Synthesis and anti-influenza virus activities of a novel class of gastrodin derivatives

Article abstract of DOI:10.3390/molecules18043789

A series of substituted aryl glycoside analogues of gastrodin have been identified as potential anti-influenza agents. The most potent inhibitor 1a exhibited moderate inhibitory activity against the A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1) strains of the influenza A virus (IC₅₀ values of 44.40 and 34.45 μM, respectively) and the oseltamivir-null B/Jifang/13/97 strain of influenza B (IC₅₀ value of 33.01 μM). In this article, multiple doses of compound 1a (80 mg/kg/day, oral administration) were used for the treatment of mice infected with influenza A/FM/1/47-MA (H1N1), and surprisingly we found that compound 1a significantly increased the number of survivors and prolonged the mean survival time. The preliminary studies on the mechanism of antiviral activity showed no interaction between compound 1a and the neuraminidase or the M2 protein. The novel target to overcome drug resistance combined with its good in vivo profile support compound 1a to be a new lead for further development of antiviral agents.

Full text of DOI:10.3390/molecules18043789

Molecules 2013, 18, 3789-3805; doi:10.3390/molecules18043789
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anti-Influenza Virus Activities of a Novel Class of
Gastrodin Derivatives
Si-Tu Xue, Wei-Ying He, Lin-Lin Ma, Hui-Qiang Wang, Bo Wang, Guang-Hui Zheng,
Xing-Yue Ji, Tian Zhang, Yu-Huan Li *, Jian-Dong Jiang and Zhuo-Rong Li *
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100050, China
* Authors to whom correspondence should be addressed; E-Mails: yuhuanlibj@126.com (Y.-H.L.);
l-z-r@263.net (Z.-R.L.); Tel.: +86-10-6301-0984 (Y.-H.L.); Fax: +86-10-6301-7302 (Y.-H.L.);
Tel.: +86-10-6302-7185 (Z.-R.L.).
Received: 16 February 2013; in revised form: 19 March 2013 / Accepted: 20 March 2013 /
Published: 26 March 2013
Abstract: A series of substituted aryl glycoside analogues of gastrodin have been
identified as potential anti-influenza agents. The most potent inhibitor 1a exhibited
moderate inhibitory activity against the A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1)
strains of the influenza A virus (IC50 values of 44.40 and 34.45 μM, respectively) and the
oseltamivir-null B/Jifang/13/97 strain of influenza B (IC50 value of 33.01 μM). In this
article, multiple doses of compound 1a (80 mg/kg/day, oral administration) were used for
the treatment of mice infected with influenza A/FM/1/47-MA (H1N1), and surprisingly we
found that compound 1a significantly increased the number of survivors and prolonged the
mean survival time. The preliminary studies on the mechanism of antiviral activity showed
no interaction between compound 1a and the neuraminidase or the M2 protein. The novel
target to overcome drug resistance combined with its good in vivo profile support
compound 1a to be a new lead for further development of antiviral agents.
Keywords: gastrodin; aryl glycoside; synthesis; influenza virus inhibitors
1. Introduction
Influenza is a respiratory disease caused by the influenza virus. According to the World Health
Organization (WHO), as of April 4, 2010, the 2009-H1N1 influenza A virus have spread to over 213

Molecules 2013, 18
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countries and resulted in over 600,000 laboratory-confirmed cases and at least 17,700 deaths (WHO,
2010). These numbers represent the tip of the iceberg with regard to the potential morbidity and
mortality associated with infection resulting from this virus [1].
Despite the extensive effort has been invested in attempting to control influenza infection, only two
classes of drugs are currently clinically available, namely inhibitors of the M2 protein
(e.g., amantadine and rimantadine) and the neuraminidase (e.g., zanamivir and oseltamivir) (Figure 1).
Clinical applications of amantadine and rimantadine have been limited as a consequence of the
increasing incidence of adamantane-resistant viruses in the general population [2,3]. Moreover,
blockers of the M2 ion channel inhibit only the replication of influenza A virus and could cause
neurological side effects [4]. The neuraminidase inhibitors, zanamivir and oseltamivirwere marketed in
1999 for the treatment and prophylaxis of influenza and have been subsequently stockpiled by many
countries for usage in the event of a pandemic. However, significant increases in the frequency of
oseltamivir-resistant seasonal influenza A (H1N1) in Europe, the United States, Oceania and South
Africa have been identified recently. By April 30, 2010, a total of 285 cases of resistance had been
reported worldwide [5], and the widespread emergence of oseltamivir-resistant strains had become
increasingly likely [6]. In addition to mutation of H275, an S274N neuraminidase mutation has also
been reported [7]. The recent pandemic of influenza A (H1N1) effectively reemphasizes the
importance of identifying new anti-influenza drugs.
Figure 1. The chemical structures of some anti-influenza drugs and gastrodin.
Gastrodia elata Blume, which is an orchid plant, is a well-known Traditional Chinese Medicine and
has been clinically used for about 2,000 years. Its medicinal part is the dry tuber, and it is widely
beleived that as a traditional medicine Gastrodia elata Blume has anti-epileptic, anti-palpitation,
anti-rheumatic, tranquil and nourishing effect. Gastrodin, which was isolated and identified from the
extracts of Gastrodia elata Blume, has been widely used in the clinic in China for the treatment of
hypertension, headache and neuralgia [8]. To the best of our knowledge, it has never been used as an
antiviral agent in clinic, and there are also no reports about its possible antiviral activities.

