Development of broad-spectrum enterovirus antivirals based on quinoline scaffold

Article abstract of DOI:10.1016/j.bioorg.2020.103981

Non-polio enteroviruses such as enterovirus A71 (EV-A71), EV-D68, and coxsackievirus B3 (CVB3) are significant human pathogens with disease manifestations ranging from mild flu-like symptoms to more severe encephalitis, myocarditis, acute flaccid paralysis/myelitis, and even death. There is currently no effective antivirals to prevent or treat non-polio enterovirus infection. In this study, we report our progress in developing potent and broad-spectrum antivirals against these non-polio enteroviruses. Starting from our previously developed lead compounds that had potent antiviral activity against EV-D68, we synthesized 43 analogs and profiled their broad-spectrum antiviral activity against additional EV-D68, EV-A71, and CVB3 viruses. Promising candidates were also selected for mouse microsomal stability test to prioritize lead compounds for future in vivo mouse model studies. Collectively, this multi-parameter optimization process revealed a promising lead compound 6aw that showed single-digit to submicromolar EC₅₀ values against two EV-D68 strains (US/KY and US/MO), two EV-A71 strains (Tainan and US/AK), and one CVB3 strain, with a high selectivity index. Encouragingly, 6aw was stable in mouse microsomes with a half-life of 114.7 min. Overall, 6aw represents one of the most potent broad-spectrum antiviral against non-polio enteroviruses, rendering it a promising lead candidate for non-polio enteroviruses with translational potential.

Full text of DOI:10.1016/j.bioorg.2020.103981

Journal Pre-proofs
Development of Broad-Spectrum Enterovirus Antivirals Based on Quinoline
scaffold
Rami Musharrafieh, Naoya Kitamura, Yanmei Hu, Jun Wang
PII:
S0045-2068(20)31278-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103981
YBIOO 103981
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 January 2020
9 April 2020
29 May 2020
Please cite this article as: R. Musharrafieh, N. Kitamura, Y. Hu, J. Wang, Development of Broad-Spectrum
Enterovirus Antivirals Based on Quinoline scaffold, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.103981
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1
Development of Broad-Spectrum Enterovirus Antivirals Based on Quinoline scaffold
Rami Musharrafieh,a,b Naoya Kitamura,a Yanmei Hu,a and Jun Wang*,a
aDepartment of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
bDepartment of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721,
United States
*Corresponding author:
Jun Wang, Tel: 520-626-1366, Fax: 520-626-0749, email: junwang@pharmacy.arizona.edu

2
Graphical Abstract
CVB3
EC50 = 0.2 ± 0.2 M
CC50 = 40.1 ± 13.2 M
SI50 = 200
EV-D68
EV-A71
Tainan
EC50 = 3.1 ± 1.0 M
CC50 = 32.4 ± 4.1 M
SI50 = 10
H
O
N
US/KY/14-18953
EC50 = 0.2 ± 0.1 M
CC50 = 32.4 ± 4.1M
SI50 = 162
N
H
N
US/AK
US/MO/14-18947
EC50 = 0.1 ± 0.02 M
CC50 = 32.4 ± 4.1 M
SI50 = 324
EC50 = 3.6 ± 0.2 M
CC50 = 32.4 ± 4.1 M
SI50 = 9
F
6aw
Mouse microsomal stability
1/2 = 114.7 mins
Lint(liver) = 47.9 ml/min/kg
T
C
Keywords: Enterovirus, A71, D68, coxsackievirus B3, quinoline, antiviral

3
ABSTRACT
Non-polio enteroviruses such as enterovirus A71 (EV-A71), EV-D68, and coxsackievirus B3
(CVB3) are significant human pathogens with disease manifestations ranging from mild flu-like
symptoms to more severe encephalitis, myocarditis, acute flaccid paralysis/myelitis, and even
death. There is currently no effective antivirals to prevent or treat non-polio enterovirus infection.
In this study, we report our progress in developing potent and broad-spectrum antivirals against
these non-polio enteroviruses. Starting from our previously developed lead compounds that had
potent antiviral activity against EV-D68, we synthesized 43 analogs and profiled their broad-
spectrum antiviral activity against additional EV-D68, EV-A71, and CVB3 viruses. Promising
candidates were also selected for mouse microsomal stability test to prioritize lead compounds for
future in vivo mouse model studies. Collectively, this multi-parameter optimization process
revealed a promising lead compound 6aw that showed single-digit to submicromolar EC50 values
against two EV-D68 strains (US/KY and US/MO), two EV-A71 strains (Tainan and US/AK), and
one CVB3 strain, with a high selectivity index. Encouragingly, 6aw was stable in mouse
microsomes with a half-life of 114.7 minutes. Overall, 6aw represents one of the most potent
broad-spectrum antiviral against non-polio enteroviruses, rendering it a promising lead candidate
for non-polio enteroviruses with translational potential.

