Synthesis of C-4-modified zanamivir analogs as neuraminidase inhibitors and their anti-AIV activities

Article abstract of DOI:10.1016/j.ejmech.2012.06.033

With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35 zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both group-1 and group-2 neuraminidase (H5N1 and H3N2) were

Full text of DOI:10.1016/j.ejmech.2012.06.033

European Journal of Medicinal Chemistry 54 (2012) 764e770
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of C-4-modified zanamivir analogs as neuraminidase inhibitors
and their anti-AIV activities
,
,
,
,
Deju Ye a 1, Woo-Jin Shin b 1, Ning Li a 1, Wei Tang a 1, Enguang Feng a, Jian Li a, Pei-Lan He a,
Jian-Ping Zuo a , Hanjo Kim , Ky-Youb Nam , Weiliang Zhu , Baik-Lin Seong , Kyoung Tai No ,
,
c
c
a,
b,
*
b
*
*
Hualiang Jiang a, Hong Liu a
,
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu chong zhi Road, Shanghai 201203, China
b Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, South Korea
c Bioinformatics & Molecular Design Research Center, Seoul 120-749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35
zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both
group-1 and group-2 neuraminidase (H5N1 and H3N2) were determined. Compound D26 exerts the
Received 23 March 2012
Received in revised form
11 June 2012
Accepted 15 June 2012
Available online 29 June 2012
most potency, with IC50 values of 0.58 and 2.72 mM against N2 and N1, respectively. Further preliminary
anti-avian influenza virus (AIV, H5N1) activities against infected MDCK cells were evaluated, and D5
exerts w58% protective against AIV infection, which was comparable to zanamivir (w67%). In a rat
pharmacokinetic study, compound D5 showed an increased plasma half-life (t1/2) compared to zanamivir
following either intravenous or oral administration. This study may represent a new start point for the
future development of improved anti-AIV agents.
Keywords:
Neuraminidase (NA)
Inhibitor
Ó 2012 Elsevier Masson SAS. All rights reserved.
Zanamivir
Avian influenza virus (AIV)
H5N1
1. Introduction
infectivity [7]. Therefore, a drug designed to selectively inhibit NA
activity may be an efficient treatment against influenza. Currently,
Influenza, a severe viral infection of the respiratory system,
remains a major threat to human health. The worldwide outbreak
of highly pathogenic H5N1 subtype of avian influenza virus (AIV)
and the recent appearance of new type human influenza A/H1N1
have heightened public awareness of potential global influenza
pandemics [1]. Vaccines are the primary method for the prevention
of influenza. However, vaccine effectiveness is significantly
hampered due to the high mutability of the virus each year [2]. As
an alternative approach, efforts have been made to develop effec-
tive antiviral drugs that lack susceptibility to mutations [3e6].
Neuraminidase (NA) is a surface glycoprotein that cleaves sialic
acid residues from the newly created virions, preventing virion
aggregation. Additionally, they cause the release of virion progeny
from infected cells, which is essential for virus replication and
a number of NA inhibitors have been reported based on the X-ray
crystal structures of NA. Two drugs, zanamivir (Relenza, 1, Fig. 1)
and oseltamivir phosphate (Tamiflu, 2), have been approved for the
treatment of influenza [2]. However, the low oral bioavailability and
rapid renal elimination of zanamivir, and the rapid emergence of
oseltamivir-resistant influenza viruses, have prompted the further
development of more potent, longer duration therapeutic drugs to
combat potential human influenza pandemics [8e10].
There are two phylogenetically distinct groups of NAs of influ-
enza A viruses: group-1 includes the N1, N4, N5, and N8 subtypes,
and group-2 includes the N2, N3, N6, N7, and N9 subtypes [11]. Both
oseltamivir and zanamivir were developed on the basis of the
crystal structures of group-2 NAs (N2 and N9). The recently solved
structures of group-1 NAs revealed that group-1 and group-2 NAs
are structurally distinct (Fig. S1). Group-1 NAs have a flexible‘150-
loop’, which can provide a novel, large cavity (150-cavity) adja-
cent to the active site, which is not present in group-2. This suggests
opportunities for the design of effective inhibitors that target this
cavity in addition to the known active site [12,13]. Based on this
idea, a C-4-modified zanamivir analog (3) [14] and a C-3-modified
* Corresponding authors. Tel./fax: þ86 21 50807042.
