Design, synthesis and biological evaluation of small molecular polyphenols as entry inhibitors against H5N1

Article abstract of DOI:10.1016/j.bmcl.2014.04.057

To find novel compounds against H₅N₁, three series of known or novel small molecular polyphenols were synthesized and tested in vitro for anti-H₅N₁ activity. In addition, the preliminary structure-antiviral activity relationships were elaborated. The results showed that some small molecular polyphenols had better anti-H₅N₁ activity, and could serve as novel virus entry inhibitors against H ₅N₁, likely targeting to HA2 protein. Noticeably, compound 4a showed the strongest activity against H₅N₁ among these compounds, and the molecular modeling analysis also suggested that this compound might target to HA2 protein. Therefore, compound 4a is well qualified to serve as a lead compound or scaffold for the further development of H ₅N₁ entry inhibitor.

Full text of DOI:10.1016/j.bmcl.2014.04.057

Accepted Manuscript
Design, synthesis and biological evaluation of small molecular polyphenols as
entry inhibitors against H5N1
Jian Yang, Jing Xiang Yang, Fang Zhang, Gang Chen, Wei Pan, RuiYu, Shuwen
Wu, Po Tien
PII:
S0960-894X(14)00405-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.057
BMCL 21550
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 November 2013
4 April 2014
16 April 2014
Please cite this article as: Yang, J., Yang, J.X., Zhang, F., Chen, G., Pan, W., Yu, R., Wu, S., Tien, P., Design,
synthesis and biological evaluation of small molecular polyphenols as entry inhibitors against H5N1, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.057
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Design, synthesis and biological evaluation of small molecular polyphenols as entry
inhibitors against H5N1
Jian Yanga, *, Jing Xiang Yanga, c, d, Fang Zhanga, c, Gang Chena, c, Wei Panb, Rui Yu a,
c, Shuwen Wu b, *, Po Tienb, *
aDepartment of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060,
China
bThe College of Life Sciences, State Key Laboratory of Virology, Modern Virology
Research Center, Wuhan University, Wuhan 430072, China
cWuhan University School of Pharmaceutical Sciences, Wuhan 430071, China
dSchool of Pharmaceutical Sciences, Hunan University of Chinese Medicine,
Changsha 410208, China
*Corresponding authors. Jian Yang is to be contacted at Department of Pharmacy,
Renmin Hospital of Wuhan University, China; Shuwen Wu, The College of Life
Sciences, State Key Laboratory of Virology, Modern Virology Research Center,
Wuhan University, China; Po Tien, The College of Life Sciences, State Key
Laboratory of Virology, Modern Virology Research Center, Wuhan University,
China.
E-mail address: Yanj502@sohu.com (J. Yang); shuwenwu@hotmail.com (S. Wu);
tienpo@sun.im.ac.cn (P. Tien).
Phone: 0086-27- 88041911- 88381.
1

Abstract: To find novel compounds against H5N1, three series of known or novel
small molecular polyphenols were synthesized and tested in vitro for anti-H5N1
activity. In addition, the preliminary structure-antiviral activity relationships were
elaborated. The results showed that some small molecular polyphenols had better
anti-H5N1 activity, and could serve as novel virus entry inhibitors against H5N1, likely
targeting to HA2 protein. Noticeably, compound 4a showed the strongest activity
against H5N1 among these compounds, and the molecular modeling analysis also
suggested that this compound might target to HA2 protein. Therefore, compound 4a is
well qualified to serve as a lead compound or scaffold for the further development of
H5N1 entry inhibitor.
Keywords: synthesis; anti-H5N1 activity; small molecular polyphenols; entry inhibitor;
HA2; molecular modeling
Influenza is considered to be one of the life threatening infectious diseases. In
some countries seasonal influenza affects annually up to 40% of the population and
500 million people die from it worldwide every year. New highly-virulent influenza
strains can arise unexpectedly to cause world-wide pandemics with markedly
increased morbidity and mortality, such as the ―avian flu‖ in 1997 and ―swine flu‖ in
1
2009. At present, only two classes of antiviral drugs, M2 ion channel blockers and
Neuraminidase (NA) inhibitors, are licensed for use against influenza A viruses, but
2
their utility has been limited by side effects and emergence of resistant viral strains.
Therefore, the development of new anti-influenza agents represents a crucial defense
2

