Discovery of novel “Dual-site” binding oseltamivir derivatives as potent influenza virus neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112147

From our research group, it was noticed that oseltamivir derivatives targeting 150-cavity of neuraminidase enzyme (NA) could significantly increase antiviral activity. Thus, we further enriched the C5–NH₂ position of oseltamivir structure to ob

Full text of DOI:10.1016/j.ejmech.2020.112147

Journal Pre-proof
Discovery of novel “Dual-site” binding oseltamivir derivatives as potent influenza virus
neuraminidase inhibitors
Jian Zhang, Wei Ai, Waleed A. Zalloum, Ruifang Jia, Srinivasulu Cherukupalli, Xiao
Ding, Zhuosen Sun, Lin Sun, Xiangyi Jiang, Xiuli Ma, Zhong Li, Defeng Wang, Bing
Huang, Peng Zhan, Xinyong Liu
PII:
S0223-5234(20)30114-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112147
EJMECH 112147
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2019
Revised Date: 12 February 2020
Accepted Date: 13 February 2020
Please cite this article as: J. Zhang, W. Ai, W.A. Zalloum, R. Jia, S. Cherukupalli, X. Ding, Z. Sun, L.
Sun, X. Jiang, X. Ma, Z. Li, D. Wang, B. Huang, P. Zhan, X. Liu, Discovery of novel “Dual-site” binding
oseltamivir derivatives as potent influenza virus neuraminidase inhibitors, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112147.
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Discovery of Novel “Dual-Site” Binding Oseltamivir Derivatives
as Potent Influenza Virus Neuraminidase Inhibitors
b#
a#
d
a
a
Jian Zhang , Wei Ai , Waleed A. Zalloum , Ruifang Jia , Srinivasulu Cherukupalli
,
a
a
a
a
c
a
Xiao Ding , Zhuosen Sun , Lin Sun , Xiangyi Jiang , Xiuli Ma , Zhong Li , Defeng
a
c,*
a,*
a,*
Wang , Bing Huang , Peng 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, P.R. China.
b
The Second Hospital of Shandong University, No. 247 Beiyuan Avenue, 250033,
Jinan, Shandong, P.R. China.
c
Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 1 Jiaoxiao
Road, Jinan, Shandong, 250023, P.R. China.
d
Department of Pharmacy, Faculty of Health Science, American University of
Madaba, P.O Box 2882, Amman, 11821, Jordan.
#
These authors contributed equally to this work and they should be considered
co-first authors
*
E-mails: hbind@163.com (Huang B.); zhanpeng1982@sdu.edu.cn (Zhan P.);
xinyongl@sdu.edu.cn (Liu X.Y.)
Abstract:
From our research group, it was noticed that oseltamivir derivatives targeting
1
50-cavity of neuraminidase enzyme (NA) could significantly increase antiviral
activity. Thus, we further enriched the C5-NH position of oseltamivir structure to
2
obtain more potent oseltamivir derivatives. In this article a series of oseltamivir
derivatives were synthesized by modifying C5-NH position of oseltamivir. All the
2
1

compounds were evaluated for in vitro antiviral activity against H5N1 and H5N8.
Encouragingly, compounds 9a and 11e were exhibited prominent activity, which is
similar to oseltamivir carboxylate (OSC) and in NAs inhibitory assay, 11e showed
remarkable potency against N1 (H5N1), N2 (H5N2), N6 (H5N6) and N8 (H5N8). In
addition, 11e demonstrated low cytotoxicity and no obvious toxicity at the dose of
1
500 mg/kg in mice. Molecular docking studies of 9a and 11e provided a plausible
rationale for the high potency against group-1 NAs. This work provided new insights
to design further neuraminidase inhibitors, which can help to investigate new potent
inhibitors for group-1 and group-2 shortly.
Keywords: Influenza virus, Neuraminidase inhibitors, Oseltamivir derivatives,
1
50-cavity, Active site.
1
. Introduction
Influenza is an acute respiratory infectious disease that can be life-threatening.
According to the specific antigens of matrix protein (M1) and nuclear protein (NP),
the influenza virus can be divided into four types: A, B, C, and D [1]. Among them,
influenza A virus has the strongest and most frequent variability and the cause of
influenza epidemic almost every year. Accordingly, influenza A virus is considered
to be the main pathogens involved in influenza outbreaks. [2, 3]. During the outbreak
of the influenza virus H1N1 in 2009, about 300,000 people died around the world
which caused great social panic [4]. Globally, from 2013 to 2017, a total of 1161
people were infected with H7N9, 433 deaths and a mortality rate of 37.3% [5]. Each
round of flu outbreaks will be unexpected, causing great social and economic loss.
According to the World Health Organization (WHO) statistical data, there are about
2
90,000-650,000 death cases every year caused by influenza [2].
The influenza virus is a negative-sense, single-stranded RNA virus with a
2