E-mail addresses: jpzuo@shcnc.ac.cn (J.-P. Zuo), wlzhu@mail.shcnc.ac.cn
(W. Zhu), blseong@yonsei.ac.kr (B.-L. Seong), hliu@mail.shcnc.ac.cn (H. Liu).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.033

D. Ye et al. / European Journal of Medicinal Chemistry 54 (2012) 764e770
765
OH
OH
O
COOH
O
COOH
NH
O
CO2Et
HO
HO
OH
AcHN
OH
AcHN
HN
HN
N
NH
NH
2 ·H3PO4
O
H2N
H2N
oseltamivir phosphate
N
O
zanamivir (Relenza)
(Tamiflu)
HN
1
2
3 (IC50 = 2.15 µM, H1N1)
OH
O
N
COOH
OH
HO
O
COOH
CH3
OH
AcHN
HO
OH
AcHN
N
OH
4 (K = 7.3 µM, H5N1)
N
5
I
HO
Fig. 1. Structures of zanamivir (1), oseltamivir phosphate (2) and reported zanamivir analogs (3e5).
sialic acid derivative (4) [15] were recently reported to inhibit
group-1 NA at <10 mM level. Further molecular modeling studies
compounds D1e30 was prepared from per-O-acetylated sialic acid
6, with a moderate yield as described previously [16,17]. The direct
reaction of 7 with substituted isocyanates and isothiocyanates
afforded intermediates A1e18, which were subsequently hydro-
lyzed to give the final compounds D1e18 in moderate to high yields.
In addition, amine 7 was coupled with different Boc protected amino
acids or dipeptides to generate intermediates A19e30. Removal of
the Boc protecting group and hydrolysis of the acetyl and methyl
ester groups afforded the desired compounds D19e30. To prepare
analogs D31e35, we reacted compound 6 with the appropriate
cyclic secondary amines using our previously reported method to
generate intermediates A31e35 with 70e80% yields [18e20].
Acetylation and subsequent reaction with trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf), resulted in compounds B31e35
which were hydrolyzed to give analogs D31e35.
and protein X-ray crystallography data indicated that these two
inhibitors can occupy the 150-cavity in the open form of group-1
NAs. We have also previously reported a series of C-4 triazole
modified zanamivir analogs that show potential as anti-AIV agents.
One compound (5) demonstrates promising inhibitory activity, and
was shown to be w61% protective against AIV (H5N1) infection,
whereas zanamivir is w86% protective in the same assay [16]. As
part of our ongoing studies to discover potent NA inhibitors, we
describe our further modifications of zanamivir at the C-4 position
with the introduction of guanidinium bioisosteres together with
scaffold hopping. The corresponding NA inhibitory and anti-AIV
(H5N1) activities are also shown.
2. Materials and methods
2.2. Biological evaluation
2.1. Chemistry
2.2.1. Neuraminidase enzyme inhibitory assay
2.1.1. Design of analogs of zanamivir
Influenza neuraminidase activity was determined using 20-(4-
Based on the structural features of zanamivir and its modeling
interactions with group-1 and group-2 NAs (Fig. S1), 35 zanamivir
analogs (D1e35, Table 1) were designed and synthesized. In order to
explore additional interactions with the 150-cavity, revealed in
group-1 NAs [12e15], a series of substituents at C-4 position of
zanamivir were introduced. Through the replacement of the C-4
guanidinium group of zanamivir with the ureido bioisostere and the
introduction of substituents at the terminal N-position, analogs
D1e6 were obtained. Further modification of the ureido function
with a substituted thioureido group resulted in compounds D7e14.
