Synthesis and Structure-Activity Relationship (SAR) Studies of Novel Pyrazolopyridine Derivatives as Inhibitors of Enterovirus Replication

Article abstract of DOI:10.1021/acs.jmedchem.7b01863

A series of novel pyrazolopyridine compounds have been designed and prepared by a general synthetic route. Their activities against the replication of poliovirus-1, EV-A71, and CV-B3 enteroviruses were evaluated. The comprehensive understanding of the structure-activity relationship was obtained by utilizing the variation of four positions, namely, N1, C6, C4, and linker unit. From the screened analogues, the inhibitors with the highest selectivity indices at 50% inhibition of viral replication (SI₅₀) were those with isopropyl at the N1 position and thiophenyl-2-yl unit at C6 position. Furthermore, the C4 position offered the greatest potential for improvement because many different N-aryl groups had better antiviral activities and compatibilities than the lead compound JX001. For example, JX040 with a 2-pyridyl group was the analogue with the most potent activity against non-polio enteroviruses, and JX025, possessing a 3-sulfamoylphenyl moiety, had the best activity against polioviruses. In addition, analogue JX037, possessing a novel pyrazolopyridine heterocycle, was also shown to have good antienteroviral activity, which further enlarges the compound space for antienteroviral drug design.

Full text of DOI:10.1021/acs.jmedchem.7b01863

Subscriber access provided by READING UNIV
Article
Synthesis and Structure-Activity Relationship (SAR) Studies of Novel
Pyrazolopyridine Derivatives as Inhibitors of Enterovirus Replication
Yanpeng Xing, Jun Zuo, Paul Krogstad, and Michael E Jung
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01863 • Publication Date (Web): 18 Jan 2018
Downloaded from http://pubs.acs.org on January 18, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis and Structure-Activity Relationship (SAR)
Studies of Novel Pyrazolopyridine Derivatives as
Inhibitors of Enterovirus Replication
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
#, ¥
‡, ¥
‡, §
#, §
Yanpeng Xing, Jun Zuo, Paul Krogstad, * and Michael E. Jung*
#
‡
§
Department of Chemistry and Biochemistry, Department of Pediatrics, and Department of
Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095
¥
Both authors contributed equally to this work
ABSTRACT: A series of novel pyrazolopyridine compounds have been designed and prepared
by a general synthetic route. Their activities against the replication of poliovirus-1, EV-A71, and
CV-B3 enteroviruses were evaluated. The comprehensive understanding of the Structure-
Activity Relationship was obtained by utilizing the variation of four positions, namely N1, C6,
C4 and linker unit. From the screened analogues, the inhibitors with the highest selectivity
indices at 50% inhibition of viral replication (SI ) were those with isopropyl at the N1 position
50
and thiophenyl-2-yl unit at C6 position. Furthermore, the C4 position offered the greatest
potential for improvement because many different N-aryl groups had better antiviral activities
and compatibilities than the lead compound JX001. For example, JX040 with a 2-pyridyl group
was the analogue with the most potent activity against non-polio enteroviruses and JX025,
possessing a 3-sulfamoylphenyl moiety, had the best activity against polioviruses. In addition,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
analogue JX037, possessing a novel pyrazolopyridine heterocycle, was also shown to have good
anti-enteroviral activity, which further enlarges the compound space for anti-enteroviral drug
design.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
■ INTRODUCTION
The human enteroviruses (EVs) are a group of more than 110 distinct viruses, each composed of
a single stranded RNA genome packaged within a protein capsid. Enteroviruses were originally
classified into groups of polioviruses, coxsackieviruses, echoviruses, and later simply assigned
1
consecutive numbers as they were discovered. Poliovirus circulation and poliomyelitis have
been nearly eliminated by immunization but other EVs, now organized into alphabetically
1
organized species , remain clinically and economically significant pathogens with global impact.
For example, enterovirus 71 (EV-A71) has been the cause of numerous epidemics of central
2
nervous system infections in Europe and the Asia-Pacific Region over the last 15 years, causing
3
an estimated 7 million cases in China between 2008 and 2012. Widespread reports of
coxsackievirus B1 (CV-B1) myocarditis in the United States in 2007 highlighted the epidemic
4
,5
potential of enteroviruses and their danger to infants. Similarly, a nationwide outbreak of
enterovirus D68 (EV-D68) occurred in the summer of 2014. Beginning with reports in the
midwest United States, EV-D68 was linked to severe respiratory illness, most often in young
children. EV-D68 was also detected in respiratory specimens of some patients with polio-like
6
-8
paralysis, meningitis, and encephalitis. In addition, enteroviruses are perennial causes of
encephalitis, acute heart failure, sepsis in newborns, and other serious and life-threatening
1
illnesses.
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
No antiviral agents are currently approved to treat enterovirus infections and supportive care is
1
,9
the mainstay of treatment.
Extensive studies in pursuit of candidate antiviral agents have
targeted the viral capsid, the virus-encoded RNA polymerase and proteases, and other viral
9
,10
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
proteins involved in replication.
Candidates that have reached preclinical or early clinical
phases of development have included the viral capsid binding agent BTA-798 (vapendavir), the
9
-11
viral protease inhibitor AG7088 (rupintrivir), and the viral 3D polymerase inhibitor DTriP-22.
Two drugs, enviroxime and pleconaril, were unable to move beyond initial clinical studies due to
10,12
limited efficacy or safety concerns, respectively.
Faced with this lack of progress, several
recent studies have been performed in hopes of repurposing medications found to have in vitro
antiviral activity against enteroviruses. For example, the selective serotonin reuptake inhibitor
fluoxetine was found to have modest anti-enteroviral activity, likely reflecting its interference
1
3,14,15
with the activity of the viral 2C protein.
We previously applied a rapid, live virus assay to identify enterovirus inhibitors from nearly
8
6,000 compounds held by the Molecular Screening Shared Resource (MSSR) core facility of
1
3,15
the CNSI (California NanoSystems Institute) at UCLA.
Using a commonly encountered
enterovirus, CV-B3), we identified a novel group of anti-enteroviral compounds: 1H-
1
5
pyrazolo[3,4-b]pyridine-4-carboxamide derivatives. These compounds are structurally unlike
9,10
(
distinct from) any previously described inhibitors of EV replication and also exhibit antiviral
1
5
activity against an array of clinically relevant enterovirus types at micromolar concentrations.
To identify the target of these compounds, we selected for resistance by intentionally exposing
the molecularly cloned CVB3-H3 virus to them at sub-inhibitory concentrations. This resistance
was genetically mapped to the coding domain for the viral 2C protein. Based on these data, we
constructed a missense mutant, CVB3-H3-C179F. This virus replicated normally and was not
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibited by these compounds, indicating that they interfere with the activities of the viral 2C
1
3-15
protein, which plays a role in viral RNA replication and other processes.
We herein describe an extensive structure-activity relationship (SAR) study of a series of
pyrazolopyridine carboxamides and their structural analogues. We have developed a simple
synthetic route to these compounds which allows one to prepare many analogues rapidly. All of
these new compounds have been tested for their ability to inhibit the growth of 3 enteroviruses
representing the major species of enteroviruses that infect humans. We have identified
compounds with higher antiviral activity and lower cytotoxicity in vitro than the original lead
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
compounds described previously.
■
RESULTS AND DISCUSSION
Chemistry. The synthesis of these molecules, which were labeled JX001 to JX076, was carried
out as shown in the various schemes. The synthesis of the 1H-pyrazolo[3,4-b]pyridines are
shown in Scheme 1. The key step was the condensation of the 1-alkyl-pyrazole-5-amine 2 with
the 4-aryl-2,4-diketoester 3 to give the 1H-pyrazolo[3,4-b]pyridine-4-carboxylic ester 11. The
components for this key coupling reaction were prepared as follows. Formation of the hydrazone
6
was easily accomplished by mixing the desired ketone 4 with 3-hydrazino-propanitrile 5.
Reaction of 6 with sodium butylate, prepared in situ, gave the desired 1-alkyl-pyrazole-5-amine
16
2
. This compound could also be prepared by another route, namely reaction of the alkyl
1
7
hydrazine hydrochloride salt 7 with commercially available 2-cholorpropenenitrile 8 to give 2.
