Discovery of novel 1,2,3-triazole oseltamivir derivatives as potent influenza neuraminidase inhibitors targeting the 430-cavity

Article abstract of DOI:10.1016/j.ejmech.2019.111940

A novel series of 1,2,3-triazole oseltamivir derivatives, which could simultaneously occupy the classical NA catalytic site and the newly reported 430-cavity, were designed, synthesized, and evaluated for their anti-influenza activities. The results demonstrated that four compounds (6g, 6l, 6y and 8c) showed robust anti-influenza potencies against H5N1, H5N2 and H5N6 strains in both enzymatic assay and cellular assay. Especially, 6l was proved to possess the most potent and broad-spectrum anti-influenza activity, with IC₅₀ values of 0.12 μM, 0.049 μM and 0.16 μM and EC₅₀ values of 2.45 μM, 0.43 μM and 2.8 μM against H5N1, H5N2 and H5N6 strains, respectively, which were slightly weaker than oseltamivir carboxylate. In addition, in the embryonated egg model, 6l achieved the similar protective effect against H9N2 strain with oseltamivir carboxylate in the tested concentrations. Preliminary structure-activity relationships (SARs), molecular modeling, and calculated physicochemical properties of selected compounds were also discussed.

Full text of DOI:10.1016/j.ejmech.2019.111940

Journal Pre-proof
Discovery of novel 1,2,3-triazole oseltamivir derivatives as potent influenza
neuraminidase inhibitors targeting the 430-cavity
Han Ju, Siyu Xiu, Xiao Ding, Min Shang, RuiFang Jia, Bing Huang, Peng Zhan,
Xinyong Liu
PII:
S0223-5234(19)31092-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111940
EJMECH 111940
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 September 2019
Revised Date: 19 November 2019
Accepted Date: 2 December 2019
Please cite this article as: H. Ju, S. Xiu, X. Ding, M. Shang, R. Jia, B. Huang, P. Zhan, X. Liu,
Discovery of novel 1,2,3-triazole oseltamivir derivatives as potent influenza neuraminidase inhibitors
targeting the 430-cavity, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
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Graphical abstract

Discovery of novel 1,2,3-triazole oseltamivirderivatives as
potent influenza neuraminidase inhibitors targeting the
4
30-cavity
a
a
a
b
a
b,
Han Ju , Siyu Xiu , Xiao Ding , Min Shang , RuiFang Jia , Bing Huang *, Peng
a,
a,
Zhan *, Xinyong Liu *
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012 Jinan, Shandong, PR China
b
Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 1, Jiaoxiao
Road, 250023 Jinan, Shandong, PR China
Abstract
A novel series of 1,2,3-triazole oseltamivir derivatives, which could simultaneously
occupy the classical NA catalytic site and the newly reported 430-cavity, were
designed, synthesized, and evaluated for their anti-influenza activities. The results
demonstrated that four compounds (6g, 6l, 6y and 8c) showed robust anti-influenza
potencies against H5N1, H5N2 and H5N6 strains in both enzymatic assay and cellular
assay. Especially, 6l was proved to possess the most potent and broad-spectrum
anti-influenza activity, with IC values of 0.12 µM, 0.049 µM and 0.16 µM and EC
5
0
50
values of 2.45 µM, 0.43 µM and 2.8 µM against H5N1, H5N2 and H5N6 strains,
respectively, which were slightly weaker than oseltamivir carboxylate. In addition, in
the embryonated egg model, 6l achieved the similar protective effect against H9N2
strain with oseltamivir carboxylate in the tested concentrations. Preliminary
structure-activity relationships (SARs), molecular modeling, and calculated

physicochemical properties of selected compounds were also discussed.
Keywords: Influenza virus, Neuraminidase inhibitors, 430-cavity, Oseltamivir
derivatives, Broad-spectrum anti-influenza activity.
1
. Introduction
Influenza A and B viruses (FluA and FluB) are respiratory pathogens that cause
an infectious disease annually associated with 290,000 to 650,000 deaths and 3 to 5
million hospitalizations worldwide, especially among high risk people such as elder
and patient with immunocompromised or cardiovascular diseases [1]. FluA can also
confer recurring pandemics, which caused devastating effects on global population in
terms of prevalence and morbidity in the past (e.g., 1918 H1N1, 1968 H3N2 and 2009
H1N1pdm09) [2]. Additionally, H5N1 and H7N9 avian FluA have crossed the species
barrier and lead to thousands of documented zoonotic outbreaks in humans with high
mortality (e.g., H5N1: 63%, H7N9: 40%) in recent years [3-5]. Although there is no
established evidence of efficient human-to-human transmission, their pandemic
potential is clear and poses a serious and constant threat to global public health.
Vaccination is the main method to prevent influenza infections, but the
continuous evolution of influenza virus to avoid host detection system by changing its
antigenicity on hemagglutinin (HA), render existing vaccines useless. And a 6-month
time gap between WHO recommendation and the actual application of vaccines
dramatically decrease its effectiveness against the strains in influenza circulation [6].

Therefore, using antiviral drugs to block the functional proteins of virus is the most
efficient method for managing influenza infections.
Neuraminidase (NA), a surface glycoprotein that facilitates the releasing and
propagating of newly formed virions, is a promising target for anti-influenza drugs
design [7]. Moreover, attributing to their low toxicity, high selectivity and potent
anti-influenza potency, neuraminidase inhibitors (NAIs: oseltamivir, zanamivir,
peramivir and laninamivir octanoate, Fig. 1) function as the only class of antiviral
available clinically [8]. However, the rapid emergence and spread of drug-resistant
influenza strains seriously compromised their clinical application. Among them,
H274Y is the most prevalent resistance-associated mutations towards oseltamivir
which was widely used as preferred drug in clinic since its approval in 1999 [9, 10].
Additionally, E119G and R292K are also observed resistance mutations in the
treatment of zanamivir and peramivir, respectively [11, 12], and one isolated I117T
mutation of NA shows cross-resistance to both oseltamivir and zanamivir [13].
Consequently, there is a clear need to develop improved NAIs for effective influenza
therapy.

