Design, synthesis and biological evaluation of “Multi-Site”-binding influenza virus neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.05.076

Encouraged by our earlier discovery of neuraminidase inhibitors targeting 150-cavity or 430-cavity, herein, to yield more potent inhibitors, we designed, synthesized, and biologically evaluated a series of novel oseltamivir derivatives via modification of C-1 and C5–NH₂ of oseltamivir by exploiting 150-cavity and/or 430-cavity. Among the synthesized compounds, compound 15e, the most potent N1-selective inhibitor targeting 150-cavity, showed 1.5 and 1.8 times greater activity than oseltamivir carboxylate (OSC) against N1 (H5N1) and N1 (H5N1–H274Y). In cellular assays, 15e also exhibited greater potency than OSC against H5N1 with EC₅₀ of 0.66 μM. In addition, 15e demonstrated low cytotoxicity in vitro and low acute toxicity in mice. Molecular docking studies provided insights into the high potency of 15e against N1 and N1–H274Y mutant NA. Overall, we envisioned that the significant breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors may lead to further investigation of more potent anti-influenza agents.

Full text of DOI:10.1016/j.ejmech.2019.05.076

Accepted Manuscript
Design, synthesis and biological Evaluation of “Multi-Site”-binding influenza virus
neuraminidase inhibitors
Ruifang Jia, Jian Zhang, Wei Ai, Xiao Ding, Samuel Desta, Lin Sun, Zhuosen Sun,
Xiuli Ma, Zhong Li, Defeng Wang, Bing Huang, Peng Zhan, Xinyong Liu
PII:
S0223-5234(19)30494-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.076
EJMECH 11386
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 November 2018
Revised Date: 22 April 2019
Accepted Date: 27 May 2019
Please cite this article as: R. Jia, J. Zhang, W. Ai, X. Ding, S. Desta, L. Sun, Z. Sun, X. Ma, Z. Li, D.
Wang, B. Huang, P. Zhan, X. Liu, Design, synthesis and biological Evaluation of “Multi-Site”-binding
influenza virus neuraminidase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.076.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of “Multi-Site”-Binding
Influenza Virus Neuraminidase Inhibitors
a
a
a
a
a
a
Ruifang Jia , Jian Zhang , Wei Ai , Xiao Ding , Samuel Desta , Lin Sun , Zhuosen
a
b
a
a
b,*
a,*
Sun , Xiuli Ma , Zhong Li , Defeng Wang , Bing Huang , Peng Zhan , Xinyong
a,*
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
Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 1 Jiaoxiao
Road, Jinan, Shandong, 250023, P.R. China
*
E-mails: hbind@163.com (Huang B.); zhanpeng1982@sdu.edu.cn (Zhan P.);
xinyongl@sdu.edu.cn (Liu X.Y.)
Abstract:
Encouraged by our earlier discovery of neuraminidase inhibitors targeting
1
50-cavity or 430-cavity, herein, to yield more potent inhibitors, we designed,
synthesized, and biologically evaluated a series of novel oseltamivir derivatives via
modification of C-1 and C5-NH of oseltamivir by exploiting 150-cavity and/or
2
4
30-cavity. Among the synthesized compounds, compound 15e, the most potent
N1-selective inhibitor targeting 150-cavity, showed 1.5 and 1.8 times greater activity
than oseltamivir carboxylate (OSC) against N1 (H5N1) and N1 (H5N1-H274Y). In
cellular assays, 15e also exhibited greater potency than OSC against H5N1 with EC50
1

ACCEPTED MANUSCRIPT
of 0.66 µM. In addition, 15e demonstrated low cytotoxicity in vitro and low acute
toxicity in mice. Molecular docking studies provided insights into the high potency of
1
5e against N1 and N1-H274Y mutant NA. Overall, we envisioned that the significant
breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors
may lead to further investigation of more potent anti-influenza agents.
Keywords: Influenza virus, Neuraminidase inhibitors, 430-cavity, 150-cavity,
Oseltamivir derivatives.
1
. Introduction
In the recent years, the Influenza A and B virus infection has become a serious
threat for human health with the potential to cause epidemics or pandemics with mass
casualties. These viruses belong to the Orthomyxoviridae family of negative sense,
single stranded, and segmented RNA viruses [1]. In April 2009, a new type Swine flu,
also known as influenza A (H1N1) rapidly spread worldwide through
human-to-human transmission, giving rise to a serious public panic [2]. H7N9, a
novel and highly virulent avian-origin influenza A virus, occurred to infect human in
eastern China in the spring 2013. By the end of January 2017, 1161 people were
infected with H7N9, with 433 deaths, and the mortality rate was as high as 37.3% [3].
In addition, the highly pathogenic avian influenza A (H5N1) virus has resulted in a
fatality rate over 60%, which is a great threat to human [4,5]. Though there is no clear
evidence of efficient human-to-human transmission [6,7], the worrying possibility
2

ACCEPTED MANUSCRIPT
remains that further adaptation of avian flu for person-to-person transmission may
lead to a global influenza pandemic.
Influenza A virus is enveloped by a lipid membrane containing two important
surface antigenic glycoproteins namely hemagglutinin (HA) and neuraminidase (NA)
[
8], which play crucial roles in the life cycle of viruses [9]. There are different
subtypes according to the distinct antigenic properties of each protein: eighteen for
HA (H1–H18) and eleven for NA (N1–N11) [10,11]. NA cleaves the connection
between the hemagglutinin and the host cell to release newly formed virion from
infected cells and facilitate propagation of the virus [12], making this enzyme a
promising target for anti-influenza drugs [13]. Currently, four neuraminidases
inhibitors (NAIs) have been developed for the prophylaxis and treatment of patients
infected with influenza A or B viruses [14,15], including oral oseltamivir (Tamiflu)
[
16], inhaled zanamivir (Relenza) [17], intravenous peramivir (Rapivab) [18] and
inhaled laninamivir octanoate (Inavir) [19] (Figure 1). Among the approved drugs,
orally administered oseltamivir (a prodrug of oseltamivir carboxylate) (OSC) has been
a first-line therapy since its approval in 1999. However, resistance to oseltamivir has
constantly been reported due to its widely used in clinic such as the most frequent NA
substitutions reported in H5N1 (H274Y) [20-22]. In addition, various mutants with
resistance to the others NA inhibitors have also appeared [20,21,23,24]. Therefore,
there is an urgent and continuing need for the next generation neuraminidase
inhibitors.
3

