Discovery of novel acylhydrazone neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.04.006

Neuraminidase (NA) plays a crucial role in the replication and transmission of influenza virus. NA inhibitors have been developed as effective treatments for influenza A and B infections. In this paper, a new lead neuraminidase inhibitor 6a (IC₅₀

Full text of DOI:10.1016/j.ejmech.2019.04.006

Accepted Manuscript
Discovery of novel acylhydrazone neuraminidase inhibitors
Zhi Xiang Zhao, Li Ping Cheng, Meng Li, Wan Pang, Fan Hong Wu
PII:
S0223-5234(19)30305-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.006
EJMECH 11241
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 December 2018
Revised Date: 12 March 2019
Accepted Date: 2 April 2019
Please cite this article as: Z.X. Zhao, L.P. Cheng, M. Li, W. Pang, F.H. Wu, Discovery of novel
acylhydrazone neuraminidase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.04.006.
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
Discovery of Novel Acylhydrazone Neuraminidase Inhibitors
A series of novel acylhydrazone neuraminidase inhibitors were designed and synthesized based on virtual
screening.

ACCEPTED MANUSCRIPT
Discovery of Novel Acylhydrazone Neuraminidase Inhibitors
Zhi Xiang Zhao, Li Ping Cheng*, Meng Li, Wan Pang*, Fan Hong Wu*
*School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China
*Corresponding author. Telephone: +86-21-6087-3250. Email: chengliping@sit.edu.cn
*Corresponding author. Telephone: +86-21-60877231. E-mail: pangwan@sit.edu.cn
*Corresponding author. Telephone: +86-21-60877231. E-mail: wfh@sit.edu.cn
1

ACCEPTED MANUSCRIPT
Abstract: Neuraminidase (NA) plays a crucial role in the replication and transmission of influenza
virus. NA inhibitors have been developed as effective treatments for influenza A and B infections. In
this paper, a new lead neuraminidase inhibitor 6a (IC50=7.10±0.2 µM) was discovered by ligand-based
virtual screening, receptor-based virtual screening, molecular dynamics simulation (MD), and bioassay
validation. MD simulation indicates that the morpholinyl group of 6a could be embedded in 430-loop
of NA. To exploit the 430-loop in the active site, a series of novel acylhydrazone NA inhibitors 6b-6g
were designed and synthesized based on the lead compound 6a. Compound 6e exerts the most potency,
with IC50 value of 2.37±0.5 µM against NA, which is lower than that of oseltamivir carboxylate (OC)
(IC50 =3.84 µM). Overall, this work provided unique insights in the discovery of potent inhibitors
against NA.
Key words: Neuraminidase inhibitor; virtual screening; 430-loop; acylhydrazone
1. Introduction
Influenza is a respiratory infectious disease which can give rise to a serious threat to public health
due to its high infectious and mortality [1]. For example, the worldwide outbreak of highly pathogenic
H5N1 had attracted wide attention due to its high virulence and lethality [2]. The most contagious flu
virus (H1N1 2009) caused more than 300,000 people death within only 18 months [3]. Currently,
prevention and treatment of influenza mainly rely on inactivated vaccines and antiviral drugs. However,
due to the high mutability of the virus each year, vaccine effectiveness is significantly hampered.
Therefore, the development of effective and safe antiviral agents, which are not susceptible to mutation,
is even more important in the event that new highly virulent strains lead to global pandemics outbreak.
Nowadays, NA inhibitors are the most widely choice for anti-influenza treatment. Two NA inhibitors,
zanamivir and oseltamivir, had been developed as most effective treatments for influenza A and B
infections and approved by the FDA. However, due to poor oral bioavailability, the application of
zanamivir is significantly restricted [4]. Oseltamivir is the most widely used antiviral agent due to its
high oral bioavailability [5]. However, oseltamivir-resistant viruses have emerged over the years [3,
6-7]. Therefore, it is imperative to develop new novel NA inhibitors to overcome drug resistance and
treat influenza infection effectively.
Neuraminidase (NA), a glycoprotein on the surface of influenza virus, which plays a crucial role in
the replication and transmission of influenza virus, has become one of the most useful targets for
design of anti-influenza drugs [8-9]. Crystal structural study showed that NA consists of four
structurally identical subunits and there is an enzyme active center in each subunit [10-11]. Recently,
two new pockets adjacent to the NA active site have been discovered and named as 150-cavity and
430-loop [1, 12-15]. Based on this discovery, a series of meaningful explorations on NA inhibitors
through targeting the 150-cavity and 430-loop had been reported [16-17]. Zhu et al. [16] modified the
carboxyl site of zanamivir to synthesize a series of compounds in an attempt to reduce the polarity of
the entire compounds and improve its bioavailability. Molecular docking indicates that the most potent
inhibitors with the elongated groups at the C-1-position being projected toward the 430-loop region.
Xie et al. [17] designed and synthesized a series of N-substituted oseltamivir derivatives targeting
150-cavity. These inhibitors showed excellent and selective inhibitory activities against H5N1
influenza neuraminidase. Therefore, 150-cavity and 430-loop are of great significance in the design of
new novel NA inhibitors.
Structure-based drug design had proven to be an effective drug discovery pathway [18]. In this study,
a pharmacophore model was firstly constructed for a rapid screening of the small-molecule database
2