Molecules 2013, 18
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In the current work, a series of substituted aryl glycosides were synthesized as analogues of
gastrodin and their anti-influenza activity was evaluated. The compounds proved to be a novel class of
potential anti-influenza agents. The most potent compound [methyl 4-fluoro-3-((2S,3R,4S,5R,6R)-
3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy) benzoate (1a)] exhibited significant
inhibitory activity against the tested influenza A strains: A/Hanfang/359/95(H3N2) and
A/FM/1/47(H1N1), with IC50 values of 44.4 and 34.4 μM, respectively. Mice infected with influenza
A/FM/1/47-MA (H1N1) were treated with multiple doses of compound 1a on a dose regimen of
80 mg/kg/day (oral administration), which led to a significant increase in the number of survivors and
a prolonged mean survival time. Herein, we reported the synthesis and biological evaluation of a series
of novel gastrodin analogues as new antiviral agents for the first time, along with a discussion of the
preliminary structure-activity relationships (SAR) study and the in vivo activity.
2. Results and Discussion
2.1. Chemistry
The chemical structures of the target analogues differed in three ways, and the modifications are as
follows: (1) changes to the substituents and their positions on the phenyl ring; (2) conversion of the
phenyl ring to a pyridyl ring; and (3) changes to the sugar moiety or protection of the hydroxyl group
by acetylation.
As shown in the Scheme 1, compound 1a was synthesized from the reaction of methyl 4-fluoro-3-
hydroxybenzoate (1) with 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl bromide using potassium
carbonate (K2CO3) as the base. The color of the reaction system darkened slightly as the reaction
proceeded. Unfortunately, we were unable to develop conditions for driving the reaction to
completion. Consequently, significant levels of the starting material were detected in the reaction
mixture, and a low yield of less than 40% was obtained. Compound 13a was obtained from 1a in a
two-step procedure involving the initial reduction of the methyl ester in 1a with lithium aluminum
hydride (LiAlH4) [9] followed by acetylation of the resulting primary alcohol with acetic anhydride.
Subsequent deprotection of the acetyl groups by saponification with sodium methoxide (CH3ONa) in
MeOH gave 14a [10] in greater than 70% overall yield from 1a (Scheme 1). Compounds 2a–5a,
7a–12a, 1b, 6b–10b were also synthesized by this route, as depicted in Scheme 1.
2.2. Evaluation of Anti-Influenzavirus Activity In Vitro
The antiviral activities of the synthesized glycoside compounds against influenza
A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1) were initially evaluated through their ability to
prevent cytopathic effects (CPE) in influenza A virus-infected Madin-Darby canine kidney (MDCK)
cells, with oseltamivir and ribavirin (RBV) as the positive controls. Except for the known compounds,
1 (the methyl 3-hydroxy-4-fluorobenzoate starting material), 5a, 7a, 11a, 6b and gastrodin, all
compounds tested in the current study were, to the best of our knowledge, synthesized and were
reported for the first time in academia. Ultimately, a total of 21 compounds were evaluated, and the
results were summarized in Table 1.

Molecules 2013, 18
3792
Scheme 1. The synthetic route to the target compounds.
O
O
R
O
O
R
AcO
AcO
a
AcO
AcO
or
R OH
OAc
OAc
OAc
OAc
O
Br
AcO
F
F
F
A:
B:
AcO
OAc
OAc
O
1b: R=
6b: R=
7b: R=
8b: R=
9b: R=
10b: R=
1a: R=
2a: R=
3a: R=
4a: R=
5a: R=
7a: R=
8a: R=
9a: R=
10a: R=
11a: R=
12a:R=
1: R=
COOMe
COOMe
COOMe
OMe
Br
AcO
AcO
OMe
COOMe
Cl
OAc
2: R=
3: R=
4: R=
5: R=
6: R=
7: R=
8: R=
9: R=
10: R=
11: R=
12: R=
COOMe
Cl
OAc
F
COOMe
OMe
Cl
OMe
COOMe
COOMe
COOC2H5
NH2
COOC2H5
Cl NH2
COOMe
F
COOMe
COOMe
F
COOC2H5
COOC2H5
F
F
COOMe
F
COOMe
COOMe
COOMe
COOMe
COOMe
F
F
COOMe
F
F
F
F
N
N
N
F
N
F
HOH2C
O
AcOH2C
O
b
c
F
F
O
O
HO
AcO
HO
OH
AcO
OAc
OH
14a
OAc
13a
Reagents and conditions: (a) K2CO3, anhydrous acetone, 56 °C; (b) LiAlH4, THF, 66 °C, acetic anhydride,
pyridine, 80 °C; (c) CH3ONa/CH3OH, CH3OH, room temperature.
As shown in Table 1, compounds 1a, 8a, 11a and 10b exhibited moderate inhibitory activity against
the A/Hanfang/359/95(H3N2) strain of the influenza virus, with IC50 values of 44.40, 44.40, 40.52 and
48.68 μM, respectively. Nevertheless, they were not as potent as the reference drugs ribavirin
(IC50 = 11.87 μM) and oseltamivir (IC50 = 2.08 μM). Of all the tested compounds, 2a showed the
highest level of inhibitory activity against the A/FM/1/47(H1N1) strain of the influenza virus, with an
IC50 value of 11.58 μM, which was close to that of the reference drug oseltamivir (IC50 value of 57.52 μM).
Furthermore, compounds 1a, 9a, 11a and 12a also exhibited moderate levels of inhibitory activity
against the A/FM/1/47(H1N1) strain of the influenza virus, with IC50 values of 34.45, 34.45, 52.23 and

Molecules 2013, 18
3793
25.24 μM, respectively. Compounds 1a and 11a showed activity against both the
A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1) strains of the influenza virus. Compounds 5a and
1b showed low levels of inhibitory activity against the A/Hanfang/359/95(H3N2) strain of the
influenza virus, with IC50 values of 93.64 and 92.38 μM, respectively. Compounds 8a and 9b showed
low levels of inhibitory activity against the A/FM/1/47(H1N1) strain of the influenza virus, with IC50
values of 83.72 and 66.56 μM, respectively. In contrast, the remaining compounds (including
compound 1 and gastrodin) showed weak or no activity against these strains under the specific
conditions used in this paper.
Table 1. Anti-influenza activities of the synthesized compounds.
A/Hanfang/359/95 a
A/FM/1/47 b
Compounds
TC50 (M)
IC50 (M)
44.40
SI
4.3
_
IC50 (M)
SI
5.6
2.0
_
1a
192.14
23.65
34.45
11.58
2a
>13.66
>362.77
>134.02
93.64
3a
>362.77
>402.05
232.53
_
>362.77
119.03
>134.29
>133.23
83.72
4a
_
>3.4
_
5a
2.5
2.5
6.2
_
7a
277.10
103.39
44.40
_
8a
277.10
3.3
>11.6
_
9a
>399.66
>438.20
226.03
>44.40
>146.07
40.52
34.45
10a
_
>146.07
52.23
11a
5.6
_
4.3
13.9
_
12a
350.06
>50.12
>129.60
>657.31
92.38
25.24
13a
>388.76
>657.31
>399.66
>414.56
>399.66
>399.66
399.66
_
>129.60
>657.31
>133.23
>138.19
133.22
>399.66
66.56
14a
_
_
1b
2.5
_
_
6b
>138.19
>133.22
>399.66
>133.22
48.68
_
7b
8b
_
>3.0
_
_
9b
_
6.0
_
10b
252.99
5.2
_
>52.23
>391.85
>232.19
13.47
1
>1175.50
>698.62
4766.99
4033.29
>1175.50
161.49
11.87
_
Gastrodin
Ribavirin
Oseltamivir
4.3
401.4
1938.5
_
353.8
2.08
57.52
70.1
a 316TCID50; b 280TCID50; Half maximal (50%) inhibitory concentration (IC) of a substance, TC50 represents
for median lethal dose, and SI represents for Selective therapeutic index.
The active compounds against the A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1) strains of the
influenza virus were further evaluated for their potential antiviral activity against an oseltamivir-null
strain of the influenza B virus, also with the means of CPE method. As summarized in Table 2,
compounds 1a, 2a and 10b also displayed antiviral activity against the influenza B oseltamivir-null
strain. Especially, compound 2a was found to be the most effective compound against the
oseltamivir-resistant strain of the influenza virus (B/Jifang/13/97), with IC50 and SI values of 6.25 μM
and 6.80, respectively.