lipid-containing envelope [6]. The viral membrane contains two important surface
glycoproteins: neuraminidase (NA) and hemagglutinin (HA) [7]. They are prone to
antigen transformation and cause mutation. NA contains 11 subtypes (N1-N11), HA
contains 18 subtypes (H1-H18), and the influenza A viruses found so far are different
combinations of HA and NA [8, 9, 10]. NA plays a crucial role in the whole
replication cycle of the virus, which has been an attractive therapeutic target in the
field of anti-influenza drugs [11]. At present, four kinds of neuraminidase inhibitors
have been approved for clinical treatment, including Oseltamivir (Tamiflu) [12],
Zanamivir (Relenza) [13], Peramivir (Rapivab) [14] and Laninamivir octanoate
(
Inavir) [15] (Figure 1). However, oseltamivir is only administered orally, also it is
the earliest clinical first-line drug to treat influenza (approved in 1999). In recent
years, the emergence of drug-resistant viral strains has restricted its clinical use,
specially N1-H274Y mutant [16, 17, 18]. In addition, the emergence of other NA
inhibitors resistant strains has also been reported. Therefore, there is an urgent need
to develop a new generation of NA inhibitors to solve these issues.
3

O
O
HO
O
O
R
O
NH2
OH
H
OH
H N
2
HN
N
H
HN
NH
OH
O
O
Oseltamivir, R = Et
Oseltamivir carboxylate (OSC), R=H
Zanamivir
O
OH
HO
O
OH
NH2
O
H
OH
H
HN
O
HN
N
NH
H
n
NH
O
O
H N
2
HN
O
O
n=6
Peramivir
Laninamivir octanoate
NA inhibitors
Figure 1. Structures of four neuraminidase inhibitors used in clinical for the treatment of flu.
The N1-N9 subtypes of influenza A virus can be divided into two groups:
group-1 NAs, which include N1, N4, N5, and N8 subtypes, and group-2 which
include N2, N3, N6, N7 and N9 subtypes [19]. X-ray crystallographic studies of
NAs showed that the 150-loop of group-1 NAs is usually present in an open
conformation while the 150-loop of group-2 NAs is present in a closed conformation
[
19]. Due to the unusual structure of these two groups of NAs 150-loop, a new
cavity (150-cavity) with a volume of 10 Å × 5 Å × 5 Å is formed near the active site
of group-1 NAs. But this cavity does not exist in group-2 NAs (Figure 2). In N2, for
example, there is a key salt bridge between conservative residues Asp147 and
His150, which is thought to be able to close 150-loop and control the formation of
1
50-cavity [20, 21]. Crystal structure studies indicate that the 150-loop of 09N1 is
closed, and it does not have an open 150-cavity [22]. This study shows that the
1
2
50-loop of 09N1 is different from group-1 NAs but similar to group-2 NAs [20, 23,
4]. Inspired by this discovery, our group previously synthesized several compounds
4

such as JMC01 and JMC02 [11] (Figure 3) by replacing the oseltamivir C-5 amino
group to target 150-cavity. From the results, it was found that, compared to
oseltamivir, the activities of the compounds against N1 (H5N1) were significantly
increased by nearly ten folds [11].
Figure 2. The crystal structures of representative group-1 NAs N1 (H5N1, PDB code: 2HU0), N8
(H5N8, PDB code: 2HT7), 09N1 (H1N1pdm09, PDB code: 3TI6), and group-2 NAs, N2 (H3N2,
PDB code: 4GZP) bound with OSC. In surface representation, it can be seen that N1 and N8 have
an open 150-cavity, but 09N1 and N2 have a closed 150-loop.
5

Figure 3. Previously designed (in our lab) oseltamivir derivatives as group-1 selective NA
inhibitors (JMC01-JMC04) [11, 25] and group-1 and -2 NAs inhibitors (JMC05 and JMC06)
[26].
Based on our earlier discovery of high activity and high selectivity oseltamivir
derivatives (JMC01 and JMC02), the 150-cavity of NAs could be further explored
to yield more potent and more anti-drug-resistant NA inhibitors. Therefore,
structures of JMC01 and JMC02 were optimized by varying substituents on the
amino group, and compounds JMC03-JMC06 were obtained [25, 26]. JMC03 and
JMC04 displayed antiviral activities similar to or better than OSC against H5N1,
H5N2, H5N6, and H5N8. Both compounds were potent group-1 NAs-selective
inhibitors which showed more potency than OSC against the N1, N8, and
N1-H274Y mutant NAs [25]. Compounds JMC05 and JMC06 showed great
activity against group-1 and group-2 NAs, especially for 09N1, N2, N6 and N9
subtypes. They also showed that the inhibitory activity of N1-H274Y and N2-E119V
6