4
Enterovirus, a genus from the Picornaviridae family, is implicated in more than 10 million
illnesses in the United States each year 1. Among the various species of enteroviruses, 7 commonly
infect humans: enterovirus A-D and rhinoviruses A-C. Since the successful eradication of
poliovirus (PV) in the developed world, the main cause of enteroviral diseases are associated with
non-polio enterovirus (EV) infections. Classification of enterovirus species are based principally
on genomic and viral protein similarities. However, many species can have distinct cell surface
receptors for entry, tissue tropism and pathogenesis. Enterovirus A serotype EV-A71 have been
associated with large-scale epidemics since the early 1970s in Europe, the United States, Australia,
and most aggressively, in the Asia-Pacific region 2. EV-A71 is associated with many neurological
complications similar to other EVs, including acute flaccid paralysis (AFM), aseptic meningitis,
encephalitis or cardiorespiratory illness, but in most cases, EV-A71 is known for causing Hand
Foot and Mouth Disease (HFMD) in children 3. Enterovirus B serotype coxsackievirus B3 (CVB3)
is commonly associated with viral heart disease, and may play a role in the onset of juvenile
diabetes mellitus 4. As of date, the most mysterious EV is the serotype enterovirus D68 (EV-D68)
from the enterovirus D species. Unlike most enteroviruses, EV-D68 was largely unstudied until
an unexpected epidemic occurred in 2014 in the United States 5. Since then the outbreak appears
to be reoccurring in a biennial pattern. Initial clinical manifestation of EV-D68 infection resembles
rhinovirus infection, with mild-to-severe upper respiratory infection in children between the ages
of 1-11 6. Unlike rhinovirus infection, recent EV-D68 strains have been linked with a large number
of patients developing acute flaccid myelitis, or AFM 7.
All EVs are non-enveloped positive-sense RNA viruses that are encapsulated by viral
proteins VP1-4. The viral genome can be translated directly into a polyprotein which is processed
by viral 2A and 3C proteases. Genome replication is mediated by the RNA-dependent RNA

5
polymerase (RDRP) 3Dpol in replication organelles. The multifunctional viral protein 2C is
involved in membrane rearrangement 8, uncoating 9, assembly 9, 10, as well as viral RNA replication
11. 2C contains Walker A,B, and C motifs that form a conserved ATPase domain that has helicase
12
activity in vitro . 2C also contains an N-terminal membrane-binding domain as well as a C-
terminal domain that may play a role in the formation of dimers or higher oligomeric species 13.
Given the multifunctional roles of 2C in viral replication, it appears to be a high profile antiviral
drug target 14.
Until vaccines can be developed, antivirals are required to alleviate the burden of disease
and reduce fatalities associated with non-polio enterovirus infections. As of date, no antiviral is
approved for the treatment of any enterovirus infection. The most promising candidates are direct-
acting antivirals targeting viral proteins such as viral VP1 capsid protein, 2A and 3C proteases, 2C
protein, and the viral 3D polymerase 1, 15-18. The viral 2C protein is a conserved viral protein, and
has been validated as a drug target by a number of potent antivirals 14, 19. Dibucaine, a quinoline
derivative commonly used as a topical anesthetic, was found to inhibit the replication of CVB3 by
20
targeting the viral 2C protein . However, it was found that dibucaine was only moderately
effective against other enteroviruses such as EV-A71 and EV-D68 20. Recently, structure-activity
relationship studies show that chemical modification of dibucaine increased its potency against
contemporary strains of EV-D68 14. However, their broad-spectrum antiviral activity against EV-
A71 and coxsackievirus B3 virus remains to be profiled. In this work, we focus on optimizing the
broad-spectrum antiviral activity and in vitro microsomal stability of quinoline analogs through
structure-activity relationship (SAR) studies and structure-property (SPR) relationship studies. In
addition, thermal shift binding assay was applied to quantify the direct binding between quinoline

6
analogs and the viral 2C protein. Overall, this study revealed several promising quinoline analogs
that had potent and broad-spectrum antiviral activity against EV-D68, EV-A71 and CVB3.
The viral 2C protein is relatively conserved among non-polio enteroviruses. Therefore, we
hypothesized that 2C inhibitors will have broad-spectrum antiviral activity against non-polio
enteroviruses. To test this hypothesis, we first started by profiling the broad-spectrum antiviral
activity of literature reported 2C inhibitors 20-24 against the non-polio enteroviruses including EV-
D68, EV-A71 and CVB3 in cell culture using cytopathic effect (CPE) assay. It was found that
dibucaine, fluoxetine, pirlindole mesylate, and formoterol all inhibited EV-D68 and CVB3, but
not the EV-A71 virus. Moreover, they all had a low selectivity index (SI50 < 50). The only
exception was guanidine, which showed broad-spectrum antiviral activity against all three viruses,
but only at very high concentrations (200 µM–900 µM). Overall, the existing 2C inhibitors appears
to lack the broad-spectrum antiviral activity against non-polio enteroviruses. Nevertheless, the
results of guanidine suggested that it might be feasible to develop broad-spectrum antivirals against
non-polio enteroviruses. As such, our focus of this study is to develop viral 2C inhibitors as broad-
spectrum antivirals against not only EV-D68, but also EV-A71 and CVB3.
Table 1. Broad-spectrum antiviral activity of known 2C inhibitors against non-polio
enteroviruses.
Antiviral activity and
selectivity index against
EV-D68 US/KY/14-
Antiviral activity and
selectivity index against
EV-A71 Tainan in RD
cells (µM)
Antiviral activity and
selectivity index against
CVB3 in Vero cells (µM)
Structure
18953 in RD cells (µM)
H
O
N
N
EC50 = 5.3 ± 1.2
CC50 = 56.6 ± 28.7
SI50 = 11
EC50 > 10.0
CC50 = 56.6 ± 28.7
SI50 = N.A.
EC50 = 1.3 ± 0.2
CC50 = 64.4 ± 2.1
SI50 = 50
N
O
Dibucaine
F
F
F
EC50 = 1.0 ± 0.1
CC50 = 11.9 ± 4.9
SI50 = 12
EC50 > 10.0
CC50 = 11.9 ± 4.9
SI50 = N.A.
EC50 = 2.1 ± 1.0
CC50 = 11.0 ± 3.1
SI50 = 5
N
O
H
Fluoxetine

