Discovery of Potent and Broad-Spectrum Pyrazolopyridine-Containing Antivirals against Enteroviruses D68, A71, and Coxsackievirus B3 by Targeting the Viral 2C Protein

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Discovery of Potent and Broad-Spectrum Pyrazolopyridine-

Containing Antivirals against Enteroviruses D68, A71, and

Coxsackievirus B3 by Targeting the Viral 2C Protein

Yanmei Hu,^†Naoya Kitamura,^†Rami Musharraﬁ eh, and Jun Wang*

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ABSTRACT: The enterovirus genus of the picornavirus family contains many important human pathogens. EV-D68 primarily

infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute ﬂaccid

myelitis (AFM). EV-A71 is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM

and death in severe cases. CVB3 infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is

currently no FDA-approved antiviral for any of these enteroviruses. In this study, we report our discovery and development of

pyrazolopyridine-containing small molecules with potent and broad-spectrum antiviral activity against multiple strains of EV-D68,

EV-A71, and CVB3. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that

these molecules target the viral protein 2C. Overall, the pyrazolopyridine inhibitors represent a promising class of candidates for the

urgently needed nonpolio enterovirus antivirals.

2014, 2016, and 2018 coincided with increasing numbers of

severe respiratory illnesses and neurological complications

such as acute ﬂaccid myelitis (AFM), meningitis, and

encephalitis.^13,15−18It has been shown in cell culture that

contemporary EV-D68 strains, but not historic strains, were

able to infect neuronal cells and cause cytopathic eﬀects.¹⁴In

studies carried out using a mouse model, the EV-D68 virus was

shown to inﬁltrate the blood−brain barrier and infect the

central nervous system (CNS) such as the spinal cord.¹⁹⁻²³

Although EV-D68 virus is rarely detected in the human patient

CNS tissues, there is nevertheless a positive correlation

between EV-D68 infection and neurological complications as

shown by a number of studies.^24,25Recent studies have shown

that antibodies reacting to the EV-D68 viral capsid protein

VP1 have been detected in high frequency in the CSF ﬂuid

samples from AFM patients.²⁵EV-A71 is one of the etiological

INTRODUCTION

■

Enteroviruses such as D68 (EV-D68), EV-A71, and coxsack-

ievirus B3 (CVB3) are important human pathogens that have a

signiﬁcant impact on human health, especially in children.¹EV-

D68, EV-A71, and CVB3 along with poliovirus and rhinovirus

belong to the Enterovirus genera of the Piconaviridae virus

family. Although poliovirus infection was nearly eliminated by

the Global Polio Eradication Initiative through eﬀective

vaccination, no vaccines are available in the United States for

the nonpolio enteroviruses such as EV-D68, EV-A71, and

CVB3. Three inactivated EV-A71 vaccines are available in

China.²⁻⁴However, their eﬃcacy is limited to certain strains

and is not broadly protective.⁵Therefore, it is imperative to

develop small molecule antivirals as therapeutics.⁵⁻⁸Drug

discovery eﬀorts on nonpolio enterovirus antivirals gained

momentum in recent years due to the increasing number of

infections and severity of the disease outcomes caused by EV-

D68, EV-A71, and CVB3.⁹⁻¹¹EV-D68 infection typically leads

to moderate ﬂu-like respiratory illness and it is self-limit-

ing.^12,13EV-D68 spreads through the respiratory tract, similar

to rhinoviruses. However, contemporary EV-D68 strains

appear to evolve and become more virulent compared to

historic strains.¹⁴For example, recent EV-D68 outbreaks in

Received: April 26, 2021

Published: June 4, 2021

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Figure 1. Structurally diverse enterovirus 2C inhibitors. The hit compound 7a identiﬁed from our high-throughput screening served as a starting

point for the lead optimization in this study.

Figure 2. Synthesis of pyrazolopyridine analogues. (A) Synthesis of 1H-pyrazolo[3,4,-b]pyridine-4-carboxylic acid. (B) Representative synthesis of

pyrazolopyridines with either tertiary amine (7g), secondary, or primary amine (7k).

agents for hand, foot, and mouth disease (HFMD) and spreads

through the fecal−oral route. Several EV-A71 outbreaks in the

Asia-Paciﬁc region have resulted in thousands of deaths.⁵

Similar to EV-D68, EV-A71 is also a neurotropic virus, and in

rare cases, EV-A71 infection can cause severe neurological

complications with a high mortality and morbidity rate in

infants and young children.^26,27CVB3 virus is the causative

agent for viral myocarditis.²⁸In addition, CVB3 infection is

also linked to type 1 diabetes mellitus and idiopathic chronic

pancreatitis due to chronic pancreatic inﬂammation induced by

the virus.²⁹

index, and antiviral spectrum.^1,5,7,30Prominent examples

include viral capsid inhibitors such as pleconaril, the viral

protease inhibitor rupintrivir, the 3D polymerase inhibitors

ribavirin and gemcitabine, as well as the viral 2C inhibitors

dibucaine and ﬂuoxetine.³¹⁻³⁶Most of these reported

compounds are either repurposed from rhinovirus antivirals

(pleconaril and rupintrivir) or FDA-approved drugs for other

disease indications (ﬂuoxetine, dibucaine, and ribavirin).³¹As

these compounds were not speciﬁcally developed for EV-D68,

EV-A71, or CVB3, their antiviral speciﬁcity, potency, and

selectivity index need to be signiﬁcantly improved for clinical

use as nonpolio enterovirus antivirals. Notably, EV-D68

antiviral development is left far behind compared to EV-A71

Several compounds have been reported in the literature as

nonpolio antivirals with varying degrees of potency, selectivity

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d

Table 1. SAR Study of Pyrazolopyridine Analogues against EV-D68, EV-A71, and CVB3

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Table 1. continued

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Table 1. continued

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Table 1. continued

a

Antiviral potency was determined in the CPE assay with EV-D68 US/KY/14-18953 virus and EV-A71 Tainan/4643/1998 virus in RD cells. For

b

c

antiviral assay with CVB3 virus, Vero cells were used. Cytotoxicity was determined using the neutral red uptake method. N.T., not tested. The

results are the mean standard deviation of three repeats. SI, selectivity index (CC₅₀/EC₅₀). JX040 also inhibited the EV-D68 US/MO/14-18947

d

strain with an EC₅₀of 0.1 0.04 μM.

and CVB3,⁷possibly because it is an emerging pathogen that

only comes to public attention in recent years.³⁷Our primary

interest is therefore to develop broad-acting nonpolio enter-

ovirus antivirals against EV-D68, EV-A71, and CVB3.

as 2C inhibitors (Figure 1).^{30−33,35,36,38}The pyrazolopyridine

compounds discovered in this study represent one of the most

potent classes of compounds with broad-spectrum antiviral

activity against nonpolio enteroviruses reported so far.

In this study, we have identiﬁed a class of pyrazolopyridine

analogues that have shown broad-spectrum antiviral activity

against EV-D68, EV-A71, and CVB3. The primary hit,

compound 7a, was identiﬁed through a cytopathic eﬀect

(CPE) assay screening against the EV-D68 virus.^38,39

Encouragingly, compound 7a was also active against EV-A71.

Subsequent structure−activity relationship (SAR) studies

yielded several lead compounds including 7d that inhibited

diﬀerent strains of EV-D68, EV-A71, and CVB3 with EC₅₀

values in the nanomolar range and a cellular selectivity index of

over 500. The antiviral activity was also consistent between the

rhabdomyosarcoma (RD) cell (muscle cell) and the SH-SY5Y

cell (neuronal cell). Mechanistic studies have shown that the

pyrazolopyridine analogues target the conserved viral 2C

protein. The EV-A71 2C protein was proposed to form a

hexamer according to the X-ray crystal structure and has been

shown to have ATPase activity and helicase activity.^40,41

Additional recognized functions of 2C include viral RNA

binding and replication, membrane remodeling, and encapsi-

dation.⁴²Structurally diverse compounds have been identiﬁed

RESULTS AND DISCUSSION

■

Chemistry. The synthesis of 1H-pyrazolo[3,4,-b]pyridine-

4-carboxylic acid 4 followed the previously reported

procedures (Figure 2A).^30,43Brieﬂy, condensation of 1-

alkylpyrazole-5-amine or 1,3-dialkylpyrazole-5-amine 1 with

the 4-alkyl-2,4-diketoester 2 in the presence of acetic acid gave

the 1H-pyrazolo[3,4,-b]pyridine-4-carboxylic ester intermedi-

ate 3. Subsequent hydrolysis using potassium hydroxide in

isopropanol gave the key intermediate 1H-pyrazolo[3,4,-

b]pyridine-4-carboxylic acid 4. Next, for ﬁnal compounds

that contain tertiary amine (7g as an example), a one-step

HATU-mediated amide coupling was used (Figure 2B). For

ﬁnal compounds that contain either primary amine or

secondary amine (7k as an example), a two-step process was

applied: the ﬁrst step was HATU-mediated amide coupling

with the mono-Boc-protected diamine, and this was followed

by TFA deprotection to give the ﬁnal product (Figure 2B).

Structure−Activity Relationship Studies. The initial hit

compound 7a was identiﬁed as an EV-D68 antiviral through a

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Figure 3. Summary of SAR studies of pyrazolopyridine. Substitutions led to compounds with a low selectivity index (SI) are labeled in red. Potent

leads have each of the shown substituents R₁, R₃, R₄, and R₆.

CPE-based phenotypic screening of the enamine compound

library.^38,39It inhibited the contemporary EV-D68 US/KY/14-

18953 strain with an EC₅₀value of 16.7 μM and a selectivity

index of 10.6 (Table 1). Gratifyingly, compound 7a also

inhibited the closely related EV-A71 (Tainan/4643/1998)

virus with an EC₅₀value of 8.1 μM. To further optimize the

antiviral potency, selectivity index, and spectrum of antiviral

activity of 7a, we subsequently initiated a structure−activity

relationship study. Speciﬁcally, we focused on diversifying the

1-alkyl R₁, 3-alkyl R₃, 4-amide R₄, and 6-alkyl R₆substitutions

on the 1H-pyrazolo[3,4,-b]pyridine core (Figure 2B).

For the initial screening, all compounds were titrated to

determine the antiviral eﬃcacy and cellular cytotoxicity in the

viral CPE assay and neutral red uptake cell viability assay,

respectively. Human rhabdomyosarcoma (RD) cells were used

for EV-D68 and EV-A71 antiviral assays, and Vero cells were

used for the CVB3 antiviral assay. For the CPE assay, virus-

infected cells were treated with serial dilutions of compounds

for 3 days, and cell viability was determined by neutral red

uptake assay. For compound 7 series with 1-isoproyl and 6-

cyclopropyl substitutions (7a−7m), it was found that the 4-

position amide substitution had a profound eﬀect on the

antiviral activity and selectivity index (Table 1). Replacing the

neutral ester in 7a with pyrrolidine gave compound 7b, which

had signiﬁcantly improved antiviral eﬃcacy. Compound 7c

with diethylamine also had submicromolar EC₅₀values against

all three viruses. Changing diethylamine to dimethylamine gave

compound 7d, which had increased selectivity index.

Compound 7e with a shorter linker between the amide and

the terminal amine was less active (7e vs 7d). Compound 7g

with a cyclized liker was highly potent (EC₅₀= 0.02 μM) but

was less selective than 7f as shown by its low CC₅₀values

(CC₅₀= 20.7 and 33.8 μM in RD and Vero cells, respectively).

