti-HA antibody, shared the same target-HA (Figure 4C).
2.2.5 Binding kinetics by surface plasmon resonance (SPR)
We then used SPR experiment to study the kinetics and quantify the interaction between the
active compounds 30 and 31 and HA protein.
The results implied that both 30 and 31 could bind to the HA protein immobilized on the
CM5 chip, and Figure 4D shows the dose-dependent responses. The KD values for 30 and 31 were
calculated as 3.76 µM and 8.04 µM, respectively. It was found that the weaker inhibitor 31 had
less effect KD than 30, suggesting that the potency of the compound against the influenza virus
was directly correlated to the inhibition of the HA-sialic acid receptor interaction. The affinity of
these compounds to HA protein was correlated well to their antiviral effects, supporting that HA
protein was a potential target for 30.
(Figure 4)
2.2.5 Blind docking calculation
Molecular docking is a frequently used tool in computer-aided structure-based rational drug
design. In this study, the newly synthesized L-ascorbic acid-triterpene conjugates were docked
into the influenza HA protein, which was obtained from the RCSB Protein Data Bank,
http://www.rcsb.org/pdb/home/home.do, using AutoDock 4.2 program.
To predict the binding mode of the newly synthesized compounds, we first performed a
docking study of compounds 30 and 31. In view of HA (HA1) as the potential interruption event,
we performed blind docking calculations to investigate such possibilities. We employed blind
docking calculations in order to figure out the potential interruption event of 30 and 31 within HA
(HA1). The docked conformations of HA-30 were determined based on the minimum free energy
analyses. The computer-aided docking data suggested that compounds 30 and 31 exhibited a
similar binding mode as Y3, occupying the binding pocket for sialic acid receptor (Figure 5) with
an estimated binding energy of -9.94 and -8.56 kcal/mol, and an inhibition constant (Ki) = 51.64
and 122.51 nM, respectively. This result suggested that the two compounds, especially 30, could
bind tightly to HA protein. One of the influenza virus receptors is the terminal sialic acid of
cellular glycoproteins, which binds the shallow depression located at the top of HA1 domain. For
all subtypes of influenza viruses, the HA protein is composed of highly conserved HA1 and HA2
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domains, which are essential for sialic acid receptor binding and viral envelope fusion,
respectively [53].
The computer-aided docking data indicated that the 3-OH of the BA moiety formed a
hydrogen bond with K222, and the carbonyl oxygen atom of BA moiety made a hydrogen bond
with H183. For the L-ascorbic acid moiety, besides the hydrophobic interaction, the C-3 O-benzyl
substitution of L-ascorbic acid occupied the narrow cavity and engaged in π-π stacking interaction
with W153 residue. Among these, H183 and L194 were critical for sialic acid binding via
hydrogen bonds. Most of the remaining interactions were within the 190 helix (residues 190-198),
the 130 loop (residues 135-138) and the 220 loop (residues 221-228), which were all important
for sialic acid binding and consistent with the former studies of Y3. These docking data were
consistent with results from SAR studies, in particular the requirement for O-benzyl groups in the
L-ascorbic acid residue and the 3-hydroxyl group in the BA moiety.
Docking studies indicated that the 3-hydroxyl group of the betulinic acid, O-benzyl groups of
the L-ascorbic acid and several other residues were critical in sialic acid binding. To make sure
the key residues could be kept in proper distance and position, the 1,2,3-triazole played an
important role. The five-membered E-ring of BA perhaps had influence on the hydrogen bond of
carbonyl oxygen atom with H183, which also explained why the L-ascorbic acid-BA conjugates
had better activity.
(Figure 5)
3. Conclusions
In conclusion, we synthesized a series of L-ascorbic acid-conjugated pentacyclic triterpene
(OA, UA, EA and BA) derivatives in the present study, and their anti-influenza activity in vitro
was tested. A systemic exploration of pharmacological activities of the compounds showed that
conjugation of triterpene with O-benzyl substitution L-ascorbic acid was the structural
requirements for maintenance of high activity. In addition to this, it was clear based on our SAR
results that the incorporation of O-benzyl substitution groups into L-ascorbic acid considerably
enhanced the anti-influenza virus potential with no cytotoxicity. Taken together, compound 30, a
BA-L-ascorbic acid conjugate, showed better anti-influenza virus activity than other types of
triterpenes. Similar to Y3, the molecular basis of its activity was potentially due to the high
affinity to HA protein, thus blocking the attachment of influenza viruses to cells. Docking studies
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suggested that these compounds occupied the conserved pocket for sialic acid receptor, which
was consistent with the SAR data. There results confirmed that the suitable incorporation of
L-ascorbic acid into the triterpene entity enabled an achievement of higher inhibitory potency of
anti-viruses. This study suggested that L-ascorbic acid-conjugated pentacyclic triterpenes were
promising anti-influenza entry inhibitors, and HA protein associated with viral entry was a
promising drug target
4. Experimental section
4.1 General information
Materials. Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293T
(HEK293T) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco BRL. Inc.,
Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories,
Linz Austria) at 37 °C under 5% CO2. The following virus was used in this study: influenza
A/WSN/33 (H1N1) virus.
Mouse anti-NP monoclonal antibody and rabbit anti-GAPDH polyclonal antibodies were
purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA. USA). Rabbit monoclonal
antibodies against influenza virus HA (H1N1) were supplied from Sino Biological Inc. (Beijing,
China). The anti-rabbit IgG and anti-mouse IgG conjugated with horseradish peroxidase (HRP)
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
OSV of 98% purity (Hoffmann-La Roche Ltd., Basel, Switzerland) was used as reference
compounds in CPE reduction assay. OA and UA were kindly supplied by Nanjing Zelang Medical
Technology Co., Ltd (Nanjing, China) and Anboruila Biotechnology Co. Ltd (Nanjing, China),
respectively. BA was supplied by Aladdin-Reagent Co. Ltd (Shanghai, China). EA was separated
from the ethanol/H2O crude extract of Gleditsia sinensis Lam, a traditional Chinese herbal
medicine. All the compounds were dissolved in DMSO to 10 mM as a stock solution.