The second component 3 was synthesized by the condensation of an alkyl or aryl methyl ketone
1
8
9
2
with diethyl oxalate 10 to give the product of the Claisen condensation, the salt 3. Addition of
1
9
and 3 in acetic acid afforded good yields of the desired heterocycle 11. Basic hydrolysis of
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the ester of 11 gave the acid 12. Formation of the acid chloride 13 with oxalyl chloride was
followed by addition of the desired aniline 14 to give the amides. These were labeled as JX
compounds starting with JX001. Overall a total of 76 analogues in all series were prepared and
tested.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R3
O
N
N
R3
H N
EtOH
N
2
+
N
H
N
H
R4
R4
1
4
5
6
Na
n-BuOH
AcONa
EtOH
1
R NHNH
+
N
2
Cl
NH2
R1=
N
N
R1
HCl
R3
7
8
R4
2
O
O
OK
O
OEt
O
t-BuOK
+
OEt
OH
R6
EtO
R6
Me
toluene
O
O
9
10
3
O
OEt
KOH
AcOH
i-PrOH;
N
2
+ 3
N
AcOH
N
R6
N
R6
N
N
R1
R1
12
11
R2
O
Cl
O
NH
ClCOCOCl
CH Cl
2 toluene
reflux
R
+
N
2
2
N
R6
N
13
R1
NH2
N
N
R6
N
14
R1
e.g., JX001
Scheme 1. Synthesis of 1H-Pyrazolo[3,4-b]pyridine-4-carboxamides, e.g., JX001.
In general, the yields of the synthesis were quite good and reasonable quantities of the materials
were easily available. Although this general synthetic route was used for the synthesis of most of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the analogues, other methods could be used for specific substitution patterns and will be given in
the experimental section. The compounds were purified by normal synthetic medicinal chemistry
means, usually column chromatography and their structures were determined by high field NMR
spectroscopy.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In addition, we also prepared several ring systems different from but similar to the 1H-
pyrazolo[3,4-b]pyridine system by an analogous chemical synthesis, as shown in Scheme 2.
Thus several 1H-pyrrolo[2,3-b]pyridine-4-carboxamides 22 were prepared by a route that
involved a protected form of 2-aminopyrrole, namely the 2-(hydroxymethyl)benzamide 17. This
compound was prepared from pyrrole itself in four steps, namely nitration and isopropylation of
1
5 to give the 2-nitropyrrole 16 followed by tin reduction in the presence of phthalic anhydride
and reduction of the imide to give 17. Condensation of 17 with the 4-(2-thiophenyl)-2,4-
OH
1
) Sn/AcOH
O
1) HNO3
phthalic
AcOH
anhydride
NO2
N
N
H
N
N
2
) K CO
2
3
toluene/∆
2) NaBH4
aq iPrOH
H
iPr-I
iPr
iPr
1
7
1
5
1
6
O
OEt
OK
O
KOH
OEt
iPrOH;
S
AcOH
17 +
AcOH
O
S
N
iPr
N
1
8
19
O
OH
O NHAr
1
) ClCOCOCl
CH Cl
2
2
2
) ArNH2
S
S
N
N
iPr
N
N
2
1
iPr
tol/∆
2
0
22
Scheme 2. Synthesis of 1H-Pyrrolo[2,3-b]pyridine-4-carboxamides, 22 (JX037, JX062-63, and
JX072).
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
diketoester 18 (prepared as in Scheme 1) gave, via the free aminopyrrole formed in situ, the 1H-
pyrrolo[2,3-b]pyridine-4-carboxylic ester 19. The remainder of the synthesis follows that of
Scheme 1, namely hydrolysis of the ethyl ester of 19 to give the acid 20 followed by activation to
the acid chloride with oxalyl chloride and final coupling with one of several anilines 21 to
affordthe analogues 22. In particular, we varied the aniline to give the following analogues: Ar =
4-FC H : JX037; 2-pyridyl: JX072; 3-pyridyl: JX062; and 4-pyridyl: JX063.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
4
In addition, one analogue in each of three additional bi-heterocyclic ring systems were prepared
and tested. Again, a very similar route was used for the preparation of each of these three new
analogues (Scheme 3). Thus beginning with the 5-amino-1-isopropyl-imidazole 23, condensation
with 18 gave the imidazopyridine 24, which, after hydrolysis to give 25 and amide formation
with 4-fluoroaniline 26, afforded the desired 3H-imidazo[4,5-b]pyridine-7-carboxamide 27
(JX034).
O
OEt
OK
O
N
N
N
OEt AcOH
S
NH2
+
N
S
N
O
iPr
iPr
2
3
18
24
O
OH
O
NH-4-FC H
6
4
1
) ClCOCOCl
CH Cl
KOH
iPrOH;
N
N
N
2
2
2
) 4-FC H NH
S
6
4
2
S
AcOH
N
N
N
2
6
iPr
iPr
tol/∆
2
5
27
Scheme 3. Synthesis of 3H-imidazo[4,5-b]pyridine-7-carboxamide, 27 (JX034).
In a similar manner (Scheme 4), the known 1-isopropyl-5-aminotriazole 28 was condensed with
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
NH-4-FC H
6
4
OK
O
N
1) AcOH
OEt 2) KOH
N
N
N
S
N
NH2
+
N
S
3) ClCOCOCl
N
O
iPr
4) 4-FC H NH
iPr
6
4
2
2
8
18
26
2
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Synthesis of 1H-Triazolo[2,3-b]pyridine-4-carboxamide, 29 (JX035).
1
8 to give, after hydrolysis and amide formation with 26, the desired 3H-[1,2,3]triazolo[4,5-
b]pyridine-7-carboxamide 29 (JX035).
Finally the 1H-pyrazolo[3,4-d]pyrimidine-4-carboxamide analogue, 37 (JX036), was prepared
by a completely different route (Scheme 5). Thus reaction of 2-(ethoxyvinylidene)malononitrile
3
0 with isopropylhydrazine followed by treatment with basic hydrogen peroxide gave the known
2
0
aminoamide 31. Condensation of this compound with methyl thiophene-2-carboxylate 32
afforded the desired 1H-pyrazolo[4,5-d]pyrimidin-ol 33. Conversion of the hydroxyl to a
bromide furnished 34 which was converted into the carboxamide 35 with copper cyanide and wet
N-methylpyrrolidone (NMP). Hydrolysis of the amide gave the carboxylic acid 36 which was
then converted, via the acid chloride, into the desired 4-fluorophenyl amide 37 (JX036).
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CONH2
1
) NaH/diox
CN 1) iPrNHNH2 .HCl
NC
N
2
2 °C/4 h
+
CO Me
2
) NH OH/H O
N
2
4
2
2
NH2
S
2) EtOH/nBuOH
80 °C/ 2 h
Et N/EtOH
3
iPr
OEt
32
30
31
OH
Br
CONH2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
POBr3
N
N
CuCN
N
N
N
S
iPr NEt
S
wet NMP
S
N
2
N
N
N
iPr
N
N
tol
iPr
iPr
3
3
34
35
CO H
O
NH-4-FC H
2
6
4
1
) ClCOCOCl
N
N
NaOH
CH Cl
2
2
N
N
N
aq EtOH
N
S
2) 4-FC H NH
6
4
2
S
N
2
6
N
iPr
tol/∆
iPr
3
6
37
Scheme 5. Synthesis of 1H-pyrazolo[3,4-d]pyrimidine-4-carboxamide, 37 (JX036)
BIOLOGICAL EVALUATION.
