Discovery of C-1 modified oseltamivir derivatives as potent influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.01.050

Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity, in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir derivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the greatest inhibition, with IC₅₀ values of 0.088 and 0.097 μM and EC₅₀ values of 4.26 and 1.31 μM against H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound 8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L. Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1 and group-2 NAs.

Full text of DOI:10.1016/j.ejmech.2018.01.050

European Journal of Medicinal Chemistry 146 (2018) 220e231
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of C-1 modified oseltamivir derivatives as potent influenza
neuraminidase inhibitors
,
**
Han Ju a, Jian Zhang a, Zhuosen Sun a, Zheng Huang a, Wenbao Qi c, Bing Huang b
,
,
, *
Peng Zhan a ***, Xinyong Liu a
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, Jinan, Shandong, 250023, PR China
c College of Veterinary Medicine, South China Agricultural University, 483, Wushan Road, Tianhe District, Guangzhou, 510642, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity,
in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir de-
rivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and
stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust
anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the
Received 6 July 2017
Received in revised form
13 January 2018
Accepted 16 January 2018
greatest inhibition, with IC50 values of 0.088 and 0.097 mM and EC50 values of 4.26 and 1.31 mM against
H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its
potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed
the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound
8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed
greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L.
Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1
and group-2 NAs.
Keywords:
Influenza virus
Neuraminidase inhibitors
430-Cavity
Oseltamivir derivatives
Embryonated egg model
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
been approved by the FDA, namely, M2 ion-channel blockers
(amantadine and rimantadine) and neuraminidase inhibitors
Influenza virus is a respiratory pathogen which can give rise to
substantial socio-economic loss and burden on medical treatment
due to its rapid global spread and high mortality [1]. For example,
the worldwide spread H1N1 influenza from the 2009 pandemic
killed 300,000 people within only 18 months and its death toll
appallingly peaked in younger persons [2], and H7N9 virus is a
lethal avian influenza virus endemic to China that has evolved to
infect humans since 2013 [3]. Moreover, it is known that the highly
pathogenic avian influenza A (H5N1) virus, with 60% mortality, is
also a great threat to humans owing to its potential propensity for
acquiring human receptor specificity [4].
(oseltamivir, zanamivir, peramivir and laninamivir octanoate)
(Fig. 1) [5]. In addition, Favipiravir (T-705), which inhibits the RNA-
dependent RNA polymerase of multiple RNA viruses, was approved
by Japan against influenza infection in 2011 [6]. The above
mentioned agents all prevent infection by targeting conserved
proteins which are functionally indispensable in lifecycle of influ-
enza virus. However, the M2 ion-channel inhibitors are no longer
recommended for treatment of influenza by Centers for Disease
Control and Prevention due to their drug resistance and severe side
effects on central nervous system, and Favipiravir is only used
against influenza virus infections when other anti-influenza drugs
are ineffective [7e9]. Thus, nowadays NA inhibitors are the most
widely choice for anti-influenza treatment.
Currently, there are two classes of anti-influenza agents have
Neuraminidase, a surface glycoprotein responsible for cutting
the connection between the hemagglutinin and the host cell to
facilitate the release of viral progeny from host cells and promoting
viral invasion in upper airways, is a promising target for anti-
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
https://doi.org/10.1016/j.ejmech.2018.01.050
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
221
Fig. 1. Three classes of approved antiviral agents for the treatment of influenza virus infection.
influenza drug design. Currently, NA inhibitors have been devel-
against all kinds of NA subtypes.
oped as effective treatments for influenza A and B infections [10].
Among them, orally administered oseltamivir is the first choice and
has been widely used since its approval in 1999. However the
possibility of widespread resistance has been a concern for several
years, for instance, oseltamivir-resistant influenza H1N1 viruses
were disseminated worldwide in 2007e2009 [11]. In addition, viral
strains that are resistant to the others NA inhibitors are also re-
ported [12,13]. Consequently, it is imperative to develop novel NA
inhibitors to overcome drug resistance and to treat influenza
infection effectively.
Inspired by our earlier studies, we focus our attention on 430-
cavity, since it widely exists in a variety of subtypes, including
group-1 and group-2, and could provide greater chemical space for
further modification [19]. The crystal structure of oseltamivir
carboxylate bounding with NA (N1 and N2) revealed that the C-1
carboxyl group was well exposed toward the newly discovered
430-cavity (Fig. 2) and could serve as the potential modification site
for designing specific inhibitors to improve the antiviral efficacy,
anti-resistance profiles and to react with various of NA subtypes. As
far as we know, little work has been done focusing on C-1 modified
oseltamivir derivatives with the exception of some ester prodrugs
and isosteric groups of carboxyl [20e22]. Therefore, the exploration
of this area is of great value and our proposed strategy is to extend
the side chain of oseltamivir carboxylate by attaching additional
groups of suitable shape, size, and/or hydrophobicity to fill the 430-
cavity and to strengthen the affinity (Fig. 3). Specifically, by intro-
ducing various substituents into the scaffold of oseltamivir
carboxylate, we have designed and synthesized 27 C-1 modified
oseltamivir analogs and evaluated their biological activity.
There are two phylogenetically distinct groups in neuramini-
dases of influenza A viruses: group-1 (N1, N4, N5, and N8 subtypes)
and group-2 (N2, N3, N6, N7, and N9 subtypes) [14]. Recently, two
new pockets adjacent to the NA active site have been discovered
and named as 150-cavity and 430-cavity [15,16]. These two cavities
are directly connected with active site and have large molecular
volumes, making them promising binding sites for inhibitor design.
Interestingly, the X-ray crystallographic structures show that
group-1 NAs have an opened 150-cavity adjacent to the active site,
but the same structure have not be found in group-2 NAs (Fig. 2)
[14]. Based on this finding, a very meaningful exploration on group-
1-specific NA inhibitors through targeting the 150-cavity has been
reported by our lab. We have identified a series of N-substituted
oseltamivir derivatives with low-nanomolar activity against H5N1
NA and H5N1-H274Y NA, and it is worth noting that the most
potent compounds JMC20l and JMC32 have potency of N1 selec-
tivity and showed an increase in inhibitory activity of about 10-fold
relative to OSC on H5N1 and H5N1-H274Y mutant strains [17].
However, a recently study revealed that the 2009 H1N1 (group-
1) influenza pandemic NA lacked the 150-cavity around its active
site [18], implying that inhibitors targeted the 150-cavity may not
be sufficient to combat all the subtype of group-1 NAs. Besides, in
theory, the modified inhibitors targeting the 150-cavity cannot
control group-2 NAs efficiently. Therefore, further optimization of
oseltamivir is initiated with the aim of obtaining improved potency
2. Results and discussion
2.1. Chemistry
The target compounds 5a-5u and 8a-8f were prepared as out-
lined in Scheme 1. All derivatives were synthesized by well-
established methods from commercially available oseltamivir
phosphate (1) as the primary starting material. Reacting with Boc-
anhydride in methanol and Et3N to produce 2, which was hydro-
lyzed by NaOH to give the key intermediate 3 [23]. In the presence
of HATU, intermediate 3 reacted with different amines to afford 4a-
4u, and eventually the target compounds 5a-5u were all prepared
as hydrochlorides with HCl/ethyl acetate. As for the route to com-
pounds 8a-8f, the pre-steps were similar until 3 was treated with
different ester-protected amino acids to obtain 6a-6f, and then

222
H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
Fig. 2. Comparison of the NA active cavities in N1 (PDB code: 2HU0) and N2 (PDB code: 4K1J) subtypes.
Fig. 3. Strategy for structure optimization of oseltamivir by targeting 430-cavity inspired by our previous work targeting 150-cavity.
Scheme 1. Reagents and conditions: (i) (Boc)2O, Et3N, methanol, RT; (ii) 4N NaOH, 3M HCl, RT; (iii) corresponding amines, HATU, Et3N, CH2Cl2, RT; (iv) HCl/ethyl acetate, RT; (v)
CF3COOH, CH2Cl2, RT.

H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
223
Table 1
hydrolyzed in the presence of NaOH to give the corresponding acids
7a-7f. Finally, 7a-7f were directly deprotected with trifluoroacetic
acid in CH2Cl2 to afford the target compounds 8a-8f.
Structures and in vitro inhibitory effects of target compounds against influenza A
NAs.
2.2. Biological activity
2.2.1. In vitro inhibitory activities on NAs
We selected H5N1 NA from group-1 and H5N6 NA from group-2
as representatives for testing, and all 27 target compounds and
oseltamivir carboxylate (OSC) were evaluated for their NA inhibi-
tion [17]. The results are summarized in Table 1. OSC was used as
Compds
R
IC50
H5N1b
(m
M)a
H5N6c
the positive control with the IC50 values of 0.023 mM and 0.0145 mM
against H5N1 and H5N6 NAs, respectively. The result showed that
the control had equal inhibition against these two NA subtypes and
did not display a certain degree of selectivity. The further structure-
activity relationship (SAR) studies revealed that the NA inhibitory
activities of these compounds depended primarily on the type of
the substituents on amide group.
The first sub-series of oseltamivir derivatives (5a-5u) were less
potent than the positive drug. It could be confirmed that
substituted phenyl fragment was not a suitable group for binding
with the 430-cavity. Compared with OSC, 5a-5u lost the interaction
between the carboxylate group and the positively charged pocket
which contains three basic residues, Arg118, Arg292, and Arg371.