7
EC50 = 8.4 ± 1.1
CC50 = 19.0 ± 1.6
SI50 = 2
EC50 > 20
CC50 = 19.0 ± 1.6
SI50 = N.A.
EC50 = 3.6 ± 0.8
CC50 = 40.5 ± 3.5
SI50 = 11
N
O
NH
OH
S
O
Pirlindole mesylate
OH
H
N
H
O
N
EC50 = 24.7 ± 1.1
CC50 = 146.3 ± 27.8
SI50 = 6
EC50 > 100
CC50 = 146.3 ± 27.8
SI50 = N.A.
EC50 = 9.9 ± 1.1
CC50 > 300.0
SI50 > 30
H
HO
O
Formoterol
NH
EC50 = 219.4 ± 15.0
CC50 > 1000.0
SI50 > 4
EC50 = 901.6
CC50 > 1000.0
SI50 > 1
EC50 = 224.5 ± 57.9
CC50 > 3000.0
SI50 > 13
H2N
NH2
Guanidine
Antiviral efficacy was determined in the CPE assay. Cytotoxicity was determined using the neutral
red method. The results are the mean ± standard deviation of three repeats. SI50 = selectivity index
(CC50/EC50). N.A. = not applicable.
Synthesis of quinoline analogs started with Suzuki-Miyaura cross-coupling of 2-
chloroquinoline-4-carboxylic acid 1 with various boronic acids 2. The carboxylic acid intermediate
3 was then reacted with various diamines to give the amide intermediate 5. Deprotection of Boc
by TFA gave the final product 6. Overall, this is a highly efficient synthesis with an overall yield
of 53.2 to 76.9%.
Scheme 1. Synthesis of quinoline analogs
O
OH
O
OH
OH
B
Pd(PPh3)4, K2CO3
OH
Ar/
Het
linker
N Boc
+
+
H2N
Dioxane/H2O
microwave 140oC, 30 mins
R1
N
Cl
X= H, Cl
N
Ar/
Het
R1
X
X
1
2
3
4
HATU, DIEA
DMF
H
H
linker
NH
linker
N Boc
N
N
O
O
TFA, DCM
N
N
Ar/
Het
Ar/
Het
R1
R1
X
X
5
6

8
The purpose of the current structure-activity relationship studies was to provide additional
lead compounds with potent antiviral activity (EC50 ≤ 1 µM) and a high selectivity index (SI50 >
100) for the following broad-spectrum antiviral activity profiling and mouse microsomal stability
test. It is expected that majority of the compounds will be filtered during this multi-parameter
optimization process; therefore it is imperative to obtain as many backup molecules as possible in
order to increase the chance of success.
The antiviral activity of all compounds was initially tested against EV-D68 US/KY/14-
18953 virus in RD cells using the CPE assay. The cytotoxicity assay was performed in parallel in
RD cells using the neutral red method. Next, compounds with potent antiviral activity (EC50 ≤ 1
µM) and a high selectivity index (SI50 > 100) against EV-D68 US/KY/14-18953 were selected for
further testing against additional EV-D68, EV-A71 and CVB3 viruses. In parallel, promising lead
compounds were profiled for mouse microsomal stability. Overall, this is a multiparameter
optimization approach with an aim of identifying lead compounds with broad-spectrum antiviral
activity and favorable in vitro pharmacokinetic properties.
Compounds 6aa and 6bf were identified as potent EV-D68 antivirals from the previous
round of structure-activity relationship studies of dibucaine 14, and both compounds were included
as references (Table 2). For the series of compounds with 1-position substitution being benzene
(6aa – 6aq), it was found that replacing the 4-position amide linkage with ester abolished the
antiviral activity (compound 6ab vs 6aa). Extending the linker between amide to terminal amine
from ethyl to propyl also reduced the antiviral activity (6aa vs 6ac and 6ad). Compound 6ae with
a terminal imidazole group was less active than compound 6aa that had a dimethyl amine group.
Compounds 6ag, 6ah, 6ai, 6aj, 6ak, 6al, and 6am all had a single-digit micromolar EC50 value,
but their low selectivity indexes (SI = 3.2 – 16.1) did not warrant further investigation of these

