Design, Synthesis, and Biological Evaluation of Novel Acylhydrazone Derivatives as Potent Neuraminidase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.0c00313

Neuraminidase (NA) is an important target for current research on anti-influenza drugs. The acylhydrazone derivatives containing the -CONHN═CH- framework have been shown to have good NA inhibitory activity. In this paper, a series of novel acylhydrazone NA inhibitors (9a-9n) were designed and synthesized, and the inhibitory activities against NA were evaluated in vitro. The NA inhibition results showed that compound 9j has the most potent inhibitory activity (IC50 = 0.6 μM) against NA, which is significantly lower than that of the positive control oseltamivir carboxylic acid (OSC) (IC50 = 17.00 μM). Molecular docking analysis indicates that the acylhydrazone group plays an important role in compound 9j, which can bind well to the residues Arg371 and Arg292 in the S1 subsite of NA. The good potency of 9j may be also ascribed to the extending of morpholinyl ring into the 430-cavity. The results of this work may contribute to the development of more potent NA inhibitors to against mutant influenza viruses.

Full text of DOI:10.1021/acsmedchemlett.0c00313

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Letter
Design, Synthesis and Biological Evaluation of Novel
Acylhydrazone Derivatives as Potent Neuraminidase Inhibitors
Meng Li, Li Ping Cheng, Wan Pang, Zhi Jian Zhong, and Ling Ling Guo
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00313 • Publication Date (Web): 24 Aug 2020
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Design, Synthesis and Biological Evaluation of Novel Acylhydrazone
Derivatives as Potent Neuraminidase Inhibitors
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Meng Li, Li Ping Cheng*, Wan Pang, Zhi Jian Zhong, Ling Ling Guo
*School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China
ABSTRACT: Neuraminidase (NA) is an important target for current research on anti-influenza drugs. The acylhydrazone
derivatives containing the -CONHN=CH- framework have been shown to have good NA inhibitory activity. In this paper, a series
of novel acylhydrazone NA inhibitors (9a-9n) were designed and synthesized, and the inhibitory activities against NA were
evaluated in vitro. The NA inhibition results showed that compound 9j has the most potent inhibitory activity (IC50 = 0.6 µM)
against NA, which is significantly lower than the positive control oseltamivir carboxylic acid (OSC) (IC50 = 17.00 µM). Molecular
docking analysis indicates that the acylhydrazone group plays an important role in compound 9j, which can bind well to the
residues Arg371 and Arg292 in the S1 subsite of NA. The good potency of 9j may be also ascribed to the extending of morpholinyl
ring into the 430-cavity. The results of this work may contribute to the development of more potent NA inhibitors to against mutant
influenza viruses.
KEYWORDS: Neuraminidase inhibitor; acylhydrazone; 430-cavity; bioassay
Influenza is a common viral respiratory disease caused by spreading of resistance to these drugs poses an urgent threat to
influenza viruses and has a strong ability to spread. Influenza public health.9,10 Therefore, it is imperative to develop novel
virus is
a negative-sense single-strand member of the drug-resistant neuraminidase inhibitors against resistant virus
Orthomyxoviridae family of RNA viruses. According to the strains.
