Antiviral activity of isoquinolone derivatives against influenza viruses and their cytotoxicity

Article abstract of DOI:10.3390/ph14070650

Influenza viruses are one of the major causative agents for human respiratory infections. Currently, vaccines and antivirals approved for preventing and treating viral infections are available. However, limited protection efficacy and frequent emergence o

Full text of DOI:10.3390/ph14070650

pharmaceuticals
Article
Antiviral Activity of Isoquinolone Derivatives against Influenza
Viruses and Their Cytotoxicity
Yejin Jang 1,†, Jinhe Han 2,†, Xiaoli Li 2, Hyunjin Shin 1, Won-Jea Cho 2,* and Meehyein Kim 1,3,
*
1
Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT),
Daejeon 34114, Korea; erythro1@krict.re.kr (Y.J.); charliezz3656@gmail.com (H.S.)
College of Pharmacy, Chonnam National University, Gwangju 61186, Korea; hjh930116@gmail.com (J.H.);
lixiaoli0425@gmail.com (X.L.)
Graduate School of New Drug Discovery and Development, Chungnam National University,
Daejeon 34134, Korea
2
3
*
†
Correspondence: wjcho@jnu.ac.kr (W.-J.C.); mkim@krict.re.kr (M.K.)
These authors contributed equally to this work.
Abstract: Influenza viruses are one of the major causative agents for human respiratory infections.
Currently, vaccines and antivirals approved for preventing and treating viral infections are available.
However, limited protection efficacy and frequent emergence of drug-resistant viruses stand for
a need for the development of antivirals with different chemical skeletons from existing drugs.
Screening of a chemical library identified an isoquinolone compound (
concentrations (EC50s) between 0.2 and 0.6 M against the influenza A and B viruses. However, it
exhibited severe cytotoxic effects with a 50% cytotoxic concentration (CC50) of 39.0 M in canine
1) as a hit with 50% effective
µ
µ
kidney epithelial cells. To address this cytotoxic issue, we synthesized an additional 22 chemical
derivatives. Through structure-activity, as well as structure-cytotoxicity relationship studies, we
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
discovered compound 21 that has higher EC50 values ranging from 9.9 to 18.5
µM, but greatly
alleviated cytotoxicity with a CC50 value over 300 M. Mode-of-action and cell type-dependent
µ
Citation: Jang, Y.; Han, J.; Li, X.; Shin,
H.; Cho, W.-J.; Kim, M. Antiviral
Activity of Isoquinolone Derivatives
against Influenza Viruses and Their
Cytotoxicity. Pharmaceuticals 2021, 14,
650. https://doi.org/10.3390/
ph14070650
antiviral experiments indicated that it targets viral polymerase activity and functions also in human
cells. Here, we present a new class of viral polymerase inhibitors with a core skeleton of isoquinolone,
of which antiviral activity could be better improved through following design and synthesis of its
derivatives for drug development.
Keywords: antiviral; influenza virus; isoquinolone
Academic Editor: Serena Massari
Received: 8 June 2021
Accepted: 3 July 2021
Published: 6 July 2021
1. Introduction
Influenza A and B viruses, belonging to the family Orthomyxoviridae, are the main
causative agents of human infectious respiratory diseases during seasonal epidemics and
unpredictable pandemics [1]. The viral genome comprises eight negative-sense RNA
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iations.
segments that are coated with nucleoprotein (NP). The 50- and 30-termini of genomic
RNA are bound to polymerase subunits composed of polymerase basic protein 2 (PB2),
PB1, and polymerase acidic protein (PA), which are required for immediate initiation
of RNA replication and transcription in the nucleus of an infected cell [2]. Currently,
quadrivalent vaccines are available to prevent infection by H1N1 and H3N2 subtypes of
influenza A virus, as well as by both the Victoria and Yamagata lineages of influenza B
virus. However, protective efficacy of these vaccines is no greater than 50% per year on
Copyright:
© 2021 by the authors.
average [3]. Moreover, several months are required to produce a vaccine against newly
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
emerging and re-emerging viruses that are less efficiently or not neutralized by the existing
vaccines. Accordingly, as an alternative way to respond to the influenza outbreaks that lack
vaccine preparedness, antiviral treatments play a pivotal role in preventing the spread of
viral infections.
Three different classes of antivirals have been developed to treat influenza viruses;
all comprise small molecules that target viral matrix protein 2 (M2), neuraminidase (NA),
Pharmaceuticals 2021, 14, 650. https://doi.org/10.3390/ph14070650
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Pharmaceuticals 2021, 14, 650
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or viral polymerase complex [
1,4
]. Adamantanes, such as amantadine and rimantadine,
are potent inhibitors that block the M2 protein of influenza A, but not influenza B, viruses.
They are no longer recommended for clinical use due to the prevalence of drug-resistant
viruses harboring single or multiple substitutions (S31N, L26I, and/or V27A) in M2. NA
inhibitors, including oseltamivir, zanamivir, peramivir, and laninamivir, are licensed in
many countries. Of these, oseltamivir phosphate, a pro-drug of oseltamivir carboxylate, is
prescribed most globally. Recently circulating influenza A and B viruses remain sensitive
to oseltamivir. However, the possible emergence of oseltamivir-resistant or less sensitive
viruses harboring the H275Y mutation or additional substitutions in NA, as occurred in
2008, is a threat to public health through increased pathogenicity and transmissibility [5].
The last class of antivirals, favipiravir and baloxavir (formally known as baloxavir mar-
boxil), affect the viral polymerase subunits PB1 (RNA-dependent RNA polymerase) and
PA (cap-dependent endonuclease) activities, respectively [
limited approval for use in pandemic situations in Japan due to concerns about its potential
embryotoxicity [ ]. Studies of the clinical safety and efficacy of baloxavir show more
promising results. Nevertheless, they have identified amino acid substitutions (mainly I38X
in PA), which may reduce drug susceptibility [ ]. These studies suggest that the influenza
6,7]. Favipiravir has gained only
8
9
viral polymerase complex is an attractive target for drug development. However, we need
to ensure the diversity of available polymerase inhibitors if we are to overcome the issues
associated with drug resistance.
We found an isoquinolone compound (1) from a cell-based, high-throughput screening
approach that shows inhibitor activity against both the influenza A and B viruses. The
compound efficiently suppresses the viral RNA replication step, but has cytotoxic effects.
To improve this limitation, we synthesized 22 chemical derivatives and identified a less
toxic antiviral agent, compound 21. Here, we show that compound 21 could act as the
backbone for development of an antiviral drug inhibiting the influenza viral polymerase
activity without exerting cytotoxic effects.
2. Results
2.1. Identification of Compound 1 with Anti-Influenza Viral Activity
To secure compounds with anti-influenza activity, we screened a chemical library from
the Korea Chemical Bank (Daejeon, Republic of Korea), which comprised 7000 different
small molecules deposited by medicinal chemists. The fluorescent diacetate-based antiviral
assay [10] identified 3-(6-ethylbenzo[d][1,3]dioxol-5-yl)-7,8-dimethoxy-2-methylisoquinolin-
1(2H) (named compound
values ranging from 0.2 to 0.6
and A/Hong Kong/8/68 (HK)] and B/Lee/40 (Lee) viruses, and a 50% cytotoxic concen-
tration (CC50) value of 39.0 M in MDCK cells (Figure 1A,B; Table 1). Despite an excellent
selective index (S.I.) of 65, incubation for 2 days led to >40% cell death at concentrations
> 0.41 M (Figure 1B). To exclude the possibility that the antiviral efficacy was mixed with
cytotoxicity, it was needed to evaluate antiviral activity of compound under subtoxic
1) as a hit compound, with 50% effective concentration (EC50)
µM against both influenza A [A/Puerto Rico/8/34 (PR8)
µ
≥
µ
1
concentrations. To find this condition, we compared its cytotoxicity between days 1 and 2
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Figure 1C).
Consistent with the fluorescein diacetate assay (Figure 1B), the MTT assay revealed that a
2-day treatment of compound
40%, while 1-day treatment below 11.1
the basis of these subtoxic concentrations, both Western blot analysis and plaque titration
assays were performed, showing that compound suppressed influenza viral infection
1
at concentrations above 0.41
µM led to cell death more than
µM resulted in a cell viability higher than 80%. On
1
by reducing the expression of viral proteins, including NP and HA, in cells, as well as by
reducing the number of viral progeny released into the culture supernatant (Figure 1D,E).