Chemistry. High-resolution mass spectra (HRMS) were obtained with an APEX IV FT_MS
(7.0 T) spectrometer (Bruker) in positive ESI mode. NMR spectra were recorded on a Bruker
1
DRX 400 spectrometer at ambient temperature. Chemical shifts of H NMR were referenced to
the internal standard TMS (δH = 0.00) or the solvent signal (δH = 3.31 for the central line of
MeOD). Chemical shifts of 13C NMR were referenced to the solvent signal (δC = 77.00 for the
central line of CDCl3, δC = 49.00 for the central line of MeOD). Reactions were monitored by
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thin-layer chromatography (TLC) on a 60 F254 plate (pre-coated with silica gel, layer thickness of
0.2 mm; E. Merck, Darmstadt, Germany) and detected by staining with a yellow solution
containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24·4H2O (24.0 g) in 6% H2SO4 (500 mL),
followed by heating.
4.2 General procedures
Compound 2 was synthesized from L-ascorbic acid, according to the procedure described by
Spickenreither and Tahir with minor modifications [47-48]. Benzylation or methylation of the
C-2 and C-3 hydroxy groups of the lactone ring in 2 was accomplished using K2CO3 and benzyl
bromide or methyl iodide in DMF to provide 3 or 4, respectively. Deblocking of
5,6-O,O-protected-L-ascorbic acid intermediates with 2 N HCl in THF-CH3OH solution gave
2,3-O,O-dialkyl-L-ascorbic acid (5 or 6). Then tosylation of C-6 hydroxy group in 5 or 6 using
TsCl in the presence of triethylamine provided 6-O-tosyl-2,3-O,O-dialkyl-L-ascorbic acid (7 or 8).
This was followed by nucleophilic substitution with sodium azide in DMF to provide the key
intermediates 9 or 10. Subsequent reduction of the azide group in 10 was carried out through the
treatment with Ph3P in THF/H2O (1:1) to yield intermediate 11 [47, 54-56]. The detailed synthesis,
1H and 13C NMR data of compounds 2-8, 10 and 11 are available in the supporting information.
Compounds 15-17, 28, 32-34 and 41 were synthesized using previously published methods
[19-23].
4.2.1 General procedure for click chemistry reaction (method A)
The starting material compounds 9 or 10 (1 equiv.), CuSO4·5H2O (1.2 equiv.) and sodium
ascorbate (2.4 equiv.) were added to a solution of compound triterpenoid substituted by alkynyl
(1.2 equiv.), respectively, in mixture solvant of THF/H2O (1:1). The mixture was stirred at room
temperature over night and monitored by TLC. The organic phase was extracted with CH2Cl2,
dried over sodium sulfate and concentrated under reduced pressure. The obtained residue was
purified by column chromatography on a silica gel using a gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent.
4.2.2 General procedure for reduction reaction (method B)
The starting material (1 equiv.) was dissolved in MeOH (6 mL), and palladium–carbon (0.1
equiv.) was added. The suspension was degassed under vacuum and urged with H2 for three times,
and then it was stirred under H2 balloon at room temperature for 24 h. The suspension was filtered
12

ACCEPTED MANUSCRIPT
through a pad of Celite, and the pad cake was washed with CH3OH. The combined filtrate was
concentrated to dryness. The residue was subjected to flash chromatography to afford the product.
4.2.3 General procedure for amide bond formation reaction (method C)
EDC (1.1 equiv.) was added to a stirred solution of compound 11 (1 equiv.) and triterpenoid
substituted by OBt (1 equiv.) and 4-DMAP (1.1 equiv.) in THF, and the mixture was then stirred
at room temperature for 12 h. The precipitate was filtered and washed with CH2Cl2. Purification
by TLC was performed on a silica gel (petroleum ether/ethyl acetate) to yield the product.
4.2.4 6-azido-6-deoxy-2,3-di-O-methoxy-L ascorbic acid (9)
To solution of compound 7 (500 mg, 1.4 mmol) in DMF, NaN3 (181 mg, 2.79 mmol) was
added at room temperature, and mixture was stirred for 30 min. Temperature was then raised to
60 °C, and reaction solution was stirred overnight. Reaction was terminated and concentrated by
evaporation in vacuo. Purification of crude product mixture was performed on silica gel column
chromatography (petroleum ether/ethyl acetate= 5:1) to yield compound 9 as white oil (240 mg,
1
75 %). H NMR (400 MHz, MeOD): δ 4.77 (s, 1H), 4.73 (d, 1H, J = 1.9 Hz), 4.18 (s, 3H), 3.96
(m, 1H), 3.79 (s, 3H), 3.50 (m, 1H), 3.38 (m, 1H); 13C NMR (100 MHz, MeOD): δ 171.68,
159.64, 124.19, 77.32, 69.33, 61.16, 60.11, 54.10; ESI-HRMS Calcd for C8H12N3O5 [M+H]+:
230.0771; found 230.0772.
4.2.5 1-[2-(3,4-bis-methoxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (18)
Compound 18 was prepared from compounds 15 (587 mg, 1.19 mmol) and 9 (227 mg, 0.99
mmol) according to general procedure A. The residue was purified by gradient mixture of
petroleum ether/ethyl acetate with increasing polarity as an eluent to yield the 18 as a white solid
(620 mg, 87 %). 1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H), 6.82 (s, 1H), 5.30 (s, 1H), 5.19 (d,
1H, J = 6.0 Hz), 4.54 (m, 3H), 4.29 (m, 3H), 4.06 (s, 3H), 3.75 (s, 3H), 3.11 (m, 1H), 2.52 (d, 1H,
J = 10.8 Hz), 1.06, 0.89, 0.82, 0.82, 0.78, 0.69, 0.42 (s, each 3H), 0.62 (d, 1H, J = 10.1 Hz); 13
C
NMR (100 MHz, CDCl3): δ 178.31, 169.11, 156.92, 144.20, 143.76, 124.17, 122.99, 122.93,
78.52, 75.45, 68.21, 60.25, 59.25, 54.85, 52.39, 50.02, 47.25, 46.29, 45.93, 41.59, 39.04, 38.47,
38.24, 36.63, 34.63, 33.82, 32.76, 32.26, 32.13, 30.42, 27.87, 26.97, 26.78, 25.53, 23.37, 23.16,
18.01, 16.23, 15.44, 15.07; ESI-HRMS Calcd for C41H63N4O7 [M+H]+: 723.4691, found
723.4685.
