Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2019.02.027

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-β gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

Full text of DOI:10.1016/j.bmcl.2019.02.027

Accepted Manuscript
Identification, Design and Synthesis of Novel Pyrazolopyridine Influenza Virus
Nonstructural Protein 1 Antagonists
Samarjit Patnaik, Dipanwita Basu, Noel Southall, Seameen Dehdashti, Kanny
K. Wan, Wei Zheng, Marc Ferrer, Mercedes Taylor, Daniel A. Engel, Juan Jose
Marugan
PII:
S0960-894X(19)30111-8
https://doi.org/10.1016/j.bmcl.2019.02.027
BMCL 26307
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2019
21 February 2019
26 February 2019
Please cite this article as: Patnaik, S., Basu, D., Southall, N., Dehdashti, S., Wan, K.K., Zheng, W., Ferrer, M.,
Taylor, M., Engel, D.A., Marugan, J.J., Identification, Design and Synthesis of Novel Pyrazolopyridine Influenza
Virus Nonstructural Protein 1 Antagonists, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.02.027
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Bioorganic & Medicinal Chemistry Letters
Identification, Design and Synthesis of Novel Pyrazolopyridine Influenza Virus
Nonstructural Protein 1 Antagonists
a,
b
a
a
a
a

Samarjit Patnaik , Dipanwita Basu , Noel Southall , Seameen Dehdashti , Kanny K. Wan , Wei Zheng ,
Marc Ferrera, Mercedes Taylora, Daniel A. Engelc,* and Juan Jose Marugana,*
aDivision of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
MD 20850.
bDepartment of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22908.
cAlexander BioDiscoveries, LLC, 530 Forrest Rd., Charlottesville, VA 22902
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and
pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report
the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently
inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic
effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN- gene
expression, and suppress the slow growth phenotype in NS1-expressing yeast. These
pyrazolopyridines will enable researchers to investigate NS1 function during infection and how
antagonists can be utilized in the next generation of treatments for influenza infection.
Keywords:
Influenza
NS1
2019 Elsevier Ltd. All rights reserved.
Antagonist
Pyrazolopyridine
Antiviral
———
 Corresponding authors. For S.P. Tel.: +1-301-827-1772; fax: +1-301-217-5736; e-mail: patnaiks@mail.nih.gov. For D.A.E Tel.: +1-434-924-
8633; fax: +1-434-982-1071; e-mail: dae2s@virginia.edu. For J.J.M. Tel.: 1-301-480-9857; fax: +1-301-217-5736; e-mail:
maruganj@mail.nih.gov.

Influenza is an acute respiratory disease caused by members
of the orthomyxoviridae family of RNA viruses. Recent analysis
by the Centers for Disease Control and Prevention (CDC)
estimates that influenza has resulted in, on average, about
600,000 hospitalization and 40,000 deaths annually in the US.1, 2
Coupled with the morbidity and mortality is the substantial
financial impact on medical cost, loss of work, and
hospitalizations. The “Spanish flu” of 1918, which caused an
estimated 20-50 million deaths worldwide, is a stark reminder of
a flu pandemic’s potential for devastation. The keystones of
current public policy remain surveillance and prevention via
administration of the seasonal vaccine containing the most
commonly circulating viral strains. However, mutations within
the viral genome lead to antigenic drift, eventually rendering the
flu shots ineffective.3 Additionally, novel viruses may be formed
by antigenic shift when segments of human viral strains reassort
with those from other animal reservoirs (e.g. birds and pigs).4-6
The danger of antigenically shifted pathogens is exemplified by
the millions of deaths in the 1957 and 1968 influenza pandemics
caused by viruses derived from avian sources for which humans
were immunologically unprepared.6 The dangerous introduction
of an antigenically novel virus for which humans have faint
immunological memory was felt again in 2009 when a version of
the 1918 H1N1 virus, after circulating in pigs for nearly 100
years, led to the emergence of a pandemic strain from Mexico
and California.7 This outbreak exposed shortcomings in global
preparedness, as it was clear that it would take at least 6 months
to generate a vaccine supply which would be useful, a timeline
which could easily be superseded by a rapidly spreading virus.
their structure-activity relationship (SAR), and their ability to
reduce virus replication, rescue infected cells from virus-induced
cytotoxicity and restore the NS1-inhibited expression of IFN
mRNA.
34
Expanding on a previous approach,29, which uses a NS1-
pYES yeast strain that exhibits a pronounced slow-growth
phenotype upon expression of NS1 protein, we have performed a
quantitative high-throughput screen (qHTS) against a library of
269,572 compounds from the NIH Molecular Libraries-Small
Molecule Repository (MLSMR) collection for activities that
specifically reversed the NS1-induced slow-growth phenotype.
The published protocol29 was adapted for 1536-well plate format
at 4 doses (100 nM-57 M). The screening effort and subsequent
cherry-picking experiments identified 200 hits, representing
0.08% of the chemical library.35 These hits were then evaluated
for their antiviral activity and cytotoxicity. Their antiviral activity
was examined by testing the compounds ability to slow
replication of the A/PR/8/34 strain of influenza A virus (H1N1)
in MDCK cells infected at a multiplicity of infection (MOI) of
0.1 for 48 hours. The reduction of viral titer was measured by a
hemagglutination assay of the supernatant and standard TCID50
analysis. Cytotoxicity was tested in MDCK cells using the
CellTiter Glo ATP cell viability assay. Twenty-three compounds
belonging to two structural clusters,
and seven singletons showed antiviral
activity. The pyrazolopyridine hit 1
(Figure 1) demonstrated the most
robust antiviral activity without
toxicity towards the host MDCK cells.
An effective response to pandemic strains will require a wider
variety of antiviral drugs than those that are currently available.
Adamantane antivirals, which target the viral M2 protein required
for viral uncoating within the host cell, are no longer
recommended for use due to widespread resistance. Inhibitors of
the neuraminidase protein (i.e. oseltamivir and zanamivir), which
block the release of new virions from the host cells, are the most
commonly prescribed treatments today.8 However, given the
emergence of oseltamivir-resistant strains8 and the rapid spread
of the highly pathogenic H5N1 strain among birds, it is vital to
continue the pursuit of new treatment modalities.9-10 Recent FDA
approval of baloxavir marboxil, which targets the viral
polymerase, highlights the need for additional viral targets to be
exploited therapeutically.11
Based on this activity and its chemical
tractability, hit 1 was chosen for
further optimization.36
Figure 1. Pyrazolopyridine
hit from qHTS screen.
In total, 40 pyrazolopyridines were
evaluated in the aforementioned
influenza virus replication and cytotoxicity assays. The non-
commercially available analogs were synthesized via the route
A proposed antiviral mechanism targets the viral nonstructural
protein 1 (NS1), which plays a key role in virus replication by
repressing the innate host immune system.12-16 NS1 is a well
conserved, 230-237 amino acid multifunctional protein that
contains an RNA binding domain and an effector domain that are
connected by a variable linker,17 and it is highly expressed during
infection.18 NS1 is the centerpiece of the viral response to the
host interferon (IFN) system, functioning as a key component in
the temporal regulation of viral RNA synthesis, its splicing, and
translation. Specifically, NS1: i) suppresses the host IFN-
response to viral infection,19-23 ii) inhibits the function of 2’-5’-
oligoadenylate synthase and protein kinase R, iii) inhibits the
maturation of host pre-mRNAs,24-25 iv) interferes with the host
RNAi pathway, adaptive immune response, and the apoptotic
response,26 and v) binds to the human PAF1 transcription
elongation complex (hPAF1C), a crucial player in transcription
of antiviral genes.27 In doing so, NS1 promotes and enhances
viral replication, making it a compelling target for influenza
shown in Scheme 1.
Scheme 1. Synthetic route to pyrazolopyridine analogs for SAR evaluation.
Most of the compounds were non-cytotoxic, and several
compounds reduced viral titer >100-fold at 10 M. Table 1
summarizes the fold reductions in viral titer after incubating the
compounds at 10 µM for 48 hours. The fold decrease in viral
replication was determined by calculating the inverse log of the
difference between the logTCID50 value for each compound and
the DMSO control. The SAR summary of the series is illustrated
in Figure 2.
treatment. As such, a number of chemical series that can disrupt
28-33
NS1 function have been reported.14,
Here we report a
structurally distinct class of pyrazolopyridine antagonists of NS1
function, discovered using a yeast-based screening strategy,29

Within the context of the 5-cyano-6-amino pyrazolopyridine
core with a catechol-like substituent in the C4 position (i.e. 2-6),
the presence of a lipophilic methyl or chloro group at R4 and/or
R5 seemed to cause potent antiviral activity, with 4 and 6
showing 256-fold reduction in virus production. Capping one of
the phenols (R1) with a methyl group (i.e. 7-11) led to
compounds that did not strictly recapitulate this trend, but still
produced 10 with similar potency. The antiviral activity of
analogs with a m,p-dimethoxy phenyl (i.e. 12-15) or m,p,m-
trimethoxy phenyl (i.e. 16-20) at C4 was attenuated in general.
Analogs with a 6-hydroxy core (i.e. 21-24) was evaluated briefly
as it was present in hit 1, and modest 16-fold reductions were
observed with compounds 21 and 23. A limited examination of a
pyridinedione tricyclic core (i.e. 25-29) with an unsubstituted
phenyl ring at N1 revealed modest antiviral activity. The
necessity of substitutions at C5 and C6 of the core was also
interrogated using synthesized analogs 30-38. Significant
reductions in viral titer were observed with 32 and 33, reiterating
the ideal presence of a small lipophilic group at R3 or R4. This
potent activity was obtained with a m-OMe-p-OH phenyl
substituent at C4, implying that the presence of a catechol-like
substituent, like that in analogs 2-6, is not necessary for potent
activity. Modest activity was also observed with a carboxylic
acid 39 or ester 40 at C5 in the context of a 4-F phenyl group at
N1.
Table 1. SAR around hit 1
Viral fold
reduction
at 10 Ma
R1
R2
R3
R4
R5
R6
Cmpd
2
3
4
5
6
7
8
9
OH
OH
OH
OH
OH
OMe OH
OMe OH
OMe OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me Me
F
Cl
H
Me
Me Me
F
Cl
H
Me
F
Cl
H
H
H
32
64
256
16
256
32
4
H
H
H
H
4
10 OMe OH
11 OMe OH
12 OMe OMe
13 OMe OMe
14 OMe OMe
15 OMe OMe
16 OMe OMe OMe
17 OMe OMe OMe Me
18 OMe OMe OMe Me Me
19 OMe OMe OMe
H
H
H
H
H
H
H
H
256
32
2
0
2
0
16
32
0
0
0
F
H
H
20 OMe OMe OMe Cl
21 OMe OH
22 OMe OMe
23 OMe OMe OMe
24 OMe OMe
H
H
H
H
H
F
H
H
H
H
H
16
2
16
0
H
H
1
OEt
OH
F
2
Our efforts at exploring the SAR of the screening hit 1 had
therefore resulted in several compounds with potent antiviral
activity. It was also noteworthy that, besides compound 37, no
other analogs exhibited cytotoxicity towards the host MDCK
cells. Key potent compounds with ≥128 fold reduction in viral
titer (i.e. 4, 6, 10, 32, and 33) were analyzed for dose response in
the TCID50 assay, and they successfully decreased viral titer with
increasing concentration. As an illustrative example, analog 32
had an IC90 of 155 nM (Figure 3), and since it did not affect cell
viability at 5, 10, 50 and 100 M, it had a greater than 100-fold
selectivity index (SI).
(hit)
25 OMe OH
26 OMe OMe
27 OMe OH
H
H
OMe
H
H
2
4
4
2
4
28 OH
OMe
29 -OCH2O-
30 OMe OH
H
H
F
0
31 OMe OH
32 OMe OH
33 OMe OH
34 OMe OH
35 OMe OH
36 OMe OH
H
H
H
H
H
H
H
H
H
CF3
H
H
OMe
F
OM
H
CF3
H
H
H
0
128
256
2
32
2
100
50
0
9
8
7
6
5
37 OMe
H
F
F
H
H
4
2
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
38 OMe OMe
Concentration (log M)
Concentration (log M)
Figure 3. The activity of 32 on viral titer. Triplicate cultures were infected
with A/PR/8/34 at a MOI of 0.1 and treated with the indicated concentrations
of compound. After 48 h (time), the supernatants were collected and analyzed
to determine the reduction in virus by assaying the supernatant infectivity.
The data are presented (left) as a percent decrease in titer back-calculated
from TCID50s where IC90 is 155 nM and (right) the actual TCID50s where 3
log units of viral suppression are observed at 1.6 µM concentration.
39
40
CO2H
CO2Et
4
16
Because the A/PR/8/34 is a mouse adapted laboratory strain,
we wanted to evaluate these compounds in other viral strains that
are more relevant to human infection. To that end, we utilized
resources available at the National Institute of Allergy and
Infectious Diseases (NIAID) and evaluated key compounds (1, 4,
6, 10, 19, 32 and 33) in an assay that measured their ability to
protect host cells from cytopathic effects of various viral strains.
The compounds were tested against 3 additional human influenza
viruses: H1N1 (strain: A/California/7/2009), H3N2 (strain:
Influenza A/Brisbane/10/20070, and H5N1 (strain: a recombinant
aMDCK cell lines infected at a MOI of 0.1 for 48 h (time) (with or without
compounds) were used to determine virus titer using TCID50 analysis.
A/PR/8/34
strain
that
encoded
NS1
from
avian
A/Vietnam/1203/2004), and the results are presented in Table 2.
The best activity was observed against a seasonal A/H1N1 strain,
Figure 2. Structure activity relationship summary for chemical series.

Table 2. Selected compounds’ ability to rescue virus-mediated cytopathy of MDCK cells
Virus Screened
Influenza A virus H1N1
Influenza A virus H3N2
Influenza A virus H5N1
Influenza A/Vietnam/1203/2004 x A/PR/8/34
(recombinant H5N1)
Virus Strain
Influenza A/California/7/2009
Influenza A/Brisbane/10/2007
a
a
a
a
cmpd
1
4
6
10
19
32
33
EC50
1.7
1.2
EC90
4.9
3.4
CC50
SI50
EC50
47.6
>50
>50
10.1
>50
17.0
2.4
EC90
>50
>50
>50
>50
>50
>50
>50
34.9
CC50
>50
39.0
20.4
45.8
6.0
27.8
48.6
>100
SI50
>1
<1
<1
5
<1
2
EC50
28.5
>50
>50
3.6
>50
>50
>50
32.2
EC90
>50
>50
>50
9.9
>50
>50
>50
68.6
CC50
>50
24.5
24.5
24.7
0.87
19.8
24.5
>100
SI50
>1
<1
<1
7
<1
<1
<1
>3
>50
39.3
31.9
48.3
5.6
25.3
49.0
>100
>29
34
<1
23
<1
38
21
>50
2.1
18.3
0.7
>50
4.6
42.4
>50
5.7
2.4
2.2
20
>3
Ribavirin
3.5
>44
28.8
aThe EC50, EC90 (Effective compound Concentration that reduces viral mediated cell death by 50% and 90% respectively) and
CC50 (compound concentration that reduces cell viability by 50%) are in μM and derived after analysis at four concentrations (0.8,
4, 20, 100 μM). The selectivity index SI50 is CC50/EC50.
with most compounds (i.e. 1, 4, 10, 33) able to rescue cytopathic
effects caused by the H1N1 virus at single digit micromolar
concentrations. Compound 32 was most potent with a sub-
micromolar EC50 value. The antiviral activity of these compounds
was in general attenuated towards H3N2 and H5N1, and all the
compounds were inactive against a common Influenza B strain,
B/Brisbane/60/2008 (data not shown). Notably, compound 10
showed broad-spectrum antiviral activity against all three strains
of influenza A viruses, and compound 33 showed potent activity
against H1N1 and H3N2 with high selectivity indices. Curiously,
we noticed that the sensitivity of this NIAID assay was reduced
compared to our primary assay.
The most promising compounds (i.e. 4, 6, 10, 32, 33) were
then cross-examined for their ability to inhibit NS1 function in
the yeast-based assay that discovered the screening hit, which
demonstrated compound-dependent restoration of yeast growth.
The data for the lead molecule 32 is presented in Figure 4
(compounds 4, 6, 10 & 33 show the same behavior and are not
shown). Examination at three time intervals (22, 42 & 60 h)
clearly indicated a reversal of the slow growth phenotype that
was observed with the NS1-pYES strain. Thus, 32 antagonized
NS1 function in yeast, confirming that structural changes to the
hit had not compromised the functional activity. Moreover,
restoration of yeast growth indicates that the compound does not
display acute toxicity in this assay.
Figure 5. A) MDCK cells were infected with A/PR/8/34 at a MOI of 2 and
incubated in the presence of 20 µM concentration of indicated compounds for
6 h (time). Cells were harvested for RT-PCR analysis of IFN-β and β-actin
mRNA. Second lane showing induction of IFN-β in the presence of poly(I:C)
represents a positive control for the RT-PCR and induction of IFN- β mRNA;
B) Cells were uninfected, except for the third lane, where cells were infected
with A/PR/8/34. Drug treatment and RT-PCR were as described for panel
(A); C) Cells were uninfected (first lane) or infected with A/PR/8/34 in the
presence of DMSO or 20 µM of the indicated compounds. Western blots
were performed for NS1 and -tubulin as loading control.
0.8
0.6
0.4
0.2
0.0
pYES: DMSO
pYES: 32
NS1-pYES: DMSO
NS1-pYES: 32
mRNA levels in infected cells. As such, compounds 4, 6, 10 and
32 were further evaluated for their ability to restore IFN-β
mRNA levels in MDCK cells. MDCK cells were infected with
A/PR/8/34, at a MOI of 2, for 6 hours (enough time to allow an
initial round of viral replication in a majority of cells), and then
harvested to detect IFN-β mRNA via RT-PCR analysis. The four
representative pyrazolopyridines significantly restored IFN-β
mRNA levels in infected cells (Figure 5A). Compounds 6 and 32
were the best, with 6 able to restore IFN-β level similar to the
known NS1 antagonist JJ329731, 37 (lane 4) or the known IFN-β
inducer poly(I:C)38 (lane 2). Importantly, the compounds did not
induce IFN-β in the absence of A/PR/8/34 infection (Figure 5B),
0
20
40
60
Time (h)
Figure 4. Compound-dependent restoration of yeast growth: Growth of the
control yeast strain pYES (without NS1) and the NS1-expressing stain NS1-
pYES measured as OD600nm at 22, 42 and 60 h. pYES shows growth in the
presence of DMSO and 32 (50 µM), while the NS1-pYES strain exhibits a
slow growth phenotype, which is reversed by the presence of 32 (50 µM).
One of the ways that NS1 enhances viral replication is by
blocking the cellular IFN response.12,
Consequently,
15, 26
antagonists of NS1 function might be expected to restore IFN-β

demonstrating that they were antagonizing specific functions of
response. Also, as shown in Figure 5C, none of the compounds
directly affected the steady-state level of NS1 protein in infected
cells, which indicates that the inhibitors act functionally, and not
at the level of NS1 production or stability.
We then set out to demonstrate that the ability for the
pyrazolopyridines to restore IFN-β mRNA levels in MDCK cells
is dependent on NS1. Poly(I:C) is a strong inducer of IFN-β
mRNA, and NS1 is known to specifically block this induction.
We recapitulated this activity by co-transfection of an NS1
expression plasmid and an IFN-β luciferase reporter plasmid,
where poly(I:C)-induced luciferase activity was abrogated in the
presence, but not in the absence, of NS1 (Figure 6A). Treatment
with NS1-inhibitory compounds in the presence of both the
plasmids efficiently restored poly(I:C)-induced luciferase
activity, demonstrating that that they block NS1 function.
Importantly, in the absence of NS1 transfection and poly(I:C)
treatment (Figure 6B), the compounds had no effect on
luciferase activity, indicating that they do not induce the IFN-β
reporter on their own and that their action strictly requires NS1
expression. Thus, the compounds are able to exert their effect
only in the presence of the NS1 protein.
Together, these experiments strongly suggest that the key
pyrazolopyridines interfere with the function of NS1, specifically
its ability to ameliorate the host innate immune response.
Because our goal is to provide the research community with tool
compounds that can be used in therapeutically relevant, advanced
in vivo, proof-of-concept studies, and because no previous NS1
antagonists have been evaluated in vivo in the literature, we
carried out preliminary examination of the key analogs’ in vitro
ADME properties. To that end, most compounds showed good
stability in mouse liver microsomes (MLM), moderate solubility,
and moderate permeability (Table 3). The compounds also
showed low efflux in a Caco-2 monolayer, though the absolute
permeability numbers in the Caco-2 assay were also low, which
is characteristic of compounds with low solubility. In order to
position this series towards in vivo evaluation, we selected 32 and
33 as two representative members for a pharmacokinetics (PK)
study.
Figure 6. A) 293 cells were co-transfected with a firefly luciferase reporter
driven by the human IFN-β promoter and NS1-encoding expression
constructs. At 16 h after transfection, cells were treated with 50 µg of
poly(I:C)/mL and the indicated compounds (20 µM), and incubated for an
additional 24 h. The cells were harvested, and their luciferase activity was
measured using a Glo-max luminometer (Promega). “con” indicates the
luciferase activity of the untransfected cells; “DMSO” indicates treatment
with 1% DMSO; B) 293 cells were transfected with the IFN-β reporter and
treated with poly(I:C), 1% DMSO, or 20 µM of the indicated compounds in
the absence of poly(I:C).
Table 3. In vitro ADME data for key analogs.
Aqueous Kinetic
Mouse Liver Microsomal Stability
Caco-2 Permeability
(10-6 cm/s after 2 h)
Efflux Ratio
Solubility
(% remaining)
15 min
cmpd
32
(µM)
3.1
0 min
100
30 min
45
Papp A to B
0.10
0.22
Papp B to A
0.23
0.27
2.29a
1.20b
2.88
7.78
66
3% BSA
33
6
4
1.7
1.9
11.6
100
100
100
100
25
93
78
74
16
90
64
60
0.13
0.34
0.38
2.64
10
3.0
aThe mass recovery was 5 and 9%, respectively
bThe mass recovery was 49 and 81%, respectively, with 3%BSA.
Table 4. Pharmacokinetics parameters for 32 and 33.
PK parameters
Unit
Plasma
Liver
Compound 32
0.250
Lung
Plasma
Liver
Lung
Compound 33
0.083
Tmax
Cmax
h (time)
ng/mL
h
0.250
2437
4.6
0.250
18567
4.5
0.083
4470
5.2
0.083
36200
5.0
49600
4.7
64000
5.3
Terminal t1/2
AUC (AUClast
)
h*ng/mL
7655
53894
80218
53854
5960
5970
119000
49100
AUCINF
h*ng/mL
%
80282
1048
53894
704
120000
2010
49100
823
AUCorgan/AUCplasma

A single intraperitoneal (IP) dose of 30 mg/kg of 32 and 33 in
male C57BL/6 mice led to an AUCINF of 53894 and 49100
h*ng/mL, respectively, and >1 M concentration in the lungs, the
primary site of infection, for over 12 hours (Table 4, Figure 7).
The overall exposures in the liver and lungs were ~10-20 and ~7-
Competing Interests Statement
The authors declare that they have no competing interests.
References and notes
8 times that in plasma. The half-life was the same (T ~ 4-5 h) in
½
all organs for both compounds in plasma, liver and lung, with no
signs of drug accumulation or toxicity. There were also no
adverse effects observed at this single dose. The reasonable PK
profile of these pyrazolopyridines should allow future in vivo
experiments to examine NS1 antagonism.
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Figure 7. Mean plasma, liver, and lung concentrations-time profiles for
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In summary, following a qHTS campaign for NS1 antagonists,
we discovered a novel class of pyrazolopyridines that inhibits
NS1 function. SAR evaluation produced several compounds that
can potently reduce virus replication, cytoprotect against seasonal
influenza A H1N1, and restore IFN- mRNA levels in infected
MDCK cells. Two of these analogs, 32 and 33, have acceptable
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Acknowledgments
This work was supported by NIH grant R03MH085680 to
DAE and R44AI084244 to D.B. This work was also supported by
contract HHSN272201100019I from the Respiratory Diseases
Branch, Division of Microbiology and Infectious Diseases,
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, USA.
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Novel pyrazolopyridine NS1-antagonists for influenza A
Reduces viral titer by 128-fold at 10 M
Inhibits cytopathic effects of a seasonal influenza A strain (EC50 = 660 nM)
Restores IFN- mRNA levels in virus-infected MDCK cells
Pharmacokinetics: > 1M concentration in lungs for over 12 h after 30mpk IP single
dose in mouse