Pharmaceuticals 2021, 14, 650
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Figure 1. Antiviral activity of compound
structure of compound 1. (B) Antiviral activity and cytotoxicity of compound 1
1
against influenza A and B viruses and its toxicity in MDCK cells. (
. MDCK cells were mock-infected (no virus)
or infected individually with A/Puerto Rico/8/34 (H1N1; PR8), A/Hong Kong/8/68 (H3N2; HK), or B/Lee/40 (Lee) in
the presence of increasing concentrations of compound . On day 2, cell viability was measured using fluorescein diacetate;
the fluorescence intensity of mock-infected DMSO-treated cells and virus-infected DMSO-treated cells was set as 100% and
0%, respectively. ( ) Comparison of the cytotoxicity of compound on different days. The percentage viability of MDCK
cells was measured in an MTT assay after treatment with increasing concentrations of compound on days 1 (black bar)
and 2 (white bar). ( ) Inhibition of viral protein expression. MDCK cells were infected with PR8 for 1 h at a multiplicity of
infection (MOI) of 0.01 and then treated with increasing concentrations of compound or 0.1 M OSV-C. On the next day,
cell lysates were harvested for Western blot analysis using anti-NP and anti-HA2 antibodies. -Actin was used as a loading
control. Each protein is denoted on the left-handed side of the blots. ( ) Plaque assay. Culture supernatants from samples
prepared in ( ) were serially diluted 10-fold and used to infect fresh MDCK cells. On day 3 post-infection, the number of
A) Chemical
1
C
1
1
D
1
µ
β
E
D
plaque-forming units (PFU) was counted by crystal violet staining. In panels (B), (C), and (E), values are expressed as the
means ± standard deviation from three different experiments. ****, p < 0.0001.
Table 1. Antiviral activity of phenylisoquinolone derivatives against influenza viruses and toxicity in MDCK cells.
EC50 (µM) b against Influenza
CC50 (µM) a
against MDCK
Cells
Substitution
Virus (S.I. c
HK e
)
Compound
1
R1
R2
R3
R4
R5
R6
R7
PR8 d
Lee f
20-Et, 40+50-
OCH2O
0.2
(195.0)
0.6
(65.0)
Me
H
H
H
OMe
OMe
39.0
0.3 (130.0)