Molecules 2013, 18
3794
Table 2. Activities of the synthesized compounds against the oseltamivir-null influenza strain.
B/Jifang/13/97 a
Compounds
TC50 (μM)
IC50 (M)
33.01
SI
6.14
3.94
_
1a
2a
202.75
24.63
6.25
9a
399.66
235.95
322.63
360.25
266.16
4921.94
3968.32
>44.40
>52.23
>50.12
>133.22
39.41
11a
_
12a
_
9b
_
10b
6.80
432.35
_
Ribavirin
Oseltamivir
11.38
>100
a 100 TCID50.
2.3. Structure-Antiviral Activity Relationships
As shown in Table 1, compounds 1a, 8a and 11a showed moderate activity against both the
A/Hanfang/359/95(H3N2) and A/FM/1/47(H1N1) strains of the influenza virus, whereas compound
10b only showed moderate activity against the A/Hanfang/359/95(H3N2) strain but no activity against
the A/FM/1/47(H1N1) strain. In contrast, compounds 2a, 9a and 12a only showed activity against the
A/FM/1/47(H1N1) strain, but no activity against the A/Hanfang/359/95(H3N2) strain. However, all of
our active compounds were less potent than the positive controls.
Relative to compound 1a, the corresponding starting material 1, which does not contain the
2,3,4,6-tetra-O-acetyl--D-glucopyranosyl moiety, showed no activity against either of the viral
strains. Additionally, when the 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl moiety was changed to the
2,3,4,6-tetra-O-acetyl--D-galactopyranosyl moiety, the antiviral activity decreased in the majority of
the derivatives and even disappeared in some cases, with only compound 10b retaining activity against
the A/Hanfang/359/95(H3N2). Those results indicated that the 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl
moiety was crucial to the antiviral activity of these compounds. When the fluorine substituent in
compound 1a was replaced with an amino group (compound 4a), the ester group in compound 1a was
replaced with an acetoxymethyl (compound 13a) or hydroxymethyl (compound 14a) group, or the
ester group was migrated to the 4-position of the benzene ring (compound 7a), the antiviral activity
was significantly reduced or even disappeared completely in some cases. Interestingly, when the
positions of the fluorine and ester groups in compound 1a were exchanged with each other
(compound 8a), the activity was retained. Taken together, it was possible that possessing hydrophobic
substituents (–F, –COOMe) both at the 2- and 5-positions simultaneously enhanced the antiviral
activity, yet this need to be confirmed by further experiment. Furthermore, it was also indicated that
the presence of a nitrogen-containing heteroaryl ring enhances antiviral activity because compounds
11a and 12a both showed antiviral activity. In summary, this is just our preliminary SAR study, and
the detailed structure optimization is ongoing in our lab.

Molecules 2013, 18
3795
2.4. Anti-Influenza Activity of Compound 1a in Vivo
We proceeded to investigate the effect of compound 1a on influenza A/FM/1/47-MA (H1N1) virus-
infected mice, with ribavirin being used as a reference standard. Two doses of 1a (80 and 40 mg/kg/day)
were administered intragastrically. The control compound ribavirin was introduced as a single dose
(100 mg/kg/day, po). All experiments were administered twice daily over a seven day period.
Against infection of the influenza A/FM/1/47-MA (H1N1) virus, the ribavirin treatment group
exhibited significant increases in mean survival time and moderate decreases in mean lung score. In
the group of mice receiving the intragastrically administered dose of 80 mg/kg/day of 1a, the numbers
of surviving animals (6/10) and the mean survival time (11.3 days) increased significantly, whereas all
of the control animals died. By contrast, in the group of mice receiving the lower dosage of 40 mg/kg/day
of 1a, only one animal survived (1/10), and the mean survival time was therefore not improved.
Therefore, the intragastricl administration of 1a provided protection against the lethal effects of the
influenza A/FM/1/47-MA (H1N1) virus in a dose-dependent fashion.
The effects of 1a on lung score and on decreasing lung consolidation at day 6 are shown in Table 3.
An 80 mg/kg/day dose of 1a provided a clear improvement in both the lung consolidation and mean
lung score relative to that of the vehicle.
a
Table 3. Effects of oral treatment with 1a on influenza A/FM/1/47-MA (H1N1) virus
infections b in mice.
Treated mice
Dosage
Compound
Mean day
to death c
11.3
Lung
consolidation d
2.25
Mean lung
score
(mg/kg/day)
% Survivors
80 (po)
40 (po)
100 (po)
60 (6/10)
10 (1/10)
1.82 ± 0.82
2.61 ± 0.85
1.31 ± 0.34 *
2.65 ± 1.06
1.01 ± 0.12 *
1a
8.0
3.56
Ribavirin
Vehicle
100 (10/10)
10 (1/10)
14.0 *
9.0
0.50 *
3.50
Normal controls
100 (10/10)
14.0 *
0.00 *
a b.i.d × 7, beginning 1 day before viral infection; b The viral infection dose was 16.32 LD50; c Mean survival
d
time of mice dying on or before day 14; Lungs were removed on day 6 post-virus exposure. Lung scores
were determined based on the percentage of the lung displaying signs of consolidation, with 0 being normal
and 4 indicative of 100% consolidation. * p < 0.05.
2.5. The Preliminary Studies on Anti-Influenza Mechanism of Compound 1a
Because compound 1a was orally active and showed potential to be developed as antiviral drug, we
researched on anti-influenza mechanism of compound 1a by neuraminidase inhibition assay,
hemagglutination assay and M2 protein expression inhibition assay.
2.5.1. Hemagglutination Inhibition Assay
The early influenza virus replication steps include virus absorption, internalization of virus to form
a virus-containing endosome, and fusion. Hemagglutination-inhibition test was used in which
aggregation of chicken erythrocytes was the consequence of the binding between viral hemagglutinin