9
compounds. Compounds 6af, 6an, and 6ap had similar antiviral activity as compound 6aa, but
their selectivity indexes did not pass the selectivity threshold (SI > 100). The only compound met
the criteria was compound 6aq (EC50 = 0.5 ± 0.3 µM; SI50 = 131.6). It is known that benzene
substituent might be easily metabolized, especially through 4-hydroxylation. We therefore made
an effort to block this metabolism by introducing either deuterium (6ar – 6au) or fluorine (6av –
6be). For compounds with deuterated benzene at 1-position (6ar – 6au), compound 6ar was the
most potent and selective candidate (EC50 = 0.3 ± 0.2 µM; SI50 = 268.7), and it was selected for
further characterization. For compounds with fluorine-substituted benzene at 1-position (6av –
6be), the four compounds 6av, 6aw, 6ax, and 6ba met the selection criteria (EC50 < 1 µM; SI50 >
100). For compounds containing thiophene at 1-position (6bf – 6bn), the five compounds 6bf,
6bg, 6bi, 6bj, and 6bl had the desired potency and selectivity index for further development. For
compounds with furan at 1-position (6bo – 6bs), only compound 6bo met the selection criteria.
Overall, in addition to the two reference compounds (6aa and 6bf), we identified 11
additional candidates (6aq, 6ar, 6av, 6aw, 6ax, 6ba, 6bg, 6bi, 6bj, 6bl, and 6bo) with high
antiviral potency (EC50 < 1 µM) and an optimal selectivity index (SI50 > 100) that warrant further
profiling of broad-spectrum antiviral activity and mouse microsomal stability.
Table 2. Antiviral activity against EV-D68 US/KY/14-18953.
H
N
H
H
N
H
H
N
O
O
O
N
O
NH2
O
N
O
N
N
N
N
N
N
N
N
N
6ab
6aa
6ae
6ac
6ad
EC50 > 50.0 µM
CC50 = 78.9 µM
SI50 = N.A.
EC50 = 0.4 ± 0.2 µM
CC50 = 73.7 ± 19.1 µM
SI50 = 184
EC50 = 11.9 ± 2.5 µM
CC50 = 134.3 ± 37.9 µM
EC50 = 8.9 ± 6.4 µM
CC50 = 93.6 ± 22.0 µM
SI50 = 11
EC50 = 7.2 ± 1.2 µM
CC50 = 168.3 ± 27.2 µM
SI50 = 23
SI50 = 11
NH
H
NH
H
H
H
H
H
O
N
O
N
O
N
O
N
O
N
N
NH
OH
NH
N
N
N
N
N
6af
EC50 = 0.9 ± 0.3 µM
6ai
6aj
EC50 = 4.2 ± 0.2 µM
6ag
EC50 = 5.8 ± 0.3 µM
6ah
EC50 = 5.1 ± 0.4 µM
EC50 = 6.0 ± 0.3 µM