differences in the antigenicity of nuclear proteins and matrix
With the further study of the crystal structure of
proteins, influenza virus are divided into three types: A, B, and
C. Influenza A viruses are prone to antigenic mutations and are
the main cause of seasonal or pandemic influenza worldwide.1-3
The most severe pandemic occurred in 1918, resulting in
approximately 40 million deaths worldwide.4 Vaccines and
antiviral drugs are currently important initiatives in the
prevention and treatment of influenza. However, due to the
long-lasting effect of the vaccine and its limited effectiveness in
patients with low immune function, the current focus is on the
development of small molecule drugs against influenza viruses.5
neuraminidase, based on the phylogenetic tree, neuraminidase
(NA) is divided into nine subtypes (N1-N9), in which N1, N4,
N5, N8 are the first group, and N2, N3, N6, N7, and N9 are the
second group. In 2006, Russell and co-workers declared that the
difference between the two groups was that the first group
contains a 150-cavity (residues 147-152), while the second
group does not.11 Recently, an additional hydrophobic binding
site adjacent to the active site had been discovered and named
as 430-cavity. 430-cavity connects directly to the active site and
has large molecular volume. Interestingly, different from 150-
cavity, 430-cavity widely and stably exists in various subtypes
Neuraminidase (NA), also known as sialidase, is
a
glycoprotein distributed on the envelope of influenza virus. It of neuraminidase, including group-1 and group-2.12,13 This
has antigenicity and can catalyze the hydrolysis of sialic acid, important discovery provides a great opportunity for inhibitor
which can help mature influenza virus to escape from its design. For example, Liu's team discovered a series of C-1
original host cells to infect new cells and play a crucial role in modified oseltamivir derivatives targeting the 430-cavity as
virus transmission. The active center of neuraminidase includes potent NA inhibitors.14 A series of zanamivir analogs with C-1
19 conserved amino acids. Among them, the eight highly and C-4 modifications had also been developed to exploit the
conserved amino acids that can directly interact with the 430-cavity.15 Therefore, as an important binding site, 430-cavity
substrate are Arg118, Arg152, Arg224, Arg292, Arg371, is of great significance for the development of new NA
Asp151, Glu276, Tyr406, respectively.6 The highly inhibitors.
conservative structure of its catalytic active site makes it a
The acylhydrazones derivatives containing the
-
superior target for antiviral drug development. So far, there are
four approved drugs of neuraminidase inhibitors, including
oseltamivir, zanamivir, peramivir and laninamivir octanoate
(Figure 1).7 Among them, oseltamivir is the only oral effective
neuraminidase inhibitor. Zanamivir can only be administered by
inhalation. Peramivir sodium chloride injection has been
approved in China. Laninamivir octanoate has recently been
authorized in Japan.8 Nevertheless, emergence and rapid
CONHN=CH- framework are the products obtained by
condensation of the acylhydrazones with the corresponding
aldehydes or ketones. They have strong biological activity and
stable structure, and have been proved to have good application
prospects in biological and drug activity, bacteriostatic and
antitumor.16-19 Besides, some acylhydrazone derivatives have
been reported to have potential anti-influenza virus activity, 20
and several of them have been evaluated for NA inhibitory
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activity. For instance, Xiao et al.21 synthesized a series of benzene ring of the acylhydrazone group is just at the position
acylhydrazone derivatives via microwave irradiation as of the hydrophobic center HY_9. There are two hydrophobic
influenza NA inhibitors. Moreover, according to our previous centers (HY_5 and HY_10) and a hydrogen bond acceptor
reports, Zhao et al.22 discovered an acylhydrazone lead center (AA_3) near the substituted phenol, indicating that some
compound as a NA inhibitor through virtual screening. Yu et hydrogen bond acceptor groups and hydrophobic groups
al.23 designed and synthesized a series of benzoylhydrazone introduced into this position may be beneficial to improve
neuraminidase inhibitors. Both series of compounds showed biological activity. Hence, we introduced some hydrogen bond
superior NA inhibitory activity to the positive control acceptor atoms or groups containing N, O and F (9a, 9b, and
oseltamivir acid. Given the highly conserved active site of 9n). Some hydrophobic groups were also introduced, such as
neuraminidase and the promising binding site of the 430-cavity, halogen atoms (9c, 9d, 9e, and 9m), nitro groups (9f, 9g, and
inspired by these studies, we tried to develop some new novel 9l), methoxy groups (9h, 9i, and 9j), and acetamide group (9k).
acylhydrazone NA inhibitors, and evaluate in vitro for Therefore, a total of 14 new compounds from 9a-9n were
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inhibitory activity assay.
designed.