SAR analysis of original compounds. We recently described the identification of an array of
1
5
chemical structures with antiviral activity against commonly encountered enteroviruses. There
were 144 1H-pyrazolo[3,4-b]pyridine-4-carboxamide compounds tested in the primary screen,
and twenty two of these were confirmed to be active against the enterovirus strain used (CVB3-
1
5
H3) (Supplementary Table S1, structures footnoted with the letter b). CV-B3 was chosen
because it has been annually reported to the Center for Disease Control since 1975 as cause of
1
severe disease, and because the availability of the molecularly cloned pathogenic variant of CV-
B3 (CVB3-H3) enhances the reproducibility of in vitro studies. The antiviral activity of this
1
6
class of compounds is greatly affected by variations at the N1 (R ), C6 (R ) and C4 positions
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Figure 1, 1). Among the 22 active library compounds, the number of variations decreased at the
order of C4 > C6 > N1. In particular, the compound N-(2-fluorophenyl)-1-(propan-2-yl)-6-
(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carboxamide (Figure 1, 1a) has shown to exhibit
activity against 12 commonly encountered members of the enterovirus B species, as well as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
enterovirus A71 (EV species A) and two polioviruses (EV species C).
F
4
3
2
5
6
R
O
NH
O
NH
3
4
3
a
5
2
N
N
N
7a N 6 R6
N
1
N
Ph
7
iPr
R1
1
1a
Figure 1. 1H-Pyrazolo[3,4-b]pyridine-4-carboxamides.
Here we used the 1H-pyrazolo[3,4-b]pyridine-4-carboxamide 1a as a reference compound and
designed new compounds in the 1H-pyrazolo[3,4-b]pyridine-4-carboxamide series 1 in order to
1
vary the four most easily altered positions, namely: 1) the alkyl group at N1 (R ); 2) the usually
6
aryl or heteroaryl ring at C6 (R ); 3) the aniline unit on the carboxamide at C4, varying the
substituents at essentially every available carbon and introducing heterocyclic amines; and 4) a
few changes in the amide linking unit between the two rings. Our goal was to identify candidate
compounds for the development of anti-enteroviral drugs with significant improvements in
potency and exhibiting reduced cytotoxicity. Specifically, we sought compounds that would
inhibit the replication of enteroviruses in the EV-A, EV-B, and EV-C species at concentrations at
less than 1 µM, yet with relatively low cytotoxicity.
1
Analogues at the N1 position (R ). For the group attached to N1, we began with the isopropyl
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
group since it was the substituent in the lead compound 1a. (henceforth referred to as JX001).
We changed the substituent and examined aryl, secondary cycloalkyl, substituted methyl units,
1
and even H. The compounds prepared with this variation were: R = phenyl: JX012; t-butyl:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
JX013; cyclobutyl: JX014; 2,2,2-trifluoroethyl: JX022; cyclopentyl: JX027; cyclohexyl:
JX028; cycloheptyl: JX029; 4-methoxybenzyl: JX031; and hydrogen: JX032. In general, those
substituents were not as favorable for activity as the isopropyl group since none of the above
analogs demonstrated antiviral activities against EV-A71, coxsackievirus B3(CV-B3), or
poliovirus-1 (PV-1). This result is consistent with the SAR analysis of the initial 144 library
compounds, in which the isopropyl group at the N1 position seemed crucial for the antiviral
activity (Supplementary Table S1). The only two exceptions were those with an ethyl group
replacing the isopropyl group, namely: N-(4-cyanophenyl)-1-ethyl-6-(thiophen-2-yl)-1H-
pyrazolo[3,4-b]pyridine-4-carboxamide
and
1-ethyl-N-(4-methyl-3-sulfamoylphenyl)-6-
(highlighted groups in
(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carboxamide
Supplementary Table S1). Therefore, we retained the isopropyl group at the N1 position in the
other analogues in this study.
6
Analogues at the 6-position (R ). The group attached at the 6-position in 1a (JX001) is a
thiophen-2-yl group; this was also present in 11 other active compounds of our primary screen
(Supplementary Table S1). Besides, cyclopropyl, phenyl and isopropyl groups were also found at
the 6-positions of active compounds. We tested the effect of variation at this position by
6
changing R to cycloalkyl, aryl, and heteroaryl units. New compounds prepared with this
6
variation were: R = cyclopropyl, JX038, JX055, JX071; phenyl, JX002, JX070; and heteroaryl,
e.g., 2-pyridyl: JX004; 3-pyridyl: JX005; 4-pyridyl: JX003; 2-thiazolyl: JX007; 5-thiazolyl:
JX010; 5-oxazolyl: JX011; 2-furyl: JX026; and thiophen-3-yl: JX030, JX057-JX061. Several
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of these compounds also had different aniline units, namely: 4-fluorophenyl in JX026, JX030
and JX038; 3-sulfamoylphenyl in JX055; 2-pyridyl in JX070 and JX071. Those changes in the
aniline units were generally associated with increased antiviral activities as discussed below. The
highest activity was seen with thiophen-2-yl unit at the 6-position although the thiophen-3-yl unit
was also associated with antiviral activity in low micromolar concentrations (Table 1). By
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
contrast, the presence of azole groups - 2-thiazolyl (JX007), thiazolyl (JX010), and 5-oxazolyl
6
(
JX011) at R clearly reduced antiviral activity. Other groups phenyl (JX002 and JX070) and
cyclopropyl (JX038 and JX055) were also associated with decreases in antiviral activities. It
seemed that cytotoxicity decreased significantly with 2-furyl (JX026) at the 6-position.
Furthermore, we found that many derivatives with a thiophen-3-yl unit replacing the thiophen-2-
yl unit: JX030, JX057, JX058, JX059, and JX060 retained antiviral activity. The direct
comparison of the effects of thiophen-2-yl and thiophen-3-yl units on antiviral activity was
manifested in the following pairs: JX017 vs. JX030, JX040 vs. JX057, JX041 vs. JX058,
JX042 vs. JX061, JX043 vs. JX059, and JX056 vs. JX060, where the thiophen-2-yl group was
generally associated with higher antiviral activities (Table 1 and Table 2).
Table 1. Antiviral activities of analogues with various substituents at the 6-position.
6
Compound
No. (JX)
R
Anti-EV-A71 ina
LLC cells (µM)
Anti- CV-B3 in a Anti-PV-1 in HeLa
a,b
HeLa cells (µM) cells (µM)
1
a (or 001) 2-thiophenyl EC50: 2.3 ± 1.2
CC : 50.0
EC50: 1.4 ± 0.6
EC50: 7.0 ± 0.0
CC : 12.5
CC : 12.5
5
0
50
50
SI : 21.7
SI : 8.9
SI : 1.8
5
0
50
50
0
02
phenyl
EC50: 5.7 ± 1.5
EC50: 4.5 ± 1.5
EC50: > 25.0
CC : > 200.0
CC : 37.5
CC : 37.5
50
50
50
SI50: > 35.1
SI : 8.3
SI : N/A
5
0
50
003
4-pyridyl
EC50: > 10.0
EC50: 5.1 ± 0.1
EC50: > 25.0
CC : 45.0
CC : 37.5
CC : 37.5
5
0
50
50
SI50: N/A
SI50: 7.4
SI50: N/A
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
0
0
04
05
2-pyridyl
3-pyridyl
2-thiazolyl
5-thiazolyl
5-oxazolyl
2-furyl
EC50: > 10.0
CC : 100.0
SI50: N/A
EC50: 6.9 ± 0.7
EC50: > 25.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI50: > 29.0
SI50: N/A
EC50: > 10.0
EC50: 7.0 ± 2.4
EC50: > 25.0
CC : 50.0
CC : 37.5
CC : 37.5
50
50
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SI50: N/A
SI50: 5.4
SI50: N/A
007
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
0
0
10
11
EC50: 3.7 ± 0.2
EC50: 3.3 ± 1.4
EC50: > 25.0
CC : 100.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI50: 27.0
SI50: > 60.6
SI50: N/A
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
SI50: N/A
SI50: N/A
SI50: N/A
026
EC50: 2.6 ± 0.3
EC50: 2.4 ± 0.0
EC50: > 25.0
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI50: > 76.9
SI50: > 83.0
SI50: N/A
0
0
30
38
3-thiophenyl EC50: 1.4 ± 0.2
EC50: 1.3 ± 0.1
EC50: > 25.0
CC : > 200.0
CC : 25.0
CC : 25.0
5
0
50
50
SI50: > 142.9
SI50: 19.2
SI50: N/A
cyclopropyl
cyclopropyl
phenyl
EC50: 3.2 ± 0.1
EC50: 3.3 ± 0.1
EC50: > 25.0
CC : 12.5
CC : 10.0
CC : 10.0
50
50
50
SI50: 3.9
SI50: 3.0
SI50: N/A
055
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
0
0
70
71
EC50: 0.7
EC50: 1.6 ± 0.0
EC50: > 25.0
CC : 12.5
CC : 18.8
CC : 18.8
5
0
50
50
SI50: 17.9
SI50: 11.8
SI50: N/A
cyclopropyl
EC50: 4.7
EC50: 4.7
EC50: > 25.0
CC : 25.0
CC : 25.0
CC : 25.0
50
50
50
SI50: 5.3
SI50: 5.3
SI50: N/A
a. For compounds that were tested against certain viruses in 3 separate experiments, means ± s.d.
are shown. Otherwise, a single value represents an average of triplicates in one experiment. b.
