Discovery of a series of novel compounds with moderate anti-avian H5N1 influenza virus activity in chick embryo

Article abstract of DOI:10.1007/s00044-012-0341-2

Enlightened by some flavonoid compounds, which had been found as influenza neuraminidase inhibitors, we designed and synthesized a series of novel compounds containing different amino acid fragments. We also reported a simple synthetic route from oseltamivir to prepare its active form which was used as the positive control. The result of enzyme inhibition assay indicated that all the designed compounds displayed weak inhibitory activity against neuraminidase. However, they showed moderate anti-avian H5N1 influenza virus activity in chick embryo. Besides interfering with the function of neuraminidase, these compounds seemed to inhibit the replication of influenza virus by some other mechanism which deserved deep study.

Full text of DOI:10.1007/s00044-012-0341-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3485–3496
DOI 10.1007/s00044-012-0341-2
ORIGINAL RESEARCH
Discovery of a series of novel compounds with moderate
anti-avian H5N1 influenza virus activity in chick embryo
•
Yuanchao Xie Bing Huang Kexiang Yu
•
•
•
Fangyuan Shi Wenfang Xu
Received: 21 August 2012 / Accepted: 7 November 2012 / Published online: 25 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Enlightened by some flavonoid compounds,
which had been found as influenza neuraminidase inhibi-
tors, we designed and synthesized a series of novel com-
pounds containing different amino acid fragments. We also
reported a simple synthetic route from oseltamivir to pre-
pare its active form which was used as the positive control.
The result of enzyme inhibition assay indicated that all the
designed compounds displayed weak inhibitory activity
against neuraminidase. However, they showed moderate
anti-avian H5N1 influenza virus activity in chick embryo.
Besides interfering with the function of neuraminidase,
these compounds seemed to inhibit the replication of
influenza virus by some other mechanism which deserved
deep study.
heightened concern. The viral surface protein, neuramini-
dase (NA), is an important target for anti-influenza drugs
design. Oseltamivir, one kind of NA inhibitors, is currently
the first line defense drug against influenza. However,
owing to the constant emergence of drug-resistant virus
strains, the anti-influenza situation becomes more critical.
It is still necessary to find novel effective anti-influenza
drugs.
Structure-based drug design has greatly contributed
the development of NA inhibitors, leading to three anti-
influenza drugs: zanamivir, oseltamivir, and peramivir (Liu
et al., 2007). Owing to low bioavailability, zanamivir and
peramivir cannot be administered orally, which is not
convenient and economical for patient care. In contrast,
oseltamivir has high bioavailability, but its wide use has
inevitably brought about drug-resistant viral strains
(Storms et al., 2012). Therefore, discovering some other
effective NA inhibitors with good pharmaceutical proper-
ties is the first concern.
Keywords Influenza ꢀ Neuraminidase ꢀ Flavonoid ꢀ
Amino acid
Introduction
According to recent studies, the design of NA inhibitors
has been in a difficult position after reaching a peak and it
seems hard to discover novel ones which could be better
than oseltamivir simply based on the NA active site.
Recently, many natural products, the structures of which
were very different from those classical NA inhibitors,
have been reported with moderate activity against NA.
Among them, flavonoids are the largest class (Liu et al.,
2008; Upadhyay et al., 2011) (Fig. 1). Unlike the classical
ones, most of these compounds acted as noncompetitive
NA inhibitors, indicating that they might inhibit the NA
activity by binding on some other sites of the enzyme,
instead of the catalytic center.
Influenza is a major health problem and can cause serious
economic loss. Recently, the worldwide spread of the
highly pathogenic avian influenza A (H5N1) virus in birds
and the increasing cases of bird-to-human transmission get
Y. Xie ꢀ F. Shi ꢀ W. Xu (&)
Department of Medicinal Chemistry, School of Pharmaceutical
Sciences, Shandong University, 44, West Culture Road,
Jinan 250012, Shandong, People’s Republic of China
e-mail: wfxu@yahoo.cn; xuwenf@sdu.edu.cn
B. Huang ꢀ K. Yu
In order to discover some kind of novel NA inhibitors
distinct from the classical ones, in this study, we con-
structed two new skeletons incorporating several structural
Institute of Poultry Science, Shandong Academy of Agricultural
Sciences, 1, Jiaoxiao Road, Jinan 250023, Shandong,
People’s Republic of China
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Med Chem Res (2013) 22:3485–3496
Fig. 1 The flavonoid compounds and mimosine tetrapeptides reported as NA inhibitors
Fig. 2 The two skeletons and
target compounds
features which were necessary for NA inhibitory activity of
some flavonoid NA inhibitors (Liu et al., 2008). Consid-
ering that the enzyme’s active site contained some polar
residues (von Itzstein, 2007), such as Glu 119, Glu 227, and
Glu 276 which preferred to bind with basic groups, and
peptides had potential to inhibit NA, we introduced dif-
ferent amino acids into the skeletons to design and syn-
thesize a series of novel compounds (Fig. 2).
synthesized by condensation of compound 4 or 5 with Boc-
protected amino acids with DCC and deprotection with
HCl/EtOAc.
Oseltamivir carboxylate (GS4071) was the active form
of oseltamivir and usually used as the positive control for
biologic evaluation. This compound was commercially
expensive and difficult to obtain by traditional methods.
We reported a simple route with high yield for preparing
GS4071 hydrochloride from oseltamivir via a three-step
sequence: protection of the amino group with Boc,
hydrolysis of the carboxylic acid ester mediated by NaOH,
and deprotection with HCl/EtOAc (Scheme 2).
Results and discussion
Chemistry
Pharmacology
The two skeletons and target compounds were synthesized
according to Scheme 1. The amino group of the starting
material, 2-amino-4-nitrophenol was first protected with
Boc, followed by nitro group reduction with hydrogen to
give the important intermediate, compound 3. (E)-3-(furan-
2-yl)acrylic acid used to construct the skeleton 4 was
obtained by the Knoevenagel condensation reaction. Caf-
feic acid was converted into acyl chloride and reacted with
compound 3. After removing the Boc-protecting group, the
skeleton 5 was obtained. The target compounds were
The target compounds were tested for their ability to inhibit
NA, Table 1. The enzyme inhibition result showed that they
displayed NA inhibitory activity with IC50 values from 57.8
to 225 lM, which was not good enough compared with
those flavonoid NA inhibitors or mimic peptides.