10
CC50 = 47.8 ± 3.6 µM
SI50 = 53
CC50 = 43.2 ± 4.5 µM
SI50 = 7
CC50 = 82.2 ± 19.9 µM
SI50 = 16
CC50 = 19.1 ± 3.7 µM
SI50 = 3
CC50 = 36.6 ± 11.2 µM
SI50 = 9
H
N
H
NH
NH2
H
N
H
H
N
O
N
O
N
H
O
H
NH2
TFA
N
O
N
O
N
N
H
TFA
N
N
N
N
N
6ak
6an
6ao
6al
6am
EC50 = 2.9 ± 1.2 µM
CC50 = 46.8 ± 13.6 µM
SI50 = 16
EC50 = 1.2 ± 0.4 µM
CC50 = 44.9 ± 8.2 µM
SI50 = 37
EC50 = 3.3 ± 1.1 µM
CC50 > 200.0 µM
SI50 > 60
EC50 = 6.9 ± 1.3 µM
CC50 = 49.5 ± 13.7 µM
SI50 = 7
EC50 = 7.1 ± 2.5 µM
CC50 = 30.1 ± 7.2 µM
SI50 = 4
H
H
H
H
H
O
N
O
N
O
N
N
N
NH2
O
N
O
N
NH2
TFA
N
H
D
D
D
D
D
D
D
D
D
N
N
N
N
N
D
D
D
D
D
D
6ap
6aq
6ar
6as
6at
EC50 = 1.2 ± 0.3 µM
CC50 = 89.1 ± 3.2 µM
SI50 = 74
EC50 = 0.5 ± 0.3 µM
CC50 = 65.8 ± 16.8 µM
SI50 = 132
EC50 = 0.3 ± 0.2 µM
CC50 = 80.6 ± 3.3 µM
SI50 = 269
EC50 = 1.1 ± 0.3 µM
CC50 = 100.8 ± 3.2 µM
SI50 = 92
EC50 = 2.2 ± 0.2 µM
CC50 = 100.3 ± 12.1 µM
SI50 = 46
NH
H
N
H
H
H
H
O
O
N
O
N
O
N
O
N
N
N
NH2
H
NH
D
D
D
N
N
N
N
N
D
F
F
F
F
D
6av
6aw
6ax
6ay
6au
EC50 = 0.1 ± 0.1 µM
CC50 = 36.2 ± 3.1 µM
SI50 = 362
EC50 = 0.2 ± 0.1 µM
CC50 = 32.4 ± 4.1 µM
SI50 = 162
EC50 = 0.4 ± 0.1 µM
CC50 = 48.2 ± 2.3 µM
SI50 = 121
EC50 = 1.1 ± 0.1 µM
CC50 = 46.7 ± 3.7 µM
SI50 = 43
EC50 = 6.4 ± 0.2 µM
CC50 = 188.5 ± 20.4 µM
SI50 = 30
H
NH
H
N
H
H
H
O
N
O
N
O
N
O
N
O
N
N
N
N
F
F
F
F
N
N
N
N
N
F
F
F
F
F
F
F
6ba
6bb
6bc
6az
6bd
EC50 = 0.3 ± 0.2 µM
CC50 = 36.7 ± 5.6 µM
SI50 = 122
EC50 = 12.1 ± 2.5 µM
CC50 = 93.0 ± 4.1 µM
SI50 = 8
EC50 = 3.6 ± 1.2 µM
CC50 = 39.2 ± 25.4 µM
SI50 = 11
EC50 = 1.2 ± 0.2 µM
CC50 = 50.6 ± 3.1 µM
EC50 > 30.0 µM
CC50 = 38.0 ± 5.2 µM
SI50 = N.A.
SI50 = 42
H
O
N
H
H
H
H
N
O
N
O
N
O
N
O
N
N
N
N
N
F
F
F
F
N
N
N
N
N
F
S
S
S
S
6bf
6bg
6bh
6bi
6be
EC50 = 0.04 ± 0.02 µM
CC50 = 42.3 ± 8.3 µM
SI50 = 1058
EC50 = 0.1 ± 0.1 µM
CC50 = 20.6 ± 0.9 µM
SI50 = 206
EC50 = 1.0 ± 0.3 µM
CC50 = 42.6 ± 4.7 µM
SI50 = 43
EC50 = 0.1 ± 0.2 µM
CC50 = 39.0 ± 3.4 µM
SI50 = 390
EC50 = 10.8 ± 2.5 µM
CC50 = 147.8 ± 14.3 µM
SI50 = 14
H
H
H
H
NH
H
O
N
O
N
O
N
O
N
O
N
NH2
N
NH2
H
NH
N
N
N
N
N
S
S
S
S
S
6bj
6bm
6bn
6bl
6bk
EC50 = 0.3 ± 0.1 µM
CC50 = 103.1 ± 49.5 µM
SI50 = 344
EC50 = 0.5 ± 0.2 µM
CC50 = 23.8 ± 2.6 µM
SI50 = 48
EC50 = 1.2 ± 0.3 µM
CC50 = 28.3 ± 7.6 µM
EC50 = 0.4 ± 0.1 µM
CC50 = 42.1 ± 13.4 µM
SI50 = 105
EC50 = 6.4 ± 2.2 µM
CC50 = 34.5 ± 6.8 µM
SI50 = 24
SI50 = 5
NH
H
H
H
H
H
O
N
O
N
O
N
O
N
O
N
N
NH2
NH2
NH
N
N
N
N
N
O
O
O
O
Cl
O
6bo
EC50 = 1.1 ± 0.4 µM
6bp
EC50 = 4.4 ± 0.8 µM
6bq
EC50 = 11.1 ± 3.2 µM
6bs
EC50 = 1.0 ± 0.1 µM
6br
EC50 = 11.5 ± 2.2 µM

11
CC50 = 198.6 ± 9.4 µM
SI50 = 181
CC50 = 146.9 ± 12.5 µM
SI50 = 33
CC50 = 160.6 ± 4.5 µM
SI50 = 15
CC50 = 170.3 ± 16.1 µM
SI50 = 15
CC50 = 50.5 ± 4.9 µM
SI50 = 51
Antiviral efficacy was determined in the CPE assay. Cytotoxicity was determined using the
neutral red method. The results are the mean ± standard deviation of three repeats. SI = selectivity
index (CC50/EC50). N.A. = not applicable.
Lead candidates with potent antiviral activity (EC50 < 1 µM) and a high selectivity index
(SI50 > 100) against EV-D68 US/KY/14-18953 virus from Table 2 were selected for further testing
against other enteroviruses, including EV-D68 US/MO/14-18947, EV-A71 Tainan/4643/98, EV-
A71 US/AK/16-19516, and CVB3 viruses. In total 13 compounds met these criteria. In parallel,
promising lead compounds were further tested for mouse microsomal stability. It was found that
all 13 compounds had potent antiviral activity against both EV-D68 strains (US/KY and US/MO)
with EC50 values ranging from single-digit to submicromolar. Encouragingly, all 13 compounds
also had potent antiviral activity against CVB3 virus, and the EC50 values were very similar to that
of EV-D68 viruses. When tested against the two EV-A71 strains, Tainan and US/AK, most of the
compounds (6aa, 6ar, 6av, 6aw, 6ax, 6ba, 6bf, 6bg, 6bi) showed potent inhibition as well with
low single-digit micromolar EC50 values. The four compounds 6aq, 6bj, 6bl and 6bo were less
potent and had high single-digit to double-digit micromolar EC50 values. Overall, the pattern of
broad-spectrum antiviral activity for compounds shown in Table 3 matches with that of known 2C
inhibitors shown in Table 1: the 2C inhibitors have potent antiviral activity against EV-D68 and
CVB3, but they are generally less active against EV-A71. Nevertheless, compounds 6ar, 6av,
6aw, 6bg, 6bi all had single-digit micromolar EC50 values against EV-A71, in addition to their
potent antiviral activity against EV-D68 and CVB3 viruses.

