Synthesis, structure activity relationship and in vitro anti-influenza virus activity of novel polyphenol-pentacyclic triterpene conjugates

Article abstract of DOI:10.1016/j.ejmech.2018.12.006

It is urgently necessary to develop more effective anti-influenza agents due to the continuous emergence of drug-resistant strains of influenza virus. Our earlier studies have identified that certain pentacyclic triterpene derivatives are effective inhibitors of influenza virus infection. In the present study, a series of C-28 modified pentacyclic triterpene derivatives via conjugation with a series of polyphenols were synthesized, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in MDCK (Madin-Darby canine kidney) cells were evaluated. Four compounds 23m, 23o, 23q and 23s displayed robust anti-influenza potency with averaged IC₅₀ values at the low-micromole level, surpassing the potency of oseltamivir. In addition, the in vitro cytotoxic activity of the four conjugates against MDCK cells showed no toxicity at 100 μM. Further mechanism studies of compound 23s, one of the best representative conjugates with IC₅₀ value of 5.80 μM and a selective index (SI) value of over 17.2, by hemagglutination inhibition (HI), surface plasmon resonance and molecular modeling indicated that this conjugate bound tightly to the viral envelope hemagglutinin (K_D = 15.6 μM), thus blocking the invasion of influenza viruses into host cells.

Full text of DOI:10.1016/j.ejmech.2018.12.006

Accepted Manuscript
Synthesis, structure activity relationship and in vitro anti-influenza virus activity of
novel polyphenol-pentacyclic triterpene conjugates
Haiwei Li, Man Li, Renyang Xu, Shouxin Wang, Yongmin Zhang, Lihe Zhang, Demin
Zhou, Sulong Xiao
PII:
S0223-5234(18)31043-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.006
EJMECH 10939
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 October 2018
Revised Date: 30 November 2018
Accepted Date: 2 December 2018
Please cite this article as: H. Li, M. Li, R. Xu, S. Wang, Y. Zhang, L. Zhang, D. Zhou, S. Xiao, Synthesis,
structure activity relationship and in vitro anti-influenza virus activity of novel polyphenol-pentacyclic
triterpene conjugates, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2018.12.006.
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ACCEPTED MANUSCRIPT
Graphic abstract
OH
OH
O
H
N
N
OH
OH
H
HO
OH
O
O
OH
R1
HO
O
O
Anti-IAV entry activity
(EC50 = 5.80 - 15.68 M)
R3
O
OH
OH
O
O
HO
R2
O
HO

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, structure activity relationship and in vitro anti-influenza virus activity
of novel polyphenol-pentacyclic triterpene conjugates
Haiwei Lia,1, Man Lia,1, Renyang Xua,1, Shouxin Wangb, Yongmin Zhangc, Lihe Zhanga,
Demin Zhoua,*, Sulong Xiaoa,b*
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
bState Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
cSchool of Pharmacy, Jining Medical University, Rizhao 276826, China
dSorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4
place Jussieu, 75005 Paris, France
Running title: polyphenol-pentacyclic triterpene conjugates
* Corresponding author. Tel.: 86-10-8280-5063; fax: 86-10-8280-5857.
E-mail address: deminzhou@bjmu.edu.cn (D. Zhou); slxiao@bjmu.edu.cn (S. Xiao)
1 These authors contributed equally to this work.
1