ACCEPTED MANUSCRIPT
SPECS. Subsequently, molecular docking was performed to reduce the size of the screened compounds
and initially explore the binding modes between the protein and ligands. Then, molecular dynamics
(MD) simulations were further carried out for a more precise prediction of binding modes and
calculation of binding free energies. The most desirable compound 6a with proper binding mode and
the lowest binding free energy was obtained. Based on this lead compound 6a, several new compounds
6b-6g were designed. Finally, the new designed compounds were further confirmed by experimental
synthesis and biological evaluation.
2. Results and discussion
2.1. Pharmacophore models generation and validation
The data set of 8 oseltamivir and zanamivir derivatives used in the study for the pharmacophore
models generation was collected from the literature [16-17, 19-20]. These inhibitors were known
to bind the similar NA target. The spatial positions and interactions between the ligands and the protein
were carefully analyzed during the preparation of data set. Inhibitory activity of these inhibitors
spans a range of four orders of magnitude from 0.0006 µM to 4.9 µM. The structures and biological
activities expressed as IC50 against influenza neuraminidase are shown in Fig. 1. Besides with high
inhibition activity, these compounds also have some common features, such as hydrophobic center,
hydrogen bond donor and acceptor groups, positive nitrogen, negative center, which are defined as
pharmacophore features and important for their efficacy [21-23].
a Oseltamivir carboxylate A and Oseltamivir Phosphonate B [17,19]. b Oseltamivir derivatives targeting 150-loop (C and D) [17].
c Zanamivir E and zanamivir phosphonate F [16, 20]. d Zanamivir derivatives targeting 430-loop (G and H) [16].
Fig. 1. Representative NA inhibitors with various chemical scaffolds.
Starting with the data set, pharmacophore models were generated by using the GALAHAD module
within the SYBYL-X 2.1 software [24]
.
Twenty pharmacophore models were generated. A desired
3

ACCEPTED MANUSCRIPT
model should have a higher SPECIFICITY value, a higher steric score, and a lower energy value. The
SPECIFICITY value is the most important parameter for distinguishing a desired model [25-28]. The
two best models model-02 and model-06 were obtained (Table 1). Then, the screening abilities of these
two models were validated by comparing their Se (true positive rate) and Sp (false positive rate). The
calculation equations for Se and Sp are shown in (1) and (2). Herein true positives (TP) and true
negatives (TN) are the results of the screening of test database and decoy database
(http://dud.docking.org/), respectively. In contrast, false negatives (FN) and false positives (FP) are the
number of discarded compounds of test database and decoy database, respectively. The higher the
number of Se is, the better the pharmacophore model is; the lower the number of Sp is, the better the
pharmacophore model is [29-30]. According to the verification results, the Se value of model-02 is
equal to that of model-06, but the Sp value of model-06 is lower than that of model-02 (Table 2).
Moreover, the SPECIFICITY of model-06 is higher. So model-06 was used for the screening of small
molecule database SPECS.
Table 1. The best two models generated by GALAHAD.
Model
SPECIFICITY
5.575
N-Hits
Feats
Energy (kcal/mol)
25.72
Sterics
1137.30
1089.40
Model-02
Model-06
7
7
8
8
5.583
46.95
Table 2. The verification results of the pharmacophore models for the test and decoy databases.
Test database
Decoy database
Model
TP
33
33
FN
16
16
Se (%)
67.3
TN
64
FP
Sp (%)
3.4
Model-02
Model-06
1810
1836
67.3
38
2.0
2.2. The features of the pharmacophore model
The characteristics of final pharmacophore were shown in Fig. 2A, including three H-bond (HB)
acceptors (AA-2, AA-3 and AA-7), one HB donor (DA-1), one hydrophobic (HY-4), two positive N
(NP-5 and NP-8), and one negative center (NC-6). As shown in Fig. 2B, The lead compound was
superimposed on the pharmacophore features. Compared with the features of model-06, the methoxy of
lead compound was readily superimposed on H-bond acceptor (AA-7). And there is an obvious
hydrophobic benzene ring was found, which is consistent with hydrophobic (HY-4). Besides, two
positive nitrogen atoms of -C=N-N- bond could be readily identified by features of NP-5 and NP-8.
Consequently, the lead compound was valuable for further study for its inhibitory activity.
4

ACCEPTED MANUSCRIPT
Fig. 2. (A) The features of pharmacophore model-06; (B) lead compound 6a superimposed onto the
pharmacophore model.
2.3. Discovery of lead compound
Subsequently, model-06 was used as an UNITY query to screen SPECS database of 670000
compounds. 379 compounds were hit docked into the crystal structure of neuraminidase 2HU0. 30
compounds with total score values more than 9 were finally obtained. MD simulations were carried out
for the screened 30 compounds to further study their binding patterns by Amber 12.0 [31]. The system
stability and overall convergence of simulations were monitored in terms of root-mean-square
deviation (RMSD) of molecule backbone atoms. The average RMSD value of each step versus time of
compound 6a was shown in Fig. 3. For comparison, the MD simulation of oseltamivir carboxylate (OC)
was also carried out. It can be seen that the fluctuations in the average RMSD values during the whole
20 ns simulation are 1.0 Å, indicating that the complexes keep the stable state. Complex 6a-protein is
more stable than OC-protein within 20 ns of simulation. The binding free energies of 6a are -16.4
kcal·mol-1 and -20.4 kcal·mol-1 via the prediction by the Molecular Mechanics/Poisson-Boltzmann
Surface Area (MM/PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)
methods (Table 3). The corresponding values for OC are -10.8 kcal·mol-1 and -14.3 kcal·mol-1, which
are consistent with the value ( -10.60 ~ 10.36 kcal·mol-1) of the reference [32]. It is known that there is
generally a good correlation between binding free energy and compound’s activity. Therefore, from the
binding free energy viewpoint, 6a may be used as a lead compound to further design and synthesize
other new potent NA inhibitors. To verify this prediction, inhibitory test against NA of 6a was
performed. The test results show that the IC50 value (7.1 µM) of 6a is comparable to that of OC (3.84
µM), and thus 6a could be used as lead compound to perform further modification to attain new
inhibitors with good inhibition activity. The binding mode of compound 6a and NA is shown in Fig. 4.
It can be seen that 6a may perfectly bounded with the interaction site. Furthermore, the structural part
of benzene ring may embed in active site and morpholine ring may embed in 430-loop simultaneously.
5