In general, all the compounds demonstrated a clear
structure–activity relationship surrounding the R1 and R2
groups. It could be found that the compounds containing a
3,4-dihydroxyphenyl group had better activity relative to
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3487
Scheme 1 The synthetic route of target compounds
Scheme 2 The synthetic route of GS4071 hydrochloride
the ones with a furyl group at R1 position, such as com-
pounds X-Val and XK-Val and compounds X-leu and XK-
Leu. The introduced amino acid fragments at R2 position
also had influence on the activity. From compounds X-lys,
X-Arg(NO2), X-c-GABA, and X-b-Ala, it was noted that
their inhibitory activity was decreased with length increase
of the side chains. This trend could also be observed on
compound X-Ala, X-thr, and X-leu. However, compound
X-Gly with the shortest side chain did not present higher
inhibitory activity than compound X-b-Ala, suggesting that
a suitable length was important. The R2 fragment bearing
large aromatic rings or rigid five-membered rings was
unfavorable to NA inhibitory activity, which could be
easily identified by compounds X-Trp, X-Phe, X-Hyp, and
X-Pro.
docking analysis (Fig. 3) performed by means of SYBYL-X
1.1 indicated that there was at least one big cavity adjacent
to the enzyme’s active center bound by oseltamivir car-
boxylate. XK-Val could occupy both the active site and an
adjacent small cavity, but could not penetrate deeply into
the enzyme compared with oseltamivir carboxylate. To a
certain extent, this may decrease the binding affinity and
led to low inhibitory activity. From the docking result, it
was also found that the region for the amino acid fragment
was very limited and could not be tolerated by large
groups, which was consistent with the enzyme inhibition
result that the compounds with bulky groups or long side
chains at R2 position were less potent.
The compounds designed in this study exhibited weak
NA inhibitory activity and could be further modified. In
fact, there were some other cavities close to the active site
according to recent studies as well as our docking result
XK-Val exhibited the best inhibitory activity in the
series against N2 (H9N2) and N1 (H5N1) NAs. The
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Table 1 The structures and IC50 values of the target compounds
OH
O
HCl
R2
R1
N
H
N
H
Compounds
X-Arg(NO2)
R1
R2
H9N2
IC50 (µM)
193
H5N1
IC50 (µM)
192
NH
O
O
O2NHN
N
H
NH2
O
O
O
X-γ-GABA
167
215
132
225
H2N
O
X-lys
H2N
NH2
O
O
X-Trp
194
189
NH2
HN
O
O
O
O
X-Phe
X-Val
X-Met
143
165
144
130
175
121
NH2
O
NH2
O
S
NH2
O
O
O
X-Ile
119
158
103
143
NH2
OH
O
X-Ser
NH2
O
O
O
X-Ala
X-Gly
91.7
175
110
139
NH2
O
H2N
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3489
Table 1 continued
Compounds
R1
R2
H9N2
H5N1
IC50 (µM)
IC50 (µM)
O
OH
O
X-Thr
X-Leu
138
154
114
135
NH2
O
O
O
NH2
O
X-β-Ala
140
200
113
187
H2N
O
O
X-Hyp
HN
OH
O
O
X-Pro
179
148
HN
O
XK-Ala
XK-Ile
118
111
108
109
HO
HO
OH
OH
NH2
O
NH2
O
XK-Val
XK-Leu
85.3
105
57.8
113
HO
HO
OH
OH
NH2
O
NH2
Oseltamivir
carboxylate
0.0017
0.012
hydrochloride
(Russell et al., 2006). It would probably be better to design
NA inhibitors considering all the potential binding sites.
Mimosine tetrapeptides were discovered as NA inhibitors
with IC50 values in the low micromolar range (Upadhyay
et al., 2011). Therefore, introducing dipeptides or tripep-
tides on the skeletons may provide improved activity;
besides, finding some new active fragments to construct
highly potent NA inhibitors could be feasible by applying
the fragment-based drug design strategy.
The chick embryo model had been reported suitably to
screen NA inhibitors for their antiviral efficacy against
influenza A virus (Sauerbrei et al., 2006). In this study,
we further evaluated the preliminary anti-influenza virus
activity of the target compounds in chick embryo,
Table 2. In previous studies, the efficacy of antiviral
compounds was determined on the basis of the survival
rate. Differently, we determined the minimal dosage of
compounds for keeping chick embryos alive and
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avian H5N1 influenza virus and the inhibitory activity
was more evident in higher concentration (some data not
showed). In general, the four compounds containing
3,4-dihydroxyphenyl group were a little more potent than
the other ones. Compared with oseltamivir or its carbox-
ylate, most of the designed compounds were only 4–8-fold
less potent, which was not consistent with the result of
enzyme inhibition assay. It seemed that these compounds
could inhibit the replication of influenza virus mainly by
some other mechanism.
Recently, besides the M2 protein, hemagglutinin (HA)
and NA, novel targets, such as the nonstructural NS1A
protein, the nucleoprotein, and the viral polymerase, have
also been identified suitably for the development of new
antivirals (Krug and Aramini, 2009). These compounds
discovered with weak NA inhibitory activity, but display-
ing effective anti-H5N1 influenza virus activity in chick
embryo deserved deep studies.
Fig. 3 The docking result of XK-Val and oseltamivir carboxylate
(the smaller compound) with the active site of NA (PDB ID:2HU0)
Table 2 Antiviral activity of the target compounds in chick embryo
Compound
Effectively inhibition of influenza virus
(mg or lmol/each chick embryo)*
Conclusion
In this study, we designed and synthesized a series of novel
NA inhibitors. Besides, we also reported a simple synthetic
route with high yield from oseltamivir to prepare its active
form which was used as the positive control for biologic
evaluation.
X-Arg(NO2)
X-c-GABA
X-lys
2.41 mg (5 lM)
1.83 mg (5 lM)
2.04 mg (5 lM)
X-Trp
1.16 mg (2.5 lM)
2.14 mg (5 lM)
X-Phe
All the compounds exhibited weak NA inhibitory activ-
ity. Among them, compound XK-Val was the best with
IC50 values of 85.3 and 57.8 lM against N2 (H9N2) and N1
(H5N1) NA, respectively. Interestingly, most of the com-
pounds displayed moderate anti-H5N1 influenza virus
activity in chick embryo, indicating that they may inhibit the
replication of influenza virus by some other mechanism.
NA was still an important target for anti-influenza drugs
design. Considering the current ani-influenza situation, it
was still necessary to discover novel NA inhibitors. In this
study though these compounds designed did not show high
inhibitory activity against NA; they really represented a
new kind of NA inhibitors and could be further modified.