strategy to combat seasonal and pandemic influenza strains.
Influenza virus entry inhibitors represent a new class of antiviral agents, which
can block virus entry into the target cells. Hemagglutinin (HA) is the important target
for developing influenza virus entry inhibitors, and HA is a trimeric glycoprotein in
2
nature and can be cleaved into HA1 and HA2 subunits by host proteases. Although
HA1 is the target for developing influenza virus entry inhibitors, the high variability
and mutation rate limit its application. Nevertheless, HA2 is for virus–endosome
membrane fusion in the acid environment after the virus has been endocytosed into
the cell and more conserved so that it may be a better target for developing influenza
virus entry inhibitors. 3, 4 Up to now, some influenza virus entry inhibitors have been
identified and discovered, which have become more and more exciting research
areas.1, 2, 5
Hydroxytyrosol (HT), 3, 4-dihydroxyphenylethanol, is one of the major
6
polyphenolic components of olive products. In several special studies, it has been
demonstrated that HT is antioxidant and antimicrobial and that it has beneficial effects
7-11
on the cardio-vascular system and in several human diseases.
Moreover, HT was
effective against the enveloped viruses, but not against the non-enveloped viruses. HT
could inhibit influenza A viruses including H1N1, H3N2, H5N1, and H9N2 subtypes.
More recently, it was reported that mechanism of the anti-H9N2 effect of HT was
involved in morphological change of the virus. 12 Recent studies also had identified
HT as the HIV-1 inhibitor, in which HT inhibited not only the fusion between viral
and cellular membranes but also the integrase activity of the virus. 13, 14 Therefore, HT
3

is an interesting scaffold for design of novel antiviral agents.
In order to further know about the antiviral activity of HT, HT was synthesized
according to previous report (Schemes.1-2)13 and was tested in vitro antiviral activity.
15, 16 In the antiviral screening experiments, we found that HT (compound 4b) has a
strong inhibitory effect on H5N1, and could inhibit H5N1 virus production most
effectively at the virus entry step of cell infection (Fig.1). Interestingly, the starting
material (compound 1b) showed no activity against H5N1, but the intermediate
(compound 3b) exhibited potent activity against H5N1. The results implied that
esterification of carboxylic acid, reduction of carboxylic acid to alcohol or other
modification of carboxylic acid in small molecular polyphenols could significantly
influence the anti-H5N1 activity. Therefore, the preliminary results stimulated interest
in exploration of HT and its analogs in which phenolic hydroxyls, the length and
functional groups of the side chain were altered to study their influence on the
anti-H5N1 activity. In the meantime, our previous results showed that some amino
acids were important moities in polyphenols as antiviral entry inhibitors, such as
gossypol derivatives.17-19
Therefore, three series of known or novel small molecular polyphenols, such as
aryl ester derivatives (compounds 2a-c and 3a-i), aromatic alcohol derivatives
(compounds 4a-c) and amino acids derivatives (compounds 5a-b, 7a-b and 8a-b),
were designed and synthesized respectively (Schemes.1-4), and their preliminary
structure-activity relationships of antiviral activity were also investigated. Small
molecular polyphenols were evaluated for their capability to inhibit the H5N1
4

(Influenza strain A/Vietnam/1194/2004) replication in MDCK cells. 15, 16 Amantidine
and Zanamivir were used as the reference drugs. The results, expressed as IC50, CC50
and SI (selectivity index), are illustrated in Table 1.
H3CO
COOCH3
A
B
H3CO
(CH2)n
HO
HO
2a. n = 0
2b. n = 1
2c. n = 2
COOH
(CH2)n
1a. n = 0
1b. n = 1
HO
HO
1c. n = 2
COOR1
(CH2)n
3a. R1= CH3, n = 0
3b. R1= CH3, n = 1
3c. R1= CH3, n = 2
3d. R1= CH2CH3, n = 0
3e. R1= CH2CH3, n = 1
3f. R1= CH2CH3, n = 2
3g. R1= CH(CH3)2, n = 0
3h. R1= CH(CH3)2, n = 1
3i. R1 =CH(CH3)2, n = 2
Scheme 1. Reagents and Conditions: (A) (CH3)2SO4, K2CO3, acetone, 70℃, 6h, 85-95% yield; (B) For
compounds 3a-c: SOCl2, methanol, rt, 8h, 95-97% yield; For compounds 3d-f: SOCl2, ethanol, rt, 8h,
90-95% yield; For compounds 3g-i: SOCl2, isopropanol, rt, 8h, 90-95% yield.
HO
HO
HO
A
CH2OH
COOCH3
HO
(CH2)n
(CH2)n
4a. n=0
3a. n=0
3b. n=1
3c. n=2
4b. n=1
4c. n=2
Scheme 2. Reagents and Conditions: (A) LiAlH4, THF, 0℃, 8h, 72-75% yield.
HO
HO
A
CONHR2
HO
COOH
HO
COOCH3
NH2
R2NH2=
R2NH2=
1a
5a.
COOCH3
NH2
5b.
Scheme 3. Reagents and Conditions: (A) EDCI, HOBT, DCM, R2NH2, 0℃-rt, 7h, 68-77% yield.
5