variants increased by nearly 4 and 2.5 times, respectively compared to OSC [26].
In addition, the studies also showed that the amino acid residues of 150-loop
and 150-cavity are quite flexible and have the possibility for further chemical
modifications on the inhibitor structures [20, 24]. By analyzing the structures of the
previously discovered active compounds, it was found that 5-amino substituents are
benzyl derivatives with a single structural type (such as compounds JMC01,
JMC04). Obviously, this cannot meet the needs of flexible 150-cavity, and the
structure-activity relationship studies of substituted benzyl sites still needs to be
further enriched. We expect to enhance the potency against both group -1 and -2
NAs and also improve the resistance for the mutant virus. Stimulated by the above
analysis, we herein more diverse fragments of C5-NH substituted oseltamivir
2
derivatives were designed and synthesized
7

Figure 4. The newly designed compounds are targeting both the active site and 150-cavity. The
benzene ring (silver) was replaced by substituted heterocycle and propenyl group,amido group
and sulfonamide groups were introduced as linkers (pastel orange ).
From the previous literature investigation, it is observed that the substitution of
aromatic rings is beneficial to occupy the space of 150-cavity effectively [11, 25, 26].
Some aromatic rings have been proved to be well adapted to the 150-cavity,
hydrophobic interaction, and van der Waals forces formed between aromatic rings
and hydrophobic amino acid residues in 150-cavity [11, 25]. On the other hand,
hydrogen-bond also formed between the amino group in the N,N-diethylamino
fragment (JMC05) and either Gly147 (of N8, 67 of N9, 66 of 09N1), Thr 361 of N6
or Asp66 of N2 in the 150-cavity [26].
Therefore, we hypothesized that many multiple N-substituted groups of OSC
may efficiently enter and enormously occupy the 150-cavity. Herein we report the
design and synthesis of four series of novel oseltamivir derivatives: 1), we decided to
incorporate some electron-withdrawing groups on the 5-amino substituted benzyl
groups; 2) according to the bioisosterism principle, the benzene or thiophene ring
(silver) in the lead compounds (JMC01 and JMC02) were replaced by the
substituted heterocycle; 3) the linker (pastel orange) between benzene ring substituent
and oseltamivir nucleus was modified by introducing double bond, amide and
sulfonamide group. Ultimately, a total of 38 compounds were designed and
synthesized and their activity were evaluated (Figure 4).
2
2
. Results and discussion
.1. Chemistry
The synthetic routes for oseltamivir derivatives (2a-2g, 4, 6, 9a-9h, 11a-11g,
1
3a-13b, 15a-15e, 17a-17g) were conducted as illustrated in Schemes 1-7. All
8

derivatives were synthesized by well-established methods using commercially
available oseltamivir phosphate as the primary starting material for all reactions. As
shown in Scheme 1, oseltamivir phosphate was reacted with a series of different
commercial aldehydes in the presence of NaBH CN to obtain the key intermediates
3
1
a-1g. Subsequently, 1a-1g were hydrolyzed in the presence of NaOH aqueous
solution and acidified by HCl aqueous solution to afford the target compounds
a-2g.
2
Scheme 2 illustrates the synthesis of compounds 4 and 6, oseltamivir phosphate
was treated with less than 1eq and more than 2eq of commercially available
thiophene-3-carboxaldehyde to obtain the intermediates 3 and 5 respectively by
Borch Reductive Amination. The target compounds 4 and 6 were prepared by direct
hydrolysis of the 3 and 5.
As shown in scheme 3, aryl bromide or bromoaryl aldehyde was coupled with
the corresponding formyl aryl boronic acid or aryl boronic acid to obtain the
intermediates 7 via Suzuki reaction. Subsequently, target compounds 9a-9h were
prepared by using the same method as in Scheme 1.
As outlined in Scheme 4, the intermediates 10a-10g were prepared by Borch
Reduction using different substituents of cinnamaldehyde reacted with oseltamivir
phosphate in the presence of NaBH CN, then they were hydrolyzed to afford the
3
target compounds 11a-11g.
As shown in scheme 5, 12a and 12b were achieved by allowing condensation
reaction between the [1, 1'-biphenyl]-4-carboxylic acid or benzoic acid and
oseltamivir phosphate in the presence of DMAP in CH Cl and Et N. And then they
2
2
3
were hydrolyzed with 4 M NaOH aqueous solution and acidified with 2 M HCl
aqueous solution to obtain the target compounds 13a and 13b.
As shown in Scheme 6, the key intermediates 14a-14e were obtained via the
9

same method as scheme 5. Subsequently, 14a-14e were hydrolyzed by following
the method as scheme 1 to afford the target compounds 15a-15e.
As outlined in scheme 7, the intermediates 16a-16g were prepared by treating
oseltamivir phosphate with a different substituted sulfonyl chloride. Further,
16a-16g were hydrolyzed using the same method in scheme 1 to afford the target
compounds 17a-17g.
Scheme 1. Reagents and conditions: (i) NaBH CN, EtOH, MeOH, r.t.; (ii) NaOH, MeOH, H O,
3
2
r.t, then HCl (2 mol/L).
Scheme 2. Reagents and conditions: (i) NaBH CN, EtOH, MeOH, r.t.; (ii) NaOH, MeOH, H O,
3
2
r.t., then HCl (2 mol/L).
1
0