X-Val
0.94 mg (2.5 lM)
2.06 mg (5 lM)
X-Met
X-Ile
0.983 mg (2.5 lM)
1.83 mg (5 lM)
X-Ser
X-Ala
1.75 mg (5 lM)
Oseltamivir carboxylate
hydrochloride
0.401–0.200 mg (1.25–0.625 lM)
X-Gly
1.68 mg (5 lM)
X-Thr
1.91 mg (5 lM)
X-Leu
1.97 mg (5 lM)
X-b-Ala
X-Hyp
1.75 mg (5 lM)
[1.97 mg (5 lM)
1.89 mg (5 lM)
X-Pro
XK-Ala
XK-Ile
1.83 mg (5 lM)
1.02 mg (2.5 lM)
0.98 mg (2.5 lM)
1.02 mg (2.5 lM)
0.513–0.257 mg (1.25–0.625 lM)
Experimental
XK-Val
XK-Leu
Oseltamivir phosphate
Chemistry
* The minimal dosage of compounds for keeping chick embryos alive
and effectively inhibiting the replication of avian H5N1 influenza
virus with a low hemagglutinin (HA) titer (0 log2) in allantoic fluid by
hemagglutination assay. The experiment was repeated three times
Compound 1, Boc-protected amino acids, oseltamivir
phosphate, and other compounds not specified in the syn-
thetic routes were commercially available. Solvent for
anhydrous reaction should be processed before use.
1H-NMR spectra were determined on a Brucker Avance
300 spectrometer or 600 using TMS as an internal standard.
The solvents for NMR were DMSO-d6 (d 2.5 for 1H),
effectively inhibiting the replication of avian H5N1
influenza virus. From table 2, it could be found that most
of the designed compounds had effective activity against
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1
3491
1
1
CD3OD (d 3.3 for H), and H2O (d 4.7 for H). HRMS
analysis was provided using Agilent 6520 Q-TOF LC/MS
spectrometer (Agilent, Germany). All reactions were
monitored by thin-layer chromatography (TLC) on
25.4 9 76.2 mm silica gel plates (GF-254). Silica gel used
for column chromatography was 200–300 mesh. Melting
points were determined on an electrothermal melting point
apparatus and were uncorrected.
gray solid product. Yield: 67.2 %, mp = 138–139 °C. H
NMR (600 MHz, DMSO-d6): d 12.42 (br, 1H), 7. 84 (s,
1H), 7.40 (d, 1H, J = 15.6 Hz), 6.93 (d, 1H, J = 2.4 Hz),
6.62–6.63 (m, 1H), 6.15 (d, 1H, J = 15.6 Hz). HRMS
calcd for C7H5O3 (M-H)- 137.0239; found: 137.0246.
(E)-N-(3-amino-4-hydroxyphenyl)-3-(furan-2-
yl)acrylamide (4)
tert-Butyl 2-hydroxy-5-nitrophenylcarbamate (2)
To a solution of (E)-3-(furan-2-yl)acrylic acid (0.83 g,
6 mmol) and catalytic amount of DMF in 20 ml anhydrous
THF was added oxalyl chloride (1.14 ml, 12 mmol) under
ice-bath. After the complete conversion of the acid to its
acyl chloride, the mixture was concentrated to half its
volume by rotary evaporation. The concentrate was added
dropwise to a mixture of compound 3 (1.34 g, 6 mmol) and
sodium bicarbonate (1.5 g, 18 mmol) in 60 ml THF/H2O
(10:1). After 4 h of stirring at rt, the reaction was complete
monitored by TLC. Remove the volatile solvent in vacuum
to leave a crude mixture which was dissolved with ethyl
acetate and washed with saturated NaHCO3, 1 M aqueous
HCl, and brine, and then dried with anhydrous MgSO4.
After concentrating to small volume, an off-white solid
could be obtained by scratching of the flask with a glass
rod.
2-amino-4-nitrophenol (50 mmol, 7.7 g) added in 200 ml
THF ? H2O (3:1) was stirred at 75 °C until the material
was dissolved. To this solution, Di-tert-butyl pyrocarbon-
ate (150 mmol, 32.7 g) was portionwise added in at least
2 h. The reaction mixture was continuously stirred at this
temperature for 5 h. After evaporation of the solvent, the
obtained residue was extracted with ethyl acetate and the
combined extracts were washed with saturated NaHCO3,
1 M aqueous HCl, brine, dried over anhydrous MgSO4, and
concentrated. The crude product was recrystallized from
ethyl acetate and petroleum ether to give a yellow solid in
50.4 % yield. Mp = 138–140 °C. 1H NMR (600 MHz,
DMSO-d6): d 11.56 (s, 1H), 8.64 (s, 1H), 8.21 (s, 1H), 7.86
(dd, 1H, J = 9.0 Hz, J = 3.0 Hz), 6.98 (d, 1H,
J = 8.4 Hz), 1.48 (s, 9H). HRMS calcd for C11H13N2O5
(M-H)- 253.0824; found: 253.0825.
Without further purification, the solid was added in
20 ml ethyl acetate HCl solution and stirred at rt overnight
to give a gray solid. The solid was neutralized with satu-
rated sodium bicarbonate solution and extracted with ethyl
acetate. Evaporation of the solvent could afford a gray
tert-Butyl 5-amino-2-hydroxyphenylcarbamate (3)
1
Compound 2 (2.54 g, 10 mmol) was added in 100 ml dried
CH3OH and hydrogenated at room temperature using
hydrogen balloon in the presence of 200 mg of 10 % Pd/C.
The reaction was monitored by TLC until the starting
material was no longer detected. Evaporation of CH3OH
could afford a light gray solid (1.7 g, yield 74 %).
Mp = 162–164 °C. 1H NMR (600 MHz, DMSO-d6): d
8.59 (s, 1H), 7.57 (s, 1H), 7.00 (s, 1H), 6.51 (d, 1H,
J = 8.4 Hz), 6.11 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz), 4.47
(s, 2H), 1.45 (s, 9H). HRMS calcd for C11H17N2O3
(M?H)? 225.1239; found: 225.1223.
solid (0.76 g, yield 53.0 %). Mp = 195–196 °C. H NMR
(600 MHz, DMSO-d6): d 9.73 (s, 1H), 8.80 (s, 1H), 7.79 (s,
1H), 7.29 (d, 1H, J = 15.6 Hz), 7.00 (d, 1H, J = 2.4 Hz),
6.79 (d, 1H, J = 3.0 Hz), 6.72 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 6.59–6.62 (m, 2H), 6.55 (d, 1H, J = 8.4 Hz),
4.58 (s, 2H). IR: (KBr) cm-1: 3450, 3368, 3209, 3169,
3061, 1621, 1550, 1453. HRMS calcd for C13H13N2O3
(M?H)? 245.0926; found: 245.0930.
(E)-N-(3-amino-4-hydroxyphenyl)-3-(3,4-
dihydroxyphenyl)acrylamide hydrochloride (5)
The synthetic procedure of (E)-3-(furan-2-yl)acrylic acid
(Jiang et al., 2009)
According to the synthetic route of compound 4, compound
5 prepared from caffeic acid (1.08 g, 6 mmol) and com-
pound 3 (1.34 g, 6 mmol) was obtained as a yellow solid in
Furan-2-carbaldehyde (1.92 g, 20 mmol) and malonic acid
(5.2 g, 50 mmol) were added in 50 ml pyridine and then
heated to reflux at 120 °C. The reaction was monitored by
TLC until the starting material was no longer detected.