Similar results were observed against anti-PV-1 and anti-PV-3 activities.
comparison of the effects of thiophen-2-yl and thiophen-3-yl units on antiviral activity was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
manifested in the following pairs: JX017 vs. JX030, JX040 vs. JX057, JX041 vs. JX058,
JX042 vs. JX061, JX043 vs. JX059, and JX056 vs. JX060, where the thiophen-2-yl group was
generally associated with higher antiviral activities (Table 1 and Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Analogues at the aniline unit. In addition, we varied extensively the substitution pattern of the
N-aryl group of the amide as shown in the Table 2. Many new compounds were prepared having
halo substituted anilines (especially fluoro substituents), sulfamoyl and carbamoyl units, and
especially heterocyclic amine units, pyridyl and pyrimidyl rings. We evaluated the effect of the
position of a single fluorine atom on the phenyl ring in JX017 and JX021. We also prepared
analogues in which the position and number of fluorine atoms were varied: JX008, JX009,
JX015, JX016, JX018, JX019, JX020, and JX050. Although nearly all of the analogues had
antiviral activity against the virus test strains, and JX017 with a 4-fluorophenyl unit had
substantially lower cytotoxicity and lower EC concentrations against EV-A71 and CV-B3
50
compared to original lead JX001 with a 2-fluorophenyl group. We also prepared and tested
compounds with a sulfamoyl unit: JX006, JX025, JX033 and JX055. Of these, the 3-sulfamoyl
analogue JX025 showed the best antiviral activity, with EC values for EV-A71 and CVB-B3
50
that were similar to those for lead JX001, in addition to a lower EC value against poliovirus,
5
0
and generally lower cytotoxicity.
In light of this result, we synthesized other sulfamoyl analogues having also a fluorine atom, e.g.,
JX068 and JX069, the 4-methylsulfonyl analogue JX047, the monomethyl- and
dimethylsulfamoyl analogues JX053 and JX052, and two carbamoyl analogues JX064 and
JX073 to further explore the effect of such substituents on activity. All of these exhibited
antiviral activity against EV-A71 and CV-B3 with the sulfone JX047 and the 4-fluoro-3-
sulfamoyl analogues, JX069, being the least cytotoxic of the compounds tested. They therefore
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
had the best selectivity indices against EV-A71 and CV-B3, but they were not active against
polioviruses. The best antiviral activity (lowest EC values) against PV-1 was seen with the
5
0
analogues JX025, JX064 and JX068, which had either sulfamoyl or carbamoyl moieties at the 3-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
position of the phenyl ring.
In addition, we prepared many compounds with heterocyclic amines in the amides, some of
which had excellent activity. The heterocyclic units prepared were: 2-pyridyl: JX040, JX057,
JX070, JX071, JX072 and JX074; 3-pyridyl: JX056, JX060, JX062 and JX065; 4-pyridyl:
JX042, JX061 and JX063; 2-pyrimidyl: JX041 and JX058; 4-pyrimidyl: JX043 and JX059;
and 5-pyrimidyl: JX066. As mentioned earlier, the five analogues JX057, JX058, JX059,
JX060 and JX061 all had a 3-thiophenyl group at the 6-position. In general, analogues with
pyridyl amides showed significantly better activity than those with substituted phenyl amides.
Among the isomeric pyridyl analogues, generally the 2-pyridyl unit was associated with
excellent antiviral activity and 4-pyridyl was associated with poor activity. In particular, the 2-
pyridyl amide JX040 had substantially lower EC values for EV-A71 and CV-B3 and higher
5
0
CC concentration than the lead 1a (JX001), resulting in a markedly higher SI . Unfortunately,
50
50
this substitution appeared to reduce activity against poliovirus. The effects of the pyrimidyl units
were complicated as they were associated with excellent EC values and below average CC
50
5
0
values. In this group, JX059 was the best analogue as it had exceptional and excellent CC50
values.
Table 2. Antiviral activities of analogues with various substituents of the aniline units.
Compd
No. (JX)
N-aryl group
Anti-EV-A71 ina
LLC cells (µM)
Anti- CV-B3 in a Anti-PV-1 in
HeLa cells (µM) HeLa cells
a,b
(µM)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
0
06
08
4-sulfamoylphenyl EC50: > 10.0
CC : N/A
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
SI : N/A
SI : N/A
50
50
50
2,4-F
2,6-F
3,4-F
3,5-F
2
2
2
2
C
6
C
6
C
6
C
6
H
H
H
H
3
3
3
3
EC : 1.6 ± 0.4
EC : 1.4 ± 0.5
EC50: > 25.0
5
0
50
CC : > 200.0
CC : > 200.0
CC : > 200.0
50
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SI : > 125.0
SI : > 142.9
SI : N/A
5
0
50
50
009
EC : 3.4 ± 0.7
EC : 3.2 ± 0.5
EC50: > 25.0
5
0
50
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : > 58.8
SI : > 62.5
SI : N/A
5
0
50
50
0
0
15
16
EC50: > 25.0
EC50: 1.3 ± 0.1
EC50: > 10.0
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : N/A
SI : > 153.8
SI : N/A
5
0
50
50
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
SI : N/A
SI : N/A
SI : N/A
5
0
50
50
017
4-FC H4
EC : 0.9 ± 0.3
EC : 0.7 ± 0.2
EC : > 25.0
6
50
50
50
CC50: > 200.0
SI : > 222.2
CC : > 200.0
CC : > 200.0
5
0
50
SI : > 285.7
SI : N/A
5
0
50
50
0
0
18
19
2,5-F
2
2
C
6
6
H
H
3
3
EC : 3.5
EC : 2.7 ± 0.4
EC50: > 25.0
5
0
50
CC : 100.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : 28.6
SI : > 74.1
SI : N/A
5
0
50
50
2,3-F
C
EC : 3.0
EC : 2.7 ± 0.4
EC50: > 25.0
5
0
50
CC : 75.0
CC : > 200.0
CC : > 200.0
50
50
50
SI : 25.0
SI : > 74.1
SI : N/A
5
0
50
50
020
2,4,6-F
3
C
6
H
2
EC : 3.0
EC : 1.8 ± 0.7
EC50: > 25.0
5
0
50
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : > 66.7
SI : > 111.1
SI : N/A
5
0
50
50
0
0
21
23
3-FC
2-CF
4-CF
6
3
3
H
4
EC50: 2.4 ± 0.8
EC50: 0.8 ± 0.3
EC50: > 25
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : > 83.3
SI : > 250.0
SI : N/A
5
0
50
50
C
C
6
6
H
4
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
SI : N/A
SI : N/A
SI : N/A
5
0
50
50
024
H
4
EC50: > 25.0
EC50: > 25.0
EC50: > 25.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
SI : N/A
SI : N/A
5
0
50
50
0
25
3-sulfamoylphenyl EC50: 1.3 ± 0.1
EC50: 1.3 ± 0.2
EC50: 5 ± 0.0
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CC : 25.0