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Abstract:
It is urgently necessary to develop more effective anti-influenza agents due to the
continuous emergence of drug-resistant strains of influenza virus. Our earlier studies
have identified that certain pentacyclic triterpene derivatives are effective inhibitors of
influenza virus infection. In the present study, a series of C-28 modified pentacyclic
triterpene derivatives via conjugation with a series of polyphenols were synthesized,
and their antiviral activities against influenza A/WSN/33 (H1N1) virus in MDCK
(Madin-Darby canine kidney) cells were evaluated. Four compounds 23m, 23o, 23q
and 23s displayed robust anti-influenza potency with averaged IC50 values at the
low-micromole level, surpassing the potency of oseltamivir. In addition, the in vitro
cytotoxicity activity of the four conjugates against MDCK cells showed no toxicity at
100 µM. Further mechanism studies of compound 23s, one of the best representative
conjugates with IC50 value of 5.80 µM and a selective index (SI) value of over 17.2,
by hemagglutination inhibition (HI), surface plasmon resonance and molecular
modeling indicated that these conjugates bound tightly to the viral envelope
hemagglutinin (KD = 15.6 µM), thus blocking the invasion of influenza viruses into
host cells.
Keywords: Influenza virus, pentacyclic triterpene, polyphenol, entry inhibitor,
hemagglutinin
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1. Introduction
As a single-stranded, helically shaped RNA virus, influenza virus belongs to the
Orthomyxoviridae family. It can be divided into three classes, A, B and C, according
to its internal proteins of nucleoprotein and matrix protein. Influenza A virus, typically
encountered more frequently than types B and C, can be further subdivided into
different subtypes according to the combinations between the hemagglutinin (HA)
(H1~H18) and neuraminidase (NA) (N1~N11), the proteins on the surface of the
virus.[1] As a result of genetic variations brought by antigenic shift and sporadic
antigenic drift, influenza A viruses possess high pathogenicity and are the main cause
of annual epidemics and occasional pandemics of respiratory diseases worldwide.[2]
In the 20th century, three influenza pandemics occurred: Spanish influenza in 1918
(~50 million deaths), Asian influenza in 1957 (two million deaths), and Hong Kong
influenza in 1968 (one million deaths).[3] The first pandemic of 21st century occurred
in April 2009, and a new influenza A virus from H1N1 subtype emerged in Mexico
and rapidly spread across the entire globe.[4] Worldwide, these annual epidemics are
estimated to result in approximately 3 to 5 million cases of severe illness, and about
290,000 to 650,000 respiratory deaths.[5] Vaccination is currently the most effective
strategy to prevent influenza infection. However, influenza viruses can be escaped
from the neutralizing activity of previously developed antibodies by antigenic drift
and antigenic shift, thus making them ineffective against the infection in the future.[2]
Two classes of anti-influenza drugs approved by FDA including M2 ion channel
inhibitors (amantadine and rimantadine) and NA inhibitors (oseltamivir, zanamivir
and peramivir) have several limitations in clinical practice, especially the rapid global
spread of drug-resistance,[6, 7] emphasizing the fact that we are constantly required to
develop new antiviral agents with different modes of action to currently used
anti-influenza drugs. The HA, a spike-shaped glycoprotein which extends from the
surface of the virus, plays an important role during the early stage of influenza
infection. It can bind to sialylglyconjugates on the surface of the host cells, causing
endocytosis of the virus and subsequently membrane fusion of the virus and the host
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cells.[8] The crucial role of it in the viral life-cycle makes it an attractive target for the
development of therapeutics to inhibit influenza virus infection.[9, 10]
Polyphenols are widely distributed in various plant species, including fruits and
vegetables, with one or more hydroxylated aromatic rings in monomer or polymer
form. As dietary antioxidants, plant polyphenols are well known for their potentials to
contribute to better health.[11] They have been shown potential antioxidant property
and can protect cell constituents against oxidative damage, thus limiting the risk of
various degenerative diseases associated with oxidative stress.[12] Recently, the
antiviral activity and the mode of action of polyphenols, such as CYSTUS052,[13]
gallic acid (1),[14] epigallocatechin gallate (EGCG, 2) and its derivatives (3 and
4),[15-18] 2,3,4,6-tetragalloylglucose (5) and its analog (6),[19, 20]
2,3,4-trihydroxybenzoyl dimer (7) and its analog (8), 2,3,4-trihydroxybenzoyl trimer
(9),[21] curcumin (10) and its derivatives (11 and 12),[22, 23] betulinic acid-gallic
acid conjugates (13 and 14), [24] have attracted increasing attention (Figure 1).
CYSTUS052, a well-known polyphenol-rich extract of Cistus incanus, can inhibit
avain and human influenza A virus replication in vitro.[13, 25] Moreover,
CYSTUS052-treated patients with influenza A and B virus infection show reduction
in symptoms and inflammatory markers in
a
prospective, randomized,
placebo-controlled clinical study.[26] EGCG (2), one of the major components of
green tea, can bind to a range of virion glycoproteins, thus inhibiting the invasion of
HCV, HIV and influenza virus into host cells.[16, 18, 27-30] Therefore, polyphenols
have been considered as a useful synthetic precursor to many molecules of potential
biological utility.[31, 32]
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Figure 1. Chemical structures of some polyphenols with antiviral activity.
Pentacyclic triterpenes are described as secondary plant metabolites formed by
the regular cyclization of squalene epoxide and widely found in a variety of
organisms ranging from bacteria, fungi, plants to mammals.[33] In recent years, the
ecological role and biological activities of pentacyclic triterpenes have begun to
attract more general interest. They are often the active constituents of many important
medicinal plants, such as Panax ginseng, Glycyrrhiza uralensis and Radix Bupleuri,
etc. Many compounds of this group show a wide range of biological activities,[34]
including hepatoprotective,[35] antiviral,[36] antitumor,[37] anti-inflammatory,[38]
antimicrobial,[39] and so on. In our previous study,[40] we have discovered that
oleanane-type triterpenoid Q8 (15) (Figure 2) shows significant activity against
A/WSN/33 (H1N1) virus invasion into MDCK host cells based on the screening of a
mini-library of triterpene-conjugates with EC50 values of 5.0 µM. Further study has
indicated that they bind tightly to the pocket of viral envelope HA protein for sialic
acid receptor, thus blocking the attachment of viruses to host cells.
Based on these literature results, and as part of our persistent efforts toward the
development of potential antiviral entry inhibitors derived from natural
product,[40-44] we described herein the design and synthesis of a total of 40
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polyphenol-pentacyclic triterpene derivatives and evaluated their in vitro
anti-influenza A virus (IAV) activities (Figure 2).
Figure 2. Structures of lead compound Q8 (15) and polyphenols-pentacyclic
triterpene derivatives.
2. Results and discussion
2.1 Chemistry
Scheme 1 depicts the synthesis of polyphenol-pentacyclic triterpene conjugates
23a-23l, in which gallic acid (16a), 3,5-dihydroxybenzoic acid (16b) and
4-hydroxybenzoic acid (16c) were attached to four pentacyclic triterpenes, including
oleanolic acid (OA), echinocystic acid (EA), ursolic acid (UA) and betulinic acid
(BA), at the C28-COOH through a flexible ethylenediamine linker. Firstly,
3,4,5-tribenzyloxybenzoic acid (19a) was prepared from commercially available
gallic acid (16a) in 56% yield via a three-step process described by Ren and
colleagues.[45]
Compound
19a
was
then
coupled
with
tert-butyl
(2-aminoethyl)carbamate using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC) and N,N-(dimethylamino)pyridine (DMAP) as the coupling agents, followed
by removal of the Boc group with 3M HCl in EtOAc to provide the key intermediate
21a, which was used directly in the next step without further purification. Compounds
22a, 22d, 22g and 22j were synthesized by condensation of 21a with four pentacyclic
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triterpene, followed by debenzylation using H2/Pd-C to afford polyphenol-pentacyclic
triterpene conjugates 23a, 23d, 23g and 23j with yields ranging from 49 to 74%.
Similarly, the conjugation of 3,5-dihydroxybenzoic acid (16b) and 4-hydroxybenzoic
acid (16c) with four pentacyclic triterpenes in a similar manner afforded conjugates
23b-23c, 23e-23f, 23h-23i and 23k-23l.
R3
R2
R3
R2
R3
R2
R3
R2
R3
R2
R1
(b)
(c)
(d)
(a)
R1
R1
R1
R1
H
N
NHBoc
COOH
COOCH3
17a: R1 = R2 = R3 = OH
16b: R1 = R3 = OH, R2 = H 17b: R1 = R3 = OH, R2 = H 18b: R1 = R3 = OBn, R2 = H
16c: R1 = R3 = H, R2 = OH 17c: R1 = R3 = H, R2 = OH 18c: R1 = R3 = H, R2 = OBn
COOCH3
COOH
O
16a: R1 = R2 = R3 = OH
18a: R1 = R2 = R3 = OBn
19a: R1 = R2 = R3 = OBn
19b: R1 = R3 = OBn, R2 = H
19c: R1 = R3 = H, R2 = OBn
20a: R1 = R2 = R3 = OBn
1 = R3 = OBn, R2 = H
20c: R1 = R3 = H, R2 = OBn
20b:
R
R5
R3
R4
R2
R1
(f)
(e)
O
H
H
N
N
R3
NH2
N
H
O
O
21a: R1 = R2 = R3 = OBn
21b: R1 = R3 = OBn, R2 = H
21c: R1 = R3 = H, R2 = OBn
R2
R6
HO
(g)
R1
22d: R1 = R2 = R3 = OBn, R4 = R6 = H, R5 = CH3
23d: R1 = R2 = R3 = OH, R4 = R6 = H, R5 = CH3
22e: R1 = R3 = OBn, R2 = R4 = R6 = H, R5 = CH3
23e: R1 = R3 = OH, R2 = R4 = R6 = H, R5 = CH3
22f: R1 = R3 = R4 = R6 = H, R2 = OBn, R5 = CH3
23f: R1 = R3 = R4 = R6 = H, R2 = OH, R5 = CH3
22g: R1 = R2 = R3 = OBn, R4 = H, R5 = CH3, R6 = OH
23g: R1 = R2 = R3 = R6 = OH, R4 = H, R5 = CH3
22h: R1 = R3 = OBn, R2 = R4 = H, R5 = CH3, R6 = OH
23h: R1 = R3 = R6 = OH, R2 = R4 = H, R5 = CH3
22i: R1 = R3 = R4= H, R2 = OBn, R5 = CH3, R6 = OH
23i: R1 = R3 = R4 = H, R2 = R6 = OH, R5 = CH3
22j: R1 = R2 = R3 = OBn, R4 = CH3, R5 = R6 = H
23j: R1 = R2 = R3 = OH, R4 = CH3, R5 = R6 = H
22k: R1 = R3 = OBn, R2 = R5 = R6 = H, R4 = CH3
23k: R1 = R3 = OH, R2 = R5 = R6 = H, R4 = CH3
22l: R1 = R3 = R5 = R6 = H, R2 = OBn, R4 = CH3
23l: R1 = R3 = R5 = R6 = H, R2 = OH, R4 = CH3
(f)
(g)
(g)
(g)
(g)
(g)
(g)
(g)
(g)
X
O
H
N
R3
R2
N
H
O
HO
(g)
R1
22a: R1 = R2 = R3 = OBn, X = double bond
23a: R1 = R2 = R3 = OH, X = single bond
22b: R1 = R3 = OBn, R2 = H, X = double bond
23b: R1 = R3 = OH, R2 = H, X = single bond
22c: R1 = R3 = H, R2 = OBn, X = double bond
23c: R1 = R3 = H, R2 = OH, X = single bond
(g)
(g)
Scheme 1. Reagents and conditions: (a) MeOH, H2SO4, 120 oC, 80-88%; (b) BnBr,
K2CO3, DMF, 80oC, 75-82%; (c) NaOH, H2O-MeOH-dioxane, reflux, 85-96%; (d)
tert-butyl (2-aminoethyl) carbamate, EDC, DMAP, DCM, rt, 80-88%; (e) 3M
HCl/EtOAc, DCM, rt, 90-95%; (f) OA, EA, UA or BA, EDC, DMAP, DCM, rt,
56-80%; (g) Pt/C, H2, 3 atm, rt, 88-93%.