And this might have largely contributed to the poor activities of
these compounds. Among them, compound 5c which had a fluorine
atom at the para-position of the phenyl group shown a moderate
5a
6.26 2.26
14.67 0.76
5b
5c
5.09 0.19
0.94 0.08
11.42 0.74
2.78 0.86
5d
5e
5f
3.02 0.79
2.91 0.89
2.07 0.53
7.86 0.72
11.04 2.56
6.01 0.54
activity against H5N1 and H5N6 (IC50 ¼ 0.94 and 2.78
mM, respec-
tively). By contrast, the activities were decreased in case that the
fluorine atom moved to the ortha- or metra-position (compounds
5a, 5b) or was replaced by other groups (compounds 5e, 5f, 5g and
5h). Elsewise, the activities were lost about 10-fold when the
fluorine atom was removed (5i), but less affected from one F atom
in the phenyl group to the compounds with two halogen atoms (5j
and 5k) against H5N1 NA. In addition, compared 5a, 5b with 5m, 5l,
the influence on the length of the carbon chain was not evident,
while bearing a methyl group in 5m (5n) could resulted in better
5g
5h
10.94 3.30
8.78 1.50
>40
>40
5i
5j
9.87 2.28
3.91 0.65
19.92 3.83
19.36 0.43
activity against both NAs (IC50 ¼ 2.65 and 2.52
mM for H5N1 and
H5N6, respectively). Within the series carrying an unsaturated
double bond at the terminal of the side chain, the activity of 5u was
particularly intriguing. Compound 5u exhibited robust inhibitory
activity against H5N6 (IC50 ¼ 0.41
dramatically against H5N1 (IC50 ¼ 5.03
compound 5q was the greatest inhibitor, showing equal inhibi-
M for H5N1 and
H5N6, respectively). It had a biphenyl group substituent and had
approximately 5-10 folds inhibitory potency than the compounds
which had other groups at the same position, like 5e, 5f, 5g and 5h.
Even if the phenyl ring was substituted with its isostere thiophene
ring (5r and 5s), the strong activity was completely lost.
m
M), but the potency decreased
mM). In this sub-series,
5k
2.22 0.77
10.42 1.62
tion to both NA subtypes (IC50 ¼ 0.96 and 0.99
m
5l
3.17 0.54
15.18 1.23
2.65 1.06
5.12 0.23
32.52 1.30
2.51 0.39
Notably, the sub-series of 8a-8f which introduced an amino acid
substituent to the carboxyl group via amide bond displayed great
5m
5n
potency. Especially, compound 8b with a
potency, with IC50 value of 0.088 and 0.097
D
-valine exerted the best
M against H5N1 and
m
H5N6 NA, respectively, which was comparable to the control. The
NA inhibitory potency of 8b may be ascribed to the retainment of
the carboxylate group. Although, compound 8c which contains a
valine motif showed weaker activity (H5N1, IC50 ¼ 0.342 M; H5N6,
IC50 ¼ 0.304 M), compared with 8b, their diastereomers mixture
8a had a medium inhibitory potency between them (H5N1,
M), and this value was
consistent with the SAR in convention and confirmed the accuracy
of the test result in a certain extent. Additionally, further conversion
of valine into alanine (8d, 8e) and glycine (8f) did not show
improved inhibition, indicating that the two methyl groups at the
L-
m
m
5o
11.66 1.37
16.27 1.97
IC50 ¼ 0.135
mM; H5N6, IC50 ¼ 0.248
m
(continued on next page)

224
H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
Table 1 (continued )
Table 2
The NA (H5N1-H274Y) inhibitory activities of selected
Compds
R
IC50
H5N1b
(m
M)a
compounds.a
H5N6c
Compds
IC50 (mM)
5p
1.46 0.36
2.27 0.02
H5N1-H274Y
5q
5u
8a
8b
8c
277.03 12.14
>400
70.88 2.19
37.83 2.63
80.09 0.84
5.05 0.19
5q
5r
0.96 0.05
0.99 0.08
OSC
a
Data are shown as mean SD of three experiments.
29.77 2.58
>40
Although the results were in consistent with the inhibition against
wild-type strain, their potency had not met the expectation that the
additional groups introduced at C-1 position would form additional
interaction with the residues around 430-cavity and improve anti-
resistance profiles.
5s
5t
7.75 0.63
4.24 0.54
10.94 0.19
12.13 0.80
2.2.2. Molecular modeling analysis
In an attempt to better understand the interactions between NA
and the newly synthesized derivatives, molecular docking studies
of the best compound 8b to the crystal structure of N1 subtype NA
extracted from the complex with oseltamivir (PDB code: 2HU0)
was conducted with SurflexeDock SYBYL-X 2.0 software, and
PyMOL was used to visualize the results. First we compared the
amino acid sequences of H5N1-1220 with the protein (2HU0) that
was used for the docking analysis. We found that amino acid
sequence of H5N1-1220 NA showed a high degree (>97%) of simi-
larity to 2HU0. Only 8 of the 368 residues were different between
the two proteins and excluded catalytic center or the 430-cavity
region, which might made a great contribution to the credibility
of the docking analysis. The docking protocol was described in the
molecular docking section.
As shown in Fig. 4a, compound 8b perfectly bounded with the
enzyme and the subject structural part of 8b interacted with the
active site in a manner similar to the binding pattern of oseltamivir
carboxylate. Notably, the elongated modification side chain was
projected toward the 430-cavity, especially the newly introduced
carboxyl group, this result was in good agreement with the purpose
of our design. And in Fig. 4b, it clearly showed that the carboxyl on
valine formed six hydrogen-bonds with Arg118, Arg371 and Try406
around 430-cavity region and the carbonyl group of the amide
could also develop one hydrogen-bond interaction with Arg152.
Compared with the binding model of oseltamivir carboxylate, the
hydrogen-bonding interactions with Arg118, Arg371 and Arg152
were still remained and a new one formed with Try 406, while the
key one with Arg292 at C-1 position was lost, and this might be the
main reason for decline in enzyme inhibitory activity. In addition,
we found the two methyl groups on valine were directed to the
newly protein-solvent interface away from 430-cavity, and this
suggested that using a hydroxyl or thiol group as hydrogen bond
donor in further optimization might obtain promising result.
5u
8a
5.03 1.27
0.41 0.03
0.135 0.023
0.248 0.022
8b
8c
0.0883 0.0133
0.342 0.0399
0.0967 0.0035
0.304 0.0399
8d
8e
8f
1.348 0.065
2.198 0.58
1.066 0.195
1.28 0.159
2.53 0.22
2.16 0.052
OSC
0.023 0.0016
0.0145 0.0016
a
Concentration required to reduce NA activity to 50% of control NA activity (IC50),
and data are shown as mean SD of three experiments.
b
A/chicken/china/1220/2012 (H5N1).
A/duck/Guangdong/674/2014 (H5N6).
c
2.2.3. In vitro anti-influenza virus activity
valine probably formed some special interactions with the enzyme.
Next, we investigated the potency of some representative
compounds (5q, 5u, 8a, 8b, 8c) against H5N1-H274Y NA by the
same assay, because the H274Y mutant NA exhibits high-level
resistance to oseltamivir, which is presently of a great concern. In
Table 2, oseltamivir carboxylate was found to be quite insensitive to
We evaluated the anti-influenza virus activity and cytotoxicity
of representative compound 8b (OSC as the positive control) by
cell-based assay [24,25]. The results (Table 3) showed that 8b
possessed in vitro anti-influenza virus (H5N1, H5N6) activity and
had no obvious cytotoxicity. The values of EC50 were comparable to
the control and correlated well with their inhibitory activities
against NAs, suggesting these compounds inhibited influenza virus
replication by binding to NAs in cells level. Additionally, the
selectivity index of compound 8b was over 200 and 750 towards
H5N1 and H5N6 virus strains.
this mutant enzyme, with an IC50 value of 5.05 mM, which was over
200-fold lower than the IC50 against the wild-type H5N1 NA.
However, these selected oseltamivir derivatives decreased the NA
inhibitory activity by at least 7 folds relative to the control.

H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
225
Fig. 4. Docking results of compounds 8b with N1 (PDB 2HU0). (a) Compared the binding mode of 8b (yellow) with OSC (azure); (b) The key residues (white) that form potential
interactions with 8b (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 3
Table 4
In vitro anti-influenza virus activities in chicken embryo fibroblast cells.
Survival number of chick embryos after inoculation of embryonated eggs with
influenza A virus and administration of 8b or OSC.a
Compds
EC50
(m
M)a
EC50
(m
M)a
CC50
(
m
M)b
SIc
H5N1d
H5N6e
H5N1, H5N6
Virus Strain
H5N1b
Drug
Therapeutic Concentration
(mmol/L)
Chick Embryos
Survived/Dead Number
8b
OSC
4.26 2.09
1.35 0.282
1.31 0.34
0.73 0.231
>1000
>1000
>200, >750
>750, >1000
no
no
8b
Without infection
Without treatment
10/0
0/5
5/0
0/5
0/5
0/5
0/5
5/0
4/1
3/2
0/5
0/5
0/5
5/0
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
a
EC50: concentration required to achieve 50% protection against virus-induced
cytopathic effect.
10
10/4
b
CC50: concentration that causes a 50% decrease in cell viability.
10/42
10/43
10/44
10
c
SI: selectivity index, the ratio of CC50/EC50
A/chicken/china/1220/2012 (H5N1).
A/duck/Guangdong/674/2014 (H5N6).