CC : > 200.0
CC : 50.0
5
0
50
50
SI : 19.2
SI : 166.7
SI : 10.0
5
0
50
50
0
33
2-sulfamoylphenyl EC50: 2.5 ± 0.7
EC50: 2.6 ± 0.4
EC50: > 25.0
CC : 25.0
CC : 10.0
CC : 10.0
50
50
50
SI : 10.0
SI : 3.8
SI : N/A
5
0
50
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
040
2-pyridyl
EC50: 0.5 ± 0.1
EC50: 0.8 ± 0.3
EC50: > 10.0
CC : > 200.0
CC : > 200.0
CC : > 200.0
5
0
50
50
SI : > 400.0
SI : > 250.0
SI : N/A
5
0
50
50
0
0
41
42
2-pyrimidyl
4-pyridyl
EC50: 0.5 ± 0.1
EC50: 0.6 ± 0.1
EC50: > 10.0
CC : 75.0
CC : 37.5
CC : 37.5
5
0
50
50
SI : 150.0
SI : 62.5
SI : N/A
5
0
50
50
EC50: 0.4 ± 0.0
CC : 6.3
EC50: 0.6 ± 0.1
EC50: > 10.0
CC : 6.0
CC : 6.0
50
50
50
SI : 15.8
SI : 10.0
SI : N/A
50
50
50
043
4-pyrimidyl
EC50: 0.5 ± 0.1
EC50: 0.6 ± 0.1
EC50: > 10.0
CC : 50.0
CC : 20.0
CC : 20.0
5
0
50
50
SI : 100.0
SI : 33.3
SI : N/A
5
0
50
50
0
45
1-isopropyl-6-(2-
thiophenyl)-1H-
pyrazolo[3,4-
b]pyridin-4-yl
4-methyl-
EC50: > 10.0
CC50: N/A
SI50: N/A
EC50: > 10.0
CC50: N/A
SI50: N/A
EC50: > 10.0
CC50: N/A
SI50: N/A
0
47
EC : 0.5 ± 0.1
EC : 1.0 ± 0.4
EC : > 10.0
50
50
50
sulfonylphenyl
CC : 50.0
CC : 200.0
CC : 200.0
5
0
50
50
SI : 100.0
SI : 200.0
SI : N/A
5
0
50
50
050
2-Br-4-FC
6
H
3
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
SI : N/A
SI : N/A
5
0
50
50
0
0
52
53
3-N,N-
dimethylsulf-
amoylphenyl
EC50: 0.8 ± 0.1
EC50: 1.0 ± 0.4
EC50: > 10.0
CC : > 200.0
CC : 12.0
CC : 12.0
5
0
50
50
SI : > 250.0
SI : 12.0
SI : N/A
50
50
50
3-N-methylsulf-
oylphenyl
EC50: 0.7 ± 0.1
EC50: 0.5 ± 0.1
EC50: > 10.0
CC : 25.0
CC : 12.0
CC : 12.0
5
0
50
50
SI : 35.7
SI : 24.0
SI : N/A
5
0
50
50
056
3-pyridyl
2-pyridyl
EC50: 0.5 ± 0.1
EC50: 0.4 ± 0.3
EC50: 10.0
CC : > 200.0
CC : 25.0
CC : 25.0
5
0
50
50
SI : > 400.0
SI : 62.5
SI : 2.5
5
0
50
50
0
57
EC50: 0.8
EC50: 1.2
EC50: > 10.0
CC : 25.0
CC : 18.0
CC : 18.0
5
0
50
50
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SI : 31.3
SI : 15.0
SI : N/A
5
0
50
50
0
0
58
59
2-pyrimidyl
4-pyrimidyl
3-pyridyl
EC50: 1.2
EC50: 1.2
EC50: > 10.0
CC : 50.0
CC : 40.0
CC : 40.0
5
0
50
50
SI : 41.7
SI : 33.3
SI : N/A
50
50
50
EC50: 0.8
EC50: 1.2 ± 0.0
EC50: > 10.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CC : > 200.0
CC : > 200.0
CC : > 200.0
50
50
50
SI : > 250.0
SI : > 166.7
SI : N/A
5
0
50
50
060
EC50: 2.4
EC50: 0.6
EC50: > 10.0
CC : 37.0
CC : 18.0
CC : 18.0
5
0
50
50
SI : 15.4
SI : 30.0
SI : N/A
5
0
50
50
0
0
61
64
4-pyridyl
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
SI : N/A
SI : N/A
50
50
50
3-carbamoylphenyl EC50: 0.8
EC50: 0.7 ± 0.1
EC50: 6.3
CC : 25.0
CC : 18.0
CC : 18.0
5
0
50
50
SI : 31.3
SI : 25.7
SI : 2.9
5
0
50
50
066
5-pyrimidyl
EC50: 0.4
EC50: 0.7 ± 0.2
EC50: 10.0
CC : 20.0
CC : 18.0
CC : 18.0
5
0
50
50
SI : 50.0
SI : 25.7
SI : 1.8
5
0
50
50
0
0
68
69
2-fluoro-5-
sulfamoylphenyl
EC50: 0.5
EC50: 0.7 ± 0.1
EC50: 6.3
CC : 25.0
CC : 18.0
CC : 18.0
5
0
50
50
SI : 50.0
SI : 25.7
SI : 2.9
50
50
50
4-fluoro-3-
sulfamoylphenyl
EC50: 0.8
EC50: 1.1 ± 0.3
EC50: > 10.0
CC : 50.0
CC : > 200.0
CC : > 200.0
50
50
50
SI : 62.5
SI : > 181.8
SI : N/A
5
0
50
50
073
2-fluoro-5-
carbamoylphenyl
EC50: 0.5
EC50: 0.8
EC50: 5.0
CC : 25.0
CC : 18.8
CC : 18.8
5
0
50
50
SI : 50.0
SI : 23.5
SI : 3.8
5
0
50
50
0
75
4-fluoro-2-pyridyl
EC50: 1.2
EC50: 0.7 ± 0.1
EC50: > 10.0
CC : > 200.0
CC : 37.5
CC : 37.5
5
0
50
50
SI : > 166.7
SI : 53.6
SI : N/A
5
0
50
50
a. For compounds that were tested against certain viruses in 3 separate experiments, means ± s.d.
are shown. Otherwise, a single value represents an average of triplicates in one experiment. b.
Similar results were observed against anti-PV-1 and anti-PV-3 activities.
Analogues at the linker unit. We also synthesized analogues with linker units other than the
original carboxamide, e.g., reverse amide: JX054; N-Me amide: JX074; imide: JX039;
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
thioamide: JX046; sulfonamide: JX049; reverse sulfonamide: JX044; and amidines: JX065,
JX067. In addition two unusual linkers were made and tested, e.g., the benzoxazole: JX051; and
the indoline amide: JX048. Testing of these analogues indicated that the normal amide linker
was crucial for the antiviral activity since many new linker units failed in the tests of antiviral
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
activity even though we put the most favorable groups at the N1, the R and the N-aryl positions.
JX054 demonstrated modest antiviral activity, while JX048 had comparable activity to the
original compound 1a (JX001) (Table 3). The indoline amide may be a promising alternative
linker unit if the issue of compound stability might not allow a carboxamide in the antiviral drug
design.
Table 3. Antiviral activities of analogues with different linker units.
Compound
No. (JX)
Structure
Anti-EV-A71ina Anti- CV-B3 in a Anti-PV-1* in
a,b
LLC cells (µM)
HeLa cells (µM)
HeLa cells (µM)
0
44
reverse
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
sulfonamide
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
0
46
thioamide
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
SI50: N/A
SI50: N/A
SI50: N/A
048
indolineamide EC50: 1.3 ± 0.2
EC50: 1.9 ± 0.6
EC50: 10.0
CC : > 200.0
CC : 18.8
CC : 18.8
5
0
50
50
SI50: > 153.4
SI50: 9.9
SI50: 1.9
0
0
49
51
sulfonamide
benzoxazole
reverse amide
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
SI50: N/A
SI50: N/A
SI50: N/A
054
EC50: 4.0
EC50: 4.7 ± 0.0
EC50: > 10.0
CC : 50.0
CC : 12.0
CC : 12.0
5
0
50
50
SI50: 12.5
SI50: 2.6
SI50: N/A
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
0
65
67
amidine
amidine
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
50
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SI50: N/A
SI50: N/A
SI50: N/A
074
N-
EC50: > 10.0
EC50: > 25.0
EC50: > 10.0
methylamide
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI50: N/A
SI50: N/A
SI50: N/A
0
76
7-azaindoline- EC50: 12.5
amide CC : 50.0
EC50: 9.4 ± 0.0
EC50: > 10.0
CC : 37.5
CC : 37.5
5
0
50
50
SI50: 4.0
SI50: 4.0
SI50: N/A
a. For compounds that were tested against certain viruses in 3 separate experiments, means ± s.d.
are shown. Otherwise, a single value represents an average of triplicates in one experiment. b.