However, the introduction of ethylenediamine at the carboxyl group of 16d and
16e in a similar manner as described in Scheme 1 failed to obtain the intermediates
21d and 21e. Then a direct and shorter synthetic route involving only one-step
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reaction and non-protected polyphenol was attempted (Scheme 2). After removal of
the Boc protecting group of 20d and 20e by treatment with 3 M HCl in EtOAc, the
intermediates were coupled with pentacyclic triterpene to afford 23m-23t with a
three-step yield of 56-72%, calculated from 16d and 16e.
Scheme 2. Reagents and conditions: (a) see Scheme 1; (b) tert-butyl (2-aminoethyl)
carbamate, EDC, DMAP, DCM, rt; (c) 3 M HCl/EtOAc, DCM, rt; (d) BA, OA, EA or
UA, EDC, DMAP, DCM, rt.
The cinnamic acid-pentacyclic triterpene conjugates 23u-23ab were obtained
readily by a procedure similar to that discussed for 23m-23t (Scheme 3).
Scheme 3. Reagents and conditions: (a) tert-butyl (2-aminoethyl) carbamate, EDC,
DMAP, DCM, rt; (b) 3 M HCl/EtOAc, DCM, rt; (c) BA, OA, EA or UA, EDC,
DMAP, DCM, rt.
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1
The H NMR, 13C NMR and HRMS spectra of the synthesized compounds
23a-23ab are shown in supplementary materials.
2.2 Biological assays
2.2.1 Anti-influenza virus activity of polyphenol-pentacyclic triterpene conjugates
The CellTiter-Glo reduction assay and the cytopathic effect (CPE) assay were
utilized in parallel to screen the synthesized conjugates as described previously.[40]
The CellTiter-Glo screening, an assay monitoring cell proliferation, was utilized to
screen and exclude compounds with significant toxicity toward MDCK cells. The
CPE screening, an assay measuring the damage to host cells during virus invasion,
was utilized to screen and identify compounds displaying a CPE reduction on
influenza A/WSN/33 virus. Figure 3A shows that no toxicity was observed when the
hydroxyl groups of polyphenol were protected by benzyl groups at the final
concentration of 50 µM (compounds 22a-22l). However, the removal of the benzyl
groups of polyphenol (23a-23aa) resulted in the significant cytotoxicity at the same
concentration, except for compounds 23f, 23m, 23o, 23q, 23s, 23u, 23v, 23y, 23z,
23aa and 23ab. indicating that the substituted groups of polyphenol had important
effect on the toxicity of polyphenol-pentacyclic triterpene conjugates. Luo et al. have
also
found
that
triterpene
derivatives,
in
which
3-(4-hydroxy-3-methoxyphenyl)acrylic acid (ferulic acid) is linked with C3-OH of
triterpene, show moderate cytotoxicity against MCF-7 (Michigan Cancer
Foundation-7) cells.[46]
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Figure 3. Inhibitory effects of polyphenol-pentacyclic triterpene conjugates against
influenza A/WSN/33 (H1N1) virus. (A) The cytotoxic effect-based screening of
conjugates 22a-22l and 23a-23ab using CellTiter-Glo® assay. DMSO acted as
negative control. (B) The CPE-based screening of conjugates 22a-22l and 23a-23ab.
MDCK was utilized as the host cell to test A/WSN/33 (H1N1) virus infection; 0.5%
DMSO (final concentration) was used as negative control, and OSV (oseltamivir) was
utilized as positive control. Error bars indicate standard deviations of triplicate
experiments.
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Except for cytotoxic compounds, the other pentacyclic triterpene-polyphenol
conjugates against the influenza A/WSN/33 (H1N1) virus were evaluated by the CPE
reduction assay. Compared with DMSO, influenza A/WSN/33 virus causes a severe
CPE in MDCK-infected cells (Figure 3B). Compounds 22d, 22g, 23a, 23m, 23o, 23q
and 23s significantly reduced the CPE induced by infection in MDCK cells,
indicating that those compounds were able to protect cells from influenza
virus-induced CPE. Analysis of the structure-activity relationships revealed that four
2-hydroxybenzoic acid-pentacyclic triterpene conjugates 23m, 23o, 23q and 23s were
active against A/WSN/33 (H1N1) virus, indicating the importance of the
2-hydroxybenzoic acid for the anti-influenza activity. In contrast, the shift of the
hydroxyl from position 2 to position 4 of benzoic acid, or introduction of an extra
hydroxyl at position 4 all showed no improvement but cytotoxic effect on potency.
The example of the effect of compound 23s on CPE reduction is shown in Figure
4.
Figure 4. Compound 23s inhibited the influenza A/WSN/33 (H1N1) virus-induced
CPE in MDCK cells. The antiviral efficacy of 23s was observed in terms of cellular
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morphology at 40 h post-infection. MDCK cells were not inflected (A-B) or infected
with influenza A/WSN/33 (H1N1) (C-D).
Seven compounds, 22j, 23m, 23o, 23q, 23s, 23v and 23y identified with high
inhibition rates in the initial screening were selected for the dose response assays. The
IC50 of OSV used as a positive control was 16.5 µM.[42] Among them, four
compounds (23m, 23o, 23q and 23s) showed much stronger anti-influenza A/WSN/33
(H1N1) activities with IC50 at micromole level (Table 1). Compound 23s showed the
strongest inhibitory activities with IC50 value of 5.80 µM in this study. In addition, all
the five compounds showed the CC50, which were all higher than 100 µM.
Table 1. In vitro anti-influenza virus activity and cytotoxicity of the active
compounds
Compounds
22j
IC50 (µM)a
32.87
9.55
CC50 (µM)b
>100
>100
23m
23o
7.65
>100
5.84
>100
23q
5.80
>100
23s
15.68
18.34
16.5
>100
23v
>100
23y
OSV c
> 200
a IC50 was measured using influenza A virus (A/WSN/33/H1N1) with MDCK cells by
b
CPE assay. The cytotoxicity (CC50) of all compounds was measured with MDCK
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cells by CellTiter-Glo assay. c Data taken from the reference.[42]
2.2.2 Conjugate 23s inhibits virus-induced hemagglutination
It is well known that influenza virus HA plays an important role during viral
invasion into host cells. HA protein can bind to sialic acid on the surface of red blood
cells
(RBCs),
causing
agglutination.
To
investigate
whether
the
polyphenol-pentacyclic triterpene conjugates blocked the ability of viral particles to
bind to cell receptors, a hemagglutination inhibition (HI) assay was performed. Like
the capability as anti-HA antibody, conjugate 23s inhibited the binding of influenza
virus A/WSN/33 to RBCs in a concentration-dependent manner, indicating that the
compound directly acted on influenza viral particles (Figure 5).
Figure 5. Hemagglutination inhibition assay. (A) Conjugate 23s was diluted as
indicated in 96-well micro titer plates using PBS (Phosphate Buffer Solution) as
negative control. (B) The HI activity of anti-HA antibody was tested in a similar
manner.
2.2.3 Conjugate 23o and 23s interact with HA
Surface plasmon resonance (SPR) was used to screen the interaction between a
variety of affinity ligands and HA protein.[47] In our study, recombinant influenza
HA proteins of influenza A/California/04/2009 (H1N1) (bought from Sino Biological
Inc, Beijing, China) were immobilized to a carboxymethylated dextran CM5 chip
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surface using an amine coupling kit (GE Healthcare, Buckinghamshire, UK). Then
compound 23o or 23s at various concentrations was passed over the immobilized HA
surface. As shown in Figure 6, the binding curves are fitted well with the Langmuir
equation for monovalent binding, which allows the determination of the apparent
dissociation constant, KD. The calculated KD values for 23o and 23s binding with HA
protein were 36.0 and 15.6 µM, respectively, indicating that 23s bound more tightly to
HA than 23o. In addition, we found compound 23o produced the similar resonance
unit signal response to 23s, but the binding of HA with 23s was faster than that of 23o.
In contrast, a lower binding affinity was observed between compound UA and HA
with a calculated KD value of 46.5 µM (SI Figure 1), almost one third as potent as that
of 23s, suggesting that 2-hydroxybenzoic acid enhanced the binding of UA with HA
protein.
Figure 6. Characterization of the affinity between active compounds (23o and 23s)
and HA protein, which were immobilized on a CM5 sensor chip, based on the SPR
assay. Their KD values are labeled on the corresponding curves.
2.2.4 Molecular docking analysis
To further understand the molecular basis of the inhibitory properties of
polyphenol-pentacyclic triterpene conjugates, we performed the docking experiments
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of the most active compound 23s and its parent compound UA with influenza HA
protein, which was obtained from the RCSB (Research Collaboratory for Structural
Bioinformatics) Protein Data Bank, http://www.rcsb.org/pdb/home/home.do, using
AutoDock 4.2 program (The scripps research institute, La Jolla, CA, USA).[40]
The docked conformations of HA-23s were determined based on the minimum
free energy analyses. As shown in Figure 7, the modeled structure of HA complexed
with 23s indicated that C3-OH of UA formed a hydrogen bond with Gln226, while the
phenolic hydroxyl of salicylic acid also formed two hydrogen bonds with Lys156 and
Gly158, respectively. According to the computer aided docking data, 23s occupied the
binding pocket for sialic acid with an estimated binding energy of -42.43 kcal/mol,
while the estimated binding energy between HA and UA, the parent compound of 23s,
is -23.50 kcal/mol as a result of no hydrogen bonds formed between UA and Lys156
and Gly158 (SI Figure 2).
3. Conclusions
In the present study, we reported the synthesis and characterization of a series of
polyphenol-pentacyclic triterpene conjugates and tested their anti-influenza
A/WSN/33 (H1N1) virus activities in MDCK cells. All the protected
polyphenol-pentacyclic triterpene conjugates 22a-22l showed no cytotoxicity to
MDCK cells, while their deprotected derivatives showed certain cytotoxicity. The
subsequent SAR studies showed that four conjugates 23a, 23m, 23o, 23q and 23s
exhibited the highest anti-influenza virus activity with IC50 at micromole level. In
particular, 23s exhibited a much greater affinity to HA with KD value of 15.6 µM. The
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results indicated that the polyphenol-pentacyclic triterpene might interfere with
influenza invasion by its interaction with the sialic acid receptor binding region of HA
protein.
Figure 7. Structural representative of 23s binding within HA protein (Protein Data
Bank: 1RVT) according to blind docking calculation. (A) Overview of HA protein.
The inhibitor pocket is highlighted in yellow square. Protein and compound 23s are
shown as orange surface and green sticks, respectively. (B) Closer view of the
inhibitor pocket. Compound 23s is shown as green sticks. (C) Modeled structure of
HA complexed with 23s. Compound 23s is shown as green sticks. Hydrogen bonds
are depicted in dashed lines. Residues in HA close to 23s are labeled in grey. (For
interpretation of the references to color in this figure legend, the red is referred to the
web version of this article.)
16