Most of the solvent (pyridine) was removed under vacuum.
Then, 20 % NaOH solution 80 ml was added and the
residual pyridine was extracted with ethyl acetate. Con-
centrated HCl was used to adjust the pH to * 5 to give the
1
48 % yield. Mp = 228–230 °C (decomposed). H NMR
(300 MHz, CD3OD): d 8.02 (d, 1H, J = 2.4 Hz), 7.53 (d,
1H, J = 15.6 Hz), 7.36 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz),
7.08 (d, 1H, J = 2.1 Hz), 6.99 (d, 1H, J = 8.7 Hz), 6.97
(dd, 1H, J = 8.1 Hz, J = 2.1 Hz), 6.80 (d, 1H,
J = 8.1 Hz), 6.56 (d, 1H, J = 15.6 Hz). HRMS calcd for
C15H15N2O4 (M?H)? 287.1032; found: 287.1024.
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Med Chem Res (2013) 22:3485–3496
The general procedure of compound X-Arg(NO2) *
X-Pro (Sheehan and Hess, 1955)
J = 9.0 Hz), 6.75 (d, 1H, J = 3.0 Hz), 6.51–6.55 (m, 2H),
4.19 (t, 1H, J = 6.6 Hz), 2.99 (t, 2H, J = 7.8 Hz), 2.00 (m,
2H), 1.72 (m, 2H), 1.54 (m, 2H). HRMS calcd for
C18H22N3O4 [M?H]? 373.1876; found: 373.1857.
To a solution of compound 4 (0.34 g, 1.5 mmol) and Boc-
L-amino acid (1.6 mmol) in 50 ml anhydrous THF, DCC
was added (0.46 g, 2.25 mmol). The reaction mixture
was stirred at room temperature for 1 h. The insoluble
N,N0-dicyclohexylurea was removed by filtration and the
solvent was removed under reduced pressure to give a
residue which was subsequently dissolved in ethyl acetate
and washed with saturated NaHCO3, 1 M aqueous HCl,
and brine. The organic phase was dried over anhydrous
MgSO4 and concentrated. Chromatographic purification of
the crude material with EtOAc and petroleum afforded the
Boc-protected target compounds which were added to
20 ml HCl/EtOAc and stirred overnight. The precipitate
was collected through filtration and washed with EtOAc to
give the target compounds in 21–55 % yield.
(S,E)-N-(3-(2-amino-3-(1H-indol-3-yl)propanamido)-4-
hydroxyphenyl)-3-(furan-2-yl)acrylamide hydrochloride
(X-Trp)
Yellow solid, decomposed *210 °C. 1H NMR (600 MHz,
D2O): d 7.47 (s, 1H),7.42 (d, 1H, J = 8.4 Hz), 7.33 (d, 1H,
J = 8.4 Hz), 7.26 (d, 1H, J = 15.6 Hz), 7.22 (s, 1H), 7.03
(t, 1H, J = 7.8 Hz), 6.95 (dd, 1H, J = 9.0 Hz,
J = 2.4 Hz), 6.90 (t, 1H, J = 7.8 Hz), 6.74 (d, 1H,
J = 9.0 Hz), 6.66 (s, 1H), 6.63 (d, 1H, J = 3.0 Hz),
6.42–6.44 (m, 1H), 6.37 (d, 1H, J = 15.6 Hz), 4.26–4.29
(m, 1H), 3.38 (dd, 1H, J = 14.4 Hz, J = 6.0 Hz), 3.30 (dd,
1H, J = 14.4 Hz, J = 9.0 Hz). HR-MS calcd for
C24H23N4O4 [M?H]? 431.1719; found: 431.1702.
(S,E)-2-amino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-5-(3-nitroguanidino)pentanamide
hydrochloride (X-arg(NO2))
(S,E)-N-(3-(2-amino-3-phenylpropanamido)-4-
hydroxyphenyl)-3-(furan-2-yl)acrylamide hydrochloride
(X-Phe)
Light yellow solid, decomposed *160 °C. 1H NMR
(300 MHz, D2O): d 7.64 (d, 1H, J = 2.4 Hz), 7.46 (d, 1H,
J = 1.8 Hz), 7.24 (d, 1H, J = 15.6 Hz), 7.07 (dd, 1H,
J = 8.7 Hz, J = 2.7 Hz), 6.83 (d, 1H, J = 8.7 Hz), 6.62
(d, 1H, J = 3.6 Hz), 6.43 (dd, 1H, J = 3.6 Hz,
J = 1.8 Hz), 6.36 (d, 1H, J = 15.6 Hz), 4.17 (t, 1H,
J = 6.3 Hz), 3.19–3.22 (m, 2H), 1.93–1.96 (m, 2H),
1.56–1.58 (m, 2H). HRMS calcd for C19H24N7O6 (M?H)?
446.1788; found: 446.1809.
1
Yellow solid, decomposed *252 °C. H NMR (600 MHz,
CD3OD): d8.21(d, 1H, J = 2.4 Hz),7.61(s, 1H),7.41(d, 1H,
J = 15.6 Hz), 7.29–7.38 (m, 5H), 7.28 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 6.82 (d, 1H, J = 8.4 Hz), 6.70 (d, 1H,
J = 3.0 Hz), 6.61 (d, 1H, J = 15.0 Hz), 6.54 (dd, 1H,
J = 3.0 Hz, J = 1.2 Hz), 4.41 (t, 1H, J = 7.2 Hz), 3.30-3.34
(m, 1H), 3.17 (dd, 1H, J = 14.4 Hz, J = 6.6 Hz). HRMS
calcd for C22H22N3O4 [M?H]? 392.1610; found: 392.1606.
(E)-4-amino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)butanamide hydrochloride (X-c-GABA)
(S,E)-2-amino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-3-methylbutanamide hydrochloride
(X-Val)
Light yellow solid, mp = 231–233 °C. 1H NMR
(600 MHz, CD3OD): d 8.10 (d, 1H, J = 2.4 Hz), 7.61 (s,
1H), 7.40 (d, 1H, J = 15.6 Hz), 7.26 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 6.83 (d, 1H, J = 8.4 Hz), 6.70 (d, 1H,
J = 3.6 Hz), 6.60 (d, 1H, J = 15.6 Hz), 6.54 (dd, 1H,
J = 3.6 Hz, J = 1.8 Hz), 3.04 (t, 2H, J = 7.2 Hz), 2.62 (t,
2H, J = 7.2 Hz), 2.02 (m, 2H). IR: (KBr) cm-1: 3324,
3158, 3159, 3051, 1674, 1641, 1532. HRMS calcd for
C17H20N3O4 [M?H]? 330.1454; found: 330.1469.