Compounds containing secondary or primary amine were less

potent than the compounds containing tertiary amine (7h, 7i

vs 7d, and 7j vs 7f). Extending the linker between the amide

and terminal amine from ethyl to propyl gave compounds 7k,

7l, and 7m, which had a reduced antiviral activity compared

with corresponding compounds with the ethyl linker (7k vs 7c,

7l vs 7d, 7m vs 7h). Overall, the most potent and selective

compounds from compound 7 series were 7b, 7d, 7f, 7g, and

7h, which had more than 100-fold improvement in antiviral

eﬃcacy compared to the original hit 7a against EV-D68 virus.

For compound 10 series (10a−10f) with 6-isopropyl

substitution, we selected the favorable amide substitutions

from compound 7 series, and compounds 10a and 10c−10f

turned out to have high antiviral potency and selectivity index

(EC₅₀< 1 μM and SI₅₀> 120). Compounds 10b with a neutral

terminal hydroxyl group had drastically reduced antiviral

activity (EC₅₀= 9.8 μM) against EV-D68, which is similar to

the antiviral activity of the screening hit 7a. These results

suggest that the terminal positive charge is critical for the

potent antiviral activity. The most potent and selective

compound from this series was compound 10a. Compounds

with 6-tert-butyl substitutions 11a and 11b also had potent

antiviral eﬃcacy but the selectivity index was lower compared

to compounds with 6-isopropyl substitution (11a vs 10a and

11b vs 10e). Compounds with 6-methyl substitution 12a−12e,

13, 14, and 15a−15b were generally less active, except

compound 12a. Next, the 1-position substitution was

examined. Compound 16 with 1-ethyl substitution was less

active than analogue 7d with 1-isopropyl substitution.

Nevertheless, the EC₅₀values were still at the single-digit

micromolar range across three diﬀerent viruses. Compound 17

with 1-methyl substitution was less active, showing an EC₅₀

value of 9.8 μM against the EV-D68 virus. Compounds 18a

and 18b with 1-tert-butyl and 3-methyl substitutions also had

potent antiviral activity but with low CC₅₀values. Compound

19 had similar antiviral activity as compound 18a against EV-

D68 but had a higher selectivity index. Compound 20 with 1-

propyl substitution, compound 21 with 1-isobutyl substitution,

and compound 22 with 1-cyclopropylethyl substitution all had

low single-digit or submicromolar EC₅₀values against all three

viruses, but their cellular cytotoxicity was higher than

compound 7d, which had 1-isopropyl substitution.

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c

Table 2. Antiviral Activity of Potent Leads against Diﬀerent Serotypes of EV-D68 and EV-A71 in RD Cells

a

b

Antiviral eﬃcacy EC₅₀was determined using the CPE assay in RD cells. Cytotoxicity CC₅₀was determined using the neutral red uptake method.

c

N.T., not tested. The results are the mean standard deviation of three repeats. SI₅₀, selectivity index (CC₅₀/EC₅₀).

It is noted that an earlier study similarly reported the

pyrazolopyridine derivatives such as JX040 (Table 1, last row)

as antiviral drugs speciﬁc for enteroviruses.³⁰Those com-

pounds had antiviral activity against EV-A71, CVB3, and

poliovirus-1, but their antiviral activity was not tested against

the EV-D68. Our study showed that JX040 also had potent

antiviral activity against two EV-D68 strains US/KY/14-18953

and US/MO/14-18947 with EC₅₀values of 0.2 and 0.1 μM,

respectively. An earlier study from the same group showed that

similar compounds inhibited the CVB3 virus by targeting the

viral 2C protein.³⁵Although the compounds described in our

study such as 7d share the same pyrazolopyridine core with

those reported earlier such as JX040, the structure−activity

relationships appear to be diﬀerent. For example, compounds

in the earlier study such as JX040 had aromatic amide

substitutions at the 4-position, while compounds from our

study such as 7d prefer a positively charged amine with a

hydrophobic alkyl linker at the same position. In addition,

compounds reported earlier had aromatic substitutions at the

6-position, while our compounds contain hydrophobic alkyl

substitutions. Given that both subtypes of compounds showed

broad-spectrum antiviral activity against nonpolio enter-

oviruses, further structural studies are needed to delineate

their binding sites in the 2C protein.

Summary of Structure−Activity Relationship. The

summary of the SAR results is shown in Figure 3. For position

1 substitution, branched alkyl substitutions such as isopropyl,

tert-butyl, isobutyl, and 1-cyclopropylethyl were favored over

methyl, ethyl, propyl, and cyclopentyl, and compounds with 1-

isobutyl and 1-(1-cyclopropylethyl) substitution had a lower

selectivity index (highlighted in red). For position 3

substitution, both methyl and hydrogen were tolerated. For

the 4-position amide, the ethyl linker was preferred over the

propyl linker. The terminal amine could be tertiary, secondary,

or primary amine. For the 6-position substitution, both small

and bulky alkyl substitutions were tolerated, and tert-butyl

substitution led to lower selectivity index (highlighted in red).

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Figure 4. Antiviral activity of compounds 7d, 7h, 10a, and 19 against EV-D68 US/MO/14-18947 in plaque assay. The plaque areas were quantiﬁed

in Image J, and EC₅₀values were determined through curve ﬁttings in Graphpad Prism 8 using log (concentration of inhibitors) vs percentage of

plaque area with variable slopes. The results are the mean standard deviation of two repeats.

Broad-Spectrum Antiviral Activity. Encouraged by the

potent and broad-spectrum antiviral activity of these

pyrazolopyridine analogues, we selected four lead candidates

7d, 7h, 10a, and 19 with the highest potency and selectivity

index from the primary screening and tested their antiviral

activity against several other contemporary EV-D68 and EV-

A71 strains (Table 2). The EV-D68 strains selected were from

the 2014 outbreak and have been linked with AFM.¹⁴

Compound 7d is the most potent lead compound with EC₅₀

values ranging from 0.04 to 0.1 μM against all EV-D68 and EV-

A71 strains tested as well as a high selectivity index of over

2000. Compounds 7h and 10a had similar antiviral activity

against diﬀerent strains of EV-D68 and EV-A71 with EC₅₀

values ranging from 0.06 to 0.3 μM and a selectivity index of

over 700. Compound 19 was less active compared to 7d, 7h,

and 10a, but it remains a potent inhibitor with broad-acting

pyrazolopyridine compounds might target a conserved viral

protein among enteroviruses or a common host signaling

pathway that is essential for the viral replication.

Antiviral Activity in Secondary Plaque Assay. The

antiviral activity of compounds 7d, 7h, 10a, and 19 against EV-

D68 US/MO/14-18947 was further conﬁrmed by the

secondary plaque assay (Figure 4). Telaprevir, an EV-D68

2A protease inhibitor,⁴⁴was included as a positive control and

showed complete inhibition of plaque formation at 3 μM. All

four compounds showed dose-dependent inhibition of plaque

formation, and the EC₅₀values for 7d, 7h, 10a, and 19 were

0.025, 0.077, 0.023, and 0.13 μM, respectively. These results

were consistent with the primary CPE assay results shown in

Table 2.

Antiviral Activity against EV-D68 in Neuronal Cells.

As contemporary EV-D68 viruses are neurotropic, we are

interested in testing whether the pyrazolopyridine compounds

can inhibit EV-D68 virus replication in neuronal cells. For this,

four lead compounds 7d, 7h, 10a, and 19 were tested against

activity against all strains tested (EC₅₀= 0.4 to 7.4 μM; SI₅₀

>

100). Overall, consistent antiviral eﬃcacy was observed across

diﬀerent strains of EV-D68 and EV-A71, suggesting that these

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Figure 5. Antiviral activity of compounds 7d, 7h, 10a, and 19 against EV-D68 US/MO/14-18947 in neuronal cell line SH-SY5Y using

immunoﬂuorescence assay. The immunoﬂuorescence signals were quantiﬁed in Image J, and EC₅₀values were determined through curve ﬁttings in

Graphpad Prism 8 using log (concentration of inhibitors) vs percentage of positive control with variable slopes. The results are the mean

standard deviation of two repeats.

the neurotropic EV-D68 strain US/MO/14-18947 in neuronal

cell line SH-SY5Y (Figure 5). Viral replication was quantiﬁed

by immunoﬂuorescence staining using an anti-VP1 antibody.

Pleconaril was included as a positive control. Pleconaril is a

known viral capsid protein VP1 inhibitor and has potent

antiviral activity against multiple EV-D68 strains. As shown in

Figure 5, pleconaril completely abolished viral replication at 50

μM as shown by the lack of VP1 ﬂuorescence signal. All four

compounds 7d, 7h, 10a, and 19 inhibited viral replication in a

dose−response manner with EC₅₀values of 0.03, 0.07, 0.03,

and 0.34 μM, respectively, which is consistent with their

antiviral eﬃcacy in RD cells (Table 2). In summary, all four

pyrazolopyridine compounds potently inhibit the neurotropic

EV-D68 virus replication in neuronal cells.

chemical probe and the EV-D68 US/MO/14-18947 virus as a

representative nonpolio enterovirus. Compound 7d inhibited

EV-D68 US/MO/14-18947 virus with an EC₅₀value of 0.07

0.02 μM in CPE assay. EV-D68 US/MO/14-18947 virus was

ampliﬁed in RD cells with escalating drug selection pressure,

starting from ∼1 × EC₅₀at passages 1 and 2 and increased to 2

× EC₅₀, 4 × EC₅₀, and 8 × EC₅₀at passages 3, 4, and 5,

respectively (Table 3). The drug sensitivity of passage 5 (P5)

virus was tested against compound 7d in CPE assay. The EC₅₀

of compound 7d against P5 virus was 1.9 0.2 μM, a 27-fold

increase compared to that of P0 virus, suggesting that

resistance might have emerged. We then sequenced the

whole viral genome and identiﬁed two mutations in the viral

2C protein, D183V and D323G. To validate the resistance

phenotype, we generated recombinant viruses that harbor

these 2C mutations and test their drug sensitivity against

compound 7d.^38,44Because the P5 virus was sequenced as a

mixture, it was unknown whether these two mutations

Mechanism of Action. To delineate the mechanism of

action of these pyrazolopyridine analogues, we ﬁrst attempted

to select drug-resistant mutant viruses through serial viral

passage experiments. For this, we chose compound 7d as a

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emerged from the same virus or diﬀerent viruses. As such, we

generated three mutant recombinant viruses, the single mutant

EV-D68/2C−D183V (rD183V) and EV-D68/2C−D323G

(rD323G) viruses and the double mutant EV-D68/2C−

D183V+D323G (rD183V/D323G) virus using the reverse

genetics system we developed earlier.^38,44In brief, we

introduced 2C−D183V, 2C−D323G, or 2C−D183V+D323G

into the EV-D68 US/MO/14-18947 genome individually via a

reverse genetics approach using a pHH21 vector. The

recombinant WT virus (rWT) was also generated for

comparison.

Table 3. Serial Viral Passage Experiments to Select Drug-

Resistant Mutants against 7d

a

drug selection

drug sensitivity

passage #

pressure 7d (μM)

EC₅₀(μM)

resistant mutant

WT

N.D.

P0

P1

P2

P3

P4

P5

0.07 0.02

N.D.

0.1

0.2

0.4

0.8

1.9 0.2

2C protein

D183V/D323G

a

N.D., not determined.

Figure 6. Mechanism of action of the broad-spectrum antiviral 7d. (A) Drug sensitivity of 7d against diﬀerent strains of recombinant EV-D68

viruses in plaque assay. (B) Thermal shift binding assay of 7d against puriﬁed EV-D68 2C protein and the mutants selected from viral passage

experiments. (C) Thermal shift binding assay of 7d against EV-D68 2C, EV-A71 2C, and CVB3 2C. (D) Melting temperature T_mand thermal shift

ΔT_mof EV-D68 2C, EV-A71 2C, and CVB3 2C in the presence of diﬀerent concentrations of 7d.