Fig. 1. Structures of approved NA inhibitors
On the basis of the X-ray crystallographic studies, newly reported 150-cavity
formed by 150-loop) and 430-cavity (formed by 430-loop) which directly connect
(
with the classical NA catalytic site are revealed as two broad regions for
accommodating further NAIs design [14-19]. In fact, in recent years, the breakthrough
has been made in our lab by modifying the C5-NH of oseltamivir for probing
2
1
50-cavity, and several obtained compounds (Fig. S1) have displayed dozens of times
greater anti-influenza activity than oseltamivir carboxylate (OSC, the active form of
oseltamivir), including against oseltamivir-resistance strains [20-22]. Our previous
work supported the idea well that addition of substituents to the oseltamivir core
could result in strong interactions with the 150-cavity and increase the affinity with
NA. However, most of the oseltamivir derivatives targeting 150-cavity cannot exert a
broad-spectrum anti-influenza activity, because 150-cavity only exists in group-1 NAs
(
containing N1, N4, N5, and N8,). This is influenced by the conformation of 150-loop,
that is, the 150-cavity will not be formed when 150-loop is at a close state. In contrast,
30-loop is always in a stably opened conformation, resulting in 430-cavitiy widely
4
existing in all group-1 and group-2 NAs (containing N2, N3, N6, N7, and N9, Fig. 2).
Therefore, in theory, modifications of NAIs targeting 430-cavity have better prospects
than targeting 150-cavity.

Fig. 2.Comparison of the crystal structures of the Group-1 NA (a, b) and Group-2 NA (c, d)
subtypes. Structures of N1 (H1N1, PDB code: 3BEQ; H5N1, PDB code: 2HU0), N2 (H3N2, PDB
code: 4GZP) and N6 (H5N6, PDB code: 5HUM) are in surface representation. The catalytic
pocket, 430-cavity and opened 150-cavity are labeled in red arrows.
Close examination of the crystal structure of OSC bound to the NA indicated that
the C-1 carboxyl group of OSC was well exposed towards the 430-cavity. Given the
attractiveness of the 430-cavity as an additional binding pocket, and the wealth of
co-crystal structures available, we chose C-1 position of OSC to focus our
optimization efforts upon. Specifically, the Cu(I)-catalyzed azide and alkyne
1
,3-dipolar cycloaddition (CuAAC), commonly known as the "click reaction",
facilitates the formation of 1,4-disubstituted 1,2,3-triazole ring which is usually used
as linker in drug optimization [23,24]. Moreover, owing to the unique properties of
1
,2,3-triazole ring, such as rigidity and stability in vivo, hydrogen bonding capability
and suitable length ( about 5Å), this unit is considered as decisive factor for
improving the biological activities of the compounds in medicinal chemistry [25].
Therefore, we decided to introduce 1,2,3-triazole unit as a linker to connect various
substituents with suitable shape, size, and/or hydrophobicity at the C-1 position of
OSC. And we hoped that the modified C-1 side chain could extend into the 430-cavity
and engender potentially additional interactions with the surrounding residue in the

cavity to enhance the affinity, the broad-spectrum and anti-resistance properties of the
compounds (Fig. 3). Eventually, 33 novel oseltamivir analogues were designed,
synthesized and evaluated for their biological activities.
Fig. 3 The design of novel C-1 modified oseltamivir derivatives targeting 430-cavity
.
2
. Results and discussion
2
.1. Chemistry
The general synthetic steps adopted to obtain the target compounds were
straightforwardly outlined in Scheme 1. These target compounds were synthesized
from the commercially available starting material oseltamivir phosphate (1). Firstly, 1
reacted with Boc anhydride in methanol and Et N to produce 2, which was
3
hydrolyzed by NaOH to give the intermediate 3 [26]. In the presence of HATU,
intermediate 3 reacted with different ynamines to afford 4. And then, 5 was generated
by CuAAC “click chemistry” reaction between the substituted azides and intermediate
4
. Finally, intermediate 5 was directly deprotected with 12 M HCl in methanol to
afford the target compounds 6a-6z, 7a-7c and 8a-8c. The newly synthesized
compounds were characterized by HRMS and NMR spectra.

Scheme 1. Reagents and conditions: (i) (Boc) O, Et N, methanol, RT; (ii) 4 M NaOH, RT; (iii)
2
3
corresponding ynamines, HATU, Et N, CH Cl , RT; (iv) RN , CuSO , L-ascorbic acid sodium salt,
3
2
2
3
4
THF, H O, RT; (v) 12 M HCl, methanol, RT.
2
2
.2. Biological activity
2
.2.1. In vitro inhibitory activities on NAs
All the newly designed oseltamivir derivatives were screened for NA inhibitory
activities using a fluorescence-based assay with MUNANA as the substrate [20-22,
6]. For broad-spectrum NA inhibition screening, we chose N1 (H5N1) from group-1,
2
N2 (H5N2) and N6 (H5N6) from group-2 as representatives for testing. OSC and
zanamivir (ZA) were run in parallel as control drugs. The measured inhibition
potencies of the synthesized compounds are summarized in Table 1. It was clear that
OSC showed great inhibitory potency toward wild-type NAs with IC values of 20
5
0
nM, 7 nM and 18 nM against H5N1, H5N2 and H5N6, respectively. Zanamivir,
another positive control drug, displayed comparable activity with oseltamivir against
H5N1 (IC = 12 nM), H5N2 (IC = 21 nM) and H5N6 NAs (IC = 22 nM), which
5
0
50
50

was consistent with reported data [21, 22].
Table 1. Structures and in vitro inhibitory effects of test compounds against influenza NAs.
a
IC (µM)
5
0
Compds
R
b
c
d
H5N1
H5N2
H5N6
6
6
a
>10
0.86±0.069
>10
b
0.58±0.033
0.98±0.035
1.19±0.06
0.86±0.07
2.85±0.34
1.17±0.09
6
c
6d
1.59±0.15
>10
1.67±0.26
6
e
1.08±0.17
2±0.18
2.2±0.11
2.0±0.18
3.2±0.15
6
f
2.11±0.16
6
6
g
0.48±0.08
7.15±1.05
3.17±0.29
0.14±0.03
0.92±0.35
1.27±0.06
0.13±0.01
2.56±0.03
3.28±0.12
h
6
i
6
j
0.64±0.02
2.29±0.38
>10
6k
4.1±0.33
2.32±0.11
4.3±0.9
Br
6
l
0.12±0.002
0.049±0.007
0.16±0.007