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4. Experimental section
4.1. General information
4.1.1. Materials
MDCK cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)
(Gibco BRL. Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine
o
serum (FBS) (PAA Laboratories, Linz Austria) at 37 C in a humidified atmosphere
containing 5% CO2. Influenza A/WSN/33 (H1N1) virus was used in this study.
OSV of 98% purity (Hoffmann-La Roche Ltd., Basel, Switzerland) was used as
reference compound 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 stock solutions.
4.1.2. 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
1
Bruker 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 =
13
3.31 for the central line of MeOD). Chemical shifts of C 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 thin-layer chromatography (TLC) on a
17

ACCEPTED MANUSCRIPT
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
Compounds 16a-c, 17a-c, 18a-c and 19a-c were synthesized according to
previously published methods.[48, 49] The synthesis of the intermediates 20a-g and
21a-g as well as the polyphenol–pentacyclic triterpene conjugates 22d-l, 23d-l, 23m-t,
23w-z, 23aa and 23a were provided in the supporting information. The synthesis of
ten polyphenol-BA conjugates, including compounds 22a-22c, 23a-23c, 23m, 23n,
23u and 23v, were obtained as follows.
4.2.1. General procedure for amide bond formation reaction (method A)
To a solution of the carboxyl compound (1.0 equiv.) in DCM, EDC (1.6 equiv.)
and DMAP (0.25 equiv.) were added slowly. Then the amino compound (1.2 equiv.)
was added to the mixture. The reaction mixture was stirred at room temperature for 1
h and then washed with saturated salt solution (50 mL × 3). The organic layer was
collected, dried over Na2SO4, filtered and concentrated. The residue was purified by
column chromatography over silica gel.
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 purged with H2 for three times, and then it was stirred under H2 balloon at room
18

ACCEPTED MANUSCRIPT
temperature for 24 h. The suspension was filtered 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.
Synthesis
of
N-(2-(3,4,5-tris(benzyloxy)benzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide
(22a)
Compound 22a was prepared from compounds 21a (208 mg, 0.43 mmol) and
BA (165 mg, 0.36 mmol) according to general procedure A. The residue was purified
by column chromatography (petroleum ether/ethyl acetate = 4:1) to yield 22a as a
white solid (288 mg, 78%), m.p. 110-112 oC. 1H NMR (400 MHz, CDCl3): δ 7.70 (t, J
= 5.5 Hz, 1H), 7.49 – 7.17 (m, 17H), 5.18 (d, J = 11.6 Hz, 2H), 5.15 (d, J = 11.6 Hz,
2H), 5.02 (s, 2H), 4.55 (s, 1H), 4.49 (s, 1H), 3.70 – 3.63 (m, 1H), 3.58 (td, J = 13.0,
2.8 Hz, 1H), 3.45 (dt, J = 13.0, 4.0 Hz, 1H), 3.01 – 3.11 (m, 2H), 2.41 (dt, J = 12.1,
3.2 Hz, 1H), 2.07 (br d, J = 13.5 Hz, 1H), 1.90 – 1.76 (m, 2H), 1.63 – 0.92 (m, other
aliphatic ring protons), 1.60, 0.89, 0.83, 0.66 (s, each 3H, 4 × CH3), 0.62 (s, 6H, 2 ×
CH3), 0.57 (d, J = 10.3 Hz, 1H); 13C NMR (100 MHz, MeOD) δ 179.16, 167.26,
152.84, 150.60, 141.31, 137.76, 136.87, 129.27, 128.63, 128.50, 128.27, 128.12,
127.95, 127.76, 109.62, 106.71, 79.06, 75.27, 71.36, 55.99, 55.43, 50.64, 50.21, 47.11,
42.80, 42.62, 40.72, 39.57, 38.93, 38.78, 38.53, 38.16, 37.22, 34.36, 33.81, 31.03,
29.61, 28.05, 27.48, 25.79, 20.95, 19.54, 18.34, 16.19, 16.03, 15.42, 14.79;
ESI-HRMS Calcd for C60H77N2O6 [M+H]+: 921.5776, found 921.5788.
4.2.4.
Synthesis
of
19