Yellow solid, mp = 199–202 °C. 1H NMR (600 MHz,
CD3OD): d 8.21 (d, 1H, J = 1.8 Hz), 7.59 (d, 1H,
J = 1.8 Hz), 7.39 (d, 1H, J = 15.6 Hz), 7.28 (dd, 1H,
J = 8.4 Hz, J = 2.4 Hz), 6.84 (d, 1H, J = 9.0 Hz), 6.68
(d, 1H, J = 3.0 Hz), 6.59 (d, 1H, J = 15.6 Hz), 6.52 (dd,
1H, J = 3.0 Hz, J = 1.2 Hz), 3.99 (d, 1H, J = 6.0 Hz),
2.26–2.28 (m, 1H), 1.11–1.13 (m, 6H). HRMS calcd for
C18H22N3O4 [M?H]? 344.1610; found: 344.1593.
(S,E)-2,6-diamino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)hexanamide hydrochloride (X-Lys)
(S,E)-2-amino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-4-(methylthio)butanamide hydrochloride
(X-Met)
Yellow solid, decomposed *189 °C. 1H NMR (600 MHz,
D2O): d 7.75 (d, 1H, J = 1.8 Hz), 7.59 (s, 1H), 7.39 (d, 1H,
J = 15.6 Hz), 7.19 (d, 1H, J = 9.0 Hz), 6.98(d, 1H,
1
Yellow solid, mp = 240–242 °C (decomposed). H NMR
(600 MHz, CD3OD): d 8.22 (s, 1H), 7.59 (d, 1H, J = 1.8 Hz),
123

Med Chem Res (2013) 22:3485–3496
3493
(E)-N-(3-(2-aminoacetamido)-4-hydroxyphenyl)-3-(furan-
2-yl)acrylamide hydrochloride (X-Gly)
7.39 (d, 1H, J = 15.6 Hz), 7.27 (d, 1H, J = 8.4 Hz), 6.84 (d,
1H, J = 8.4 Hz), 6.68 (d, 1H, J = 3.6 Hz), 6.59 (d, 1H,
J = 15.6 Hz), 6.52 (dd, 1H, J = 3.6 Hz, J = 1.8 Hz), 4.26
(t, 1H, J = 6.6 Hz), 2.63–2.69 (m, 2H), 2.19–2.25 (m, 2H),
2.13 (s, 3H). HRMS calcd for C18H22N3O4S [M?H]?
376.1331; found: 376.1319.
1
Yellow solid, mp = 216–219 °C (decomposed). H NMR
(600 MHz, D2O): d 7.64 (d, 1H, J = 2.4 Hz), 7.43 (s, 1H),
7.18 (d, 1H, J = 15.6 Hz), 7.03 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 6.79 (d, 1H, J = 8.4 Hz), 6.58 (d, 1H,
J = 3.6 Hz), 6.39 (dd, 1H, J = 3.6 Hz, J = 1.8 Hz), 6.29
(d, 1H, J = 15.6 Hz), 3.88 (s, 2H). IR: (KBr) cm-1: 3474
(br), 3129, 3059, 1673, 1638, 1548, 1509. 13C NMR
(75 MHz, D2O): d 166.3, 165.7, 150.7, 145.3, 145.1, 129.9,
128.4, 123.7, 119.9, 117.3, 117.1, 116.1, 115.0, 112.4,
40.9. HRMS calcd for C15H16N3O4 [M?H]? 302.1141;
found: 302.1132.
(2S)-2-amino-N-(5-((E)-3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-3-methylpentanamide hydrochloride
(X-Ile)
Yellow solid, mp = 256–258 °C. 1H NMR (600 MHz,
CD3OD): d 8.23 (d, 1H, J = 2.4 Hz), 7.61 (s, 1H), 7.40 (d,
1H, J = 15.6 Hz), 7.29 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz),
6.85 (d, 1H, J = 8.4 Hz), 6.70 (d, 1H, J = 3.6 Hz), 6.60
(d, 1H, J = 15.6 Hz), 6.54 (dd, 1H, J = 3.6 Hz, J =
1.8 Hz), 4.05 (d, 1H, J = 6.0 Hz), 2.01–2.04 (m, 1H),
1.67–1.70 (m, 1H), 1.30–1.33 (m, 1H), 1.11 (d, 3H,
J = 6.6 Hz), 1.02 (t, 3H, J = 7.2 Hz). HRMS calcd for
C19H24N3O4 [M?H]? 358.1767; found: 358.1784.
(2S)-2-amino-N-(5-((E)-3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-3-hydroxybutanamide hydrochloride (X-
Thr)
1
Yellow solid, mp = 199–201 °C (decomposed). H NMR
(600 MHz, CD3OD): d 8.28 (d, 1H, J = 2.4 Hz), 7.59 (s,
1H), 7.39 (d, 1H, J = 15.6 Hz), 7.26 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 6.83 (d, 1H, J = 8.4 Hz), 6.69 (d, 1H,
J = 3.0 Hz), 6.59 (d, 1H, J = 15.6 Hz), 6.52 (dd, 1H,
J = 3.6 Hz, J = 1.8 Hz), 4.11–4.14 (m, 1H), 3.99 (d, 1H,
J = 5.4 Hz), 1.35 (d, 3H, J = 6.6 Hz). HRMS calcd for
C17H20N3O5 [M?H]? 346.1403; found: 346.1402.
(S,E)-N-(3-(2-amino-3-hydroxypropanamido)-4-
hydroxyphenyl)-3-(furan-2-yl)acrylamide hydrochloride
(X-Ser)
1
Yellow solid, mp = 162–164 °C (decomposed). H NMR
(600 MHz, D2O): d 7.63 (s, 1H), 7.43 (s, 1H), 7.20 (d, 1H,
J = 15.6 Hz), 7.05 (d, 1H, J = 8.4 Hz), 6.80 (d, 1H,
J = 8.4 Hz), 6.59 (s, 1H), 6.40 (s, 1H), 6.32 (d, 1H,
J = 15.6 Hz), 4.20 (t, 1H, J = 4.8 Hz), 3.91–3.97 (m, 2H).
IR: (KBr) cm-1: 3400 (br), 3259, 3044, 2885, 1685, 1662,
1620, 1542. 13C NMR (75 MHz, D2O): d 166.5, 150.7,
145.6, 145.1, 129.9, 128.6, 123.6, 120.5, 117.6, 117.3,
116.2, 115.1, 112.5, 60.3, 54.9. HRMS calcd for
C16H18N3O5 [M?H]? 332.1246; found: 332.1236.