Similar results were observed against anti-PV-1 and anti-PV-3 activities.
New Heterocyclic Ring Systems. Finally, we also prepared several new heterocyclic ring
systems other than the 1H-pyrazolo[3,4-b]pyridine system shown in compound 1. In particular,
we developed syntheses of the 1H-pyrrolo[2,3-b]pyridine-4-carboxamides, 22 (JX037, JX062,
JX063 and JX072), as shown in Scheme 2. We also prepared the 3H-imidazo[4,5-b]pyridine-7-
carboxamide, 27 (JX034), depicted in Scheme 3 and the 1H-1,2,3-triazolo[4,5-b]pyridine-4-
carboxamide, 29 (JX035), as shown in Scheme 4, changing the pyrazole unit for an imidazole
and a 1,2,3-triazole, respectively. Finally a different synthetic route was used to make the 1H-
pyrazolo[3,4-d]pyrimidine-4-carboxamide, 37 (JX036), depicted in Scheme 5, in which the
pyrimidine unit was substituted for the pyridine. These changes also had profound impact on the
activity. The first three heterocyclic ring systems - the imidazopyridine, the triazolopyridine, and
the pyrazolopyrimidine abrogated antiviral activity in our test system. By contrast,
pyrrolopyridine analogues were active with the best analogue, JX062, having EC50
concentrations for EV-A71 and CV-B3 that were similar to the original lead 1a (Table 4).
Table 4. Antiviral activities of analogues with new heterocyclic ring systems.
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound Structure
No. (JX)
Anti-EV-
A71in LLC
cells (µM)
Anti- CV-B3 in Anti-PV-1 in
HeLa cells
(µM)
HeLa cells
(µM)
a
a
a,b
0
34
3H-imidazo[4,5- EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
b]pyridine-7-
carboxamide
CC : N/A
CC : N/A
CC : N/A
50
50
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SI : N/A
SI : N/A
SI : N/A
5
0
50
50
035
1H-triazolo[4,5- EC50: > 10.0
EC50 > 10.0
EC50: > 10.0
b]pyridine-4-
carboxamide
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
5
0
SI : N/A
50
SI : N/A
50
0
0
36
37
1H-pyrazolo[3,4- EC50: > 10.0
d]pyrimidine-4-
carboxamide
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
5
0
SI : N/A
50
SI : N/A
50
1H-pyrrolo[2,3-
b]pyridine-4-
carboxamide
EC50: > 25.0
EC50: 3.1 ± 0.1 EC50: > 25.0
CC : > 200.0
CC : > 200.0
CC : > 200.0
50
50
50
SI : N/A
5
0
SI : 64.5
50
SI : N/A
50
062
1H-pyrrolo[2,3-
b]pyridine-4-
carboxamide
EC50: 0.8
EC50: 1.3 ± 0.1 EC50: > 10.0
CC : 25.0
CC : 18.0
CC : 18.0
5
0
50
50
SI : 31.3
5
0
SI : 13.8
50
SI : N/A
50
0
0
63
72
1H-pyrrolo[2,3-
b]pyridine-4-
carboxamide
EC50: > 10.0
EC50: > 10.0
EC50: > 10.0
CC : N/A
CC : N/A
CC : N/A
5
0
50
50
SI : N/A
5
0
SI : N/A
50
SI : N/A
50
1H-pyrrolo[2,3-
b]pyridine-4-
carboxamide
EC50: 1.2
EC50: 2.2 ± 0.4 EC50: > 10.0
CC : > 200.0
CC : 18.8
CC : 18.8
50
50
50
SI : > 166.7
5
0
SI : 8.5
50
SI : N/A
50
a. For compounds that were tested against certain viruses in 3 separate experiments, means ± s.d.
are shown. Otherwise, a single value represents an average of triplicates in one experiment. b.
Similar results were observed against anti-PV-1 and anti-PV-3 activities.
The direct ofꢀcomparison of pyrazolopyridine and pyrrolopyridine was shown in the following
pairs: JX017 vs. JX037, JX040 vs. JX072, JX042 vs. JX063 and JX056 vs. JX062 (Tables 2
and 4). The pyrrolopyridine analogues with either 4-fluorophenyl (JX037) or 3-pyridyl (JX062)
had good antiviral activity against CV-B3, a commonly encountered representative of the EV-B
1
species. Thus, this new heterocyclic ring system represents a new class of antiviral compounds
with a different heterocyclic core than the original lead 1a.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
■
CONCLUSIONS
In this report, we reported the synthesis and in vitro testing of novel pyrazolopyridine analogues
for possible development into antiviral drugs for the treatment or prevention of enterovirus
infections. We modified four sites around the core structure and carried out antiviral testing to
establish a structure-activity relationship for this system. The best analogues with the highest SI50
values were those with isopropyl group at the 1-position and a thiophen-2-yl unit at the 6-
position. The 4-position allowed the most variation since many different N-aryl groups had equal
or better antiviral activity than 2-fluorophenyl unit in the lead compound 1a (JX001). The 4-
fluorophenyl group (JX017) had the best antiviral activity in its class while the 3-
sulfamoylphenyl moiety (JX025) also exhibited antiviral activity against polioviruses, albeit
with an EC of 5 µM, which fell short of our target (1µM). Furthermore, several heterocyclic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
amines as the N-aryl group also had very favorable antiviral activities. Of all the pyridine and
pyrimidine analogues, those with the 2-pyridyl group had perhaps the best overall activity, e.g.,
JX040 had the greatest antiviral activity against non-polio enteroviruses, although it had weak
antiviral activity against polioviruses. Given the fact that enteroviruses have more than 110 types
and that many important pathogenic viruses may belong to different species (A-D), the diversity
at the N-aryl position may provide options against different enteroviruses. The antiviral breadth
of these variations at the N-aryl position will be studied in further research. We also screened
different linker units and, while most of those had reduced activity compared to carboxamides,
the indolineamide, reverse amide, and imide showed some activity. Finally, we changed the core
structure from pyrazolopyridine to pyrrolopyridine with minimal loss in activity, thus further
expanded the compound space for anti-enteroviral drugs. We infected cells with CVB3-H3 or
CVB3-H3-C179F, which has a missense mutation in the 2C coding domain. As previously
ACS Paragon Plus Environment

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
15
described , CVB3-H3-C179F was resistant to JX001, which inhibited the wildtype virus.
Similarly, CVB3-H3-C179F was not inhibited by JX017, JX034, and JX048 (data not shown),
indicating that these compounds also target activities of the 2C protein.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We acknowledge that in vitro studies of the biological activity of antiviral compounds may not
predict in vivo efficacy or toxicity; additional in vitro characterization and animal model studies
1
0
are key to the pre-clinical development of antiviral agents. In addition, we focused in this study
on examining one representative each of the EV-A, EV-B, and EV-C species of enteroviruses,
1
which are the most commonly encountered types in most of the world. The recent outbreak of
EV-D68 in the US re-emphasizes the need for anti-enteroviral drugs and several studies have
1
0
looked at existing candidates such as fluoxetine. Pyrazolopyridine analogues, which also target
15
the viral 2C protein, represent a novel class of antiviral candidates and their activity against
EV-D68 warrants additional studies.