12
Next, selected lead compounds with potent and broad-spectrum antiviral activity against
EV-D68, EV-A71, and CVB3 were profiled for mouse microsomal stability 25, 26. Compound 6af
was included to investigate the influence of the terminal amine substitution on microsomal stability.
In general, compounds with terminal monoalkylamine and dialkylamine had longer half-lives than
compounds with trialkylamine (6af vs 6aa; 6aw, 6ax vs 6av; 6bj, 6bl vs 6bf). Installing a fluorine
at the 4-position of benzene indeed increased the microsomal stability (6aw vs 6af). Collectively,
the most promising candidate is 6aw, which not only had potent antiviral activity against EV-D68,
EV-A71 and CVB3 viruses, but also had a long half-life in mouse microsomes with T1/2 of 114.7
minutes.
Table 3. Broad-spectrum antiviral activity against EV-D68, EV-A71, CVB3, and mouse
microsomal stability.
Mouse
Anti-CVB3
in Vero C1008 cells
(µM)
Anti-EV-D68
in RD cells (µM)
Anti-EV-A71
in RD cells (µM)
microsomal
stability T1/2
(mins)
CLint(liver)
(ml/min/kg)
Structure
US/KY/14-18953
EC50 = 0.4 ± 0.2
CC50 = 73.7 ± 19.1
SI50 = 184
US/MO/14-18947
EC50 = 0.2 ± 0.07
CC50 = 73.7 ± 19.1
SI50 = 369
US/KY/14-18953
EC50 = 0.9 ± 0.3
CC50 = 47.8 ± 3.6
SI50 = 53
US/MO/14-18947
EC50 = 0.6 ± 0.3
CC50 = 47.8 ± 3.6
SI50 = 80
US/KY/14-18953
EC50 = 0.5 ± 0.3
CC50 = 65.8 ± 16.8
SI50 = 132
US/MO/14-18947
EC50 = 0.3 ± 0.1
CC50 = 65.8 ± 16.8
SI50 = 219
US/KY/14-18953
EC50 = 0.3 ± 0.2
CC50 = 80.6 ± 3.3
SI50 = 269
US/MO/14-18947
EC50 = 0.2 ± 0.1
CC50 = 80.6 ± 3.3
SI50 = 403
Tainan
EC50 = 4.2 ± 0.5
CC50 = 73.7 ± 19.1
SI50 = 18
H
O
N
N
EC50 = 0.3 ± 0.2
CC50 = 116.2 ± 56.3
SI50 = 387
22.4
60.2
N.T.
23.6
245.5
N
US/AK
EC50 > 10
6aa
Tainan
EC50 = 7.8
CC50 = 47.8 ± 3.6
SI50 = 6
H
N
O
NH
EC50 = 1.1 ± 0.2
CC50 > 30.0
SI50 > 27
91.2
N
US/AK
EC50 > 10
6af
Tainan
EC50 = 18.3 ± 4.2
CC50 = 65.8 ± 16.8
SI50 = 4
H
N
O
N
EC50 = 2.0 ± 0.2
CC50 = 185.9 ± 13.9
SI50 = 93.0
N.T.
US/AK
N
EC50 = 11.7 ± 3.0
CC50 = 65.8 ± 16.8
SI50 = 6
6aq
Tainan
H
N
O
EC50 = 5.2 ± 0.2
CC50 = 80.6 ± 3.3
SI50 = 16
N
EC50 = 0.4 ± 0.1
CC50 = 128.8 ± 25.8
SI50 = 322.0
D
D
D
232.2
N
US/AK
D
D
EC50 = 4.9 ± 0.2
CC50 = 80.6 ± 3.3
SI50 = 16
6ar