Molecules 2013, 18
3796
and sialic acid receptors on the surface of the cells [11]. At 2 fold serial diluted compound 1a (from
40 μg/mL) (Figure 2), the compound 1a did not show an inhibitory effect on hemagglutination (data
not shown), suggesting that these compounds do not interfere with virus-receptor interaction.
Figure 2. Hemagglutination inhibitory activity of 1a.
μg/mL
Oseltamivir
Ribavirin
g/mL
1a
C
VC
500
40
100
20
20
4
10
5
2.5.2. Neuraminidase Inhibition Assay
Neuraminidase inhibition assay were evaluated for in vitro inhibitory actions using the method
according to the reference with slight modification [12]. This spectrofluorometric assay (excitation
wavelength: 355 nm and emission wavelength: 460 nm) uses 2′-(4-methylumbelliferyl)-α-D-
acetylneuraminic acid (MUNANA) as the enzyme substrate, which is converted to a fluorescent
product upon cleavage by NA. The intensity of fluorescence can reflect the activity of NA sensitively.
The compound 1a did not have inhibitory effect (IC50 > 40 μg/mL) on neuraminidase of the two
influenza virus A strain. Oseltamivir has a strong inhibitory effect (IC50 = 0.02 μg/mL) on
neuraminidase of influenza virus A/Wuhan/359/95.
2.5.3. M2 Protein Expression Inhibition Assay
The effect of compounds on M2 protein expression level were detected by immunoboltting assay [13].
The compound 1a had no effect on influenza virus M2 protein expression (Figure 3). Amantadine at
concentration of 25 μg/mL significantly inhibited influenza virus M2 protein expression. Oseltamivir
at concentration of 10 μg/mL has inhibitory effect on influenza virus M2 protein expression, but not so
good as amantadine (25 μg/mL). Ribavirin at concentration of 10 μg/mL has significant inhibitory
effect on influenza virus M2 protein expression, because at concentration of 10 μg/mL, ribavirin
completely inhibits influenza virus induced cytopathic effect.
Figure 3. The effect of 1a on M2 protein expression in MDCK cell infected with influenza
virus A/Wuhan/359/95.
VC
CC
Oseltamivir Ribavirin
10 10
1a
40
Amantadine
25
μg/mL
Ab: anti-β-actin
(1:5000)
Ab: anti-M2
(1:400)

Molecules 2013, 18
3797
In summary, compound 1a tested in hemagglutination inhibition assay, neuraminidase inhibition
assay and M2 protein expression inhibition assay have no effect on the three antiviral target of
influenza virus. Although this series novel compounds were not as potent as reference compounds, but
they had different mechanism against influenza virus. With these results in hand, the future research
became more meaningful. Further mechanism studies are now in progress.
3. Experimental
3.1. Chemistry
1H-NMR spectra were recorded in CDCl3 on a 400 or 500 MHz Varian Inova spectrometer.
Chemical shifts have been reported in parts per million (ppm) relative to tetramethylsilane (TMS) as
the internal standard. The melting points were determined with an X6 microscope melting point
apparatus and have been reported uncorrected. Electrospray ionization (ESI) mass spectra and
high-resolution mass spectra (HRMS) were obtained on an MDS SCIEX Q-Trap mass spectrometer.
3.2. Synthesis
Methyl 4-fluoro-3-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (1a). Methyl 4-fluoro-3-hydroxybenzoate (1, 1.7 g, 10 mmol), 2,3,4,6-tetra-O-acetyl--
D-glucopyranosyl bromide (5 g, 12 mmol) and K2CO3 (1.7 g, 12 mmol) were added to anhydrous
acetone (75 mL), and the resulting mixture was stirred at 56 °C for 16 h before being cooled to
ambient temperature and filtered. The solvent was then evaporated in vacuo to give a sticky residue,
which was partitioned between dichloromethane (60 mL) and a 0.1 N NaOH solution (20 mL). The
organic phase was collected and concentrated, and the resulting solid was purified by column
chromatography on silica gel to yield compound 1a (1.65 g, 36%). White solid; m.p.: 144–146 °C;
1H-NMR (400 MHz, CDCl3) δ (ppm): 7.86 (1H, d, J = 7.6 Hz, PhH), 7.79 (1H, m, PhH), 7.14 (1H, t,
J = 8.0 Hz, PhH), 5.15 (4H, m, CH), 4.28 (2H, m, CH), 3.93 (1H, d, J = 4.0 Hz, CH), 3.90 (3H, s,
OCH3), 2.10 (3H, s, CH3), 2.07 (3H, s, CH3), 2.02 (6H, s, CH3); ESI-MS (m/z): 523 [M+Na]+;
HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14078.
Methyl 2,6-dimethoxy-4-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-
2-yloxy)benzoate (2a). Compound 2a was synthesized using a method similar to that described above
for 1a. Yield: 35%, white solid; m.p.: 121–123 °C; 1H-NMR (400 MHz, CDCl3) δ (ppm): 6.50 (2H, s,
PhH), 5.28 (3H, m, CH), 5.15 (1H, d, J = 7.2 Hz, CH), 4.22 (1H, m, CH), 4.09 (1H, d, J = 8.4 Hz,
CH), 3.89 (3H, s, OCH3), 3.85 (6H, s, OCH3), 3.69 (1H, m, CH), 2.04 (3H, s, CH3), 2.01 (9H, s, CH3);
ESI-MS (m/z): 565 [M+Na]+; HRMS-ESI (m/z): calculated for C24H31O14 [M+H]+: 543.17138;
measured: 543.17148.
Ethyl 3,5-dichloro-4-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (3a). Compound 3a was synthesized using a method similar to that described above for
1
1a. Yield: 32%, white solid; m.p.: 112–114 °C; H-NMR (400 MHz, CDCl3) δ (ppm): 7.93 (2H, s,
PhH), 6.39 (1H, s, CH), 5.30 (3H, m, CH), 4.33 (3H, m, CH), 4.11 (2H, m, CH), 2.06 (3H, s, CH3),