In order to obtain analogs with diverse structures, we subsequently
used the scaffold hopping method, to obtain a series of derivatives
methylumbelliferyl)-a-D-N-acetylneuraminic acid (MU-NANA;
Sigma Chemical Co., St. Louis, Mo.) as a substrate, as described
previously but with slight modification [21]. The NA activity of each
virus was determined before use in NA inhibition tests. Briefly, 50 ml
of virus was serially diluted in enzyme buffer [sodium acetate
buffer 150 mM (pH 7.0) containing 1 mM calcium chloride] and
50
tration of 100
for 30 min. The reactions were then stopped by the addition of
100 l of 0.014 N NaOH in 83% ethanol to each well. The fluores-
m
l of substrate in enzyme buffer to give a final MUN concen-
m
M. The reaction mixtures were incubated at 37 ꢀC
m
cence of the released 4-methylumbelliferone was quantified in
a fluorescence spectrophotometer (excitation wavelength, 355 nm;
emission wavelength, 460 nm).
D15e26 with
a-L-amino acid moieties in place of ureido group in
compounds D1e6. On the basis of the structural feature in
compound D26, we designed analogs D27e30 with the introduction
of D-Asn, L-Gln or dipeptide groups. Finally, compounds D31e35
NA inhibition was determined by measurement of the drug
concentration required to reduce NA activity to 50% of control NA
activity (IC50). Each compound was diluted five-fold (concentration
were obtained by replacing the guanidinium group at C-4 position
with the corresponding cyclic secondary amino substituents.
range from 10
incubated with 25
m
M to 128 nM) and 25
ml of each dilution was
ml of virus containing allantoic fluid at a standard
amount of NA activity (150 relative fluorescence units). After
2.1.2. Synthesis of target compounds
incubation of the mixture at 37 ꢀC for 1 h, the enzymatic reaction
The synthetic pathway to zanamivir analogs D1e35 is shown in
Scheme 1. The key intermediate 7, required for the synthesis of
was initiated by the addition of substrate (50
ml) in enzyme buffer at
a final concentration of 100 M. The reaction was stopped after
m

766
D. Ye et al. / European Journal of Medicinal Chemistry 54 (2012) 764e770
Table 1
Structures and inhibition of NA of target compounds D1-D35 and their anti-AIV activities.
OH
OH
OH
H
H
H
O
CO2H
O
CO2H
O
O
R
CO2H
HO
HO
HO
HO
HO
AcHN
HO
AcHN
AcHN
HN
HN
X
R
HN
R
R = cyclic amine
D31-D35
R = amino acid
D1-D6: X = O
D19-D30
D7-D18: X = S
Compd
R
NA Inhibition
IC50
M) a
Anti-AIV
Compd
R
NA Inhibition
Anti-AIV
(
m
Protective rate (%) b
IC50
(m
M) a
Protective rate (%) b
c
c
H3N2
H5N1d
AIV (H5N1)
H3N2
H5N1d
AIV (H5N1)
e
f
D1
D2
D3
D4
D5
D6
D7
D8
p-Me-phenyl
p-Cl-phenyl
p-F-phenyl
p-MeO-phenyl
