Identification, synthesis, and evaluation of new neuraminidase inhibitors

Article abstract of DOI:10.1021/ol502410x

High-throughput screening was performed on ～6800 compounds to identify KR-72039 as an inhibitor of H1N1 and H5N1 neuraminidases (NAs). Structure-activity relationship studies led to 3e, which inhibited H5N1 NA with an IC₅₀ of 2.8 μM and blocked viral replication. Docking analysis shows that compounds bind to loop-430 around the NA active site. Compound 3l additionally inhibited H7N9 NA, making it a dual inhibitor of N1- and N2-type NAs.

Full text of DOI:10.1021/ol502410x

Letter
pubs.acs.org/OrgLett
Identification, Synthesis, and Evaluation of New Neuraminidase
Inhibitors
Vathan Kumar,†,‡,§ Chih-Kang Chang,§ Kian-Pin Tan,‡ Young-Sik Jung,∥ Shih-Hsun Chen,§
Yih-Shyun E. Cheng,⊥ and Po-Huang Liang*,†,‡,§
†Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan
‡Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
§Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
∥Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea
⊥Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
S
* Supporting Information
ABSTRACT: High-throughput screening was performed on ∼6800
compounds to identify KR-72039 as an inhibitor of H1N1 and H5N1
neuraminidases (NAs). Structure−activity relationship studies led to 3e,
which inhibited H5N1 NA with an IC50 of 2.8 μM and blocked viral
replication. Docking analysis shows that compounds bind to loop-430
around the NA active site. Compound 3l additionally inhibited H7N9
NA, making it a dual inhibitor of N1- and N2-type NAs.
because the existing drug treatment, oseltamivir, has become
ineffective.17,18
avian influenza has sporadically caused pandemic out-
Abreaks. In early 2003, there was an AI H5N1 outbreak in
South and Southeast Asia1 and, more recently, the emergence
of the novel AI strain H7N9 in March 2013.2 Such a sporadic
upsurge continues to pose a serious health threat. Unlike the
highly pathogenic AI H1N1 virus, H5N1 and H7N9 viruses
are less likely to undergo interhuman transmission. However,
if the H5N1 virus acquires the ability for interhuman
transmission, by either reassortment or mutations as
demonstrated in laboratories recently, it would lead to a
pandemic that could be much worse than H1N1.3−7 H7N9
infection could go unnoticed until it reaches humans due to
its low pathogenicity in birds.8 Such a deceptive transmission
could be more perilous and has already claimed 44 lives.9
Vaccines, as therapeutic agents, suffer support challenges
during pandemics which include a longer production time,
shorter shelf lives, and the need for annual validation due to
continual antigenic variance.10 Marketed chemotherapeutics
targeting M2 ion channels have become obsolete in terms of
treating H5N1 and H7N9 infections owing to resistance, thus
leaving neuraminidase inhibitors as the only choice.11,12
Neuraminidase (NA, EC 3.2.1.18) facilitates the detachment
of the viral scions from the infected host cell surface in the
final step of viral replication and thus serves as the target for
anti-influenza drugs oseltamivir and zanamivir. However, the
frequent usage of these drugs has led to the development of
resistance, for example, to oseltamivir by H5N1 due to
H274Y mutation.13,14 Recently, the outbreak of H7N9 in
China15,16 and the emergence of A/Shanghai/1/2013 H7N9
virus with R292K mutation without the loss of in vivo
virulence or transmissibility has caused a serious concern
Although loop-150 has been considered an ideal region for
exploring the further design of the transition-state ana-
logues,19−21 so far no fruitful results against drug-resistant
mutant NAs have been obtained. This necessitates the need to
have a newer scaffold with the feasibility to yield a wide array
of analogues to facilitate the structure−activity relationship
(SAR) studies. Recent research has shown some non-
transition-state analogues with the ability to inhibit NAs and
even drug resistant NAs, though not very effectively or
selectively.22−25 The recent reports focus on increasing the
binding affinity by the development of mechanism-based
covalent inhibitors, which are attacked by the nucleophilic
catalytic residue Tyr406.26,27
As reported here, we performed high throughput screening
against H1N1 and H5N1 NAs using our compound library.28
By screening ∼6800 compounds at 100 μM we were able to
obtain one hit (KR-72039) (structure shown in Table 1),
which inhibited half of the H1N1 and H5N1 NA activity
(IC50) at 37.1
2.3 and 49.5
2.2 μM, respectively.
Compared to the known transition-state analogues (e.g.,
oseltamivir), which has complex synthetic routes,29−31 this
scaffold can be easily synthesized and modified as described in
Scheme 1.