13

ACCEPTED MANUSCRIPT
4.2.6
1-[2-(3,4-bis-methoxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β,16α-
dihydroxy- -olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (19)
Compound 19 was prepared from compounds 17 (293 mg, 0.58 mmol) and 9 (110 mg, 0.48
mmol) according to general procedure A. The residue was purified by gradient mixture of
petroleum ether/ethyl acetate with increasing polarity as an eluent to yield 19 as a white solid
(359 mg, 84 %). 1H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H), 6.97 (s, 1H), 5.45 (s, 1H), 4.87 (s,
1H), 4.53 (m, 3H), 4.29 (s, 4H), 4.07 (s, 3H), 3.76 (s, 3H), 3.14 (d, 1H, J = 6.5 Hz), 2.67 (d, 1H, J
= 12.3 Hz), 2.18 (t, 1H, J = 13.0 Hz), 1.28, 0.90, 0.84, 0.82, 0.79, 0.69, 0.45 (s, each 3H), 0.64 (d,
1H, J = 11.6 Hz); 13C NMR (100 MHz, CDCl3): δ 178.33, 169.33, 157.06, 144.18, 143.37, 124.08,
123.40, 123.16, 78.73, 75.70, 74.79, 68.53, 60.43, 59.48, 55.06, 53.36, 52.59, 48.89, 46.73, 46.58,
41.66, 41.43, 39.43, 38.64, 38.47, 36.80, 35.14, 35.03, 34.20, 32.51, 30.14, 29.57, 27.97, 27.00,
26.65, 24.99, 23.25, 18.14, 16.57, 15.56, 15.49; ESI-HRMS Calcd for C41H63N4O8 [M+H]+:
739.4640, found 739.4637.
4.2.7 1-[2-(3,4-bis-methoxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
urs-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (20)
Compound 20 was prepared from compounds 17 (291 mg, 0.59 mmol) and 10 (112 mg, 0.49
mmol) according to general procedure A. The residue was purified by gradient mixture of
petroleum ether/ethyl acetate with increasing polarity as an eluent to yield 20 as a white solid
(290 mg, 68 %). 1H NMR (400 MHz, CDCl3): δ 7.72 (s, 1H), 6.79 (s, 1H), 5.25 (s, 1H), 5.19 (m,
1H), 4.55 (s, 2H), 4.47 (m, 1H), 4.25 (m, 3H), 4.06 (s, 3H), 3.75 (s, 3H), 3.11 (br s, 1H), 2.38 (s,
1H), 0.99, 0.89, 0.86, 0.79, 0.77, 0.69, 0.44 (s, each 3H), 0.61 (d, 1H, J = 11.2 Hz); 13C NMR
(100 MHz, CDCl3): δ 178.15, 169.11, 156.91, 144.17, 138.56, 125.92, 124.05, 123.02, 78.54,
75.49, 68.23, 60.25, 59.26, 54.88, 53.15, 52.43, 50.04, 47.35, 47.25, 42.00, 39.39, 39.23, 38.67,
38.47, 38.41, 36.78, 36.61, 34.54, 32.50, 30.59, 27.92, 27.51, 26.83, 24.45, 23.07, 20.96, 18.00,
16.91, 16.25, 15.49, 15.20; ESI-HRMS Calcd for C41H63N4O7 [M+H]+: 723.4691, found
723.4692.
4.2.8 1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (21)
Compound 21 was prepared from compounds 15 (211 mg, 0.43 mmol) and 10 (136 mg, 0.36
mmol) according to general procedure A. The residue was purified by gradient mixture of
14

ACCEPTED MANUSCRIPT
petroleum ether/ethyl acetate with increasing polarity as an eluent to yield 21 as a white solid
(266 mg, 85 %). 1H NMR (400 MHz, CDCl3): δ 7.36-7.32 (m, 8H), 7.22 (m, 2H), 6.74 (br s, 1H),
5.51 (s, 1H), 5.22 (d, 1H, J = 11.8 Hz), 5.06 (s, 2H), 4.54 (s, 1H), 4.35 (br s, 1H), 3.95 (br s, 1H),
3.52 (m, 1H), 3.35 (m, 1H), 3.21 (m, 1H), 2.78 (d, 1H, J = 12.0 Hz), 2.20 (t, 1H, J = 13.3 Hz),
1.34, 0.99, 0.94, 0.90, 0.88, 0.78, 0.74 (s, each 3H); 13C NMR (100 MHz, CDCl3): δ 179.55,
169.54, 156.99, 144.30, 143.48, 135.90, 135.31, 129.00, 128.64, 128.58, 127.75, 125.54, 123.34,
121.26, 78.85, 76.60, 74.86, 73.88, 73.44, 68.94, 55.15, 49.25, 46.71, 43.08, 41.84, 41.63, 39.61,
38.72, 38.53, 36.89, 35.23, 35.04, 32.50, 30.12, 29.15, 28.02, 27.09, 26.82, 25.32, 23.37, 18.19,
17.98, 17.00, 15.60; ESI-HRMS Calcd for C53H71N4O7 [M+H]+: 875.5317, found 875.5318.