Furan-2-ylmethyl 6.6
776.7 ꢂ 363.6 933.5 ꢂ 97.2
0
0
0
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
Gly
529.7 ꢂ 115.9 112.5 ꢂ 19.3
6.2 ꢂ 0.2
e
>1000.0
>1000.0
>1000.0
>1000.0
L-Ala-
L-Val-
L-Met-
L-Phe-
L-Pro-
L-Thr-
L-Asn-
D-Asn-
L-Gln-
Gly-Gly-
663.0 ꢂ 74.5
690.1 ꢂ 19.7
917.2 ꢂ 47.8
405.6 ꢂ 19.8
> 1000.0
0 f
0 f
0 f
0 f
0 f
0 f
e
54.35
413.2 ꢂ 34.6
628.8
e
848.1 ꢂ 112.5 910.5 ꢂ 103.1
38.4
704.7 ꢂ 274.0 > 1000.0
>1000.0 > 1000.0
6.9 ꢂ 7.8
e
58.0 ꢂ 19.3
Benzyl
Phenyl
ND g
573.7
> 1000.0
e
13.6 ꢂ 2.8
487.8 ꢂ 51.9
0.58 ꢂ 0.13
22.3 ꢂ 16.1
58.1 ꢂ 2.8
68.5 ꢂ 3.3
101.3 ꢂ 15.7
566.8 ꢂ 76.9
392.3 ꢂ 52.9
2.72 ꢂ 0.58
77.3 ꢂ 11.2
103.5 ꢂ 15.7
70.4 ꢂ 10.2
125.9 ꢂ 62.4
517.0 ꢂ 105.1
e
f
p-CF3-phenyl
m-CF3-phenyl
m-MeO-phenyl
o-F-phenyl
o-MeO-phenyl
o-Br-phenyl
893.6 ꢂ 110.9 > 1000.0
34.4 ꢂ 13.1
8.7 ꢂ 2.1
e
D9
>1000.0
>1000.0
57.1
> 1000.0
> 1000.0
395.5 ꢂ 17.5
0
0
ND g
ND g
e
D10
D11
D12
D13
e
f
14.8 ꢂ 0.7
37.1 ꢂ 6.6
e
89.6
578.3 ꢂ 200.6 11.7 ꢂ 1.8
L-Ala-Gly-
ND g
e
f
>1000.0
> 1000.0
2.9 ꢂ 8.7
26.8 ꢂ 3.4
N
O
e
ND g
COOH
D14
D15
1-Naphthyl
Ethyl
674.4 ꢂ 182.2 900.6 ꢂ 52.1
611.6 ꢂ 157.6 297.8 ꢂ 20.0
0
D32
D33
495.6 ꢂ 94.7
531.8 ꢂ 53.7
N
N
O
O
f
27.8 ꢂ 11.9
510.9 ꢂ 222.2 738.27 ꢂ 107.6 0 f
f
D16
D17
2-Cl-ethyl
100.0 ꢂ 58.8
64.5 ꢂ 5.3
67.2 ꢂ 2.9
21.4 ꢂ 6.5
20.3 ꢂ 9.8
D34
D35
836.3 ꢂ 267.2 760.8 ꢂ 30.5
271.3 ꢂ 153.3 122.3 ꢂ 60.5
ND g
N
N
N
CF3
f
n-Butyl
32.6 ꢂ 3.1
ND g
N
f
e
D18
Cyclopropyl
549.9 ꢂ 169.8 295.2 ꢂ 9.0
11.3 ꢂ 2.1
zanamivir
e
0.0014
0.012
67.2 ꢂ 13.8
a
NA inhibition was assayed by determining the drug concentration required to reduce NA activity to 50% of control NA activity (IC50).
b
c
d
e
f
Antiviral efficacy was assessed in vitro by neutral red (NR) uptake assay and expressed as percent protective rate against virus (H5N1) infected cells (MDCK cell).
H3N2 (A/Sydney/5/97).
X-31 ca virus based on H5N1.
Protective rates were tested at 5
Protective rates were tested at 4
Not determined.
m
m
M.
M.
g
30 min at 37 ꢀC. Standard curves were constructed by plotting the
percentage of fluorescence inhibition relative to control group
activity against the log of inhibitor concentration. The IC50s were
obtained from the graphs by extrapolation.
2.2.3. Pharmacokinetic studies
Compound D5 was tested in pharmacokinetic studies on Sprague
DawleyÒ (SD) rats weighing 200e250 g, with four rats (male) in each
group. The test compound was administered orally (po) or intrave-
nously (iv) at a dose of 10 mg/kg. Serial samples (0.3 ml) were
collected via the retrobulbar vein, and quantificationwas performed
by LCeMS. Pharmacokinetic parameters were calculated from the
mean plasma concentration by noncompartmental analysis.
2.2.2. Anti-AIV (H5N1) activity assay
Confluent MDCK cells were cultured in 96-well flat bottomed
microplates at a density of 2 ꢁ 104 cells/well for 24 h at 37 ꢀC in 5%
CO2. Following infection of the cells with 100 times of TCID50 A/
H5N1 virus (dilutions of viral stocks), varying concentrations of the
compounds and trypsin were added. Uninfected cells and infected
cells without compound were performed as controls. Antiviral
efficacy of test compounds was assessed in vitro by Neutral Red
(NR) uptake assay. After 2 days post-infection, cells were incubated
with NR dye for 2e3 h at 37 ꢀC. Free dye was washed from the wells
and the uptake dye was quantified using a microplate reader at
absorbance of 540 nm. Absorbance values were expressed as
percentages of control.
3. Results and discussion
3.1. Inhibitory activities against NAs
All 35 target compounds were initially evaluated for their NA
inhibitory activities using 20-(4-methylumbelliferyl)-
a-D-N-ace-
tylneuraminic acid (MU-NANA) as the substrate. With the aim of
achieving uniform activity, we simultaneously selected N1 (H5N1)
from group-1 and N2 (H3N2) from group-2 as representatives for

D. Ye et al. / European Journal of Medicinal Chemistry 54 (2012) 764e770
767
OH
OAc
H
O
CO2H
X
H
HO
O
CO2Me
AcO
OH
a
OAc
b
AcHN
AcHN
HN
R
HN
R
X
OAc
H
NH
O
CO2Me
X = O, S
NH
AcO
D1-18
OAc
AcHN
A1-18
NH2
7
OAc
OH
H
H
O
CO2Me
O
CO2H
AcO
HO
d
c
OAc
OH
AcHN
AcHN
HN
O
HN
O
OAc
OAc
H
O
COOMe
AcO
AcO
BocHN
R'
H2N
R'
OAc
A19-30
AcHN
D19-30
OAc
6
e
OAc
H
OAc
OH
H
OH
H
O
N
COOMe
O
COOMe
O
N
COOH
AcO
HO
g
OAc
AcHN
OAc
OH
f
AcHN
AcHN
N
X
X
B31-35
X
D31-35
X = O, NR1, CHR2
A31-35
Scheme 1. Synthesis of compounds D1e35. Reagents and conditions: (a) RNCX, CH2Cl2, rt.; (b) (i) 5% NaOH, CH3OH, rt., overnight; (ii) Hþ resin; (c) Boc-amino acid, EDCI, HOBt, TEA,
CH2Cl2, rt.; (d) (i) TFA, CH2Cl2; (ii) OH- resin; (iii) 5% NaOH, CH3OH, rt., overnight; (iv) Hþ resin; (e) pyridine, cyclic secondary amine, r.t.; (f) (i) Ac2O, DMAP, pyridine, rt., overnight;
(ii) TMSOTf, CH3CN, 0 ꢀC; (g) (i) 5% NaOH, CH3OH, rt., overnight; (ii) Hþ resin.
testing, and the results are summarized in Table 1. Preliminary
results showed that most compounds displayed decreased inhibi-
tory activities against both of N1 and N2 compared with zanamivir,
and all compounds were more potent against N2 than that against
N1. The replacement of the guanidinium group in zanamivir with
bioisosteres [22], such as substituted urea or thiourea, in
compounds D1e4 and D6e18 showed no significant activity
assay [16] with MDCK cells. The protective rates of test compounds
against infected cells are summarized in Table 1. Most test
compounds showed no significant anti-AIV activity (protective rate
<50% at tested concentration). Compound D26, which has the best
NA inhibitory activity (IC50 ¼ 2.72
AIV activity against infected cells (8.7% at 4
compounds tested, D5 with moderate inhibition of N1
(IC50 ¼ 38.38 M), showed the most potent anti-AIV efficacy, with
a protective rate of w58% at 5 M, comparable to zanamivir (w67%
at 5 M). This may be ascribed to the higher cell membrane
mM, N1), only showed week anti-
mM). However, of all the
a
(IC50 > 50
substituent at the terminal N-position of group urea improved the
NA inhibitory activities considerably (IC50 of 6.6 M against N2). In
mM), whereas compound D5 bearing a furan-2-ylmethyl
m
m
m
m
an effort to gain more potency, we subsequently used the scaffold
hopping method [23] to transform the substituted ureido groups in
permeability of D5 with a hydrophobic furan-2-ylmethyl substit-
uent, and therefore, gave rise to a higher cell uptake of D5 than that
of D26.
D1e5 into
hydrophobic amino acid side chains, did not show improved inhi-
bition. Compound D26 bearing an -Asn substituent was the most
potent inhibitor of all the 35 analogs, with IC50 values of 0.58
(N2) and 2.72 M (N1), respectively. Further conversion of -Asn
moiety in D26 into corresponding -Asn, -Gln or dipeptide groups
a-amino acid moieties. Compounds D20e25, with
L
3.3. Pharmacokinetic (PK) profiles of the selected anti-AIV agent
mM
m
L
It is reported that zanamivir has poor bioavailability and is
rapidly eliminated (short plasma half-life t1/2) following oral
administration [8,9]. To understand the pharmacokinetic parame-
ters of C-4-modified zanamivir analogs, we selected compound D5
(the most potent antiviral activity in MDCK cells) as representative
for further investigations following intravenous (iv) and oral (po)
administration in rats. For comparison, we also show the reported
literature values for zanamivir [24] in Table 2. The results show that
D5 has no improved bioavailability following oral administration
compared to zanamivir (2.73% vs 2.7%). However, D5 exhibited
a much longer plasma t1/2 after both iv and po dosing, indicating
D
L
(D27e30) did not translate into improved inhibitory activity.
Finally, only weak NA inhibitory activity was observed for
compounds D31e35 incorporating cyclic secondary amines
substituted at C-4 position.
3.2. Anti-AIV activity
In addition to NA enzyme inhibition, we investigated anti-
influenza viral efficacy (AIV/H5N1) using a cytopathic effect (CPE)

768
D. Ye et al. / European Journal of Medicinal Chemistry 54 (2012) 764e770
Table 2
for interaction with different NA inhibitors (Fig. 3) [3,7,8]. Site S1 is
comprised of three positively charged residues (Arg118, 292 and
371) and interacts with anionic substituents (carboxylate) of the
inhibitors. Site S2 is a negatively charged region and composed of
Glu119, Asp151 and Glu227, which participates in a network of
hydrogen bonding and ionic interactions with a guanidine or an
amine group. Site S3 contains a small hydrophobic region formed
from the side chains of Trp178 and Ile222 adjacent to Arg152,
providing hydrophobic interactions as well as a hydrogen-bond
with acetyl substituent of the inhibitors. Site S4 is primarily
a hydrophobic region derived from the side chains of Ile222, Ala246
(Ser246 in N1) and the hydrophobic face of Arg224. In site S5, the
carboxylate of Glu276 functions as a hydrogen bond acceptor, with
the alkyl region of the side chain together with Ala246 (Ser246 in
N1) forming an additional hydrophobic pocket.
Fig. 2A and B indicates that zanamivir adopts a similar binding
mode in the active site. In subtype N2, the guanidinium group of
zanamivir is directed toward site S2 and makes tight salt-bridge
interactions with Glu119, Asp151 and Glu227. In the open form of
N1, the acidic residues Glu119 and Asp151 are further from zana-
mivir than those in N2, resulting in the distance between guani-
dinium group and Asp151 being too large to form strong
interactions. This change may explain the reduced potency of
zanamivir and its analogs against N1 compared to N2. When the
Comparison of pharmacokinetic parameters for compound D5 and zanamivir in rat.
Parameter
D5a
Zanamivir
Dosed intravenously
(iv)
Dosed orally
(po)
iv
po
dose (mg/Kg)
Cmax (ng/ml)b
Tmax (h)c
10
e
10
2
e
10
e
74.9 ꢂ 24.0
1.31 ꢂ 0.85
5.67 ꢂ 2.63
381 ꢂ 129
7.82 ꢂ 2.86
27.7 ꢂ 4.0
e
e
e
e
t1/2 (h)d
4.02 ꢂ 1.05
13,969 ꢂ 1703
0.982 ꢂ 0.110
0.723 ꢂ 0.085
4.16 ꢂ 1.00
e
0.4
e
0.25
e
AUC0et (ng h/ml)e
MRT (h)f
e
e
Cl (l/h/kg)g
Vd (l/kg)h
0.085
0.03
e
e
F (%)i
2.73b
2.7
a
Data are means of four independent experiments.
The maximum plasma concentration (Cmax).
The time to reach Cmax (Tmax).
Terminal half time (t1/2).
The area under the concentrationetime curve (AUC0et).
Mean resident time (MRT).
Clearance (Cl).
Volume of distribution (Vd).
Percentage bioavailability (F (%)).
b
c
d
e
f
g
h
i
that D5 may serve as a possible lead compound with increased
duration of action in anti-influenza drug development.
guanidine group is replaced by
-Asn moiety (D26) or a bulky N-3-
L
(furan-2-ylmethyl)ureido group (D5), two different binding modes
are observed, which decreases the interaction between inhibitors
and NA, and hence results in a reduction in inhibitory activity. As
shown in Fig. 2C, D26 binds to N2 and moves to accommodate the
3.4. Binding models
To better understand the activity discrepancies of analogs
D1e35 with zanamivir, we compared 3D models of zanamivir with
those of compounds D5 and D26 binding to N2 (PDB ID: 2AEP) [25]
and N1 (PDB ID: 2HU0) [11] in an open form based on the docking
simulation (Fig. 2). This revealed that the active site of NAs could be
empirically divided into five major binding sites which are critical
slightly larger L-Asn moiety in the S2 pocket. The L-Asn moiety of
D26 electrostatically bonds to Asp151, Glu227, and Glu277 in site
S2. An additional hydrogen-bond between D26 and the backbone
carbonyl of Trp178 is also observed. The glycerol side chain of D26 is
distant from Glu276 in site S5, and only forming two hydrogen
Fig. 2. Detailed interactions of zanamivir (A, B), D26 (C, D), and D5 (E, F) binding to the active sites of group-2 neuraminidase (N2: A, C, E) and group-1 neuraminidase (N1: B, D, F) in
the open form. Zanamivir and compounds D26 and D5 are indicated by cyan, marine, and marine stick models, respectively. The neuraminidases are shown in gray, whereas the key
residues in the active site are highlighted in atom-color stick model. Hydrogen-bonds are represented by purple dotted lines. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

D. Ye et al. / European Journal of Medicinal Chemistry 54 (2012) 764e770
769
without further purification. Analytical thin-layer chromatography
(TLC) was performed on HSGF 254 (150e200 m thickness, Yantai
Glu 276
m
Huiyou Company, China). Yields were not optimized. Melting
points were measured in capillary tube on a SGW X-4 melting point
apparatus without correction. Column chromatography was per-
formed with CombiFlashÒ Companion system (Teledyne Isco, Inc.).
Nuclear magnetic resonance (NMR) spectra were performed on
a Brucker AMX-400 and AMX-300 NMR (IS as TMS). Chemical shifts
Site S5
Arg 292
Ala 246
OH
Arg 371
Arg118
Arg 224
OH
Site S1
O
Site S4
O
HO
HN
OH
were reported in parts per million (ppm, d) downfield from tetra-
Ile 222
methylsilane. Proton coupling patterns were described as singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br).
