Syntheses of triazole-modified zanamivir analogues via click chemistry and anti-AIV activities

Article abstract of DOI:10.1016/j.bmcl.2006.07.047

Sixteen novel 4-triazole-modified zanamivir (1) analogues were synthesized using the click reactions, and their inhibitory activities against avian influenza virus (AIV, H5N1) were determined. Compound 3b exerts promising inhibitory activity with EC₅₀ of 6.4 μM, which is very close to that of zanamivir (EC₅₀ = 2.8 μM). Molecular modeling provided the information about the binding model between inhibitors and neuraminidase, which are in good agreement with inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2006.07.047

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5009–5013
Syntheses of triazole-modified zanamivir analogues via click
chemistry and anti-AIV activities
Jian Li,a Mingyue Zheng,a Wei Tang,b Pei-Lan He,b Weiliang Zhu,a Tianxian Li,c
a,
a,d,
*
Jian-Ping Zuo,b,* Hong Liu and Hualiang Jiang
*
aDrug Discovery and Design Centre, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
bLaboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
cWuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
dSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Received 27 March 2006; revised 5 June 2006; accepted 17 July 2006
Available online 28 July 2006
Abstract—Sixteen novel 4-triazole-modified zanamivir (1) analogues were synthesized using the click reactions, and their inhibitory
activities against avian influenza virus (AIV, H5N1) were determined. Compound 3b exerts promising inhibitory activity with EC50
of 6.4 lM, which is very close to that of zanamivir (EC50 = 2.8 lM). Molecular modeling provided the information about the bind-
ing model between inhibitors and neuraminidase, which are in good agreement with inhibitory activities.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Recently, the H5N1 subtype of avian influenza virus
(AIV) has been spreading in Southern China, Africa,
and European Union mainly imputing to migratory
birds.1 More than 200 human cases of AIV infection
mainly as a result of poultry-to-human transmission
have been reported with a >50% case fatality rate for
H5N1 infections.2 A mutant virus capable of efficient
human-to-human transmission could trigger another
influenza pandemic.2,3 Whereas H5N1 vaccines have
been significantly hampered by the high mutability of
the virus.4 Accordingly, discovering effective drugs,
which are not susceptible to mutation, is an urgent need.
OH OH
H
H
O
COOH
NH
O
COOEt
H3PO4
OH
AcHN
AcHN
HN
NH2
H2N
Zanamivir, 1
Oseltamivir phosphate, 2
Figure 1. The structures of zanamivir (1) and oseltamivir phosphate (2).
aimed at developing more potent, long-lasting therapeu-
tic drugs for anti-AIV infection.7 For example, Wood-
hall et al.8 have synthesized a series of zanamivir
mimetics based on sialic acid aldolase.
There are currently only a few drugs available for AIV
treatment. The licensed existing drugs are the zanamivir5
(1) and oseltamivir phosphate6 (2) (Fig. 1) as specific
neuraminidase (NA) inhibitors. However, these drugs
are not effectively used owing to the rapid emergence
of resistant virus during treatment. Many groups have
designed and synthesized some zanamivir analogues
Click chemistry, coined by Barry K. Sharpless,9 is expe-
riencing a growing popularity.10 The process of click
chemistry is modular, wide in scope, high yielding, and
requires only simple reaction and purification condi-
tions. Such kind of chemistry is possible to generate a
plethora of new compounds reliably and thereby has
been applied in lead discovery and optimization. The
Huisgen 1,3-dipolar cycloaddition of azides and alky-
nes11 is regarded as the ‘cream of the crop’ of concerted
Keywords: Zanamivir analogues; Avian influenza virus; Click chemis-
try; Molecular modeling.
*
Corresponding authors. Tel.: +86 21 50806600x1210; fax: +86 21
50807088; e-mail addresses: jpzuo@mail.shcnc.ac.cn; hliu@mail.
shcnc.ac.cn; hljiang@mail.shcnc.ac.cn
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.047