OH
O
H
O
O
HO
OH
O
O
O
OH
HN
N
HN
NH
H
O
NH2
NH2
Zanamivir
Oseltamivir
O
O
O
H
O
Figure 2. (a) The features of pharmacophore model; (b)
Superposition of compound 9j onto the pharmacophore model.
O
HN
O
OH
H
H
HO
HN
H2N
N
The synthesis of acylhydrazones (9a-9n) was accomplished
in six steps as outlined in Scheme 1.24-26 Firstly,
isophthalaldehyde (1) was subjected to a single reduction under
the action of sodium borohydride to obtain 3-(hydroxymethyl)
benzaldehyde 2. Then 3-(hydroxymethyl) benzaldehyde 2 was
brominated under the action of phosphorus tribromide (PBr3) to
form 3-(bromomethyl) benzaldehyde 3. Next, compound 3 was
etherified with substituted phenol 4 by Williamson ether
synthesis to obtain compounds 5a-5n. Then diethyl oxalate 6
reacted with hydrazine hydrate to prepare monoethyl oxalate
hydrazine 7, which was stirred with compounds 5a-5n at room
temperature to produce compounds 8a-8n. Finally, compounds
8a-8n were refluxed with N-(3-aminopropyl) morpholine in an
organic solvent to obtain target compounds 9a-9n.
HN
OH
HN
NH
O
NH2
O
HO
Laninamivir octanoate
Peramaivir
Figure 1. NA inhibitors had been approved for the treatment of
influenza viruses.
Based on the features derived from the pharmacophoric
model in our previous study,22 a series of novel acylhydrazone
neuraminidase inhibitors 9a-9n (see Scheme 1) were designed.
The superimposition of the new designed compound 9j onto the
pharmacophore model is shown in Figure 2. It can be seen that
the two nitrogen atoms in the acylhydrazone moiety coincides
with two positive nitrogen centers (NP_6 and NP_7), and the
O
(ii)
O
HO
O
Br
(i)
O
O
N
2
3
O
1
O
O
NH
OH
(iii)
R
Br
HN
O
O
R
O
N
O
O
HN
5a-5n
4
3
(vi)
(v)
N
O
O
H
(iv)
H2N
N
N
O
O
NH2
O
O
O
O
O
7
6
O
9k
:R=4-acetylamino
9a
:R=2- fluorine
9f
:R=2-nitro
9g
R
9l
:R=2-methoxy,4-nitro
9b
9c
:R=4- fluorine
:R=2-chlorine
:R=4-nitro
8a-8n
9m
9n
:R=2-methoxy,5-bromo
:R=2-nitro,5-fluorine
9h
:R=2-methoxy
:R=3-methoxy
R
9d
:R=3-chlorine
9i
9j
9a-9n
9e
:R=4-chlorine
:R=4-methoxy
Scheme 1. Synthesis of compounds 9a-9n. Regents and conditions: (i) NaBH4, C2H5OH, THF, 0 °C; (ii) PBr3, CH2Cl2, 0 °C; (iii)
.
Cs2CO3, KI, DMF, 60 °C; (iv) NH2NH2 H2O, absolute ethanol, 0 °C; (v) H2O, rt; (vi) MeOH, 70 °C refluex.
Biological activity against NA for the new synthesized
compounds was further tested and listed in Table 1. It can be
seen that several of the target compounds show good
inhibitory potency. For example, compounds 9b, 9e, 9j, and
9m, with IC50 values of 5.29 µM, 6.20 µM, 0.60 µM, and 7.31
µM, respectively, which are significantly superior to the
positive control OSC (IC50 = 17.00 µM). Especially,
compound 9j exerts the most potency. The activities of 9i (IC50
= 13.60 µM) and 9n (IC50 = 17.82 µM) are comparable to that
of OSC. Meanwhile, as shown in Table 1, the activity of
compounds with the same substituents at the 4-position of the
phenol ring is generally higher than that at the 2-position. This
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was supported by the comparison of inhibitory activity of
compound 9a (IC50 = 21.57 µM) with 9b (IC50 = 5.29 µM),
compound 9c (IC50 = 20.40 µM) with 9e (IC50 = 6.20 µM),
compound 9f (IC50 = 85.97 µM) with 9g (IC50 = 27.84 µM)
and compound 9h (IC50 = 47.75 µM) with 9j (IC50 = 0.60 µM).