ACCEPTED MANUSCRIPT
Besides, a total of four hydrogen bonds were formed. As shown in Fig. 4, four hydrogen bonds formed
by 6a and key residues (Arg74, Glu37). The hydrogen bond distances captured are 2.4 Å
(CH=N---Arg74), 1.8 Å ((NH)(C)C=O----Arg74), 1.9 Å ((NH)(C)C=O----Glu37), and 1.8 Å
((CH2)(C)NH----Glu37), respectively.
Fig. 3. Molecular Dynamics Simulation of new compound 6a and OC.
Fig. 4. The binding conformation (A) and interaction with major residues (B) of compound 6a and
neuraminidase.
2.4. Designing new scaffolds for NA inhibitors
According to the features derived from the best pharmacophore model-06, some new NA inhibitors
were designed (Table 3). Lead compound 6a was taken as a template to design new compounds
.
Considering the features of H-bond acceptors (AA-2, AA-3 and AA-7) and donor (DA-1) in the moiety
of phenyl, some H-bond acceptor groups (methoxy and nitryl) and donor group (hydroxyl) were
introduced to the benzene ring. On the other hand, the moiety of phenyl in 6a could bind to the active
site (Fig. 4A), but Fig. 4B shows that there is not any interactions between phenyl and amino acids.
Methoxy, hydroxyl, and nitryl groups readily form hydrogen bonds with amino acids at the active site,
which may increase the interactions between substitute groups and protein, and thus improve
inhibitor’s activity. Then MD simulations were carried out for these new designed compounds to
6

ACCEPTED MANUSCRIPT
further predict the binding mode of inhibitor-enzyme. Stable RMSD trajectory within 20 ns was shown
in Fig. S1, and the fluctuations of inhibitors 6b-6g in the average RMSD values within 20 ns are
between 1.5Å-3.5Å, indicating that these compounds were able to bind to enzyme stably [33-34].
Binding free energy calculation had been proved to be very useful and successful in designing new
potent drug molecules [35]. The binding free energies of compounds 6b-6g were also calculated and
the corresponding results are shown in Table 3. As seen from Table 3, most compounds have more
negative binding free energies than OC, indicating their good inhibitory activity.
Table 3. The prediction binding free energies via MM/PBSA and MM/GBSA of OC and compounds
6a-6g.
MM/GBSA
(kcal/mol)
MM/PBSA
(kcal/mol)
Entry
OC
6a
-14.3
-20.4
-10.8
-16.4
6b
6c
6d
-27.8
-27.4
-17.8
-15.7
-25.8
-13.0
6e
6f
-36.7
-20.3
-27.8
-12.8
6g
-21.9
-17.0
7

ACCEPTED MANUSCRIPT
2.5. Synthesis and biological assay
Based on binding pattern analysis and MD simulation of inhibitors and NA, compounds 6a-6g were
synthesized by feasible synthetic strategies in Fig. 5. Their activities against NA were further
biologically tested. The corresponding results are displayed in Table 4. As seen from Table 4, The
inhibitory activities of all compounds 6b-6g are better than that of the lead compound 6a. Compounds
6b, 6e and 6g show better inhibition activities than oseltamivir carboxylate (OC) (IC50 =3.84 µM).
Compound 6e exhibits the most potent inhibitory activity, with IC50 value of 2.37 µM against NA.
Specifically, 6e is about 3.0-fold, 1.8-fold, 2.7-fold, and 2.0-fold more active against NA compared
with 6a, 6c, 6d and 6f respectively. Whereas its activity is comparable to those of 6b (IC50 = 2.61 µM)
and 6g (IC50 = 3.15 µM). The inhibitory profiles of the best NA inhibitors 6b and 6e are shown in Fig.
6.
Fig. 5. Synthesis of compounds 6a-6g.
Table 4. The inhibition IC50 value of inhibitors 6a-6g and OC against NA.
Inhibitors 6a
6b
6c
6d
6e
6f
6g
OC
7.10±0.2
2.61±0.7
4.37±0.07
6.48±0.7
2.37±0.5
4.67±0.6
3.15±0.6
3.84±0.8
IC50(µM)
Fig.6. The inhibitory profiles of inhibitors 6b and 6e.
To better understand the discrepancy in activity between compounds, a series of molecular docking
simulations were carried out. Fig. 7 shows the 3D binding modes of the representative 6c, 6e and OC at
8

ACCEPTED MANUSCRIPT
the binding site of neuraminidase (PDB ID: 2HU0). Specifically, the molecular docking experiments
give three different binding modes for this series of acylhydrazone compounds. In one category, the
aryl moiety of compounds 6a, 6d and 6f is located in NA active site (Fig. 4 and Fig. S2). In another
category, the aryl moiety of compounds 6b, 6e and 6g is located in 430-loop (Fig. 7 and Fig. S2). For
compound 6c, both the aryl and morpholine moieties are located in the active site. For the most potent
6e, the interactions between ligand and protein includes five hydrogen bonds with ARG152, ARG371,
TYR406 and ARG292. It can be seen that 6e can bind to active site as well as 430-loop. Notably, the
3,4-dimethoxyphenyl moiety of 6e was projected toward the 430-loop, while the morpholine ring
interacts with the active site in a manner similar to the binding pattern of OC. However, compared with
6e, both OC and 6c could only occupy the active site but could not reach the 430-loop. Therefore, the
higher NA inhibitory potency of inhibitor 6b, 6e, and 6g may be ascribed to the elongation the
molecule to the 430-loop to form strong hydrophobic interactions with NA.
Fig. 7. Docking results of compounds 6c, 6e and OC with NA (PDB 2HU0).
9

ACCEPTED MANUSCRIPT
3. Conclusion
A virtual screening strategy combined with pharmacophore model screening, molecular docking,
and MD simulations, as well as bioassay was performed for discovery of novel NA inhibitors. 6a was
picked out as a lead compound of virtual screening from SPECS database. Six new inhibitors 6b-6g
were designed and synthesized based on the structure of 6a. The inhibitors were then analyzed for their
bioactivity against NA. Three synthesized inhibitors exhibit better inhibition than oseltamivir
carboxylate against NA. Particularly, compound 6e exerts the most potency, with IC50 value of 2.37 µM
against NA. Molecular docking analysis indicated that the good inhibitory activity of 6e may be
ascribed to the elongation of the 3,5-dimethoxyphenyl moiety of the molecule to the 430-loop. So
430-loop maybe play an important role in discovering novel NA inhibitors.
4. Experimental section
4.1. Chemistry
All
chemical
reagents
were
purchased
from
commercial
suppliers
Tansoole
(http://www.tansoole.com/index.html) without further purification. Experiments were monitored by thin
layer chromatography (TLC) and the TLC was performed on pre-coated silica gel plates. Flash column
chromatography was performed using silica gel (200~300 mesh) purchased from Tansoole. The 1H and
13C NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer with CDCl3, MeOD or
Dimethyl Sulfoxide-D6 as the solvent and tetramethylsilane (TMS) as an internal standard at room
temperature. Chemical shifts are given in d relative to TMS, and the coupling constants J values are given
in Hertz (Hz). Mass spectra (MS) were obtained from solariX.
4.1.1. Synthesis of monoethyl oxalate hydrazide 2
Diethyl oxalate 1 (0.03 mol) and hydrazine hydrate (0.01 mol) were dissolved in 10 ml of C2H5OH,
respectively. The ethanol solution of hydrazine hydrate was slowly added dropwise to compound 1 in
ethanol. Then, the mixture was stirred at room temperature overnight. Filtration was performed to
remove insoluble white impurities and concentrate residual mixture at reduced pressure. The desired
compound 2 was separated from the excess ester through partition water and diethyl ether by throwing
away the organic phase and collecting the aqueous one. The organic layer containing excess diester was
extracted four times with water and discarded. Compound 2 was temporarily saved in water [43].
4.1.2. Synthesis of Monoalkyl Oxalate-[(aryl)viny]hydrazide compounds (4a-4g)
The corresponding benzaldehyde derivatives 3a-3g (0.6 mmol) were added to 20 ml aqueous solution
of monoethyl oxalate hydrazide, stirred at 25 -60 for 10 min-5 h. Solids were collected by filtration
and filtrate was washed by water. The product was drained by vacuum pump to afford desired
compound. All yields were calculated relative to the benzaldehyde derivatives [44].
4.1.2.1. Ethanedioic acid,1-ethyl ester,2-[2-[(2-methoxylphenyl)methylene]hydrazide](4a)
White solid. Yield 77.8%, mp 131-132 . 1H NMR (500 MHz, Methanol-d4) δ 8.83 (s, 1H), 8.06 (d,
J = 7.7 Hz, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H), 4.39 (q, J =
7.1 Hz, 2H), 3.90 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, Methanol-d4) δ 159.66 ,
158.86 , 154.37 , 148.16 , 132.40 , 126.36 , 121.68 , 120.50 , 111.07 , 62.71 , 54.85 , 12.84 . HRMS
(ESI): m/z calcd for C12H14O4N2 [M+H],251.1026; found,251.1034.
4.1.2.2. Ethanedioic acid,1-ethyl ester,2-[2-[(3,4,5-trimethoxyphenyl)methylene]hydrazide] (4b)
10

ACCEPTED MANUSCRIPT
White solid. Yield 73.3%, mp 150-151 . 1H NMR (500 MHz, Chloroform-d) δ 10.82 (s, 1H), 8.39
(s, 1H), 7.03 (s, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.93 (d, J = 6.4 Hz, 9H), 1.45 (t, J = 7.1 Hz, 3H). 13C
NMR (125 MHz, Chloroform-d) δ 160.19, 153.43, 152.88, 151.80, 140.65, 128.36, 105.15, 63.55,
60.92, 56.26, 13.93 HRMS (ESI): m/z calcd for C14H18O6N2 [M+H], 311.1237; found, 311.1237.
4.1.2.3. Ethanedioic acid,1-ethyl ester,2-[2-[(3-nitro-4-hydroxyphenyl)methylene]hydrazide] (4c)
Yellow solid. Yield 71.4%, mp 178-180 .1H NMR (500 MHz, Methanol-d4) δ 8.44 (s, 1H), 8.38 (s,
1H), 8.14 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz,
3H). 13C NMR (125 MHz, Methanol-d4) δ 159.50, 155.65, 154.48, 149.53, 134.87, 134.40, 126.09,
125.12, 120.20, 62.79, 12.84. HRMS (ESI): m/z calcd for C11H11O6N3 [M+H], 282.0720; found,
282.0724.
4.1.2.4. Ethanedioic acid,1-ethyl ester,2-[2-[(2-hydroxyphenyl)methylene]hydrazide] (4d)
White solid. Yield 66.7%, mp 152-153 . 1H NMR (500 MHz, Methanol-d4) δ 8.68 (s, 1H), 7.55 (d, J
= 7.5 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.06 – 7.00 (m, 2H), 4.50 (q, J = 7.1 Hz, 2H), 1.51 (t, J = 7.1 Hz,
3H). 13C NMR (125 MHz, Methanol-d4) δ 159.39 , 158.30 , 153.76 , 153.10 , 132.10 , 130.59 , 119.27 ,
117.78 , 116.38 , 62.77 , 12.85 . HRMS (ESI): m/z calcd for C11H12O4N2 [M+H], 237.0869; found,
237.0874.
4.1.2.5. Ethanedioic acid,1-ethyl ester,2-[2-[(3,4-dimethoxyphenyl)methylene]hydrazide] (4e)
White solid. Yield 98%, mp 159-160 . 1H NMR (500 MHz, Chloroform-d) δ 10.39 (s, 1H), 8.28 (s,
1H), 7.41 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 3.89 (d, J
= 3.4 Hz, 6H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, Chloroform-d) δ 160.38, 152.54, 151.96,
151.78, 149.50, 125.95, 123.58, 110.58, 108.55, 63.47, 56.10, 13.94. HRMS (ESI): m/z calcd for
C13H16O5N2 [M+H], 281.1131; found, 281.1134.
4.1.2.6. Ethanedioic acid,1-ethyl ester,2-[2-[(3-methoxy-4-hydroxyphenyl)methylene]hydrazide] (4f)
Yellow solid. Yield 88.4%, mp 190-192 . 1H NMR (500 MHz, Methanol-d4) δ 8.38 (s, 1H), 7.75 (s,
1H), 7.22 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 4.03 (s, 3H), 1.51 (t, J
= 7.1 Hz, 3H). 13C NMR (125 MHz, Methanol-d4) δ 159.69, 154.29, 152.63, 149.91, 148.24, 125.40,
123.77, 114.66, 108.78, 62.72, 55.06, 12.85. HRMS (ESI): m/z calcd for C12H14O5N2 [M+H], 235.1071;
found, 235.1078.
4.1.2.7. Ethanedioic acid,1-ethyl ester,2-[2-[(3,5-dimethoxy-4-hydroxyphenyl)methylene]hydrazide]
(4g)
Yellow solid. Yield 60.7%, mp 202-204 . 1H NMR (500 MHz, Methanol-d4) δ 8.27 (s, 1H), 7.18 (s,
2H), 4.40 (q, J = 7.2 Hz, 2H), 3.91 (s, 6H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, Methanol-d4)
δ 159.67, 154.29, 152.62, 148.09, 138.78, 124.24, 105.33, 62.73, 55.46, 12.85. HRMS (ESI): m/z calcd
for C13H16O6N2 [M+H], 297.1081; found, 297.1084.
4.2. Synthesis of Acetic acid,2-[[3-(4-morpholinyl)propyl]amino]-2-oxo-,2-[(n-phenyl)methylene]
hydrazide (6a-6g)
To dissolve compounds 4a-4g (1 mmol) and 3-Morpholinopropylamine (1 mmol) in 20 ml of ethanol,
the mixture was refluxed at 85 for 3 h-6 h. The final mixture was purified by column chromatography
using silica gel as stationary phase and CH2Cl2-methanol as mobile phase.
11