6
m
2.45±0.5
4.05±0.07
1.53±0.11
2.14±0.17
0.66±0.008
0.36±0.024
0.82±0.1
1±0.19
0.57±0.02
2.8±0.7
6
n
o
p
6
6
6
2.42±0.27
2.4±0.08
q
2.72±0.14
3.86±0.14
1.41±0.15
1.96±0.15
2.18±0.19
1.13±0.06
2.86±0.03
5.5±0.24
2.2±0.43
6
r
6
s
6
t
3.12±0.26
3.17±0.03
3.96±0.22
1.58±0.07
1.89±0.06
0.53±0.05
2.78±0.17
3.44±0.16
4.54±0.1
3.1±0.12
4.59±0.2
6.34±0.66
2.01±0.1
1.4±0.16
0.79±0.01
6
u
v
6
6w
1.6±0.16
6
6
x
y
0.78±0.007
0.38±0.04
N
O
O
6
7
z
0.44±0.07
0.69±0.082
0.29±0.05
HO
O
NH
a
4.63±0.33
>10
3.48±0.22
8.86±1.12
7.6±0.55
>10
7b
7
8
c
7.69±0.25
5.66±0.27
>10
NO2
a
7.8±0.5
>10
5.23±0.4
4.05±1.2
>10
>10
8b

8c
0.46±0.06
0.49±0.03
0.58±0.13
OSC
ZA
------
0.020±0.002
0.012±0.001
0.007±0.001
0.018±0.001
0.022±0.001
-------
0.021±0.00008
a
Concentration required to reduce NA activity to 50% of control NA activity (IC ). Data are
5
0
shown as mean ± SD of three experiments.
b
A/Goose/Guangdong/SH7/2013 (H5N1).
c
A/Chicken/Hebei/LZF/2014 (H5N2).
d
A/Duck/Guangdong/674/2014 (H5N6).
As shown in Table 1, the biological results clearly showed that most of the
newly synthesized compounds exhibited anti-NAs activities from micromolar to
sub-micromolar ranges. Impressively, several of the designed compounds (6c, 6g, 6l,
6
n, 6y and 6z) displayed robust inhibitory potencies against all three NAs, and in
particular, 6l achieved the most potent activity with the IC values of 0.12 M, 0.049
M against H5N1, H5N2 and H5N6 NAs, respectively, being slightly
µ
5
0
µM and 0.16 µ
weaker than that of OSC and ZA. On the basis of the above results, the preliminary
SARs could be concluded in terms of different terminal substitution groups and the
length of the side chain.
For the former, an aromatic ring might be sterically and electronically
complementary to the 430-cavity, and compounds 6a-6v were designed with various
substituted benzene rings. Detailed comparison of the data of compounds 6a-6l, we
found that variation of the position of the identical substituent in the benzene (from
the ortho-position to the para-position) resulted in different inhibitory activities. As
for fluorine atom substituent, compounds 6d-6f had similar inhibition against three
NAs with the IC values ranging from 1
µM to 3
µM no matter which position the
5
0
fluorine atom was introduced. However, in the case of bearing a nitro group or a

chlorine atom, the order of potencies against NAs was 6b (3-NO ) ≈ 6c (4-NO ) > 6a
2
2
(
2-NO ) and 6g (2-Cl) > 6i (4-Cl) > 6h (3-Cl). Surprisingly, compound 6l with a
2
bromine atom at para-position of benzene ring showed the greatest activities for all
NAs in this series, but the activities decreased sharply when moved the bromine atom
to ortho- (6j) or meta-position (6k). Likewise, replacing 4-Br of 6l with 4-nitrile (6q),
4
-methyl (6r), 4-phenyl (6s) or non-substituent (6m) also resulted in activities decline
against all the NAs. Next, comparison of activities of 6g and 6l with 6o (IC50
.82-2.8 M) and 6p (IC = 2.14-2.4 M) indicated that mono-substituted derivatives
=
0
µ
µ
5
0
of halogen atom were preferred than di-substituted ones distinctly. In addition,
pairwise comparison of the activities (compounds 6m vs 6n and 6l vs 6v) suggested
that the length of side chain at the end of 1,2,3-triazole could not govern the anti-NA
activities proportionally or inversely.
To extend the SAR information, we designed compounds 6w-6z by replacing the
benzene ring with heteroatomic naphthenes. Among them, compounds 6y and 6z
bearing a morpholine ring and a Zidovudine motif, respectively, were more
advantageous for inhibitory activities against all three NAs than benzene-contained
derivatives except for compounds 6h and 6l. Thus, it appeared that a moderately
flexible group at the end of side chain might more sensitive to NA.
Inspired by the result of 6a-6z, we further designed two subseries of compounds
7
a-7c and 8a-8c with the C-1 modification at the position between the amide bond
and 1,2,3-triazole linker. However, with a negative impact on the anti-NA activities,
most of the compounds lost the potency (IC >10 M) against H5N1 and H5N6 NAs,
µ
5
0

and as for H5N2 NA, their inhibition were also very weak. But encouragingly,
compound 8c exhibited considerably enhanced anti-NA activity with the IC of 0.46
5
0
µM, 0.49
µM and 0.58 µM against H5N1, H5N2 and H5N6 NAs, respectively.
Next, we selected compounds 6c, 6g, 6l, 6n, 6y, 6z and 8c as representatives to
investigate their potencies against H5N1-H274Y NA, because the clinical strain with
this NA mutant exhibits high-level resistance to oseltamivir, which is presently of a
great concern. We used the same assay as above and the data was shown in Table 2.
In agreement with previous reports [21, 22], this mutant conferred resistance to OSC
with the potency (IC = 3.38
µM) over 160-fold weaker than that of wild-type H5N1
5
0
NA, but its susceptibility to zanamivir (IC = 0.013 µM) was not altered. Apart from
5
0
6
g and 6l, all the tested compounds showed weak inhibitory activities (IC >100
µ
M)
against H5N1-H274Y mutation. But notably, compound 6l displayed robust activity
against H5N1-H274Y, with IC50 values of 23.8 M, which was 7 times weaker
5
0
µ
relative to OSC. These results were in consistent with the inhibitory effects against
wild-type H5N1 NA, but their potencies did not support the design strategy well that
modification at C-1 side chain targeting 430-cavity could improve their anti-resistance
profiles.
a
Table 2. NA (H5N1-H274Y) inhibitory activities of selected compounds
IC (µM)
5
0
Compds
H5N1-H274Y
>150
6c
6
g
79.2±1.9
23.8±2.1
6
l