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Table 1. Cont.
EC50 (µM) b against Influenza
CC50 (µM) a
against MDCK
Cells
Substitution
Virus (S.I. c
HK e
)
Compound
R1
R2
R3
R4
R5
R6
R7
PR8 d
Lee f
45.0
88.1
2
H
H
H
H
H
H
H
H
>300.0
>300.0
>300.0
120.4
73.4 (>4.1)
(>6.7)
(>3.4)
57.0
(>5.3)
103.4
(>2.9)
65.0
(>7.7)
3
20-OH
20-CH2OH
20-OMe
30-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
52.4
(>5.7)
17.2
(>17.4)
4
H
81.3 (>3.7)
>120.4
>120.4
(n.d.)
1.4
(86.0)
5
6
H
H
H
H
H
g
(n.d.
)
>300.0
(n.d.)
>300.0
(n.d.)
58.7
(>5.1)
H
H
NMe2
NMe2
NMe2
NMe2
NMe2
H
H
H
>300.0
>300.0
>300.0
>300.0
>300.0
>300.0
>300.0
>300.0
>300.0
>300.0
148.5
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
7
H
40-Me
H
H
H
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
8
H
40-Cl
H
H
H
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
9
H
40-Br
H
H
H
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
10
H
40-OMe
30,40-(OMe)2
20,60-Me2
30,40-(OMe)2
20,60-Me2
20-CHO
20-OH
H
H
H
32.3
(>9.3)
74.8
(>4.0)
11
H
H
Me
Me
Me
Me
H
H
94.5 (>3.2)
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
12
H
H
H
H
177.0
(>1.7)
172.5
(>1.7)
>300.0
(n.d.)
13
H
Br
Br
H
H
H
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
14
H
H
H
>300.0
(n.d.)
>300.0
(n.d.)
>300.0
(n.d.)
15
Me
Me
Me
H
H
>148.5
(n.d.)
>148.5
(n.d.)
>148.5
(n.d.)
16
CHO
H
H
H
H
20-Et, 40+50-
OCH2O
36.8
(3.0)
>109.0
(n.d.)
17
H
H
OMe
109.0
5.8 (18.8)
>300.0
(n.d.)
1.6
>300.0
(n.d.)
AMT h
RBV i
>300.0
>300.0
>300.0
(>187.5)
25.0
(>16.2)
43.8
(>19.7)
48.4
(>30.6)
0.05
(>6000)
0.03
(>10,000)
0.37
(>810.8)
OSV-C j
a
b
50% cytotoxic concentration, concentration at which cell viability was reduced by 50%; 50% effective concentration, concentration
c
that is required to improve the viability of influenza virus-infected MDCK cells by 50%; Selectivity index, the ratio of CC50/EC50
;
d
A/Puerto Rico/8/34 (H1N1); e A/Hong Kong/8/68 (H3N2); f B/Lee/40; g not determined; h amantadine hydrochloride; i ribavirin;
j
oseltamivir carboxylate.
2.2. Compound 1 Inhibits Viral Polymerase Activity
Next, we performed a time-of-addition experiment to determine the step in the in-
fluenza virus life cycle targeted by compound
exposure to compound (10
M) during viral adsorption (0–1 h) at 4 ◦C failed to induce
antiviral activity, whereas EGCG, an entry inhibitor, blocked PR8 infection completely at
the same condition (Figure 2A). However, treatment with compound starting at any time
point until 7 h post-infection consistently inhibited plaque formation by over 80%. This
suggests that compound could target virus penetration into cells or genome replication,
rather than its attachment to cellular sialic acid receptors. To further evaluate this hypothe-
1. Plaque titration assays revealed that
1
µ
1
1