0
Scheme 3. Reagents and conditions: (i) DMSO, K CO , Pd(PPh ) , N , 100 C. (ii) NaBH CN,
2
3
3 4
2
3
EtOH, MeOH, rt; (iii) NaOH, MeOH, H O, r.t., then HCl (2 mol/L).
2
Scheme 4. Reagents and conditions: (i) NaBH CN, EtOH, MeOH, r.t.; (ii) NaOH, MeOH, H O,
3
2
r.t., then HCl (2 mol/L).
Scheme 5. Reagents and conditions: (i) [1,1'-biphenyl]-4-carboxylic acid or benzoic acid, HBTU
or TBTU, DMAP, Et N, CH Cl , rt; (ii) 4M NaOH, CH OH, rt, then HCl (2 mol/L).
3
2
2
3
1
1

Scheme 6. Reagents and conditions: (i) Substituted cinnamic acid, HBTU or TBTU, DMAP,
Et N, CH Cl , rt; (ii) 4M NaOH, CH OH, rt, then HCl (2 mol/L).
3
2
2
3
Scheme 7. Reagents and conditions: (i) substituted benzenesulfonyl chloride, Et N, CH Cl , rt;
3
2
2
(
ii) 4M NaOH, CH OH, rt, then HCl (2 mol/L).
3
2
2
.2. Biological Activity
.2.1. Anti-Influenza Virus Activity in Cell Culture
In order to study the efficacy of newly synthesized compounds against
influenza virus infection, we evaluated their inhibition abilities against avian
influenza virus proliferation by cell-based assays (chicken embryo fibroblasts, CEFs),
Using A/ goose/ Guangdong/ SH7/ 2013 (H5N1) and A/goose/ Jiangsu/ 1306/ 2014
(H5N8) represent influenza virus of group 1, A/chicken/Hebei/LZF/2014 (H5N2)
and A/Duck/Guangdong/674/2014 (H5N6) represent influenza virus of group 2.
JMC02 oseltamivir carboxylic acid (OSC), Zanamivir (ZAN), and Ribavirin (Rib)
were used as control drugs in parallel. The values of IC (effectiveness of inhibition
5
0
of avian influenza virus proliferation) and CC (cytotoxicity) for the synthesized
5
0
compounds were reported in Table 1. All compounds showed no significant
cytotoxic activities at the highest test concentration (CC > 200 µM) and exhibited
5
0
antiviral activity in CEFs. OSC and ZAN as neuraminidase inhibitors have high
1
2

potency to inhibit the proliferation of four influenza virus subtypes. Furthermore,
they have different inhibitory abilities to N1 and N2 subtypes of influenza viruses,
which is consistent with the literature reports [27]; Rib has moderate (H5N1, H5N6,
H5N8) or poor (H5N2) potency against influenza virus proliferation. It is consistent
with the results of our previous tests [25, 26], which proves that this method has
good reliability.
We focused on the novel oseltamivir derivatives (2a-2g), where the thienyl
group of the 4-benzyl moiety of JMC02 was replaced with various
electron-withdrawing groups containing nitro, cyano group and a fluorine atom. We
also incorporated trifluoromethyl, nitro and fluorine atom at the meta-position of
phenyl (2a, 2c, 2f) to explore the effect of withdrawing groups at different positions.
The results showed that these oseltamivir derivatives were less potent than OSC and
ZAN, especially 2a-2f were almost lost the inhibition activity against H5N1 (IC >
5
0
1
0 μΜ). Further, when the electron-withdrawing group on the benzene ring was
replaced by a bromine atom (2g), little improvement in antiviral activity was
observed and it is still less than OSC and ZAN. Therefore, it concluded that
electron-withdrawing groups would reduce the activity.
To explore whether the various substituents at C5-NH position of oseltamivir
2
will affect the activity, we introduced a thiophen-3-methyl (4) and two
thiophen-3-methyl (6). The results exhibited that these two compounds have equal
inhibition activities to H5N1, H5N2, and H5N6, and as for H5N8, the inhibition
activity of compound 4 surpasses compound 6 more than ten times. However, these
results were not prominent and still lower than OSC and ZAN.
Inspired by the structure of JMC01-JMC04, we designed and synthesized
compounds 9a-9h. The potency of 9a against H5N1 and H5N6 is lower than that of
JMC02, but encouragingly it displayed almost equal activity against H5N2 and
H5N8. Also 9a displayed a prominent selective inhibition to H5N1 and H5N8
compared to OSC and ZAN. It showed similar inhibition activity to OSC and ZAN
1
3