13
US/KY/14-18953
EC50 = 0.1 ± 0.1
CC50 = 36.2 ± 3.1
SI50 = 362
US/MO/14-18947
EC50 = 0.05 ± 0.02
CC50 = 36.2 ± 3.1
SI50 = 724
US/KY/14-18953
EC50 = 0.2 ± 0.1
CC50 = 32.4 ± 4.1
SI50 = 162
US/MO/14-18947
EC50 = 0.1 ± 0.02
CC50 = 32.4 ± 4.1
SI50 = 324
US/KY/14-18953
EC50 = 0.4 ± 0.1
CC50 = 48.2 ± 2.3
SI50 = 121
US/MO/14-18947
EC50 = 0.3 ± 0.2
CC50 = 48.2 ± 2.3
SI50 = 161
US/KY/14-18953
EC50 = 0.3 ± 0.2
CC50 = 36.7 ± 5.6
SI50 = 122
US/MO/14-18947
EC50 = 0.2 ± 0.1
CC50 = 36.7 ± 5.6
SI50 = 184
US/KY/14-18953
EC50 = 0.04 ± 0.02
CC50 = 42.3 ± 8.3
SI50 = 1058
US/MO/14-18947
EC50 = 0.05 ± 0.03
CC50 = 42.3 ± 8.3
SI50 = 846
US/KY/14-18953
EC50 = 0.1 ± 0.1
CC50 = 20.6 ± 0.9
SI50 = 206
US/MO/14-18947
EC50 = 0.1 ± 0.03
CC50 = 20.6 ± 0.9
SI50 = 206
US/KY/14-18953
EC50 = 0.1 ± 0.2
CC50 = 39.0 ± 3.4
SI50 = 390
US/MO/14-18947
EC50 = 0.06 ± 0.03
CC50 = 39.0 ± 3.4
SI50 = 650
Tainan
EC50 = 2.2 ± 0.1
CC50 = 36.2 ± 3.1
SI50 = 17
H
O
O
O
O
N
N
EC50 = 0.06 ± 0.04
CC50 = 48.8 ± 17.7
SI50 = 813
34.2
114.7
114.1
N.T.
26.6
160.5
US/AK
N
EC50 = 2.3 ± 0.1
CC50 = 36.2 ± 3.1
SI50 = 16
F
6av
Tainan
H
N
EC50 = 3.1 ± 1.0
CC50 = 32.4 ± 4.1
SI50 = 11
N
H
EC50 = 0.2 ± 0.2
CC50 = 40.1 ± 13.2
SI50 = 200.5
47.9
US/AK
N
EC50 = 3.6 ± 0.2
CC50 = 32.4 ± 4.1
SI50 = 9
F
6aw
Tainan
H
N
EC50 = 9.0 ± 0.3
CC50 = 48.2 ± 2.3
SI50 = 5
NH2
EC50 = 0.4 ± 0.1
CC50 = 52.3 ± 5.0
SI50 = 131
48.1
US/AK
N
EC50 = 14.0 ± 2.1
CC50 = 48.2 ± 2.3
SI50 = 3
F
6ax
H
N
Tainan
EC50 = 7.9 ± 0.5
CC50 = 36.7 ± 5.6
SI50 = 5
N
EC50 = 0.4 ± 0.2
CC50 = 92.3 ± 3.2
SI50 = 231
N.T.
206.6
N.T.
N.T.
N
US/AK
EC50 > 10
F
F
6ba
H
N
Tainan
EC50 = 2.9 ± 0.2
CC50 = 42.3 ± 8.3
SI50 = 15
O
N
EC50 = 0.07 ± 0.0 µM
CC50 = 94.5 ± 4.1
SI50 = 1350
N
S
US/AK
EC50 > 10
6bf
Tainan
EC50 = 7.9 ± 0.4
CC50 = 20.6 ± 0.9
SI50 = 3
H
N
O
N
EC50 = 0.4 ± 0.2
CC50 = 111.8 ± 7.6
SI50 = 280
N.T.
N.T.
US/AK
N
S
EC50 = 8.8 ± 0.8
CC50 = 20.6 ± 0.9
SI50 = 2
6bg
Tainan
H
N
EC50 = 6.3 ± 0.6
CC50 = 39.0 ± 3.4
SI50 = 6
O
N
EC50 = 0.2 ± 0.1
CC50 = 90.3 ± 5.9
SI50 = 452
US/AK
N
EC50 = 8.0 ± 0.5
CC50 = 39.0 ± 3.4
SI50 = 5
S
6bi
US/KY/14-18953
EC50 = 0.3 ± 0.1
CC50 = 103.1 ± 49.5
SI50 = 344
US/MO/14-18947
EC50 = 0.2 ± 0.1
CC50 = 103.1 ± 49.5
SI50 = 516
US/KY/14-18953
EC50 = 0.4 ± 0.1
CC50 = 42.1 ± 13.4
SI50 = 105
Tainan
H
N
EC50 = 8.4 ± 2.6
CC50 = 103.1 ± 49.5
SI50 = 12
O
N
H
EC50 = 0.3 ± 0.05
CC50 = 70.7 ± 4.8
SI50 = 236
75.4
72.8
54.5
US/AK
N
S
EC50 = 11.5 ± 0.8
CC50 = 103.1 ± 49.5
SI50 = 9
6bj
H
N
Tainan
O
NH2
EC50 = 38.3 ± 6.5
CC50 = 42.1 ± 13.4
SI50 = 1
EC50 = 0.6 ± 0.2
CC50 = 63.7 ± 3.2
SI50 = 106
100.8
N
US/MO/14-18947
US/AK
S