6
n
y
>150
106.1±5.1
>150
6
6
z
c
8
122.7+3.7
3.38±0.07
0.013±0.00035
OSC
ZA
a
Data are shown as mean ± SD of three experiments.
2
.2.2 Molecular modeling analysis
In order to obtain further insight into the binding of 1,2,3-triazole oseltamivir
derivatives to the biologically relevant chemical space of NA, by means of software
SYBYL-X 2.0, the most potent compound 6l was subjected to further docking studies
utilizing the structures of co-crystal of NA (PDB code: 2HU0 (Group-1, N1) and
5
HUM (Group-2, N6)) [26,27]. The amino acid sequences of tested NAs are highly
similar to that of PDBs (N1>90% and N6>99%, shown in supplementary material),
and these inconsistent amino acid residues are far from NA catalytic center or the
4
30-cavity, which greatly increases the credibility of the docking analysis. Our
docking results were visualized by PyMOL.
As shown in Fig. 4a, the docking simulations of 6l with N1 revealed that the
binding mode resembled its lead compound OSC in active center, and of particular
note was that the elongated C-1 side chain was projected toward the 430-cavity. In
Fig. 4b, compound 6l developed four hydrogen bonds with Arg371, Tyr406 and
Arg152 in active center, and the 1,2,3-triazole group also formed additional double
hydrogen bonds with Arg118. Meanwhile, the 1,2,3-triazole linked 4-bromobenzyl
group stretched into the 430-cavity and occupied a small hydrophobic pocket

consisted of Pro431, Lys432, Ser370 and the backbone of Arg371. These docking
results showed that 6l could simultaneously occupy the classical NA catalytic site and
4
30-cavity which was consistent with our original design intention and thereby
displaying great NA inhibition.
As observed in the X-ray crystal structure of N6 bound to 6l (Fig. 4c), this binding
mode resembled OSC in parent scaffold and the 4-bromobenzyl group also extended
into the 430-cavity as the fragment located. Similarly, compound 6l maintained the
typical hydrogen bonds with the same residues around active center and 430-cavity
like that of N1, though their residue serial number were different in 2HU0 and 5HUM
(Fig. 4d). Comparing the above two binging modes, we found that the modified C-1
amide bond developed four hydrogen bonds with residues Arg214, Arg293 and
Tyr327 in N6, but only three hydrogen bonds were observed in the same region in N1.
However, the 1,2,3- triazole group of compound 6l could form one more hydrogen
bond with Arg118 in N1 than in N6, which might compensate for affinity loss at the
amide bond. Therefore, the similar binding modes adopted and the same number of
hydrogen bonds formed in the binding pockets could provide a rational explanation of
the equally broad-spectrum NA inhibitory activity for this molecule. On the other
hand, compared with OSC, the hydrogen bonds with Arg292 and Tyr347 were not
observed in the modes of 6l, which might the main reason for the reduced anti-NA
activity of 6l relative to OSC (Fig. S2). To sum up, the molecular modeling analysis
explained the theoretical binding mode and the potent activity of compound 6l, which
was consistent with our design and would assist further structural optimization.

Fig.4. Predicted binding modes of compound 6l (green) with Group-1 NA (H5N1, PDB code:
HU0) and Group-2 NA (H5N6, PDB code: 5HUM). (a) Superposition of the binding modes of 6l
green) and OSC (yellow)) in H5N1 NA; (b) The key residues of H5N1 (azure) that form potential
interactions with compound 6l; (c) Superposition of the binding modes of 6l (green) and OSC
yellow)) in H5N6 NA; (d) The key residues of H5N6 (azure) that form potential interactions with
2
(
(
compound 6l. Hydrogen bonds between inhibitor and amino acid residues are indicated with
dashed lines (red).
2
.2.3 In vitro anti-influenza virus activity
To validate the efficacy of the synthesized compounds against influenza virus
infection, we employed cell-based assay (chicken embryo fibroblasts, CEFs) that

addressed the cytopathic effect (CPE) of influenza virus infection using the same
virus strains with anti-NA assay [21, 27-28]. We further chose 6g, 6l, 6y and 8c as
representatives, and used oseltamivir carboxylate (OSC) as reference compound in
parallel. The results were expressed as EC50 and CC50 summarized in Table 3.
Notably, all the tested compounds exhibited no appreciable cytotoxicity at the highest
tested concentrations (CC > 1000 µM) in CEFs.
50
Table 3. In vitro anti-influenza virus activities in CEFs
a
g
EC (µM)
EC (µM)
EC (µM)
SI
5
0
50
50
b
Compds
CC (µM)
50
c
d
e
H5N1
H5N2
H5N6
(H5N1)
>300
6g
3.32±0.07
2.45±0.2
3.1±0.2
0.47±0.06
0.43±0.04
1±0.15
8.6±4.2
2.8±0.5
3.78±0.2
>20
>1000
>1000
>1000
>1000
>1000
6l
>400
6y
>330
8c
6.28±0.8
0.22±0.01
0.53±0.02
0.18±0.02
>160
OSC
0.43±0.12
>4500
a
EC : concentration required to achieve 50% protection against virus-induced cytopathic effect.
50
b
c
CC : concentration of 50% cellular toxicity.
5
0
A/Goose/Guangdong/SH7/2013 (H5N1).
d
A/Chicken/Hebei/LZF/2014 (H5N2).
e
A/Duck/Guangdong/674/2014 (H5N6)
g
SI: selectivity index, the ratio of CC /EC .
5
0
50
In the case of the H5N1 virus, 6l displayed the greatest activity (EC = 2.45 µM),
5
0
which was about 11 folds weaker than OSC (EC = 0.22 µM), and the values of other
50
compounds were 15-30 times lower than positive control. Additionally, we obtained
similar results against H5N6 virus, that 6l also exerted the best activity (EC = 2.8
50