against H5N1 and H5N8 but it is less active against H5N2 and H5N6.
We further exchanged the positions of thiophene and benzene groups of
compound JMC02 to obtain 9b, which slightly reduced the antiviral activity in
contrast to JMC02. And we replaced phenyl of JMC02 to thienyl to afford
compound 9c, which showed the activity against H5N1 and H5N6 was less than
JMC02, especially to H5N1, it was observed nearly lacked inhibition activity.
However, it exhibited similar potency against H5N2 and H5N8 as JMC02. The
substituents at the C5-NH position of 9d and 9e are 2-(p-tolyl)-thiazole and
2
2
-methyl-5-phenylthiazole respectively, and 9d and 9e are a pair of isomers, both
exhibited lower anti-viral activities than OSC, ZAN and JMC02.
The terminal aromatic rings of JMC01, JMC02 and 9a, were re-positioned
from the para-position to the meta-position of the benzene ring to obtain 9f, 9g and
9
h respectively. Among these compounds, 9f displayed similar potency to OSC and
ZAN for H5N1, but lower than that of JMC02. In contrast, 9f showed weaker
activity against H5N1, H5N2 and H5N6 strains compared to OSC, ZAN and
JMC02 while, 9g and 9h displayed similar potency to OSC, ZAN and JMC02
against H5N8. Their activities against H5N1, H5N2 and H5N6 were slightly reduced
compared to OSC and ZAN. The activity of 9h was similar to JMC02 for H5N2 and
H5N6 inhibition, but slightly less for H5N1. The activity of compound 9g against
H5N1, H5N2 and H5N6 was slightly less compared to JMC02.
To enrich the structure-activity relationship studies, we synthesized
phenylpropene derivatives to modify C5-NH of OSC (compounds 11a-11g).
2
However, 11b and 11c, 11e-11g exhibited slightly reduced anti-H5N1 activity
compared to OSC and ZAN. 11d showed considerably decreased potency against
H5N1compared to other compounds; 11a displayed similar potency to OSC of
H5N1.
Notably, from 11a-11g, compound 11e was the most potent compound against
1
4

H5N2, H5N6 and H5N8 strains except 11b (the IC of 11b was 0.065µM against
5
0
H5N2). Moreover, 11e showed comparable activity to JMC02 against H5N2 and
H5N8 and displayed greater potency than JMC02 against H5N6. 11b showed
similar potency to JMC02 against H5N2, H5N6, and H5N8. Compounds 11a, 11c,
11d, 11f and 11g were all less potent than JMC02, OSC, and ZAN for inhibition of
H5N2, H5N6 and H5N8, displayed moderate ability to inhibit viral proliferation.
Further, we inserted an acyl or sulfonyl between the position of C5-NH and
2
aromatic group to obtain compounds 13a, 13b, 15a-15e, and 17a-17g respectively.
Unfortunately, the anti-viral activities of these compounds were sharply reduced or
lost.
Overall, the arylmethyl and phenylpropenyl substituted derivatives at the
C5-NH position of OSC displayed greater activities towards H5N1, H5N2, H5N6,
2
and H5N8 compared to amide and sulfonamide derivatives. Among arylmethyl
substituted OSC derivatives, the stronger the electron-withdrawing ability of the
substituent on the aryl group, the weaker the ability to inhibit virus proliferation.
And, the anti-viral activities of 2a-2f were less than 2g. Biaryl group substituents
consist of benzene and thiophene have similar ability to inhibit viral proliferation
regardless of their relative positions. When the compounds were substituted by
smaller groups of dithiophene or thiazole, the ability to inhibit H5N1 were
significantly reduced.
Among the phenylpropenyl substituted derivatives at the C5-NH position of
2
OSC, most compounds have moderate inhibitory ability against the four strains
except individual compounds that have antiviral activity close to OSC and ZAN for
some strains, such as 11a to H5N1.
1
5

Table 1. Anti-Influenza Virus Activity and Cytotoxicity of Oseltamivir Derivatives in CEFs.
a
IC50 values (µM) against influenza virus
b
Compound
R1
R2
CC50
c
d
e
f
H5N1
H5N2
H5N6
H5N8
Group-1
Group-2
Group-2
Group-1
g
2
2
a
H
＞10
0.34±0.02
3.17±0.23
0.75±0.073
> 200
b
H
H
＞10
＞10
0.21±0.0045
0.24±0.063
2.16±0.025
2.16±0.35
0.42±0.022
0.70±0.19
>200
>200
2
c
2
d
H
＞10
0.38±0.089
＞10
0.68±0.21
>200
1
6