ACCEPTED MANUSCRIPT
Figure 1. Structures of approved NA inhibitors for the treatment of influenza virus infection.
With the exception of N10, recently identified in a bat influenza A virus genome
[
25], the N1−N9 subtypes can be categorized phylogenetically into two groups:
group-1 (N1, N4, N5, and N8 subtypes), and group-2 (N2, N3, N6, N7, and N9
subtypes) [26]. The X-ray crystallographic structures show that in group-1 NAs
[
except N1 of 2009pdmH1N1 (09N1)] [27], there is a large cavity termed the
1
1
50-cavity (formed by an open conformation of the 150-loop containing residues
47–151) adjacent to the active site, which is absent in group-2 NAs [28] (Figure 2).
Based on this finding, several C-5 amino-substituted oseltamivir derivatives JMC32,
JMC20I [29], and JMC21h [30] (Figure 3) have been identified to inhibit H5N1
(group-1 NA) at low-nanomolar level (approximately 8-fold, 9-fold and 28-fold more
potent than OSC, respectively) through targeting the 150-cavity in our lab. In 2013,
4

ACCEPTED MANUSCRIPT
Wu and co-workers demonstrated for the first time that oseltamivir carboxylate could
induce the rigid closed N2 150-loop into a half-open one [25]. Thus, there was
another significant discovery in our lab that a series of N-substituted oseltamivir
derivatives such as JMC15b and JMC15c (Figure 3) showed exceptionally active
against both group-1 and -2 NAs, especially for 09N1, N2, N6, and N9 subtypes
(6.8−12.5 and 1.2−3.9 times greater activity than OSC). They also showed an increase
in inhibitory activity of about 4.2-fold and 2.5-fold relative to OSC toward H274Y
and E119V variant, respectively [31].
Moreover, there has also been evidenced the existence of another auxiliary
binding site adjacent to sialic acid binding site called “430-cavity” (formed by the
4
30-loop comprising residues Arg430-Thr439 [32]) (Figure 2) which could be
exploited for the design of new antivirals has been revealed [33]. It was worth noting
that, 430-cavity adopted a more open conformation in various group-1 and -2
subtypes, and could provide greater chemical space for further modification [34,35].
In 2018, our lab reported a panel of C-1 modified oseltamivir derivatives via targeting
4
30-cavity. Among them, compound EJMC8b (Figure 3) exhibited the best
inhibitory activity with IC values of 0.088 µM and 0.097 µM against H5N1 and
5
0
H5N6 NAs, respectively. Besides this, EJMC5c (Figure 3) also showed a moderate
activity against H5N1 and H5N6 (IC = 0.94 and 2.78 µM, respectively) [36].
5
0
5

ACCEPTED MANUSCRIPT
Figure 2. Comparison of the crystal structures of N1 (H5N1, PDB ID: 2HU0), 09N1
(H1N1pdm09, PDB ID: 3TI6), N8 (H5N8, PDB ID: 2HT7) and N2 (H3N2, PDB ID: 4GZP)
bound with OSC. It can be seen that 09N1, along with N2 have a closed 150-loop, but N1 and N8
have an open 150-cavity. Moreover, 430-cavity widely exists in a variety of subtypes including
group-1 (N1, 09N1 and N8) as well as group-2 (N2).
O
OH
O
OH
O
OH
N
O
N
O
H
N
O
HN
H
H
HN
R
HN
R'
Ar
O
O
O
R =
N
S
N
R' =
Ar =
S
JMC15b
JMC15c
JMC32
JMC20I
JMC21h
IC50 (JMC15b) =
IC50 (JMC15c) =
IC50 (JMC21h) =
.96 nM (N1, H5N1)
96.9 nM (N2, H5N2)
3065 nM (N6, H5N6)
1.89 nM (N8, H5N8)
0
0
.55 nM (N1, 2009H1N1)
.35 nM (N2, H9N2)
,26 nM (N6, H5N6)
.34 nM (N8, H5N8)
1,79 nM (N9, H7N9)
2.31 nM (N1, 2009H1N1)
2.13 nM (N2, H9N2)
10.85 nM (N6, H5N6)
4.06 nM (N8, H5N8)
6.51 nM (N9, H7N9)
IC50 (JMC32) =
.1 nM (N1, H5N1)
10 nM (N2, H9N2)
60 nM (N1, H5N1-H274Y)
IC50 (JMC20I) =
1.9 nM (N1, H5N1)
580 nM (N2, H9N2)
0
8
2
2
1
1
5
1160 nM (N1, H5N1-H274Y)
32.81 nM (N1, H5N1-H274Y)
OH
387.07 nM(N1, H5N1-H274Y) 663.90 nM(N1, H5N1-H274Y)
F
O
H
N
O
NH
O
H N
O
2
H N
2
O
HN
HN
O
IC50 (EJMC5c) =
.94 µM (N1, H5N1)
.78 µM (N6, H5N6)
IC50 (EJMC8b) =
.0883 µM (N1, H5N1)
.0967 µM (N6, H5N6)
O
0
2
0
0
EJMC8b
EJMC5c
Figure 3. Structures of the group-1 selective NA inhibitors (JMC32, JMC20I and JMC21h) and
nonspecific group-1 and group-2 NA inhibitors (JMC15b and JMC15c) targeting 150-cavity, as
well as oseltamivir derivatives (EJMC8b and EJMC5c) targeting 430-cavity.
6