Low- and high-resolution mass spectra (LRMS and HRMS) were
given with electric, electrospray and matrix-assisted laser desorp-
tion ionization (EI, ESI and MALDI) produced by Finnigan MAT-95,
LCQ-DECA spectrometer and IonSpec 4.7 Tesla. LCeMS analysis
were conducted on Agilent 1100 Series HPLC with an Agilent Zor-
HN
NH2
Asp 151
O
Site S3
H2N
Trp 178
Glu 119
Site S2
Arg 152
bax Eclipse XDB-C18 (4.6 ꢁ 50 mm, 5
mm) reversed phase column.
Glu 227
Compounds D1eD35 were confirmed ꢃ95% purity (Supporting
information, Table S1). The details for purity analyses of
compounds D1eD35 are described in the Supporting Information.
Fig. 3. Active site of neuraminidase (representative as zanamivir binding to N2).
bonds with residues Arg292 and Asn294 around S5 pocket,
compared with additional hydrogen bonds observed in zanamivir
binding to N2 of the same binding site (Fig. 2A). When binding to
N1 in open form (Fig. 2D), D26 sits deeper within the pocket than
5.2. Viruses and cells
Influenza virus A/Sydney/5/97 (H3N2) and recombinant virus
containing the HA and NA genes from A/Indonesia/5/2005 (H5N1)
virus in the background of X-31 ca virus were propagated in 11-day-
old embryonated chicken eggs as described elsewhere [27,28].
MadineDarby canine kidney (MDCK) cells were obtained from
American Type Culture Collection (ATCC) and were cultured as
monolayers in the minimal essential medium (MEM) supple-
mented with 10% heat-inactivated fetal bovine serum (FBS), 50
zanamivir, and the L-Asn moiety establishes multi hydrogen bonds
and electrostatic interactions with Glu119, Asp151, Glu227 and
Arg118. However, only one hydrogen bond with Ser246 in S5 pocket
was observed, resulting in reduced NA inhibition compared to
zanamivir. In compound D5, the N-3-(furan-2-ylmethyl)ureido
group is too large to be accommodated in S2 pocket of both N2 and
N1, hence the dihydropyran ring flips, adopting a reversed orien-
tation relative to zanamivir (Fig. 2E and F). The glycerol side chain is
embedded within S2 pocket interacting with Glu119, Arg156 and
Glu227, while the furan ring is able to extend towards the S4 site,
providing further hydrophobic interactions with Ile222, Ala246 and
the hydrophobic face of Arg224. As a result, the original hydrogen
bonds between the glycerol side chain and residues in S5 pocket of
N2 or N1 cannot occur, potentially explaining the reduced activity
of D5 compared to D26 or zanamivir. Further evidence for this
alternative binding mode in which X-ray crystal structures showing
complexes of NA with some inhibitors has also been reported by
Stoll et al. [26].
units of penicillin, 50 mg streptomycin, and 25 mg of amphotericin.
Acknowledgments
We gratefully acknowledge financial support from National
Basic Research Program of China (Grants 2009CB918502 and
2012CB518005), the National Natural Science Foundation of China
(Grants 20721003 and 81025017), National S&T Major Projects
(2012ZX09103-101-072), Silver Project (260644), the R&D Program
of MKE/KEIT (10031969), Translational Research Center for Protein
Function Control, NSF (2011-0001244) and a grant from the Korea
Healthcare Technology R&D project, Ministry for Health, Welfare
Family Affairs, Republic of Korea (A085105).
4. Conclusions
Appendix A. Supplementary information
In conclusion, we have designed and synthesized 35 zanamivir
analogs with a modification at the C-4 position using bioisostere
replacement and scaffold hopping strategies. Compound D26
exhibited strong potency against both group-2 (H3N2) and group-1
Supplementary data (Fig. S1, Table S1 for HPLC purity, and
experimental details) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.
06.033.
(H5N1) NAs, with IC50 ¼ 0.58 and 2.72
mM, respectively. Moreover,
compound D5 was shown to have good anti-AIV activity against
infected MDCK cells and a prolonged plasma half-life following
either intravenous or oral dosing in rats. Molecular modeling
results revealed two different binding modes between inhibitors
and NAs, which may aid the identification and future development
of improved inhibitors against both group-1 and group-2 NAs.
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