4.2.9
1-[2-(3,4-dihydroxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (22)
Compound 22 was prepared from 21 (80 mg, 0.09 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 22 as a white solid
(50 mg, 80 %). 1H NMR (400 MHz, MeOD): δ 7.85 (s, 1H), 7.73 (t, 1H, J = 5.3 Hz), 5.34 (s, 1H),
4.68 (m, 2H), 4.51 (m, 1H), 4.39 (s, 2H), 4.30 (br s, 1H), 3.14 (dd, 1H, J = 4.3, 10.8 Hz), 2.78 (d,
1H, J = 10.2 Hz), 2.05 (m, 1H), 1.88 (m, 2H), 1.77 (t, 1H, J = 13.6 Hz), 1.15, 0.96, 0.94, 0.91,
0.91, 0.77, 0.54 (s, each 3H), 0.72 (d, 1H, J = 11.3 Hz); 13C NMR (100 MHz, MeOD): δ 180.28,
172.84, 153.61, 146.06, 144.97, 125.61, 124.16, 120.20, 79.63, 77.41, 69.22, 56.66, 53.84, 49.85,
48.94, 47.60, 47.45, 42.81, 42.50, 40.56, 39.80, 38.06, 35.79, 35.04, 34.10, 33.75, 33.60, 31.60,
28.76, 28.43, 27.81, 26.52, 24.49, 24.04, 19.43, 17.52, 16.35, 15.94; ESI-HRMS Calcd for
C39H59N4O7 [M+H]+: 695.4378; found 695.4368.
4.2.10
1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β,16α-
dihydroxy-olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (23)
Compound 23 was prepared from 17 (211 mg, 0.43 mmol) and 10 (136 mg, 0.36 mmol)
according to general procedure A. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield the 23 as a white solid (266 mg,
85 %). 1H NMR (400 MHz, CDCl3): δ 7.73 (br s, 1H), 7.37-7.32 (m, 8H), 7.20 (br s, 2H), 6.98 (s,
1H), 5.52 (s, 1H), 5.12 (d, 2H, J = 11.7 Hz), 5.08 (d, 2H, J = 11.4 Hz), 4.57 (m, 3H), 4.35 (m, 4H),
3.20 (m, 1H), 2.73 (d, 1H, J = 12.6 Hz), 2.26 (t, 1H, J = 13.3 Hz), 1.34, 0.97, 0.91, 0.89, 0.85,
0.75, 0.53 (s, each 3H); 13C NMR (100 MHz, CDCl3): δ 178.32, 169.35, 156.53, 144.27, 143.42,
15

ACCEPTED MANUSCRIPT
135.76, 135.08, 129.02, 128.77, 128.73, 128.65, 127.76, 125.92, 123.57, 121.40, 78.86, 76.01,
75.08, 73.97, 73.66, 68.57, 55.13, 52.73, 48.99, 46.82, 46.65, 41.75, 41.56, 39.52, 38.71, 38.52,
36.87, 35.24, 35.02, 32.56, 32.51, 30.22, 29.62, 28.01, 27.11, 26.73, 25.04, 23.33, 18.20, 16.67,
15.56; ESI-HRMS Calcd for C53H71N4O8 [M+H]+: 891.5266, found 891.5266.
4.2.11
1-[2-(3,4-dihydroxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β,16α-
dihydroxy-olean-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (24)
Compound 24 was prepared from 23 (104 mg, 0.12 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 24 as a white solid
(65 mg, 78 %). 1H NMR (400 MHz, MeOD): δ 7.84 (s, 1H), 7.56 (t, 1H, J = 5.2 Hz), 5.46 (s, 1H),
4.69 (dd, 1H, J = 4.4, 13.9 Hz), 4.63 (d, 1H, J = 1.5 Hz), 4.51 (m, 1H), 4.37 (s, 3H), 4.29 (m, 1H),
3.14 (dd, 1H, J = 4.8, 11.3 Hz), 2.87 (m, 1H), 2.37 (t, 1H, J = 13.1 Hz), 1.36, 0.96, 0.95, 0.92,
0.88, 0.77, 0.56 (s, each 3H), 0.73 (d, 1H, J = 11.1 Hz); 13C NMR (100 MHz, MeOD): δ 180.10,
172.98, 154.12, 145.82, 144.82, 125.54, 124.24, 120.07, 79.62, 77.44, 75.55, 69.21, 56.74, 53.84,
49.92, 49.85, 48.08, 48.02, 42.73, 42.24, 40.69, 39.89, 39.80, 38.03, 36.31, 35.90, 33.88, 33.29,
31.83, 31.20, 28.72, 27.84, 27.31, 25.35, 24.43, 19.39, 17.60, 16.34, 16.13; ESI-HRMS Calcd for
C39H59N4O8 [M+H]+: 711.4327, found 711.4311.
4.2.12
1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-
hydroxy-urs-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (25)
Compound 25 was prepared from 17 (193 mg, 0.38 mmol) and 10 (123 mg, 0.32 mmol)
according to general procedure A. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield 25 as a white solid (236 mg,
84 %). 1H NMR (400 MHz, CDCl3): δ 7.76 (s, 1H), 7.35-7.18 (m, 10 H), 6.81 (br s, 1H), 5.32 (s,
1H), 5.16 (d, 1H, J = 11.7 Hz), 5.05 (s, 2H), 4.52 (m, 6H), 3.16 (m, 1H), 1.06, 0.95, 0.92, 0.85,
0.83, 0.74, 0.52 (s, each 3H) , 0.67 (d, 1H, J = 11.6 Hz); 13C NMR (100 MHz, CDCl3): δ 178.20,
169.26, 156.59, 144.19, 138.67, 135.67, 135.02, 128.77, 128.40, 127.49, 125.97, 124.15, 121.21,
78.64, 75.84, 73.78, 73.36, 68.31, 54.92, 53.25, 52.54, 47.41, 47.29, 42.06, 39.45, 39.27, 38.71,
38.50, 38.41, 36.83, 36.64, 34.62, 32.53, 30.63, 27.95, 27.57, 26.89, 24.53, 23.11, 21.01, 18.04,
16.96, 16.33, 15.51, 15.25; ESI-HRMS Calcd for C53H71N4O7 [M+H]+: 875.5317, found
875.5317.