ACCEPTED MANUSCRIPT
The main objective of our research was to discover potent group-1 selective NA
inhibitors functioning as anti-influenza agents via exploiting the 150-cavity and/or
4
30-cavity adjacent to the NAs active site of influenza A. Recently, several group-1
selective NA inhibitors were discovered by structure-based design targeting the
activity site and 150-cavity simultaneously. Notably, JMC20I and JMC21h,
containing hydrophobic p-phenyl and p-phenylthiobenzyl groups, respectively,
showed more potent N1-selective inhibitory activity than OSC [29,30]. Besides,
another report revealed that a series of C-5 amino-substituted oseltamivir derivatives
such as JMC15b and JMC15c bearing N,N-diethylamino and 1-pyrrolidine groups,
respectively, exhibited excellent inhibitory activity against both group-1 and -2 NAs
[
31]. On the other hand, the exploration of 430-cavity resulted in the discovery of
EJMC8b (IC : 0.088 and 0.097 µM) and EJMC5c (IC : 0.94 and 2.78µM) exerted
5
0
50
the greatest and moderate inhibition against H5N1 and H5N6, respectively [36].
Encouraged by these results, in this manuscript, using molecular hybridization
strategy, the well-matched privileged moieties of 150-cavity and 430-cavity were
introduced into C-5 and C-1 positions of oseltamivir core structure simultaneously to
fill these two cavities (Figure 4). Finally, 21 novel oseltamivir analogues were
designed, synthesized, and evaluated their biological activity.
7

ACCEPTED MANUSCRIPT
Figure 4. Strategy for structure optimization of oseltamivir analogues via targeting 150-cavity
and/or 430-cavity.
2
2
. Results and discussion
.1. Chemistry
The synthetic route of C-5 and/or C-1 modified oseltamivir derivatives (6a–e,
0a–e, 16a–e, 18a–e) and intermediate (13) were conducted following reactions
1
depicted in Scheme 1, 2 and 3. All derivatives were synthesized by well-established
methods using commercially available oseltamivir phosphate 1 as the primary starting
material. In Scheme 1, oseltamivir phosphate 1 was treated with Boc-anhydride and
DMAP in CH Cl and Et N to afford 2, which was hydrolyzed in the presence of 4 M
2
2
3
NaOH aqueous solution and acidified with 3 M HCl aqueous solution to give the key
intermediate 3. Subsequently, the intermediate 3 reacted with corresponding aminesin
8

ACCEPTED MANUSCRIPT
the presence of HBTU and DMAP to afford 4 and 7, and then prepared as
hydrochlorides 5and 8, respectively, with ethyl acetate solution of hydrogen chloride.
Subsequently, the target compounds 6a–b and 9a–b were prepared via reacting with
corresponding aldehydes in the presence of NaBH CN, and eventually 9a–b were
3
hydrolyzed by NaOH to give target compounds 10a–b.
As shown in Scheme 2, the 4-(phenylthio) benzaldehyde (13) were formed by
reaction of benzenethiol (11) with 4-fluorobenzaldehyde (12) in the presence of
potassium carbonate.
As outlined in Scheme 3, the synthesis of compounds 6c–e, 10c–e, 16a–e and
1
8a–e were carried out starting with oseltamivir phosphate 1. 1 was treated with a
series of different aldehydes in the presence of NaBH CN to afford 14a–e, which
3
followed by hydrolyzation in the presence of 4 M NaOH to give the key intermediates
1
5a–e. The target compounds (6c–e, 16a–e) and intermediates (9c–e and 17a–e) were
prepared via similar procedures (different corresponding amines) as compounds 4 and
, and finally the intermediates (9c–e and 17a–e) were hydrolyzed in the presence of
7
NaOH to result in the corresponding acids 10c–e and 18a–e.
9

ACCEPTED MANUSCRIPT
O
O
O
O
O
OH
i
ii
Boc
Boc
iii
H
2
N
O
N
O
N
O
H
H
HN
HN
HN
H PO
3
4
O
O
O
3
1
2
F
F
F
H
N
H
N
H
O
O
O
N
iv
iii
Boc
v
R2
H
2
N
O
N
O
N
O
H
H
HN
HCl HN
HN
O
O
O
4
5
6
a-b
O
O
O
O
O
OH
O
O
O
NH
O
NH
O
NH
O
NH
vi
iv
v
Boc
R
2
R
2
N
O
N
O
N
O
H N
2
O
H
H
H
HN
HN
HN
HCl
HN
O
O
O
O
1
0a-b
9a-b
8
7
R :
2
N
N
a
b
Scheme 1. Reagents and conditions: (i) (Boc) O, Et N, CH Cl , DMAP, rt; (ii) 4M NaOH,
2
3
2
2
CH OH, rt, then 3M HCl; (iii) corresponding amines, HBTU, DMAP, Et3N, CH CN, rt; (iv)
3
3
HCl/ethyl acetate, rt; (v) corresponding aldehydes, NaBH CN, CH OH, rt; (vi) 4M NaOH,
3
3
CH OH, rt, then 3M HCl.
3
Scheme 2. Reagents and conditions: (i) K CO , DMF, 120°C.
2
3
1
0