Particularly, the activity of 9j is about 79-fold more active
against NA compared with 9h. In addition, the introduction of
groups containing N, O and F, such as methoxy, nitro group,
and F atoms, etc., is easy to form hydrogen bonds with amino
acids at the active site, which may increase the activity of the
inhibitors to a certain extent.
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Table 1. NA inhibition activity of target compounds 9a-9n.
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9
Com
R group
2-F
IC50 (µM)
Com
R group
IC50 (µM)
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9a
21.57±0.43
9i
3-OCH3
13.60±0.5
4
9b
9c
4-F
5.29±0.14
9j
4-OCH3
4-NHAc
0.60±0.16
2-Cl
20.40±0.86
9k
73.39±2.9
3
9d
3-Cl
22.13±0.68
9l
2-OCH3-4-NO2
28.38±2.8
6
9e
9f
4-Cl
6.20±0.47
9m
9n
2-OCH3-5-Br
5-F-2-NO2
7.31±0.23
2-NO2
85.97±2.19
17.82±3.5
8
-
9g
9h
4-NO2
27.84±2.36
47.75±0.05
OSC
17.00±1.4
1
2-OCH3
To better understand the discrepancy in activity between
compounds, molecular docking was performed to explore the
binding affinities between inhibitors and NA. The Surflex-
Dock module of SYBYL-X 2.1 was used for molecular
docking.27 The crystal structure of the neuraminidase protein
complex was taken from the RCSB protein database (PDB ID:
2HU0). The neuraminidase activity site is highly polar. It was
suggested that the active site of NA could be divided into five
regions empirically, termed subsites S1−S5, which are critical
for the interactions with different NA inhibitors (Figure 3).28
Figure 4 shows the binding patterns of compounds OSC, 9j,
and 9f with NA. The docking results of the remaining
compounds with NA are shown in Figure S1. As seen from
Figure 4, for OSC, its whole skeleton is completely embedded
in the active site of neuraminidase, which can form multiple
hydrogen bonding interactions with some key amino acids,
such as Arg118, Arg371, Arg292, Arg152, and Asp151.
Among them, the C-1 carboxylate can form strong
electrostatic interactions and three hydrogen bonds with the
three positively charged arginine residues (Arg118, Arg292,
Arg371) in the S1 site, which are essential for the NA
inhibitory activity.29 The N atom of C-5 amino group forms a
hydrogen bond with the Asp151 residues of the S2 site. The
carbonyl oxygen atom of acetamido may form a hydrogen
bond with the Arg152 residue of the S3 site and strong
hydrophobic interaction with the Trp178 residue. For the most
potent 9j, the acylhydrazone (-CONHN=CH-) group and its
adjacent carbonyl group form four hydrogen bonds with some
key residues Arg118, Arg371 and Arg292 of the S1 site, in a
similar pattern with that of 1-carboxyl group of OSC. In
addition, the carbonyl oxygen atom of the acylhydrazone
group forms a hydrogen bond with the Tyr406 residue. The
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amide nitrogen atom forms a hydrogen bond with the Tyr347
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residue. The benzene ring of the acylhydrazone moeity forms
hydrophobic interaction with the hydrophobic faces of
residues Arg224 and Glu276 of the S4 and S5 sites. The 4-
OCH3 substituted phenol may form strong hydrophobic
interactions with the hydrophobic faces of residues Asp151,
Glu119, and Glu227 of the S2 site. It is worth noting that the
morpholine ring of compound 9j can extend into the 430-
cavity and form strong hydrophobic interaction with residues
Lys432, Pro431, and Ser370, which may explain its higher
activity than the positive control OSC. Meanwhile, as shown
in Figure 4, compound 9f can also well occupy the active site.