(S,E)-2-amino-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-4-methylpentanamide hydrochloride
(X-leu)
Yellow solid, mp = 250–252 °C. 1H NMR (600 MHz,
CD3OD): d 8.22 (d, 1H, J = 1.8 Hz), 7.59 (d, 1H,
J = 1.8 Hz), 7.39 (d, 1H, J = 15.6 Hz), 7.26 (dd, 1H,
J = 8.4 Hz, J = 2.4 Hz), 6.84 (d, 1H, J = 8.4 Hz), 6.69
(d, 1H, J = 3.0 Hz), 6.59 (d, 1H, J = 15.0 Hz), 6.53 (dd,
1H, J = 3.0 Hz, J = 1.8 Hz), 4.18 (t, 1H, J = 7.2 Hz),
1.75–1.84 (m, 3H), 1.02–1.05 (m, 6H). IR: (KBr) cm-1
:
3393, 3255, 2958, 2919, 1691, 1664, 1619, 1536. 13C NMR
(75 MHz, DMSO-d6): d 167.9, 164.9, 151.3, 145.4, 144.5,
130.5, 127.7, 124.6, 118.4, 118.2, 115.6, 115.0, 113.8,
111.9, 52.2, 40.5, 24.2, 21.7, 20.9. HRMS calcd for
C19H24N3O4 [M?H]? 358.1767; found: 358.1760.
(S,E)-N-(3-(2-aminopropanamido)-4-hydroxyphenyl)-3-
(furan-2-yl)acrylamide hydrochloride (X-Ala)
Yellow solid, decomposed *196 °C. 1H NMR (600 MHz,
D2O): d 7.73 (d, 1H, J = 1.8 Hz), 7.57 (s, 1H), 7.35 (d, 1H,
J = 15.6 Hz), 7.19 (d, 1H, J = 8.4 Hz), 6.95 (d, 1H,
J = 8.4 Hz), 6.73 (s, 1H), 6.54 (s, 1H), 6.47 (d, 1H,
J = 15.6 Hz), 4.29 (q, 1H, J = 7.2 Hz), 1.64 (d, 3H,
J = 7.2 Hz). IR: (KBr) cm-1: 3453 (br), 3256, 3126, 2918,
2850, 1635, 1557, 1514. 13C NMR (75 MHz, D2O): d
169.4, 166.3, 150.7, 145.6, 145.0, 129.9, 128.4, 123.5,
120.2, 117.4, 117.3, 116.2, 115.0, 112.4, 49.5, 16.6. HRMS
calcd for C16H18N3O4 [M?H]? 316.1297; found:
316.1288.
(E)-N-(3-(3-aminopropanamido)-4-hydroxyphenyl)-3-
(furan-2-yl)acrylamide hydrochloride (X-b-Ala)
Yellow solid, mp = 219–224 °C (decomposed). 1H NMR
(600 MHz, DMSO-d6): d 10.09 (s, 1H), 9.63 (s, 1H), 9.54
(s, 1H), 8.13 (d, 1H, J = 2.4 Hz), 7.93 (br, 3H), 7.81 (s,
1H), 7.33 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz), 7.32 (d, 1H,
J = 15.6 Hz), 6.83 (d, 1H, J = 8.4 Hz), 8.81 (d, 1H,
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Med Chem Res (2013) 22:3485–3496
(S,E)-N-(3-(2-aminopropanamido)-4-hydroxyphenyl)-3-
(3,4-dihydroxyphenyl)acrylamide hydrochloride (XK-Ala)
J = 3.0 Hz), 6.63 (d, 1H, J = 15.6 Hz), 6.61 (dd, 1H,
J = 3.0 Hz, J = 1.8 Hz), 3.07 (t, 2H, J = 6.6 Hz), 2.80 (t,
2H, J = 6.6 Hz). HRMS calcd for C16H18N3O4 [M?H]?
316.1297; found: 316.1287.
Yellow solid, mp = 220–224 °C. 1H NMR (600 MHz,
D2O): d 7.52 (s, 1H), 7.21 (d, 1H, J = 15.6 Hz), 6.96 (d,
1H, J = 8.4 Hz), 6.87 (s, 1H), 6.82 (d, 1H, J = 7.8 Hz),
6.73 (d, 1H, J = 8.4 Hz), 6.65 (d, 1H, J = 8.4 Hz), 6.23
(d, 1H, J = 15.6 Hz), 4.12 (q, 1H, J = 6.6 Hz), 1.47 (d,
3H, J = 6.6 Hz). IR: (KBr) cm-1: 3418 (br), 3299 (br),
1684, 1645, 1600, 1550, 1509. 13C NMR (75 MHz, D2O):
d 169.4, 166.6, 146.2, 145.4, 143.9, 141.6, 130.0, 127.2,
123.4, 122.1, 120.1, 117.4, 117.2, 116.1, 115.9, 114.7,
49.5, 16.6. HRMS calcd for C18H20N3O5 [M?H]?
358.1403; found: 358.1377.
(2S,4R)-N-(5-((E)-3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)-4-hydroxypyrrolidine-2-carboxamide
hydrochloride (X-Hyp)
Yellow solid, decomposed *140 °C, mp = 164–166 °C.
1H NMR (300 MHz, CD3OD): d 8.27 (d, 1H, J = 2.4 Hz),
7.62 (d, 1H, J = 1.5 Hz), 7.43 (d, 1H, J = 15.6 Hz), 7.29
(dd, 1H, J = 8.7 Hz, J = 2.4 Hz), 6.87 (d, 1H,
J = 8.7 Hz), 6.72 (d, 1H, J = 3.6 Hz), 6.62 (d, 1H,
J = 15.6 Hz), 6.56 (dd, 1H, J = 3.6 Hz, J = 1.8 Hz), 4.78
(dd, 1H, J = 7.8 Hz, J = 10.2 Hz), 4.65–4.68 (m, 1H),
3.50 (dd, 1H, J = 12.0 Hz, J = 3.6 Hz), 3.35–3.39 (m,
1H), 2.57–2.66 (m, 1H), 2.13–2.26 (m, 1H). HRMS calcd
for C18H20N3O5 [M?H]? 358.1403; found: 358.1378.