Molecules 2013, 18
3798
2.03 (9H, s, CH3), 1.33 (3H, s, CH3); ESI-MS (m/z): 587 [M+Na]+; HRMS-ESI (m/z): calculated for
C22H25Cl2O12 [M+H]+: 551.07231; measured: 551.07226.
Methyl 4-amino-3-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (4a). Compound 4a was synthesized using a method similar to that described above for
1
1a. Yield: 29%; brown solid; m.p.: 126–128 °C; H-NMR (400 MHz, CDCl3) δ (ppm): 7.84 (1H, d,
J = 6.0 Hz, PhH), 7.77 (1H, m, PhH), 7.10 (1H, t, J = 8.8. Hz, PhH), 5.54 (1H, t, J = 8.4 Hz, CH), 5.44
(1H, d, J = 3.2 Hz, CH), 5.14 (1H, dd, J = 8.8 Hz, J = 3.2 Hz, CH), 5.02 (1H, d, J = 8.4 Hz, CH), 4.21
(2H, m, CH), 4.05 (1H, t, J = 6.4 Hz, CH), 3.90 (3H, s, OCH3), 3.74 (2H, s, NH2), 2.19 (3H, s, CH3),
2.10 (3H, s, CH3), 2.07 (3H, s, CH3), 2.00 (3H, s, CH3); ESI-MS (m/z): 520 [M+Na]+; HRMS-ESI
(m/z): calculated for C22H28NO12 [M+H]+: 498.16115; measured: 498.16102.
Methyl 4-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate
(5a). Compound 5a was synthesized using a method similar to that described above for 1a. Yield:
1
30%; white solid; m.p.: 147–149 °C; H-NMR (400 MHz, CDCl3) δ (ppm): 8.00 (2H, d, J = 8.4 Hz,
PhH), 7.00 (2H, d, J = 8.4 Hz, PhH), 5.53 (1H, d, J = 7.6 Hz, CH), 5.27 (2H, m, CH), 5.14 (1H, t,
J = 6.4 Hz, CH), 5.02 (1H, d, J = 7.2 Hz, CH), 4.25 (4H, m, CH), 2.08 (3H, s, CH3), 2.05 (6H, s, CH3),
2.04 (3H, s, CH3), 1.38 (3H, s, CH3); ESI-MS (m/z): 519 [M+Na]+; HRMS-ESI (m/z): calculated for
C23H29O12 [M+H]+: 497.16590; measured: 497.16594.
Methyl 3-fluoro-4-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (7a). Compound 7a was synthesized using a method similar to that described above for
1
1a. Yield: 31%; white solid; m.p.: 121–123 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.83 (1H, t,
J = 6.5 Hz, PhH), 6.90 (1H, dd, J = 10.5 Hz, J = 2.5 Hz, PhH), 6.83 (1H, t, J = 8.0 Hz, PhH), 5.57 (1H,
d, J = 4.0 Hz, CH), 5.21 (4H, m, CH), 4.37 (2H, m, CH), 3.90 (3H, s, OCH3), 2.10 (6H, s, CH3), 2.06
(6H, s, CH3); ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+:
501.14083; measured: 501.14091.
Ethyl 4-fluoro-2-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)-
benzoate (8a). Compound 8a was synthesized using a method similar to that described above for 1a.
Yield: 30%; white solid; m.p.: 136–138 °C; 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.80 (1H, t, J = 7.5 Hz,
PhH), 6.95 (1H, dd, J = 8.0 Hz, J = 2.0 Hz, PhH), 6.84 (1H, m, PhH), 5.92 (1H, t, J = 9.0 Hz, CH),
5.48 (1H, d, J = 3.5 Hz, CH), 5.08 (2H, m, CH), 4.17 (3H, m, CH), 3.85 (3H, s, OCH3), 2.16 (3H, s,
CH3), 2.09 (3H, s, CH3), 2.05 (3H, s, CH3), 2.01 (3H, s, CH3); ESI-MS (m/z): 523 [M+Na]+;
HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14082.
Methyl 2-fluoro-6-((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (9a). Compound 9a was synthesized using a method similar to that described above for
1
1a. Yield: 35%; white solid; m.p.: 130–132 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.31 (1H, q,
J = 8.5 Hz, PhH), 6.90 (1H, d, J = 8.5 Hz, PhH), 6.86 (1H, t, J = 8.0 Hz, PhH), 5.27 (2H, m, CH), 5.18
(1H, t, J = 6.5 Hz, CH), 5.05 (1H, d, J = 7.5 Hz, CH), 4.25 (3H, m, CH), 3.89 (3H, s, OCH3), 2.11 (3H,
s, CH3), 2.08 (3H, s, CH3), 2.05 (3H, s, CH3), 2.02 (3H, s, CH3); ESI-MS (m/z): 523 [M+Na]+;
HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14077.