H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
HO
HO
A
C
B
COOCH3
CONHR2
COOH
CONHR2
COOCH3
NH2
COOCH3
NH2
2b
6
R2NH2=
R2NH2=
R2NH2=
R2NH2=
8a.
7a.
7b.
COOCH3
NH2
COOCH3
NH2
8b.
Scheme 4. Reagents and Conditions: (A) 20% Na2CO3, THF, 80℃, 10h, then 4M aq. HCl, 93-95%
yield; (B): EDCI, HOBT, TEA, DCM, R2NH2, 0℃-rt, 7h, 78-81% yield; (C) BBr3, DCM, -78℃, 8h,
72-78% yield.
Table 1
Inhibition of small molecular polyphenols on replication of H5N1 (Influenza strain
A/Vietnam/1194/2004) in MDCK cells
ICa50
CCb
50
Compound
Amantidine
SIc
(μM)
(μM)
10.78
218.92
20.27
>113.64
NA
Zanamivir
0.88
—
>100.03
—
1a
1b
—
—
NA
—
—
—
—
NA
NA
NA
1c
2a
2b
>169.99
>158.65
—
>148.73
>183.07
>169.99
>237.98
NA
2c
3a
3b
3c
18.27
19.53
23.78
>10.02
>8.70
>10.01
16.11
16.86
21.47
14.89
>169.99
>158.64
>216.34
>158.65
>10.55
>9.41
3d
3e
3f
>10.08
>10.65
3g
6

15.22
19.52
1.78
>148.72
>198.30
>142.66
>172.11
>9.77
>10.16
>80.15
>9.82
3h
3i
4a
4b
4c
17.53
22.91
22.23
—
>198.31
>118.57
>105.77
>8.66
>5.33
NA
5a
5b
7a
7b
8a
8b
—
—
—
NA
NA
—
41.78
—
>183.07
>169.99
>4.38
NA
aIC50: compound concentration required to achieve 50% inhibition of replication of H5N1, as determined by plaque
reduction assays.
bCC50: compound concentration required to cause 50% death of uninfected MDCK cells, as determined by the
MTT method.
cSI: selectivity index as CC50/ IC50 . NA: no activity. —: no calculation.
All data represent average values for three separate experiments. The variation of these results under the standard
procedures is below ±10%.
As shown in Table 1, aromatic acid derivatives (compounds 1a-c) had no activity
against H5N1. However, the derivatives of aryl ester (compounds 3a-i) and aromatic
alcohol (compounds 4a-c) exhibited potent inhibitory activity, while some amino
acids derivatives (compounds 5a and 8a) showed moderate activity against H5N1. The
results verified the hypothesis that esterification of carboxylic acid or
reduction of carboxylic acid to alcohol in small molecular polyphenols could
significantly enhance the anti-H5N1 activity besides amino acid-activated amidation of
7