ACCEPTED MANUSCRIPT
4.2.1. Acetic acid, 2-[ [3-(4-morpholinyl) propyl]amino]-2-oxo-,2-[(2-methoxylphenyl) methylene]
hydrazide (6a)
1
White solid, yield 63%. mp: 171-172 , purity:99.58%. H NMR (500 MHz, DMSO-d6) δ 12.15 (s,
1H), 9.10 (t, J = 5.4 Hz, 1H), 8.92 (s, 1H), 7.84 (d, J = 7.0 Hz, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.10 (d, J
= 8.3 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 3.85 (s, 3H), 3.62 – 3.57 (m, 4H), 3.25 (q, J = 6.4 Hz, 2H), 2.33
(dd, J = 12.8, 5.7 Hz, 6H), 1.69 – 1.62 (m, 2H).13C NMR (125 MHz, DMSO) δ 160.01, 158.52, 157.00,
146.83, 132.47, 126.26, 122.59, 121.16, 112.39, 66.62, 56.78, 56.23, 53.79, 38.39, 25.61. HRMS (ESI):
m/z calcd for C17H24N4O4 [M+H], 349.1870; found, 349.1887.
4.2.2. Acetic acid, 2-[[3-(4-morpholinyl)propyl]amino]-2-oxo-,2-[(3,4,5-trimethoxyphenyl)
methylene]hydrazide (6b)
1
White solid, yield 60%. mp: 206 , purity:99.25%. H NMR (500 MHz, DMSO-d6) δ 12.11 (s, 1H),
9.13 (t, J = 5.7 Hz, 1H), 8.47 (s, 1H), 6.98 (s, 2H), 3.83 (s, 6H), 3.71 (s, 3H), 3.62 – 3.58 (m, 4H), 3.26
(q, J = 6.5 Hz, 2H), 2.39 – 2.32 (m, 6H), 1.66 (p, J = 6.7, 6.0 Hz, 2H).13C NMR (125 MHz, DMSO) δ
159.98, 156.88, 153.65, 151.18, 129.83, 105.02, 66.58, 60.60, 56.78, 56.45, 53.75, 38.45, 25.52.HRMS
(ESI): m/z calcd for C19H28N4O6 [M+H], 409.2082; found, 409.2099.
4.2.3. Acetic acid, 2-[ [3-(4-morpholinyl) propyl]amino]-2-oxo-,2-[(3-nitro-4-hydroxyphenyl)
methylene]hydrazide (6c)
1
Red solid, yield 58%. mp: 230-232 . purity:94.38%. H NMR (500 MHz, DMSO-d6) δ 12.23 (s,
1H), 9.24 (t, J = 5.8 Hz, 1H), 8.60 (s, 1H), 8.22 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 8.7 Hz,
1H), 3.73 – 3.70 (m, 5H), 3.35 (q, J = 6.4 Hz, 2H), 2.52 (dd, J = 15.9, 8.9 Hz, 6H), 1.78 (p, J = 6.7 Hz,
2H). 13C NMR (125 MHz, DMSO-d6) δ 160.01, 156.84, 149.43, 137.52, 133.38, 125.10, 121.03, 66.35,
56.53, 53.57, 40.35, 38.24, 25.40. HRMS (ESI): m/z calcd for C16H21N5O6 [M+H], 380.1565; found,
380.1584.
4.2.4. Acetic acid, 2-[ [3-(4-morpholinyl) propyl]amino]-2-oxo-,2-[(2-hydroxyphenyl) methylene]
hydrazide (6d)
1
White solid, yield 69%. purity:96.34%. mp: 180-182 . H NMR (500 MHz, DMSO-d6) δ 12.53 (s,
1H), 11.14 (s, 1H), 9.25 (t, J = 5.6 Hz, 1H), 8.87 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.7 Hz,
1H), 7.02 (t, J = 8.5 Hz, 2H), 3.73 – 3.66 (m, 4H), 3.36 (q, J = 6.3 Hz, 2H), 2.44 (dd, J = 13.3, 6.1 Hz,
6H), 1.76 (p, J = 6.6 Hz, 2H).13C NMR (125 MHz, DMSO-d6) δ 159.66 , 158.08 , 156.88 , 151.52 ,
132.32 , 130.07 , 119.86 , 118.98 , 116.93 , 66.59 , 56.73 , 53.75 , 38.40 , 25.58 . HRMS (ESI): m/z
calcd for C16H22N4O4 [M+H], 335.1714; found, 335.1712.
4.2.5. Acetic acid, 2-[ [3-(4-morpholinyl) propyl]amino]-2-oxo-,2-[(3,4-dimethoxyphenyl) methylene]
hydrazide (6e)
1
White solid, yield 70%. mp: 210-211 . purity:94.33%. H NMR (500 MHz, DMSO-d6) δ 12.10 (s,
1H), 9.25 – 9.19 (m, 1H), 8.59 (s, 1H), 7.40 (s, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H),
3.91 (d, J = 2.7 Hz, 6H), 3.70 (s, 4H), 3.36 (q, J = 6.2 Hz, 2H), 2.50 – 2.40 (m, 6H), 1.76 (p, J = 6.2 Hz,
2H).13C NMR (125 MHz, DMSO) δ 160.07, 156.71, 151.60, 151.39, 149.56, 127.06, 122.79, 111.97,
108.93, 66.61, 56.81, 56.06, 55.98, 53.78, 38.46, 25.56. HRMS (ESI): m/z calcd for C18H26N4O5
[M+H], 379.1976; found, 379.1976.
12