µM) which however was 6 folds weaker than OSC (EC50 = 0.43 µM), and the
potencies of other compounds were sharply reduced. These results were in line with
the data in enzymatic assay and compound 6l still displayed the best antiviral potency
towards H5N1 and H5N6 strains.
Surprisingly, against H5N2 virus, compounds 6g, 6l, and 8c shown great
antiviral activities, with EC values of 0.43 µM, 0.47 µM and 0.53 µM, which was
50
comparable to OSC (EC = 0.18 µM). And the weakest compound tested also exerted
5
0
moderate antiviral activity (6y, EC50 = 1 µM)). The results indicated that these
,2,3-triazole derivatives were more sensitive to H5N2 virus than other two strains at
1
least in cellular level.
Overall, the antiviral activities of oseltamivir derivatives 6g, 6l, 6y and 8c
against H5N1 and H5N6 were consistent with their NA-inhibitory potencies and their
activities against H5N2 were comparable to OSC. Moreover, it was worth noting that
compound 6l displayed the best inhibition towards all three virus strains, reflecting a
great broad-spectrum anti-influenza ability with minimal cytotoxicity and high
selectivity index values (SI>400).
2
.2.4 In silico prediction of physicochemical properties
An overall assessment of the physicochemical properties of compounds 6g, 6h,
y and 8z were further conducted by using free molinspiration software
6
(http://www.molinspiration.com/). As depicted in Table 4, the results suggested that
compounds 6g and 6l could theoretically meet the general requirements for useful

drugs, since size, polarity, volume and potentially reactive groups were consistent
with the Lipinski’s “rule of five” except for the slight deviation of molecular weight
of 6l. However, the parameters of 6y and 8z, especially hydrogen bond acceptors
(nON) and tPSA, go beyond the normal scope and reflect undesired physicochemical
properties. Of note, the topological polar surface area (tPSA), which is characterized
by the absorption and membrane permeability of molecules, showed 6g and 6l had a
2
value of both 124.17 Å , confirming their advantage for intestinal absorption (<140
2
Å ) and inability to penetrate the blood-brain barrier, averting the central nervous
2
system toxicity (>60 Å ) [29].
Table 4. Physicochemical properties of representative compounds
Parameter items
6g
6l
6y
8z
natoms
34
489.02
9
34
533.47
9
35
491.59
12
44
616.72
15
MW (< 500 Da)
nON (
nOHNH (
Nrotb (
≤
10)
5)
10)
≤
4
4
4
6
11
≤
10
10
10
2
tPSA (<140 A )
MV
124.17
124.17
153.71
208.50
446.47
1.5
451.01
1.68
1
458.84
-1.53
2
564.28
-0.83
5
miLog p (< 5)
nViol
0
natoms = no. of non
hydrogen atoms; MW= molecular weight; nON = no. of hydrogen bond
acceptors; nOHNH = no. of hydrogen bond donors; nrotb = no. of rotatable bonds; tPSA =
topological polar surface area; MV = molar volume; miLogP = molinspiration predicted LogP;
nViol = number of violations.
2
.2.5 In vivo anti-influenza activity in Specific Pathogen Free (SPF) chicken
embryonated egg
Based on the results of above experiments, in further study, we used an

embryonated egg model which was regarded to have no conflict with ethical and legal
aspects of animal protection to test the anti-influenza activity of compound 6l [22, 26,
3
0].We infected the embryonated eggs with H5N2 virus and another N2 subtype virus
(H9N2) and treated them with injecting different concentrations of 6l. Also, OSC was
administrated as control. After 72 h, we recorded the number of the survival and death
of the embryonated eggs in Table 5. Unexpectedly, as for H5N2 virus, treating with
the concentration of 10 mM resulted in 80 % survival (survived/dead = 4/1) of OSC
and only 20 % survival (survived/dead = 1/4) of compound 6l, and other lower
concentrations had no protective effects for chicken embryos with both tested drugs.
By contrast, compound 6l could alleviate mortality in this infection model, as
demonstrated by against H9N2 strain. The results clearly showed that the protective
potency of 6l for chicken embryos was comparable to OSC at the same concentration
with over 80 % survival achieved.
Table 5. Survival number of chick embryos after inoculation of embryonated eggs with influenza
a
virus and administration of 6l or OSC .
Therapeutic
Chick Embryos
Virus Strain
Drug
Concentration
Survived/ Dead
(
mM
)
Number
10/0
0/5
no
no
Without infection
Without treatment
10
b
H5N2
6l
1/4
2
.5
0/5
0
.625
.156
10
0/5
0
0/5
OSC
4/1
2
.5
0/5
0
0
.625
.156
0/5
0/5
c
H9N2
no
Without treatment
10
2/3
6l
5/0

2
.5
4/1
4/1
4/1
5/0
5/0
4/1
3/2
0
.625
.156
10
0
OSC
2
.5
0
0
.625
.156
a
The 200 TCID50 virus solution was mixed with an equal volume of tested compounds and then
incubated for 1 h before inoculation.
b
A/Chicken/Hebei/LZF/2014 (H5N2).
c
A/Chicken/China/415/2013 (H9N2).
3
. Conclusions
We designed and expeditiously synthesized a series of 1,2,3-triazole oseltamivir
derivatives via CuAAC “click reaction” targeting the newly reported 430-cavity. The
biological assays demonstrated that the majority of these compounds showed
moderate NA inhibition and four compounds (6g, 6l, 6y and 8c) showed robust
anti-influenza potencies against H5N1, H5N2 and H5N6 strains in both enzymatic
assay and cellular assay. Especially, 6l possessed the most potent and broad-spectrum
anti-influenza activity, with IC values of 0.12 µM, 0.049 µM and 0.16 µM and EC
5
0
50
values of 2.45 µM, 4.3 µM and 2.8 µM against H5N1, H5N2 and H5N6 strains,
respectively, which were slightly weaker than that of OSC. Besides, molecular
modeling revealed that the 1,2,3-triazole linked side chain could extend into the
4
30-cavity and interact with the amino acid residues around this region in both
Group-1 and Group-2 NAs as our original design. More encouragingly, 6l could
achieve the similar protective effect against H9N2 virus with OSC in the embryonated
egg model and possessed desirable druglike properties in silico prediction. To sum up,

the biological activities and docking studies indicates that the 430-cavity of NA can
accommodate various substituents and are worth further exploring for discovering
novel and broad-spectrum NAIs.
4
. Experimental section
4
.1 Chemistry
1
13
H NMR and C NMR spectra were acquired on a Bruker AV-400 spectrometer using
d -DMSO and D O as solvents. Chemical shifts were reported in δ values (ppm) with
6
2
TMS as the internal standard, and J values were reported in hertz (Hz). Melting points
mp) were determined on a micromelting point apparatus (Tian Jin Analytical
(
Instrument Factory, Tianjin, China). High-resolution mass spectra (HRMS) were
measured using an Agilent 6520 Q-TOF LC/MS spectrometer (Agilent, Germany).
Reaction progress was routinely monitored using thin-layer chromatography (TLC)
analysis on silica gel GF254, and spots were visualized by irradiation with UV light (λ
=
254 nm). Flash column chromatography was performed on columns packed with
silica gel (200-300 mesh), purchased from Qingdao Haiyang Chemical Company. The
key reactants oseltamivir phosphate was provided by Shandong Qidu Pharmaceutical.
Solvents were of reagent grade and were purified and dried by standard methods
when necessary. The solvents of CH Cl , THF and MeOH were obtained from
2
2
Sinopharm Chemical Reagent Co., Ltd (SCRC).
4
.1.1
General
procedure
for
the
synthesis
of
compound

ethyl(3R,4R,5S)-4-acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-carboxylate (2)
To a solution of commercially available oseltamivir phosphate (1) (5 g, 12.2 mmol) in
MeOH (50 mL) was added di-tert-butoxycarbonyl anhydride ((Boc) O, 5 g, 22.3
2
mmol) and triethylamine (TEA, 2 mL), and the mixture was stirred at room
temperature for 12 h. Subsequently, the reaction mixture was transferred to a separator
funnel, and the residue was washed with water to afford 2 as a white solid, yield
9
6.3 %, mp: 149-150 °C.
1
H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.1 Hz, 1H), 6.61 (d, J = 6.9 Hz, 2H), 4.21
3.99 (m, 3H), 3.75 - 3.61 (m, 1H), 3.57 (td, J = 15.6, 10.1 Hz, 1H), 3.38 (dd, J =
-
1
0.9, 5.5 Hz, 1H), 2.46 (d, J = 4.7 Hz, 1H), 2.25 (dd, J = 17.5, 10.3 Hz, 1H), 1.78 (s,
13
3
H), 1.52 - 1.28 (m, 13H), 1.22 (t, J = 7.1 Hz, 3H), 0.80 (dt, J = 26.5, 7.3 Hz, 6H); C
NMR (100 MHz, DMSO) δ 169.66 (s), 165.99 (s), 155.78 (s), 138.66 (s), 129.08 (s),
8
2
1.49 (s), 78.04 (s), 75.44 (s), 60.88 (s), 54.61 (s), 49.31 (s), 30.61 (s), 28.65 (s),
6.17 (s), 25.65 (s), 23.31 (s), 14.52 (s), 9.88 (s), 9.43 (s). HRMS: m/z 413.2644 [M +
+
H] , C H N O (412.2573).
2
1
36
2
6
4
.1.2 General procedure for the synthesis of compound (3R, 4R,
S)-4-acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)cyclohex-1-ene-1
5
-carboxylic acid (3)
Intermediate 2 (5 g, 12.1 mmol) was dissolved in 50 mL MeOH, and 4 M NaOH
aqueous solution was added until pH to 13. Then the solution was stirred at room

temperature for 2 h. The reaction solution was evaporated under reduced pressure to
remove MeOH, and then 3M HCl was added until no more precipitate formed. The
precipitate was filtrated and washed with H O to afford 3 as white solid, yield 93.3 %,
2
mp: 208-209 °C
.
1
H NMR (400 MHz, DMSO) δ 12.55 (s, 1H), 7.81 (d, J = 9.1 Hz, 1H), 6.65 - 6.52 (m,
2
H), 4.06 (d, J = 7.5 Hz, 1H), 3.69 (dd, J = 19.4, 9.2 Hz, 1H), 3.56 (dt, J = 16.1, 10.2
Hz, 1H), 3.37 (dd, J = 11.4, 5.9 Hz, 1H), 2.46 (dd, J = 17.9, 4.9 Hz, 1H), 2.21 (dd, J =
1
7.5, 10.3 Hz, 1H), 1.78 (s, 3H), 1.52 - 1.28 (m, 13H), 0.80 (dt, J = 26.1, 7.3 Hz, 6H);
C NMR (100 MHz, DMSO) δ 169.71 (s), 167.70 (s), 155.80 (s), 138.13 (s), 129.71
1
3
(s), 81.44 (s), 78.04 (s), 75.55 (s), 54.70 (s), 49.44 (s), 30.73 (s), 28.66 (s), 26.19 (s),
+
2
5.63 (s), 23.32 (s), 9.89 (s), 9.42 (s). HRMS: m/z 385.2338 [M + H] , C H N O
1
9
32
2
6
(384.2260).
4
.1.3 General procedure for the synthesis of compound 4.
To a solution of acid 3 (0.38 g, 1mmol), HATU (0.38 g, 1 mmol), and Et N (1mL) in
3
2
0 mL dry CH Cl was added an equimolar amount of the appropriate ynamine
2 2
(1mmol). The mixture was kept stirring at room temperature for 12 h. After TLC
detection to show no starting materials, the mixture was extracted with 100 mL ethyl
acetate and washed with saturated NaHCO (3 × 30 mL) and saturated NaCl (1 × 50
3
mL). The organic layers were dried over MgSO , filtered and evaporated under
4
vacuum. The crude product was purified by column chromatography with
MeOH:CH Cl = 1:30 to obtain intermediate 4.
2
2