The nitro group of the phenol ring forms two hydrogen bonds
with the Arg292 residue of the S1 site, and two hydrogen
bonds with the Tyr347 and Tyr406 residues respectively. The
phenol ring forms hydrophobic interactions with the
hydrophobic face of Arg151 of the S2 site. The acylhydrazone
moiety forms a hydrogen bond with Arg152 of the S3 site. The
morpholine ring forms strong hydrophobic interaction with the
residues Trp178 and Ile222 of the S3 site as well as the
hydrophobic face of residue Arg224 of the S4 site. A total of
five hydrogen bonds were formed. Although 9f forms as many
hydrogen bonds in the active pocket of NA as OSC, its activity
is not better than that of OSC, perhaps because the binding
affinity of the inhibitor with residues Arg152 and Tyr406 is
not as strong as that with residues Arg118 and Arg292. In
addition, compared with 9j, compound 9f could occupy the
active site but it has no group to extend into the 430-cavity.
Therefore, the higher NA inhibitory potency of 9j may be
mainly ascribed to the elongation of the molecule to the 430-
cavity to form strong hydrophobic interactions with NA.
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Figure 4. The binding models of compounds OSC, 9j, and 9f
with NA (PDB: 2HU0).
To clarify the dynamic interaction patterns between the
inhibitors and NA protein and to verify the reliability of
molecular docking results, molecular dynamics (MD)
simulations were conducted for NA-inhibitor complexes by
using Amber 12.0 software package.30 The RMSD value (Å)
with respect to simulation time (ns) is shown in Figure 5. It
can be seen that most of the complexes are stable after 10 ns
of the simulation runtime, and the fluctuation of the average
RMSD value during the whole simulation is between 2.0 Å
and 4.5 Å, indicating that the complexes keep the proximate
native state. Figure 6 depicts the superposition of the
previously docked conformation (in green) and the
conformations after the stabilization of RMSD values (in
magenta) of 9j–2HU0 complex. It can be seen that except for a
slight drift and rotation of bonds, the two conformers are in a
nearly parallel poses and there seems to be no significant
difference. This confirms that the binding pocket and the poses
of the ligand are stable and the docking results are reliable.
Glu227
Arg152
Trp178
Glu119
S2
O
S3
OH
Asp151
HN
Ile222
Arg118
HO
HO
O-
O
S1
O
S4
Arg 371
Arg224
OH
Arg292
Ala246
S5
Glu276
Figure 3. Schematic diagram of S1-S5 region division and the
combination of sialic acid (Neu5Ac) and NA active sites.
Based on MD simulations, the binding free energies (ΔGbind
)
of the 14 new inhibitors with NA were calculated and listed in
Table 2. In this research, ΔGbind was calculated by MM/PBSA
and MM/GBSA methods. It was worth mentioned that
MM/PBSA is generally considered superior to MM/GBSA in
calculating binding free energy.31 As shown in Table 2, the
smaller the ΔGbind of inhibitor-NA is, the stronger the activity
of the inhibitor is for most inhibitors. For instance, the binding
free energies of compounds 9e, 9j and 9m were predicted by
MM/PBSA methods to be -24.31 kcal.mol-1, -22.66 kcal.mol-1
and -26.40 kcal.mol-1, respectively. They are all significantly
lower than that of OSC (-10.34 kcal.mol-1). Correspondingly,
these three compounds have better experimental inhibitory
activity, with IC50 values of 6.20 µM, 0.60 µM and 7.31 µM,
which are significantly lower than that of OSC (IC50 = 17.00
µM). Moreover, the binding free energy of compound 9i (-
11.23 kcal.mol-1) is similar to that of OSC. They also have
comparable inhibitory activity, with IC50 values of 13.60 µM
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and 17.00 µM, respectively. The same rule also applies to
compounds 9a and 9g, and compounds 9f and 9k. Hence one
can see that the theoretical calculation results from MD
simulation are well consistent with the experimental results.