2
e
H
H
H
＞10
＞10
0.11±0.087
0.14±0.012
4.57±0.13
3.87±0.34
1.99±0.32
0.52±0.094
0.49±0.091
0.25±0.017
>200
>200
>200
2
f
2
g
2.53±0.099
0.11±0.000071
4
H
＞10
0.13±0.029
3.58±0.069
0.081±0.0032
>200
6
＞10
0.15±0.011
0.10±0.025
0.12±0.0084
3.03±0.24
5.5±0.21
3.02±0.32
0.88±0.27
0.051±0.0019
0.098±0.013
>200
>200
>200
9
a
H
H
0.88±0.018
1.59±0.037
9
b
9
c
H
H
＞10
＞10
0.083±0.0081
0.20±0.0051
7.12±0.00
3.23±0.27
0.072±0.011
0.059±0.018
>200
>200
9
d
1
7

9
e
H
H
＞10
0.41±0.043
4.29±0.11
0.49±0.045
0.65±0.13
>200
>200
9
f
0.84±0.094
0.23±0.0011
4.49±0.017
9
9
g
H
0.97±0.35
0.22±0.025
6.71±0.00
0.089±0.0038
>200
h
H
H
H
1.25±0.23
0.79±0.25
1.29±0.27
0.070±0.0079
0.16±0.013
2.62±0.36
3.07±0.26
0.056±0.036
0.61±0.077
0.88±0.11
>200
>200
>200
11a
11b
0.065±0.0014
2.78±0.0042
1
1c
H
H
1.20±0.15
7.56±0.48
0.13±0.0012
0.21±0.029
3.07±0.26
0.089±0.012
0.64±0.15
>200
>200
11d
4.07±0.057
1
8

1
1e
H
H
3.4±1.24
0.094±0.023
0.79±0.29
0.077±0.013
>200
11f
0.96±0.011
0.24±0.0095
3.93±0.37
0.53±0.029
>200
11g
H
2.99±0.42
0.29±0.00
5.52±0.34
0.13±0.023
>200
1
3a
H
H
＞10
＞10
＞10
＞10
＞10
＞10
＞10
>200
>200
1
3b
3.54±0.59
1
5a
H
H
＞10
＞10
＞10
＞10
＞10
＞10
5.32±0.48
4.76±0.50
>200
>200
1
5b
1
5c
H
＞10
＞10
＞10
4.37±0.16
>200
1
9

1
5d
H
H
H
H
H
H
H
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
8.48±0.66
5.16±0.44
4.82±0.23
3.12±0.59
5.29±0.56
4.76±0.31
4.25±0.39
>200
>200
>200
>200
>200
>200
>200
1
5e
7a
1
1
7b
1
7c
1
7d
1
7e
2
0

1
7f
H
H
＞10
＞10
＞10
3.23±0.39
>200
1
7g
＞10
＞10
＞10
6.79±1.35
>200
>200
JMC02
0.29±0.044
0.079±0.021
2.39±0.14
0.053±0.0069
OSC
ZAN
Rib
---
---
---
0.69±0.013
0.42±0.026
4.01±0.31
0.0042±0.000025
0.013±0.00078
＞10
0.097±0.0034
0.097±0.0034
6.68±0.34
0.037±0.0028
0.041±0.0017
1.96±0.046
>200
>200
>200
a
IC : Concentration of a compound required to achieve 50% inhibition of influenza virus proliferation in CEFs, presented as the mean ± standard deviation
50
(SD).
b
c
g
CC : Concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the CCK-8 method.
50
d
e
f
A/goose/Guangdong/SH7/2013. A/Chicken/Hebei/LZF/2014. A/duck/Guangdong/674/2014. A/goose/Jiangsu/1306/2014.
Highest tested concentration.
2
1