(S,E)-2-amino-N-(5-(3-(3,4-dihydroxyphenyl)acrylamido)-
2-hydroxyphenyl)-4-methylpentanamide hydrochloride
(XK-Leu)
Yellow solid, mp = 233–236 °C. 1H NMR (600 MHz,
D2O): d 7.51 (s, 1H), 7.22 (d, 1H, J = 15.6 Hz), 6.97 (d,
1H, J = 8.4 Hz), 6.89 (s, 1H), 6.84 (d, 1H, J = 7.8 Hz),
6.76 (d, 1H, J = 8.4 Hz), 6.67 (d, 1H, J = 7.8 Hz), 6.25
(d, 1H, J = 15.6 Hz), 4.05 (t, 1H, J = 7.2 Hz), 1.60–1.69
(m, 3H), 0.81–0.84 (m, 6H). HRMS calcd for C21H26N3O5
[M?H]? 400.1872; found: 400.1859.
(S,E)-N-(5-(3-(furan-2-yl)acrylamido)-2-
hydroxyphenyl)pyrrolidine-2-carboxamide hydrochloride
(X-Pro)
Yellow solid, mp = 170–174 °C. 1H NMR (300 MHz,
D2O): d 7.64 (d, 1H, J = 2.4 Hz), 7.46 (d, 1H, J = 1.8 Hz),
7.22 (d, 1H, J = 15.6 Hz), 7.08 (dd, 1H, J = 8.7 Hz,
J = 2.4 Hz), 6.83 (d, 1H, J = 8.7 Hz), 6.61 (d, 1H,
J = 3.3 Hz), 6.43 (dd, 1H, J = 3.3 Hz, J = 1.8 Hz), 6.33
(d, 1H, J = 15.6 Hz), 4.44-4.50 (m, 1H), 3.28–3.44 (m, 2H),
2.38–2.50 (m, 1H), 1.95–2.16 (m, 3H). HRMS calcd for
C18H20N3O4 [M?H]? 342.1454; found: 342.1445.
(2S)-2-amino-N-(5-((E)-3-(3,4-dihydroxyphenyl)
acrylamido)-2-hydroxyphenyl)-3-methylpentanamide
hydrochloride (XK-Ile)
Yellow solid, mp = 208–210 °C. 1H NMR (300 MHz,
D2O):
d 7.55 (d, 1H, J = 2.4 Hz), 7.18 (d, 1H,
J = 15.6 Hz), 6.97 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz), 6.85
(d, 1H, J = 1.8 Hz), 6.84 (dd, 1H, J = 8.4 Hz, J =
1.8 Hz), 6.75 (d, 1H, J = 8.7 Hz), 6.65 (d, 1H, J =
8.4 Hz), 6.18 (d, 1H, J = 15.6 Hz), 3.96 (d, 1H,
J = 5.4 Hz), 1.95–1.99 (m, 1H), 1.42–1.56 (m, 1H),
1.14–1.24 (m, 1H), 0.96 (d, 3H, J = 6.9 Hz), 0.85 (t, 3H,
J = 7.2 Hz). HRMS calcd for C21H26N3O5 [M?H]?
400.1872; found: 400.1864.
The general procedure of compound XK-Ala * XK-Val
To a solution of compound 5 (0.34 g, 1.5 mmol), Et3N
(0.21 ml, 1.5 mmol) and Boc-L-amino acid (1.6 mmol) in
50 ml anhydrous THF, DCC was added (0.46 g,
2.25 mmol). The reaction mixture was stirred at room
temperature for 1 h. The insoluble N,N0-dicyclohexylurea
was removed by filtration and the solvent was removed
under reduced pressure to give a residue, which was sub-
sequently dissolved in ethyl acetate and then washed with
saturated NaHCO3, 1 M aqueous HCl, and brine. The
organic phase was dried over anhydrous MgSO4 and con-
centrated. Chromatographic purification of the crude
material with EtOAc and petroleum afforded the Boc-
protected target compounds which were added to 20 ml
HCl/EtOAc and stirred overnight. The precipitate was
collected through filtration and washed with EtOAc to give
the target compounds in 25–42 % yield.
(S,E)-2-amino-N-(5-(3-(3,4-dihydroxyphenyl)acrylamido)-
2-hydroxyphenyl)-3-methylbutanamide hydrochloride
(XK-Val)
Yellow solid, decomposed *194 °C, mp = 204 °C. 1H
NMR (600 MHz, CD3OD): d 8.23 (d, 1H, J = 2.4 Hz),
7.48 (d, 1H, J = 15.6 Hz), 7.28 (dd, 1H, J = 8.4 Hz,
J = 2.4 Hz), 7.04 (d, 1H, J = 1.8 Hz), 6.95 (dd, 1H,
J = 8.4 Hz, J = 1.8 Hz), 6.85 (d, 1H, J = 8.4 Hz), 6.78
(d, 1H, J = 8.4 Hz), 6.53 (d, 1H, J = 15.6 Hz), 3.99 (d,
1H, J = 6.0 Hz), 2.27–2.31 (m, 1H), 1.14 (t, 6H,
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Med Chem Res (2013) 22:3485–3496
3495
J = 7.2 Hz). IR: (KBr) cm-1: 3447 (br), 2968, 1656, 1609,
1508. 13C NMR (75 MHz, D2O): d 168.1, 166.5, 146.2,
145.7, 143.9, 141.5, 130.0, 127.1, 123.1, 122.0, 120.3,
117.3, 116.1, 115.9, 114.7, 58.9, 30.1, 17.7, 16.8. HRMS
calcd for C20H24N3O5 [M?H]? 386.1716; found:
386.1719.
Pharmacology
Influenza A (H9N2 and H5N1) NA inhibition assay
The NA inhibition assay was performed according to a
standard method (Liu et al., 2008). Influenza virus sus-
pensions (H9N2 virus: A/Chicken/Shandong/LY/08 and
H5N1 virus: A/duck/China/QJ/01) obtained from the
allantoic fluid of embryonated chicken eggs were used as
the enzyme on the basis that NA was present on the viral
surface. The substrate, 20-(4-methylumbelliferyl)-a-D-
acetylneuraminic acid sodium salt hydrate (4-MU-NANA)
(Sigma, M8639), was cleaved by NA to yield a fluorescent
product which can be quantified. Compounds tested were
dissolved in DMSO and diluted to the corresponding
concentrations in MES buffer (32.5 mM 2-(N-morpho-
lino)-ethanesulfonic acid, 4 mM CaCl2, pH 6.5), if not
dissolved in MES directly. In a 96-well plate, 10 ll of the
diluted virus supernatant, 70 ll of MES buffer, and 10 ll
of compounds at different concentration were added suc-
cessively and then incubated for 5 min at 37 °C. The
reaction was started by the addition of the substrate. After
incubation for 30–60 min, the reaction was terminated by
adding 150 ll 0.2 M glycine–NaOH (pH 10.2) or 0.034 M
NaOH in water. Fluorescence was recorded (excitation at
360 nm and emission at 450 nm), and substrate blanks
were subtracted from the sample readings. The 50 %
inhibitory concentration (IC50) was calculated by plotting
percent inhibition of NA activity versus the inhibitor
concentration.