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(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(3-fluoro-2-methylphenoxy)-tetrahydro-2H-pyran-3,4,5-triyl
triacetate (10a). Compound 10a was synthesized using a method similar to that described above for
1
1a. Yield: 33%; white solid; m.p.: 127–129 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.62 (1H, m,
PhH), 7.21 (1H, m, PhH), 6.90 (1H, dd, J = 9.5 Hz, J = 3.5 Hz, PhH), 6.33 (1H, d, J = 3.5 Hz, CH),
5.72 (1H, d, J = 8.0 Hz, CH), 5.50 (2H, m, CH), 4.57 (1H, t, J = 7.0 Hz, CH), 4.22 (2H, m, CH), 2.40
(3H, s, CH3), 2.07 (6H, s, CH3), 2.02 (6H, s, CH3); ESI-MS (m/z): 479 [M+Na]+; HRMS-ESI (m/z):
calculated for C21H26FO10 [M+H]+: 457.15100; measured: 457.15095.
(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(pyridin-4-yloxy)-tetrahydro-2H-pyran-3,4,5-triyl triacetate
(11a). Compound 11a was synthesized using a method similar to that described above for 1a. Yield:
27%; colorless solid; m.p.: 132–134 °C; 1H-NMR (500 MHz, CDCl3) δ (ppm): 8.30 (2H, d, J = 9.0 Hz,
PhH), 6.93 (2H, d, J = 9.0 Hz, PhH), 5.50 (1H, dd, J = 10.5 Hz, J = 3.0 Hz, CH), 5.18 (1H, d, J = 3.0 Hz,
CH), 5.05 (1H, t, J = 7.0 Hz, CH), 4.15 (4H, m, CH), 2.08 (3H, s, CH3), 2.04 (3H, s, CH3), 2.02 (6H, s,
CH3); ESI-MS (m/z): 448 [M+Na]+; HRMS-ESI (m/z): calculated for C19H24NO10 [M+H]+: 426.14002;
measured: 426.14006.
(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(3-fluoropyridin-2-yloxy)-tetrahydro-2H-pyran-3,4,5-triyl
triacetate(12a). Compound 12a was synthesized using a method similar to that described above for 1a.
1
Yield: 31%; pale yellow solid; m.p.: 140–142 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.92 (1H, d,
J = 5.0 Hz, PhH), 7.39 (1H, t, J = 8.5 Hz, PhH), 6.99 (1H, m, PhH), 6.18 (1H, d, J = 7.5 Hz, CH), 5.35
(2H, m, CH), 5.24 (1H, t, J = 9.5 Hz, CH), 4.30 (1H, dd, J = 12.5 Hz, J = 4.5 Hz, CH), 4.14 (1H, d,
J = 12.5 Hz, CH), 3.94 (1H, m, CH), 2.10 (3H, s, CH3), 2.07 (3H, s, CH3), 2.04 (3H, s, CH3), 2.02 (3H,
s, CH3); ESI-MS (m/z): 466 [M+Na]+; HRMS-ESI (m/z): calculated for C19H23FNO10 [M+H]+:
444.13060; measured: 444.13051.
(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(5-(acetoxymethyl)-2-fluorophenoxy)-tetrahydro-2H-pyran-
3,4,5-triyl triacetate (13a). Compound 1a (500 mg, 1 mmol) was added to THF (25 mL), and solid
LiAlH4 was added in a portion-wise manner to the resulting solution. Upon completion of the addition,
the mixture was stirred at 66 °C for 3 h before being cooled to ambient temperature and quenched by
the dropwise addition of CH3OH (10 mL). The reaction mixture was then stirred at room temperature
for 10 min and concentrated to dryness under vacuum. The resulting residue was then dissolved in a
mixture of acetic anhydride (20 mL) and pyridine (1 mL). The reaction mixture was stirred at 80 °C
for 4 h and then concentrated to dryness in vacuo. The residue was added to ice water and stirred until
precipitation occurred. The mixture was then filtered, and the filter-cake was collected and purified by
flash chromatography over silica gel, affording the title compound (401 mg, 78%). White solid; m.p.:
125–127 °C; 1H-NMR (500 MHz, CDCl3) δ (ppm): 7.80 (1H, d, J = 8.0 Hz, PhH), 7.74 (1H, m, PhH),
7.10 (1H, t, J = 8.5 Hz, PhH), 5.89 (1H, d, J = 6.5 Hz, CH), 5.30 (1H, dd, J = 10.5, 4.0 Hz, CH), 5.07
(1H, t, J = 11.0 Hz, CH), 5.19 (2H, s, CH), 4.36 (1H, m, CH), 4.14 (3H, m, CH), 2.11 (3H, s, CH3),
2.08 (6H, s, CH3), 2.06 (3H, s, CH3), 2.04 (3H, s, CH3); ESI-MS (m/z): 537 [M+Na]+; HRMS-ESI
(m/z): calculated for C23H28FO12 [M+H]+: 515.15648; measured: 515.15652.
(2S,3R,4S,5S,6R)-2-(2-Fluoro-5-(hydroxymethyl)phenoxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-
3,4,5-triol (14a). To a solution of 13a (510 mg, 1 mmol) in CH3OH (10 mL) was added a 0.2 N