2
.2.2. In Vitro Inhibitory Activities of Neuraminidases
We selected 10 representative compounds for further enzyme assay against
influenza neuraminidases N1 (H5N1), N2 (H5N2), N6 (H5N6), N8 (H5N8), and
N1-H274Y to validate their binding target. OSC and ZAN were used as control drugs.
The results were summarized in Table 2, and each data was measured after three
parallel tests. From the test results, we found that OSC and ZAN displayed very high
activity against the four wild-type NAs, and it was clear that OSC and ZAN did not
show equal potency to NAs but have some selectivity. For example, OSC displayed
less potency against N1 (H5N1) than ZAN, but showed more activity against N2
(H5N2), and ZAN exhibited high activity against N1-H274Y mutant but OSC did not,
which was consistent with reported data [11, 28]. This reflected the reliability of the
activity test method which we have established.
It was found that the biarylmethyl-substituted derivatives at the position of
5
-amino group showed significant inhibition for group-1 NAs (H5N1 and H5N8). The
order of their potency is 9f > 9g > 9a >9b >9d, which is consistent with the sequence
of anti-H5N1 strain activity at their cellular level. But these five compounds showed
poor activities to inhibit group-2 NAs (H5N2 and H5N6), only at the micromolar
level, this is because N2 and N6 do not have the opened 150-loop. Among the
phenylpropenyl substituted derivatives at the 5-amino group, 11a, 11f and 11g showed
significant selective inhibition of group-1 NAs relative to OSC and ZAN.
Compounds 11a (H5N1, IC = 57.66 nM), 11f (H5N1, IC = 22.39 nM), and OSC
5
0
50
(H5N1, IC50 = 20.61 nM), which have comparable activities, cannot form good
interactions to N2 and N6 due to the closed conformation of 150-loop. Overall, the
inhibitory effects of these three compounds for the four wild-type NAs (H5N1, H5N2,
H5N6 and H5N8) were weaker than the OSC. These results were consistent with their
trend in antiviral activity in cells. 11c and 11e showed strong potency to all four
wild-type NAs, and their inhibitory activity against H5N2 was even comparable to
OSC (11c, IC = 21.37 nM; 11e, IC = 16.81 nM; OSC, IC = 16.22 nM). They did
5
0
50
50
not show significant selective inhibition, which were also consistent with their cellular
activities.From the results, it was concluded that the inhibitory activity of the above
1
0 representative compounds was weaker than OSC against N1-H274Y
[
A/Anhui/1/2005 (H5N1-H274Y)].
2
2

In conclusion, the test results of the representative compounds indicated that
most of them have obvious selective inhibition of group-1 NAs, and the selectivity of
biarylmethyl group at C5-NH position derivatives are particularly noticeable. The
2
phenylpropenyl group substituted derivatives, such as 11a, 11f, and 11g, also have
significant group-1 NAs selective inhibition. Moreover, 11a and 11f inhibitory
activities against N1 have achieved a similar level as OSC. 11c and 11e have strong
inhibitory effects against four wild-type NAs, especially for N2 which have similar
activity to OSC. Unfortunately, the inhibition of the selected representative
compounds against N1-H274Y was significantly reduced. This could be due to that
the substitution group at the position of C5-NH of OSC affects the binding mode of
2
the compounds with the mutant enzyme.
2
3

Table 2. Neuraminidase (NA) Inhibition of Oseltamivir Derivatives in Chemiluminescence-Based Assay
NA-Inhibitory Activity, IC (nM)
a
5
0
Compound
R1
R2
b
c
d
e
f
H5N1
H5N2
H5N6
Group-2
H5N8
H5N1-H274Y
Group-1
Group-2
Group-1
Group-1
9
a
H
H
118.17±9.56
1442.6±121.99
34543.33±2849.81
78.06±10.64
10260±493.25
9b
264.03±5.35
391.5±37.68
1100.66±103.24
7881.67±1061.17
1743.67±350.44
90.06±28.19
31213.33±2826.42
9d
H
H
9595±784.88
108.57±9074
83.13±10.08
＞100000
9
f
53.99±5.73
13700±854.34
39073.33±3780.86
2922.67±333.41
9
g
H
H
74.82±3.45
57.66±2.38
11996.67±1803.62
952.45±89.87
16110±339.41
55.25±4.76
92.61±1.76
7384.33±1007.34
5994±278.60
11a
5622.33±466.57
2
4

1
1c
H
H
H
254.2±11.79
33.26±2.16
22.39±1.28
21.37±0.14
16.81±0.73
155.43±20.25
45.46±9.17
62.09±8.22
33.02±8.78
174.4±18.98
10431±670.39
5270.33±210.02
8336.5±848.09
11e
1
1f
255.33±43.24
809.05±140.36
11g
H
129.28±9.98
869.03±159.98
10674±1592.40
309±44.97
28340±1936.28
OSC
ZAN
H
H
20.61±0.73
1.66±0.44
16.22±0.91
33.66±2.99
14.42±0.25
24.23±1.59
8.81±0.10
9.99±0.46
1826.5±234.05
12.02±0.29
----
----
a
Concentration required to reduce NA activity to 50% of the control NA activity (IC ). Values are the mean of three experiments, presented as the mean ± standard
5
0
b
c
d
e
f
deviation (SD). A/goose/Guangdong/SH7/2013. A/Chicken/Hebei/LZF/2014. A/duck/Guangdong/674/2014. A/goose/Jiangsu/1306/2014. A/Anhui/1/2005.
2
5