4
.1.4 General procedure for the synthesis of compound 5.
The key intermediate 4 (1.0 equiv), azide compounds (1.2 to 1.5 equiv), ascorbic acid
sodium (0.6 equiv) and CuSO ·5H O (0.3 equiv) were dissolved in the solution of
4
2
THF/water (V:V= 1:1). The resulting mixture was stirred at room temperature for 6 h.
After TLC detection to show no starting materials, 100 mL saturated NaCl was added
and the reaction mixture was washed with ethyl acetate (3 × 50 mL). The combined
organic phase was dried over MgSO , filtered, and concentrated under reduced
4
pressure to give the corresponding crude product, which was purified by flash column
chromatography MeOH:CH Cl = 1:30 to afford product 5.
2
2
4
.1.5 General procedure for the synthesis of target compounds
The intermediate 5 (0.2 g, about 0.4 mmol) was dissolved in 3 mL MeOH and then 3
mL of 12 M HCl was added. The mixture was stirred at room temperature for 1 h.
After TLC detection to show no starting materials, the solvent was evaporated and
purified by column chromatography with MeOH:CH Cl = 1:10 to get target
2
2
compounds 6a-6z, 7a-7c and 8a-8c as white or pale yellow solid.
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)met
hyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6a): Recrystallized from
1
isopropyl ether as a yellow solid, yield 64 %, mp: 155-156 °C. H NMR (400 MHz,

D O) δ 8.03 (dd, J = 8.2, 1.1 Hz, 1H), 7.86 (s, 1H), 7.61 (td, J = 7.6, 1.1 Hz, 1H), 7.55
2
-
7.45 (m, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.33 (s, 1H), 5.83 (s, 2H), 4.43 (s, 2H), 4.20
d, J = 8.9 Hz, 1H), 3.96 (dd, J = 11.7, 9.0 Hz, 1H), 3.51 (td, J = 10.8, 5.6 Hz, 1H),
(
3
.39 (dd, J = 11.1, 5.6 Hz, 1H), 2.80 (dd, J = 16.8, 5.6 Hz, 1H), 2.54 - 2.39 (m, 1H),
.00 (s, 3H), 1.38 (dtd, J = 28.1, 14.2, 7.0 Hz, 4H), 0.73 (dd, J = 13.1, 7.3 Hz, 6H);
2
1
3
C NMR (100 MHz, D O) δ 175.23 (s), 168.88 (s), 147.20 (s), 144.63 (s), 134.80 (s),
2
1
1
2
32.78 (s), 131.03 (s), 130.25 (d, J = 29.5 Hz), 130.11 - 129.92 (m), 129.50 (s),
25.54 (s), 124.71 (s), 84.27 (s), 75.07 (s), 52.67 (s), 51.32 (s), 49.13 (s), 34.60 (s),
8.38 (s), 25.42 (s), 25.05 (s), 22.36 (s), 8.47 (s)，8.44 (s). HRMS: m/z 500.2611
+
[
M+H] , C H N O (499.2543).
24
33
7
5
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)met
hyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6b): Recrystallized from
1
isopropyl ether as a yellow solid, yield 74 %, mp: 157-160°C. H NMR (400 MHz,
D O) δ 7.89 (t, J = 7.7 Hz, 3H), 7.53 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 6.24
2
(
s, 1H), 5.54 (s, 2H), 4.40 (d, J = 15.9 Hz, 2H), 4.12 (d, J = 8.6 Hz, 1H), 3.89 (dd, J =
1.5, 9.1 Hz, 1H), 3.45 (td, J = 11.0, 5.5 Hz, 1H), 3.36 - 3.23 (m, 1H), 2.92 - 2.62 (m,
H), 2.57 - 2.24 (m, 1H), 1.95 (s, 3H), 1.45 - 1.12 (m, 4H), 0.64 (dt, J = 17.9, 7.4 Hz,
1
1
6
1
1
3
H); C NMR (100 MHz, D O) δ 175.19 (s), 168.73 (s), 147.82 (s), 136.55 (s),
2
34.64 (s), 132.84 (s), 130.28 (s), 130.20 (s), 124.26 (s), 123.49 (s), 122.70 (s), 84.17
(s), 74.97 (s), 52.84 (s), 52.61 (s), 49.08 (s), 34.54 (s), 28.33 (s), 25.37 (s), 25.00 (s),
+
2
2.34 (s), 8.46 (s), 8.38 (s). HRMS: m/z 500.2618 [M+H] , C H N O (499.2543).
24
33
7
5

(3R,4R,5S)-4-acetamido-5-amino-N-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)met
hyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6c): Recrystallized from
1
isopropyl ether as a yellow solid, yield 58 %, mp: 169-171 °C. H NMR (400 MHz,
D O) δ 7.94 (dd, J = 8.0, 3.1 Hz, 3H), 7.29 (d, J = 8.2 Hz, 2H), 6.28 (s, 1H), 5.59 (s,
2
2
H), 4.42 (s, 2H), 4.16 (d, J = 8.5 Hz, 1H), 3.92 (dd, J = 11.6, 9.0 Hz, 1H), 3.49 (td, J
10.9, 5.6 Hz, 1H), 3.43 - 3.26 (m, 1H), 2.78 (dd, J = 17.0, 5.2 Hz, 1H), 2.51 - 2.33
=
1
3
(
m, 1H), 1.97 (s, 3H), 1.34 (tdt, J = 21.5, 14.2, 7.0 Hz, 4H), 0.77 - 0.57 (m, 6H); C
NMR (100 MHz, D O) δ 175.21 (s), 168.78 (s), 147.35 (s), 142.10 (s), 132.91 (s),
2
1
3
5
30.28 (s), 128.78 (s), 124.00 (s), 84.21 (s), 75.00 (s), 53.00 (s), 52.64 (s), 49.10 (s),
4.54 (s), 28.36 (s), 25.40 (s), 25.03 (s), 22.36 (s), 8.48 (s)，8.41 (s). HRMS: m/z
+
00.2611 [M+H] , C H N O (499.2543).
24
33
7
5
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)me
thyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6d): Recrystallized from
1
isopropyl ether as a white solid, yield 70 %, mp: 100-102 °C. H NMR (400 MHz,
D O) δ 7.79 (s, 1H), 7.36 - 7.06 (m, 2H), 6.94 (dt, J = 18.6, 8.3 Hz, 2H), 6.24 (s, 1H),
2
5
3
1
1
.40 (s, 2H), 4.12 (d, J = 8.4 Hz, 1H), 4.01 - 3.73 (m, 1H), 3.62 - 3.38 (m, 1H), 3.38 -
.19 (m, 1H), 2.87 - 2.58 (m, 1H), 2.52 - 2.23 (m, 1H), 1.95 (s, 3H), 1.31 (tdt, J = 21.3,
1
3
4.2, 6.9 Hz, 4H), 0.86 - 0.44 (m, 6H); C NMR (100 MHz, D O) δ 175.20 (s),
2
1
68.66 (s), 160.48 (d, J = 246.7 Hz), 132.90 (s), 131.22 (d, J = 8.4 Hz), 130.73 (d,
CF
J = 3.1 Hz), 130.27 (s), 124.82 (d, J = 3.6 Hz), 124.19 (s), 121.42 (d, J = 14.8 Hz),