(3R,4R,5S)-4-acetamido-5-((tert-butoxycarbonyl)amino)-
3-(pentan-3-yloxy)cyclohex-1-enecarboxylic acid
(Boc-GS4071)
To a mixture of oseltamivir phosphate (2.05 g, 5 mmol)
and NaHCO3 (2.1 g, 25 mmol) in 60 ml THF ? H2O (5:1)
(Boc)2O (1.31 g, 6 mmol) was added in one portion. The
reaction was allowed to stand for 6 h at rt. The solvent was
removed in vacuum followed by extraction of the residues
with EtOAc. After concentration, the obtained oil product
was dissolved in 30 ml CH3OH ? H2O (3:1) and NaOH
(1.6 g, 40 mmol) was added. The reaction mixture was
stirred at room temperature for 1 h. After evaporation of
CH3OH, 15 ml H2O was added. The undissolved substance
was filtered and the clear filtrate was neutralized with 1 M
HCl to give a white solid (1.40 g, yield = 72.9 %).
Mp = 212–214 °C. 1H NMR (600 MHz, DMSO-d6): d
12.57 (br, 1H), 7.80 (d, 1H, J = 9.0 Hz), 6.59–6.62 (m,
2H), 4.04–4.06 (m, 1H), 3.65–3.71 (m, 1H), 3.52–3.58 (m,
1H), 3.35–3.39 (m, 1H), 2.45 (dd, 1H, J = 17.4 Hz,
J = 5.4 Hz), 2.21 (dd, 1H, J = 17.4 Hz, J = 10.8 Hz),
1.77 (s, 3 H), 1.33–1.48 (m, 13H), 0.83 (t, 3H, J = 7.2 Hz),
0.76 (t, 3H, J = 7.2 Hz). HRMS calcd for C19H31N2O6
(M-H)- 383.2182; found: 383.2177.
Evaluation of anti-influenza virus activity in chick embryo
(3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-
yloxy)cyclohex-1-enecarboxylic acid hydrochloride
(GS4071-HCl)
The test for activity of target compounds against avian
H5N1 influenza virus in chick embryo was conducted
based on a reported method with a slight modification
(Sauerbrei et al., 2006). The target compound dissolved in
phosphate buffered saline (PBS) or DMSO was serially
diluted by twofold in PBS. Then, 100 ll of compound at
different concentrations was mixed with an equal volume
of 100 TCID50 (50 % tissue culture infective dose) of
H5N1 virus (A/duck/China/QJ/01). After 1-h incubation at
37 °C, the compound–virus preparation was injected into
11-day old chick embryos, followed by incubation at 37 °C
for 5 days. Define the minimal effective dosage for keeping
the eggs alive and inhibiting the viral replication with a low
HA titer (0 log2) in allantoic fluid determined by hemag-
glutination assay. (The eggs of blank control groups were
all dead within 36 h).
Boc-GS4071 (0.38 g, 1 mmol) was added to 20 ml HCl/
EtOAc and stirred overnight. The precipitate was collected
through filtration and washed with EtOAc to give a white
solid (0.26 g, yield = 81.2 %). Mp = 186–188 °C. 1H
NMR (600 MHz, D2O): d 6.67 (1H, s), 4.15 (d, 1H,
J = 7.8 Hz), 3.88 (dd, 1H, J = 11.4 Hz, J = 9.0 Hz),
3.35-3.44 (m, 2H), 2.76 (dd, 1H, J = 17.4 Hz,
J = 5.4 Hz), 2.32 (dd, 1H, J = 17.4 Hz, J = 10.8 Hz),
1.90 (s, 3H), 1.35–1.42 (m, 3H), 1.26–1.31 (m, 1H), 0.70 (t,
3H, J = 7.2 Hz), 0.66 (t, 3H, J = 7.2 Hz). IR:
(KBr) cm-1: 3466 (br), 3040, 2971, 2877, 1703, 1653,
1549. 13C NMR (75 MHz, D2O): d 175.2, 168.9, 138.4,
127.5, 84.3, 75.1, 52.5, 49.2, 28.1, 25.4, 25.1, 22.4, 8.51,
8.48. HRMS calcd for C14H25N2O4 [M?H]? 285. 1814;
found: 285.1810.
Acknowledgments This work was supported by the 12th Five Years
National Science and Technology Major Project (2011ZX09401-015)
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Med Chem Res (2013) 22:3485–3496
and Doctoral Fund of Ministry of Education of China (No.
20110131110037).
avian influenza neuraminidase suggests new opportunities for
drug design. Nature 443(7107):45–49
Sauerbrei A, Haertl A, Brandstaedt A, Schmidtke M, Wutzler P
(2006) Utilization of the embryonated egg for in vivo evaluation
of the anti-influenza virus activity of neuraminidase inhibitors.
Med Microbiol Immunol 195(2):65–71
References
Sheehan JC, Hess GP (1955) A new method of forming peptide
bonds. J Am Chem Soc 77(4):1067–1068
Jiang D, Wang YY, Xu YN, Dai LY (2009) Doebner condensation in
ionic liquids [Bmim] BF4 and [Bpy] BF4 to synthesize
[alpha],[beta]-unsaturated carboxylic acid. Chin Chem Lett
20(3):279–282
Krug RM, Aramini JM (2009) Emerging antiviral targets for influenza
A virus. Trends Pharmacol Sci 30(6):269–277
Liu Y, Zhang J, Xu W (2007) Recent progress in rational drug design
of neuraminidase inhibitors. Curr Med Chem 14(27):2872–2891
Liu AL, Wang HD, Lee SM, Wang YT, Du GH (2008) Structure-
activity relationship of flavonoids as influenza virus neuramin-
idase inhibitors and their in vitro anti-viral activities. Bioorg
Med Chem 16(15):7141–7147
Storms AD, Gubareva LV, Su S, Wheeling JT, Okomo-Adhiambo M,
Pan CY, Reisdorf E, St George K, Myers R, Wotton JT,
Robinson S, Leader B, Thompson M, Shannon M, Klimov A,
Fry AM (2012) Oseltamivir-resistant pandemic (H1N1) (2009)
virus infections, United States, 2010–11. Emerg Infect Dis
18(2):308–311
Upadhyay A, Chompoo J, Taira N, Fukuta M, Gima S, Tawata S
(2011) Solid-phase synthesis of mimosine tetrapeptides and their
inhibitory activities on neuraminidase and tyrosinase. J Agric
Food Chem 59(24):12858–12863
von Itzstein M (2007) The war against influenza: discovery and
development of sialidase inhibitors. Nat Rev Drug Discov
6(12):967–974
Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM,
Hay AJ, Gamblin SJ, Skehel JJ (2006) The structure of H5N1
123