ACCEPTED MANUSCRIPT
Scheme 3. Reagents and conditions: (i) corresponding aldehydes, NaBH CN, CH OH, rt; (ii) 4M
3
3
NaOH, CH OH, rt, then 3M HCl; (iii) corresponding amines, HBTU, DMAP, Et N, CH CN, rt.
3
3
3
2
2
.2. Biological activity
.2.1. In vitro inhibitory activities of Influenza Neuraminidases
For NA inhibition screening against N1 (H5N1) from group-1 and N2 (H5N2)
from group-2, and all the newly synthesized 21 oseltamivir derivatives and
oseltamivir carboxylate (OSC) were evaluated using a chemiluminescence-based
assay with 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) as
the substrate [29]. The measured inhibition potencies of the synthesized compounds
toward N1 (H5N1), N2(H5N2) as well as N1 (H5N1-H274Y) were summarized in
Table 1. It was clear that OSC showed more potency against N2 (IC = 0.0045 µM)
5
0
than N1 (IC50 = 0.067 µM), meanwhile, it exhibited less potency toward the
H5N1-H274Y mutant (IC = 2.45 µM), which was consistent with reported data
50
1
1

ACCEPTED MANUSCRIPT
[
29,30,31,36].
As shown in Table 1, the series of oseltamivir derivatives (6a–e, 10a–e, 16a–e,
8a–e) exhibited significantly decreased activities compared with the positive drug
1
OSC and “dual-site”-binding NA inhibitors, confirming that “three-site”-binding
oseltamivir analogues were not suitable for binding with NAs. Compared with OSC,
6
a–e and 10a–e demonstrated less potent or no activities against H5N1, H5N2 and
H5N1-H274Y. Among 6a–e, compounds 6c–e with R group of 4-phenylthio benzyl,
2
4
-phenyl benzyl and 4-methylsulfanyl benzyl, respectively, showed better inhibitory
activities against N1 (H5N1) (IC = 8.33 µM, 22.14 µM and 34.64 µM, respectively)
5
0
than N2 (H5N2) (IC50 > 100 µM). In contrast, 6b replaced by 4-(pyrrolidin-1-yl)
benzyl at the R position resulted in similar activities for both H5N1 and H5N2. The
2
inhibitory effects of 10a–e bearing D-valine of R group toward the three NA
1
subtypes performed unfavorable activities, and similarly, 10c–e were N1-selective
inhibitors but 10b was 23-fold more potent against H5N2 compared to H5N1.
Impressively, compound 10e exerted robust inhibitory activity against H5N1 with
IC50 value of 0.21 µM. As for compounds 16a–e with 4-chloro benzyl at the R1
position, all the five inhibitor molecules were showing much lower potency than
OSC. Among them, compounds 16b and 16e bearing 4-(pyrrolidin-1-yl) benzyl and
4
-methylsulfanyl benzyl groups, respectively, exhibited selective inhibition against
H5N2 (with IC50 value of 0.94 µM) and H5N1 (with IC50 value of 27.39 µM),
respectively. In addition, when the R group was substituted by 4-carboxy benzyl, the
1
1
2

ACCEPTED MANUSCRIPT
resulting compounds 18a–e appeared to have a similar effect on the potency as 6a–e
and 10a–e. It was worth noting that IC values of 18a and 18b were lower against N2
5
0
(
H5N2) than against N1 (H5N1) of group-1. Indeed, the IC value of 18c against
50
NAs of H5N1 and H5N1-H274Y were approximately 46 and 3 times lower than OSC
for the same NAs, respectively.
Fortunately, the inhibitory effects of 15e bearing hydroxyl and 4-methylsulfanyl
benzyl at the R and R -position, respectively, had equal inhibitory potency to OSC.
1
2
In particular, 15e showed a high degree of selectivity for N1 over N2. Compound 15e
was approximately 1.5- and 1.8-fold more potent against N1 (H5N1) and N1
(H5N1-H274Y) than the OSC and was deemed a suitable lead compound for further
investigation.
Table 1. NA Inhibitory Activities of Target Compounds 6a–e, 10a–e, 15e,16a–e and
1
8a–e.
a
NA Enzyme-Inhibitory Assay, IC (µM)
5
0
b
c
d
compds
R1
R2
H5N1
H5N2
H5N1-H274Y
group-1
group-1
group-2
1
3

ACCEPTED MANUSCRIPT
6
6
a
> 100
> 100
> 100
b
20.17 ± 1.87
8.33 ± 0.76
22.14 ± 1.50
34.64 ± 5.64
> 100
48.43 ± 2.92
> 100
> 100
> 100
> 100
> 100
> 100
6
c
6
d
> 100
6
e
> 100
1
0a
0b
> 100
1
56.58 ± 7.35
9.34 ± 0.77
70.03 ± 8.52
2.41 ± 0.23
> 100
> 100
48.82 ± 3.30
> 100
1
0c
1
0d
> 100
1
0e
5e
0.21 ± 0.0012
0.044 ± 0.0049
> 100
> 100
8.34 ± 0.69
> 100
> 100
1.40 ± 0.061
> 100
1
1
6a
6b
1
31.81 ± 2.59
> 100
0.94 ± 0.09
> 100
> 100
> 100
> 100
> 100
1
6c
1
6d
> 100
> 100
1
6e
27.39 ± 0.61
> 100
1
4

ACCEPTED MANUSCRIPT
1
8a
8b
41.02 ± 6.44
13.28 ± 0.73
> 100
1
15.53 ± 2.83
3.07 ± 0.53
> 100
4.70 ± 0.31
> 100
> 100
8.49 ± 0.57
> 100
1
8c
1
8d
> 100
1
8e
8.38 ± 1.19
> 100
> 100
OSC
0.067 ± 0.0095 0.0045 ± 0.31
2.45 ± 0.31
a
Concentration required to reduce NA activity to 50% of control NA activity (IC ) and values are
5
0
shown as mean ± SD of three experiments.
b
c
d
A/goose/Guangdong/SH7/2013. A/Chicken/Hebei/LZF/2014. A/Anhui/1/2005.
Next, we investigated the potency of some representative compounds (10c,
1
0e,15e, 16e) against influenza B neuraminidase by the same assay, and data was
shown in Table 2. Compound 15e exhibited weaker activity against influenza B NA,
approximately 215-fold lower than OSC. The other compounds 10c, 10e and 16e
showed no activities toward influenza B NA. The X-ray crystallographic structural
information supports the conclusion that influenza B neuraminidases are similar to the
group-2 enzymes [26].
a
Table 2. The Influenza Virus B NA Inhibitory Activities of Selected Compounds.
compds
10c
10e
15e
16e
OSC
b
IC (µM)
> 100
> 100
7.97 ± 1.27
> 100
0.037 ± 0.0014
5
0
1
5