We also tested compounds 9a, 9b, 9d, 9g, 11a, 11e, 11f, and 11g inhibit N1
H1N1pdm09) and N2 (H3N2) to validate their binding target by
(
chemiluminescence-based assay. OSC was used as positive control for inhibition. The
values of IC of the selected oseltamivir derivatives and the positive control drugs are
5
0
summarized in Table 3. But all the tested compounds exhibited weaker potency
against N1 (0.087 µM-1.67 µM) and N2 (0.24 µM-17.69 µM) compared to OSC (N1:
0
.041 µM, N2: 0.016 µM), which can be easily explained by the structural features of
N1 (H1N1pdm09) and N2 (H3N2). The closed 150-loop and the lack of 150-cavity in
N1 (H1N1pdm09) and N2 (H3N2) (Figure 2) indicate that the cavity would not well
accommodate the selected oseltamivir derivatives bearing large groups at C-5-NH2
position.
Table 3. N1 (H1N1pdm09) and N2 (H3N2) Inhibition of Oseltamivir Derivatives
in Chemiluminescence-Based Assay
NA-Inhibitory Activity, IC (µM)
50
Compound
R1
R2
a
b
N1 (H1N1pdm09)
N2 (H3N2)
Group-1
Group-2
9
9
a
H
H
0
.48±0.029
12.22±0.66
0.91±0.06
b
0
.31±0.12
9
9
d
H
H
1
.67±0.48
10.21±0.11
7.64±1.49
g
0
.60±0.066
1
1a
H
H
0
.43±0.16
.43±0.15
4.64±0.09
3.47±0.39
11e
0
11f
H
0
.84±0.0039
1.56±0.16
2
6

1
1g
H
H
0
.087±0.0056
.041±0.0082
17.69±1.51
OSC
H
0
0.016±0.0004
a
b
A/California/04/2009. A/Babol/36/2005.
2.3. Molecular Docking
To further study the binding modes of the best active compounds in the
1
50-cavity of Group-1 NAs, we selected 9a and 11e for molecular docking with N1
(PDB code: 2HU0) and N8 (PDB code: 2HT7), respectively. The Surflex-Dock
module of SYBYL-X 1.3, PyMOL and Ligplot softwares were used for docking and
graphic display respectively.
As shown in Figure 5, it can be observed from A, C, E, and G that the
oseltamivir carboxylic acid part and the position of C-5-NH substituent of 9a and 11e
2
occupy the active sites and 150-cavities of N1 and N8, respectively, and the
oseltamivir carboxylic acid moieties of 9a and 11e were highly coincident with the
OSC. The above two points are the basis of the effective inhibition of N1 and N8 by
9
a and 11e, and also consistent with the idea of two-site binding design. Because the
N1 and N8 structures are highly similar, the binding modes and interactions between
9
a and 11e are also very similar.
The interactions between the part of C5-NH substituents of 9a and 11e
2
(thiophenylbiphenyl and p-fluorophenylpropenyl) and the 150-cavities of N1 and N8
are shown in figure B, D, F, H: The hydrophobic amino acids or amino acid residues
with hydrophobic side chains (Arg118, Gly147, Val149, Asp151 Arg156 and Thr439
for N1，Glu119, Gly147, Asp151 Arg156 and Thr439 for N8) in N1 and N8
1
50-cavities interact with the thiophene biphenyl to form hydrophobic and van der
Waals interactions. The guanidine group in Arg181 and the benzene group in
thiophene biphenyl are parallel to each other, forming cation-π interactions. These
interactions closely bind thiophene biphenyl to 150-cavity. However, from figures B
and F, it was found that the hydrogen bonds formed by the C-5 amino group of 9a
with Glu119 and Asp151 disappeared, possibly because the presence of
2
7

thiophenebiphenyl changes the conformation of oseltamivir carboxylic acid ring in
NAs, and weakened the binding force of 9a to the active site to some extent. In
addition, the whole conformation changes occurred when the molecule 9a combined
with N1-H274Y, which reduced the inhibitory activity to N1-H274Y. Similarly, the
hydrophobic amino acids in 150-cavity of N1 and N8 or the amino acid residues
containing hydrophobic side chains (Arg118,Gln136 Gly147, Val149, Asp151 and
Arg156 for N1 ， Asp151 Arg156 and Thr439 for N8) interacted with
p-fluorophenylene propylene groups in 11e to form hydrophobicity and van der Waals
interactions. Although the propene group was beneficial to the cross-linking of
p-fluorophenylpropene group with 150-cavity, the interaction between propene group
and 150-loop was weak. In summary, the ability of 11e to inhibit N1 and N8 is similar
to that of OSC. Because of the slender p-fluorophenylpropenyl group, 11e may have
little effect on the change of the conformation of oseltamivir carboxylic acid in N8, so
the hydrogen bond between C5-NH and Glu119 of N8 exists, but the hydrogen bond
2
in N1 disappears. In addition, the interaction between p-fluorophenylpropenyl and
1
50-cavity is weak, so the inhibitory effect of 11e on N1-H274Y is not enhanced.
2
8