ACCEPTED MANUSCRIPT
4.2.6. Acetic acid, 2-[ [3-(4-morpholinyl) propyl]amino]-2-oxo-,2-[(3-methoxy-4-hydroxyphenyl)
methylene]hydrazide (6f)
1
White solid, yield 59%. mp: 209-210 . purity:96.47% H NMR (500 MHz, DMSO-d6) δ 12.04 (s,
1H), 9.75 (s, 1H), 9.21 (t, J = 5.6 Hz, 1H), 8.54 (s, 1H), 7.37 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.94 (d,
J = 8.1 Hz, 1H), 3.93 (s, 3H), 3.73 – 3.67 (m, 4H), 3.35 (q, J = 6.3 Hz, 2H), 2.49 – 2.40 (m, 6H), 1.76
(p, J = 6.4 Hz, 2H).13C NMR (125 MHz, DMSO) δ 160.11, 156.62, 151.72, 149.89, 148.51, 125.75,
123.04, 115.94, 109.63, 66.58, 56.81, 56.06, 53.76, 38.45, 25.53. HRMS (ESI): m/z calcd for
C17H24N4O5 [M+H], 365.1819; found, 365.1821.
4.2.7. Aceticacid, 2-[[3-(4-morpholinyl) propyl] amino]-2-oxo-,2-[(3,5-dimethoxy-4-hydroxyphenyl)
methylene]hydrazide (6g)
1
White solid, yield 55%. mp: 175-175 . purity:96.55%. H NMR (500 MHz, DMSO-d6) δ 12.09 (s,
1H), 9.21 (t, J = 5.7 Hz, 1H), 9.11 (s, 1H), 8.52 (s, 1H), 7.04 (s, 2H), 3.92 (s, 6H), 3.70 (t, J = 4.0 Hz,
4H), 3.35 (q, J = 6.4 Hz, 2H), 2.49 – 2.40 (m, 6H), 1.76 (p, J = 6.4 Hz, 2H).13C NMR (125 MHz,
DMSO) δ 160.10, 156.66, 151.82, 148.60, 138.89, 124.56, 105.44, 66.60, 56.82, 56.54, 53.77, 38.47,
25.54. HRMS (ESI): m/z calcd for C18H26N4O6 [M+H], 395.1925; found, 395.1945.
4.3. Neuraminidase inhibitory activity assay
A
commercially available neuraminidase inhibitor screening kit (P0309 Beyotime,
http://www.beyotime.com/index.htm.) was used to assay inhibitory activity for compounds 6a-6g. The
inhibition assay was conducted following the standard strategy. The inhibitory activity of oseltamivir
carboxylate was measured as reference. Firstly, 70 µL neuraminidase buffer and 10 µL neuraminidase
were added to each well of 96-well black microtiter plate. Then, inhibitors were dissolved in DMSO in
different concentration was added to neuraminidase. After shaking and mixing evenly, the enzymatic
reaction was performed at 37 for 10 min. Subsequently, 10 µL fluorescent substrate was added to the
mixture and incubate at 37 °C for 30 min. Next, 50 µL stop solution was added to each well of 96-well
black microtiter plate. After shaking and mixing evenly, the fluorescence intensity was measured by
fluorescence microplate, while the excitation and emission wavelengths were set at 355 nm and 450 nm,
respectively. IC50 values were calculated based on fluorescence intensity.
It should be mentioned that bacterial neuraminidase is extracted from not influenza strain but
clostridium perfringens. Since the sequences are highly consistent with H5N1, this kit is suitable for the
high-throughput screening of NA inhibitors in vitro. However, since there is obvious difference in IC50
values between the neuraminidase detecting system and the virus detecting system, the IC50 value of
OC against avian influenza virus is as low as nM level in some references, while the detection value for
neuraminidase detecting system may reach 20 µM. Therefore, it is very meaningful if one can screen
out a inhibitor with IC50 value close to 20 µM by using Beyotime kit P0309 according to the
specification.
Acknowledgments
Thanks for financial support given by Middle and Youth teachers Scientific and Technological
Talents Developing Fund (No. ZQ 2018-20), Natural Science Foundation of Shanghai (No.
15ZR1440400), Collaborative Innovation Fund (No. XTCX2016-14), National Youth Fund Project of
China (No. 21602135), Shanghai Municipal Education Commission (Plateau Discipline Construction
Program).
13