.
d
e
OSC
10/4
10/42
10/43
10/44
Without treatment
10
2.2.4. In vivo anti-influenza virus activity in embryonated egg
In further study, we tested anti-influenza virus activity of
compound 8b with an embryonated egg model which was regar-
ded to have no conflict with ethical and legal aspects of animal
protection [26]. We infected the embryonated eggs with H5N1 and
H5N6 virus strains and treated them with injecting different con-
centrations of 8b. Also, OSC was administrated as control. At 72 h
post infection and treatment, we recorded the number of the sur-
vival and death to evaluate the anti-influenza virus activity of the
H5N6c
no
8b
10/4
10/42
10/43
10/44
10
OSC
10/4
10/42
10/43
10/44
compound. As shown in Table
4 (Figure data shown in
supplementary material), all embryos were survived without
infected any influenza virus (survived/dead ¼ 10/0), and in other
two groups all were dead after infection without any treatment
(H5N1: survived/dead ¼ 0/5; H5N6: survived/dead ¼ 0/5), sug-
gesting the test procedure was standard. In the condition of
infecting H5N1 virus, all embryos were survived in curing with 8b
and OSC in concentration of 10 mmol/L. But with the decrement of
the drugs concentration, the number of death was raised. And the
embryos were entirely dead at the concentration of 10/4 mmol/L in
treating with 8b, while the same result occurred at the concen-
tration of 10/43 mmol/L in OSC, suggesting 8b was not sensitive
toward H5N1 strain relative to OSC. Notably, 8b could protect the
embryos more efficient than OSC against H5N6 strain, as compound
8b can make all embryos live (survived/dead ¼ 5/0) at the con-
centration of 10 mmol/L while OSC cannot (survived/dead ¼ 0/5).
Although this cannot consist with the values of IC50 and EC50 tested
toward H5N6, the result is inspiring and we guess that the unex-
pected cellular localization, endogenous contacts with other
a
The virus solution was mixed with an equal volume of tested compounds and
then incubated for 1 h before inoculation.
b
A/chicken/china/1220/2012 (H5N1).
A/duck/Guangdong/674/2014 (H5N6).
c
proteins or some active metabolites generated in the organism
perhaps are the reasons for such a leftward shift in potency.
3. Conclusion
In summary, we designed and synthesized 27 oseltamivir ana-
logs by modification at the C-1 position to explore the chemical
space around 430-cavity. Among which, compound 8b exerted the
best inhibitory activity against H5N1 and H5N6 NAs, with IC50
values of 0.088 mM and 0.097 mM, respectively. Moreover, cell-
based assay further illustrated that compound 8b was safe and
could inhibit influenza virus replication in vitro, with EC50 values of

226
H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
4.26
mM and 1.35
mM against H5N1 and H5N6, which was similar to
room temperature for 2 h. The reaction solution was evaporated
positive drug OSC (1.31
m
M and 0.73 M, respectively) and corre-
m
under reduced pressure to remove methanol, then 3M HCl was
added until no more precipitate formed. The precipitate was fil-
trated, washed with H2O and dry to afford 3 as white solid, yield
lated well with its inhibitory activities against NAs. The equally
inhibitory against these two virus strains suggests that our strategy
for targeting the 430-cavity to discover novel NA inhibitors against
both group-1 and group-2 NAs is rational. Furthermore, it is worth
noting that compound 8b displayed more potent anti-influenza
virus effect against H5N6 strain than OSC at the concentration of
10 mmol/L in the embryonated egg model. According to these
excellent in vitro and in vivo results, we hope that the further
derivatization of oseltamivir based on this strategy might lead to
more potent compounds against both group-1 and group-2 NAs.
93%. 1H NMR (400 MHz, DMSO-d6)
d 12.55 (s, 1H), 7.81 (d,
J ¼ 9.1 Hz, 1H), 6.65e6.52 (m, 2H), 4.06 (d, J ¼ 7.5 Hz, 1H), 3.69 (dd,
J ¼ 19.4, 9.2 Hz, 1H), 3.56 (dt, J ¼ 16.1, 10.2 Hz, 1H), 3.37 (dd, J ¼ 11.4,
5.9 Hz, 1H), 2.46 (dd, J ¼ 17.9, 4.9 Hz, 1H), 2.21 (dd, J ¼ 17.5, 10.3 Hz,
1H), 1.78 (s, 3H), 1.52e1.28 (m, 13H), 0.80 (dt, J ¼ 26.1, 7.3 Hz, 6H);
13C NMR (100 MHz, DMSO-d6)
d 169.71 (s), 167.70 (s), 155.80 (s),
138.13 (s), 129.71 (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), 25.63 (s), 23.32 (s), 9.89 (s), 9.42 (s).
HRMS (ESI): calcd for C19H32N2O6 [MþH]þ 385.2338; found m/z:
385.2338.
4. Experimental section
4.1. Chemistry
4.1.3. General procedure for the synthesis of compounds 4aꢀ4u
To a solution of acid 3 (0.384 g, 1 mmol), HATU (0.38 g, 1 mmol),
and Et3N (1 mL) in 20 mL of dry CH2Cl2 was added an equimolar
amount of the appropriate amine. The mixture was kept stirring at
room temperature for 24 h. After TLC detection to show no starting
materials, the mixture was extracted with 100 mL ethyl acetate and
washed with saturated NaHCO3 (3 ꢁ 30 mL) and saturated NaCl
(1 ꢁ 50 mL). The organic layers were dried over MgSO4 and evap-
orated under vacuum. The crude product was purified by column
chromatography with methanol: CH2Cl2 ¼ 1: 30 to obtain in-
termediates 4a¡4u.
1H NMR and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer using solvents as indicated (DMSO-d6 or D2O).
Chemical shifts were reported in
thylsilane as the internal reference, and J values were reported in
hertz (Hz). Melting points (mp) were determined on a micro-
melting point apparatus. TLC was performed on Silica Gel GF254 for
TLC (Merck) and spots were visualized by iodine vapor or by irra-
diation with UV light (l ¼ 254 nm). Flash column chromatography
was performed on column packed with Silica Gel60 (200e300
mesh).
d values (ppm) with tetrame-
Thin layer chromatography was performed on pre-coated
HUANGHAI_HSGF254, 0.15-0.2 mm TLC-plates. Solvents were of
reagent grade and were purified and dried by standard methods
when necessary. The key reactants oseltamivir phosphate was
provided by Shandong Qidu Pharmaceutical. The solvents of
CH2Cl2, Et3N and methanol were obtained from Sinopharm
Chemical Reagent Co., Ltd (SCRC), which were of AR grade. Chem-
icals HATU, corresponding amines and amino acids, TFA, etc. were
obtained from Beijing Innochem Science & Technology Co.,Ltd,
which were of CP grade.
4.1.4. General procedure for the synthesis of compounds 5aꢀ5u
The compound 4a (0.1 g, 0.2 mmol) was dissolved in 20.0 mL
HCl/ethyl acetate and the mixture was stirred at room temperature
for 1 h. The precipitate was filtered and then dried to get target
compound 5a as a white solid. Compound 5b-5u was prepared
using the same method in good yield.
(3R,4R,5S)-4-acetamido-5-amino-N-(2-fluorobenzyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5a):
white solid yield 80%, mp: 150e152 ꢂC. 1H NMR (400 MHz, D2O)
d
7.21 (t, J ¼ 7.0 Hz, 2H), 7.14e6.91 (m, 2H), 6.31 (s, 1H), 4.43e4.25
(m, 2H), 4.18 (d, J ¼ 8.5 Hz, 1H), 4.05e3.85 (m, 1H), 3.48 (td, J ¼ 11.0,
5.6 Hz, 1H), 3.42e3.35 (m, 1H), 2.76 (dd, J ¼ 16.9, 5.3 Hz, 1H),
2.53e2.35 (m, 1H), 1.96 (s, 3H), 1.53e1.23 (m, 4H), 0.73 (dd, J ¼ 17.6,
4.1.1. General procedure for the synthesis of ethyl (3R, 4R, 5S)-4-
acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)
cyclohex-1-ene-1-carboxylate (2)
To a solution of commercially available oseltamivir phosphate
(1) (10 g, 24.4 mmol) in 100 mL methanol was added diter-
butoxycarbonyl anhydride ((Boc)2O, 10 g) and triethylamine (TEA,
4 mL) at room temperature, and the mixture was stirred for 12 h.
Subsequently, the solution was evaporated under reduced pressure
to remove methanol and was transferred to a separators funnel, and
the residue was washed with water to afford 2 as a white solid,
7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.22 (s), 169.02 (s), 160.54
(d, 1 J ¼ 244.8 Hz), 132.53 (s), 130.55 (s), 129.51 (d, J ¼ 3.9 Hz),
CF
124.37 (d, J ¼ 3.6 Hz), 124.25 (s), 115.46 (s), 115.25 (s), 84.34 (s),
75.11 (s), 52.64 (s), 49.12 (s), 37.51 (d, J ¼ 4.2 Hz), 28.39 (s), 25.41 (s),
25.08 (s), 22.33 (s), 8.47 (s), 8.43 (s). HRMS (ESI): calcd for
C
21H30FN3O3 [MþH]þ 392.2349; found m/z: 392.2349.