carboxylic acid in small molecular polyphenols. Among amino acid derivatives, it
was also observed that instead of the L-Phenylalanine methyl ester derivatives, the
L-Leucine methyl ester derivatives could enhance the anti-H5N1 activity.
Interestingly, methylation of diphenol hydroxyls at the 3 and 4 positions of
benzyl ring of aryl ester derivatives abolished their anti-H5N1 activity, such as
compounds 2a-c. Likewise, methylation of diphenol hydroxyls at the 3 and 4
positions of benzyl ring of amino acid derivatives also led inactive, such as compound
7a. The results suggested that phenol hydroxyls might play a very important role in
the anti-H5N1 activity of small molecular polyphenols, whereas introduction of alkyl
groups in the hydroxyls should be avoided.
Among aryl ester derivatives, it was observed that the increase of the chain
length at the 1 position of phenyl ring from one carbon to three carbons resulted in
slightly decrease in activity against H5N1, while increasing the length or bulk of the
O-substituted (R2) of the aryl ester derivatives from methyl group to isopropyl group
resulted in a slightly increased activity against H5N1. Noticeably, there were not
significant difference in anti-H5N1 activity and selectivity index among these
compounds, but compound 3g (IC50=14.89μM) profiled relatively better anti-H5N1
activity.
However, increasing the chain length at the 1 position of phenyl ring from one
carbon to three carbons resulted in significantly decreasing the activity against H5N1
among aromatic alcohol derivatives. Amino acids derivatives also had the similar
antiviral effect. For example, compound 4a (IC50=1.78μM) was almost 10-fold or
8

12-fold more active against H5N1 than compound 4b (IC50=17.53μM) or compound
4c (IC50=22.91μM) respectively, and compound 5a (IC50=22.23μM) was also almost
2-fold more active against H5N1 than compound 8a (IC50=41.78μM).
Notably, an aromatic alcohol derivative, compound 4a (IC50=1.78μM, SI>80.15),
exhibited excellent activity against H5N1 among these derivatives, which was superior
to Amantidine (IC50=10.78μM, SI=20.27) and next to Zanamivir (IC50=0.88μM,
SI>113.64).
Compounds 3g, 4a-b and 5a had better selectivity indexes and comparable
potency in the corresponding derivatives, thus, compounds 3g, 4a-b and 5a were
chosen to investigate their mechanism of action against H5N1.
To determine whether Neuraminidase protein was the target of these compounds,
compounds 3g, 4a-b and 5a were tested in vitro for the inhibition of Neuraminidase
activity using Neuraminidase Inhibitors Identification Kit P0309 (Beyotime Institute
of Biotechnology). The preliminary results demonstrated that these compounds could
not significantly inhibit the Neuraminidase activity at concentrations of up to 20μg/ml,
indicating that Neuraminidase protein might not be the primary target of small
molecular polyphenols.
Therefore, time-of-addition assay was carried out to investigate the possible
target phase of small molecular polyphenols in the H5N1 life cycle.20 Compounds 3g,
4a-b and 5a were added to the confluent monolayers of MDCK cells at three time
points: before viral adsorption (pre-treatment), at the same time as virus infection
(simultaneous treatment) and after the viral adsorption (post treatment).
9

As shown in Fig.1, MDCK cells pre-treated with the compounds 4a and 4b prior
to infection had slightly virus inhibition at -4 h and -2 h point. But compounds 3g and
5a had no inhibition effect when they were added to cells before virus infection. The
result suggested that compounds 4a and 4b could slightly provoke the cell factors to
resist the virus.
Interestingly, these compounds all had the significant inhibitory effect on H5N1
when they were added to the cells with virus simultaneously. Compound 4a almost
reached the 100% inhibition effect. And the three others also had the inhibition effect
over 50% at this concentration.
However, these compounds have no significant inhibitory activity on H5N1 if
they were added 2 h or longer after virus adsorption. These compounds had only 20%
inhibition effect 2 h after infection. And when they were added to the infected cells 4
h or 8 h after infection, all of them had no inhibition effect at all. That means that
these compounds had not effect on H5N1 virus replication stage.
The data from the timing assays suggested that small molecular polyphenols
inhibited H5N1 virus production most effectively at the virus entry step of cell
infection, and probably affected the attachment protein, such as HA protein.
Figure 1. Compounds 3g, 4a-b and 5a inhibited H5N1 (Influenza strain A/Vietnam/1194/2004) entry.
Inhibition of H5N1 entry was determined by a time-of-addition assay.20
10