ACCEPTED MANUSCRIPT
Supplementary Material
Supplementary materials related to this article include materials and methods, Fig. S1, Fig. S2,
MASS spectra, 1H NMR, and 13C NMR spectra for all the compounds.
Reference
1. J. Kirchmair, J. M. Rollinger, K. R. Liedl, N. Seidel, A. Krumbholz, M. Schmidtke. Novel
neuraminidase inhibitors: identification, biological evaluation and investigations of the binding mode,
Future Med. Chem. 3 (2011) 437-450.
2. R. G. Webster, E. J. Walker, Influenza-the world is teetering on the edge of a pandemic that could kill
a large fraction of the human population, Am. Sci. 91 (2003) 122–129.
3. K. Chairat, J. Tarning, N. J. White, N. Lindegardh, Pharmacokinetic properties of anti-Influenza
neuraminidase inhibitors, J. Clin. Pharmacol. 53 (2013) 119-139.
4. C. J. Dunn, K. L. Goa, Zanamivir, Drugs. 58 (1999) 761-784.
5. K. McClellan, C. M. Perry, Oseltamivir: a review of its use in influenza, Drugs. 61 (2001) 263.
6. R. A. Bright, M. Medina, X. Y. Xu, G. Perez-Oronoz, T. R. Wallis, X. M. Davis, L. Povinelli, N. J.
Cox, A. I. Klimov, Incidence of adamantane resistance among influenza A (H3N2) viruses isolated
worldwide from 1994 to 2005: a cause for concern, Lancet. 366 (2005)1175-1181.
7. F. Hayden, Developing new antiviral agents for influenza treatment: what does the future hold, Clin.
Infect. Dis. 48 (2009) S3-S13.
8. N. M. Bouvier, P. Palese, The biology of influenza viruses, Vaccine. 26 (2008) D49-D53.
9. M. Ohuchi, N. Asaoka, T. Sakai, R. Ohuchi, Roles of neuraminidase in the initial stage of influenza
virus infection, Microbes. Infect. 8 (2006) 1287-1293.
10. J. N. Varghese, L. G. Laver, P. M. Colman, Structure of the influenza virus glycoprotein antigen
neuraminidase at 2.9 |[angst]| resolution, Nature. 303 (1983) 35-40.
11. P. M. Colman, J. N. Varghese, W. G. Laver, Structure of the catalytic and antigenic sites in influenza
virus neuraminidase, Nature. 303 (1983) 41-44.
12. R. E. Amaro, X. L. Cheng, I. Ivanov, D. Xu, J. A. McCammon, Characterizing loop dynamics and
ligand recognition in human- and avian-type Influenza neuraminidases via generalized born molecular
dynamics and end-point free energy calculations, J. Am. Chem. Soc. 131 (2009) 4702–4709.
13. N. Han, X. Liu, Y. Mu, Exploring the mechanism of zanamivir resistance in a neuraminidase
mutant: a molecular dynamics study, Plos One. 7 (2012) e44057.
14. V. Kumar, C. Chang, K. Tan, Y. Jung, S. Chen, Y. E. Cheng, P. Liang, Identification, synthesis, and
evaluation of new neuraminidase inhibitors, Org. Lett. 16 (2014) 5060-5063.
15. R. E. Amaro, D. D. L. Minh, L .S. Cheng, W. M. Lindstrom, A. J. Olson, J. Lin, W. W. Li, J. A.
McCammon, Remarkable loop flexibility in avian influenza N1 and its implications for antiviral drug
design, J. Am. Chem. Soc. 129 (2007) 7764-7765.
16. E. G. Feng, W. Shin, X. L. Zhu, J. Li, D. J. Ye, J. Wang, M. Y. Zheng, J. P. Zuo, K. T. No, X. Liu, W.
L. Zhu, W. Tang, B. Seong, H. L. Jiang, H. Liu, Structure-based design and synthesis of C-1- and
C-4-modified analogs of zanamivir as neuraminidase inhibitors, J. Med. Chem. 56 (2013) 671−684.
17. Y. C. Xie, D. Q. Xu, B. Huang, X. L. Ma, W. B. Qi, F. Y. Shi, X. Y. Liu, Y. J. Zhang, W. F. Xu,
Discovery of N-substituted oseltamivir derivatives as potent and selective inhibitors of H5N1 influenza
neuraminidase, J. Med. Chem. 57 (2014) 8445−8458.
18. A. C. Anderson, The process of structure-based drug design, Chem. Biol. 10 (2003) 787-797.
14