16

ACCEPTED MANUSCRIPT
4.2.13
1-[2-(3,4-dihydroxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
urs-12-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (26)
Compound 26 was prepared from 25 (200 mg, 0.23 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 26 as a white solid
1
(141 mg, 89 %). H NMR (400 MHz, MeOD): δ 7.85 (s, 1H), 5.32 (s, 1H), 4.68 (d, 1H, J = 1.9
Hz), 4.50 (m, 1H), 4.37 (d, 2H, J = 2.0 Hz), 4.31 (m, 1H), 3.15 (dd, 1H, J = 5.0, 11.3 Hz), 2.13 (d,
1H, J = 10.6 Hz), 2.07 (t, 1H, J = 9.7 Hz), 1.10, 0.96, 0.92, 0.90, 0.89, 0.77, 0.56 (s, each 3H),
0.71 (d, 1H, J = 11.3 Hz); 13C NMR (100 MHz, MeOD): δ 180.05, 172.93, 154.03, 145.90,
139.59, 127.26, 125.54, 120.05, 79.58, 77.46, 69.24, 56.61, 54.06, 53.83, 49.85, 48.86, 48.75,
43.15, 40.72, 40.68, 40.16, 39.91, 39.75, 38.42, 37.97, 35.69, 34.06, 31.83, 28.80, 27.80, 25.23,
24.28, 24.14, 21.65, 19.36, 17.77, 17.50, 16.41, 16.06; ESI-HRMS Calcd for C39H59N4O7 [M+H]+:
695.4378; found 695.4363.
4.2.14 1-[2-(3,4-bis-methoxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
lup-20(29)-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (29)
Compound 29 was prepared from 28 (222 mg, 0.45 mmol) and 9 (86 mg, 0.38 mmol)
according to general procedure A. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield the 29 as a white solid (157 mg,
58 %). 1H NMR (400 MHz, CDCl3): δ 7.88 (br s, 1H), 7.05 (br s, 1H), 4.70 (m, 2H), 4.57 (m, 2H),
4.43 (s, 2H), 4.33 (m, 1H), 4.14 (s, 3H), 3.84 (s, 3H), 3.18 (m, 2H), 3.05 (m, 1H), 2.31 (t, 1H, J =
10.8 Hz), 1.65, 0.95, 0.93, 0.78, 0.75, 0.73 (s, each 3H), 0.65 (d, 1H, J = 9.2 Hz); 13C NMR (100
MHz, CDCl3): δ 176.83, 169.20, 156.89, 150.78, 145.78, 123.24, 123.09, 109.36, 78.87, 75.61,
68.61, 60.46, 59.55, 55.63, 55.29, 53.39, 52.94, 50.50, 49.99, 46.71, 42.36, 40.66, 38.80, 38.67,
38.14, 37.73, 37.11, 34.23, 33.29, 30.76, 29.35, 27.94, 27.27, 25.55, 20.83, 19.37, 18.25, 16.09,
15.69, 15.37, 14.58; ESI-HRMS Calcd for C41H63N4O7 [M+H]+: 723.4691, found 723.4688.
4.2.15
1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-
hydroxy-lup-20(29)-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (30)
Compound 30 was prepared from 28 (233 mg, 0.47 mmol) and 5 (150 mg, 0.39 mmol)
according to general procedure A. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield 30 as a white solid (201 mg,
59 %). 1H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H), 7.37-7.27 (m, 8H), 7.20 (m, 2H), 6.67 (br s,
17

ACCEPTED MANUSCRIPT
1H), 5.15 (d, 1H, J = 11.7 Hz), 5.08 (d, 1H, J = 11.2 Hz), 4.72 (s, 1H), 4.59 (s, 2H), 4.53 (m, 2H),
4.41 (m, 2H), 4.35 (s, 1H), 3.16 (dd, 1H, J = 4.7, 11.0 Hz), 3.08 (m, 1H), 2.35 (t, 1H, J = 10.2 Hz),
1.66, 0.94, 0.94, 0.79, 0.78, 0.73 (s, each 3H), 0.64 (d, 1H, J = 8.6 Hz); 13C NMR (100 MHz,
CDCl3): δ 176.71, 169.29, 156.51, 150.74, 145.18, 135.74, 135.08, 129.03, 128.73, 128.69,
128.61, 127.79, 123.94, 121.31, 109.41, 78.92, 75.82, 73.92, 73.60, 68.37, 55.63, 55.30, 52.69,
50.52, 50.00, 46.71, 42.39, 40.68, 38.78, 38.65, 38.18, 37.74, 37.11, 34.58, 34.26, 33.39, 30.79,
29.36, 27.92, 27.29, 25.56, 20.85, 19.40, 18.23, 16.09, 15.81, 15.34, 14.58; ESI-HRMS Calcd for
C53H71N4O7 [M+H]+: 875.5317, found 875.5314.
4.2.16
1-[2-(3,4-dihydroxy-5-oxo-2,5-dihydro-furan-2-yl)-2-hydroxy-ethyl]-4-[N-(3β-hydroxy-
lup-20(29)-en-28-oyl)-amino]methyl-1H-1,2,3-triazole (31)
Compound 31 was prepared from 30 (143 mg, 0.16 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 31 as a white solid
(100 mg, 89 %). 1H NMR (400 MHz, MeOD): δ 7.78 (br s, 1H), 4.68 (m, 2H), 4.66 (m, 1H), 4.56
(m, 2H), 4.40 (br s, 2H), 4.29 (br s, 1H), 3.12 (dd, 1H, J = 4.8, 11.1 Hz), 2.48 (t, 1H, J = 10.4 Hz),
2.27 (br s, 1H), 2.14 (d, 1H, J = 13.2 Hz), 1.67, 0.97, 0.94, 0.84, 0.83, 0.75 (s, each 3H), 0.68 (d,
1H, J = 8.1 Hz); 13C NMR (100 MHz, MeOD): δ 180.86, 172.89, 153.57, 152.22, 146.08, 125.34,
120.23, 109.96, 79.65, 77.40, 69.24, 56.89, 53.96, 52.05, 51.39, 48.04, 43.45, 41.96, 40.09, 39.92,
39.17, 38.91, 38.28, 35.53, 34.01, 31.89, 30.53, 28.61, 28.01, 26.96, 22.10, 19.67, 19.41, 16.78,
16.64, 16.11, 15.09; ESI-HRMS Calcd for C39H59N4O7 [M+H]+: 695.4384; found 695.4382.