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Figure 7. (A) Competition growth assay of rD183V/D323G with the rWT EV-D68 virus. Electropherogram traces of 2C protein coding region

were exported from Chromas Lite 2.6, and residues 183 and 323 from viruses at each passage were shown on the left side. Codon GAT is for Asp,

condon GTT is for Val, and codon GGT is for Gly. The percentages of the mutant and WT virus populations at both positions were determined by

measuring the height of the nucleotide peaks in the sequencing trace and shown on the right side. (B) Stability of recombinant virus rD183V/

D323G propagated in cell culture. The recombinant virus rD183V/D323G was propagated in RD cells sequentially for four passages, and 2C

protein coding regions from the viruses collected from passages 2, 3, and 4 (P2, P3, P4) were sequenced. Electropherogram traces at residues 183

and 323 from viruses at each passage were shown.

To determine which recombinant virus(s) confers the

phenotypic drug resistance, we titrated 7d against these viruses

in the plaque assay (Figure 6A). It was found that the drug

sensitivity of 7d against rD183V virus was decreased by 22-fold

compared to that of the rWT virus (rD183V EC₅₀= 0.66 μM

vs rWT EC₅₀= 0.03 μM), suggesting that the 2C−D183V is

the predominant drug-resistant mutant. In contrast, the 2C−

D323G only conferred moderate drug resistance and the drug

sensitivity of 7d against rD323G virus was decreased by 3-fold

(rD323G EC₅₀= 0.09 μM vs rWT EC₅₀= 0.03 μM). The

double mutant virus rD183V/D323G had more profound

resistance and decreased the drug sensitivity by 33-fold

(rD183V/D323G EC₅₀= 1.00 μM vs rWT EC₅₀= 0.03

μM). These results suggest that the viral 2C protein might be

the drug target of 7d, and 2C−D183V mutant and to a less

extent 2C−D323G mutant confer the phenotypic drug

resistance in cell culture.

To further conﬁrm that viral 2C protein is the direct drug

target of 7d, we performed the diﬀerential scanning ﬂuorimetry

(DSF) assay,⁴⁵⁻⁴⁸also called thermal shift assay, to character-

ize the direct binding of 7d to 2C protein and its mutants

(Figure 6B). For this, we expressed the EV-D68 US/MO/14-

18947 2C protein as well as the 2C−D183V, 2C−D323G, and

2C−D183V/D323G mutants. Binding of 7d to WT 2C

protein is evident as shown by stabilization of the protein by

4.93 °C in T_mshift (Figure 6B). In contrast, no binding was

observed for the 2C−D183V mutant (ΔT_m= 0.01 °C). The

2C−D323G mutant can also be stabilized by 7d, and the ΔT_m

was 3.92 °C, which is comparable to that of the WT 2C

protein. The 2C−D183V/D323G double mutant had drasti-

cally reduced binding to 7d compared to WT 2C (ΔT_m= 0.61

vs 4.93 °C) (Figure 6B). Consistent with plaque assay results

described above for recombinant EV-D68 viruses (Figure 6A),

the DSF assay results showed that the 2C−D183V is the

predominant drug-resistant mutant, while the 2C−D323G

only confers moderate drug resistance, and a combination of

these two mutants further increased the degree of drug

resistance.

As compound 7d and its analogues have broad-spectrum

antiviral activity against EV-A71 and CVB3 in addition to EV-

D68, we further expressed the EV-A71 and CVB3 2C proteins

and performed DSF assay to conﬁrm the direct binding of 7d

to these 2C proteins (Figure 6C). As expected, 7d showed

dose-dependent binding to EV-A71 and CVB3 2C proteins,

similar to EV-D68 2C (Figure 6D). This result implies that 7d

inhibits EV-A71 and CVB3 by similarly targeting their viral 2C

proteins.

Drug-Resistant rD183V/D323G Virus Had a Reduced

Fitness of Replication Compared to WT Virus. To proﬁle

the ﬁtness of replication of the rD183V/D323G virus, we

conducted viral competition growth assay with the rWT virus.

In the viral competition assay, the rWT and rD183V/D323G

viruses were mixed at a 1:100 ratio at passage 0 to favor the

mutant virus, and the mixture was cocultured in RD cells for

several passages. At each passage, the viral 2C protein was

sequenced to determine the relevant ratios of the substitutions

at residues 183 and 323. It was found that the rWT completely

overtook the rD183V/D323G mutant virus at passage 3

(Figure 7A). To rule out the possibility that the rD183V/

D323G virus might revert to WT by itself without the selection

pressure, we passaged rD183V/D323G virus alone four times

and found no change on the viral 2C protein (Figure 7B),

suggesting that the mutant virus is stable in the absence of

competition from WT virus. Taken together, these results

suggest that rD183V/D323G virus had a reduced ﬁtness of

replication compared to rWT.

CONCLUSIONS

■

Nonpolio enteroviruses are important human pathogens and

present a challenge for public health, especially in children and

immunocompromised adults. Therefore, there is a dire need to

develop eﬀective antivirals targeting nonpolio enteroviruses

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1-alkylpyrazole-5-amine or the 1,3-dialkylpyrazole-5-amine (1 mmol)

and the 4-alkyl-2,4-diketoester (1 mmol) were dissolved in acetic acid

(10 mL). The resulting solution was reﬂuxed for 5 h under a nitrogen

atmosphere. After cooling down to ambient temperature, the solvent

was removed in vacuo and the resulting residue was puriﬁed by silica

gel ﬂash column chromatography (10−50% ethyl acetate/hexane) to

give the ﬁnal product.

General Procedure for the Synthesis of 1H-Pyrazolo[3,4-

b]pyridine-4-carboxylic Acid Intermediate 4. Ester 3 (1 mmol)

was dissolved in isopropanol (5 mL). Potassium hydroxide (2 mmol)

was added in portion, and the resulting solution was stirred at ambient

temperature until complete disappearance of the starting material, as

monitored by TLC (2−4 h). The solvent was removed in vacuo, and

the resulting residue was extracted with dichloromethane and 3 N

HCl. The organic layer was dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (1−10% CH₃OH/CH₂Cl₂) to give

the ﬁnal product.

General Procedure of Amide Coupling. To a DMF solution of

carboxylic acid (1 mmol) were added HATU (1 mmol) and DIEA

(1.2 mmol). After stirring for 2 min, amine (1 mmol) was added. The

resulting solution was stirred overnight at ambient temperature. The

reaction was diluted with dichloromethane and extracted with

aqueous NaHCO₃solution, followed by brine. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The crude product was puriﬁed by ﬂash column

chromatography (1−10% CH₃OH/CH₂Cl₂) to give the ﬁnal product.

General Procedure of TFA Deprotection of Boc. The Boc-

protected amide intermediate (1 mmol) was dissolved in dichloro-

methane (5 mL), and TFA (2 mL) was added cautiously. The

resulting mixture was stirred at ambient temperature for 5 h, and then,

the solvent was removed and the ﬁnal product was puriﬁed by ﬂash

column chromatography (1−10% CH₃OH/CH₂Cl₂).

including EV-D68, EV-A71, and CVB3. In this study, we

report our discovery of a series of pyrazolopyridine compounds

with potent and broad-spectrum antiviral activity against

nonpolio enteroviruses. Starting from a screening hit 7a, we

conducted structure−activity relationship studies, which

yielded several lead compounds including 7d, 7h, 10a, and

19 with signiﬁcantly improved antiviral activity and cellular

selectivity. The mechanism of action was delineated through

resistance selection using compound 7d as a chemical probe

and the EV-D68 US/MO/14-18947 virus as a representative

nonpolio enterovirus. Two mutations at the viral 2C protein

were identiﬁed, 2C−D183V and 2C−D323G. Using the

reverse genetics system we developed earlier, we generated

the recombinant EV-D68 viruses with either single or double

2C mutants selected from the passage experiment. It was found

that the D183V is the dominant drug-resistant mutant, while

D323G only confers moderate resistance. This was further

supported by the thermal shift assay results. In addition, we

have shown that compound 7d similarly binds to EV-A71 and

CVB3 2C proteins, implying that its antiviral activity against

EV-A71 and CVB3 was through targeting the viral 2C proteins.

Although resistance could be isolated in cell culture against 7d,

it was found that the rD183V/D323G mutant virus had

reduced ﬁtness of replication compared with WT, as shown by

the competition growth assay results (Figure 7).

Despite a number of 2C protein inhibitors being developed

including those described here, the high-resolution X-ray

crystal structure of the EV-D68 2C protein has not been

solved. It is critical to obtain the drug-bound complexes of 2C

with diﬀerent inhibitors, which will greatly facilitate the

rational drug design. To the best of our knowledge, the

compounds reported herein represent the most potent

antivirals against nonpolio enteroviruses with a high cellular

selectivity index. Further optimization might yield drug

candidates for in vivo animal model studies as well as chemical

probes for in vivo drug target validation.

The following compounds were synthesized using the same

procedure as shown for compound 7g: 7a, 7b, 7c, 7d, 7f, 7g, 7k,

7l, 10a, 10b, 10c, 11a, 12a, 12b, 12c, 13, 14, 15a, 16, 17, 18a, 19, 20,

21, 22, and JX040.

The following compounds were synthesized using the same

procedure as shown for compound 7k: 7e, 7h, 7i, 7j, 7m, 10d, 10e,

10f, 11b, 12d, 12e, 15b, and 18b.

Compound Characterization. 6-Cyclopropyl-1-(propan-2-yl)-

1

EXPERIMENTAL SECTION

1H-pyrazolo[3,4-b]pyridine-4-carboxylic Acid (4a). Yield: 35%. H

■

NMR (400 MHz, DMSO-d₆) δ 13.73 (brs, 1H), 8.22 (s, 1H), 7.59 (s,

1H), 5.22−5.02 (m, 1H), 2.44−2.27 (m, 1H), 1.47 (d, J = 6.7 Hz,

6H), 1.15−0.98 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.3,

162.9, 150.2, 131.7, 131.6, 116.3, 111.1, 48.2, 21.8, 17.1, 11.1.

C₁₃H₁₅N₃O₂, EI-MS: m/z (M − H⁺): 244.3 (calculated), 244.3

(found).

General Chemical Methods. All chemicals were purchased from

commercial vendors and used without further puriﬁcation unless

otherwise noted. ¹H and ¹³C NMR spectra were recorded on a

Bruker-400 or -500 NMR spectrometer. Chemical shifts are reported

in parts per million referenced with respect to the residual solvent

(CD₃OD) 3.31 ppm, (DMSO-d₆) 2.50 ppm, and (CDCl₃) 7.26 ppm

or from internal standard tetramethylsilane (TMS) 0.00 ppm. The

following abbreviations were used in reporting spectra: s, singlet; d,

doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets;

ddd, doublet of doublet of doublets. All reactions were carried out

under a N₂atmosphere, unless otherwise stated. High-performance

liquid chromatography (HPLC)-grade solvents were used for all

reactions. Flash column chromatography was performed using silica

gel (230−400 mesh, Merck). High-resolution mass spectra were

obtained using an OrbitrapTM for all of the compounds obtained in

an ion cyclotron resonance (ICR) spectrometer. The purity was

assessed by using a Shimadzu UPLC with a Shimadzu C18-AQ

column (4.6 mm × 150 mm P/N #227-30767-05) at a ﬂow rate of 1

mL/min; λ = 254 and 220 nm; mobile phase A, 0.1% triﬂuoroacetic

acid in H₂O; and mobile phase B, 0.1% triﬂuoroacetic acid in 90%

CH₃CN and 10% H₂O. The gradients are 0−2 min 10% B, 2−15 min

10−100% B, 15−18 min, 100% B, and 18.1−20 min 10% B. All

compounds submitted for testing were conﬁrmed to be >95.0% purity

by HPLC traces. All ﬁnal products were characterized by proton and

carbon NMR, HPLC, and electrospray ionization mass spectrometry

(ESI-MS).