Furthermore, we investigated whether these small molecular polyphenols could
inhibit hemagglutination(HA) by interfering with the H5 HA adsorption to RBCs. The
hemagglutination inhibition assay was employed to evaluate the effects of compounds
3g, 4a-b and 5a on viral adsorption to target cells.18 Both H5 standard antigen and
antiserum were provided by Beijing KangNong XingMu technology development
center, China. Inhibitory activities of these compounds were tested at a serial 2-fold
dilution (ranging from 100 to 0.098μg/ml). The results showed that compounds 3g,
4a-b and 5a could not inhibit the adsorption of H5 HA to chicken RBCs even at the
highest concentration of 100μg/ml, while the H5-positive control antisera could
effectively inhibit H5 HA adsorption to chicken RBCs with a titer of 1:128. The
results suggested that small molecular polyphenols, such as compounds 3g, 4a-b and
5a, might not target the receptor binding domain of the HA1 subunit. On the other
hand, HT (compound 4b) is active against enveloped viruses, but not against
nonenveloped viruses. HT could inhibit the fusion between HIV-1 and cellular
membranes.13 Taken together, we postulated that small molecular polyphenols could
target to the membranes fusion step medicated by the more conserved hemagglutinin
transmembrance subunit HA2, and not HA1.
On the basis of the results, we used the neutral-pH crystal structure of the HA of
H5N1 (Influenza strain A/Vietnam/1194/2004, PDB: 2IBX) 21 as a reference for our
modeling work. Using Molegro Virtual Docker (version 5.0), the predicted binding
modes of compounds 4a and 4b are shown in Fig.2, respectively. The docking results
of compounds 4a and 4b are summarized in Table 2, which shows that compounds 4a
11

and 4b can fit inside the same cavity in the stem region of the HA2 near the fusion
peptide through Van der Waals interactions, hydrophobic contacts, and hydrogen
bonding networks. The MolDockScore of compound 4b was very close to that of
compound 4a, but compound 4a formed more favorable hydrogen bonding networks
with HA protein residues than compound 4b, which helped to enhance the binding
affinities to HA protein. As shown in Fig.2, at the top of cavity, compound 4a
occupied the binding site with the diphenol hydroxyls forming stable hydrogen bonds
with Leu2, Asp109, and Arg322, respectively. Hydroxyl group at compound 4a also
could form stable hydrogen bonds with Arg322 and Leu108, respectively. However,
one of phenol hydroxyls at the compound 4b only formed stable hydrogen bonds with
Asn117 and Asp120 at the bottom of cavity respectively. As expected, the analysis of
binding models also gave our results support. Our study results suggested that phenol
hydroxyls might play a very important role in the anti-H5N1 activity of small
molecular polyphenols, whereas introduction of alkyl groups in the hydroxyls should
be avoided. These interactions, identified by docking simulations, rationalized the
anti-H5N1 activity of small molecular polyphenols, such as compounds 4a and 4b.
The results also show that compound 4a can be potential anti-H5N1 entry
inhibitors, likely targeting to the HA2 protein. Noticeably, compound 4a is a small
molecule with molecular weight of 140.14 and is different from the other known entry
inhibitors.1, 2, 5
12

Table 2
Results of the molecular modeling study for compounds 4a and 4b
Compound
MolDock Score
Total Energy
E-Intra
Steric Energy
Hbond number
Hbond Energy
Hbond Interaction residues
4a
4b
-70.174
-70.1735
-68.6509
9.46595
8.30568
-70.1618
-74.7874
5
2
-9.47759
-2.16921
Leu2, Leu108, Asp109, Arg322(2)
Asn117, Asp120
-68.0979
MolDock Score: The energy score using docking (arbitrary units); E-Intra: energy between a ligand atom and the other atoms in the ligand; Steric Energy: steric energy; Hbond energy: Hydrogen bonding
energy.
Figure 2. The binding modes of compound 4a (left) or compound 4b (right) with H5 HA protein. The
modeling is based on the published crystal structure of HA of H5N1 (Influenza strain
A/Vietnam/1194/2004, PDB: 2IBX). The HA1 subunit (A, C, E chains) is colored in blue. The HA2
subunit (B, D, F chains) is colored in red. Compounds 4a and 4b are shown in green stick model and
hydrogen bonds with compounds 4a and 4b are in blue dashed lines. Residues Leu2, Leu108, Asp109,
Asn117, Asp120 and Arg322 are indicated on the protein structure and other residues are not shown.
Interestingly, it was reported that the structure of H9N2 virus could be disrupted
by HT. Electron microscopic analysis revealed morphological abnormalities in the
12
HT-treated H9N2 virus. In the time-of-addition assay, however, the addition of HT
to the cells simultaneously with the viral inoculation caused only a marginal antiviral
effect, and Pretreatment of the cells with HT did not affect the titer of the H9N2 virus
13