Molecules 2013, 18
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solution of CH3ONa in CH3OH, and the resulting mixture was stirred at room temperature for 1 h and
then allowed to stand at ambient temperature overnight. The reaction mixture was filtered, and the
filter cake was collected and recrystallized from anhydrous ethanol to afford the title compound (280 mg,
1
90%). White solid; m.p.: 134–136 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.15 (1H, t, J = 9.5 Hz,
PhH), 6.95 (1H, dd, J = 9.5 Hz, J = 4.0 Hz, PhH), 6.91 (1H, m, PhH), 5.60 (1H, s, CH), 4.66 (2H, s,
CH), 4.20 (1H, s, CH), 3.47 (5H, m, CH), 2.76 (1H, s, OH), 2.16 (1H, s, OH), 1.78 (2H, s, OH), 0.99
(1H, s, OH); ESI-MS (m/z): 327 [M+Na]+; HRMS-ESI (m/z): calculated for C13H18FO7 [M+H]+:
305.10366; measured: 305.10362.
Methyl 4-fluoro-3-((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (1b). Compound 1b was synthesized using a method similar to that described above for
1
1a. Yield: 38%; white solid; m.p.: 143–145 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.88 (1H, d,
J = 6.5 Hz, PhH), 7.78 (1H, m, PhH), 7.14 (1H, t, J = 9.0 Hz, PhH), 5.53 (1H, t, J = 8.5 Hz, CH), 5.46
(1H, d, J = 3.0 Hz, CH), 5.11 (1H, dd, J = 10.0, 3.0 Hz, CH), 5.01 (1H, d, J = 8.0 Hz, CH), 4.20 (2H,
m, CH), 4.07 (1H, t, J = 6.5 Hz, CH), 3.90 (3H, s, OCH3), 2.19 (3H, s, CH3), 2.10 (3H, s, CH3), 2.07
(3H, s, CH3), 2.00 (3H, s, CH3); ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for
C22H26FO12 [M+H]+: 501.14083; measured: 501.14086.
Methyl 3-((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yloxy)benzoate
(6b). Compound 6b was synthesized using a method similar to that described above for 1a. Yield:
1
36%; white solid; m.p.: 154–156 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.75 (1H, d, J = 8.0 Hz,
PhH), 7.67 (1H, s, PhH), 7.37 (1H, t, J = 8.0 Hz, PhH), 7.20 (1H, dd, J = 8.0, 2.5 Hz, CH), 5.50 (2H,
m, CH), 5.12 (2H, m, CH), 4.16 (3H, m, CH), 3.91 (3H, s, OCH3), 2.19 (3H, s, CH3), 2.07 (6H, s,
CH3), 2.00 (3H, s, CH3); ESI-MS (m/z): 505 [M+Na]+; HRMS-ESI (m/z): calculated for C22H27O12
[M+H]+: 483.15025; measured: 483.15030.
Methyl 3-fluoro-4-((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (7b). Compound 7b was synthesized using a method similar to that described above for
1
1a. Yield: 32%; white solid; m.p.: 121–123 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.77 (2H, m,
PhH), 7.21 (1H, t, J = 8.5 Hz, PhH), 5.52 (1H, t, J = 8.0 Hz, CH), 5.45 (1H, d, J = 3.0 Hz, CH), 5.11
(1H, dd, J = 10.5, 3.0 Hz, CH), 5.01 (1H, d, J = 7.5 Hz, CH), 4.20 (2H, m, CH), 4.05 (1H, m, CH),
3.90 (3H, s, OCH3), 2.15 (3H, s, CH3), 2.08 (3H, s, CH3), 2.03 (3H, s, CH3), 1.99 (3H, s, CH3);
ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083;
measured: 501.14076.
Methyl 4-fluoro-2-((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (8b). Compound 8b was synthesized using a method similar to that described above for
1
1a. Yield: 30%; white solid; m.p.: 136–138 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.83 (1H, t,
J = 7.0 Hz, PhH), 6.92 (1H, dd, J = 8.5, 2.0 Hz, PhH), 6.83 (1H, m, PhH), 5.60 (1H, t, J = 8.0 Hz, CH),
5.47 (1H, d, J = 3.0 Hz, CH), 5.11 (1H, dd, J = 10.5, 3.5 Hz, CH), 5.04 (1H, d, J = 8.0 Hz, CH), 4.17
(3H, m, CH), 3.85 (3H, s, OCH3), 2.18 (3H, s, CH3), 2.08 (3H, s, CH3), 2.06 (3H, s, CH3), 2.02 (3H, s,
CH3); ESI-MS (m/z): 523 [M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083;
measured: 501.14074.

Molecules 2013, 18
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Methyl 2-fluoro-6-((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-
yloxy)benzoate (9b). Compound 9b was synthesized using a method similar to that described above for
1
1a. Yield: 35%; white solid; m.p.: 130–132 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.31 (1H, q,
J = 8.5 Hz, PhH), 6.92 (1H, d, J = 8.0 Hz, PhH), 6.84 (1H, t, J = 8.5 Hz, PhH), 5.47 (2H, m, CH), 5.07
(1H, dd, J = 10.5 Hz, J = 3.0 Hz, CH), 5.00 (1H, d, J = 8.0 Hz, CH), 4.16 (3H, m, CH), 3.90 (3H, s,
OCH3), 2.21 (3H, s, CH3), 2.12 (3H, s, CH3), 2.06 (3H, s, CH3), 2.03 (3H, s, CH3); ESI-MS (m/z): 523
[M+Na]+; HRMS-ESI (m/z): calculated for C22H26FO12 [M+H]+: 501.14083; measured: 501.14077.
(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(3-fluoro-2-methylphenoxy)-tetrahydro-2H-pyran-3,4,5-triyl
triacetate (10b). Compound 10b was synthesized using a method similar to that described above for 1a.
1
Yield: 36%; white solid; m.p.: 127–129 °C; H-NMR (500 MHz, CDCl3) δ (ppm): 7.60 (1H, m, PhH),
7.20 (1H, m, PhH), 6.93 (1H, dd, J = 9.5, 3.5 Hz, PhH), 6.32 (1H, d, J = 3.5 Hz, CH), 5.74 (1H, d,
J = 8.0 Hz, CH), 5.50 (2H, m, CH), 4.54 (1H, t, J = 7.0 Hz, CH), 4.24 (2H, m, CH), 2.40 (3H, s, CH3),
2.09 (3H, s, CH3), 2.07 (3H, s, CH3), 2.04 (3H, s, CH3), 2.02 (3H, s, CH3); ESI-MS (m/z): 479
[M+Na]+; HRMS-ESI (m/z): calculated for C21H26FO10 [M+H]+: 457.15100; measured: 457.15098.
3.3. Antiviral Assays
MDCK cells, influenza A (A/Hanfang/359/95, H3N2), influenza A (A/FM/1/47, H1N1) and
influenza B (B/Jifang/13/97) were obtained from the Institute of Virology of the Chinese Academy of
Preventive Medicine (Beijing, China).
3.3.1. Cytotoxicity Assay
The cytotoxicities of the compounds in the presence of MDCK cells were monitored by CPE.
MDCK cells (2.5 × 104/well) were plated into a 96-well plate. After 24 h, the monolayer cells were
incubated in the presence of a variety of different concentrations of the test compounds. After 48 h of
culture at 37 °C and under a 5% CO2 atmosphere in a carbon dioxide incubator, the cells were
monitored by CPE. The median toxic concentrations (TC50) were determined using a Reed and
Muench calculation.
3.3.2. Anti-Influenza Assays
Confluent MDCK cells grown in 96-well microplates were infected with 100 median tissue culture
infective dose units (100TCID50) of the test strains. Following 2 h of adsorption at 37 °C, the
monolayers were washed with phosphate-buffered saline (PBS) and incubated at 37 °C in the
maintenance medium either in the presence or in the absence of different concentrations of the test
compounds. The viral CPE was observed when the viral control group reached 4, and the antiviral
activities (IC50) of the bisaryl amide derivatives were determined using a Reed and Muench
calculation. The SI value was calculated from TC50/IC50.