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Figure 5. Diagram of RMSD value (Å) with respect to
simulation time (ns).
Table 2. The calculated binding free energies (ΔGbind, kcalmol-1) of ligand-2HU0 complexes along with the different energy
(kcalmol-1) contributions.
Com.
OSC
9a
VDW
-24.43
-29.60
-25.42
-43.81
-32.83
-45.83
-40.20
-37.88
-24.27
-33.06
-46.65
-35.30
-30.91
-51.77
-23.46
EEL
ΔGgas
ΔGGB
26.42
22.50
30.50
46.30
29.07
38.18
49.43
34.28
20.43
26.23
48.25
38.42
29.62
38.74
54.39
ΔGSA
-3.43
-4.33
-3.52
-5.99
-4.76
-5.53
-5.32
-5.03
-3.13
-4.19
-6.57
-4.36
-4.33
-6.15
-3.37
ΔGsolv(GB)
22.99
18.17
26.98
40.31
24.31
32.65
44.10
29.26
17.31
22.04
41.69
34.06
25.29
32.59
51.02
ΔGbind(GB)
-14.66
-22.66
-14.80
-28.11
-17.79
-34.57
-25.49
-27.45
-15.43
-15.98
-41.36
-19.67
-16.82
-35.02
-20.70
ΔGPB
30.26
24.37
29.46
51.79
32.98
47.08
51.91
40.77
20.97
30.47
65.40
36.80
29.64
46.31
54.63
ΔGSA ΔGsolv(PB)
ΔGbind(PB)
-10.34
-19.84
-15.74
-21.53
-13.48
-24.31
-21.92
-19.85
-14.81
-11.23
-22.66
-20.92
-16.56
-26.40
-20.33
-13.22
-11.23
-16.36
-24.61
-9.27
-37.65
-40.83
-41.78
-68.42
-42.10
-67.23
-69.59
-56.70
-32.74
-38.02
-83.05
- 53.73
-42.11
-67.61
-71.72
-2.96
-3.38
-3.42
-4.89
-4.36
-4.16
-4.24
-3.91
-3.03
-3.68
-5.02
-3.99
-4.09
-5.10
-3.25
27.31
20.99
26.04
46.89
28.62
42.92
47.67
36.85
17.93
26.79
60.39
32.81
25.55
41.21
51.38
9b
9c
9d
9e
-21.40
-29.39
-18.82
-8.47
9f
9g
9h
9i
-4.97
9j
-36.40
-18.43
-11.20
-15.84
-48.26
9k
9l
9m
9n
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(No. ZQ 2018-20), and Shanghai Municipal Education
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Commission (Plateau Discipline Construction Program).
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Notes
The authors declare that there is no financial/commercial conflict
of interest.
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ABBREVIATIONS
NA, neuraminidase; RMSD, root mean square deviation;
MM/GBSA, Molecular Mechanics/Generalized Born Surface
Area; MM-PBSA, Molecular Mechanics/Poisson-Boltzmann
Surface Area.
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Figure 6. The superposition of the previously docked
conformation (in green) and the conformations after the
stabilization of RMSD values (in magenta) of 9j–2HU0
complex.
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In this research,
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Corresponding Author
*Phone: +86-21-6087-3250. Email: chengliping@sit.edu.cn
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Teachers Scientific and Technological Talents Developing Fund
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Insert Table of Contents artwork here
A series of novel acylhydrazone neuraminidase inhibitors were designed and synthesized according to
the characteristics of pharmacophore model.
Compound 9j IC50=0.6 µM
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