The pyrazolone core was synthesized by the condensation
of substituted hydrazines with various β-ketoesters. The
substituted aldehydes were synthesized by Meerwein arylation
of substituted diazonium salts with furfural.32 Finally,
Received: August 14, 2014
Published: September 17, 2014
© 2014 American Chemical Society
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Letter
Table 1. Results of Neuraminidase Inhibition Assay
number
R1
R2
R3
H1N1 IC50 (μM) H5N1 IC50 (μM) H7N9-Anhui IC50 (μM) H7N9-Shanghai IC50 (μM)
KR-72039
3a
2-COOH, 5-Cl-Ph
2-COOH, 5-Cl-Ph
2-COOH, 5-Cl-Ph
H
CH3
CF3
Ph
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
H
37.1 2.3
>50
49.5 2.2
3.3 0.2
37.1 0.3
>50
>50
107.5 1.6
>200
>50
3b
>50
>50
>200
3c
Ph
>50
>50
>200
3d
3e
H
CH3
Ph
>50
>50
>50
>200
2-COOH, 5-Cl-Ph
2-COOH, 5-Cl-Ph
2-COOH, -Ph
2-COOH, -Ph
2-COOH, -Ph
Ph
>50
2.8 0.1
2.9 0.1
54.94 7.2
13.7 1.2
3.9 0.1
>50
>50
>200
3f
Ph
4-F
32.4 3.8
44.6 4.3
28.8 1.6
>50
>50
>200
3g
CH3
Ph
3-COOH
H
>50
>200
3h
3i
>50
>200
CF3
CH3
Ph
3-COOH
3-COOH
3-COOH
4-F
>50
109.1 3.7
>200
3j
>50
>50
3k
Ph
>50
>50
>50
>200
3l
2-COOH, -Ph
2-COOH, 5-Cl-Ph
Ph
Ph
>50
10.8 0.1
>50
28.1 3.7
>50
89.6 5.9
114.7 2.7
>200
3m
3n
3o
3p
3q
3r
CH3
Ph
H
>50
H
>50
>50
>50
2-COOH, 5-Cl-Ph
2-COOH, -Ph
2-COOH, -Ph
3,5-COOH
Ph
4-CH(CH3)2
4-CN
>50
>50
>50
>200
Ph
>50
>50
>50
>200
Ph
4-OCH3
3-COOH
>50
>50
34.5 1.9
42.7 5.5
108.6 8.1
63.1 0.9
CH3
>50
>50
Knoevenagel condensation of substituted aldehydes with
various pyrazolones in the presence of a catalytic amount of
sodium acetate in acetic acid yielded the final compounds
(50−88%). The methanolic wash yielded pure compounds in
most cases, thus avoiding the need for chromatographic
purification.
the Z isomer (Z/E ratio 3:1) based on the NOE observed
between the methyl of pyrazolone (2.53 ppm) and olefinic
proton (7.69 ppm) in NMR (Figure S1). Therefore, the IC50
in Table 1 for those compounds should be theoretically 4-fold
smaller, if the E-isomer is contributing to the inhibitory
activity.
For the ease of understanding, we have divided the
molecules under study into rings A, B, C, and D (Table 1).
In the case of H5N1 inhibition, replacement of the methyl
substituent at R2 by a phenyl ring (3b) resulted in a marginal
increase of inhibitory activity (IC50 = 37.1 μM). Drastic
improvement was observed (IC50 = 3.3 μM) on isosteric
replacement of the methyl substitution at R2 by trifluor-
omethyl (3a).
Scheme 1. Synthesis of Substituted Pyrazolones
The removal of ring A was not tolerated as demonstrated
by the loss of activity in the case of 3c and 3d irrespective of
R2 substitution. The loss of activity could be due to the
absence of a carboxylate on ring A. The removal of
carboxylate from ring A also resulted in the loss of activity
as seen with 3j, 3k, and 3n. Similar results were obtained
when KR-72039 was esterified (data not shown). This
demonstrated that the carboxylate on ring A is a critical
component.
Interestingly, while elimination of the carboxylate from ring
D abolished the activity of 3m (R2 = methyl), a 15-fold
increase in the potency (IC50 = 2.8 μM) was observed for the
molecule with a phenyl substituent as R2 (3e). In fact, 3e is
the most potent inhibitor of H5N1 NA in the series.
Apparently, the phenyl group as R2 seems to increase the
binding affinity with H5N1 NA. It is also worth noting here
that while the original hit (KR-72039) was not selective
between NAs from H1N1 and H5N1, the best compound 3e,
The pyrazolones with phenyl as R2 were obtained as a
single E-isomer, as there was no NOE signal observed
between the olefinic proton and the R2 phenyl protons in
NMR (Figures S5−S8). However, the compounds were
obtained as geometrical isomers (E/Z) when R2 was methyl,
which were inseparable due to the rapid interconversion in
solution. Such isomerization has also been reported on a
similar system that inhibited heptosyltransferases, such as
WaaC, of Gram-negative bacteria.33 The compounds with
methyl substitution were determined to have a higher ratio of
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Letter
along with 3a, 3i, and 3l, selectively inhibited the H5N1
enzyme at a low micromolar range.
the potency specifically against R292K mutant. Two
compounds with R2 phenyl substitution (3l, 3q) and the
one with R2 methyl substitution (3r) inhibited both the wild-
type and the mutant H7N9 NA.
With an attempt to reduce the molecular weight, we
eliminated chlorine from the ring A. This caused about a 4-
fold drop in the activity when R2 was a phenyl group
(comparing 3e, 3f with 3h, 3l respectively). In contrast, there
was no significant drop in the activity when 3a was converted
to 3i (R2 = CF3).
We further determined the mode of inhibition of our best
inhibitor, namely 3e, using a Lineweaver−Burk plot (Figure
1A). This compound exhibited a mixed-mode inhibition on
Interestingly, 3l was a common inhibitor for both H5N1
and H7N9 NAs. It is also worthwhile to note that 3l inhibits
H7N9-R292K mutant NA from H7N9-Shanghai with an IC50
of 90 μM, merely 3-fold higher than that in inhibiting wild-
type H7N9 NA from H7N9-Anhui. In contrast, oseltamivir
inhibits H7N9-R292K mutant NA with an IC50 of 9 μM but
with a >1000-fold difference between inhibiting the wild-type
and the mutant H7N9 NAs.37 To the best of our knowledge,
this is the first nontransition-state analog reported to inhibit
H7N9 NAs. We are continuing our effort to understand exact
binding of the molecule in N1 and N2 NAs by X-ray
crystallography. This will provide us with a better under-
standing of the binding modes in these NAs and facilitate
further modification.
In summary, this study resulted in the discovery of a
pyrazolone scaffold with dual advantages: a simple synthetic
approach and larger molecular diversity. Modifications led to
3a, 3b, and 3e, selective low micromolar inhibitors of H5N1
NA, with 3e as the most potent one in vitro and in vivo.
Moreover, we identified 3l, a dual inhibitor of H5N1 and
recently emerged H7N9 NAs. Since our compounds have
been verified for in vitro and in vivo efficacy and are devoid of
toxicity at doses as high as 200 μM, they could act as suitable
leads for anti-AI virus drug development.
Figure 1. (A) Lineweaver−Burk plot to confirm mixed-type
inhibition of NA hydrolase activity by 3e. (B) Docking analysis of
3e (PDB ID: 2HTY).
H5N1 NA with a 2-fold higher affinity to free enzyme (Kic =
2.9 μM) than to the enzyme−substrate complex (Kiu = 5.6
μM) (Table S1).
ASSOCIATED CONTENT
* Supporting Information
Synthesis details, 1H and 13C NMR of synthesized
compounds, and assay methods. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
We then performed molecular docking simulations to
understand the possible binding interactions of our com-
pounds with H5N1 NA. Since the enzyme kinetics experiment
showed that the compound 3e has higher affinity toward free
enzyme (Kic = 2.9 μM), we used an apo-form NA crystal
structure (PDB ID: 2HTY) for our docking analysis. The
docking simulation option (Accurate docking) of iGemdock
v2.1 was used to generate 20 solutions. The inhibitors with R2
as the phenyl group prefer to occupy loop-430, an allosteric
site of NA, and extend partly into the active site to interact
with the residues therein. This is consistent with the mixed-
type inhibition pattern determined experimentally. For
example, in 3e as shown in Figure 1B, R2 phenyl mainly
interacts with the hydrophobic side chain of I427, P431, and
K432 along with the additional weak π−π interaction with
W403 in loop-430. The carboxylate moiety on ring A forms a
H-bond with R292 at the catalytic site and appears to be
necessary for activity. When this interaction was lost, as seen
with 3h and 3l that interact with R156, there was about a
30% drop in activity (Figure S2). The chlorine on the ring A
can help to keep the ring in a proper orientation. Additionally,
there are some other reports that corroborate the binding of a
noncompetitive inhibitor around the NA active site.34−36
A cell-based assay using live H5N1 virus showed 3e, 3f, and
3h effectively inhibited the virus with an EC50 of 27, 32, and
24 μM, respectively. This demonstrated NA as the genuine
target of our compounds. All effective inhibitors were
nontoxic toward HEK-293 cells at 100 μM, and their CC50
were larger than 200 μM except in the case of 3f (Figures S3
and S4).
S
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: phliang@gate.sinica.edu.tw.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Academia Sinica. We thank
Yueh-Kuei Lo (Institute of Biological Chemistry, Academia
Sinica) for assistance in high-throughput screening and Shi-
Yun Wang (Genomic Research Center, Academia Sinica) for
assistance in antiflu assay.
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