ACCEPTED MANUSCRIPT
a
b
B/PHUKET/3073/2013.
Concentration required to reduce NA activity to 50% of control NA activity (IC ). Values are
5
0
shown as mean ± SD of three experiments.
2
.2.2. In vitro anti-influenza virus activity
Anti-influenza virus potency of representative compounds 10e, 15e and 16b was
evaluated in Chicken Embryo Fibroblast cells infected with
A/goose/Guangdong/SH7/2013 (H5N1) as well as A/Chicken/Hebei/LZF/2014
H5N2), and oseltamivir carboxylate (OSC) were selected as reference compounds in
parallel. The values of EC50 (antiavian influenza A virus potency) and CC50
cytotoxicity) of the selective compounds were summarized in Table 3. Notably, all
(
(
the tested compounds with the exception of 16b exhibited no appreciable cytotoxicity
at the highest tested concentrations (CC > 200 µM) in chicken embryo fibroblasts
5
0
(CEFs).
In case of the H5N1 viruses, the activity of 15e (EC = 0.66µM) was superior to
5
0
that of OSC (EC50 = 0.82 µM). As for H5N2 virus, 15e performed poor potent
inhibitory activity than OSC, which is in accordance with their lower potency for NA
of H5N2. In contrast, 10e and 16b displayed no or little antiviral activity against both
H5N1 and H5N2. Overall, the anti-H5N1 and -H5N2 activities of the novel
oseltamivir derivatives were consistent with the results of the NAs (H5N1 and
H5N2)-inhibitory activities, suggesting these compounds inhibited influenza virus
replication by binding to NAs in cellular level.
1
6

ACCEPTED MANUSCRIPT
Table 3. Anti-Influenza Virus Activity and Cytotoxicity of Oseltamivir Derivatives.
a
EC50 values (µM) towards influenza viruses
c
d
b
Compds
H5N1
H5N2
CC50
group-1
> 100
group-2
26.56 ± 2.40
0.48 ± 0.10
> 100
1
1
0e
5e
> 200
> 200
0.66 ± 0.13
> 100
16b
77.13 ± 20.83
> 200
OSC
0.82 ± 0.07
0.17 ± 0.07
a
EC : concentration of compound required to achieve 50% protection of CEF cultures against
50
influenza virus-induced cytotoxicity, presented as the mean ± standard deviation (SD) and
determined by the CCK-8 method.
b
CC : concentration required to reduce the viability of mock-infected cell cultures by 50%, as
5
0
determined by the CCK-8 method.
c
d
A/goose/Guangdong/SH7/2013. A/Chicken/Hebei/LZF/2014.
2
.3. Molecular modeling
To postulate the interactions of the newly synthesized compounds with the target
and to account for the experimental results, molecular docking studies of
representative compounds 15e and 10e to the N1 NA (PDB 2HU0) were conducted
using SurflexeDock SYBYL-X 2.0 software. PyMOL was used to visualize the
results.
Firstly,
by
comparing
the
amino
acid
sequences
of
A/goose/Guangdong/SH7/2013 (H5N1)-N1 with the protein as used for the docking
studies, we found that amino acid sequence of A/goose/Guangdong/SH7/2013
1
7

ACCEPTED MANUSCRIPT
(H5N1)-N1 showed a high degree (> 95%) of similarity to 2HU0. Only 19 of the 387
residues were different between the two proteins, which might make a great
contribution to the credibility of the docking studies. The docking protocol was
described in the molecular docking section.
As shown in Figure 5, the 4-methylthiobenzyl group of compound 15e (Figure
5
A) occupied the 150-cavity while the other structural elements of this compound
interacted with the active site in a manner similar to the binding pattern of oseltamivir
carboxylate, and the “three-site”-binding compound 10e (Figure 5C) bounded with
active site, 150-cavity and 430-cavity simultaneously. This result was in good
agreement with the purpose of our design. As can be seen in Figure 5B, compound
1
5e showed key interactions in both the active site and the 150-cavity as expected.
These key interactions include the electrostatic interaction between the carboxylate
group of 15e and Arg292, Tyr347 and Tyr406, as observed in the X-ray crystal
structure of N1 bound to OSC. Moreover, 15e formed a strong hydrogen-bonding
interaction between the 5-amino linker and Asp151, which was not observed in OSC.
Furthermore, the 4-methylthiobenzyl group of 15e was well adapted to the 150-cavity
of NA, which was surrounded mostly by polar and non-polar residues. It was clearly
shown in Figure 5D that the carboxyl on valine of compound 10e formed four
hydrogen-bonds with Arg292, Tyr347, Tyr406 and Arg18 around 430-cavity region,
which had less hydrogen-bonds than C1-carboxyl of compound 15e. Meanwhile, the
hydrogen-bond with Asp151 at C5-NH- of 10e was not observed, which might be
another reason for the decreased enzyme inhibitory activity. Thus, the molecular
1
8

ACCEPTED MANUSCRIPT
modeling helped to interpret the binding mode of the newly designed compounds with
NA, and explained the reason why compound 15e exhibited high potency but 10e
showed less potency against NA.
Figure 5. Binding modes of compounds 15e (green, B) and 10e (green, D) with N1 (PDB code:
2
HU0) and superposition with the binding mode of OSC (cyan, A and C).
2
.4. Safety assessment
A single-dose toxicity test of compound 15e was conducted in Kunming mice.
After intragastric administration of 15e at a dose of 2 g/kg, no death occurred and
1
9

ACCEPTED MANUSCRIPT
there was no abnormity of body weight increase over the subsequent week as shown
in Figure 6 and Table 4.
Figure 6. There were no obvious differences in the weight gain from the two different treatment
groups. A: body weight of female mice (g)-time (day); B: body weight of male mice (g)-time
(day).
Table 4. Clinical Behaviors after Administration of 15e in Mice
Day, number of mice
1
2
3
4
5
6
7
Dose of 15e
Clinical behaviors
10min
30min 1h 3h 6h
(g/kg)
Female mice
Blank control
No abnormality
Death
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
5
0
0
0
0
Lethargy
Clonic convulsion
Hunched posture
2
0

ACCEPTED MANUSCRIPT
Piloerection
No abnormality
Death
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
0
0
0
0
2
lethargy
Clonic convulsion
Hunched posture
Piloerection
Male mice
Blank control
No abnormality
Death
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
5
0
0
0
0
0
Lethargy
Clonic convulsion
Hunched posture
piloerection
No abnormality
Death
2
lethargy
Clonic convulsion
Hunched posture
Piloerection
3
. Conclusion
2
1

ACCEPTED MANUSCRIPT
In conclusion, to explore the chemical space of 430-cavity and 150-cavity in
NAs, a series of novel oseltamivir derivatives were designed, synthesized and
evaluated by modifying C1-COOH and C5-NH2 of OSC. Among 20
“three-site”-binding inhibitors, we found that the NA-inhibitory potency were sharply
decreased in comparison with OSC and “dual-site”-binding compounds, suggesting
that it was unreasonable to introduce large groups to bind with 150-cavity and
4
30-cavity simultaneously. On the other hand, 15e, group-1-specific NA inhibitor
targeting 150-cavity, exhibited similar or greater inhibitory potency against N1
H5N1) and N1 (H5N1-H274Y), with IC50 value of 0.044 µM and 1.40 µM,
respectively, compared to the activities against N2 (H5N2) and influenza B virus NA
IC value of 8.34 µM and 7.97 µM, respectively). In cellular assay, 15e displayed
(
(
5
0
better activity than OSC against H5N1, in accordance with the results of the
NA-inhibitory activities. Moreover, 15e demonstrated favorable pre-clinical safety
profile in Kunming mice by intragastric administration. Considering these in vitro and
in vivo results, we envision that the further optimization of oseltamivir targeting 150-
and 430-cavities via introducing suitable size groups will afford more potent
compounds with improved drug-resistance profile.
4
4
. Experimental section
.1. Chemistry
The key chemical reactant oseltamivir phosphate was provided by Shandong
Qidu Pharmaceutical Co., Ltd. The other common chemicals and reagents such as
2
2

ACCEPTED MANUSCRIPT
HBTU, DMAP, corresponding amines and amino acids, etc. were purchased from
Aladdin, TCI, J&K, ENERGY CHEMICAL, and Sino pharm Chemical Reagent Co.,
Ltd. with purities of at least 97%. Solvents were obtained from commercial suppliers
and were purified and dried by means of standard methods when necessary. Thin
layer chromatography (TLC) was performed using plates coated with Silica Gel
GF254 for TLC (Merck), and spots were detected under UV light (254 nm). Flash
column chromatography was conducted on Silica Gel (200-300 mesh), purchased
from Qingdao Haiyang Chemical Company. Melting points (mp) were measured on a
micro melting point apparatus (RY-1G, Tianjin TianGuang Optical Instruments) and
1
13
were uncorrected. H-NMR and C-NMR spectra were acquired on a Bruker AV-400
spectrometer in CD OD or DMSO-d with TMS as the internal standard. Coupling
3
6
constants (J) were reported in hertz (Hz), and chemical shifts were given in parts per
million (ppm) from TMS. ESI-MS was carried out using an API 4000 LC/MS
spectrometer (Applied Biosystems, USA). High resolution mass spectrometry
(
HRMS) was obtained on an Agilent 6520 Q-TOF LC/MS spectrometer (Agilent,
Germany).
.1.1. General procedure for the synthesis of ethyl (3R, 4R, 5S)-4-
4
acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carbo
xylate (2)
To a solution of commercially available oseltamivir phosphate 1 (8.2 g, 0.02
mol, 1 equiv) in 50 mL dichloromethane was added di-tert-butyl dicarbonate
2
3

ACCEPTED MANUSCRIPT
(
(Boc) O, 8.72 g, 2 equiv), DMAP (0.2 g) and triethylamine (TEA, 30 mL) at room
2
temperature, and the mixture was stirred for 2 h. Subsequently, the solution was
evaporated under reduced pressure to remove dichloromethane and TEA and then
saturated citric acid solution was added. After that, the mixture was extracted with
ethyl acetate, and the organic phase was dried over anhydrous MgSO , and
4
concentrated under reduced pressure to give the crude product, which was purified by
recrystallization form isopropyl ether to afford 2 as a white powder, yield 87.2%. mp:
1
1
49.1–150.2°C. H NMR (400 MHz, CD OD) δ: 6.76 (s, 1H, CH), 4.20 (q, J = 7.1
3
Hz, 2H, CH ), 4.10 (d, J = 8.3 Hz, 1H, CH), 3.85 (dd, J = 11.0, 8.7 Hz, 1H, CH), 3.76
2
–
3.66 (m, 1H, CH), 3.40 (p, J = 5.6 Hz, 1H, CH), 2.68 (dd, J = 17.7, 5.1 Hz, 1H,
CH), 2.30 – 2.18 (m, 1H, CH), 1.95 (s, 3H, CH ), 1.56 – 1.41 (m, 13H, 2CH , 3CH ),
3
2
3
1
3
1
.29 (t, J = 7.1 Hz, 3H, CH ), 0.95 – 0.83 (m, 6H, 2CH ). C NMR (100 MHz,
3 3
CD OD) δ: 172.34, 166.13, 156.67, 137.67, 129.05, 82.33, 78.85, 75.71, 60.68, 54.77,
3
4
8.95, 30.48, 27.33, 25.87, 25.40, 21.60, 13.09, 8.46, 8.26. ESI-MS: m/z 413.5 [M +
+
H] , C H N O (412.52).
2
1
36
2
6
4
.1.2.
General
procedure
for
the
synthesis
of
(3R,4R,5S)-
4
-acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3-yloxy)cyclohex-1-ene-1-car
boxylic acid (3)
To a solution of intermediate 2 (5 g, 12.1 mmol) was dissolved in 70 mL
methanol, and 4N NaOH aqueous solution was added until the pH to 12, and then the
solution was stirred at room temperature for 2 h. The reaction solution was evaporated
2
4

ACCEPTED MANUSCRIPT
under reduced pressure to remove methanol, then the residue was taken up in water
(30 mL), and the pH was adjusted to 2 with 3 M HCl aqueous solution. This solution
was extracted with ethyl acetate and tetrahydrofuran (V:V = 2:1, 4 × 30 mL). The
combined organic phase was washed with saturated sodium chloride (2 × 30 mL) and
water (30 mL), then dried over anhydrous MgSO , and concentrated under reduced
4
1
pressure to afford 3 as white powder, yield 80.37%. mp: 208.9–209.8°C. H NMR
(
400 MHz, CD OD) δ: 6.78 (s, 1H, CH), 4.11 (d, J = 8.3 Hz, 1H, CH), 3.85 (dd, J =
3
1
2
–
1.1, 8.7 Hz, 1H, CH), 3.71 (td, J = 10.7, 5.4 Hz, 1H, CH), 3.46 – 3.36 (m, 1H, CH),
.68 (dd, J = 17.7, 5.1 Hz, 1H, CH), 2.30 – 2.17 (m, 1H, CH), 1.96 (s, 3H, CH ), 1.56
3
1
3
1.40 (m, 13H, 2CH , 3CH ), 0.96 – 0.84 (m, 6H, 2CH ). C NMR (100 MHz,
2
3
3
CD OD) δ: 172.42, 168.00, 156.73, 137.71, 129.30, 82.37, 78.89, 75.81, 54.84, 49.01,
3
+
3
0.56, 27.33, 25.88, 25.40, 21.61, 8.37. ESI-MS: m/z 385.4 [M + H] , C H N O
19
32
2
6
(384.47).
4
.1.3. General procedure for the synthesis of compounds 4 and 7.
To a solution of acid 3 (1.25 g, 3.1 mmol), HBTU (1.6 g, 3.7 mmol), and Et N
3
(10 mL) in 20 mL acetonitrile was added 1.2 equimolar amount of the appropriate
amine. The mixture was kept stirring at room temperature for 5 h. TLC detection
found that the reaction was complete, then the mixture was evaporated under reduced
pressure to remove solvent. The residue was taken up saturated sodium chloride and 1
mL 3N HCl and extracted with ethyl acetate (30 mL × 3), and the combined organic
layers were washed two times with saturated sodium chloride and 1 mL 3N HCl. The
2
5

ACCEPTED MANUSCRIPT
organic layers were dried (MgSO ), filtered, and solvent was removed under reduced
4
pressure at 40°C to give the crude product, which was purified by column
chromatography to produce the corresponding intermediate, 4 and 7.
Tert-butyl((1S,5R,6R)-6-acetamido-3-((4-fluorobenzyl)carbamoyl)-5-(pentan-3-
yloxy)cyclohex-3-en-1-yl)carbamate (4). White powder, 75.4% yield, mp: 186.1–
1
1
88.2°C. H NMR (400 MHz, CD OD) δ: 7.30 (dd, J = 8.4, 5.5 Hz, 2H, 2Ph-H), 7.03
3
(
t, J = 8.8 Hz, 2H, 2Ph-H), 6.47 (s, 1H, CH), 4.45 – 4.34 (m, 2H, CH ), 4.10 (d, J =
2
8
.2 Hz, 1H, CH), 3.86 (dd, J = 11.0, 8.6 Hz, 1H, CH), 3.73 (td, J = 10.5, 5.3 Hz, 1H,
CH), 3.46 – 3.36 (m, 1H, CH), 2.64 (dd, J = 17.3, 5.0 Hz, 1H, CH), 2.38 – 2.25 (m,
H, CH), 1.96 (s, 3H, CH ), 1.57 – 1.39 (m, 13H, overlapped, 2CH , 3CH ), 0.89 (dt,
1
3
2
3
1
3
J = 12.4, 7.4 Hz, 6H, 2CH ). C NMR (100 MHz, CD OD) δ: 172.40, 168.05, 163.25,
3
3
1
4
60.82, 156.64, 134.78, 132.57, 132.07, 129.02, 82.33, 78.87, 75.79, 54.83, 49.05,
+
2.12, 30.75, 27.32, 25.85, 25.34, 21.58, 8.36. ESI-MS: m/z 492.4 [M + H] ,
C H FN O (491.60).
26
38
3
5
Methyl((3R,4R,5S)-4-acetamido-5-((tert-butoxycarbonyl)amino)-3-(pentan-3ylo
xy)cyclohex-1-ene-1-carbonyl)-D-valinate (7). White powder, 42.8% yield, mp:
1
1
1
3
=
95.1–196.2°C. H NMR (400 MHz, CD OD) δ: 6.42 (s, 1H), 4.32 (d, J = 6.8 Hz,
3
H), 4.11 (d, J= 8.2 Hz, 1H), 3.87 (dd, J = 10.9, 8.6 Hz, 1H), 3.80 – 3.68 (m, 4H),
.47 – 3.39 (m, 1H), 2.63 (dd, J = 17.4, 5.1 Hz, 1H), 2.39 – 2.26 (m, 1H), 2.18 (dq, J
1
3
13.6, 6.8 Hz, 1H), 1.96 (s, 3H), 1.59 – 1.39 (m, 13H), 1.01 – 0.82 (m, 12H). C
NMR (100 MHz, CD OD) δ: 172.30, 168.83, 156.65, 132.52, 132.13, 82.34, 78.85,
3
2
6

ACCEPTED MANUSCRIPT
7
1
4
5.76, 58.35, 54.84, 51.10, 48.98, 30.76, 30.20, 27.32, 25.82, 25.40, 21.58, 18.16,
+
7.60, 8.37. ESI-MS: m/z 498.5 [M + H] , C H N O (497.62).
2
5
43
3
7
.1.4. General procedure for the synthesis of compounds 5 and 8.
The compound 4 (0.6 g) was dissolved in 30.0 mL ethyl acetate solution of
hydrogen chloride and the mixture was stirred at room temperature for 2 h. When the
reaction was complete, the mixture was evaporated under reduced pressure to remove
solvent and was recrystallized from isopropyl ether to afford 5. Compound 8 was
prepared using the same method as 5.
(3R,4R,5S)-4-acetamido-5-amino-N-(4-fluorobenzyl)-3-(pentan-3-yloxy)cyclohe
1
x-1-ene-1-carboxamide (5). White powder, 72.1% yield, mp: 83.1–84.2°C. H NMR
(
400 MHz, CD OD) δ: 7.32 (dd, J = 8.4, 5.5 Hz, 2H, 2Ph-H), 7.03 (dd, J = 8.7 Hz,
3
2
H, 2Ph-H), 6.50 (s, 1H, CH), 4.41 (s, 2H, CH ), 4.26 (d, J = 8.6 Hz, 1H, CH), 3.98
2
(dd, J = 11.3, 8.5 Hz, 1H, CH), 3.54 (td, J = 10.6, 5.6 Hz, 1H, CH), 3.46 (p, J = 5.6
Hz, 1H, CH), 2.86 (dd, J = 17.6, 5.1 Hz, 1H, CH), 2.58 – 2.44 (m, 1H, CH), 2.05 (s,
13
3
H, CH ), 1.52 (qd, J = 14.1, 7.0 Hz, 4H, 2CH ), 0.93 – 0.85 (m, 6H, 2CH ). C
3 2 3
NMR (100 MHz, CD OD) δ: 173.39, 167.53, 160.85, 134.70, 132.09, 130.34, 129.12,
3
1
8
14.83, 114.61, 82.33, 74.31, 53.03, 49.49, 42.15, 28.43, 25.75, 25.16, 21.84, 8.40,
+
.18. ESI-MS: m/z 392.4 [M + H] , C H FN O (391.48).
21
30
3
3
Methyl((3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-ene-1-c
1
arbonyl)-D-valinate (8). Pale yellow sticky substance, 70.1% yield. H NMR (400
MHz, CD OD) δ: 6.51 (s, 1H, CH), 4.33 (d, J = 6.8 Hz, 1H, CH), 4.27 (d, J = 8.3 Hz,
3
2
7

ACCEPTED MANUSCRIPT
1
H, CH), 3.99 (dd, J = 14.0, 5.4 Hz, 1H, CH), 3.72 (s, 3H, CH ), 3.59 – 3.42 (m, 2H,
3
overlapped, 2CH), 2.83 (dd, J = 17.3, 5.3 Hz, 1H, CH), 2.58 – 2.44 (m, 1H, CH), 2.19
(
dq, J = 13.6, 6.8 Hz, 1H, CH), 2.05 (s, 3H, CH ), 1.63 – 1.45 (m, 4H, 2CH ), 1.01 –
3 2
1
3
0
1
2
4
.86 (m, 12H, overlapped, 4CH ). C NMR (100 MHz, CD OD) δ: 173.38, 172.17,
3
3
68.29, 132.32, 130.16, 82.37, 74.27, 58.42, 53.06, 51.13, 49.49, 30.18, 28.47, 25.72,
+
5.22, 21.82, 18.15, 17.62, 8.30. ESI-MS: m/z 398.5 [M + H] , C H N O (397.51).
20
35
3
5
.1.5. General procedure for the synthesis of compounds 6a–b, 9a–b.
To a solution of compound 5 or 8 (2.0 mmol, 1 equiv) and aldehydes (2.4 mmol,
1
.2 equiv) in 30 mL ethanol, NaBH CN (4.0 mmol, 2 equiv) was slowly added. The
3
mixture was stirred at room temperature for 6 h and then concentrated. To the residue,
0 mL saturated NaCl and 10 mL saturated Na CO solution was added, and the
2
2
3
mixture was extracted with EtOAc. The combined extracts were dried over anhydrous
MgSO and concentrated to give the crude product, which was purified by column
4
chromatography to produce the corresponding target compounds 6a–b and
intermediates 9a–b.
(3R,4R,5S)-4-acetamido-5-((4-(diethylamino)benzyl)amino)-N-(4-fluorobenzyl)-
3
-(pentan-3-yloxy)cyclohex-1-ene-1-carboxamide (6a). Yellow powder, 75.3% yield,
1
mp: 82.6–84.3°C. H NMR (400 MHz, CD OD) δ: 7.31 (dd, J = 8.2, 5.6 Hz, 2H,
3
2
8
Ph-H), 7.10 (d, J = 8.5 Hz, 2H, 2Ph-H), 7.03 (t, J = 8.7 Hz, 2H, 2Ph-H), 6.66 (d, J =
.6 Hz, 2H, 2Ph-H), 6.48 (s, 1H, CH), 4.41 (s, 2H, CH ), 4.03 (d, J = 8.1 Hz, 1H,
2
CH), 3.96 – 3.87 (m, 1H, CH), 3.74 (d, J = 12.5 Hz, 1H, CH), 3.55 (d, J = 12.5 Hz,
2
8