subsequently inoculated onto the cells, 12 which were different from our timing assays
against H5N1. The results implied that the antiviral effects of HT on the H9N2 virus
might be different from those of small molecular polyphenols on H5N1 virus.
Therefore, more investigation must be taken to elucidate the entry mechanism of
small molecular polyphenols against H5N1.
In summary, three series of known or novel small molecular polyphenols were
designed and synthesized, and further screened in vitro for their anti-H5N1activity.
The results verified the hypothesis that esterification of carboxylic acid or
reduction of carboxylic acid to alcohol in small molecular polyphenols could
significantly enhance the anti-H5N1 activity besides amino acid-activated amidation of
carboxylic acid in small molecular polyphenols. On the other hand, the preliminary
SAR studies showed that the anti-H5N1 activity of small molecular polyphenols
depended on the availability of the phenolic groups, carbon chain length and the
carbon chain functional groups at small molecular polyphenols. Small molecular
polyphenols impaired the virus entry step of cell infection, likely targeting to HA2
protein, but the detail mechanism of action against H5N1 need further study. Among
these derivatives, an aromatic alcohol derivative, compound 4a, exhibits excellent
activity against H5N1 and may serve as a lead compound or scaffold for the further
development of H5N1 entry inhibitor.
Acknowledgment
We gratefully acknowledge financial support of the National Natural Science
Foundation of China (Grant No. 30770228).
14

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20. Time-of-addition assay：The time-of-addition experiment was carried out with
MDCK cells. The MDCK cells were plated in 24-wells plates (2×104 cells/well)
and incubated at 37℃/5% CO2 for 24 h to allow the cells to form a monolayer on
the bottom of the well. Then the compounds (the final concentration of 30μM)
were added to the medium at the time point -4 h, -2 h (pre-treatment group), 0 h
(simultaneous treatment group), 2 h, 4 h, 8 h (post treatment group). The untreated
wells infected with virus at the same dilution were taken as the control group. In
pre-treatment group, compounds were incubated with cell for 2 h and were
removed 2 h or 4 h prior to virus infection. Then the medium with compounds
were removed and the MDCK cells were washed twice with PBS. The H5N1 virus
(80 PFU/well) were added to the cells and incubated at 37℃/5 % CO2 for 2 h. In
16

simultaneous treatment group, the virus (80 PFU/well) and compounds were
added to the cells simultaneously, and then incubated at 37℃/5 % CO2 for 2 h. In
post treatment group, the cells were first infected with virus (80 PFU/well) for 2 h.
Then 2 h, 4 h and 8 h after the infection, the virus suspension was aspirated and
cells were washed twice with PBS. The plaque medium containing test
compounds (30μM) was added to cells, and remained throughout the time of
addition experiments. After those treatments, the inhibitor rate of virus was
determined by plaque assay. The cells were washed twice with PBS, overlaid with
plaque medium (medium with 1% penicillin-streptomycin, 0.5% BSA, 1μg/ml
TPCK and 1% agarose), and incubated at 37℃/ 5% CO2 for 48 h. After the
agarose was removed, the cells were stained with crystal violet. Plaques were
counted to measure the effect of compounds. Every condition was tested in
triplicate. The inhibition ratio was obtained using the equation: Inhibition (%) =
(1- C
/ C control) × 100%, C
is the count of plaques in compounds added
test
test
group; C control is the count of plaques in the control group.
21. Yamada, S.; Suzuki, Y.; Suzuki, T.; Le, M. Q.; Nidom, C. A.; Sakai-Tagawa, Y.;
Muramoto, Y.; Ito, M.; Kiso, M.; Horimoto, T.; Shinya, K.; Sawada, T.; Kiso, M.;
Usui, T.; Murata, T.; Lin, Y.; Hay, A.; Haire, L. F.; Stevens, D. J.; Russell, R. J.;
Gamblin, S. J.; Skehel, J. J.; Kawaoka, Y. Nature. 2006, 444, 378.
17

HO
HO
CH2OH
compound 4a
Compound 4a exhibits excellent activity against H5N1 and may serve as a lead
compound or scaffold for the further development of H5N1 entry inhibitor.