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3.4. Antiviral Assays in Vivo
The effect of compound 1a on influenza A/FM/1/47-MA (H1N1) virus-infected mice was evaluated
during the course of the current study, with ribavirin being used as a reference standard. Mice (female,
5–6 weeks of age and 14–16 grams in weight) were purchased from the National Institute for the
Control of Pharmaceutical and Biological Products (Beijing, China).
Ether-anesthetized mice were infected intranasally with a 50 times LD50 dose of mouse-adapted
influenza A/FM/1/47-MA (H1N1) virus. Compound 1a, prepared as a suspension in 5% tween-80, or
5% tween-80 alone (vehicle group), was administered intragastrically twice daily for seven days, with
the dose regimen being initiated one day prior to viral infection. The 10 mice involved in each group
were observed twice daily for 14 days during the experiment for the occurrence of death. The mean
day-to-death and survival times were then calculated (Table 3).
In a separate study, all eight of the mice involved in each group were killed when deaths began to
occur in the virus control group (day 6 post-virus exposure), and their lungs were removed aseptically.
Visual evidence of lung consolidation was scored on a blind basis. Lung scores were based on the
percentage of the lung displaying signs of consolidation, with 0 being normal and 4 being indicative of
100% consolidation. Lung consolidation scores were evaluated using ranked sum analysis (Table 3).
3.5. Mechanism Studies
3.5.1. Hemagglutination Inhibition Assay
Hemagglutination inhibition (HI) assay was employed to evaluate the effect of a compound on virus
adsorption to target cells according to the reference with slight modification. Briefly, compound in
PBS was mixed with an equal volume of influenza virus that was serially diluted by two-fold in PBS.
After 30 min incubation at 4 °C, 100 µL of compound-virus preparation was mixed with an equal
volume of 1% chicken erythrocyte suspension in PBS in a U bottom 96-well plate. The mixture was
incubated for 1 h at room temperature before observing erythrocyte aggregation on the plate.
3.5.2. Neuraminidase Inhibition Assay
Neuraminidase inhibition assay were evaluated for in vitro inhibitory actions using the method
according to the reference with slight modification. This spectrofluorometric assay (excitation
wavelength: 355 nm and emission wavelength: 460 nm) uses 2′-(4-methylumbelliferyl)-α-D-
acetylneuraminic acid (MUNANA) as the enzyme substrate, which is converted to a fluorescent
product upon cleavage by NA. The intensity of fluorescence can reflect the activity of NA sensitively.
The enzyme reaction system included 10 μL sample, 30 μL enzyme and 60 μL substrate buffer mix
(30 μM MUNAN, 33 mM MES buffer (pH 3.5), 4 mM CaCl2, DDW). The terminal volume was 100 μL.
Two-fold serially diluted compound samples and 30 μL enzyme incubated 1 hour at room temperature,
and 60 μL substrate buffer mix was added. After 15 min at 37 °C, 100 μL of 14 mM NaOH in 83%
ethanol was added to the reaction mixture to terminate the reaction. The intensity of the fluorescence
was quantitated in fluorescence read on Enspire (Perkin Elmer, Waltham, MA, USA) (excitation,
355 nm; emission, 460 nm), and substrate blanks were subtracted from the sample readings. The IC50

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was calculated by plotting percent inhibition versus the inhibitor concentration, and determination of
each point was performed in duplicate.
3.5.3. M2 Protein Expression Inhibition Assay
The effect of compounds on M2 protein expression level were detected by immunoboltting assay.
The immunoboltting experiment was employed according to the reference with slight
modification.MDCK cells were infected with influenza virus A/Wuhan/359/95 at an 300TCID50, and
treated with the indicated concentration of compounds. 24 hours post infection, the cells were lysed
with lysis buffer (1% Triton, 150 mM NaCl, 20 mM HEPES pH7.5, 10% Glycerol, 1 mM EDTA) with
protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The lysates were then performed to
immunoblotting with M2 andβ-actin antibodies (Influenza A m2 Antibody from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA, USA) and β-Actin Mouse mAb from Cell Signaling Technology
Inc. (Danvers, MA, USA). Protein was detected using Immobilon Western Chemiluminescent HRP
Substrate (Millipore, Inc., Billerica, MA, USA) with ChemiDoc XRS and Image Acquisition (BioRad,
Inc., Hercules, CA, USA). Most studies of the activity and drug sensitivity of A/M2 channels have
employed electrophysiology techniques, but since establishment of this technology takes time, we just
detected M2 protein expression levels.
4. Conclusions
Gastrodin is a traditional Chinese herbal medicine that has been used clinically in China for
thousands of years, mainly for the treatment of neurasthenia. To the best of our knowledge, there have
been no reports documenting the use of gastrodin or any of its derivatives as antiviral agents. In our
study, a novel series of gastrodin analogues was found to exhibit potent anti-influenza activity. A
series of substituted aryl glycoside compounds were synthesized and evaluated for their anti-influenza
virus activities, and preliminary structure-activity relationships (SAR) were analyzed.
A total of 15 novel glycoside compounds were synthesized and screened for their antiviral activity
against influenza A virus, among which the most potent inhibitor 1a exhibited moderate inhibitory
activity against the virus. Herein, we also report the first total synthesis of 1a and its associated in vivo
anti-influenza data. In this experiment, a dose regimen of 80 mg/kg/day of compound 1a
(oral administration) yielded a significant increase in the survival numbers and prolonged the mean
survival time of the animals tested.
Furthermore, the data were analyzed to produce a preliminary understanding of the
structure-activity relationships. Seven compounds exhibited clear antiviral activity against the tested
strain of the influenza A virus. The IC50 and SI values of the most effective compound, 12a, against the
influenza A (A/FM/1/47(H1N1)) virus were 25.24 μM and 13.9, respectively. Unfortunately, the SI
values for all the compounds tested were much lower than those of the reference compounds.
Remarkably, compounds 1a, 2a and 10b also displayed antiviral activity against the oseltamivir-null
strain of influenza B. In addition, there was an increasing need for the development of antiviral along
with novel antiviral mechanism. Compound 1a had a different mechanism of action from oseltamivir
and ribavirin against influenza virus, so future research may be more meaningful with this result.

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Undoubtedly, further exploration will be needed to produce more potent compounds with higher SI
values and to deduce the detailed mechanism of action of this novel class of glycosides compounds
against the influenza virus. Besides, since compound 1a has been demonstrated that it has no
interaction with NA or M2, it probably has novel targets which might be an important breakthrough in
viral study.
Acknowledgments
This work was supported by the National S&T Major Special Project for Major New Drugs
Innovation (2012ZX09301002-001-017).
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