115.74 (s), 115.54 (s), 84.10 (s), 74.97 (s), 52.65 (s), 49.11 (s), 48.03 (s), 34.47 (s),
+
2
8.39 (s), 25.41 (s), 25.01 (s), 22.40 (s), 8.48 (s). HRMS: m/z 473.2676 [M+H] ,
C H FN O (472.2598).
24
33
6
3
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)me
thyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6e): Recrystallized from
1
isopropyl ether as a white solid, yield 67 %, mp: 160-162 °C. H NMR (400 MHz,
D O) δ 7.84 (s, 1H), 7.28 (dd, J = 14.4, 7.5 Hz, 1H), 6.99 (t, J = 8.0 Hz, 2H), 6.92 (d,
2
J = 9.7 Hz, 1H), 6.32 (s, 1H), 5.48 (s, 2H), 4.42 (s, 2H), 4.19 (d, J = 8.9 Hz, 1H), 3.96
(
(
(
dd, J = 11.7, 9.0 Hz, 1H), 3.51 (td, J = 10.9, 5.6 Hz, 1H), 3.43 - 3.32 (m, 1H), 2.79
dd, J = 17.1, 5.2 Hz, 1H), 2.54 - 2.37 (m, 1H), 2.00 (s, 3H), 1.50 - 1.27 (m, 4H), 0.73
13
1
td, J = 7.4, 4.7 Hz, 6H); C NMR (100 MHz, D O) δ 175.23 (s), 168.88 (d, J
=
CF
2
2
45.2 Hz), 163.84 (s), 161.41 (s), 132.78 (s), 130.84 (d, J = 8.4 Hz), 130.37 (s),
23.73 (d, J = 2.9 Hz), 115.55 (s), 115.34 (s), 114.81 (s), 114.58 (s), 53.21 (s), 52.64
1
(s), 49.12 (s), 34.61 (s), 28.35 (s), 25.41 (s), 25.05 (s), 22.35 (s), 8.45 (d, J = 4.6 Hz).
+
HRMS: m/z 473.2673 [M+H] , C H FN O (472.2598).
2
4
33
6
3
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)me
thyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6f): Recrystallized from
1
isopropyl ether as a white solid, yield 71 %, mp: 128-130 °C. H NMR (400 MHz,
D O) δ 7.79 (s, 1H), 7.19 (dd, J = 8.4, 5.5 Hz, 2H), 6.96 (t, J = 8.8 Hz, 2H), 6.28 (s,
2
1
H), 5.41 (s, 2H), 4.37 (s, 2H), 4.16 (d, J = 8.8 Hz, 1H), 3.93 (dd, J = 11.6, 9.1 Hz,

1
2
H), 3.57 - 3.41 (m, 1H), 3.41 - 3.26 (m, 1H), 2.75 (dd, J = 17.0, 5.4 Hz, 1H), 2.52 -
1
3
.33 (m, 1H), 1.97 (s, 3H), 1.36 (tt, J = 21.1, 7.2 Hz, 4H), 0.88 - 0.56 (m, 6H); C
1
NMR (100 MHz, D O) δ 175.20 (s), 168.82 (s), 162.47 (d, J = 245.0 Hz), 132.78
2
CF
(s), 130.63 (d, J = 3.1 Hz), 130.31 (s), 130.14 (s), 130.05 (s), 123.87 (s), 115.85 (s),
115.63 (s), 84.27 (s), 75.02 (s), 53.16 (s), 52.61 (s), 49.08 (s), 34.53 (s), 28.32 (s),
+
2
5.37 (s), 25.01 (s), 22.32 (s), 8.45 (s), 8.39 (s). HRMS: m/z 473.2672 [M+H] ,
C H FN O (472.2598).
24
33
6
3
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)me
thyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6g)：Recrystallized from
1
isopropyl ether as a white solid, yield 73 %, mp: 144-147 °C. H NMR (400 MHz,
D O) δ 7.82 (s, 1H), 7.33 - 7.10 (m, 4H), 6.28 (s, 1H), 5.53 (s, 2H), 4.40 (s, 2H), 4.17
2
(d, J = 8.8 Hz, 1H), 3.94 (dd, J = 11.7, 9.0 Hz, 1H), 3.49 (td, J = 11.0, 5.6 Hz, 1H),
3
1
1
1
4
4
.40 - 3.27 (m, 1H), 2.78 (dd, J = 16.8, 5.4 Hz, 1H), 2.53 - 2.38 (m, 1H), 1.99 (s, 3H),
13
.46 - 1.26 (m, 4H), 0.72 (tt, J = 9.7, 4.9 Hz, 6H); C NMR (100 MHz, D O) δ
2
75.23 (s), 168.79 (s), 133.45 (s), 132.78 (s), 131.80 (s), 130.96 (s), 130.62 (s),
30.36 (s), 129.84 (s), 127.65 (s), 124.39 (s), 84.20 (s), 75.01 (s), 52.65 (s), 51.78 (s),
9.10 (s), 34.53 (s), 28.37 (s), 25.42 (s), 25.04 (s), 22.36 (s), 8.47 (s). HRMS: m/z
+
89.2379 [M+H] , C H ClN O (488.2303). HPLC purity: 99.99%.
24
33
6
3
(3R,4R,5S)-4-acetamido-5-amino-N-((1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)me
thyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6h)：Recrystallized from