■
EXPERIMENTAL SECTION
General: Toluene was distilled from sodium under an argon atmosphere. Dichloromethane was
distilled from calcium hydride under an argon atmosphere. All other solvents or reagents were
1
purified according to literature procedures. H NMR spectra were recorded on Bruker
1
spectrometers at 400 MHz and are reported relative to deuterated solvent signals. Data for H
NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant
(
Hz) and integration. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet;
1
3
q, quartet; m, multiplet; and br, broad. C NMR spectra were recorded on Bruker Spectrometers
13
at 100 MHz. Data for C NMR spectra are reported as follows: chemical shift (δ ppm),
1
multiplicity and coupling constant (Hz). Splitting patterns are designated as the same in H
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR. High resolution mass spectrometry was taken on a Thermo Fisher Scientific Exactive Plus
mass spectrometer equipped with an IonSense ID-CUBE DART ion source. The purity of all
final compounds was determined to be >95% by analytical HPLC analysis. For most final
compounds, purity was determined using a Shimadzu LC-20 HPLC with a Nova-Pak Silica 60Å
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
µm HPLC Column (3.9 x 150 mm, Waters) and UV 254 nm detection. Elution at 0.5 mL/min
with a mixture of CH Cl (A) and EtOAc (B) isocratic at 90% A and 10% B, or CH Cl (A) and
2
2
2
2
MeOH (B) isocratic at 90% A and 10% B. The purity of compounds JX042, JX056, JX060-063,
and JX066-067 was determined using a Waters Acquity UPLC connected to a Waters LCT-
Premier XE Time of Flight Instrument with an Acquity BEH C18 1.7µm UPLC Column (2.1 x
2
50 mm, Waters). Elution with a gradient of 0.4 mL/min H O/MeCN/0.3% Formic acid with a
gradient of 3 to 90% MeCN between 0 and 5 min. Mass spectra were recorded from 70 to 2000
Daltons. All solvents were LC-MS/MS Grade and purchased from Fisher Scientific.
General procedure for the preparation of 2:
Method A: To a solution of compound 5 (10.0 mmol) in ethanol (10.0 mL) cooled to 0 °C , was
added dropwise compound 4 (10.0 mmol) with stirring. The mixture was stirred overnight at 21
°C, then the solvent was evaporated in vacuo to give product 6 in almost quantitative yield. The
product 6 was added to a solution of sodium (12.0 mmol) in n-butanol (20.0 ml), and the
resulting mixture was refluxed for 12 h under an argon atmosphere, then cooled and the solvent
evaporated in vacuo. The resulting residue was purified by flash column chromatography on
silica gel (hexanes/ethyl acetate) to give the desired product 2.
Method B: To 20.0 ml of ethanol were added successively 10.0 mmol of compound 7, 20.0
mmol of sodium acetate, and 10.0 mmol of compound 8 at 21 °C, followed by stirring the
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
reaction mixture at 80 °C for 12 h under an argon atmosphere. After removing the solvent in
vacuo, water was added to the residue. The mixture was neutralized with sodium bicarbonate,
and extracted with ethyl acetate. The combined ethyl acetate solution was washed with brine,
dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo. The
resulting residue was purified by flash column chromatography on silica gel (hexanes/ethyl
acetate) to give the desired product 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General procedure for the preparation of 3: Potassium tert-butoxide (24.0 mmol) was added
to a solution of the substrate 9 (20.0 mmol) in anhydrous toluene (100 ml) at 0 °C under an argon
atmosphere in one portion. The mixture was stirred at 0 °C for 15 min. Then diethyl oxalate 10
(4.0 ml) was added via syringe, and the resulting mixture was stirred at 21 °C for 12 h. The
precipitated product was collected by filtration, washed with toluene and dried in vacuo to give
the desired product 3.
General procedure for the preparation of 11: To 25.0 ml of acetic acid were added
successively 5.0 mmol of compound 2 and 5.0 mmol of compound 3 at 21 °C. The resulting
mixture was stirred at 21 °C for 15 min, then refluxed for 4 h under an argon atmosphere. After
removing the solvent in vacuo, the resulting residue was purified by flash column
chromatography on silica gel (hexanes/ethyl acetate) to give the desired product 11.
General procedure for the preparation of 12: To a solution of compound 11 (3.0 mmol) in 2-
propanol (15.0 ml), was added potassium hydroxide (6.0 mmol) in one portion. The resulting
mixture was stirred at 21 °C for 2 h. After removing the solvent in vacuo, the resulting residue
was dissolved in water (100 ml) and neutralized with acetic acid. The precipitated product was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
collected by filtration and dried in vacuo over phosphorus pentoxide to give the desired product
1
2.
General procedure for the preparation of JX001 to JX076: To a solution of the substrate 12
0.5 mmol) in anhydrous dichloromethane (5.0 ml) cooled to 0 °C was added dropwise oxalyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
chloride (1.0 ml, 2.0 M in dichloromethane) with stirring under an argon atmosphere. Then a
catalytic amount of DMF was added. The resulting mixture was stirred at 21 °C for 2 h. After
removing the solvent in vacuo, product 13 was obtained in almost quantitative yield. The product
1
3 was dissolved in anhydrous toluene (15.0 ml) and compound 14 (2.5 mmol) was added at 21
°
C. The resulting mixture was refluxed for 12 h under an argon atmosphere, then cooled to 21
°C, diluted with ethyl acetate, washed successively with 2 M hydrochloric acid and brine, dried
over anhydrous sodium sulfate and the solvent was evaporated in vacuo. The resulting residue
was purified by flash column chromatography on silica gel (hexanes/ethyl acetate) to give the
desired JX compounds (JX001 to JX076). The yields are generally quite good: for example, the
yield of JX001 in this coupling of 13 and 14 was 82%.
Characterization data for JX001 to JX076:
N-(2-Fluorophenyl)-1-isopropyl-6-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX001. H NMR (400 MHz, CDCl ) δ 8.49 (t, J = 7.8 Hz, 1H), 8.34 (br s, 1H), 8.30 (s,
3
1
H), 7.91 (s, 1H), 7.76 (d, J = 2.8 Hz, 1H), 7.46 (d, J = 4.8 Hz, 1H), 7.25-7.12 (m, 4H), 5.41-
1
3
5.34 (m, 1H), 1.64 (d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ) δ 163.3, 152.8 (d, J = 242.4
3
Hz), 151.6, 150.2, 144.3, 135.9, 130.5, 128.9, 128.2, 126.6, 125.8 (d, J = 10.3 Hz), 125.3 (d, J =
7
.6 Hz), 124.8 (d, J = 3.8 Hz), 122.0, 115.0 (d, J = 18.8 Hz), 111.7, 110.7, 49.2, 22.0; HRMS
-
(
ESI, m/z): calcd for C H FN OS ([M-H] ): 379.1029, Found: 379.1031. mp 171-172 °C.
20 16
4
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N-(2-Fluorophenyl)-1-isopropyl-6-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide,
1
JX002. H NMR (400 MHz, CDCl ) δ 8.51 (td, J = 8.0, 1.2 Hz, 1H), 8.37 (m, 2H), 8.19-8.16 (m,
3
2H), 8.03 (s, 1H), 7.54-7.45 (m, 3H), 7.25-7.14 (m, 3H), 5.52-5.45 (m, 1H), 1.67 (d, J = 6.8 Hz,
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
H); C NMR (100 MHz, CDCl ) δ 163.5, 156.6, 152.8 (d, J = 242.5 Hz), 150.7, 138.5, 136.0,
3
1
30.4, 129.8, 128.9, 127.5, 125.9 (d, J = 10.0 Hz), 125.3 (d, J = 7.7 Hz), 124.8 (d, J = 3.4 Hz),
122.0, 115.0 (d, J = 18.8 Hz), 112.9, 110.9, 49.0, 22.1 ; HRMS (ESI, m/z): calcd for C H FN O
2
2
18
4
-
(
[M-H] ): 373.1465, Found: 373.1465.
N-(2-Fluorophenyl)-1-isopropyl-6-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX003. H NMR (400 MHz, CDCl ) δ 8.71 (d, J = 4.4 Hz, 2H), 8.62 (br s, 1H), 8.43-
3
8.39 (m, 2H), 8.06 (s, 1H), 8.01 (d, J = 5.6 Hz, 2H), 7.22-7.13 (m, 3H), 5.48-5.41 (m, 1H), 1.65
13
(
d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ) δ 163.2, 153.4, 153.0 (d, J = 242.9 Hz), 150.5,
3
1
50.4, 145.6, 136.5, 130.6, 125.65 (d, J = 10.3 Hz), 125.59 (d, J = 7.7 Hz), 124.8 (d, J = 3.5 Hz),
122.4, 121.4, 115.1 (d, J = 19.1 Hz), 112.7, 112.1, 49.3, 22.0; HRMS (ESI, m/z): calcd for
-
C H FN O ([M-H] ): 374.1417, Found: 374.1414.
2
1
17
5
N-(2-Fluorophenyl)-1-isopropyl-6-(pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX004. H NMR (400 MHz, CDCl ) δ 8.71-8.70 (m, 2H), 8.58 (d, J = 8.0 Hz, 1H), 8.48-
3
8
1
.44 (m, 3H), 7.86 (td, J = 7.8, 1.6 Hz, 1H), 7.38-7.34 (m, 1H), 7.24-7.13 (m, 3H), 5.48-5.42 (m,
1
3
H), 1.67 (d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ) δ 163.6, 155.3, 155.1, 153.0 (d, J =
3
242.5 Hz), 150.4, 149.2, 137.0, 136.0, 131.8, 125.8 (d, J = 10.3 Hz), 125.3 (d, J = 7.7 Hz), 124.7
(d, J = 3.8 Hz), 124.4, 122.4, 121.5, 115.1 (d, J = 18.8 Hz), 112.9, 111.9, 49.0, 22.1; HRMS
-
(
ESI, m/z): calcd for C H FN O ([M-H] ): 374.1417, Found: 374.1419.
2
1
17
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-(2-Fluorophenyl)-1-isopropyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX005. H NMR (400 MHz, CDCl ) δ 9.37 (s, 1H), 8.67 (d, J = 4.0 Hz, 1H), 8.54 (d, J =
3
1.6 Hz, 1H), 8.48-8.42 (m, 2H), 8.38 (s, 1H), 8.03 (s, 1H), 7.44-7.41 (m, 1H), 7.24-7.14 (m, 3H),
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.48-5.41 (m, 1H), 1.65 (d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ) δ 163.3, 153.8, 152.9
3
(d, J = 242.4 Hz), 150.6, 150.5, 148.8, 136.5, 134.7, 134.1, 130.5, 125.8 (d, J = 9.9 Hz), 125.5
(d, J = 7.6 Hz), 124.8 (d, J = 3.5 Hz), 123.6, 122.2, 115.1 (d, J = 19.2 Hz), 112.5, 111.4, 49.3,
-
2
2.0; HRMS (ESI, m/z): calcd for C H FN O ([M-H] ): 374.1417, Found: 374.1417.
2
1
17
5
1
-Isopropyl-N-(4-sulfamoylphenyl)-6-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-
1
carboxamide, JX006. H NMR (400 MHz, DMSO-d ) δ 10.96 (s, 1H), 8.30 (s, 1H), 8.25 (s,
6
1H), 8.05 (d, J = 3.2 Hz, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 4.8
13
Hz, 1H), 7.29 (s, 2H), 7.23 (t, J = 4.2 Hz, 1H), 5.24-5.18 (m, 1H), 1.52 (d, J = 6.8 Hz, 6H); C
NMR (100 MHz, DMSO-d ) δ 164.4, 151.6, 150.0, 144.3, 141.9, 139.9, 137.1, 132.4, 130.3,
6
129.1, 128.3, 127.1, 120.7, 112.1, 111.8, 49.0, 22.4; HRMS (ESI, m/z): calcd for C H N O S
3 2
2
0
18
5
-
(
[M-H] ): 440.0851, Found: 440.0849.
N-(2-Fluorophenyl)-1-isopropyl-6-(thiazol-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX007. H NMR (400 MHz, CDCl ) δ 8.48-8.44 (m, 3H), 8.37 (br s, 1H), 7.98 (d, J = 3.2
3
Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.25-7.15 (m, 3H), 5.43-5.36 (m, 1H), 1.67 (d, J = 6.8 Hz,
1
3
6
H); C NMR (100 MHz, CDCl ) δ 168.6, 163.0, 153.0 (d, J = 242.8 Hz), 150.1, 150.0, 144.2,
3
136.4, 132.0, 125.7 (d, J = 9.9 Hz), 125.5 (d, J = 7.6 Hz), 124.8 (d, J = 3.8 Hz), 122.4, 122.3,
15.1 (d, J = 19.1 Hz), 113.5, 110.8, 49.4, 22.1; HRMS (ESI, m/z): calcd for C H FN OS ([M-
1
1
9
15
5
-
H] ): 380.0981, Found: 380.0984.
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N-(2,4-Difluorophenyl)-1-isopropyl-6-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX008. H NMR (400 MHz, CDCl ) δ 8.46-8.40 (m, 1H), 8.28 (s, 1H), 8.21 (br s, 1H),
3
7.90 (s, 1H), 7.76 (dd, J = 3.6,1.2 Hz, 1H), 7.47 (dd, J = 5.2 Hz, 1.2 Hz, 1H), 7.15-7.13 (m, 1H),
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
1
.99-6.93 (m, 2H), 5.41-5.34 (m, 1H), 1.65 (d, J = 6.4 Hz, 6H); C NMR (100 MHz, CDCl ) δ
3
63.4, 159.2 (dd, J = 246.3, 11.5 Hz), 153.0 (dd, J = 245.1, 11.9 Hz), 151.7, 150.2, 144.2, 135.7,
130.4, 129.0, 128.3, 126.6, 123.2 (dd, J = 9.2, 2.0 Hz), 122.1 (dd, J = 10.3, 3.8 Hz), 111.7, 111.5
(dd, J = 21.5, 3.5 Hz), 110.7, 104.0 (dd, J = 26.5, 23.0 Hz), 49.3, 22.0; HRMS (ESI, m/z): calcd
-
for C H F N OS ([M-H] ): 397.0935, Found: 397.0936.
2
0
15
2
4
N-(2,6-Difluorophenyl)-1-isopropyl-6-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX009. H NMR (400 MHz, CDCl ) δ 8.31 (s, 1H), 7.92 (s, 1H), 7.76 (dd, J = 3.6, 0.8
3
Hz, 1H), 7.73 (br, 1H), 7.47 (dd, J = 5.2, 1.2 Hz, 1H), 7.32-7.27 (m, 1H), 7.15-7.13 (m, 1H),
1
3
7
.05-7.00 (m, 2H), 5.41-5.34 (m, 1H), 1.64 (d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ) δ
3
163.9, 157.8 (dd, J = 250.1, 4.6 Hz), 151.6, 150.2, 144.3, 134.9, 131.1, 128.9, 128.3 (t, J = 9.8
Hz), 126.5, 113.3 (t, J = 16.1 Hz), 112.0, 111.9 (d, J = 18.4 Hz), 111.8, 111.2, 49.2, 22.1; HRMS
-
(
ESI, m/z): calcd for C H F N OS ([M-H] ): 397.0935, Found: 397.0936.
20 15
2
4
N-(2-Fluorophenyl)-1-isopropyl-6-(thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-
1
amide, JX010. H NMR (400 MHz, CDCl ) δ 8.87 (s, 1H), 8.50 (s, 1H), 8.46 (t, J = 8.2 Hz, 1H),
3
8
.42 (s, 1H), 8.33 (s, 1H), 7.93 (s, 1H), 7.25-7.14 (m, 3H), 5.39-5.32 (m, 1H), 1.64 (d, J = 6.8
1
3
Hz, 6H); C NMR (100 MHz, CDCl ) δ 163.0, 155.4, 152.9 (d, J = 242.4 Hz), 150.1, 149.3,
3
1
1
42.0, 140.1, 136.4, 130.6, 125.7 (d, J = 9.9 Hz), 125.5 (d, J = 8.1 Hz), 124.8 (d, J = 3.9 Hz),
22.2, 115.1 (d, J = 19.2 Hz), 112.3, 111.3, 49.4, 22.0; HRMS (ESI, m/z): calcd for C H FN OS
1
9
15
5
-
(
[M-H] ): 380.0981, Found: 380.0986.
ACS Paragon Plus Environment