(3R,4R,5S)-4-acetamido-5-amino-N-(3-fluorobenzyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5b):
yield 91%. 1H NMR (400 MHz, DMSO-d6)
d
7.80 (d, J ¼ 9.1 Hz, 1H),
white solid yield 80%, mp: 169e171 ꢂC. 1H NMR (400 MHz, D2O)
6.61 (d, J ¼ 6.9 Hz, 2H), 4.21e3.99 (m, 3H), 3.75e3.61 (m, 1H), 3.57
(td, J ¼ 15.6, 10.1 Hz, 1H), 3.38 (dd, J ¼ 10.9, 5.5 Hz, 1H), 2.46 (d,
J ¼ 4.7 Hz, 1H), 2.25 (dd, J ¼ 17.5, 10.3 Hz, 1H), 1.78 (s, 3H), 1.52e1.28
(m, 13H), 1.22 (t, J ¼ 7.1 Hz, 3H), 0.80 (dt, J ¼ 26.5, 7.3 Hz, 6H); 13C
d
7.25 (dd, J ¼ 14.5, 7.9 Hz,1H), 6.96 (dd, J ¼ 26.6, 8.2 Hz, 3H), 6.35 (s,
1H), 4.44e4.24 (m, 2H), 4.20 (d, J ¼ 8.4 Hz, 1H), 4.04e3.81 (m, 1H),
3.49 (td, J ¼ 11.0, 5.6 Hz, 1H), 3.44e3.33 (m, 1H), 2.78 (dd, J ¼ 16.8,
5.1 Hz, 1H), 2.58e2.37 (m, 1H), 1.97 (s, 3H), 1.52e1.15 (m, 4H), 0.73
NMR (100 MHz, DMSO-d6)
d
169.66 (s),165.99 (s),155.78 (s),138.66
(dt, J ¼ 10.7, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.21 (s),
(s), 129.08 (s), 81.49 (s), 78.04 (s), 75.44 (s), 60.88 (s), 54.61 (s),
49.31 (s), 30.61 (s), 28.65 (s), 26.17 (s), 25.65 (s), 23.31 (s), 14.52 (s),
9.88 (s), 9.43 (s). HRMS (ESI): calcd for C21H36N2O6 [MþH]þ
413.2651; found m/z: 413.2644.
168.93 (s), 162.68 (d, 1 J CF ¼ 243.7 Hz), 140.43 (d, J ¼ 7.2 Hz), 132.70
(s), 130.44 (s), 130.36 (s), 122.81 (d, J ¼ 2.5 Hz), 114.20 (s),
114.20e113.71 (m), 113.63 (s), 84.25 (s), 75.09 (s), 52.65 (s), 49.12
(s), 42.68 (s), 28.40 (s), 25.43 (s), 25.06 (s), 22.35 (s), 8.49 (s), 8.47
(s). HRMS (ESI): calcd for C21H30FN3O3 [MþH]þ 392.2349; found m/
z: 392.2348.
4.1.2. General procedure for the synthesis of compound (3R,4R,5S)-
4-acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)
cyclohex-1-ene-1-carboxylic acid (3)
Without further purification, intermediate 2 (5 g, 12.1 mmol)
was dissolved in 50 mL methanol, and 4N NaOH aqueous solution
was added until the pH to 12, and then the solution was stirred at
(3R,4R,5S)-4-acetamido-5-amino-N-(4-fluorobenzyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5c):
white solid yield 48%, mp: 108e110 ꢂC. 1H NMR (400 MHz, D2O)
d
7.33e7.16 (m, 2H), 7.03 (ddd, J ¼ 8.9, 5.9, 2.5 Hz, 2H), 6.37 (s, 1H),
4.39e4.26 (m, 2H), 4.23 (d, J ¼ 9.0 Hz, 1H), 3.99 (dd, J ¼ 11.7, 8.9 Hz,

H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
227
1H), 3.53 (td, J ¼ 10.8, 5.6 Hz, 1H), 3.44 (dd, J ¼ 10.9, 5.6 Hz, 1H),
2.86e2.75 (m, 1H), 2.50 (ddt, J ¼ 16.8, 10.5, 3.0 Hz, 1H), 2.01 (s, 3H),
1.56e1.29 (m, 4H), 0.77 (dt, J ¼ 11.0, 7.4 Hz, 6H); 13C NMR (100 MHz,
(m, 4H), 0.75 (dd, J ¼ 16.9, 7.5 Hz, 6H); 13C NMR (100 MHz, D2O)
d
175.23 (s), 168.87 (s), 158.09 (s), 132.36 (s), 130.59 (d, J ¼ 19.2 Hz),
128.79 (s), 114.18 (s), 84.34 (s), 75.13 (s), 55.35 (s), 52.67 (s), 49.14
(s), 42.58 (s), 28.42 (s), 25.42 (s), 25.07 (s), 22.32 (s), 8.46 (s). HRMS
D2O)
d
175.26 (s), 168.98 (s), 161.84 (d, 1
J
¼ 242.6 Hz), 133.69 (d,
CF
J ¼ 2.9 Hz), 132.54 (s), 130.61 (s), 129.05 (d, J ¼ 8.3 Hz), 115.47 (s),
115.25 (s), 84.36 (s), 75.15 (s), 52.69 (s), 49.16 (s), 42.57 (s), 28.43 (s),
25.45 (s), 25.11 (s), 22.38 (s), 8.49 (s). HRMS (ESI): calcd for
(ESI): calcd for
404.2544.
C
22H33N3O4 [MþH]þ 404.2549; found m/z:
(3R,4R,5S)-4-acetamido-5-amino-N-benzyl-3-(pentan-3-yloxy)
C
21H30FN3O3 [MþH]þ 392.2349; found m/z: 392.2341.
cyclohex-1-ene-1-carboxamide hydrochloride (5i): white solid
(3R,4R,5S)-4-acetamido-5-amino-N-(3-chlorobenzyl)-3-(pen-
yield 69%, mp: 142e143 ꢂC. 1H NMR (400 MHz, D2O)
d 7.26 (dt,
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5d):
J ¼ 13.2, 4.7 Hz, 5H), 6.35 (s, 1H), 4.40e4.28 (m, 2H), 4.21 (d,
J ¼ 9.0 Hz, 1H), 3.96 (dd, J ¼ 11.7, 9.0 Hz, 1H), 3.50 (td, J ¼ 10.8,
5.6 Hz, 1H), 3.42 (dt, J ¼ 11.1, 5.6 Hz, 1H), 2.79 (dd, J ¼ 16.9, 5.5 Hz,
1H), 2.48 (ddt, J ¼ 16.7, 10.5, 2.9 Hz, 1H), 1.98 (s, 3H), 1.49e1.28 (m,
white solid yield 64%, mp: 185e187 ꢂC. 1H NMR (400 MHz, D2O)
d
7.23 (dd, J ¼ 6.3, 4.5 Hz, 3H), 7.16e7.07 (m, 1H), 6.36 (s, 1H),
4.37e4.26 (m, 2H), 4.21 (d, J ¼ 8.9 Hz, 1H), 3.96 (dd, J ¼ 11.7, 9.0 Hz,
1H), 3.51 (td, J ¼ 10.9, 5.7 Hz, 1H), 3.41 (dd, J ¼ 10.4, 5.4 Hz, 1H), 2.79
(dd, J ¼ 16.9, 5.5 Hz, 1H), 2.48 (ddt, J ¼ 16.7, 10.5, 2.9 Hz, 1H), 1.98 (s,
3H), 1.51e1.31 (m, 4H), 0.75 (dt, J ¼ 12.5, 7.4 Hz, 6H); 13C NMR
4H), 0.75 (dt, J ¼ 12.1, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.22
(s), 169.00 (s), 137.83 (s), 132.45 (s), 130.62 (s), 128.79 (s), 127.48 (s),
127.16 (s), 84.36 (s), 75.13 (s), 52.65 (s), 49.14 (s), 43.14 (s), 28.41 (s),
25.42 (s), 25.07 (s), 22.33 (s), 8.47(s), 8.45(s). HRMS (ESI): calcd for
(100 MHz, D2O) d 175.23 (s), 168.97 (s), 139.98 (s), 133.82 (s), 132.68
(s), 130.47 (s), 130.18 (s), 127.37 (s), 126.91 (s), 125.48 (s), 84.34 (s),
75.11 (s), 52.66 (s), 49.12 (s), 42.65 (s), 28.39 (s), 25.43 (s), 25.08 (s),
22.34 (s), 8.50 (s), 8.46 (s). HRMS (ESI): calcd for C21H30ClN3O3
[MþH]þ 408.2054; found m/z: 408.2052.
C
21H31N3O3 [MþH]þ 374.2443; found m/z: 374.2439.
(3R,4R,5S)-4-acetamido-5-amino-N-(2-chloro-4-fluorobenzyl)-
3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride
(5j): yellow solid yield 74%, mp: 214e216 ꢂC. 1H NMR (400 MHz,
(3R,4R,5S)-4-acetamido-5-amino-N-(4-methylbenzyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5e):
white solid yield 77%, mp: 119e120 ꢂC. 1H NMR (400 MHz, D2O)
D2O)
d
7.30 (dd, J ¼ 8.6, 6.1 Hz, 1H), 7.22 (dd, J ¼ 8.8, 2.6 Hz, 1H),
7.08e6.94 (m, 1H), 6.37 (s, 1H), 4.43 (q, J ¼ 15.3 Hz, 2H), 4.24 (d,
J ¼ 8.8 Hz, 1H), 4.00 (dd, J ¼ 11.7, 9.0 Hz, 1H), 3.55 (td, J ¼ 10.9,
5.6 Hz, 1H), 3.49e3.41 (m, 1H), 2.89e2.78 (m, 1H), 2.60e2.45 (m,
d
7.13 (s, 4H), 6.32 (s, 1H), 4.37e4.23 (m, 2H), 4.13 (d, J ¼ 8.7 Hz, 1H),
3.79 (ddd, J ¼ 9.1, 7.3, 2.7 Hz, 1H), 3.44e3.35 (m, 1H), 3.16 (td,
J ¼ 10.8, 5.3 Hz, 1H), 2.68 (dd, J ¼ 17.0, 4.9 Hz, 1H), 2.38e2.26 (m,
1H), 2.22 (s, 3H), 2.00 (s, 3H), 1.50e1.30 (m, 4H), 0.77 (dt, J ¼ 11.1,
1H), 2.02 (s, 3H),1.57e1.34 (m, 4H), 0.79 (dt, J ¼ 10.7, 7.4 Hz, 6H); 13
C
NMR (100 MHz, D2O)
d
175.20 (s), 169.00 (s), 161.63 (d, 1J
¼ 247.1 Hz), 133.59 (d, J ¼ 10.8 Hz), 132.65 (s), 130.79 (s), 130.54
CF
7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d
175.00 (s), 169.39 (s), 137.59
(s), 116.85 (s), 116.60 (s), 114.16 (s, J ¼ 21.3 Hz), 113.95 (s), 84.18 (s),
75.10 (s), 52.67 (s), 49.12 (s), 41.04 (s), 28.44 (s), 25.45 (s), 25.28 (s),
22.37 (s), 8.52 (s), 8.49 (s). HRMS (ESI): calcd for C21H29ClFN3O3
[MþH]þ 426.1959; found m/z: 426.1955.