Pharmaceuticals 2021, 14, 650
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sis, we compared the intracellular distribution of NP at 5 h post-infection in the presence
or absence of compound
1. Confocal microscopy showed few or no NP-positive cells when
compound was treated at concentrations of 3 or 30
1
µM (Figure 2B). To collect a more
direct evidence, we adopted a cell-based reporter assay for influenza viral RdRp activity in
0
which a negative strand of the firefly luciferase (FLuc) RNA transcript flanked with viral 5
and 30 UTRs is used as a template for synthesis of positive sense RNA, as well as mRNA,
in the presence of viral polymerase complex. To address a concern that antiviral function
observed could be induced limitedly in canine cells in a species-specific manner (Figure 1),
a human cell line, HeLa, was applied to this polymerase assay. The results of the dual
luciferase assay clarified that compound
1 suppressed viral RNA replication/transcription
in a similar way to RBV (Figure 2C). Taken together, these mode-of-antiviral action experi-
ments suggested that compound 1 works at the viral genome replication step.
Figure 2. Inhibition of viral genome replication by compound
plates were infected with PR8 (MOI, 0.01) at 4 C for 1 h. Unabsorbed virus was removed by washing with PBS. Compound
1
. (
A
) Time-of-addition experiment. MDCK cells in 48-well
◦
1
(10
µ
M) was added at different time points. EGCG (1
µ
M), which blocks virus entry, was used as a control. At 7 h
◦
post-infection, the supernatant was removed and overlay medium containing 0.6% CMC was overlaid for 3 days at 33 C.
Plaque numbers in DMSO-treated samples under each condition were set as 100%. (B) Confocal microscopy showing nuclear
localization of NP. MDCK cells were infected with PR8 at an MOI of 3 in the presence of DMSO, compound
1 (3 or 30 µM),
◦
or ribavirin (RBV; 100
µM) at 33 C for 5 h. Viral NP was visualized using an anti-NP antibody and Alexa 488-conjugated
goat anti-mouse IgG (green), while cellular nuclei were counterstained with DAPI (blue). Original magnification,
×
60.
(C
) Inhibition of viral polymerase activity. HeLa cells were transfected with plasmids expressing viral polymerase complex,
including pVP-PB2, -PB1, -PA, or -NP (200 ng in well of a 24-well plate), together with reporter plasmids pHH21-FLuc
(200 ng per well) and phRL-CMV (10 ng per well). At 6 h after transfection, cells were treated with increasing concentrations
of compound
pHH21-FLuc [normalized to Renilla luciferase (RLuc) expression from phRL-CMV]. In (
the mean ± standard deviation from three different experiments. **, p < 0.01; ****, p < 0.0001.
1
or RBV. On day 1, viral polymerase activity (%) was determined by measuring FLuc expression from
A
) and ( ), values are expressed as
C