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N-(2-(3,4,5-trihydroxybenzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23a)
Compound 23a was prepared from compound 22a (100 mg, 0.11 mmol)
according to general procedure B. The residue was purified by column
chromatography (DCM/MeOH = 15:1) to yield 23a as a white solid (62 mg, 88%).
o
1
Mp: 215-217 C. H NMR (400 MHz, MeOD): δ 6.88 (s, 2H), 3.54 – 3.40 (m, 3H), ,
3.10 (dd, J = 11.3, 4.9 Hz, 1H), 2.40 (dt, J = 12.2, 3.6 Hz, 1H), 2.34 – 2.29 (m, 1H),
2.10 (br d, J = 12.8 Hz, 1H), 1.78 – 0.63 (m, other aliphatic ring protons), 0.93, 0.92
(s, each 3H, 2 × CH3), 0.85 (d, J = 6.8 Hz, 3H, CH3), 0.78 (s, 3H, CH3), 0.75 (d, J =
6.8 Hz, 3H, CH3), 0.74, 0.73, 0.63 (s, each 3H, 2 × CH3), 0.64 (d, J = 8.9 Hz, 1H); 13C
NMR (100 MHz, MeOD) δ 180.30, 170.65, 146.67, 138.28, 125.55, 107.90, 79.72,
57.49, 56.90, 51.83, 50.93, 45.39, 43.60, 41.87, 41.39, 40.34, 40.09, 39.93, 39.71,
38.95, 38.26, 35.51, 33.96, 31.20, 30.68, 28.63, 28.31, 28.04, 23.96, 23.38, 22.10,
19.38, 16.69, 16.65, 16.11, 14.98, 14.95; ESI-HRMS Calcd for C39H61N2O6 [M+H]+:
653.4524, found 653.4532.
4.2.5.
Synthesis
of
N-(2-(3,5-bis(benzyloxy)benzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (22b)
Compound 22b was prepared from compounds 21b (208 mg, 0.55 mmol) and
BA (279 mg, 0.61 mmol) according to general procedure A. The residue was purified
by column chromatography (petroleum ether/ethyl acetate = 4:1) to yield 22b as a
white solid (346 mg, 77%), m.p. 123-125 oC. 1H NMR (400 MHz, CDCl3) δ 7.94 (br s,
1H), 7.42 – 7.29 (m, 11H), 7.16 (d, J = 2.2 Hz, 2H), 6.71 (t, J = 2.2 Hz, 1H), 6.68 (br
s, 1H), 5.06 (d, J = 11.7 Hz, 2H), 5.03 (d, J = 11.7 Hz, 2H), 4.59 (s, 1H), 4.51 (s, 1H),
20

ACCEPTED MANUSCRIPT
3.56 – 3.55 (m, 3H), 3.41 – 3.37 (m, 1H), 3.15 – 3.09 (m, 2H), 2.33 (dt, J = 9.5, 2.6
Hz, 1H), 2.00 (d, J = 13.7 Hz, 1H), 1.89 – 0.80 (m, other aliphatic ring protons), 1.60,
13
0.92, 0.90, 0.69, 0.66, 0.63 (s, each 3H, 6 × CH3); C NMR (100 MHz, CDCl3) δ
178.93, 167.59, 160.06, 150.74, 136.56, 136.08, 128.62, 128.13, 127.65, 109.42,
106.12, 105.43, 78.91, 70.26, 55.81, 55.36, 50.58, 50.15, 46.90, 42.47, 42.29, 40.58,
39.46, 38.86, 38.73, 38.43, 37.98, 37.13, 34.24, 33.53, 30.96, 29.55, 28.04, 27.41,
25.69, 20.85, 19.50, 18.25, 16.10, 15.85, 15.45, 14.68; ESI-HRMS Calcd for
C53H71N2O5 [M+H]+: 815.5358, found 815.5357.
4.2.6.
Synthesis
of
N-(2-(3,5-dihydroxybenzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23b)
Compound 23b was prepared from compound 22b (100 mg, 0.12 mmol)
according to general procedure B. The residue was purified by column
chromatography (DCM/MeOH = 15:1) to yield 23b as a faint yellow solid (66 mg,
85%), m.p. 175-177 oC. 1H NMR (400 MHz, Pyr-d5) δ 9.28 (s, 1H), 8.38 (s, 1H), 7.68
(d, J = 1.4 Hz, 2H), 7.13 (s, 1H), 3.86 (br s, 3H), 3.72 (br s, 1H), 3.45 (t, J = 7.5 Hz,
1H), 2.92 (t, J = 9.8 Hz, 1H), 2.76 (t, J = 10.4 Hz, 1H), 2.05 (br d, J = 13.0 Hz, 1H),
2.01 (dd, J = 11.4, 7.1 Hz, 1H), 1.86 – 1.16 (m, other aliphatic ring protons), 1.12,
1.05 (s, each 3H, 2 × CH3), 1.01 (s, 6H, 2 × CH3), 0.92 (d, J = 6.6 Hz, 3H, CH3), 0.87
(s, 3H, CH3), 0.82 – 0.81 (m, 4H); 13C NMR (100 MHz, Pyr-d5) δ 178.84, 169.18,
160.85, 138.58, 107.35, 107.18, 78.67, 56.96, 56.51, 51.40, 50.72, 44.94, 43.41, 42.17,
42.04, 41.69, 40.65, 40.04, 39.83, 39.59, 38.24, 38.06, 35.40, 34.00, 30.81, 30.51,
29.23, 28.81, 28.12, 24.12, 23.81, 21.83, 19.36, 17.04, 16.99, 16.93, 15.47, 15.29;
21