ACCEPTED MANUSCRIPT
19. T. J. R. Cheng, S. Weinheimer, E. B. Tarbet, J. T. Jan, Y. S. E. Cheng, J. J. Shie, C. L. Chen, C. A.
Chen, W. C. Hsieh, P. W. Huang, W. H. Lin, S. Y. Wang, J. M. Fang, O. Y. P. Hu, C. H. Wong,
Development of oseltamivir phosphonate congeners as anti-influenza agents, J. Med. Chem. 55 (2012)
8657-8670.
20. J. J. Shie, J. M. Fang, P. T. Lai, W. H. Wen, S. Y. Wang, Y. S. E. Cheng, K. C. Tsai, A. S. Yang, C. H.
Wong, A practical synthesis of zanamivir phosphonate congeners with potent anti-influenza activity, J.
Am. Chem. Soc. 133 (2011) 17959-17965.
21. C. Zhang, L. J. Feng, Y. Y. Huang, D. Y. Wu, Z. Li, Q. Zhou, Y. N. Wu, H. B. Luo, Discovery of
novel phosphodiesterase-2A inhibitors by structure-based virtual screening, structural optimization, and
bioassay, J. Chem. Inf. Model. 57 (2017) 355−364.
22. J. K. Shepphird, R. D. Clark, A marriage made in torsional space: using GALAHAD models
to drive pharmacophore multiplet searches, J. Comput-Aided. Mol. Des. 20 (2006) 763–771.
23. G. Kothandan, T. Madhavan, C. G. Gadhe, S. J. Cho, A combined 3D QSAR and
pharmacophore-based virtual screening for the identification of potent p38 MAP kinase inhibitors: an
in silico approach, Med. Chem. Res. 22 (2013) 1773–1787.
24. H. Z. Zhong, J. P. Bowen, GALAHAD, J. Am. Chem. Soc. 129 (2007) 5780-5780.
25. N. J. Richmond, C. A. Abrams, P. R. N. Wolohan, E. Abrahamian, P. Willett, R. D. Clark,
GALAHAD: 1. Pharmacophore identification by hypermolecular alignment of ligands in 3D, J.
Comput-Aided Mol. Des. 20 (2006) 567–587.
26. H. D. Xie, K. X. Qiu, X. G. Xie, Pharmacophore modeling, virtual screening, and 3D-QSAR
studies on a series of non-steroidal aromatase inhibitors, Med. Chem. Res. 24 (2015) 1901–1915.
27. Y. X. Huang, J. Zhao, Q. H. Song, L. H. Zheng, C. Fan, T. T. Liu, Y. L. Bao, L. G. Sun, L. B. Zhang,
Y. X. Li, Virtual screening and experimental validation of novel histone deacetylase Inhibitors, BMC
Pharmacol. Toxicol. 17 (2016) 32.
28. J. Caballero, 3D-QSAR (CoMFA and CoMSIA) and pharmacophore (GALAHAD) studies on the
differential inhibition of aldose reductase by flavonoid compounds, J. Mol. Graph. Model. 29 (2010)
363–371.
29. J. K. Shepphird, R. D. Clark. A marriage made in torsional space: using GALAHAD models to
drive pharmacophore multiplet searches, J. Comput-Aided. Mol. Des. 20 (2006) 763–771.
30. N. Triballeau, F. Acher, I. Brabet, J. P. Pin, H. O. Bertrand, Virtual screening workflow
development guided by the “receiver operating characteristic” curve approach. application to
high-throughput docking on metabotropic glutamate receptor subtype 4, J. Med. Chem 48 (2005)
2534-2547.
31. N. Huang, B. K. Shoichet, J. J. Irwin, Benchmarking sets for molecular docking, J. Med. Chem. 49
(2006) 6789-6801.
32. Park. J. W, Jo. W. H, Computational design of novel, high-affinity neuraminidase inhibitors for
H5N1 avian influenza virus, Eur. J. Med. Chem. 45 (2010) 536-541.
33. A. Pabis, I. Geronimo, D. M. York, P. Paneth, Molecular dynamics simulation of nitrobenzene
dioxygenase using AMBER force field, J. Chem. Theory. Comput. 10 (2014) 2246−2254.
34. J. C. Baber, D. C. Thompson, J. B. Cross, C. Humblet, GARD: A generally applicable replacement
for RMSD, J. Chem. Inf. Model. 49 (2009) 1889–1900.
35. M. R. Lee, D. Baker, P. A. Kollman, 2.1 and 1.8 Å average CR RMSD structure predictions on two
small proteins, HP-36 and S15, J. Am. Chem. Soc. 123 (2001) 1040-1046.
15

ACCEPTED MANUSCRIPT
36. P. A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. H. Huo, L. L. Chong, M. Lee, T. S. Lee, Y. Duan,
W. Wang, O. Donini, P. Cieplak, J. Srinivasan, D. A. Case, T. E. Cheatham, Calculating structures and
free energies of complex molecules: combining molecular mechanics and continuum models, Acc.
Chem. Res. 33 (2000) 889-897.
37. L. Fu, Y. H. Bi, Y. Wu, S. S. Zhang, J. X. Qi, Y. Li, X. C. Lu, Z. N. Zhang, X. Lv, J. H. Yan, G. F.
Gao, X. B. Li, Structure-based tetravalent zanamivir with potent inhibitory activity against
drug-resistant influenza viruses, J. Med. Chem. 59 (2016) 6303-6012.
38. T. Luchko, S. Gusarov, D. R. Roe, C. Simmerling, D. A. Case, J. Tuszynski, A. Kovalenko,
Three-dimensional molecular theory of solvation coupled with molecular dynamics in amber, J. Chem.
Theory. Comput. 6 (2010) 607-624.
39. S. K. Sadiq, D. Wright, S. J. Watson, S. J. Zasada, I. Stoica, P. V. Coveney, Automated molecular
simulation based binding affinity calculator for ligand-bound HIV-1 proteases, J. Chem. Inf. Model. 48
(2008) 1909-1919.
40. J. Wang, R. M. Wolf, D. A. Case, P. A. Kollman, Development and testing of a general AMBER
force field (GAFF), J. Comput. Chem. 25 (2004) 1157–1174.
41. Y. Duan, C. Wu, S. Chowdhury, M .C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T.
Lee, A point-charge force field for molecular mechanics simulations of proteins based on
condensedphase quantum mechanical calculations, J. Comput. Chem. 24 (2003) 1999–2012.
42. W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, Comparison of simple
potential functions for simulating liquid water, J. Chem. Phys. 79 (1983) 926–935.
43. M. D. Braccio, G. Grossi, G. Roma, D. Piras, F. Mattioli, M. Gosmar, 1,8-Naphthyridines VI.
synthesis and anti-inflammatory activity of 5-(alkylamino)-N,N-diethyl[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6-carboxamides with a new substitution pattern on the triazole ring, Eur. J. Med.
Chem. 43 (2008) 584-594.
44. Z. X. Zhao, L. P. Cheng, W. Pang, Green synthesis of ethyl oxalate benzylidinyl hydrazides,
Tetrahedron Lett. 59 (2018) 2079-2081.
16

ACCEPTED MANUSCRIPT
ò
Discovery of novel acylhydrazone neuraminidase(NA) inhibitors targeting 430-loop based on
virtual screening.
ò
ò
ò
The synthesized inhibitors show excellent inhibitory activity against NA.
430-loop plays an important role in discovery of novel acylhydrazone NA inhibitors.
This work provides unique insights in the discovery of potent inhibitors against NA.