(s), 134.87 (s), 132.55 (s), 131.60 (s), 129.33 (s), 127.27 (s), 84.15 (s),
75.74 (s), 55.06 (s), 49.09 (s), 42.84 (s), 30.53 (s), 25.47 (s), 25.13 (s),
22.34 (s), 20.10 (s). HRMS (ESI): calcd for C22H33N3O3 [MþH]þ
388.2600; found m/z: 388.2599.
(3R,4R,5S)-4-acetamido-5-amino-N-(3,5-difluorobenzyl)-3-
(3R,4R,5S)-4-acetamido-5-amino-N-(4-bromobenzyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5f):
white solid yield 66%, mp: 163e165 ꢂC. 1H NMR (400 MHz, D2O)
(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide
(5k): white solid yield 59%, mp: 190e191 ꢂC. 1H NMR (400 MHz,
D2O)
hydrochloride
d
7.24 (dd, J ¼ 15.3, 8.6 Hz, 1H), 6.86 (dd, J ¼ 14.6, 8.0 Hz, 2H),
d
7.36 (d, J ¼ 7.8 Hz, 2H), 7.08 (d, J ¼ 7.7 Hz, 2H), 6.30 (s, 1H), 4.25 (t,
6.33 (s, 1H), 4.48e4.25 (m, 2H), 4.21 (d, J ¼ 8.6 Hz, 1H), 3.96 (dd,
J ¼ 11.5, 9.2 Hz, 1H), 3.58e3.48 (m, 1H), 3.45e3.33 (m, 1H), 2.78 (dd,
J ¼ 17.1, 5.3 Hz, 1H), 2.54e2.36 (m, 1H), 1.99 (s, 3H), 1.38 (ddd,
J ¼ 21.2, 13.4, 6.3 Hz, 4H), 0.75 (dd, J ¼ 17.3, 7.4 Hz, 6H); 13C NMR
J ¼ 12.1 Hz, 2H), 4.23e4.14 (m, 1H), 3.96 (t, J ¼ 10.2 Hz, 1H), 3.52 (s,
1H), 3.36 (s, 1H), 2.80 (d, J ¼ 14.5 Hz, 1H), 2.60e2.40 (m, 1H), 2.00 (s,
3H), 1.38 (t, J ¼ 14.9 Hz, 4H), 0.73 (s, 6H); 13C NMR (100 MHz, D2O)
d
175.17 (s), 168.85 (s), 137.10 (s), 132.54 (s), 131.57 (s), 130.64 (s),
(100 MHz, D2O)
d
175.23 (s), 169.02 (s), 161.36 (dd, 1J ¼ 246.6,
CF
129.09 (s), 120.66 (s), 83.96 (s), 75.03 (s), 52.73 (s), 49.14 (s), 42.56
(s), 28.52 (s), 25.54 (s), 25.14 (s), 22.43 (s), 8.71 (s), 8.54 (s). HRMS
174.2 Hz), 132.58 (s), 130.59 (dd, J ¼ 8.8, 4.8 Hz), 120.47 (dd, J ¼ 14.9,
3.7 Hz), 103.98 (s), 103.73 (s), 103.47 (s), 84.36 (s), 75.12 (s), 52.65
(s), 49.12 (s), 37.13 (d, J ¼ 3.3 Hz), 28.38 (s), 25.42 (s), 25.09 (s), 22.34
(s), 8.47 (s), 8.43 (s). HRMS (ESI): calcd for C21H29F2N3O3 [MþH]þ
410.2255; found m/z: 410.2253.
(ESI): calcd for
452.1545.
C
21H30BrN3O3 [MþH]þ 452.1549; found m/z:
(3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)-N-(4-
(trifluoromethyl)benzyl)cyclohex-1-ene-1-carboxamide
hydro-
(3R,4R,5S)-4-acetamido-5-amino-N-(2-fluorophenethyl)-3-
chloride (5g): white solid yield 85%, mp: 164e166 ꢂC. 1H NMR
(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide
(5l): white solid yield 86%, mp: 159e161 ꢂC. 1H NMR (400 MHz,
D2O)
hydrochloride
(400 MHz, D2O)
d
7.51 (d, J ¼ 8.2 Hz, 2H), 7.31 (d, J ¼ 8.1 Hz, 2H), 6.33
(s, 1H), 4.38 (q, J ¼ 15.7 Hz, 2H), 4.20 (d, J ¼ 8.7 Hz, 1H), 3.97 (dd,
J ¼ 11.6, 9.0 Hz, 1H), 3.53 (td, J ¼ 11.0, 5.7 Hz, 1H), 3.43e3.30 (m, 1H),
2.82 (dd, J ¼ 16.9, 5.4 Hz, 1H), 2.51 (d, J ¼ 2.9 Hz, 1H), 2.00 (s, 3H),
1.48e1.21 (m, 4H), 0.72 (td, J ¼ 7.3, 2.0 Hz, 6H); 13C NMR (100 MHz,
d
7.25e7.12 (m, 2H), 7.04 (ddd, J ¼ 18.7, 8.9, 4.7 Hz, 2H), 6.05 (s,
1H), 4.15 (d, J ¼ 9.0 Hz, 1H), 3.91 (dd, J ¼ 11.7, 9.0 Hz, 1H), 3.54e3.30
(m, 4H), 2.81 (t, J ¼ 6.4 Hz, 2H), 2.66 (dd, J ¼ 16.9, 5.5 Hz, 1H), 2.37
(ddt, J ¼ 16.7, 10.5, 2.9 Hz, 1H), 1.98 (s, 3H), 1.53e1.28 (m, 4H), 0.76
D2O)
d
175.18 (s), 169.03 (s), 142.16 (s), 132.59 (s), 130.62 (s), 128.82
(dt, J ¼ 14.8, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.22 (s),
(d, 2J CF ¼ 32.2 Hz), 127.41 (s), 125.45 (dd, J ¼ 7.5, 3.7 Hz), 122.82 (s),
84.04 (s), 75.06 (s), 52.73 (s), 49.15 (s), 42.72 (s), 28.50 (s), 25.46 (s),
25.11 (s), 22.41 (s), 8.52 (s), 8.41 (s). HRMS (ESI): calcd for
169.27 (s), 161.18 (d, 1J
¼ 242.6 Hz), 131.60 (s), 131.48 (d,
CF
J ¼ 4.9 Hz), 130.92 (s), 128.58 (d, J ¼ 8.2 Hz), 125.71 (d, J ¼ 16.0 Hz),
124.32 (d, J ¼ 3.4 Hz), 115.24 (s), 115.02 (s), 84.40 (s), 75.13 (s), 52.67
(s), 49.13 (s), 39.37 (s), 28.39 (s), 28.02 (s), 25.48 (s), 25.03 (s), 22.34
(s), 8.54 (s), 8.48 (s). HRMS (ESI): calcd for C22H32FN3O3 [MþH]þ
406.2506; found m/z: 406.2505.
C
21H30ClN3O3 [MþH]þ 442.2318; found m/z: 442.2307.
(3R,4R,5S)-4-acetamido-5-amino-N-(4-methoxybenzyl)-3-
(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide
(5h): white solid yield 80%, mp: 115e117 ꢂC. 1H NMR (400 MHz,
D2O)
hydrochloride
(3R,4R,5S)-4-acetamido-5-amino-N-(3-fluorophenethyl)-3-
d
7.18 (d, J ¼ 8.3 Hz, 2H), 6.88 (d, J ¼ 8.2 Hz, 2H), 6.33 (s, 1H),
(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide
(5m): white solid yield 53%, mp: 116e117 ꢂC. 1H NMR (400 MHz,
D2O)
7.25 (ddd, J ¼ 8.9, 7.5, 6.4 Hz, 1H), 7.03e6.82 (m, 3H), 6.04 (s,
1H), 4.15 (d, J ¼ 8.9 Hz, 1H), 3.92 (dd, J ¼ 11.7, 9.0 Hz, 1H), 3.51e3.32
hydrochloride
4.28 (d, J ¼ 3.4 Hz, 2H), 4.20 (d, J ¼ 8.5 Hz, 1H), 3.96 (dd, J ¼ 11.6,
9.0 Hz, 1H), 3.72 (s, 3H), 3.55e3.45 (m, 1H), 3.44e3.37 (m, 1H), 2.79
(dd, J ¼ 16.9, 5.4 Hz, 1H), 2.55e2.38 (m, 1H), 1.99 (s, 3H), 1.50e1.31
d

228
H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
(m, 4H), 2.77 (t, J ¼ 6.5 Hz, 2H), 2.67 (dd, J ¼ 16.9, 5.5 Hz, 1H),
2.44e2.32 (m, 1H), 1.98 (s, 3H), 1.51e1.30 (m, 4H), 0.75 (dt, J ¼ 9.9,
3.92 (s, 1H), 3.84e3.67 (m, 1H), 3.39 (d, J ¼ 6.1 Hz, 1H), 2.93 (s, 1H),
2.80e2.60 (m, 1H), 2.39 (s, 1H), 1.89 (s, 3H), 0.98 (t, J ¼ 45.4 Hz, 5H),
7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d
175.22 (s), 169.24 (s), 162.58
0.57e0.20 (m, 7H); 13C NMR (100 MHz, D2O)
d 174.83 (s), 168.49 (s),
(d, 1J
¼ 237.0 Hz), 141.75 (d, J ¼ 7.5 Hz), 131.68 (s), 130.92 (s),
141.24 (s), 137.09 (s), 133.92 (s), 132.07 (s), 130.91 (s), 129.72 (s),
127.88 (s), 126.58 (s), 126.05 (d, J ¼ 6.5 Hz), 125.82 (s), 119.97 (s),
82.76 (s), 74.72 (s), 63.99 (s), 52.74 (s), 49.01 (s), 25.51 (s), 25.08 (s),
22.65 (s), 22.48 (s), 8.93 (s), 8.43 (s). HRMS (ESI): calcd for
CF
130.13 (d, J ¼ 8.5 Hz),124.82 (d, J ¼ 2.6 Hz), 84.27 (s), 75.09 (s), 52.66
(s), 49.12 (s), 40.07 (s), 34.10 (s), 28.39 (s), 25.44 (s), 25.03 (s), 22.33
(s), 8.49 (s), 8.47 (s). HRMS (ESI): calcd for C22H32FN3O3 [MþH]þ
406.2506; found m/z: 406.2505.
C
25H33N3O3S [MþH]þ 456.2321; found m/z: 456.2317.
(3R,4R,5S)-4-acetamido-5-amino-N-(3-fluorobenzyl)-N-
methyl-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide hydro-
chloride (5n): grey solid yield 46%, mp: 117e119 ꢂC. 1H NMR
(3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)-N-(4-
(thiophen-2-yl)benzyl)cyclohex-1-ene-1-carboxamide hydrochlo-
ride (5s): green solid yield 71%, mp: 172e174 ꢂC. 1H NMR (400 MHz,
(400 MHz, D2O)
d
7.13 (dd, J ¼ 14.2, 7.6 Hz, 1H), 6.99e6.69 (m, 3H),
D2O)
d
6.81 (d, J ¼ 7.5 Hz, 2H), 6.64 (d, J ¼ 7.9 Hz, 2H), 6.52 (s, 1H),
5.96 (s, 1H), 4.04 (t, J ¼ 22.9 Hz, 1H), 3.92e3.74 (m, 1H), 3.53e3.13
(m, 4H), 2.86e2.50 (m, 3H), 2.33 (dd, J ¼ 15.0, 12.1 Hz, 1H), 1.93 (s,
3H),1.43e1.13 (m, 4H), 0.76e0.54 (m, 6H); 13C NMR (100 MHz, D2O)
6.39 (d, J ¼ 4.6 Hz, 1H), 6.25 (d, J ¼ 3.4 Hz, 1H), 5.72 (s, 1H), 3.85 (dd,
J ¼ 44.9, 15.0 Hz, 2H), 3.69 (s, 1H), 3.62e3.45 (m, 1H), 3.16 (d,
J ¼ 7.0 Hz, 1H), 2.72 (s, 1H), 2.58e2.40 (m, 1H), 2.16 (s, 1H), 1.67 (s,
3H), 0.97e0.68 (m, 4H), 0.20 (dt, J ¼ 49.7, 6.9 Hz, 6H); 13C NMR
d
175.16 (s), 169.10 (s), 162.59 (d, 1 J
¼ 243.2 Hz), 141.76 (d,
CF
J ¼ 7.3 Hz), 131.26 (s), 130.91 (s), 130.08 (d, J ¼ 8.5 Hz), 124.77 (d,
J ¼ 2.4 Hz), 115.59 (s), 115.39 (s), 113.22 (s), 113.01 (s), 84.10 (s),
75.06 (s), 52.69 (s), 49.07 (s), 40.26 (s), 34.15 (s), 28.47 (s), 25.48 (s),
25.05 (s), 22.41 (s), 8.61 (s),8.49 (s). HRMS (ESI): calcd for
(100 MHz, DMSO-d6) d 171.04 (s), 166.57 (s), 143.67 (s), 139.57 (s),
132.78 (s), 132.39 (s), 130.89 (s), 128.92 (s), 128.45 (s), 125.91 (s),
125.80 (s), 123.95 (s), 81.50 (s), 74.72 (s), 52.95 (s), 49.59 (s), 42.47
(s), 29.18 (s), 26.16 (s), 25.44 (s), 23.83 (s), 9.89 (s), 9.39 (s). HRMS
(ESI): calcd for
456.2318.
C
22H32FN3O3 [MþH]þ 406.2506; found m/z: 406.2505.
C
25H33N3O3S [MþH]þ 456.2321; found m/z:
(3R,4R,5S)-4-acetamido-5-amino-N-(4-fluorophenyl)-3-(pen-
tan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride (5o):
(3R,4R,5S)-4-acetamido-5-amino-N-(2-morpholinoethyl)-3-
(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide hydrochloride
(5t): yellow solid yield 33%, mp: 179e181 ꢂC. 1H NMR (400 MHz,
D2O)
yellow solid yield 70%, mp: 120e122 ꢂC. 1H NMR (400 MHz, D2O)
d
7.42e7.19 (m, 2H), 7.06 (t, J ¼ 8.8 Hz, 2H), 6.43 (s, 1H), 4.19 (d,
J ¼ 9.0 Hz,1H), 3.86 (dd, J ¼ 11.4, 9.2 Hz,1H), 3.49e3.38 (m,1H), 3.27
(td, J ¼ 11.0, 5.5 Hz, 1H), 2.78 (dd, J ¼ 17.0, 5.3 Hz, 1H), 2.47e2.30 (m,
1H), 2.00 (s, 3H), 1.42 (dtd, J ¼ 40.1, 14.2, 7.4 Hz, 4H), 0.77 (dt,
d
6.43 (s, 1H), 4.25 (d, J ¼ 8.9 Hz, 1H), 4.14e3.89 (m, 3H), 3.76
(t, J ¼ 12.3 Hz, 2H), 3.64e3.40 (m, 6H), 3.30 (t, J ¼ 6.0 Hz, 2H), 3.16 (t,
J ¼ 10.9 Hz, 2H), 2.82 (dd, J ¼ 16.8, 5.4 Hz, 1H), 2.59e2.39 (m, 1H),
2.00 (s, 3H), 1.43 (dtd, J ¼ 21.3, 14.2, 7.1 Hz, 4H), 0.94e0.63 (m, 6H);
J ¼ 14.5, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.05 (s), 168.34
(s), 160.27 (d, 1 J ¼ 242.9 Hz), 133.17 (s), 132.33 (d, J ¼ 2.8 Hz),
13C NMR (100 MHz, D2O)
d 175.27 (s), 169.62 (s), 133.42 (s), 129.99
CF
131.68 (s), 125.02 (d, J ¼ 8.0 Hz), 115.86 (s), 115.63 (s), 84.33 (s),
75.63 (s), 54.61 (s), 49.10 (s), 30.15 (s), 25.45 (s), 25.05 (s), 22.35 (s),
8.49 (s). HRMS (ESI): calcd for C20H28FN3O3 [MþH]þ 378.2193;
found m/z: 378.2183.
(s), 84.53 (s), 75.12 (s), 63.68 (s), 56.24 (s), 52.66 (s), 52.01 (s), 49.12
(s), 33.99 (s), 28.37 (s), 25.44 (s), 25.05 (s), 22.39 (s), 8.51 (s), 8.49
(s). HRMS (ESI): calcd for C20H36N4O4 [MþH]þ 397.2815; found m/z:
397.2813.
(3R,4R,5S)-4-acetamido-5-amino-N-(4-(3-oxomorpholino)
phenyl)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide hydro-
chloride (5p): white solid yield 80%, mp: 193e195 ꢂC. 1H NMR
(3R,4R,5S)-4-acetamido-N-allyl-5-amino-3-(pentan-3-yloxy)
cyclohex-1-ene-1-carboxamide hydrochloride (5u): white solid
yield 89%, mp: 149e152 ꢂC. 1H NMR (400 MHz, D2O)
d 6.37 (s, 1H),
(400 MHz, D2O)
d
7.54e7.44 (m, 2H), 7.39e7.27 (m, 2H), 6.51 (s, 1H),
5.79 (ddd, J ¼ 15.7, 10.2, 5.0 Hz, 1H), 5.10 (dd, J ¼ 7.3, 1.3 Hz, 1H), 5.06
(s, 1H), 4.24 (d, J ¼ 9.0 Hz, 1H), 4.00 (dd, J ¼ 11.7, 9.0 Hz, 1H), 3.79 (d,
J ¼ 5.0 Hz, 2H), 3.54 (td, J ¼ 10.9, 5.6 Hz, 1H), 3.45 (dd, J ¼ 11.2,
5.6 Hz, 1H), 2.81 (dd, J ¼ 16.9, 5.4 Hz, 1H), 2.59e2.36 (m, 1H),
2.07e1.93 (m, 3H), 1.43 (ddt, J ¼ 28.2, 14.2, 7.3 Hz, 4H), 0.78 (dt,
4.31 (s, 3H), 4.12e3.98 (m, 3H), 3.73 (dd, J ¼ 9.8, 4.9 Hz, 2H),
3.63e3.56 (m, 1H), 3.53e3.46 (m, 1H), 2.92 (dd, J ¼ 17.0, 5.5 Hz, 1H),
2.60 (ddt, J ¼ 16.7, 10.6, 2.9 Hz, 1H), 2.03 (s, 3H), 1.47 (dtd, J ¼ 21.1,
14.2, 7.1 Hz, 4H), 0.85e0.74 (m, 6H); 13C NMR (100 MHz, D2O)
d
175.27 (s), 169.68 (s), 167.86 (s), 137.52 (s), 136.36 (s), 133.18 (s),
J ¼ 14.8, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.25 (s), 169.01
131.11 (s), 126.91 (s), 123.34 (s), 84.47 (s), 75.18 (s), 67.07 (s), 63.62
(s), 52.73 (s), 49.80 (s), 49.18 (s), 28.51 (s), 25.48 (s), 25.05 (s), 22.42
(s), 8.51 (s). HRMS (ESI): calcd for C24H34N4O5 [MþH]þ 459.2607;
found m/z:459.2603.
(s), 133.29 (s), 132.35 (s), 130.62 (s), 115.58 (s), 84.41 (s), 75.16 (s),
52.70 (s), 49.18 (s), 41.73 (s), 28.45 (s), 25.45 (s), 25.09 (s), 22.38 (s),
8.50 (s), 8.47 (s). HRMS (ESI): calcd for C17H29N3O3 [MþH]þ
324.2287; found m/z: 324.2279.
(3R,4R,5S)-N-([1,10-biphenyl]-4-ylmethyl)-4-acetamido-5-
amino-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide hydro-
chloride (5q): yellow solid yield 35%, mp: 150e152 ꢂC. 1H NMR
4.1.5. General procedure for the synthesis of compounds 8aꢀ8f
To a solution of acid 3 (0.38 g, 1 mmol), HATU (0.38 g, 1 mmol),
and Et3N (1 mL) in 20 mL of dry CH2Cl2 was added an equimolar
amount of the appropriate amino acid methyl ester. The mixture
was kept stirring at room temperature for 48 h. After TLC detection
to show no starting materials, the mixture was extracted with
100 mL ethyl acetate and washed with saturated NaHCO3
(3 ꢁ 30 mL) and saturated NaCl (1 ꢁ 50 mL). The organic layers
were dried with MgSO4 and evaporated under vacuum. The crude
product was purified by column chromatography with ethyl ace-
tate: petroleum ether ¼ 1: 1 to obtain intermediates 6a-6f. To the
mixture solution of 6a (0.2 g, 0.4 mmol) in methanol, 4N NaOH
aqueous solution was added until the pH to 12, and then the so-
lution was stirred at room temperature for 2 h. The reaction solu-
tion was evaporated under reduced pressure to remove methanol,
then 3M HCl was added until no more precipitate formed. The
precipitate was filtrated and washed with H2O, dried to afford 7a-7f
(400 MHz, D2O)
d
6.98 (dd, J ¼ 15.7, 8.7 Hz, 6H), 6.77 (dt, J ¼ 26.7,
7.2 Hz, 3H), 5.99 (s, 1H), 4.26 (d, J ¼ 15.2 Hz, 1H), 4.11 (d, J ¼ 14.9 Hz,
1H), 4.01 (d, J ¼ 7.8 Hz, 1H), 3.91e3.79 (m, 1H), 3.59e3.38 (m, 1H),
3.03e2.87 (m, 1H), 2.76 (dd, J ¼ 26.3, 17.0 Hz, 1H), 2.48 (s, 1H),
2.14e1.83 (m, 3H), 1.16e0.89 (m, 4H), 0.52 (dd, J ¼ 14.7, 7.3 Hz, 3H),
0.35 (t, J ¼ 7.1 Hz, 3H); 13C NMR (100 MHz, D2O)
d 174.90 (s), 168.68
(s), 140.11 (s), 139.05 (s), 137.49 (s), 132.11 (s), 131.05 (s), 128.44 (s),
127.86 (s), 126.60 (d, J ¼ 17.2 Hz), 82.77 (s), 74.78 (s), 52.85 (s), 49.09
(s), 42.46 (s), 28.83 (s), 25.53 (s), 25.14 (s), 22.54 (s), 8.90 (s), 8.48
(s). HRMS (ESI): calcd for C27H35N3O3 [MþH]þ 450.2756; found m/
z: 450.2750.
(3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)-N-(4-
(thiophen-3-yl)benzyl)cyclohex-1-ene-1-carboxamide hydrochlo-
ride (5r): yellow solid yield 57%, mp: 164e166 ꢂC. 1H NMR
(400 MHz, D2O) d 7.11e6.61 (m, 7H), 5.93 (s, 1H), 4.23e3.96 (m, 2H),

H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
229
as white solid. A solution of compound 7a (0.1 g, 0.2 mmol) and TFA
(3.0 mL, 39.0 mmol) in CH2Cl2 (3 mL) was stirred at room temper-
ature for 12 h. The solvent was evaporated and purified by column
chromatography with methanol: CH2Cl2 ¼ 1: 10. The solvent was
evaporated and dried under vacuum to get solid compounds 8a-8f.
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carbonyl)valine acid trifluoroacetate salt (8a): white
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carbonyl)glycine acid trifluoroacetate salt (8f): white
solid yield 58%, mp: 81e83 ꢂC. 1H NMR (400 MHz, D2O)
6.46 (s,
d
1H), 4.26 (d, J ¼ 8.7 Hz, 1H), 4.09e3.98 (m, 1H), 3.91 (s, 2H), 3.55 (td,
J ¼ 11.1, 5.6 Hz, 1H), 3.47 (dd, J ¼ 11.2, 5.6 Hz, 1H), 2.85 (dd, J ¼ 16.8,
5.4 Hz, 1H), 2.58e2.46 (m, 1H), 2.02 (s, 3H), 1.45 (dtd, J ¼ 21.2, 14.2,
7.0 Hz, 4H), 0.79 (dt, J ¼ 14.9, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
solid yield 78%, mp: 130e133 ꢂC. 1H NMR (400 MHz, D2O)
d
6.35 (d,
d 196.96 (s), 175.24 (s), 169.16 (s), 133.09 (s), 130.12 (s), 84.42 (s),
J ¼ 2.1 Hz,1H), 4.19 (dd, J ¼ 10.4, 6.3 Hz, 2H), 4.05e3.93 (m,1H), 3.51
(td, J ¼ 10.8, 5.2 Hz,1H), 3.46 (dd, J ¼ 10.6, 5.2 Hz,1H), 2.86e2.71 (m,
1H), 2.60e2.41 (m, 1H), 2.20e2.07 (m, 1H), 1.99 (d, J ¼ 7.0 Hz, 3H),
1.57e1.33 (m, 4H), 0.98e0.80 (m, 6H), 0.77 (dd, J ¼ 15.8, 7.9 Hz, 6H);
75.13 (s), 52.64 (s), 49.14 (s), 28.29 (s), 25.43 (s), 25.04 (s), 22.35 (s),
8.48 (s), 8.44 (s). HRMS (ESI): calcd for C16H27N3O5 [MþH]þ
342.2029; found m/z: 342.2028.
13C NMR (100 MHz, D2O)
d
175.36 (s), 175.26 (d, J ¼ 6.5 Hz), 169.87
4.2. In vitro NA inhibitory assay
(s) 132.67 (d, J ¼ 10.5 Hz), 130.52 (d, J ¼ 4.1 Hz), 84.36 (d, J ¼ 2.7 Hz),
75.11 (d, J ¼ 10.5 Hz), 58.91 (d, J ¼ 16.6 Hz), 52.66 (s), 49.08 (s), 29.78
(d, J ¼ 11.8 Hz), 28.44 (d, J ¼ 6.5 Hz), 25.40 (s), 25.11 (d, J ¼ 8.0 Hz),
22.33 (s), 18.34 (d, J ¼ 6.2 Hz), 17.55 (d, J ¼ 11.4 Hz), 8.46 (s). HRMS
(ESI): calcd for C19H33N3O5 [MþH]þ 384.2498; found m/z: 384.2497.
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
Materials Used for Enzyme Inhibitory Assay: Influenza virus A/
chicken/china/1220/2012(H5N1) and A/duck/Guangdong/674/2014
(H5N6) were kindly provided by the College of Veterinary Medi-
cine, South China Agricultural University, P. R. China. The H5N1-
H274Y NA was obtained from Sino Biological Inc. The substrate
hex-1-ene-1-carbonyl)-
D
-valine acid trifluoroacetate salt (8b):
used in the enzyme inhibition assay, 2'-(4-methylumbelliferyl)-
a-
white solid yield 74%, mp: 126e128 ꢂC. 1H NMR (400 MHz, D2O)
D-acetylneuraminic acid sodium salt hydrate (4-MU-NANA) (Sigma,
d
6.35 (s, 1H), 4.21 (dd, J ¼ 13.3, 7.7 Hz, 2H), 4.04e3.95 (m, 1H),
M8639), was purchased from Sigma. Influenza virus suspensions
were obtained from the allantoic fluid of the chicken embryo layer.
And the substrate, 4-MU-NANA, was cleaved with NA to yield a
quantifiable fluorescent product. The tested compounds were dis-
solved and diluted to the corresponding concentrations in MES
buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid, 4 mM
3.58e3.50 (m, 1H), 3.49e3.40 (m, 1H), 2.82 (dd, J ¼ 17.2, 5.4 Hz, 1H),
2.55e2.45 (m, 1H), 2.14 (dd, J ¼ 13.4, 6.7 Hz, 1H), 2.00 (s, 3H),
1.52e1.36 (m, 4H), 0.90 (d, J ¼ 6.8 Hz, 6H), 0.82e0.74 (m, 6H); 13C
NMR (100 MHz, D2O)
d 175.24 (s), 175.20 (s), 169.86 (s), 132.64 (s),
130.54 (s), 84.37 (s), 75.07 (s), 58.93 (s), 52.65 (s), 49.09 (s), 29.72
(s), 28.43 (s), 25.41 (s), 25.08 (s), 22.34 (s), 18.31 (s), 17.62 (s), 8.46
(s). HRMS (ESI): calcd for C19H33N3O5 [MþH]þ 384.2498; found m/
z: 384.2488.
CaCl2, pH 6.5). In a 96-well plate, 10
mL of the diluted virus super-
natant, 70 mL of MES buffer, and 10 mL of compounds at different
concentrations were added successively and incubated at 37 ꢂC for
10 min. Then the reaction was started by the addition of the sub-
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carbonyl)-
L
-valine acid trifluoroacetate salt (8c):
strate. After incubation for 40 min, 150 mL of 0.2M glycine-NaOH
white solid yield 70%, mp: 118e120 ꢂC. 1H NMR (400 MHz, D2O)
(pH 10.2) were added in the solution to terminate the reaction
solution. Fluorescence was recorded (excitation at 365 nm and
emission at 455 nm), and substrate blanks were subtracted from
the sample readings. The 50% inhibitory concentration (IC50) was
calculated by plotting the percent inhibition of NA activity versus
the inhibitor concentration [17].
d
6.38 (s, 1H), 4.23 (dd, J ¼ 16.2, 7.2 Hz, 2H), 4.01 (dd, J ¼ 11.5, 9.0 Hz,
1H), 3.58e3.50 (m, 1H), 3.47 (dd, J ¼ 10.7, 5.3 Hz, 1H), 2.79 (dd,
J ¼ 17.1, 5.4 Hz, 1H), 2.58e2.47 (m, 1H), 2.16 (td, J ¼ 13.3, 6.6 Hz, 1H),
2.02 (s, 3H), 1.43 (ddd, J ¼ 21.1, 13.4, 6.9 Hz, 4H), 0.98e0.83 (m, 6H),
0.79 (dt, J ¼ 15.1, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 175.56 (s),
175.24 (s), 169.81 (s), 132.69 (s), 130.54 (s), 84.34 (s), 75.18 (s), 58.97
(s), 52.68 (s), 49.09 (s), 29.91 (s), 28.49 (s), 25.42 (s), 25.16 (s), 22.34
(s), 18.42 (s), 17.49 (s), 8.47 (s). HRMS (ESI): calcd for C19H33N3O5
[MþH]þ 384.2498; found m/z: 384.2493.
4.3. Molecular docking
Molecular modeling of molecule 8b was performed using the
Tripos molecular modeling packages Sybyl-X 2.0 [27]. Through the
Tripos force field, 8b was optimized for 2000-generations till the
maximum derivative of energy became 0.005 kcal/(mol*A). The
flexible docking method (Surflex-Dock) docked the ligand auto-
matically into the receptor ligand binding site by the use of
protocol-based approach and empirically derived scoring function.
The protein was prepared by removing the ligand, water molecules,
and other unnecessary small molecules from the crystal structure
of the ligand NA complex (PDB code 2HU0) prior to docking; polar
hydrogen atoms and charges were added to the protein. Surflex-
Dock default settings were used for other parameters, such as the
maximum number of rotatable bonds per molecule (set to 100) and
the maximum number of poses per ligand (set to 20). The atomic
charges were recalculated using the Kollman all-atom approach for
the protein and the Gasteig-Hückel approach for the ligand. And
the binding interaction energy was calculated, including van der
Waals, electrostatic, and torsional energy terms defined in the
Tripos force field. After the protocol was generated, the optimized
8b was docked into the binding pocket and to define the binding
interaction.
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carbonyl)-
D
-alanine acid trifluoroacetate salt (8d):
yellow solid yield 66% mp: 160e162 ꢂC. 1H NMR (400 MHz, D2O)
d
6.42 (s, 1H), 4.34 (q, J ¼ 7.3 Hz, 1H), 4.24 (d, J ¼ 8.9 Hz, 1H), 4.00
(dd, J ¼ 11.7, 8.9 Hz, 1H), 3.54 (td, J ¼ 10.9, 5.6 Hz, 1H), 3.46 (dd,
J ¼ 11.1, 5.6 Hz, 1H), 2.88e2.77 (m, 2H), 2.57e2.45 (m, 1H), 2.01 (s,
3H), 1.48 (dd, J ¼ 13.0, 6.1 Hz, 4H), 1.38 (d, J ¼ 7.3 Hz, 3H), 0.79 (dt,
J ¼ 14.9, 7.4 Hz, 6H); 13C NMR (100 MHz, D2O)
d 176.66 (s), 175.25
(s), 169.01 (s), 133.03 (s), 130.18 (s), 84.43 (s), 75.12 (s), 52.66 (s),
49.18 (s), 49.13 (s), 28.34 (s), 25.44 (s), 25.03 (s), 22.36 (s), 15.91 (s),
8.48 (s), 8.44 (s). HRMS (ESI): calcd for C17H29N3O5 [MþH]þ
356.2185; found m/z: 356.2179.
((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carbonyl)-
L
-alanine acid trifluoroacetate salt (8e):
white solid yield 75%, mp: 96e99 ꢂC. 1H NMR (400 MHz, D2O)
d
6.40
(s, 1H), 4.34 (q, J ¼ 7.3 Hz, 1H), 4.24 (d, J ¼ 9.0 Hz, 1H), 4.00 (dd,
J ¼ 11.7, 8.9 Hz, 1H), 3.53 (td, J ¼ 10.8, 5.6 Hz, 1H), 3.49e3.39 (m, 1H),
2.81 (dd, J ¼ 16.9, 5.4 Hz, 1H), 2.50 (ddt, J ¼ 16.7, 10.4, 3.0 Hz, 1H),
2.00 (s, 3H), 1.56e1.30 (m, 7H), 0.78 (dt, J ¼ 14.9, 7.4 Hz, 6H); 13C
NMR (100 MHz, D2O)
d 176.50 (s), 175.20 (s), 169.10 (s), 132.91 (s),
130.15 (s), 84.44 (s), 75.09 (s), 52.61 (s), 49.07 (s), 49.01 (s), 28.31 (s),
25.38 (s), 24.96 (s), 22.29 (s), 15.77 (s), 8.43 (s), 8.37 (s). HRMS (ESI):
calcd for C17H29N3O5 [MþH]þ 356.2185; found m/z: 356.2184.
Amino acid sequences of 2HU0 compared with that of H5N1-
1220 NA (tested by our cooperator Institute of Poultry Science,
Shandong Academy of Agricultural Sciences, the different residues

230
H. Ju et al. / European Journal of Medicinal Chemistry 146 (2018) 220e231
were marked in red and in bold).
the chick embryos was checked by candling the eggs, and reaf-
firmed through observing the breakdown of the visible blood vessel
structures of the chorioallantoic membrane, the appearance of
disseminated coagulation, blood leakage into the yolk or the
allantoic fluid areas, the absence of movement of the embryo, and
extensive autolysis. At the same time, to study the effect of the
operational procedure on the viability of the chick embryos, eggs
were handled as the same as the treatment experiments, but only
treated with 0.9% NaCl (no virus and compounds), and as control
experiments, the eggs were inoculated by the mixture of virus and
0.9% NaCl. Finally, the number of the survival and death were
recorded to evaluate the anti-influenza virus activity of the com-
pound [26].
NA, 2HU0 (87~454):
GNSSLCPINGWAVYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLN
DKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSWLTI
GISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPS
NGQASYKIFKMEKGKVVKSVELDAPNYHYEECSCYPNAGEITCVCRDNWHG
SNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSF
KYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYS
GSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVG
H5N1-1220 NA (87~454):
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
GNSSLCPI
DKHSNGTVKDRSPHR
IGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDG
PSNGQASYKIFK EKGKVVKSVELDAPNYHYEECSCYPDSGEITCVCRDNWH
GSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVS NGAYGVKGF
SGWAVH
SKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLN
A
LMSCPVGEAPSPYNSRFESVAWSASACHDGT WLT
G
This work was supported by National Natural Science Founda-
tion of China (NSFC No. 81773574), the Key Project of NSFC for In-
ternational Cooperation (No. 81420108027), the Science and
Technology Development Project of Shandong Province (No.
2014GSF118175), and Major Project of Science and Technology of
Shandong Province (2015ZDJS04001), Key research and develop-
ment project of Shandong Province (No. 2017CXGC1401).
L
P
SFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSG
YSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVG.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.01.050.
4.4. In vitro anti-influenza virus assay and cytotoxicity assay
The in vitro anti-influenza virus activities of NA inhibitors were
measured by the EC50 values, the concentrations of the compound
required for 50% protection of the influenza virus infection-
mediated cytopathic effects (CPE). Chicken embryo fibroblast cells
were grown in 96-well plates for 24 h at 37 ꢂC. The compounds
dissolved in water were serially diluted by 3-fold in DMEM (con-
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