ACCEPTED MANUSCRIPT
ESI-HRMS Calcd for C39H61N2O5 [M+H]+: 637.4575, found 637.4569.
4.2.7.
Synthesis
of
N-(2-(4-(benzyloxy)benzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (22c)
Compound 22c was prepared from compounds 21c (150 mg, 0.56 mmol) and
BA (284 mg, 0.62 mmol) according to general procedure A. The residue was purified
by column chromatography (petroleum ether/ethyl acetate = 4:1) to yield 22c as a
white solid (315 mg, 80%), m.p. 122-124 oC. 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J
= 8.8 Hz, 2H), 7.43 – 7.31 (m, 5H), 7.00 (m, J = 8.8 Hz, 2H), 6.35 (t, J = 5.2 Hz, 1H),
5.09 (s, 2H), 4.72 (s, 1H), 4.59 (s, 1H), 3.59 – 3.56 (m, 3H), 3.49 (dd, J = 10.6, 5.5
Hz, 1H), 3.17 – 3.10 (m, 2H), 2.30 (dt, J = 12.1, 3.5 Hz, 1H), 1.96 (td, J = 13.8, 3.0
Hz, 1H), 1.91 – 0.76 (m, other aliphatic ring protons), 1.66, 0.93, 0.92, 0.72, 0.69,
0.68 (s, each 3H, 6 × CH3), 0.62 (d, J = 10.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
178.77, 167.66, 161.54, 150.95, 136.48, 129.09, 128.76, 128.27, 127.56, 126.55,
114.57, 109.46, 79.01, 70.20, 55.86, 55.43, 50.63, 50.26, 46.99, 42.53, 41.96, 40.68,
39.59, 38.93, 38.80, 38.47, 38.02, 37.23, 34.30, 33.59, 31.00, 29.62, 28.08, 27.48,
25.71, 20.95, 19.62, 18.32, 16.16, 16.07, 15.50, 14.71; ESI-HRMS Calcd for
C46H65N2O4 [M+H]+: 709.4939, found 709.4940.
4.2.8.
Synthesis
of
N-(2-(4-hydroxybenzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23c)
Compound 23c was prepared from compound 22c (120 mg, 0.19 mmol)
according to general procedure B. The residue was purified by column
chromatography (DCM/MeOH = 15:1) to yield 23c as a white solid (92 mg, 88%),
22

ACCEPTED MANUSCRIPT
1
m.p. 159-161 oC. H NMR (400 MHz, Pyr-d5) δ 9.11 (br s, 1H), 8.43 (br s, 1H), 8.36
(s, 1H), 8.34 (s, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 3.93-3.85 (m, 3H), 3.75 (t, J = 5.6 Hz,
1H), 3.47 (t, J = 7.6 Hz, 1H), 2.95 (t, J = 10.5 Hz, 1H), 2.77 (t, J = 9.7 Hz, 1H), 2.51 –
2.47 (m, 1H), 2.03 (dd, J = 10.8, 7.3 Hz, 1H), 1.87 – 0.82 (m, m, other aliphatic ring
protons), 1.24 (s, 3H, CH3), 1.03 (s, 9H, 3 × CH3), 0.92 (d, J = 6.3 Hz, 3H, CH3), 0.85
– 0.82 (m, 6H, 2 × CH3); 13C NMR (100 MHz, Pyr-d5) δ 178.80, 168.50, 162.47,
130.51, 126.85, 116.42, 78.61, 56.89, 56.42, 51.28, 50.68, 44.99, 43.35, 42.20, 41.60,
40.74, 39.99, 39.73, 39.50, 38.17, 37.98, 35.31, 33.92, 30.72, 30.45, 29.15, 28.81,
28.06, 24.04, 23.80, 21.76, 19.26, 16.97, 16.89, 16.85, 15.35, 15.21; ESI-HRMS
Calcd for C39H61N2O4 [M+H]+: 621.4626, found 621.4625.
4.2.9.
Synthesis
of
N-(2-(2-hydroxybenzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23m)
Compound 23m was prepared from compounds 21d (100 mg, 0.55 mmol) and
BA (278 mg, 0.61 mmol) according to general procedure A. The residue was purified
by column chromatography (petroleum ether/ethyl acetate = 2:1) to yield 23m as a
white solid (135 mg, 51%), m.p. 142-144 oC. 1H NMR (400 MHz, CDCl3) δ 12.54 (s,
1H), 8.17 (s, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 8.3
Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H), 6.33 (t, J = 5.6 Hz, 1H), 4.73 (s, 1H), 4.59 (s, 1H),
3.67 – 3.47 (m, 4H), 3.19 – 3.13 (m, 2H), 2.23 – 2.17 (m, 1H), 1.94 (d, J = 14.0 Hz,
1H), 1.86 (q, J = 10.8 Hz, 1H), 1.74 – 0.81 (m, other aliphatic ring protons), 1.66 (s,
3H, 2 × CH3), 0.93 (s, 6H, 2 × CH3), 0.71 (s, 3H, CH3), 0.65 (s, 9H, 3 × CH3 ), 0.60 (d,
J = 10.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 179.69, 170.91, 161.75, 150.73,
23

ACCEPTED MANUSCRIPT
134.22, 126.42, 118.76, 118.45, 114.18, 109.61, 79.09, 55.97, 55.46, 50.62, 50.34,
47.15, 42.90, 42.58, 40.71, 38.95, 38.87, 38.81, 38.46, 38.30, 37.22, 34.34, 33.64,
31.07, 29.57, 28.10, 27.51, 25.74, 20.87, 19.67, 19.29, 18.31, 16.07, 15.95, 15.48,
14.76; ESI-HRMS Calcd for C39H59N2O4 [M+H]+: 619.4469, found 619.4449.
4.2.10.
Synthesis
of
N-(2-(2,4-dihydroxybenzamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23n)
Compound 23n was prepared from compounds 21e (108 mg, 0.55 mmol) and
BA (27 mg, 0.61 mmol) according to general procedure A. The residue was purified
by column chromatography (petroleum ether/ethyl acetate = 1:1) to yield 23n as a
o
white solid (148 mg, 56%), m.p. 186-188 C. 1H NMR (400 MHz, MeOD) δ 7.53 (d,
J = 8.8 Hz, 1H), 6.29 (dd, J = 8.8, 2.2 Hz, 1H), 6.24 (s, 1H), 4.65 (s, 1H), 4.53 (s, 1H),
3.57 – 3.50 (m, 2H), 3.38 (dd, J = 9.6, 2.7 Hz, 1H), 3.22 – 3.19 (m, 1H), 3.06 (dd, J =
10.7, 5.7 Hz, 2H), 2.32 (td, J = 11.6, 3.1 Hz, 1H), 2.05 (d, J = 13.4 Hz, 1H), 1.89 –
0.57 (m, other aliphatic ring protons), 1.63, 0.89, 0.88, 0.71, 0.69, 0.66 (s, 3H each, 6
× CH3); 13C NMR (100 MHz, MeOD) δ 179.79, 171.72, 163.65, 163.56, 151.84,
129.80, 109.91, 108.37, 108.03, 103.87, 79.46, 56.80, 56.56, 51.63, 51.16, 47.96,
43.23, 41.50, 40.31, 39.81, 39.69, 39.05, 38.86, 38.01, 35.03, 33.87, 31.75, 30.35,
28.51, 27.76, 26.62, 21.75, 19.71, 19.46, 19.16, 16.58, 16.42, 15.99, 15.07;
ESI-HRMS Calcd for C39H59N2O5 [M+H]+: 635.4418, found 635.4405.
4.2.11. Synthesis of N-(2-cinnamamidoethyl)-3β-hydroxy-lup-20(29)-en-28-amide (23u)
Compound 23u was prepared from compounds 21f (100 mg, 0.53 mmol) and BA
(266 mg, 0.58 mmol) according to general procedure A. The residue was purified by
24

ACCEPTED MANUSCRIPT
column chromatography (petroleum ether/ethyl acetate = 4:1) to yield 23u as a white
solid (245 mg, 74%). m.p. 164-166 oC. 1H NMR (400 MHz, MeOD) δ 7.59 (t, J = 4.6,
1H), 7.56 – 7.51 (m, 3H), 7.40 – 7.33 (m, 3H), 6.60 (d, J = 15.8 Hz, 1H), 4.69 (s, 1H),
4.57 (s, 1H), 3.57 – 3.52 (m, 2H), 3.40 – 3.33 (m, 1H), 3.23 – 3.20 (m, 1H), 3.10 –
3.05 (m, 2H), 2.50 (dt, J = 12.8, 2.9 Hz, 1H), 2.11 (d, J = 12.8 Hz, 1H), 1.94 – 1.79
(m, 2H), 1.69 – 0.80 (m, other aliphatic ring protons), 1.66, 0.93, 0.87, 0.86, 0.72,
13
0.67 (s, 3H each, 6 × CH3), 0.58 (d, J = 11.4, 1H); C NMR (100 MHz, MeOD) δ
179.58, 168.92, 152.17, 141.84, 136.17, 130.84, 129.96, 128.94, 121.94, 110.02,
79.55, 57.00, 56.76, 51.93, 51.35, 48.05, 43.45, 41.90, 40.39, 40.16, 40.03, 39.86,
39.30, 38.85, 38.23, 35.33, 34.12, 31.94, 30.72, 28.65, 28.01, 26.88, 22.06, 19.76,
19.25, 16.83, 16.75, 16.15, 15.15; ESI-HRMS Calcd for C41H61N2O3 [M+H]+:
629.4677, found 629.4676.
4.2.12.
Synthesis
of
N-(2-((E)-3-(4-hydroxyphenyl)acrylamido)ethyl)-3β-hydroxy-lup-20(29)-en-28-amide
(23v)
Compound 23v was prepared from compounds 21g (50 mg, 0.24 mmol) and BA
(11 mg, 0.22 mmol) according to general procedure A. The residue was purified by
column chromatography (petroleum ether/ethyl acetate = 1:1) to yield 23v as a white
o
1
solid (70 mg, 49%), m.p. 204-206 C. H NMR (400 MHz, MeOD) δ 7.53 (t, J = 4.4
Hz, 1H), 7.46 – 7.40 (m, 3H), 6.80 (d, J = 8.7 Hz, 2H), 6.39 (d, J = 15.8 Hz, 1H), 4.69
(d, J = 2.9 Hz, 1H), 4.56 (s, 1H), 3.63 – 3.54 (m, 2H), 3.19 – 3.05 (m, 3H), 2.48 (td, J
= 12.9, 3.5 Hz, 1H), 2.10 (d, J = 13.2 Hz, 1H), 1.90 – 0.57 (m, other aliphatic ring
25

ACCEPTED MANUSCRIPT
protons), 1.67, 0.93, 0.88, 0.85, 0.74, 0.68 (s, each 3H, 6 × CH3); 13C NMR (100 MHz,
MeOD) δ 178.34 , 168.32, 159.28, 150.91, 140.70, 129.35, 126.21, 117.02, 115.36,
108.56, 78.27, 55.67, 55.40, 50.55, 49.97, 42.06, 40.52, 38.88, 38.78, 38.61, 38.49,
37.92, 37.51, 36.85, 33.87, 32.73, 30.52, 29.37, 27.17, 26.61, 25.49, 20.63, 18.22,
17.83, 15.35, 15.29, 14.66, 13.60; ESI-HRMS Calcd for C41H61N2O4 [M+H]+:
645.4623, found 645.4626.
4.3 Biological assays
4.3.1. CPE reduction assay
The assay was performed as previously described 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, test compound and 2 mg/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 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. HI assay
Compound from a 3-fold serial dilution in saline was mixed with an equal
26

ACCEPTED MANUSCRIPT
volume of influenza virus (2 HA units) in the V-bottomed 96-well microplates.
Subsequently, 50 µL of freshly prepared chicken RBCs (cRBCs) (1% v/v in saline)
was added to each well. The mixture was incubated at room temperature for 30 min
before observing cRBC aggregation on the plate.
4.3.4. SPR
Interactions between the influenza HA and the compounds were analyzed using
o
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 mL/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.
4.3.5. Docking simulation
The protein structure, co-crystallized with LSTC (LS-Tetrasaccharide C), was
downloaded from PDB (PDB code: 1rvt). Protein Preparation Wizard of the
Schrödinger Suite was used to prepare protein ensuring that the downloaded X-ray
structure was reliable and qualitatively considerable for further studies by
27