4.2.17 N-(3β-hydroxy-olean-12-en-28-oyl)-6-amino-6-deoxy-2,3-bis-benzyloxy-L-ascorbic acid
(35)
Compound 35 was prepared from 32 (245 mg, 0.54 mmol) and 11 (173 mg, 0.49 mmol)
according to general procedure C. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield the 35 as a white solid (187 mg,
48 %). 1H NMR (400 MHz, CDCl3): δ 7.36-7.32 (m, 8H), 7.21 (m, 2H), 6.75 (br s, 1H), 5.51 (br s,
1H), 5.17 (d, 2H, J = 11.8 Hz), 5.06 (s, 2H), 4.54 (s, 1H), 4.35 (s, 1H), 3.95 (br s, 1H), 3.51 (m,
1H), 3.35 (m, 1H), 3.22 (m, 1H), 2.78 (d, 1H, J = 11.3 Hz), 2.22 (t, 1H, J = 13.3 Hz), 1.35, 0.99,
0.94, 0.90, 0.88, 0.78, 0.74 (s, each 3H); 13C NMR (100 MHz, CDCl3): δ 179.48, 169.54, 156.99,
143.45, 135.88, 135.29, 128.96, 128.55, 127.69, 123.28, 121.23, 78.81, 76.58, 74.83, 73.86, 73.74,
73.39, 68.88, 55.12, 49.20, 46.68, 43.06, 41.80, 41.57, 39.57, 38.69, 38.51, 36.85, 35.20, 35.01,
18

ACCEPTED MANUSCRIPT
32.50, 30.10, 29.18, 28.00, 27.05, 26.80, 25.29, 23.34, 18.17, 16.98, 15.58; ESI-HRMS Calcd for
C50H68NO7 [M+H]+: 794.4996; found 794.4969.
4.2.18 N-(3β-hydroxy-olean-12-en-28-oyl)-6-amino-6-deoxy-L-ascorbic acid (36)
Compound 36 was prepared from 35 (40 mg, 0.05 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 36 as a white solid
1
(23 mg, 74 %). H NMR (400 MHz, MeOD): δ 7.31 (br s, 1H), 5.50 (br s, 1H), 4.53 (br s, 1H),
4.32 (br s, 1H), 3.92 (br s, 1H), 3.46 (m, 1H), 3.15 (d, 1H, J = 10.4 Hz), 2.91 (d, 1H, J = 13.0 Hz),
2.35 (t, 1H, J = 12.2 Hz), 1.37, 0.97, 0.97, 0.97, 0.89, 0.83, 0.78 (s, each 3H); 13C NMR (100
MHz, MeOD): δ 180.10, 173.47, 157.75, 144.98, 124.05, 121.26, 80.12, 79.65, 68.46, 56.70,
47.57, 47.46, 42.80, 42.36, 40.66, 38.11, 35.17, 34.26, 33.86, 33.67, 31.63, 28.77, 27.85, 26.54,
24.53, 24.20, 23.87, 19.47, 17.78, 16.35, 15.94; ESI-HRMS Calcd for C36H56NO7 [M+H]+:
614.4057; found 614.4061.
4.2.19 N-(3β,16α-dihydroxy-olean-12-en-28-oyl)-6-amino-6-deoxy-2,3-bis-benzyloxy-L-ascorbic
acid (37)
Compound 37 was prepared from 33 (257 mg, 0.55 mmol) and 11 (176 mg, 0.50 mmol)
according to general procedure C. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield 37 as a white solid (209 mg,
52 %). 1H NMR (400 MHz, CDCl3): δ 7.36-7.31 (m, 8H), 7.22 (m, 2H), 6.77 (t, 1H, J = 5.3 Hz),
5.51 (m, 1H), 5.17 (d, 1H, J = 11.8 Hz), 5.06 (s, 2H), 4.54 (d, 1H, J = 1.4 Hz), 4.35 (br s, 1H),
3.95 (br s, 1H), 3.51 (m, 1H), 3.37 (m, 1H), 3.21 (m, 1H), 2.79 (d, 1H, J = 10.6 Hz), 2.20 (t, 1H, J
= 13.4 Hz), 1.34, 0.98, 0.94, 0.90, 0.87, 0.77, 0.74 (s, each 3H); 13C NMR (100 MHz, CDCl3): δ
179.44, 169.59, 157.04, 143.42, 135.86, 135.28, 128.93, 128.53, 127.66, 123.24, 121.21, 78.79,
76.58, 74.75, 73.86, 73.36, 68.78, 55.11, 49.18, 46.66, 43.01, 41.78, 41.52, 39.55, 38.67, 38.49,
36.83, 35.18, 34.98, 32.49, 30.08, 29.15, 27.99, 27.02, 26.78, 25.30, 23.32, 18.16, 16.96, 15.57;
ESI-HRMS Calcd for C50H68NO8 [M+H]+: 810.4939, found 810.4930.
4.2.20 N-(3β,16α-dihydroxy-olean-12-en-28-oyl)-6-amino-6-deoxy-L-ascorbic acid (38)
Compound 38 was prepared from 37 (100 mg, 0.12 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 38 as a white solid
(62 mg, 80 %). 1H NMR (400 MHz, MeOD): δ 7.31 (t, 1H, J = 5.1 Hz), 5.50 (s, 1H), 4.58 (d, 1H,
J = 1.6 Hz), 4.32 (br s, 1H), 3.93 (br s, 1H), 3.44 (m, 1H), 3.16 (dd, 1H, J = 4.8, 11.1 Hz), 2.91
19

ACCEPTED MANUSCRIPT
(dd, 1H, J = 3.1, 13.6 Hz), 2.33 (t, 1H, J = 13.2 Hz), 1.38, 0.98, 0.98, 0.96, 0.89, 0.83, 0.78 (s,
each 3H); 13C NMR (100 MHz, MeOD): δ 180.81, 173.16, 154.88, 144.77, 124.28, 119.84, 79.62,
78.20, 75.44, 68.93, 56.76, 50.21, 49.85, 48.20, 47.98, 42.85, 42.38, 40.81, 39.94, 39.81, 38.07,
36.23, 36.10, 33.94, 33.27, 31.39, 31.15, 28.73, 27.84, 27.38, 25.62, 24.49, 19.43, 17.86, 16.33,
16.17; ESI-HRMS Calcd for C36H56NO8 [M+H]+: 630.4000, found 630.3991.
4.2.21 N-(3β-hydroxy-urs-12-en-28-oyl)-6-amino-6-deoxy-2,3-bis-benzyloxy-L-ascorbic acid (39)
Compound 39 was prepared from 34 (165 mg, 0.36 mmol) and 11 (117 mg, 0.34 mmol)
according to general procedure C. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield 39 as a white solid (109 mg,
40 %). 1H NMR (400 MHz, CDCl3): δ 7.38-7.32 (m, 8H), 7.22 (m, 2H), 6.34 (t, 1H, J = 5.4 Hz),
5.31 (br s, 1H), 5.15 (d, 2H, J = 11.7 Hz), 5.09 (s, 2H), 4.55 (d, 1H, J = 2.0 Hz), 3.95 (br s, 1H),
3.52 (m, 1H), 3.31 (m, 1H), 3.21 (m, 2H), 1.09, 0.99, 0.95, 0.87, 0.86, 0.78, 0.72 (s, each 3H); 13C
NMR (100 MHz, CDCl3): δ 180.03, 169.25, 156.86, 139.42, 135.93, 135.35, 129.07, 128.63,
128.59, 127.74, 125.92, 121.26, 78.92, 76.55, 73.84, 73.41, 69.37, 55.09, 53.76, 47.80, 47.47,
43.06, 42.39, 39.68, 39.49, 38.97, 38.71, 38.58, 37.30, 36.88, 32.67, 30.79, 28.10, 27.78, 27.14,
24.74, 23.35, 23.27, 21.17, 18.21, 17.17, 16.85, 15.60, 15.44; ESI-HRMS Calcd for C50H68NO7
[M+H]+: 794.4990, found 794.4979.
4.2.22 N-(3β-hydroxy-urs-12-en-28-oyl)-6-amino-6-deoxy-L-ascorbic acid (40)
Compound 40 was prepared from 39 (85 mg, 0.11 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 40 as a white solid
(57 mg, 86 %). 1H NMR (400 MHz, MeOD): δ 7.30 (t, 1H, J = 5.2 Hz), 5.36 (br s, 1H), 4.60 (br s,
1H), 3.95 (m, 1H), 3.38 (m, 2H), 3.15 (dd, 1H, J = 4.3, 10.9 Hz), 2.14 (d, 1H, J = 10.7 Hz), 2.07
(dd, 1H, J = 4.0, 13.5 Hz), 1.13, 0.98, 0.96, 0.92, 0.91, 0.82, 0.78, 0.74 (s, each 3H); 13C NMR
(100 MHz, MeOD): δ 180.98, 173.07, 154.33, 139.86, 127.46, 120.05, 79.69, 78.02, 69.03, 56.70,
54.33, 43.56, 43.35, 40.89, 40.22, 39.99, 39.83, 38.55, 38.09, 34.18, 31.91, 29.00, 28.77, 27.89,
25.34, 24.40, 24.04, 21.56, 19.44, 17.93, 17.69,16.36, 16.03; ESI-HRMS Calcd for C36H56NO7
[M+H]+: 614.4051, found 614.4057.
4.2.23 N-(3β-hydroxy-lup-20(29)-en-28-oyl)-6-amino-6-deoxy-2,3-bis-benzyloxy-L-ascorbic acid
(42)
20

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Compound 42 was prepared from 41 (230 mg, 0.72 mmol) and 11 (179 mg, 0.50 mmol)
according to general procedure C. The residue was purified by gradient mixture of petroleum
ether/ethyl acetate with increasing polarity as an eluent to yield 42 as a white solid (210 mg,
53 %). 1H NMR (400 MHz, CDCl3): δ 7.38-7.34 (m, 8H), 7.23 (m, 2H), 6.11 (br s, 1H), 5.20 (m,
4H), 4.73 (s, 1H), 4.59 (s, 1H), 4.56 (m, 1H), 4.12 (dd, 1H, J = 7.1, 14.3 Hz), 3.97 (m, 1H), 3.57
(m, 1H), 3.42 (m, 1H), 3.17 (dd, 2H, J = 4.9, 11.0 Hz), 3.07 (td, 1H, J = 4.3, 11.9 Hz), 2.42 (td,
1H, J = 3.2, 12.6 Hz), 1.68, 1.26, 0.96, 0.95, 0.87, 0.79, 0.75 (s, each 3H), 0.67 (d, 1H, J = 6.1
Hz ); 13C NMR (100 MHz, CDCl3): δ 178.31, 169.28, 156.89, 150.75, 135.93, 135.35, 129.07,
128.71, 128.63, 128.60, 127.85, 121.30, 109.42, 78.96, 73.82, 73.52, 69.54, 60.39, 55.73, 55.35,
50.59, 50.04, 46.62, 42.69, 42.45, 40.70, 38.83, 38.69, 38.22, 37.69, 37.17, 34.35, 33.67, 30.82,
29.68, 29.45, 29.32, 27.96, 27.37, 26.89, 25.58, 21.03, 20.90, 19.47, 18.27, 16.12, 16.00, 15.36,
14.62, 14.17; ESI-HRMS Calcd for C50H68NO7 [M+H]+: 794.4990; found 794.4980.