5010
J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5009–5013
Table 1. Chemical Structures of Compounds 3a–p and their anti-AIV
activities
reactions, therefore it has been a general and standard
reaction of click chemistry.9,10
OH
H
Compound 9, a key intermediate for synthesizing
zanamivir (Scheme 1), contains an azide group. Accord-
ingly, this compound is a good starting material to syn-
thesize zanamivir analogues by using click reactions.
Thus, we designed and synthesized a series of zanamivir
analogues (3a–p) with different substituted triazoles and
tested their preliminary anti-AIV activities. This reac-
tion produces a unique substituted position for R3. To
increase the structural diversity of the analogues, we
selected R3 with different size. Scheme 1 depicts the se-
quence of reactions that led to the preparation of com-
pounds 3a–p using N-acetylneuraminic acid (NANA,
4) as the starting material. Meanwhile, taking com-
pounds 3b and 3i as examples, the interaction models
between inhibitors and neuraminidase were studied.
O
N
COOR4
HO
OH
AcHN
N
N
R3
Compound R3
R4
Protective ratea
at 50 lM
3a
3b
3c
3d
3e
3f
C6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
18.7
> 61.4
8.9
CH(OH)C2H5
Cyclopropyl
CH2CH2OH
(CH2)3OH
14.5
6.3
Pyridin-2-yl
C6H4–4-F
NAb
NAb
34.4
40.2
22.1
19.1
34.8
17.3
NAb
3g
3h
3i
As shown in Scheme 1, compound 4 was esterified with
methanol in the presence of an acidic ion exchange res-
in.12 Oxazoline 7 was prepared using a modification of
the procedure described by Chandler et al.13 Compound
6 was obtained using acetic anhydride in pyridine with
4-(dimethylamino)pyridine (DMAP) catalysis, then
was treated with trimethylsilyl trifluoromethanesulfo-
nate (TMSOTf) in ethyl acetate at 52 ꢁC to give 7 in high
yield. Compound 7 was hydrolyzed without purification.
Reacted with diphenylphosphoryl azide (DPPA) and
1,8-diazabicyclo[5,4,0]undecen-7-ene (DBU), the alco-
hol 8 was converted to the inverted azide 9, the key
intermediate, applying Scheigetz methods.14 Compound
10 was obtained by a click reaction between azide 9 and
C6H4–4-C2H5
C6H4–4-OC3H7
C6H4–4-C3H7
C6H4–4-C6H5
CH2OC6H5
CH2N(C2H4)2O
CH2NH2
3j
3k
3l
3m
3n
3o
3p
CH(OH)C2H5
COOCH3
(CH2)2CH(CH3)2 8.9
CH3 9.9
a Antiviral efficacy of test compounds was assessed in vitro by Neutral
Red (NR) uptake assay and expressed as percent protective rate
against virus infection.15 Data are means of three independent
experiments. The protective rate for zanamivir at 50 lM is 86.1%.
b No activity at 50 lM.
H
OAc
CO2Me
H
OH
CO2Me
R2
O
H
OH
COOH
R1
O
R1
O
a
b
AcHN
OAc
AcHN
AcHN
OAc
6
OH
5
OH
4
HO
OH
= R1
= R2
AcO
OH
OAc
H
R2
N
O
O
CO2Me
H
H
R2
AcHN
O
CO2Me
R2
AcHN
O
CO2Me
H
R1
AcHN
O
COOH
NH
c
d
e
OH
8
N3
9
H3C
HN
H2N
7
f
Zanamivir
O
H
R1
O
H
H
R1
O
N
COOH
R2
AcHN
O
CO2Me
O
g
i
AcHN
AcHN
N
N
N
N
N
N
N
N
h
OH
R3
R3
3a-n
10
3o
H
R1
O
COOMe
AcHN
N
N
N
COOMe
3p
Scheme 1. Reagents and conditions: (a) H+ Resin, CH3OH, 25 ꢁC (91%); (b) Ac2O, pyridine, DMAP (85%); (c) TMSOTf, EtOAc, N2 (84%); (d)
AcOH, EtOAc, H2O (42%); (e) DPPA, DBU, benzene (85%); (f) R3C „ CH, sodium ascorbate, CuSO4, C2H5OH/H2O = 1:1, 25 ꢁC (60–80%); (g) (1)
NaOH, CH3OH, 25 ꢁC, (2) H+ Resin (95%); (h) (1) NaOCH3, CH3OH, 25 ꢁC, (2) H+ Resin (90%); (i) 3-methyl-1-butanol, H+ Resin, THF (50%).

J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5009–5013
5011
alkyne in 60–80% yield, and then hydrolyzed with
NaOH/methanol and NaOCH3/methanol to give 3a–n
and 3p, respectively. 3o was synthesized by the corre-
sponding acid reacting with 3-methyl-1-butanol in the
presence of an acidic ion exchange resin.
Comparing compound 3a with compounds 3g–k, the
substitutions on the phenyl ring substantially play an
important role in the potency activity of compounds.
Generally, alkyl (3h and 3j) and alkoxy (3i) substituents
are favorable to the activities of these compounds, how-
ever, introductions of large aromatic (3k) or halogen
(3g) substituents are improper. Among non-aromatic
substituents of R3, propyl alcohol of 3b is favorable to
increase activity.
Protective rates15 of compounds 3a–p against infected
cell (MDCK cell) at 50 lM are summarized in Table
1. EC50 values (the concentration of a compound in
the media which reduced virus-infected cell numbers
by 50% of the untreated control value) of compound
3b and zanamivir (1) were determined as 6.4 and
2.8 lM, respectively. Although general structure–activi-
ty relationship of those compounds is not evident, the
bioassay results still provide some information for fur-
ther structural modification. Replacement of carboxyl
acid group with esters cannot be tolerated (compounds
3b and 3o), leading to a remarkable loss of activity.
To better understand the activity discrepancy of com-
pounds 3a–h with zanamivir, we compare the 3D bind-
ing models of zanamivir16 to NA (Fig. 2A) obtained
from the crystal structure with that of inhibitors 3b
and 3i to NA derived from docking simulation. Molec-
ular docking was performed by using the AutoDock3.05
program.17 The crystal structure of a wild-type NA in
complex with zanamivir was retrieved from Protein
Figure 2. Detailed interactions of zanamivir (A), 3b (B), and 3i (C) to the binding sites of NA. Hydrogen-bonds are represented by blue dotted lines;
Docking models of 3b (D) and 3i (E) overlaid with the binding conformation of zanamivir (1) into the active sites of NA. The NA surface was colored
by electrostatic potential. These images were generated using the PyMol program (http://www.pymol.org/). Compounds 3b, 3i, and zanamivir (1) are
indicated by green, green, and yellow thick sticks, respectively.

5012
J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5009–5013
Figure 2. (continued)
Data Bank (PDB entry 1NNC).16 During the docking
simulation, the protonation states of the residues of
NA at pH 7.0 were used, and the docking parameters
in our previous study18 were adopted. For each inhibi-
tor, conformation with the lowest predicted binding free
energy to NA was selected for further analysis. Figures
2B–E show the binding models of compounds 3b and
3i with NA.
In summary, 16 novel 4-triazole-modified zanamivir
analogues were designed and synthesized by using the
click reactions. Some compounds exert moderate inhibi-
tion against AIV (H5N1), compound 3b is almost as ac-
tive as zanamivir (1). Molecular modeling result
provides the information about the binding model be-
tween inhibitors and neuraminidase, and is helpful for
future inhibitors’ design.
The binding site of NA has been characterized as highly
polar by virtue of the 10 charged (Arg 118, 152, 224,
292, 371, Glu 119, 227, 276, 277, and Asp 151) and only
four hydrophobic (Trp 178, Ile 222, Ala 246, and Tyr
406) constitutional amino acids.19 Figure 2A indicates
that the carboxylic acid of zanamivir (1) forms strong
hydrogen-bonds with two arginine residues (Arg118
and Arg371) in the S1 region (Fig. 2D). Around the
guanidinyl moiety of zanamivir, there is a spacially
restricted region (S2, Fig. 2D) with negatively charged
electrostatic environment caused by three acidic resi-
dues, Glu119, Asp151, and Glu227 (Fig. 2A). Actually,
two different orientations of R3 group are observed
when the guanidinyl is replaced by a bulky and less po-
lar triazole ring, which more or less decreased the inter-
action between inhibitors and NA, and hence resulted in
a drop of inhibitory activity. In Figures 2B and D, the
binding conformation of compound 3b, which has a
smaller propyl alcohol group at R3 moiety, moves up
and only forms a hydrogen-bond with residue Arg152
in the S2 region (guanidinyl moiety of zanamivir forms
five hydrogen-bonds in S2 region, Fig. 2A). The R3 of
3i is too large to be accommodated in the subsite S2,
and hence the six-membered ring flips over and takes a
reversed orientation of zanamivir (Figs. 2C and E).
Accordingly, being devoid of hydrogen-bond interaction
with S2 (Fig. 2C), this series of compounds are generally
less potent than 3b and zanamivir. This alternative bind-
ing model (Fig. 2E) can also be seen in the X-ray crystal
structures of the complexes of NA with some inhibitors
discovered by Stoll et al.20
Acknowledgments
We gratefully acknowledge financial support from the
State Key Program of Basic Research of China (Grant
2002CB512802), the National Natural Science Founda-
tion of China (Grants 20372069 and 20472094), the Ba-
sic Research Project for Talent Research Group from
the Shanghai Science and Technology Commission,
the Key Project from the Shanghai Science and Technol-
ogy Commission (Grant 02DJ14006), the Key Project
for New Drug Research from CAS, and the 863 Hi-Tech
Programm
2003AA235030).
(Grants
2002AA233061
and
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