14
6bl
EC50 = 0.2 ± 0.1
CC50 = 42.1 ± 13.4
SI50 = 211
EC50 > 30
US/KY/14-18953
EC50 = 1.1 ± 0.4
CC50 = 198.6 ± 9.4
SI50 = 181
US/MO/14-18947
EC50 = 1.1 ± 0.1
CC50 = 198.6 ± 9.4
SI50 = 181
Tainan
EC50 = 30.9 ± 1.3
CC50 = 198.6 ± 9.4
SI50 = 6
H
N
O
N
EC50 = 1.2 ± 0.1
CC50 > 300.0
SI50 > 250
N.T.
N.T.
US/AK
N
EC50 = 20.9 ± 8.2
CC50 = 198.6 ± 9.4
SI50 = 10
O
6bo
Antiviral efficacy was determined in the CPE assay. Cytotoxicity was determined using the neutral
red method. The results are the mean ± standard deviation of three repeats. SI = selectivity index
(CC50/EC50). T1/2 was obtained using mouse liver microsomes. N.T. = not tested.
To provide direct evidence that the quinoline compounds target the viral 2C protein, we
expressed the CVB3 2C protein and quantified the drug binding using the differential scanning
fluorimetry (DSF), or thermal shift assay (TSA) 16, 24. In this assay, a temperature gradient is
applied to unfold a given protein, and when the protein unfolds, it exposes the hydrophobic region
that can bind to a fluorescence dye, resulting in increased fluorescence emission. When the protein
is stabilized by a small molecule binder, the melting temperature, Tm, will increase. For this
experiment, we included two known 2C inhibitors, fluoxetine and guanidine as positive controls,
and we tested the most promising lead candidate 6aw in three different concentrations. As shown
in Fig. 1, CVB3 2C protein was stabilized by both fluoxetine and guanidine with Tm shifts of 1.31
and 1.77 oC when tested at 100 µM and 1 mM, respectively (Figs. 1A&1B). Similarly, compound
6aw also increased the thermal stability of CVB3 2C in a concentration dependent manner and had
a more profound effect than fluoxetine at 100 µM (Figs. 1C&1D), which suggests that compound
6aw binds with a higher affinity than fluoxetine to 2C protein. This result is consistent with the
higher antiviral efficacy of 6aw compared to fluoxetine. Overall, the TSA assay provided direct

15
evidence that quinoline compound 6aw binds directly to the viral 2C protein and the binding
affinity is correlated with the cellular antiviral efficacy.
Figure 1. Binding of fluoxetine, guanidine, and 6aw to CVB3 2C protein using differential
scanning fluorimetry. CVB3 (4.5 µM) 2C was incubated with DMSO (black), 100 µM fluoxetine
(A), 1 mM guanidine (B), or different concentrations of 6aw (C), and the raw DSF data were
plotted and curve fitting were performed using the Boltzmann sigmoidal equation in Prism (v5)
software to determine the Tm and ΔTm (D).
Non-polio enteroviruses are significant human pathogens for which we do not have any
treatment available for now. More alarmingly, there have been an increasing trend of severe
infections including EV-A71 and EV-D68 associated neurological complications such as acute
flaccid myelitis in recently years 27, 28. Although the detailed mechanism regarding how these
contemporary viruses lead to neurological infections is still under debate 29, 30, there is nevertheless
a consensus among the scientific community that EV-A71 and EV-D68 are the etiological agents
causing the neurological complications based on clinical data as well as animal model studies 31,

16
32. As such, developing effective antivirals against these non-polio enteroviruses is a valid
approach to prevent and treat these viral infections.
In this study, we aim to develop broad-spectrum antivirals with a high selectivity index as
well as a long half-life in mouse microsomes. This is a multi-parameter optimization approach and
the focus was on balancing different properties. To achieve this goal, we adapted a stepwise
optimization strategy. Specifically, we started with structure-activity relationship studies of a
quinoline lead compound 6aa, and generated a number of candidates with potent antiviral activity
and a high selectivity index against the EV-D68 US/KY/14-18953 virus. Next, 13 prioritized lead
compounds were further profiled for broad-spectrum antiviral activity against an additional strain
of EV-D68, the EV-D68 US/MO/14-18947 virus, as well as two EV-A71 viruses (Tainan/4643/98
and US/AK/16-19516) and one CVB3 virus. In parallel, compounds with potent and broad-
spectrum antiviral activity were evaluated for mouse liver microsomal stability. Overall, this multi-
parameter optimization strategy yielded the promising candidate 6aw that not only showed potent
and broad-spectrum antiviral activity against all five strains of non-polio enteroviruses tested, but
also displayed a high microsomal stability with a T1/2 of 114.7 minutes. In summary, this study is
a step forward towards developing the urgently needed antivirals against non-polio enteroviruses,
and the results presented herein might be informative in guiding other medicinal chemistry projects,
such as optimization of microsomal stability.
ASSOCIATED CONTENT
Supporting information

17
The supporting information is available free of charge on the ACS Publications website: synthesis
procedures; characterization of compounds; antiviral and cytotoxicity assays; mouse microsomal
stability assay; thermal shift assay.
AUTHOR INFORMATION
Corresponding Author
*E-mail: junwang@pharmacy.arizona.edu. Tel: +1 (520)-626-1366.
ORCID
Jun Wang: 0000-0002-4845-4621
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. Conceived project (J. W.), performed experiments (R. M.,
N. K., Y. H.), analyzed data (R. M., N. K., Y. H., J. W.), and prepared manuscript (R. M., J. W.).
All authors reviewed the manuscript.
Funding
This research is supported by the NIH grant AI147325, and the Arizona Biomedical Research
Centre Young Investigator grant ADHS18-198859 to J.W.
ABBREVIATIONS
AFM: acute flaccid myelitis, EV-D68: enterovirus D68, EV-A71: enterovirus A71, SI:
selectivity index, TSA: thermal shift assay.

18
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24
Highlights
 Compound 6aw inhibits multiple strains of EV-D68, EV-A71 and CVB3
 Compound 6aw has a high selectivity index
 Compound 6aw is stable in mouse liver microsomes
 Compound 6aw targets viral 2C protein as confirmed by thermal shift binding assay

25
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: