Divalent oseltamivir analogues as potent influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.carres.2019.03.012

A panel of divalent oseltamivir and guanidino oseltamivir analogues with esterification on the carboxyl acid group as potent inhibitors of influenza virus neuraminidase was prepared via click reaction. The primary structure activity relationship study demonstrated that appropriate distance between two oseltamivir monomers around 30 ? can crosslink two adjacent neuraminidase tetramers on the virion surface and result in highly effective NA inhibitors against three strains of influenza virus and H7N9 virus like particle. This strategy also provides a basis for the multivalent modification on oseltamivir.

Full text of DOI:10.1016/j.carres.2019.03.012

Accepted Manuscript
Divalent oseltamivir analogues as potent influenza neuraminidase inhibitors
Zhong-Li Yan, Ao-Yun Liu, Xia-Xia Wei, Zhuang Zhang, Long Qin, Qun Yu, Peng Yu,
Kui Lu, Yang Yang
PII:
S0008-6215(19)30093-X
https://doi.org/10.1016/j.carres.2019.03.012
CAR 7694
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 13 February 2019
Revised Date: 21 March 2019
Accepted Date: 28 March 2019
Please cite this article as: Z.-L. Yan, A.-Y. Liu, X.-X. Wei, Z. Zhang, L. Qin, Q. Yu, P. Yu, K. Lu, Y. Yang,
Divalent oseltamivir analogues as potent influenza neuraminidase inhibitors, Carbohydrate Research
(2019), doi: https://doi.org/10.1016/j.carres.2019.03.012.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Divalent oseltamivir analogues as potent
influenza neuraminidase inhibitors
Leave this area blank for abstract info.
Zhong-Li Yana, Ao-Yun Liub, Xia-Xia Weib, Zhuang Zhangb, Long Qinb, Qun Yub, Peng Yub, Kui Lub*,
Yang Yanga, b*
a Research Centre of Modern Analytical Technology, Tianjin University of Science & Technology, No. 29, 13th Avenue, TEDA, Tianjin
300457,China
b China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of
Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, TEDA, Tianjin 300457, China
Highlights
• Novel divalent oseltamivir analogues were synthesized using Click chemistry.
• Distance between two oseltamivir monomers ~30 Å can enhance antiviral activity.
• Esterification of oseltamivir carboxylic acid can be further developed for improving efficacy.

ACCEPTED MANUSCRIPT
Carbohydrate Research
journal homepage: www.elsevier.com
Divalent oseltamivir analogues as potent influenza neuraminidase inhibitors
Zhong-Li Yana, Ao-Yun Liub, Xia-Xia Weib, Zhuang Zhangb, Long Qinb, Qun Yub, Peng Yub, Kui Lua, 
Yang Yanga, b
a Research Centre of Modern Analytical Technology, Tianjin University of Science & Technology, No. 29, 13th Avenue, TEDA, Tianjin 300457,China
b China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin
University of Science and Technology, No. 29, 13th Avenue, TEDA, Tianjin 300457, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A panel of divalent oseltamivir and guanidino oseltamivir analogues with esterification on the
carboxyl acid group as potent inhibitors of influenza virus neuraminidase was prepared via click
reaction. The primary structure activity relationship study demonstrated that appropriate distance
between two oseltamivir monomers around 30 Å can crosslink two adjacent neuraminidase
tetramers on the virion surface and result in highly effective NA inhibitors against three strains
of influenza virus and H7N9 virus like particle. This strategy also provides a basis for the
multivalent modification on oseltamivir.
Available online
Keywords:
Cluster effect
Oseltamivir
2009 Elsevier Ltd. All rights reserved.
Guanidino oseltamivir
Click chemistry
Neuraminidase inhibitor
resistant strains9 has stressed the urgent need for the development
of new antiviral agents in recent years.
1. Introduction
Neuraminidases (NAs) or sialidases, catalyze the hydrolysis of
α-glycosidic linkages of terminal sialic acid (SA) residues in
glycoconjugates and are widespread in many tissues of animals,
pathogens and viruses1-2. Several human pathologies are strongly
associated with abnormal NAs activity3, and some bacteria and
viruses utilize their NAs for host cell infections4. Therefore,
much attention has been paid on the design and synthesis of
efficient NA inhibitors for the therapeutic applications and
functional studies5. One of the most successful structure-based
designs of potent NA inhibitor is the discovery of Zanamivir
(RelenzaTM, ZA) (Fig. 1.), which mimics the transition state of
the influenza NA-catalyzed sialoside hydrolysis reaction and has
been used as the first-line drugs for the treatment of viral flu6.
However, due to the low oral bioavailability and rapid renal
elimination of ZA, Oseltamivir (TamifluTM, OS) (Fig. 1.) was
developed7. Compared with ZA, the pyran ring, C-4 guanidino
group and glycerol side chain are replaced with cyclohexene,
amine and 3-pentyl ether, respectively. After further
esterification of the carboxyl group, OS as the orally available
therapeutic prodrug is usually prescribed in the clinic for combat
human influenza pandemics8. Unfortunately, the frequent
mutations of NA, which leads to the emergence of new OS-
Figure 1. Structures of two marketed NA inhibitors, ZA and OS.
It is now well known that the binding conformation of 3-
pentyl ether side chain in OS affected by NA mutations is the
main reason for the unbeneficial interactions, which results in
drug-resistance10. In order to compensate this decreased binding
affinity, many attempts of chemical modifications on C-5 amine11,
moving the location of C–C double bond12 or replacing the C-3
pentyl group13 have been applied. Generally speaking, this
strategy more or less enhances the binding to NA in vitro, but
does not lead to better inhibitors in vivo. Recently, L-amino acid
ester linked Oseltamivir Carboxylate (OC) esters, which are
———
 Corresponding author. e-mail: lukui@tust.edu.cn
 Corresponding author. e-mail: yyang@tust.edu.cn

ACCEPTED MANUSCRIPT
2. Results and discussion
facilitated by the peptide transporter–cellular activation as
prodrugs show improved oral absorption and systemic
availability14. In another example, OC hydroxamate and acyl
sulfonamide derivatives15 were also prepared and have been
shown to have anti-influenza activity against wild-type H1N1
and OS-resistant virus. Building on these reports, we proposed
that the esterification of the carboxyl group may be a target for
the chemical structure modification and lead to obtain better NA
inhibition activity.
2.1. Chemical synthesis
The synthesis of divalent oseltamivir and guanidino
oseltamivir analogues with different linker lengths were
illustrated in Scheme 1. At first, OS was treated with di-tert-butyl
dicarbonate (Boc)2O and further hydrolyzed to obtain the
corresponding Boc protected carboxylic acid35 (OC). Different
lengths of hydrophobic or hydrophilic azidoalcohols were reacted
with the acid under typical esterification condition to afford the
azide moiety 2-9 in reasonable yields for the next Click
chemistry36-39. Meanwhile, the alkyne part was prepared from
ethylene glycol reacted with propargyl bromide under basic
condition. With the two desired components in hand, 1, 3 dipolar
Huisgen cycloaddition (Click reaction) was applied for the dimer
synthesis. Typically, catalytic quantity of CuSO4 ·5H2O was
added to the solution of bis-alkyne scaffold and different azide
ester in tetrahydrofuran (THF) and water followed by sodium
ascorbate. After purification by chromatography, the Boc group
was removed with trifluoroacetic acid (TFA) to afford the final
divalent OC analogues 10-17. A Boc protected guanidino group
was introduced by reaction with N, N'-bis-(tert-butoxycarbonyl)-
S-methylisothiourea in the presence of HgCl2 and Et3N to provide
divalent Boc-guanidino OC derivatives40. Treatment with TFA
afforded free 18-25 in good yield. All intermediates and final
products were characterized by spectroscopic properties.
A different approach for the design of potent NA-inhibitors
is to introduce “cluster effects”16-18, which has been validated by
the success of synthesizing multivalent ZA conjugates19-29. As
four NA monomers form a mushroom-like tetramere anchored on
viral surface30, various ZA with appropriate distance attached on
the multivalent backbones can simultaneously bind to more than
one NA monomer within a single or different tetramers on the
same or even separated virions. Structurally, these strategies use
C-7 hydroxyl group of ZA, which has no direct interaction to the
active site of NA for the linker attachment31 and then assemble
ZA on the multivalent scaffolds. However, due to the lack of
hydroxyl group in the glycerol side chain of OS, multivalent OS
conjugates have not been pursued.
Herein, we want to combine with “cluster effect”32-34 and ester
prodrug masked strategy together for increasing the binding
affinity of OS to NA. As the initial investigation, a panel of
different lengths of the linker carrying two OC or guanidino OC
monomers was designed and synthesized. Furthermore, a primary
relationship between the distance of two monomers in the
divalent analogues and the NA inhibition activity is discussed.
Scheme 1. Synthesis of divalent OC analogues. Reagents and conditions: (a) (i) (Boc)2O, trimethylamine (Et3N), 98%; (ii) NaOH,
tetrahydrofuran (THF)/H2O 95%; (b) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine
(DMAP), N, N-diisopropylethylamine (DIPEA), 85-89%; (c) CuSO4·5H2O, sodium ascorbate (VcNa), THF/H2O; (d) trifluoroacetic acid
(TFA)/CH2Cl2 1:1; (e) MeSC(=NBoc)NHBoc, HgCl2, Et3N, CH2Cl2, rt, 12 h.

ACCEPTED MANUSCRIPT
2.2. Biological Assays
With the divalent OC and guanidino OC analogues in hand,
we evaluated NA inhibition activity using a typical fluorogenic
substrate 2-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid
(MUNANA) against three viral strains [A/Hecheng, Hunan
Province/SWL1331/2014(H1N1); A/Puerto Rico/8/1934 (H1N1);
A/Chicken/Beijing/AT609/2014 (H9N2)] and H7N9 virus like
particles (VLP)41. To compare the inhibition activity
enhancement, OS and guanidino OS were selected as positive
control, respectively. The IC50 values of each divalent analogues
and potency per one corresponding OS monomer (γ)29 are
summarized in Table 1.
It is gratifying to note that the esterification on the carboxylate
has little effect on NA inhibition activity, as all of the divalent
analogues had comparable IC50 to their corresponding monomers.
Additionally, introduction of the guanidino group increased the
inhibition activities of
18-25; decreased IC50 values in
submicromolar than corresponding animo divalent analogues 10-
17 were observed. This is due to the more basic gaunidino group,
which has strong electrostatic interactions with the acidic peptide
residues in the active site of NA42.
We were very interested in whether these divalent OC
analogues exhibit a bidentate binding effect similar to the
reported dimeric ZA26, 43-44 with ten to hundred times decreased
EC50 or S-sialoside40 clusters with ten times decreased IC50
compared to their corresponding monomers. Based on the X-ray
crystal structure of influenza NA, it has been demonstrated that
the distance of two NA active sites in a tetramer or two different
neighboring tetramers on the same virion is approximately ~50 Å
and ~30 Å, respectively (Fig. 2.)19, 26. Only the dimension of the
two monomers in the divalent analogues, which matches the NA
tetramer distribution can improve the binding affinity. To
measure the distance between the two OC monomers in our
synthetic dimers, the low-energy conformations of 10-17 and 18-
25 were simulated using Discovery Studio 3.1 and the primary
relationship between the lengths of the linker and the antiviral
activity was determined (Table 1.).
Figure 2. Schematic representation of distance between two NA
monomer active sites on a same virion and the low-energy extended
conformation of 17.
Table 1. Neuraminidase inhibition activity of divalent OC and guanidino OC analogues
Distance between
IC50 (µM)
Compound
number
two OC ananlogs
monomer (Å)
H1N1a (γd)
H1N1 b (γ)
H7N9 VLP (γ)
H9N2 c (γ)
10
11
12
13
14
15
16
17
OS
19.30
24.60
25.90
28.86
27.48
32.80
25.30
32.28
1.10 (1)
3.63 (0.4)
0.52 (2)
3.79 (0.3)
0.84 (1)
0.22 (6)
0.93 (1)
0.24 (5)
2.56 (1)
1.30 (2)
5.41 (0.6)
0.73 (4)
4.76 (0.7)
1.21 (3)
0.37 (9)
1.48 (2)
0.048 (68)
6.49 (1)
0.73 (1)
2.10 (0.5)
0.35 (3)
1.80 (1)
0.45 (2)
0.11 (9)
0.55 (2)
0.10 (10)
2.06 (1)
0.18 (2)
0.59 (0.6)
0.11 (3)
0.71 (0.5)
0.13 (3)
0.03 (11)
0.14 (2)
0.034 (10)
0.69 (1)
18
19
20
21
22
23
24
25
19.70
24.60
26.06
28.98
27.27
32.98
25.50
31.32
0.24 (4)
0.13 (8)
0.59 (2)
0.10 (10)
0.21 (5)
0.32 (3)
0.10 (10)
0.08 (12)
2.01 (1)
0.31 (9)
0.17 (17)
0.71 (4)
0.13 (22)
0.27 (11)
0.51 (6)
0.16 (18)
0.13 (22)
5.87 (1)
0.26 (2)
0.16 (3)
0.50 (1)
0.08 (6)
0.19 (2)
0.26 (2)
0.13 (4)
0.08 (6)
0.95 (1)
0.26 (7)
0.14 (13)
0.62 (3)
0.30 (6)
0.24 (8)
0.23 (8)
0.11 (17)
0.08 (23)
3.74 (1)
Guanidino OS
a A/Hecheng, Hunan Province/SWL1331/2014(H1N1);
b A/Puerto Rico/8/1934 (H1N1);
c A/Chicken/Beijing/AT609/2014 (H9N2);
d γ=IC50 corresponding monomer/ (2×IC50 divalent analogue).

35
ACCEPTED MANUSCRIPT
Our modeling study predicted that the distances between two
OC monomers in the prepared divalent analogues are ranging
from 20 Å to 33 Å. In 15, 17 and 25, the intermolecular space of
the two OC monomeric analogues is ~32 Å, which can bind two
neighboring NA resulting in bidentate binding effect and have
stronger inhibition activity against all of the four strains, leading
to decreased IC50 values. In contrast, the other divalent analogues
with two OC monomer spacing less than 30 Å is not sufficient to
achieve the bivalent binding requirement leading to less effective
To a solution of the Boc protected oseltamivir acid (78 mg,
0.2 mmol) in CH2Cl2 (2 mL) were added alcohol (0.24 mmol),
EDCI (91 mg, 0.24 mmol), DIPEA (0.1 mL, 0.6 mmol) and
DMAP (7.4 mg, 0.02 mmol). The mixture was stirred at room
temperature for 10 h, diluted with CH2Cl2 and then washed with
1 M HCl, saturated NaHCO3 and brine. The organic layer was
dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by flash silica gel column
chromatography to give Boc protected OC monomers with
different length linkers as pale yellow oil.
NA inhibition activity, which is in agreement with other
published results19,
40. To clearly compare the activity
26,
4.2.1.1. 2-azidoethyl (3R, 4R, 5S)-4-acetamido-5-((tert-
enhancement, a γ factor indicates the potency per OC monomer
was used with OS and guanidino OS as positive control,
respectively (Table 1). The most potent NA inhibitor 17 exhibits
approximately 68-fold increase in inhibition against H1N1 strain
compared with its OS monomer, which further stresses the
importance of the linker for the dimer attachment.
butoxycarbonyl)
carboxylate (2)
amino)-3-(1-ethylpropoxy)-1-cyclohexene-1-
According to the general procedure, 2 was synthesized in
85% yield. H NMR (400 MHz, CDCl3) δ 6.82 (s, 1H), 6.06 (s,
1
1H), 5.22 (s, 1H), 4.31 (t, J = 4.1 Hz, 2H), 4.09–3.95 (m, 2H),
3.79 (dd, J = 10.2, 5.1 Hz, 1H), 3.55–3.46 (m, 2H), 3.37–3.27 (m,
1H), 2.77 (dd, J = 6.4, 18.7 Hz, 1H), 2.30 (dd, J = 17.7, 10.0 Hz,
1H), 1.97 (s, 3H), 1.52–1.44 (m, 4H), 1.41 (s, 9H), 0.87 (q, J =
7.6 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 170.87, 165.54,
156.36, 139.10, 128.53, 82.26, 79.67, 75.85, 63.69, 54.46, 49.80,
49.10, 30.91, 28.33, 26.10, 25.58, 23.34, 9.50, 9.14.
3. Conclusions
We have introduced “cluster effect” principle to prepare a
45
panel of divalent OC and guanidino OC ester analogues with
different linker lengths. A primary relationship between the
length of the linker and NA inhibition activity was identified.
The appropriate distance between two OS monomers to achieve
divalent NA active site binding is ~32 Å, which is essential for
the divalent NA inhibitor design. With these encouraging results,
we are currently choosing new scaffolds to obtain multi or
polyvalent OC or guanidino OC conjugates as more potent NA
inhibitors. Furthermore, the inhibition activities of these synthetic
dimers on cell or animal level are also under investigation in our
lab. We believe that this research will provide a basis for
investigating multivalent OC conjugates not only as therapeutics
agents for wide strains of influenza virus, but also against drug-
resistant strains.
4.2.1.2. 3-azidopropyl (3R, 4R, 5S)-4-acetamido-5-((tert-
butoxycarbonyl)
carboxylate (3)
amino)-3-(1-ethylpropoxy)-1-cyclohexene-1-
According to the general procedure, 3 was synthesized in
87% yield. H NMR (400 MHz, CDCl3) δ 6.72 (s, 1H), 6.40 (br,
1
1H), 5.36 (br, 1H), 4.23–4.16 (m, 2H), 4.08–3.94 (m, 2H), 3.81–
3.71 (m, 1H), 3.37 (t, J = 6.6 Hz, 2H), 3.34–3.27 (m, 1H), 2.68
(d, J = 17.6 Hz, 1H), 2.27 (dd, J = 17.6, 10.1 Hz, 1H), 1.95 (s,
3H), 1.93–1.88 (m, 2H), 1.48–1.43 (m, 4H), 1.39 (s, 9H), 0.88–
0.81 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 170.89, 165.72,
156.36, 138.33, 128.94, 82.31, 79.65, 75.90, 61.86, 54.51, 49.14,
48.19, 30.92, 28.35, 28.13, 26.13, 25.72, 23.33, 9.57, 9.21.
HRMS (ESI): m/z calcd for C22H37N5O6Na [M+Na]+: 490.2636,
found: 490.2624.
4. Experimental
4.1. General methods
4.2.1.3. 6-azidohexyl (3R, 4R, 5S)-4-acetamido-5-((tert-
All the materials and solvents were obtained from commercial
suppliers and used without further purification. Oseltamivir
phosphate was purchased from Hangzhou Rongda Pharm &
Chem Co, Ltd. China. Thin-layer chromatography (TLC) was
purchased from EMD Co. Ltd. (German). All compounds were
stained with iodine vapor. Flash column chromatography was
performed on silica gel 200-300 mesh. All of the final
compounds were purified using SephadexTM LH-20 (GE
Healthcare). NMR spectra were recorded on Bruker AVANCE
III (400 MHz) instrument. Chemical shifts (δ) were reported in
parts per million downfield from TMS, the internal standard; J
values were given in Hertz. The molecular weights of the
compounds were confirmed by electrospray ionization mass
spectra (ESI-MS) on a hybrid IT-TOF mass spectrometer
(Shimadzu LCMS-IT-TOF, Kyoto, Japan). Fluorescence
intensity was measured using the SynergyTM H1/H1MF
microplate reader (BioTek Instruments, Inc. USA).
butoxycarbonyl)
amino)-3-(1-ethylpropoxy)-1-cyclohexene-1-
carboxylate (4)
According to the general procedure, 4 was synthesized in
1
86% yield. H NMR (400 MHz, CDCl3) δ 6.69 (s, 1H), 6.47 (br,
1H), 5.38 (br, 1H), 4.09–3.97 (m, 4H), 3.72 (br, 1H), 3.30–3.22
(m, 3H), 2.65 (d, J = 17.4 Hz, 1H), 2.25 (dd, J = 17.4, 10.2 Hz,
1H), 1.93 (s, 3H), 1.62–1.37 (m, 21H), 0.90–0.73 (m, 6H). 13C
NMR (100 MHz, CDCl3) δ 170.87, 165.90, 156.33, 137.89,
129.16, 82.24, 79.44, 75.83, 64.72, 54.55, 51.29, 49.31, 30.84,
28.71, 28.43, 28.32, 26.35, 26.13, 25.71, 25.54, 23.24, 9.54, 9.21.
HRMS (ESI): m/z calcd for C25H43N5O6Na [M+Na]+: 532.3106,
found: 532.3091.
4.2.1.4. 12-azidododecyl (3R, 4R, 5S)-4-acetamido-5-((tert-
butoxycarbonyl)
amino)-3-(1-ethylpropoxy)-1-cyclohexene-1-
carboxylate (5)
According to the general procedure, 5 was synthesized in
1
86% yield. H NMR (400 MHz, CDCl3) δ 6.73 (s, 1H), 6.22 (br,
The influenza viruses were obtained from National Institute for
Viral Disease Control and Prevention, China CDC and
propagated in 9 days old embryonated chicken eggs. The
allantoic fluid was collected, then centrifuged at 3000 rpm/min
for 10 min and the supernatant was stored at -80 ℃ before use.
1H), 5.25 (s, 1H), 4.12–3.95 (m, 4H), 3.80–3.71 (m, 1H), 3.36–
3.28 (m, 1H), 3.23 (t, J = 6.9 Hz, 2H), 2.69 (dd, J = 17.7, 4.1 Hz,
1H), 2.26 (dd, J = 19.5, 11.8 Hz, 1H), 1.95 (s, 3H), 1.66–1.52 (m,
4H), 1.51–1.43 (m, 4H), 1.39 (s, 9H), 1.31–1.27 (m, 16H), 0.88–
0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 170.85, 165.98,
156.34, 137.69, 129.29, 82.20, 79.54, 77.39, 77.07, 76.75, 75.94,
65.07, 54.50, 51.47, 49.21, 30.94, 29.51, 29.48, 29.44, 29.24,
29.13, 28.82, 28.58, 28.32, 26.69, 26.14, 25.95, 25.73, 23.30,
9.54, 9.21.
4.2. Chemistry
4.2.1. General procedure for the linker attachment

®
ACCEPTED MANUSCRIPT
4.2.1.5. 2-(2-azidoethoxy)-ethyl (3R, 4R, 5S)-4-acetamido-5-
((tert-butoxycarbonyl) amino)-3-(1-ethylpropoxy)-1-cyclohexene-
1-carboxylate (6)
CupriSorb . After the purification step, TFA (5 ml) was added to
a solution of click reaction product in CH2Cl2 (5 ml). The
o
reaction mixture was stirred at 0 C for 10 mins and another 1 h
According to the general procedure, 6 was synthesized in
89% yield. H NMR (400 MHz, CDCl3) δ 6.81 (s, 1H), 5.95 (br,
at room temperature, and then the solvent was evaporated. The
resulting syrup was dissolved in distilled water and purified by
Sephadex® LH-20 then lyophilisation to afford divalent
Oseltamivir derivatives as foam and stored at 4 °C until use. No
[M+Cu] ion peak of the final product in the mass spectrum was
detected, and the potential content of Cu was not analyzed.
1
1H), 5.18 (br, 1H), 4.31 (br, 2H), 4.09–3.94 (m, 2H), 3.79–3.65
(m, 6H), 3.40–3.33 (m, 3H), 2.73 (d, J = 17.6 Hz, 1H), 2.30 (dd,
J = 17.6, 9.6 Hz, 1H), 1.96 (s, 3H), 1.49–1.41 (m, 14H), 0.87 (q,
J = 7.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 170.75, 165.83,
156.27, 138.18, 128.92, 82.13, 79.60, 75.77, 70.07, 69.02, 63.87,
54.22, 50.60, 48.89, 30.79, 28.29, 26.04, 25.62, 23.31, 9.47, 9.14.
HRMS (ESI): m/z calcd for C23H39N5O7Na [M+Na]+: 520.2742,
found: 520.2726.
4.2.2.1. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)ethyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (10)
According to the general procedure, 10 was synthesized in
1
4.2.1.6. 2-(2-(2-azidoethoxy)ethoxy)ethyl (3R, 4R, 5S)-4-
acetamido-5-((tert-butoxycarbonyl) amino)-3-(1-ethylpropoxy)-
1-cyclohexene-1-carboxylate (7)
61% yield. H NMR (400 MHz, D2O): δ 7.98 (s, 2H), 6.59 (s,
2H), 4.54–4.51 (m, 5H), 4.17 (d, J = 8.8 Hz, 2H), 3.93–3.88 (m,
2H), 3.59 (s, 4H), 3.48–3.36 (m, 4H), 2.76 (dd, J = 17.2, 5.5 Hz,
2H), 2.37–2.29 (m, 2H), 1.97 (s, 6H), 1.45–1.29 (m, 8H), 0.71 (t,
J = 7.3 Hz, 12H). 13C NMR (100 MHz, D2O): δ 175.26, 166.13,
144.22, 138.77, 126.75, 125.23, 84.16, 75.01, 68.84, 63.28,
63.02, 52.55, 49.26, 49.03, 28.05, 25.47, 25.16, 22.37, 8.65, 8.58,
8.47, 8.41. HRMS (ESI): m/z calcd for C40H66N10O10 [M+2H]2+:
423.2476, found: 423.2467.
According to the general procedure, 7 was synthesized in
86% yield. 1H NMR (400 MHz, CDCl3) δ 6.82 (s, 1H), 5.87 (d, J
= 9.1 Hz, 1H), 5.16 (d, J = 9.1 Hz, 1H), 4.30 (br, 2H), 4.07 (dd, J
= 18.2, 9.1 Hz, 1H), 3.96–3.94 (m, 1H), 3.83–3.73 (m, 3H),
3.68– 3.65 (m, 6H), 3.45–3.26 (m, 3H), 2.74 (dd, J = 17.7, 4.4
Hz, 1H), 2.31 (dd, J = 17.3, 9.5 Hz, 1H), 1.98 (s, 3H), 1.52–1.41
(m, 13H), 0.89 (q, J = 7.6 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ 170.82, 165.89, 156.33, 138.15, 129.04, 82.19, 79.70, 75.89,
70.71, 70.70, 70.14, 69.16, 64.11, 54.34, 50.68, 48.91, 30.91,
28.33, 26.09, 25.69, 23.36, 9.53, 9.19. HRMS (ESI): m/z calcd
for C25H43N5O8Na [M+Na]+: 564.3004, found: 564.2997.
4.2.2.2. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)propyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (11)
According to the general procedure, 11 was synthesized in
1
51% yield. H NMR (400 MHz, D2O): δ 7.97 (s, 2H), 6.64 (s,
2H), 4.56 (s, 2H), 4.48 (t, J = 6.52 Hz, 3H), 4.21–4.12 (m, 6H),
3.93 (dd, J = 9.2, 11.9 Hz, 2H), 3.62 (s, 4H), 3.51–3.41 (m, 4H),
2.79 (dd, J = 17.3, 5.5 Hz, 2H), 2.39–2.32 (m, 2H), 2.28–2.22 (m,
4H), 1.98 (s, 6H), 1.48–1.34 (m, 8H), 0.80–0.72 (m, 12H). 13C
NMR (100 MHz, D2O): δ 175.30, 166.71, 162.99, 162.63,
138.37, 127.08, 117.79, 114.89, 84.13, 75.01, 69.02, 62.99,
62.85, 52.61, 49.08, 47.95, 28.19, 28.07, 25.51, 25.18, 22.40,
8.65, 8.48. HRMS (ESI) m/z calcd for C42H70N10O10 [M+2H]2+
437.2633, found 437.2624.
4.2.1.7. 14-azido-3,6,9,12-tetraoxatetradecyl (3R, 4R, 5S)-4-
acetamido-5-((tert-butoxycarbonyl) amino)-3-(1-ethylpropoxy)-
1-cyclohexene-1-carboxylate (8)
According to the general procedure, 8 was synthesized in
87% yield. 1H NMR (400 MHz, CDCl3) δ 6.81 (s, 1H), 5.80 (d, J
= 9.1 Hz, 1H), 5.14 (d, J = 9.1 Hz, 1H), 4.29 (t, J = 4.6 Hz, 2H),
4.06 (dd, J = 18.3, 9.3 Hz, 1H), 3.95 (br, 1H), 3.83–3.75 (m, 1H),
3.72 (t, J = 4.68 Hz, 2H), 3.68–3.64 (m, 14H), 3.39–3.33 (m,
3H), 2.73 (dd, J = 17.7, 4.9 Hz, 1H), 2.30 (dd, J = 17.7, 9.8 Hz,
1H), 1.97 (s, 3H), 1.55–1.44 (m, 4H), 1.41 (s, 9H), 0.88 (q, J =
7.4 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 170.78, 165.90,
156.32, 138.12, 129.06, 82.17, 79.69, 77.24, 75.90, 70.70, 70.67,
70.63, 70.60, 70.03, 69.06, 64.13, 54.32, 50.70, 48.86, 30.89,
28.33, 26.09, 25.68, 23.38, 9.53, 9.19.
4.2.2.3. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)hexyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (12)
According to the general procedure, 12 was synthesized in
1
55% yield. H NMR (400 MHz, D2O): δ 7.93 (s, 2H), 6.75 (s,
2H), 4.56 (s, 2H), 4.36–4.24 (m, 6H), 4.08–3.94 (m, 6H), 3.62–
3.44 (m, 10H), 2.87 (d, J = 16.9 Hz, 2H), 2.43 (br, 2H), 2.02 (s,
6H), 1.82 (br, 4H), 1.57–1.21 (m, 22H), 0.77 (s, 12H). 13C NMR
(100 MHz, D2O): δ 175.27, 167.24, 138.07, 127.49, 84.08, 75.13,
68.69, 65.87, 63.01, 52.77, 50.32, 49.14, 29.25, 25.50, 25.21,
25.10, 24.64, 22.37, 8.66, 8.46. HRMS (ESI) m/z calcd for
C48H82N10O10 [M+2H]2+ 479.3102, found 479.3098.
4.2.1.8. 17-azido-3,6,9,12,15-pentaoxaheptadecyl (3R, 4R, 5S)-4-
acetamido-5-((tert-butoxycarbonyl) amino)-3-(1-ethylpropoxy)-
1-cyclohexene-1-carboxylate (9)
According to the general procedure, 9 was synthesized in
85% yield. 1H NMR (400 MHz, CDCl3) δ 6.80 (s, 1H), 5.87 (d, J
= 8.9 Hz, 1H), 5.18 (d, J = 9.1 Hz, 1H), 4.28 (t, J = 4.64 Hz, 2H),
4.05 (dd, J = 18.2, 9.2 Hz, 1H), 3.95 (br, 1H), 3.82–3.74 (m, 1H),
3.71 (t, J = 4.64 Hz, 2H), 3.67–3.63 (m, 18H), 3.38–3.33 (m,
3H), 2.72 (dd, J = 17.8, 4.7 Hz, 1H), 2.29 (dd, J = 17.8, 9.5 Hz,
1H), 1.96 (s, 3H), 1.53–1.44 (m, 4H), 1.40 (s, 9H), 0.87 (q, J =
7.3 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 170.76, 165.89,
156.29, 138.09, 129.05, 82.16, 79.63, 77.25, 75.84, 70.69, 70.66,
70.63, 70.62, 70.60, 70.58, 70.56, 70.02, 69.05, 64.12, 54.28,
50.69, 48.88, 30.83, 28.33, 26.08, 25.69, 23.36, 9.52, 9.20.
4.2.2.4. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy) dodecyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (13)
According to the general procedure, 13 was synthesized in
1
52% yield. H NMR (400 MHz, CDCl3): δ 7.56 (s, 2H), 6.77 (s,
2H), 6.34 (br, 2H), 4.67 (s, 4H), 4.32 (t, J = 7.2 Hz, 4H), 4.27–
4.04 (m, 5H), 3.70–3.64 (m, 5H), 3.35–3.32 (m, 3H), 2.79 (br,
2H), 2.21 (br, 2H), 2.03 (s, 6H), 1.90–1.86 (m, 4H), 1.68–1.59
(m, 4H), 1.54–1.44 (m, 8H), 1.30–1.25 (m, 36H), 0.91–0.85 (m,
16H). 13C NMR (100 MHz, CDCl3): δ 171.44, 166.30, 144.92,
137.93, 129.21, 122.48, 81.93, 69.69, 65.02, 64.68, 50.38, 30.27,
29.70, 29.40, 29.37, 29.30, 29.12, 28.94, 28.56, 26.48, 26.23,
25.90, 25.74, 9.56, 9.33. HRMS (ESI) m/z calcd for
C60H106N10O10 [M+2H]2+ 563.4041, found 563.4038
4.2.2. General procedure for the preparation of divalent
Oseltamivir derivatives
To a stirring solution of azide (2.5 eq.) and 1, 2-bis (prop-2-
yn-1-yloxy) ethane (1eq) in THF/ water (1:1), CuSO4 (cat.) was
added along with sodium L-ascorbate (cat.). The reaction was
stirred at r.t. for 12 h. Solvent was removed in vacuum and the
residue was purified using flash column chromatography. The
copper complex in 13 and 17 were further absorbed by

ACCEPTED MANUSCRIPT
4.2.2.5. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
The reaction was stirred at r.t. for 12 h. The reaction mixture was
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)
-2-(2-ethoxy)-
washed with HCl (1M, 25 mL), extracted with CH2Cl2 (3×10 mL)
and the product was purified by column chromatography to give
the Boc protected divalent guanidino Oseltamivir derivatives,
which were further dissolved in CH2Cl2. CH2Cl2/TFA (1:1)
was added dropwise under 0 ℃, the reaction mixture was
stirred at room temperature for 3 h. The solvent was
evaporated to dryness and the crude products were purified by
SephendexTM LH-20. No [M+Cu] or [M+Hg] ion peak of the
final product in the mass spectrum was detected, and the
potential content of Cu or Hg was not analyzed.
ethyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane (14)
According to the general procedure, 14 was synthesized in
55% yield. H NMR (400 MHz, D2O): δ 7.93 (s, 2H), 6.69 (s,
2H), 4.69–4.36 (m, 4H), 4.21–4.12 (m, 6H), 3.94–3.88 (m, 7H),
3.65– 3.40 (m, 14H), 2.80 (dd, J = 17.1, 4.8 Hz, 2H), 2.39 (d, J =
10.8 Hz, 2H), 1.98 (s, 6H), 1.46–1.34 (m, 9H), 0.78–0.70 (m,
12H). 13C NMR(100 MHz, D2O): δ 175.22, 166.62, 138.45,
126.98, 125.25, 84.10, 74.96, 68.78, 68.28, 64.44, 63.04, 52.50,
50.05, 49.04, 28.05, 25.41, 25.05, 22.31, 8.55, 8.39. HRMS (ESI)
m/z calcd for C44H74N10O12 [M+2H]2+ 467.2738, found 467.2740.
1
4.2.3.1. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- guanidino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)ethyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (18)
4.2.2.6. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)
2-(2-(2-
ethoxy)ethoxy)ethyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane, (15)
According to the general procedure, 15 was synthesized in
According to the general procedure, 18 was synthesized in
1
52% yield. H NMR (400 MHz, D2O): δ 7.98 (s, 2H), 6.57 (s,
1
50% yield. H NMR (400 MHz, D2O): δ 7.96 (s, 2H), 6.75 (s,
2H), 4.53–4.49 (m, 5H), 4.17 (d, J = 8.6 Hz, 2H), 3.79–3.60 (m,
5H), 3.57 (s, 4H), 3.39–3.34 (m, 2H), 2.63 (dd, J = 17.5, 4.9 Hz,
2H), 2.26–2.16 (m, 2H), 1.92 (s, 6H), 1.45–1.25 (m, 9H), 0.71 (q,
J = 7.1 Hz, 12H). 13C NMR (100 MHz, D2O) δ 174.71, 166.41,
156.75, 138.61, 127.87, 125.24, 84.14, 75.29, 68.71, 63.12,
62.92, 54.74, 50.44, 49.23, 46.63, 29.73, 25.56, 25.23, 21.93,
8.59, 8.50, 8.18. HRMS (ESI) m/z calcd for C42H70N14O10
[M+2H]2+: 465.2694, found: 465.2699.
2H), 4.64–4.44 (m, 6H), 4.28–4.10 (m, 6H), 3.94 (dd, J = 11.7,
9.0 Hz, 2H), 3.88–3.80 (m, 4H), 3.61–3.39 (m, 22H), 2.84 (dd, J
= 17.2, 5.4 Hz, 2H), 2.41 (dd, J = 10.3, 6.9 Hz, 2H), 1.97 (s, 6H),
1.45–1.32 (m, 9H), 0.73 (q, J = 7.3 Hz, 12H). 13C NMR (100
MHz, D2O): δ 175.27, 166.86, 143.94, 138.49, 127.19, 125.42,
84.16, 75.05, 69.75, 69.66, 68.91, 68.75, 68.47, 64.67, 63.11,
52.65, 50.10, 49.11, 28.19, 25.47, 25.09, 22.38, 8.60, 8.45.
HRMS (ESI) m/e calcd for C48H82N10O14 [M+2H]2+ 511.3001,
found 511.2987.
4.2.3.2. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)propyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (19)
4.2.2.7. 1, 2-Bis-[[3-(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)-3,6,9,12-
tetraoxatetradecyl]-1H-1,2,3-triazol-4-yl-methoxyl ]ethane (16)
According to the general procedure, 16 was synthesized in
According to the general procedure, 19 was synthesized in
51% yield. 1H NMR (400 MHz, D2O) δ 7.94 (s, 2H), 6.59 (s, 2H),
4.48 (s, 2H), 4.46 (t, J = 6.3 Hz, 3H), 4.19 (br, 2H), 4.10 (t, J =
5.6 Hz, 4H), 3.79–3.65 (m, 5H), 3.61 (s, 4H), 3.41–3.38 (m, 2H),
2.60 (dd, J = 17.4, 4.9 Hz, 2H), 2.26–2.15 (m, 6H), 1.93 (s, 6H),
1.47–1.30 (m, 8H), 0.78–0.71 (m, 12H). 13C NMR (100 MHz,
D2O) δ 174.78, 167.12, 156.83, 138.23, 128.30, 84.12, 75.31,
68.96, 63.11, 62.09, 54.90, 50.53, 47.96, 29.79, 28.18, 25.66,
25.31, 22.02, 8.65, 8.56. HRMS (ESI) m/z calcd for
C44H74N14O10 [M+2H]2+: 479.2851, found: 479.2861.
1
54% yield. H NMR (400 MHz, D2O): δ 7.97 (s, 1H), 6.78 (s,
1H), 4.58–4.50 (m, 5H), 4.25–4.21 (m, 6H), 3.94 (dd, J = 11.6,
9.1 Hz, 2H), 3.85 (t, J = 4.96 Hz, 4H), 3.72–3.70 (m, 5H), 3.61–
3.41 (m, 35H), 2.86 (dd, J = 17.2, 5.4 Hz, 2H), 2.45–2.38 (m,
2H), 1.98 (s, 6H), 1.48–1.31 (m, 8H), 0.78–0.71 (m, 12H). 13
C
NMR (100 MHz, D2O): δ 175.20, 166.87, 143.82, 138.43,
127.16, 125.44, 84.14, 75.03, 69.74, 69.61, 69.56, 69.54, 69.43,
68.84, 68.70, 68.47, 64.60, 63.03, 52.63, 49.99, 49.05, 28.19,
25.42, 25.03, 22.32, 8.57, 8.40. HRMS (ESI) m/z calcd for
C56H98N10O18 [M+2H]2+ 599.3525, found 599.3520.
4.2.3.3. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)hexyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (20)
4.2.2.8. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5- amino-3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)-3,6,9,12,15-
pentaoxaheptadecyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane (17)
According to the general procedure, 17 was synthesized in
According to the general procedure, 20 was synthesized in
1
54% yield. H NMR (400 MHz, D2O) δ 6.68 (s, 3H), 4.47 (br,
2H), 4.34 (br, 4H), 4.22 (d, J = 8.0 Hz, 2H), 4.06– 3.99 (m, 4H),
3.82–3.69 (m, 9H), 3.42–3.38 (m, 2H), 2.73 (dd, J = 17.5, 4.4 Hz,
2H), 2.31–2.23 (m, 2H), 1.93 (s, 6H), 1.80 (br, 4H), 1.52–1.14
(m, 21H), 0.73 (q, J = 7.3 Hz, 12H). 13C NMR (100 MHz, D2O):
δ 174.81, 167.61, 156.89, 138.03, 128.66, 84.05, 75.43, 65.83,
54.93, 50.64, 29.98, 29.14, 27.50, 25.70, 25.39, 25.17, 24.68,
22.05, 8.72, 8.58. HRMS (ESI) m/z calcd for C50H86N14O10
[M+2H]2+: 521.3320, found: 521.3338.
1
50% yield. H NMR (400 MHz, D2O): δ 7.97 (s, 2H), 6.79 (s,
2H), 4.58–4.51 (m, 5H), 4.27–4.22 (m, 6H), 3.96 (dd, J = 11.6,
9.0 Hz, 2H), 3.92–3.79 (m, 6H), 3.78–3.66 (m, 6H), 3.61–3.42
(m, 52H), 2.87 (dd, J = 17.1, 5.1 Hz, 2H), 2.44 (dd, J = 10.1, 7.0
Hz, 2H), 1.98 (s, 6H), 1.48–1.31 (m, 8H), 0.84–0.64 (m, 12H).
13C NMR(100 MHz, D2O): δ 175.26, 166.92, 143.93, 138.49,
127.17, 125.48, 84.17, 75.06, 69.79, 69.68, 69.61, 69.50, 68.90,
68.76, 68.53, 64.67, 63.10, 52.59, 50.05, 49.12, 28.13, 25.49,
25.11, 22.38, 8.63, 8.46. HRMS (ESI) m/z calcd for
C60H106N10O20 [M+2H]2+ 643.3787, found 643.3774.
4.2.3.4. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy) dodecyl]-1H-1,2,3-
triazol-4-yl-methoxyl]ethane (21)
According to the general procedure, 21 was synthesized in
52% yield. H NMR (400 MHz, MeOD) δ 7.97 (s, 1H), 6.82 (s,
4.2.3. General procedure for the preparation of divalent
guanidino oseltamivir derivatives
1
2H), 4.62 (s, 4H), 4.39 (t, J = 7.1 Hz, 4H), 4.28 (br, 2H), 4.16 (q,
J = 6.3 Hz, 4H), 3.91 (br, 4H), 3.46–3.40 (m, 2H), 3.31–3.30 (m,
3H), 2.90–2.78 (m, 2H), 2.40–2.29 (m, 2H), 1.99 (s, 6H), 1.92–
1.85 (m, 4H), 1.70–1.64 (m, 4H), 1.54–1.49 (m, 8H), 1.28 (br,
34H), 0.94–0.87 (m, 12H). 13C NMR (100 MHz, MeOD) δ
172.75, 165.83, 157.17, 144.51, 137.57, 128.15, 123.61, 82.38,
74.69, 69.33, 64.85, 63.62, 54.46, 50.31, 49.97, 29.96, 29.87,
29.21, 29.17, 29.09, 28.64, 28.30, 26.05, 25.88, 25.70, 25.52,
Divalent Oseltamivir analogs were dissolved in CH2Cl2.
TFA/CH2Cl2 (1:1) was added dropwise under 0 ℃, the reaction
mixture was stirred at room temperature for 3 h. The solvent was
evaporated to dryness and the crude products were dissolved in
CH2Cl2 (5 ml), Et3N (1 mL) was added and the solution was
stirred at rt for 30 mins. HgCl2 (0.6 eq.) and 1, 3-Bis (tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea (3 eq.) was added.

ACCEPTED MANUSCRIPT
21.50, 8.51, 8.31. HRMS (ESI) m/z calcd for C62H110N14O10
conducted in 384-well plates containing 15 μL diluted virus, 15
μL compounds in the buffer. The mixture was incubated for 30
[M+2H]2+: 605.4259, found: 605.4239.
o
min at 37 C, and then added with 30 μL 4-MUNANA substrate
4.2.3.5. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
per well in the buffer. The enzymatic reactions were carried out
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)
-2-(2-ethoxy)-
o
for 2 hours at 37 C. The fluorescent signal was monitored using
ethyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane (22)
According to the general procedure, 22 was synthesized in
51% yield. H NMR (400 MHz, D2O) δ 8.13 (br, 1H), 6.66 (s,
the kinetics function of SynergyTM H1/H1MF microplate reader
with excitation and emission wavelengths of 360 and 440 nm,
respectively. The buffer instead of the inhibitor was used as
control and the concentration of the virus was determined as the
hydrolysis reaction was completed in 30 min and the final
fluorescent unit (FU) is stable at 10000. The IC50 was calculated
as the concentration of inhibitor resulting in a 50% reduction in
FU compared to the control.
1
2H), 4.52 (br, 4H), 4.22 (d, J = 8.4 Hz, 2H), 4.12 (br, 4H), 3.98–
3.53 (m, 17H), 3.44–3.38 (m, 2H), 2.66 (q, J = 4.6 Hz, 2H), 2.65
(dd, J = 4.8, 17.6 Hz, 2H), 2.27–2.20 (m, 2H), 1.93 (s, 6H), 1.48–
1.30 (m, 8H), 1.17 (t, J = 7.3 Hz, 2H), 0.74 (q, J = 7.5 Hz, 12H).
13C NMR (100 MHz, D2O) δ 174.72, 167.01, 156.79, 138.33,
128.20, 84.11, 75.28, 68.71, 68.28, 64.36, 54.83, 50.50, 46.63,
29.78, 25.58, 25.21, 21.94, 8.56, 8.50, 8.18. HRMS (ESI) m/z
calcd for C46H78N14O12 [M+2H]2+: 509.2956, found: 509.2955.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (81773583). The authors are
thankful to the Research Centre of Modern Analytical
Technology, Tianjin University of Science and Technology for
NMR measurements and HRMS analysis. The authors also
appreciate National Institute for Viral Disease Control and
Prevention, China CDC for the viral strains.
4.2.3.6. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)
2-(2-(2-
ethoxy)ethoxy)ethyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane (23)
According to the general procedure, 23 was synthesized in
1
50% yield. H NMR (D2O, 400 MHz) δ 6.71 (br, 3H), 4.77 (br,
4H), 4.52 (br, 4H), 4.23–4.11 (m, 8H), 3.86–3.53 (m, 20H),
3.43–3.48 (m, 2H), 2.73 (d, J = 12.9 Hz, 2H), 2.30 (br, 2H), 1.92
(s, 6H), 1.52–1.29 (m, 13H), 0.83–0.64 (m, 12H). 13C NMR
(D2O, 100 MHz) δ 174.78, 167.25, 156.84, 138.38, 128.38,
84.16, 75.36, 69.77, 69.66, 68.96, 68.75, 68.47, 64.59, 63.13,
54.87, 50.58, 50.14, 29.92, 25.64, 25.27, 22.02, 8.63, 8.55.
HRMS (ESI) m/z calcd for C50H86N14O14 [M+2H]2+: 553.3218,
found: 553.3210.
Supplementary Material
Supplementary data (1H and 13C spectra) related to this
article can be found at http://
References and notes
4.2.3.7. 1, 2-Bis-[[3-(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-
(1-ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)-3,6,9,12-
tetraoxatetradecyl]-1H-1,2,3-triazol-4-yl-methoxyl ]ethane (24)
According to the general procedure, 24 was synthesized in
1. Schwerdtfeger, S. M.; Melzig, M. F., Sialidases in biological
systems. Die Pharmazie 2010, 65 (8), 551–561.
2. Taylor, G., Sialidases: structures, biological significance and
therapeutic potential. Curr. Opin. Struct. Biol. 1996, 6 (6), 830–
837.
1
48% yield. H NMR (D2O, 400 MHz) δ 8.02 (br, 2H), 6.75 (s,
2H), 4.55–4.52 (m, 4H), 4.24 (br, 6H), 3.86–3.42 (m, 44H), 2.77
(d, J = 17.3 Hz, 2H), 2.35–2.28 (m, 2H), 1.96 (s, 6H), 1.52–1.27
(m, 9H), 0.78–0.71 (m, 12H). 13C NMR (D2O, 100 MHz) δ
174.77, 167.33, 156.84, 138.37, 128.41, 84.16, 75.38, 69.82,
69.70, 69.61, 69.52, 68.95, 68.75, 68.53, 64.62, 54.88, 50.58,
50.12, 29.91, 25.65, 25.28, 22.02, 8.65, 8.57. HRMS (ESI) m/z
calcd for C58H102N14O18 [M+2H]2+: 641.3743, found: 641.3755.
3. Christopher, W., Inhibitors of the human neuraminidase
enzymes. Med. Chem. Commun. 2014, 5 (8), 1067–1074.
4. Buschiazzo, A.; Alzari, P. M., Structural insights into sialic acid
enzymology. Curr. Opin. Chem. Biol. 2008, 12 (5), 565–572.
5. Asano, N., Glycosidase inhibitors: update and perspectives on
practical use. Glycobiology 2003, 13 (10), 93R–104R.
6. McKimm-Breschkin, J.; Trivedi, T.; Hampson, A.; Hay, A.;
Klimov, A.; Tashiro, M.; Hayden, F.; Zambon, M.,
Neuraminidase sequence analysis and susceptibilities of
influenza virus clinical isolates to zanamivir and oseltamivir.
Antimicrob. Agents Chemother. 2003, 47 (7), 2264–2272.
7. Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Li, W.; Sidwell, R. W.;
Huffman, J. H.; Sweet, C.; Jakeman, K. J.; Merson, J.; Lacy, S.
A., Oral administration of a prodrug of the influenza virus
neuraminidase inhibitor GS 4071 protects mice and ferrets
against influenza infection. Antimicrob. Agents Chemother. 1998,
42 (3), 640–646.
8. De Clercq, E., Strategies in the design of antiviral drugs. Nat.
Rev. Drug Discov. 2002, 1 (1), 13–25.
9. Samson, M.; Pizzorno, A.; Abed, Y.; Boivin, G., Influenza virus
resistance to neuraminidase inhibitors. Antiviral Res. 2013, 98
(2), 174–185.
10. Ives, J. A.; Carr, J. A.; Mendel, D. B.; Tai, C. Y.; Lambkin, R.;
Kelly, L.; Oxford, J. S.; Hayden, F. G.; Roberts, N. A., The
H274Y mutation in the influenza A/H1N1 neuraminidase active
site following oseltamivir phosphate treatment leave virus
severely compromised both in vitro and in vivo. Antiviral Res.
2002, 55 (2), 307–317.
11. Mohan, S.; McAtamney, S.; Haselhorst, T.; von Itzstein, M.;
Pinto, B. M., Carbocycles related to oseltamivir as influenza
virus group-1-specific neuraminidase inhibitors. Binding to N1
enzymes in the context of virus-like particles. J. Med. Chem.
2010, 53 (20), 7377–7391.
4.2.3.8. 1, 2-Bis-[[(((3R, 4R, 5S)-4-acetamido-5-guanidino -3-(1-
ethylpropoxy)-1-cyclohexene-1-carboxyl)oxy)-3,6,9,12,15-
pentaoxaheptadecyl]-1H-1,2,3-triazol-4-yl-methoxyl]ethane (25)
According to the general procedure, 25 was synthesized in
50% yield. 1H NMR (D2O, 400 MHz): δ 7.97 (s, 1H), 6.74 (s, 2H),
4.58–4.50 (m, 5H), 4.23 (br, 7H), 3.86–3.70 (m, 16H), 3.60–3.05
(m, 54H), 3.72 (q, J = 7.3 Hz, 6H), 2.76 (dd, J = 17.4, 4.3 Hz,
2H), 2.39–2.24 (m, 2H), 1.92 (s, 6H), 1.46–1.29 (m, 9H), 1.16 (t,
J = 7.3 Hz, 9H), 0.78–0.70 (m, 12H); 13C NMR (D2O ,100 MHz):
δ 174.69, 167.23, 163.11, 162.76, 156.76, 138.34, 128.34, 125.46,
117.80, 114.90, 84.12, 75.33, 69.74, 69.63, 69.57, 69.53, 69.46,
68.86, 68.71, 68.46, 64.54, 63.05, 54.82, 50.52, 50.00, 46.63,
29.87, 25.60, 25.22, 21.96, 8.61, 8.19. HRMS (ESI) m/z calcd for
C62H110N14O20 [M+2H]2+: 685.4005, found: 685.4022.
4.3. Neuraminidase Inhibition Assay
The virus used as the source of NAs is inactivated by the
addition of β-propiolactone (β-PL). Enzyme inhibition assays
were measured using 4-methylumbelliferyl -α-D-N –acetylneura-
minic acid sodium salt (4-MUNANA) in the enzyme buffer (100
mM sodium acetate pH 5.5 and 10 mM CaCl2) as the substrate.
All compounds were dissolved in buffer and diluted to the
corresponding concentrations. The enzyme inhibition assay was

12. Albohy, A.; Mohan, S.; Zheng, R. B.; Pinto, BA. MC.;CCEairPo,TCE. WD., MANdUosiSngCrRegIimPeTn. Antimicrob. Agents Chemother. 2004, 48 (12),
Inhibitor selectivity of a new class of oseltamivir analogs against
viral neuraminidase over human neuraminidase enzymes. Bioorg.
Med. Chem. 2011, 19 (9), 2817–2822.
4542–4549.
27. Wen, W. H.; Lin, M.; Su, C. Y.; Wang, S. Y.; Cheng, Y. S.;
Fang, J. M.; Wong, C. H., Synergistic effect of zanamivir-
porphyrin conjugates on inhibition of neuraminidase and
inactivation of influenza virus. J. Med. Chem. 2009, 52 (15),
4903–4910.
28. Zhao, T. F.; Qin, H. J.; Yu, Y.; Yang, M. B.; Chang, H.; Guo, N.;
He, Y.; Yang, Y.; Yu, P., Multivalent zanamivir-bovine serum
albumin conjugate as a potent influenza neuraminidase inhibitor.
J. Carbohyd. Chem. 2017, 1–12.
29. Yang, Z.-L.; Zeng, X.-F.; Liu, H.-P.; Yu, Q.; Meng, X.; Yan, Z.-
L.; Fan, Z.-C.; Xiao, H.-X.; Iyer, S. S.; Yang, Y.; Yu, P.,
Synthesis of multivalent difluorinated zanamivir analogs as
potent antiviral inhibitors. Tetrahedron Lett. 2016, 57 (24),
2579–2582.
30. Das, K.; Aramini, J. M.; Ma, L. C.; Krug, R. M.; Arnold, E.,
Structures of influenza A proteins and insights into antiviral drug
targets. Nat. Struct. Mol. Biol. 2010, 17 (5), 530–538.
31. Taylor, N. R.; Cleasby A Fau - Singh, O.; Singh O Fau -
Skarzynski, T.; Skarzynski T Fau - Wonacott, A. J.; Wonacott Aj
Fau - Smith, P. W.; Smith Pw Fau - Sollis, S. L.; Sollis Sl Fau -
Howes, P. D.; Howes Pd Fau - Cherry, P. C.; Cherry Pc Fau -
Bethell, R.; Bethell R Fau - Colman, P.; Colman P Fau -
Varghese, J.; Varghese, J., Dihydropyrancarboxamides related to
zanamivir: a new series of inhibitors of influenza virus sialidases.
2. Crystallographic and molecular modeling study of complexes
of 4-amino-4H-pyran-6-carboxamides and sialidase from
influenza virus types A and B. J. Med. Chem. 1998, 41 (6), 798–
807.
32. Kanfar, N.; Bartolami, E.; Zelli, R.; Marra, A.; Winum, J.-Y.;
Ulrich, S.; Dumy, P., Emerging trends in enzyme inhibition by
multivalent nanoconstructs. Org. Biomol. Chem. 2015, 13 (39),
9894-9906.
33. Gouin, S. G., Multivalent Inhibitors for Carbohydrate-Processing
Enzymes: Beyond the “Lock-and-Key” Concept. Chem. Eur. J.
2014, 20 (37), 11616-11628.
13. Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.;
Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendel, D. B.;
Tai, C. Y.; Laver, W. G.; Stevens, R. C., Influenza
Neuraminidase Inhibitors Possessing a Novel Hydrophobic
Interaction in the Enzyme Active Site:ꢀ Design, Synthesis, and
Structural Analysis of Carbocyclic Sialic Acid Analogues with
Potent Anti-Influenza Activity. J. Am. Chem. Soc. 1997, 119 (4),
681–690.
14. Gupta, D.; Varghese Gupta, S.; Dahan, A.; Tsume, Y.; Hilfinger,
J.; Lee, K. D.; Amidon, G. L., Increasing oral absorption of polar
neuraminidase inhibitors: a prodrug transporter approach applied
to oseltamivir analogue. Mol. Pharm. 2013, 10 (2), 512–522.
15. Hong, B. T.; Chen, C. L.; Fang, J. M.; Tsai, K. C.; Wang, S. Y.;
Huang, W. I.; Cheng, Y. S.; Wong, C. H., Oseltamivir
hydroxamate and acyl sulfonamide derivatives as influenza
neuraminidase inhibitors. Bioorg. Med. Chem. 2014, 22 (23),
6647–6654.
16. Cecioni, S.; Imberty, A.; Vidal, S., Glycomimetics versus
Multivalent Glycoconjugates for the Design of High Affinity
Lectin Ligands. Chem. Rev. 2015, 115 (1), 525–561.
17. Lundquist, J. J.; Toone, E. J., The Cluster Glycoside Effect.
Chem. Rev. 2002, 102 (2), 555–578.
18. Brissonnet, Y.; Assailly, C.; Saumonneau, A.; Bouckaert, J.;
Maillasson, M.; Petitot, C.; Roubinet, B.; Didak, B.; Landemarre,
L.; Bridot, C.; Blossey, R.; Deniaud, D.; Yan, X.; Bernard, J.;
Tellier, C.; Grandjean, C.; Daligault, F.; Gouin, S. G.,
Multivalent Thiosialosides and Their Synergistic Interaction with
Pathogenic Sialidases. Chem. Eur. J. 2019, 25 (9), 2358-2365.
19. Fu, L.; Bi, Y.; Wu, Y.; Zhang, S.; Qi, J.; Li, Y.; Lu, X.; Zhang,
Z.; Lv, X.; Yan, J.; Gao, G. F.; Li, X., Structure-Based
Tetravalent Zanamivir with Potent Inhibitory Activity against
Drug-Resistant Influenza Viruses. J. Med. Chem. 2016, 59 (13),
6303–6312.
20. Haldar, J.; Alvarez de Cienfuegos, L.; Tumpey, T. M.; Gubareva,
L. V.; Chen, J.; Klibanov, A. M., Bifunctional polymeric
inhibitors of human influenza A viruses. Pharm. Res. 2010, 27
(2), 259–263.
21. Honda, T.; Yoshida, S.; Arai, M.; Masuda, T.; Yamashita, M.,
Synthesis and anti-influenza evaluation of polyvalent sialidase
inhibitors bearing 4-guanidino-Neu5Ac2en derivatives. Bioorg.
Med. Chem. Lett. 2002, 12 (15), 1929–1932.
22. Lee, C. M.; Weight, A. K.; Haldar, J.; Wang, L.; Klibanov, A.
M.; Chen, J., Polymer-attached zanamivir inhibits synergistically
both early and late stages of influenza virus infection. Proc. Natl.
Acad. Sci. USA 2012, 109 (50), 20385–20390.
23. Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M.;
Honda, T., Synthesis and anti-influenza evaluation of polyvalent
sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives.
Chem. Pharm. Bull. 2003, 51 (12), 1386–1398.
24. Weight, A. K.; Belser, J. A.; Tumpey, T. M.; Chen, J.; Klibanov,
A. M., Zanamivir conjugated to poly-L-glutamine is much more
active against influenza viruses in mice and ferrets than the drug
itself. Pharm. Res. 2014, 31 (2), 466–474.
25. Weight, A. K.; Haldar, J.; Alvarez de Cienfuegos, L.; Gubareva,
L. V.; Tumpey, T. M.; Chen, J.; Klibanov, A. M., Attaching
zanamivir to a polymer markedly enhances its activity against
drug-resistant strains of influenza a virus. J. Pharm. Sci. 2011,
100 (3), 831–835.
34. Compain, P.; Bodlenner, A., The Multivalent Effect in
Glycosidase Inhibition: A New, Rapidly Emerging Topic in
Glycoscience. ChemBioChem 2014, 15 (9), 1239-1251.
35. Gunasekera, D. S., Formal Synthesis of Tamiflu: Conversion of
Tamiflu into Tamiphosphor. Synlett 2012, 2012 (04), 573–576.
36. Meng, X.; Yang, M.; Li, Y.; Li, X.; Jia, T.; He, H.; Yu, Q.; Guo,
N.; He, Y.; Yu, P.; Yang, Y., Multivalent neuraminidase
hydrolysis resistant triazole-sialoside protein conjugates as
influenza-adsorbents. Chinese Chem. Lett. 2018, 29 (1), 76–80.
37. Yang, Y.; Liu, H. P.; Yu, Q.; Yang, M. B.; Wang, D. M.; Jia, T.
W.; He, H. J.; He, Y.; Xiao, H. X.; Iyer, S. S.; Fan, Z. C.; Meng,
X.; Yu, P., Multivalent S-sialoside protein conjugates block
influenza hemagglutinin and neuraminidase. Carbohydr. Res.
2016, 435, 68–75.
38. Zhang, W. Q.; He, Y.; Yu, Q.; Liu, H. P.; Wang, D. M.; Li, X.
B.; Luo, J.; Meng, X.; Qin, H. J.; Lucchi, N. W.; Udhayakumar,
V.; Iyer, S. S.; Yang, Y.; Yu, P., Polyvalent effect enhances
diglycosidic antiplasmodial activity. Eur. J. Med. Chem. 2016,
121, 640–648.
39. Tiwari, V. K.; Mishra, B. B.; Mishra, K. B.; Mishra, N.; Singh,
A. S.; Chen, X., Cu-Catalyzed Click Reaction in Carbohydrate
Chemistry. Chem. Rev. 2016, 116 (5), 3086-3240.
40. Yang, Y.; He, Y.; Li, X.; Dinh, H.; Iyer, S. S., Bifunctional
thiosialosides inhibit influenza virus. Bioorg. Med. Chem. Lett.
2014, 24 (2), 636–643.
26. Macdonald, S. J.; Watson, K. G.; Cameron, R.; Chalmers, D. K.;
Demaine, D. A.; Fenton, R. J.; Gower, D.; Hamblin, J. N.;
Hamilton, S.; Hart, G. J.; Inglis, G. G.; Jin, B.; Jones, H. T.;
McConnell, D. B.; Mason, A. M.; Nguyen, V.; Owens, I. J.;
Parry, N.; Reece, P. A.; Shanahan, S. E.; Smith, D.; Wu, W. Y.;
Tucker, S. P., Potent and long-acting dimeric inhibitors of
influenza virus neuraminidase are effective at a once-weekly
41. Liu, H.-P.; Meng, X.; Yu, Q.; Tao, Y.-C.; Xu, F.; He, Y.; Yu, P.;
Yang, Y., Synthesis of S-sialyl polymers as efficient polyvalent
influenza inhibitors and capturers. J. Carbohyd. Chem. 2018, 37
(1), 18–29.
42. Taylor, N. R.; von Itzstein, M., Molecular modeling studies on
ligand binding to sialidase from influenza virus and the
mechanism of catalysis. J. Med. Chem. 1994, 37 (5), 616–624.

43. Macdonald, S. J.; Cameron, R.; Demaine, DA. AC.;CFeEntPonT, RE.DJ.; MANdUimSerCs aRs IhPigThly potent inhibitors of influenza A and B. Med.
Foster, G.; Gower, D.; Hamblin, J. N.; Hamilton, S.; Hart, G. J.;
Hill, A. P.; Inglis, G. G.; Jin, B.; Jones, H. T.; McConnell, D. B.;
McKimm-Breschkin, J.; Mills, G.; Nguyen, V.; Owens, I. J.;
Parry, N.; Shanahan, S. E.; Smith, D.; Watson, K. G.; Wu, W. Y.;
Tucker, S. P., Dimeric zanamivir conjugates with various linking
groups are potent, long-lasting inhibitors of influenza
neuraminidase including H5N1 avian influenza. J. Med. Chem.
2005, 48 (8), 2964–2971.
Chem. Commun. 2013, 4 (2), 383–386.
45. Fasting, C.; Schalley, C. A.; Weber, M.; Seitz, O.; Hecht, S.;
Koksch, B.; Dernedde, J.; Graf, C.; Knapp, E. W.; Haag, R.,
Multivalency as a chemical organization and action principle.
Angew. Chem. Int. Ed. 2012, 51 (42), 10472–10498.
44. Fraser, B. H.; Hamilton, S.; Krause-Heuer, A. M.; Wright, P. J.;
Greguric, I.; Tucker, S. P.; Draffan, A. G.; Fokin, V. V.;
Sharpless, K. B., Synthesis of 1,4-triazole linked zanamivir
Click here to remove instruction text...