4.2.24 N-(3β-hydroxy-lup-20(29)-en-28-oyl)- 6-amino-6-deoxy-L-ascorbic acid (43)
Compound 43 was prepared from 42 (100 mg, 0.13 mmol) according to general procedure B.
The residue was purified by column chromatography (CH2Cl2/MeOH) to yield 43 as a white solid
1
(57 mg, 72 %). H NMR (400 MHz, MeOD): δ 7.75 (t, 1H, J = 5.5 Hz), 4.60 (s, 1H), 3.95 (m,
1H), 3.44 (m, 1H), 3.13 (dd, 1H, J = 5.0, 11.2 Hz), 2.59 (td, 1H, J = 3.5, 12.8 Hz), 2.28 (m, 1H),
2.14 (m, 1H), 0.99, 0.97, 0.95, 0.88, 0.86, 0.77, 0.75 (s, each 3H), 0.71 (d, 1H, J = 9.4 Hz); 13C
NMR (100 MHz, MeOD): δ 180.18, 173.33, 157.97, 119.94, 79.69, 77.90, 69.26, 57.47, 56.89,
51.95, 50.93, 49.85, 45.31, 43.69, 43.07, 42.08, 40.12, 39.96, 39.69, 38.77, 38.34, 35.71, 34.00,
31.22, 30.69, 28.61, 28.42, 28.05, 23.98, 23.40, 22.27, 19.47, 16.88, 16.74, 16.11, 14.94;
ESI-HRMS Calcd for C36H58NO7 [M+H]+: 616.4208; found 616.4204.
4.3 Biological Assays
4.3.1 CPE reduction assay
The assay was performed as previously described [50] with some modifications. MDCK
cells were seeded into 96-well plates, incubated overnight and infected with influenza virus (MOI
= 0.1). Cells were suspended in DMEM supplemented with 1% FBS, containing test compound
and 2 ꢀg/mL TPCK-treated trypsin, and a final DMSO concentration of 1% was added in each
well. After 40 h of incubation, CellTiter-Glo reagent (Promega Corp., Madison, WI, USA) was
21

ACCEPTED MANUSCRIPT
added, and the plates were read using a plate reader (Tecan Infinite M2000 PRO; Tecan Group
Ltd., Mannedorf, Switzerland).
4.3.2 Cytotoxicity test
Cells were grown in 96-well plates containing 1% FBS overnight and then cultured with
increasing amounts of the test compounds for 40 h. Cytotoxicity was assessed with the
CellTiter-Glo assay as above described.
4.3.3 Time-of-addition experiment
MDCK cells were seeded into 6-well plates at a density of 5×105 cells per well 24 h prior to
infection and incubated at 37 °C under 5% CO2. Then MDCK cells were infected with WSN virus
at an MOI of 1. Compound was added at 0-10, 0-2, 2-5, 5-8 or 8-10 h post infection. The cell
lysates were harvested and applied to Western blotting for NP analysis.
4.3.4 HI assay
Compound from a 3-fold serial dilution in saline was mixed with an equal volume of
influenza virus (2 HA units) in the V-bottomed 96-well microplates. Subsequently, 50 ꢀL of
freshly prepared chicken red blood cells (cRBC) (1% v/v in saline) was added to each well. The
mixture was incubated for 30 min at room temperature before observing cRBC aggregation on the
plate.
4.3.5 SPR
Interactions between the influenza HA and the compounds were analyzed using the Biacore
T200 system (GE Healthcare, Uppsala, Sweden) at 25 °C. Recombinant influenza HA (Sino
Biological Inc., Beijing, China) was immobilized on a sensor chip (CM5) using an amine
coupling kit (GE Healthcare, Buckinghamshire, UK). Final HA immobilized levels were typically
~16,000 RU. Subsequently, compounds were injected as analytes at various concentrations, and
PBS-P (10 mM phosphate buffer with 2.7 mM KCl and 137 mM NaCl, 0.05% surfactant P20, pH
4.5) was used as running buffer. For binding studies, analytes were applied at corresponding
concentrations in running buffer at a flow rate of 30 ꢀL/min with a contact time of 60 s and a
dissociation time of 60 s. Chip platforms were washed with running buffer and 50% DMSO. Data
were analyzed with the Biacore evaluation software (T200 version 1.0) by curve fitting using a
binding model of 1:1.
4.3.6 Docking simulation
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The docking simulation was performed via AutoDock 4.2 (Scripps Research Institute) [28],
and assessed by OpenSource PyMOL 1.5.x (Schrödinger, LLC) and PoseView web service
(Universität Hamburg) [39]. Afterwards, structure of the protein (Protein Data Bank: 1RVT) was
obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Protein
preparation was performed as follows. After the removal of HETATM atoms, hydrogen atoms
and the crystal cells, the protein was further processed via AutoDock Tools 1.5.6 rc3 (ADT,
Scripps Research Institute) to add Polar hydrogen and Kollman charge. Ligand preparation was
performed as follows. Polar hydrogen and Gasteiger charge were given, and there was no further
adjustment on the ligand torsion tree. Grid box covering the HA head was prepared with ADT
with parameters as follows: grid box center coordinate: 87.320, -9.696, -34.751; box size: 74 × 78
× 80; grid point spacing: 0.375 Å. Lamarckian genetic algorithm was applied to the docking
simulation with modifications of the parameters described below due to the compounds’ highly
flexible nature: number of individuals in population: 300; maximum number of energy
evaluations: 25,000,000; number of GA runs: 100. A hundred docking output conformations for
one compound were clustered with a maximum RMS tolerance of 2.0 Å. To avoid isolated
conformations, the best binding conformation from the largest cluster was used for further
analysis.
Acknowledgements
This work was supported by the National Basic Research Program of China (973 Program;
Grant No. 2010CB912300) and the National Natural Science Foundation of China (Grants Nos.
81361168002, 81373271, 81573269 and 21572015).
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