1,6-Bis(propan-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carboxylic

1

Acid (4b). Yield: 22%. H NMR (400 MHz, DMSO-d₆) δ 13.75 (s,

1H), 8.27 (s, 1H), 7.60 (s, 1H), 5.33−5.11 (m, 1H), 3.35−3.12 (m,

1H), 1.50 (d, J = 6.7 Hz, 7H), 1.31 (d, J = 6.9 Hz, 7H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 166.7, 166.3, 149.9, 132.2, 131.6, 115.7,

111.3, 48.2, 35.8, 22.4, 21.9. C₁₃H₁₇N₃O₂, EI-MS: m/z (M − H⁺):

246.3 (calculated), 246.3 (found).

6-tert-Butyl-1-(propan-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-car-

boxylic Acid (4c). Yield: 31%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.26

(s, 1H), 7.75 (s, 1H), 5.33−5.08 (m, 1H), 1.52 (d, J = 6.7 Hz, 6H),

1.41 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.4, 166.3, 149.4,

132.2, 131.3, 113.7, 110.9, 48.5, 37.8, 29.9, 21.7. C₁₄H₁₉N₃O₂, EI-MS:

m/z (M − H⁺): 260.3 (calculated), 260.3 (found).

6-Methyl-1-(propan-2-yl)-1H-pyrazolo[3,4-b]pyridine-4-carbox-

1

ylic Acid (4d). Yield: 27%. H NMR (400 MHz, DMSO-d₆) δ 13.75

(brs, 1H), 8.26 (s, 1H), 7.55 (s, 1H), 5.39−5.09 (m, 1H), 2.67 (s,

3H), 1.48 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ

166.2, 158.1, 149.9, 131.7, 131.6, 117.9, 110.8, 47.8, 24.4, 21.9.

C₁₁H₁₃N₃O₂, EI-MS: m/z (M − H⁺): 218.2 (calculated), 218.3

(found).

1-Cyclopentyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic

Synthesis Procedures. General Procedure for the Synthesis of

1H-Pyrazolo[3,4-b]pyridine-4-carboxylic Ester Intermediate 3. The

1

Acid (4e). Yield: 41%. H NMR (400 MHz, DMSO-d₆) δ 8.26 (s,

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1H), 7.55 (s, 1H), 5.47−5.29 (m, 1H), 2.67 (s, 3H), 2.18−2.06 (m,

2H), 2.06−1.78 (m, 4H), 1.78−1.60 (m, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 166.2, 158.2, 150.5, 132.0, 131.7, 117.9, 110.8, 56.6,

32.0, 24.4, 24.2. C₁₃H₁₅N₃O₂, EI-MS: m/z (M − H⁺): 244.3

(calculated), 244.3 (found).

6-Methyl-1-propyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic Acid

(4f). Yield: 38%. ¹H NMR (400 MHz, DMSO-d₆) δ 13.76 (brs, 1H),

8.25 (s, 1H), 7.55 (s, 1H), 4.40 (t, J = 7.0 Hz, 2H), 2.66 (s, 3H),

2.01−1.75 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 166.1, 158.4, 150.8, 131.8, 131.7, 117.8, 110.6, 47.9,

24.4, 22.5, 11.0. C₁₁H₁₃N₃O₂, EI-MS: m/z (M − H⁺): 218.2

(calculated), 218.2 (found).

1-Ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic Acid

(4g). Yield: 27%. ¹H NMR (400 MHz, DMSO-d₆) δ 13.77 (brs,

1H), 8.25 (s, 1H), 7.55 (s, 1H), 4.48 (q, J = 7.2 Hz, 2H), 2.67 (s,

3H), 1.42 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

166.1, 158.4, 150.3, 131.8, 131.7, 117.8, 110.7, 41.4, 24.4, 14.7.

C₁₀H₁₁N₃O₂, EI-MS: m/z (M − H⁺): 204.2 (calculated), 204.2

(found).

6-Cyclopropyl-1-ethyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic

Acid (4h). Yield: 36%. ¹H NMR (400 MHz, DMSO-d₆) δ 13.78 (brs,

1H), 8.21 (s, 1H), 7.59 (s, 1H), 4.42 (q, J = 7.2 Hz, 2H), 2.44−2.27

(m, 1H), 1.40 (t, J = 7.2 Hz, 3H), 1.19−0.98 (m, 4H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 166.2, 163.1, 150.4, 131.7, 131.6, 116.2,

110.8, 41.4, 17.1, 14.6, 11.0. C₁₂H₁₃N₃O₂, EI-MS: m/z (M − H⁺):

230.3 (calculated), 230.2 (found).

6-Cyclopropyl-N-(2-methoxyethyl)-1-(propan-2-yl)-1H-pyrazolo-

[3,4-b]pyridine-4-carboxamide (7a). Yield: 75%. ¹H NMR (400

MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.20 (d, J = 0.5 Hz, 1H), 7.42 (s,

1H), 5.21−5.00 (m, 1H), 3.57−3.42 (m, 4H), 3.29 (s, 3H), 2.34−

2.16 (m, 1H), 1.47 (d, J = 6.7 Hz, 6H), 1.13−1.02 (m, 4H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 166.45, 164.90, 149.58, 136.33,

131.55, 112.73, 110.96, 59.50, 47.93, 42.08, 36.08, 22.36, 21.81.

C₁₆H₂₂N₄O₂, EI-MS: m/z (M + H⁺): 303.4 (calculated), 303.4

(found). HPLC purity: 100%.

6-Cyclopropyl-1-(propan-2-yl)-N-[2-(pyrrolidin-1-yl)ethyl]-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7b). Yield: 72%. H NMR

(400 MHz, DMSO-d₆) δ 9.02 (t, J = 5.7 Hz, 1H), 8.24 (s, 1H), 7.56

(s, 1H), 5.21−5.00 (m, 1H), 3.67−3.48 (m, 2H), 3.14−2.79 (m, 6H),

2.32−2.21 (m, 1H), 1.91−1.71 (m, 4H), 1.46 (d, J = 6.7 Hz, 7H),

1.17−1.03 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.90,

162.64, 149.83, 135.37, 131.58, 113.08, 110.70, 53.66, 53.24, 47.89,

36.89, 22.79, 21.74, 17.44, 10.78. C₁₉H₂₇N₅O, EI-MS: m/z (M + H⁺):

342.5 (calculated), 342.5 (found). HPLC purity: 97.8%.

6-Cyclopropyl-N-[2-(diethylamino)ethyl]-1-(propan-2-yl)-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7c). Yield: 81%. H NMR

(400 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.22 (s, 1H), 7.40 (s, 1H),

5.20−5.04 (m, 1H), 3.51−3.31 (m, 2H), 2.71−2.61 (m, 2H), 2.61−

2.52 (m, 4H), 2.33−2.20 (m, 1H), 1.47 (d, J = 6.7 Hz, 6H), 1.14−

1.03 (m, 4H), 0.99 (t, J = 7.1 Hz, 6H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 164.66, 162.56, 149.82, 135.89, 131.49, 112.87, 110.67, 51.10,

47.89, 46.63, 37.22, 21.72, 17.35, 11.58, 10.76. C₁₉H₂₉N₅O, EI-MS:

m/z (M + H⁺): 344.5 (calculated), 344.5 (found). HPLC purity:

98.0%.

6-Cyclopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic

1

Acid (4i). Yield: 22%. H NMR (400 MHz, DMSO-d₆) δ 13.80 (brs,

1H), 8.21 (s, 1H), 7.60 (s, 1H), 4.00 (s, 3H), 2.44−2.32 (m, 1H),

1.18−1.00 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.15,

163.23, 150.96, 131.68, 131.62, 116.17, 110.69, 33.51, 17.09, 10.99.

C₁₁H₁₁N₃O₂, EI-MS: m/z (M − H⁺): 216.2 (calculated), 216.2

(found).

1-tert-Butyl-6-cyclopropyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-

4-carboxylic Acid (4j). Yield: 21%. ¹H NMR (400 MHz, DMSO-d₆) δ

13.67 (s, 1H), 7.46 (s, 1H), 2.53 (s, 3H), 2.40−2.21 (m, 1H), 1.70 (s,

9H), 1.13−0.94 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.96,

161.25, 151.40, 137.42, 133.83, 114.86, 109.86, 58.78, 28.65, 16.68,

15.45, 11.01. C₁₅H₁₉N₃O₂, EI-MS: m/z (M − H⁺): 272.3

(calculated), 272.3 (found).

1-tert-Butyl-3,6-dimethyl-1H-pyrazolo[3,4-b]pyridine-4-carbox-

ylic Acid (4k). Yield: 18%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.34 (s,

1H), 2.62 (s, 3H), 2.53 (s, 3H), 1.73 (s, 9H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 166.97, 156.46, 151.29, 137.34, 134.29, 115.96, 109.75,

59.00, 28.74, 24.52, 15.44. C₁₃H₁₇N₃O₂, EI-MS: m/z (M − H⁺):

246.3 (calculated), 246.3 (found).

6-Cyclopropyl-N-[2-(dimethylamino)ethyl]-1-(propan-2-yl)-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7d). Yield: 79%. H NMR

(400 MHz, DMSO-d₆) δ 8.73 (t, J = 5.7 Hz, 1H), 8.22 (s, 1H), 7.44

(s, 1H), 5.23−5.02 (m, 1H), 3.53−3.37 (m, 2H), 2.56 (t, J = 6.7 Hz,

2H), 2.28 (s, 7H), 1.47 (d, J = 6.7 Hz, 6H), 1.08 (d, J = 6.4 Hz, 4H).

¹³C NMR (101 MHz, DMSO-d₆) δ 164.71, 162.56, 149.83, 135.77,

131.53, 112.97, 110.71, 57.60, 47.88, 44.78, 36.92, 21.73, 17.35, 10.75.

C₁₇H₂₅N₅O, EI-MS: m/z (M + H⁺): 316.4 (calculated), 316.4

(found). HPLC purity: 96.9%.

N-(Azetidin-3-yl)-6-cyclopropyl-1-(propan-2-yl)-1H-pyrazolo[3,4-

b]pyridine-4-carboxamide (7e). TFA salt. Yield: 65%. ¹H NMR (400

MHz, DMSO-d₆) δ 9.57 (d, J = 6.7 Hz, 1H), 9.16 (s, 2H), 8.25 (s,

1H), 7.52 (s, 1H), 5.24−5.03 (m, 1H), 4.99−4.79 (m, 1H), 4.35−

4.05 (m, 4H), 2.37−2.19 (m, 1H), 1.48 (d, J = 6.7 Hz, 6H), 1.18−

1.04 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.79, 162.71,

149.88, 134.65, 131.52, 113.24, 110.56, 51.83, 48.02, 41.52, 21.76,

17.40, 10.85. C₁₆H₂₁N₅O, EI-MS: m/z (M + H⁺): 300.4 (calculated),

300.4 (found). HPLC purity: 98.6%.

6-Cyclopropyl-1-propyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic

1

6-Cyclopropyl-N-[1-(dimethylamino)propan-2-yl]-1-(propan-2-

Acid (4l). Yield: 35%. H NMR (400 MHz, DMSO-d₆) δ 13.76 (brs,

1

yl)-1H-pyrazolo[3,4-b]pyridine-4-carboxamide (7f). Yield: 86%. H

1H), 8.22 (s, 1H), 7.59 (s, 1H), 4.35 (t, J = 6.8 Hz, 2H), 2.44−2.30

(m, 1H), 1.95−1.76 (m, 2H), 1.18−0.98 (m, 4H), 0.78 (t, J = 7.4 Hz,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.16, 163.06, 150.98,

131.74, 131.58, 116.23, 110.68, 47.85, 22.42, 17.03, 11.06.

C₁₃H₁₅N₃O₂, EI-MS: m/z (M − H⁺): 244.3 (calculated), 244.3

(found).

NMR (400 MHz, DMSO-d₆) δ 8.52 (d, J = 8.2 Hz, 1H), 8.22 (s, 1H),

7.46 (s, 1H), 5.24−5.03 (m, 1H), 4.34−4.16 (m, 1H), 2.63−2.50 (m,

1H), 2.42−2.18 (m, 8H), 1.48 (d, J = 6.7 Hz, 6H), 1.19 (d, J = 6.6

Hz, 3H), 1.15−1.04 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ

164.18, 162.48, 149.81, 136.13, 131.60, 113.13, 110.79, 63.65, 47.86,

45.03, 42.83, 21.74, 21.71, 18.73, 17.30, 10.73, 10.71. C₁₈H₂₇N₅O, EI-

MS: m/z (M + H⁺): 330.4 (calculated), 330.4 (found). HPLC purity:

98.4%.

1-(Butan-2-yl)-6-cyclopropyl-1H-pyrazolo[3,4-b]pyridine-4-car-

1

boxylic Acid (4m). Yield: 38%. H NMR (400 MHz, DMSO-d₆) δ

8.24 (s, 1H), 7.60 (s, 1H), 4.97−4.79 (m, 1H), 2.44−2.30 (m, 1H),

2.03−1.75 (m, 2H), 1.47 (d, J = 6.7 Hz, 3H), 1.18−0.98 (m, 4H),

0.62 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.22,

162.79, 150.90, 131.74, 116.21, 110.79, 53.73, 28.79, 20.04, 17.01,

11.07, 11.05, 10.62. C₁₄H₁₇N₃O₂, EI-MS: m/z (M − H⁺): 258.3

(calculated), 258.3 (found).

6-Cyclopropyl-N-(1-methylpyrrolidin-3-yl)-1-(propan-2-yl)-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7g). Yield: 77%. H NMR

(400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.42 (s,

1H), 5.27−5.13 (m, 1H), 5.04−4.84 (m, 1H), 3.56−3.40 (m, 1H),

3.38−3.22 (m, 1H), 2.96−2.83 (m, 1H), 2.67 (s, 3H), 2.65−2.48 (m,

2H), 2.27−2.15 (m, 1H), 2.15−2.02 (m, 1H), 1.55 (d, J = 6.7 Hz,

6H), 1.22−1.13 (m, 2H), 1.10−0.99 (m, 2H). ¹³C NMR (101 MHz,

CDCl₃) δ 165.43, 163.48, 150.69, 134.62, 131.61, 113.55, 111.00,

62.30, 54.87, 49.01, 48.64, 41.15, 32.51, 22.08, 17.94, 11.19.

C₁₈H₂₅N₅O, EI-MS: m/z (M + H⁺): 328.4 (calculated), 328.4

(found). HPLC purity: 95.6%.

6-Cyclopropyl-1-(1-cyclopropylethyl)-1H-pyrazolo[3,4-b]-

1

pyridine-4-carboxylic Acid (4n). Yield: 29%. H NMR (400 MHz,

DMSO-d₆) δ 8.25 (s, 1H), 7.58 (s, 1H), 4.34−4.15 (m, 1H), 2.44−

2.27 (m, 1H), 1.61 (d, J = 6.8 Hz, 3H), 1.46−1.28 (m, 1H), 1.14−

0.95 (m, 4H), 0.65−0.49 (m, 1H), 0.47−0.37 (m, 1H), 0.37−0.13

(m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.36, 162.63, 150.51,

131.72, 116.19, 111.16, 57.16, 19.63, 17.23, 16.98, 10.96, 10.91, 3.70,

3.55. C₁₅H₁₇N₃O₂, EI-MS: m/z (M − H⁺): 270.3 (calculated), 270.3

(found).

6-Cyclopropyl-N-[2-(methylamino)ethyl]-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (7h). TFA salt. Yield: 72%.

¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (t, J = 5.7 Hz, 1H), 8.77 (s,

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2H), 8.25 (s, 1H), 7.49 (s, 1H), 5.23−5.01 (m, 1H), 3.63 (q, J = 5.9

Hz, 2H), 3.23−3.07 (m, 2H), 2.62 (s, 3H), 2.35−2.18 (m, 1H), 1.47

(d, J = 6.7 Hz, 6H), 1.16−1.01 (m, 4H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 165.51, 162.60, 149.90, 135.20, 131.68, 113.25, 110.69,

47.97, 47.66, 35.69, 32.59, 21.76, 17.40, 10.77. C₁₆H₂₃N₅O, EI-MS:

m/z (M + H⁺): 302.4 (calculated), 302.4 (found). HPLC purity:

96.9%.

59.50, 47.93, 42.08, 36.08, 22.36, 21.81. C₁₅H₂₂N₄O₂, EI-MS: m/z (M

+ H⁺): 291.4 (calculated), 291.4 (found). HPLC purity: 100.0%.

N-[1-(Dimethylamino)propan-2-yl]-1,6-bis(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (10c). Yield: 75%. ¹H NMR

(400 MHz, CD₃OD) δ 8.29 (s, 1H), 7.51 (s, 1H), 5.43−5.24 (m,

1H), 4.53−4.32 (m, 1H), 3.31−3.19 (m, 1H), 2.83−2.65 (m, 1H),

2.51−2.29 (m, 7H), 1.59 (d, J = 6.7, 1.5 Hz, 6H), 1.42 (d, J = 6.9, 1.5

Hz, 6H), 1.31 (d, J = 6.6, 1.5 Hz, 3H). ¹³C NMR (101 MHz,

CD₃OD) δ 168.93, 167.84, 151.45, 137.96, 132.96, 114.36, 112.48,

65.31, 49.90, 45.74, 44.78, 38.04, 22.91, 22.89, 22.23, 19.34.

C₁₈H₂₉N₅O, EI-MS: m/z (M + H⁺): 332.5 (calculated), 332.4

(found). HPLC purity: 98.8%.

N-(2-Aminoethyl)-6-cyclopropyl-1-(propan-2-yl)-1H-pyrazolo-

1

[3,4-b]pyridine-4-carboxamide (7i). TFA salt. Yield: 68%. H NMR

(400 MHz, CD₃OD) δ 8.26 (s, 1H), 7.46 (s, 1H), 5.28−5.10 (m,

1H), 3.77 (t, J = 5.9 Hz, 2H), 3.28 (t, J = 5.9 Hz, 2H), 2.31−2.18 (m,

1H), 1.51 (d, J = 6.8 Hz, 6H), 1.22−1.00 (m, 4H). ¹³C NMR (101

MHz, CD₃OD) δ 168.93, 165.18, 151.68, 136.17, 133.00, 132.91,

114.94, 112.00, 49.84, 40.66, 38.69, 22.13, 18.42, 11.57. C₁₅H₂₁N₅O,

EI-MS: m/z (M + H⁺): 288.4 (calculated), 288.4 (found). HPLC

purity: 97.6%.

N-(1-Aminopropan-2-yl)-6-cyclopropyl-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (7j). TFA salt. Yield: 81%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J = 8.1 Hz, 1H), 8.24 (s,

1H), 8.13 (s, 3H), 7.56 (s, 1H), 5.21−5.04 (m, 1H), 4.45−4.24 (m,

1H), 3.12−2.93 (m, 2H), 2.35−2.22 (m, 1H), 1.48 (dd, J = 6.7, 1.2

Hz, 6H), 1.26 (d, J = 6.7 Hz, 3H), 1.18−1.04 (m, 4H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 164.98, 162.54, 149.91, 135.54, 131.75,

113.47, 110.83, 47.96, 43.57, 43.06, 21.78, 21.76, 17.96, 17.38, 10.78,

10.68. C₁₆H₂₃N₅O, EI-MS: m/z (M + H⁺): 302.4 (calculated), 302.4

(found). HPLC purity: 100.0%.

N-[2-(Methylamino)ethyl]-1,6-bis(propan-2-yl)-1H-pyrazolo[3,4-

1

b]pyridine-4-carboxamide (10d). TFA salt. Yield: 71%. H NMR

(400 MHz, DMSO-d₆) δ 9.18 (t, J = 5.6 Hz, 1H), 9.00 (s, 2H), 8.30

(s, 1H), 7.61 (s, 1H), 5.32−5.08 (m, 1H), 3.65 (q, J = 5.9 Hz, 2H),

3.28−3.09 (m, 3H), 2.62 (s, 3H), 1.49 (d, J = 6.7 Hz, 6H), 1.33 (d, J

= 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.55, 165.52,

149.68, 135.63, 131.69, 113.11, 110.97, 48.02, 47.62, 36.14, 35.72,

32.52, 22.35, 21.83. C₁₆H₂₅N₅O, EI-MS: m/z (M + H⁺): 304.4

(calculated), 304.4 (found). HPLC purity: 97.5%.

N-(2-Aminoethyl)-1,6-bis(propan-2-yl)-1H-pyrazolo[3,4-b]-

pyridine-4-carboxamide (10e). TFA salt. Yield: 80%. ¹H NMR (400

MHz, DMSO-d₆) δ 9.09 (t, J = 5.6 Hz, 1H), 8.29 (s, 1H), 8.12 (brs,

3H), 7.59 (s, 1H), 5.35−5.09 (m, 1H), 3.59 (q, J = 6.0 Hz, 2H),

3.29−3.12 (m, 1H), 3.13−2.96 (m, 2H), 1.50 (d, J = 6.7 Hz, 6H),

1.34 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.53,

165.49, 149.64, 135.72, 131.65, 113.06, 110.93, 48.00, 38.34, 37.10,

36.13, 22.36, 21.84. C₁₅H₂₃N₅O, EI-MS: m/z (M + H⁺): 290.4

(calculated), 290.4 (found). HPLC purity: 97.2%.

N-(1-Aminopropan-2-yl)-1,6-bis(propan-2-yl)-1H-pyrazolo[3,4-

b]pyridine-4-carboxamide (10f). TFA salt. Yield: 73%. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.78 (d, J = 8.1 Hz, 1H), 8.27 (s, 1H), 8.04

(brs, 3H), 7.57 (s, 1H), 5.31−5.10 (m, 1H), 4.46−4.20 (m, 1H),

3.29−3.13 (m, 1H), 3.11−2.94 (m, 2H), 1.50 (d, J = 6.7, 1.3 Hz, 6H),

1.34 (d, J = 6.9 Hz, 6H), 1.26 (d, J = 6.8 Hz, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 166.41, 165.00, 149.63, 135.99, 131.68, 113.10,

111.02, 47.98, 43.56, 43.09, 36.09, 22.36, 21.82, 21.79,

17.94.C₁₆H₂₅N₅O, EI-MS: m/z (M + H⁺): 304.4 (calculated), 304.4

(found). HPLC purity: 99.0%.

6-tert-Butyl-N-[2-(dimethylamino)ethyl]-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (11a). Yield: 83%. ¹H NMR

(400 MHz, CD₃OD) δ 8.27 (s, 1H), 7.68 (s, 1H), 5.38−5.20 (m,

1H), 3.72−3.56 (m, 2H), 2.79 (t, J = 6.6 Hz, 2H), 2.47 (s, 6H),

1.62−1.51 (m, 6H), 1.46 (d, J = 1.4 Hz, 9H). ¹³C NMR (101 MHz,

CD₃OD) δ 168.93, 165.18, 151.68, 136.17, 133.00, 132.91, 114.94,

112.00, 49.84, 40.66, 38.69, 22.13, 18.42, 11.57. C₁₈H₂₉N₅O, EI-MS:

m/z (M + H⁺): 332.5 (calculated), 332.4 (found). HPLC purity:

97.9%.

6-Cyclopropyl-N-[3-(diethylamino)propyl]-1-(propan-2-yl)-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7k). Yield: 86%. H NMR

(400 MHz, CD₃OD) δ 8.24 (s, 1H), 7.46 (s, 1H), 5.34−5.11 (m,

1H), 3.58 (t, J = 6.6 Hz, 2H), 3.30−3.13 (m, 6H), 2.38−2.23 (m,

1H), 2.20−1.98 (m, 2H), 1.55 (d, J = 6.8 Hz, 6H), 1.35 (t, J = 7.3 Hz,

6H), 1.25−1.02 (m, 4H). ¹³C NMR (101 MHz, CD₃OD) δ 168.59,

165.26, 151.77, 136.56, 132.85, 132.77, 114.83, 112.06, 50.85, 49.92,

48.43, 37.97, 25.43, 22.15, 18.46, 11.61, 9.27. C₂₀H₃₁N₅O, EI-MS: m/

z (M + H⁺): 358.5 (calculated), 358.5 (found). HPLC purity: 97.8%.

6-Cyclopropyl-N-[3-(dimethylamino)propyl]-1-(propan-2-yl)-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (7l). Yield: 79%. H NMR

(400 MHz, DMSO-d₆) δ 8.78 (t, J = 5.6 Hz, 1H), 8.19 (s, 1H), 7.39

(s, 1H), 5.12 (hept, J = 6.7 Hz, 1H), 3.46−3.20 (m, 2H), 2.36−2.22

(m, 3H), 2.16 (s, 6H), 1.78−1.63 (m, 2H), 1.47 (d, J = 6.7 Hz, 6H),

1.15−1.01 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.68,

162.56, 149.84, 135.85, 131.53, 112.92, 110.71, 56.31, 47.88, 44.46,

37.39, 26.27, 21.73, 17.37, 10.76. C₁₈H₂₇N₅O, EI-MS: m/z (M + H⁺):

330.4 (calculated), 330.4 (found). HPLC purity: 97.4%.

6-Cyclopropyl-N-[3-(methylamino)propyl]-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (7m). TFA salt. Yield: 65%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.98 (t, J = 5.7 Hz, 1H), 8.79 (s,

2H), 8.22 (s, 1H), 7.46 (s, 1H), 5.21−5.03 (m, 1H), 3.40 (q, J = 6.4

Hz, 2H), 3.05−2.89 (m, 2H), 2.65−2.54 (m, 3H), 2.40−2.21 (m,

1H), 1.90 (p, J = 6.9 Hz, 2H), 1.46 (d, J = 6.7 Hz, 6H), 1.08 (d, J =

6.4 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.13, 162.66,

149.91, 135.59, 131.60, 113.07, 110.73, 47.97, 46.18, 36.32, 32.41,

25.70, 21.76, 17.38, 10.80. C₁₇H₂₅N₅O, EI-MS: m/z (M + H⁺): 316.4

(calculated), 316.4 (found). HPLC purity: 98.3%.

N-[2-(Dimethylamino)ethyl]-1,6-bis(propan-2-yl)-1H-pyrazolo-

[3,4-b]pyridine-4-carboxamide (10a). Yield: 84%. ¹H NMR (400

MHz, DMSO-d₆) δ 8.72 (t, J = 5.7 Hz, 1H), 8.28 (s, 1H), 7.50 (s,

1H), 5.31−5.13 (m, 1H), 3.53−3.39 (m, 2H), 3.27−3.13 (m, 1H),

2.56−2.49 (m, 2H), 2.25 (s, 6H), 1.51 (d, J = 6.7 Hz, 6H), 1.35 (d, J

= 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.44, 164.69,

149.57, 136.29, 131.48, 112.69, 110.93, 57.81, 47.92, 45.00, 37.14,

36.04, 22.35, 21.79. C₁₇H₂₇N₅O, EI-MS: m/z (M + H⁺): 318.4

(calculated), 318.4 (found). HPLC purity: 96.3%.

N-(2-Aminoethyl)-6-tert-butyl-1-(propan-2-yl)-1H-pyrazolo[3,4-

1

b]pyridine-4-carboxamide (11b). TFA salt. Yield: 78%. H NMR

(400 MHz, DMSO-d₆) δ 9.11 (t, J = 5.6 Hz, 1H), 8.29 (s, 1H), 8.06

(brs, 3H), 7.74 (s, 1H), 5.30−5.09 (m, 1H), 3.60 (q, J = 6.1 Hz, 2H),

3.14−2.98 (m, 2H), 1.51 (d, J = 6.7 Hz, 6H), 1.43 (s, 9H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 168.43, 165.62, 149.20, 135.52, 131.50,

111.43, 110.56, 48.33, 38.37, 37.97, 37.09, 29.99, 21.74. C₁₆H₂₅N₅O,

EI-MS: m/z (M + H⁺): 304.4 (calculated), 304.4 (found). HPLC

purity: 96.8%.

N-[2-(Dimethylamino)ethyl]-6-methyl-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (12a). Yield: 77%. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.73 (t, J = 5.7 Hz, 1H), 8.26 (s, 1H), 7.47

(s, 1H), 5.30−5.08 (m, 1H), 3.52−3.36 (m, 2H), 2.66 (s, 3H), 2.58

(t, J = 6.6 Hz, 2H), 2.30 (s, 6H), 1.48 (d, J = 6.7 Hz, 6H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 164.68, 157.80, 149.71, 135.97, 131.57,

114.96, 110.45, 57.49, 47.61, 44.67, 36.81, 24.49, 21.89. C₁₅H₂₃N₅O,

EI-MS: m/z (M + H⁺): 290.4 (calculated), 290.4 (found). HPLC

purity: 99.1%.

N-(2-Hydroxyethyl)-1,6-bis(propan-2-yl)-1H-pyrazolo[3,4-b]-

1

pyridine-4-carboxamide (10b). Yield: 73%. H NMR (400 MHz,

DMSO-d₆) δ 8.74 (t, J = 5.6 Hz, 1H), 8.26 (d, J = 0.5 Hz, 1H), 7.51

(s, 1H), 5.30−5.08 (m, 1H), 4.79 (t, J = 5.6 Hz, 1H), 3.57 (q, J = 5.9

Hz, 2H), 3.40 (q, J = 6.0 Hz, 2H), 3.19 (h, J = 6.9 Hz, 1H), 1.50 (d, J

= 6.7 Hz, 6H), 1.34 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 166.45, 164.90, 149.58, 136.33, 131.55, 112.73, 110.96,

6-Methyl-N-(1-methylpyrrolidin-3-yl)-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (12b). Yield: 65%. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.94 (d, J = 7.0 Hz, 1H), 8.24 (s, 1H), 7.55

(s, 1H), 5.30−5.08 (m, 1H), 4.59−4.41 (m, 1H), 3.02−2.92 (m, 1H),

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2.92−2.78 (m, 1H), 2.78−2.69 (m, 1H), 2.69−2.59 (m, 4H), 2.42 (s,

3H), 2.34−2.16 (m, 1H), 2.00−1.82 (m, 1H), 1.48 (d, J = 6.7 Hz,

6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.54, 157.76, 149.70,

135.67, 131.61, 115.14, 110.53, 61.04, 54.40, 48.95, 47.60, 41.19,

31.05, 24.46, 21.89. C₁₆H₂₃N₅O, EI-MS: m/z (M + H⁺): 302.4

(calculated), 302.4 (found). HPLC purity: 100.0%.

6-Methyl-N-(1-methylazetidin-3-yl)-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (12c). Yield: 65%. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.20 (d, J = 7.0 Hz, 1H), 8.24 (s, 1H), 7.53

(s, 1H), 5.33−5.13 (m, 1H), 4.62−4.42 (m, 1H), 3.77−3.60 (m, 2H),

3.23−3.12 (m, 2H), 2.67 (s, 3H), 2.34 (s, 3H), 1.48 (d, J = 6.7 Hz,

6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.31, 157.79, 149.71,

135.41, 131.58, 115.05, 110.45, 62.15, 47.62, 45.04, 39.81, 24.48,

21.89. C₁₅H₂₁N₅O, EI-MS: m/z (M + H⁺): 288.4 (calculated), 288.3

(found). HPLC purity: 98.5%.

6-Methyl-N-[2-(methylamino)ethyl]-1-(propan-2-yl)-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (12d). TFA salt. Yield: 74%.

¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (t, J = 5.6 Hz, 1H), 8.86 (brs,

2H), 8.29 (s, 1H), 7.52 (s, 1H), 5.33−5.07 (m, 1H), 3.72−3.55 (m,

2H), 3.22−3.09 (m, 2H), 2.66 (s, 3H), 2.62 (s, 3H), 1.48 (d, J = 6.7

Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.44, 157.79, 149.79,

135.44, 131.73, 115.21, 110.46, 47.69, 47.61, 35.70, 32.54, 24.48,

21.91. C₁₄H₂₁N₅O, EI-MS: m/z (M + H⁺): 276.4 (calculated), 276.3

(found). HPLC purity: 97.6%.

2H), 2.35−2.21 (m, 7H), 1.39 (t, J = 7.2 Hz, 3H), 1.12−1.01 (m,

4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.66, 162.84, 150.19,

135.75, 131.73, 112.96, 110.55, 57.57, 44.73, 41.22, 36.89, 17.38,

14.61, 10.75. C₁₆H₂₃N₅O, EI-MS: m/z (M + H⁺): 302.4 (calculated),

302.4 (found). HPLC purity: 100%.

6-Cyclopropyl-N-[2-(dimethylamino)ethyl]-1-methyl-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (17). Yield: 76%. H NMR

(400 MHz, DMSO-d₆) δ 8.73 (t, J = 5.7 Hz, 1H), 8.22 (s, 1H), 7.45

(s, 1H), 4.00 (s, 3H), 3.49−3.39 (m, 2H), 2.56−2.48 (m, 2H), 2.35−

2.28 (m, 1H), 2.26 (s, 6H), 1.18−1.04 (m, 4H). ¹³C NMR (101

MHz, DMSO-d₆) δ 164.60, 162.98, 150.74, 135.74, 131.70, 112.88,

110.42, 57.76, 44.97, 37.12, 33.41, 17.41, 10.70. C₁₅H₂₁N₅O, EI-MS:

m/z (M + H⁺): 288.4 (calculated), 288.4 (found). HPLC purity:

98.2%.

1-tert-Butyl-6-cyclopropyl-N-[2-(dimethylamino)ethyl]-3-meth-

yl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide (18a). Yield: 69%. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.56 (t, J = 5.7 Hz, 1H), 7.07 (s, 1H),

3.45−3.29 (m, 2H), 2.46 (t, J = 6.7 Hz, 2H), 2.39 (s, 3H), 2.30−2.23

(m, 1H), 2.21 (s, 6H), 1.70 (s, 9H), 1.13−0.96 (m, 4H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 166.10, 161.15, 150.94, 138.87, 137.32,

112.40, 109.80, 58.62, 57.71, 44.98, 37.01, 28.67, 16.81, 13.87, 10.80.

C₁₉H₂₉N₅O, EI-MS: m/z (M + H⁺): 344.5 (calculated), 344.5

(found). HPLC purity: 98.3%.

1-tert-Butyl-6-cyclopropyl-3-methyl-N-[2-(methylamino)ethyl]-

1H-pyrazolo[3,4-b]pyridine-4-carboxamide (18b). TFA salt. Yield:

53%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92−8.81 (m, 3H), 7.23 (s,

1H), 3.60 (q, J = 6.1 Hz, 2H), 3.15−3.04 (m, 2H), 2.63 (s, 3H), 2.39

(s, 3H), 2.29−2.15 (m, 1H), 1.70 (s, 9H), 1.16−0.87 (m, 4H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 166.66, 161.17, 151.06, 138.00,

137.46, 112.78, 109.78, 58.71, 48.53, 47.33, 35.49, 32.49, 28.70, 16.91,

14.36, 10.82. C₁₈H₂₇N₅O, EI-MS: m/z (M + H⁺): 330.4 (calculated),

330.4 (found). HPLC purity: 98.0%.

N-(2-Aminoethyl)-6-methyl-1-(propan-2-yl)-1H-pyrazolo[3,4-b]-

pyridine-4-carboxamide (12e). TFA salt. Yield: 65%. ¹H NMR (400

MHz, DMSO-d₆) δ 9.00 (t, J = 5.6 Hz, 1H), 8.29 (s, 1H), 8.09 (s,

3H), 7.52 (s, 1H), 5.29−5.09 (m, 1H), 3.64−3.53 (m, 2H), 3.13−

2.98 (m, 2H), 2.66 (s, 3H), 1.48 (d, J = 6.7 Hz, 6H). ¹³C NMR (101

MHz, DMSO-d₆) δ 165.41, 157.81, 149.79, 135.54, 131.72, 115.22,

110.46, 47.70, 38.34, 37.10, 24.49, 21.92. C₁₃H₁₉N₅O, EI-MS: m/z

(M + H⁺): 262.3 (calculated), 262.3 (found). HPLC purity: 98.5%.

1-Cyclopentyl-N-[2-(dimethylamino)ethyl]-6-methyl-1H-

1

1-tert-Butyl-N-[2-(dimethylamino)ethyl]-3,6-dimethyl-1H-

pyrazolo[3,4-b]pyridine-4-carboxamide (13). Yield: 69%. H NMR

1

pyrazolo[3,4-b]pyridine-4-carboxamide (19). Yield: 83%. H NMR

(400 MHz, DMSO-d₆) δ 8.75 (t, J = 5.6 Hz, 1H), 8.26 (s, 1H), 7.47

(s, 1H), 5.48−5.25 (m, 1H), 3.53−3.38 (m, 2H), 2.66 (s, 3H), 2.62

(t, J = 6.6 Hz, 2H), 2.32 (s, 6H), 2.16−2.04 (m, 2H), 2.04−1.81 (m,

4H), 1.80−1.59 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.71,

157.85, 150.28, 135.93, 131.67, 115.00, 110.49, 57.41, 56.45, 44.55,

36.71, 31.97, 24.48, 24.22. C₁₇H₂₅N₅O, EI-MS: m/z (M + H⁺): 316.4

(calculated), 316.4 (found). HPLC purity: 100.0%.

N-[2-(Dimethylamino)ethyl]-6-methyl-1-propyl-1H-pyrazolo-

[3,4-b]pyridine-4-carboxamide (14). Yield: 70%. ¹H NMR (400

MHz, DMSO-d₆) δ 8.73 (t, J = 5.7 Hz, 1H), 8.25 (s, 1H), 7.47 (s,

1H), 4.39 (t, J = 7.0 Hz, 2H), 3.53−3.35 (m, 2H), 2.65 (s, 3H), 2.56

(t, J = 6.7 Hz, 2H), 2.28 (s, 6H), 1.95−1.79 (m, 2H), 0.82 (t, J = 7.4

Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.64, 158.06, 150.62,

135.98, 131.70, 114.91, 110.25, 57.57, 47.81, 44.74, 36.89, 24.49,

22.48, 11.01. C₁₅H₂₃N₅O, EI-MS: m/z (M + H⁺): 290.4 (calculated),

290.4 (found). HPLC purity: 100.0%.

(400 MHz, DMSO-d₆) δ 8.60 (t, J = 5.7 Hz, 1H), 7.02 (s, 1H), 3.49−

3.37 (m, 3H), 2.60 (s, 3H), 2.54 (t, J = 6.7 Hz, 2H), 2.41 (s, 3H),

2.28 (s, 6H), 1.74 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ

166.11, 156.35, 150.88, 139.06, 137.27, 113.62, 109.67, 58.86, 57.46,

44.69, 36.72, 28.77, 24.64, 13.95. C₁₇H₂₇N₅O, EI-MS: m/z (M + H⁺):

318.4 (calculated), 318.4 (found). HPLC purity: 97.8%.

6-Cyclopropyl-N-[2-(dimethylamino)ethyl]-1-propyl-1H-

1

pyrazolo[3,4-b]pyridine-4-carboxamide (20). Yield: 83%. H NMR

(400 MHz, DMSO-d₆) δ 8.79 (t, J = 5.7 Hz, 1H), 8.22 (s, 1H), 7.46

(s, 1H), 4.35 (t, J = 6.8 Hz, 2H), 3.47 (q, J = 6.4 Hz, 2H), 2.62 (t, J =

6.7 Hz, 2H), 2.33 (s, 6H), 2.31−2.20 (m, 1H), 1.92−1.78 (m, 2H),

1.17−1.01 (m, 4H), 0.79 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 164.74, 162.85, 150.75, 135.70, 131.75, 113.00, 110.38,

57.44, 47.73, 44.57, 36.72, 22.44, 17.37, 11.05, 10.79. C₁₇H₂₅N₅O, EI-

MS: m/z (M + H⁺): 316.4 (calculated), 316.4 (found). HPLC purity:

98.9%.

N-[2-(Dimethylamino)ethyl]-1-ethyl-6-methyl-1H-pyrazolo[3,4-

1

b]pyridine-4-carboxamide (15a). Yield: 73%. H NMR (400 MHz,

1-(Butan-2-yl)-6-cyclopropyl-N-[2-(dimethylamino)ethyl]-1H-

1

DMSO-d₆) δ 8.80 (t, J = 5.7 Hz, 1H), 8.26 (s, 1H), 7.51 (s, 1H), 4.47

(q, J = 7.2 Hz, 2H), 3.52−3.39 (m, 2H), 2.66 (s, 3H), 2.61 (t, J = 6.6

Hz, 2H), 2.32 (s, 6H), 1.41 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 164.64, 158.06, 150.11, 135.93, 131.73, 114.97, 110.38,

57.42, 44.56, 41.27, 36.72, 24.47, 14.74. C₁₄H₂₁N₅O, EI-MS: m/z (M

+ H⁺): 276.4 (calculated), 276.3 (found). HPLC purity: 98.9%.

1-Ethyl-6-methyl-N-[2-(methylamino)ethyl]-1H-pyrazolo[3,4-b]-

pyridine-4-carboxamide (15b). TFA salt. Yield: 63%. ¹H NMR (400

MHz, DMSO-d₆) δ 9.04 (t, J = 5.5 Hz, 1H), 8.29 (s, 1H), 8.14 (brs,

2H), 7.54 (s, 1H), 4.47 (q, J = 7.2 Hz, 2H), 3.59 (q, J = 6.0 Hz, 2H),

3.16−2.94 (m, 2H), 2.66 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 165.35, 158.06, 150.17, 135.54, 131.87,

115.19, 110.38, 41.34, 38.31, 37.11, 24.46, 14.77. C₁₃H₁₉N₅O, EI-MS:

m/z (M + H⁺): 262.3 (calculated), 262.3 (found). HPLC purity:

98.9%.

pyrazolo[3,4-b]pyridine-4-carboxamide (21). Yield: 78%. H NMR

(400 MHz, DMSO-d₆) δ 8.71 (t, J = 5.7 Hz, 1H), 8.24 (s, 1H), 7.43

(s, 1H), 5.00−4.79 (m, 1H), 3.45 (q, J = 6.4 Hz, 2H), 2.58−2.48 (m,

2H), 2.36−2.19 (m, 7H), 2.04−1.90 (m, 1H), 1.90−1.73 (m, 1H),

1.47 (d, J = 6.7 Hz, 3H), 1.21−0.95 (m, 4H), 0.63 (t, J = 7.3 Hz, 3H).

¹³C NMR (101 MHz, DMSO-d₆) δ 164.72, 162.56, 150.66, 135.84,

131.69, 112.97, 110.47, 57.75, 53.54, 44.95, 37.09, 28.78, 20.07, 17.32,

10.80, 10.76, 10.62. C₁₈H₂₇N₅O, EI-MS: m/z (M + H⁺): 330.4

(calculated), 330.4 (found). Purity: 98.2%.

6-Cyclopropyl-1-(1-cyclopropylethyl)-N-[2-(dimethylamino)-

ethyl]-1H-pyrazolo[3,4-b]pyridine-4-carboxamide (22). Yield: 71%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (t, J = 5.7 Hz, 1H), 8.23 (s,

1H), 7.43 (s, 1H), 4.41−4.18 (m, 1H), 3.50−3.37 (m, 2H), 2.58−

2.47 (m, 2H), 2.34−2.19 (m, 7H), 1.62 (d, J = 6.8 Hz, 3H), 1.46−

1.29 (m, 1H), 1.13−0.98 (m, 4H), 0.67−0.50 (m, 1H), 0.49−0.36

(m, 1H), 0.36−0.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ

164.71, 162.52, 150.24, 135.88, 131.49, 113.11, 110.61, 57.76, 57.03,

44.99, 37.13, 19.63, 17.27, 17.24, 10.79, 10.72, 3.65, 3.57. C₁₉H₂₇N₅O,

6-Cyclopropyl-N-[2-(dimethylamino)ethyl]-1-ethyl-1H-pyrazolo-

[3,4-b]pyridine-4-carboxamide (16). Yield: 86%. ¹H NMR (400

MHz, DMSO-d₆) δ 8.77 (t, J = 5.7 Hz, 1H), 8.22 (s, 1H), 7.46 (s,

1H), 4.42 (q, J = 7.2 Hz, 2H), 3.52−3.37 (m, 2H), 2.57 (t, J = 6.7 Hz,

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EI-MS: m/z (M + H⁺): 342.5 (calculated), 342.4 (found). HPLC

purity: 97.4%.

the Avicel overlay. Plaque areas were quantiﬁed by Image J and

plotted with drug concentration for the calculation of EC₅₀values.

Diﬀerential Scanning Fluorimetry (DSF). The binding of

compound 7d to EV-A71 Tainan/4643/1998, EV-D68 US/MO/14-

18947, and CVB3 Nancy 2C proteins was detected in DSF using a

Thermal Fisher QuantStudio 5 RealTime PCR System, as previously

described.⁴⁶EV-A71, EV-D68, and CVB WT 2C proteins (4 μM)

were incubated with compound 7d at 10, 30, 100, and 300 μM

concentrations in a buﬀer containing 20 mM Hepes (pH7.5), 300

mM NaCl at 37 °C for 1 h. For the EV-D68 2C−D183V, D323G, and

D183V/D323G mutant 2C proteins, 7d was tested at 100 μM. 1 ×

SYPRO orange (Thermal Fisher) was added, and the ﬂuorescent

signals were monitored under a temperature gradient ranging from 30

to 90 °C (incremental steps of 0.05 °C/s). As the temperature

increases, test proteins gradually denatured and the melting

temperature (T_m) was calculated as the mid-log of the transition

phase from the native to the denatured protein using a Boltzmann

model in Protein Thermal Shift Software v1.3. ΔT_mwas calculated by

subtracting the reference melting temperature of proteins in the

presence of DMSO from the T_min the presence of an indicated

concentration of 7d. Curve ﬁttings were performed using the

Boltzmann sigmoidal equation in Prism 8 software.

Immunoﬂuorescence Staining Assay. Immunoﬂuorescence

staining was performed similarly, as previously described,^38,39,45with

minor modiﬁcations. Neuronal cell line SH-SY5Y growing on

coverslips (Nunc Thermanox) was infected with EV-D68 US/MO/

14-18947 at an MOI of 1. Viruses were ampliﬁed in the presence of

DMSO or indicated concentrations of testing compounds. At 18 hpi,

cells were ﬁxed with 4% formaldehyde for 10 min, followed by

permeabilization with 0.2% Triton X-100 for another 10 min. After

blocking with 3% bovine serum albumin (Sigma-Aldrich) at 4 °C

overnight, cells were stained with a rabbit anti-VP1 antibody at room

temperature for 2 h and then with an antirabbit immunoglobulin

secondary antibody conjugated to Alexa Fluor 488. The nuclei were

stained with 300 nM DAPI after secondary antibody incubation.

Fluorescent images were acquired using a ZOE Fluorescent Cell

Imager (Bio-Rad).

Serial Viral Passage Experiment and Viral 2C Gene

Sequencing. Serial viral passage experiments were carried out in

the presence of compound 7d by doubling the concentration from the

previous passage, starting with a concentration of ∼1 × EC₅₀at P1.

RD cells were infected with EV-D68 US/MO/14-18947 at an MOI of

0.1, and the ampliﬁed virus in the cell culture supernatant was

collected after approximately 3 days when a signiﬁcant cytopathic

eﬀect was observed, and viral titer was quantiﬁed by plaque assay.

Drug sensitivity was tested in the P5 virus by determining the EC₅₀

value through the CPE assay. The viral genome RNA was puriﬁed

using the QIAGEN viral RNA mini kit, followed by reverse

transcription using SuperScript III ﬁrst-strand reverse transcriptase

(Invitrogen) with an oligo(dT) primer. The whole viral genome was

sequenced via 14 sequencing reactions by Eton Biosciences, Inc. The

sequencing primers were reported before.³⁹Speciﬁcally, the fragment

that contains the whole 2C gene was PCR-ampliﬁed using primers

(forward: 5′- GTTAGGTACACATATTGTTTGG-3′ and reverse:

5′-CTTTAGGTTTAG GATTGG GGATTCCTG-3′) and se-

quenced by Eton Bioscience, Inc. using primers (5′-CAAGCCT-

TATTC AACAACGTCC-3′ and 5′- CTTTAGGTTTAG GATTGG

GGATTCCTG-3′).

1-(Propan-2-yl)-N-(pyridin-2-yl)-6-(thiophen-2-yl)-1H-pyrazolo-

1

[3,4-b]pyridine-4-carboxamide (JX040). Yield: 85%. H NMR (400

MHz, DMSO-d₆) δ 11.30 (s, 1H), 8.47 (s, 1H), 8.46−8.42 (m, 1H),

8.36 (s, 1H), 8.29 (d, J = 1.0 Hz, 1H), 8.12 (dd, J = 3.8, 1.2 Hz, 1H),

7.96−7.85 (m, 1H), 7.75 (dd, J = 5.0, 1.1 Hz, 1H), 7.28−7.17 (m,

2H), 5.23 (hept, J = 6.6 Hz, 1H), 1.55 (d, J = 6.7 Hz, 6H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 164.0, 151.7, 151.2, 149.7, 148.1, 144.0,

138.4, 135.9, 132.1, 129.8, 128.6, 128.0, 120.4, 115.0, 111.9, 111.6,

48.5, 21.9. HPLC purity: 100%.

Cell Lines and Viruses. Rhabdomyosarcoma (RD, ATCC, CCL-

136), A172 (ATCC, CRL-1620), and SH-SY5Y (ATCC, CRL-2266)

were maintained in a 37 °C incubator in a 5% CO₂atmosphere. RD

cell was cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM)

with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin

(P/S). SH-SY5Y were cultured in 10% FBS and 1% P/S with 50%

DMEM and 50% F-12 medium. All of the following EV-D68 strains

used in this study were purchased from ATCC: US/KY/14-18953

(ATCC, NE-49132), US/MO/14-18947 (ATCC, NR-49129), US/

MO/14-18949 (ATCC, NR-49130), US/IL/14-18952 (ATCC, NR-

49131), and US/IL/14-18956 (ATCC, NR-49133). All of the

following EV-A71 strains used in this study were purchased from

ATCC or BEI Resources: Tainan/4643/1998 (BEI Resources, NR-

471) and enterovirus 71, MP4 (BEI Resources, NR-472). Enterovirus

A71 US/CT/2016-19519 was obtained from Dr. William Nix at the

Centers for Disease Control and Prevention under a material transfer

agreement. All viruses were ampliﬁed in RD cells prior to infection

assays.

CPE Assays. CPE assays were carried out similarly, as previously

described.^33,49For EV-D68 CPE assays, RD cells were grown to over

90% conﬂuency after seeding in a 96-well plate for 18−24 h, growth

medium was aspirated, and cells were washed with 200 μL of PBS

buﬀer supplemented with magnesium and calcium. Cells were

infected with EV-D68 viruses diluted in DMEM with 2% FBS and

30 mM MgCl₂at an MOI of 0.01 and incubated at a 33 °C incubator

in a 5% CO₂atmosphere for 1−2 h. Testing compounds diluted in

DMEM with 2% FBS and 30 mM MgCl₂were added, and cells were

incubated in a 33 °C incubator in a 5% CO₂atmosphere for 3 days to

develop complete CPE in virus-infected cells. For EV-A71 CPE

assays, similar procedures were performed on RD cells except that the

30 mM MgCl₂was not included in the medium and infected cells

were incubated at 37 °C instead of 33 °C, and the incubation time

was typically 2.5 days for EV-A71 virus to develop complete CPE. For

the CVB3 virus CPE assay, Vero cells were used for infection with

CVB3 Nancy virus at an MOI of 0.3 with a similar procedure as EV-

A71. For all CPE assays, growth media was aspirated and 50 μg/mL

of neutral red staining solution was used to stain viable cells in each

well. Absorbance at 540 nm was measured using a Multiskan FC

Microplate Photometer (Thermo Fisher Scientiﬁc). The EC₅₀values

were calculated from best-ﬁt dose−response curves using GraphPad

Prism 8.

Cytotoxicity Assay. The cytotoxicity of each compound was

determined using the neutral red cell viability assay. The assay was

performed under similar conditions (incubation temperature, time,

and media) as the CPE assay but excluded viral infection. Data

acquisition and analysis (CC₅₀) were performed similarly to the

antiviral CPE assay, and all values are from triplicate experiments.

Plaque Assay. Plaque assay for EV-D68 US/MO/14-18947 was

performed as previously described.^38,39RD cells were grown to more

than 90% conﬂuent and washed with PBS supplemented with

magnesium and calcium after removing the growth medium. Cells

were infected by EV-D68 virus and incubated at a 33 °C incubator for

1−2 h to allow virus attachment. After washing the unbound viruses,

an overlay containing the indicated concentration of test compounds

and 1.2% Avicel (FMC BioPolymer, Philadelphia, PA) in DMEM

supplemented with 2% FBS and 30 mM MgCl₂was added with 4 mL

per well. The plates were incubated at a 33 °C (5% CO₂) incubator

for 3 days, and the cells were stained with crystal violet after removing

Generation of EV-D68 Virus-Containing 2C Mutants by

Reverse Genetics. A plasmid-based reverse genetic system for EV-

D68 US/MO/14-18947 was generated in the pHH21 plasmid, as

previously described.^38,39,44The 2C mutations were introduced via

site-directed mutagenesis using Agilent Technologies QuikChange II

XL kit according to the manufacturer’s instructions, and the inserted

mutations were conﬁrmed by sequencing. RD cells or HEK293T cells

were transfected with the pHH21 plasmids containing US/MO/14-

18947 genome (WT or 2C−D183V, D323G, D183V/D323G) using

Lipofectamine 3000 Transfection Reagent (Thermo Fisher) accord-

ing to the manufacturer’s instructions. The generated viruses in the

cell culture medium were collected (3−5 days post transfection) and

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ampliﬁed on RD cells. The mutations in the viral 2C genes of the

ampliﬁed viruses were conﬁrmed by sequencing.

(ADHS18-198859) to J.W. Y.H. was supported by the NIH

training grant T32 GM008804.

Virus Growth Competition Assay. To compare the ﬁtness of

replication of WT virus (rWT) with the 2C mutant virus (r2C−

D183V/D323G), virus growth competition assay was carried out. RD

cells were infected by a virus mixture containing rWT and r2C−

D183V/D323G at a ratio of 1:100 (MOI = 0.1). The ampliﬁed virus

from the culture medium was collected 3 days post infection, and viral

titers were quantiﬁed by plaque assay and were used for the next

round of infection. After 3 passages, the viral 2C gene from each

passage of the virus was sequenced as described in the serial passage

experiment. The percentages of WT and 2C−D183V and D323G in

each passage were estimated by measuring the height of the

nucleotide peaks in the sequencing trace, as previously described.⁵⁰

ABBREVIATIONS USED

■

AFM, acute ﬂaccid myelitis; CNS, central nervous system;

CVB3, coxsackievirus B3; DSF, diﬀerential scanning ﬂuorim-

etry; EV-A71, enterovirus A71; EV-D68, enterovirus D68

REFERENCES

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ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00758.

■

sı

HPLC traces for the ﬁnal products (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

■

Jun Wang − Department of Pharmacology and Toxicology,

College of Pharmacy, The University of Arizona, Tucson,

Arizona 85721, United States; orcid.org/0000-0002-

4845-4621; Phone: 520-626-1366; Email: junwang@

pharmacy.arizona.edu; Fax: 520-626-0749

Authors

Yanmei Hu − Department of Pharmacology and Toxicology,

College of Pharmacy, The University of Arizona, Tucson,

Arizona 85721, United States

Naoya Kitamura − Department of Pharmacology and

Toxicology, College of Pharmacy, The University of Arizona,

Tucson, Arizona 85721, United States

Rami Musharraﬁeh − Department of Pharmacology and

Toxicology, College of Pharmacy, The University of Arizona,

Tucson, Arizona 85721, United States

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Enterovirus and Encephalitis. Front. Microbiol. 2020, 11, 261.

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D68 in the United States and implications for acute flaccid myelitis.

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00758

Author Contributions

^†Y.H. and N.K. contributed equally.

Author Contributions

J.W. designed the experiments. Y.H. and R.M. performed the

CPE assay, plaque assay, immunoﬂuorescence assay, serial

passage experiments, thermal shift binding assay, and

competition growth assay. N.K. synthesized and characterized

the compounds. J.W. wrote the manuscript with the inputs

from others.

Notes

The authors declare the following competing ﬁnancial

interest(s): J. W. and N. K. are inventors of a pending patent

that claimed the use of the described compounds as potential

broad-spectrum antivirals against non-polio enteroviruses.

ACKNOWLEDGMENTS

■

This research was supported by the National Institute of

Allergy and Infectious Diseases of Health (NIH) (grants

AI147325 and AI157046) and the Arizona Biomedical

Research Commission Centre Young Investigator grant

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NOTE ADDED AFTER ASAP PUBLICATION

■

This article published June 4, 2021 with errors throughout the

text, in Table 1, and an incorrect version of the Supporting

Information. The corrected article published June 10, 2021.

8774

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J. Med. Chem. 2021, 64, 8755−8774

Article Doi

Discovery of Potent and Broad-Spectrum Pyrazolopyridine-Containing Antivirals against Enteroviruses D68, A71, and Coxsackievirus B3 by Targeting the Viral 2C Protein

DOI: 10.1021/acs.jmedchem.1c00758

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