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2.3. Synthesis of Isoquinolone Derivatives
Among 23 isoquinolone compounds,
1
and 17–19 have been synthesized according to
our established protocol (Supplementary Scheme S1) [11
22 without the methylenedioxy group at R2 were separately synthesized (Supplementary
Scheme S2) [13 16]. A newly designed synthetic method was used for phensylisoquinolone
,12]. Compounds 2, 3, 13–15 and
–
derivatives 7–10 and 21, and these compounds were prepared in a one-step cyclization
reaction using n-BuLi to link the N,N-diethyl-2-methylbenzamide and benzonitrile moieties
(Scheme 1). These products were characterized by 1H NMR (Supplementary Figures S1 to
S5). The remaining compounds,
4, 5, 6, 11, 12, 20, and 23, were also synthesized according
to Scheme 1 and our previous reports [15–19].
Scheme 1. Synthesis of phenylisoquinolones 4–12, 20, 21, and 23.
2.4. Structure-Activity Relationship: Isoquinolone Derivatives
As mentioned above, although compound
showed severe cytotoxic effects after treatment for 2 days (Figure 1B,C). Thus, we wondered
which moiety within compound was responsible for the cytotoxicity, and whether it could
1 is a highly potent antiviral agent, it
1
be improved by chemical modifications. To investigate the structure-activity relationship
(SAR), as well as the structure-cytotoxicity relationship analyses, we chemically modified
three rings (A, B, and C) of phenylisoquinolone, either independently or simultaneously
(Table 1).
Taking the simplest compound with a basic skeleton, 3-phenylisoquinolone (
sertion of substituents into Ring C revealed that a hydroxyl ( ) or hydroxymethyl group
) at the ortho position inhibited replication of the PR8, HK, and Lee influenza viral
strains, with less cytotoxicity than compound . The methoxy group ( ) suppressed the
Lee strain slightly, but was still cytotoxic. When the dimethylamino group was introduced
at R4 in phenyl Ring C, the m-methyl derivative (
) was effective against Lee, but not
against PR8 and HK. Interestingly, antiviral activity disappeared in derivatives with a
para-substitution in Ring C (e.g., p-methyl ( ), p-chloro ( ), p-bromo ( ), or p-methoxy (10
substitutions). Although none of these dimethylamino-substituted compounds showed cy-
totoxicity at the maximum concentration of 300 M, additional dimethylamino derivatives
2) in-
3
(
4
1
5
6
7
8
9
)
µ
were not considered for further modification and testing due to expectation of their loss of
antiviral activities.
To investigate the influence of substitutions at R5 on cytotoxicity and antiviral activ-
ity, a methyl group was first introduced at that position, while R2 was modified. When
dimethoxy moieties were substituted at the 30 and 40 positions of R2, the resulting com-
pound 11 was non-toxic and showed considerable antiviral activity against the PR8 strain
(S.I., >9.3), but its activity against the HK and Lee strains was weak (S.I., >3.2). Meanwhile,
as shown for compound 12, dimethyl modification at the 20 and 60 positions of the phenyl
ring led to complete loss of antiviral activity. Bromination at R3 in compounds 11 and 12

Pharmaceuticals 2021, 14, 650
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generated 13 and 14, respectively, which did not show significant antiviral activity. As
another approach, derivatives methylated at R1 but not at R5 were synthesized, resulting
0
in compounds 15
,
16, and 17. Compound 15 with a formyl group at the 2 position showed
neither antiviral activity nor severe toxicity. Even though its cytotoxicity was not fully
reflected in the CC50 calculation, approximately 30–40% of cells were dead at concentra-
tions > 100 µ
M (data not shown). Compound 16, with a hydroxyl substitution at 20 but a
formyl group at R3, was not effective at subtoxic concentrations. Compound 17, with a
methoxy group at R6, was partially active against influenza A viral strains (EC50 values
between 5.8 and 36.8
(CC50, 109.0 M) was lower than that of compound
results from active but less toxic compounds 2, 3, 4,
µ
M) but not against the influenza B virus. Notably, its cytotoxicity
(CC50, 39.0 M). Taken together, the
11, and 17 suggest that the intrinsic
µ
1
µ
antiviral activity of the hit compound 1 can be maintained with much lower cytotoxicity.
2.5. Structure-Activity Relationship: Dimethoxy Isoquinolone Derivatives
Based on previous conjecture, we asked if it is possible to discover an active poly-
merase inhibitor (S.I., >15) without cytotoxicity at 300
interesting that compound 17 exhibited considerable antiviral activity against the HK strain
(S.I., 18.8) and was less cytotoxic than compound (Table 1). From a comparison of their
µM, as observed in RBV. It was
1
chemical structures between compounds
1 and 17, we assumed that deleting the methoxy
group at R7 would be associated directly with increased cell viability while maintaining
antiviral activity. For an in depth SAR analysis, we focused on synthesizing additional
dimethoxy derivatives of phenylisoquinolone, starting from compound
ethyl group at the 20 position of R2 in compound
was replaced by ethylene (18) and hy-
droxyethyl (19) groups. As expected, compound 18 had properties very similar to those of
compound , i.e., it was active but highly toxic, with a CC50 value of 40.3 M. Surprisingly,
compound 19 was not only active against all three strains, even with relatively high EC50
values ranging from 22.2 to 59.9 M, but also highly improved the cytotoxicity profile
1 (Table 2). The
1
1
µ
µ
(CC50, >300
µ
M). A comparative SAR analysis of 19 wit0h
1 or with 18 indicated that the
magnitude of R7-OMe-derived cytotoxicity depends on 2 modification of R2. Notably, the
demethylated derivative at R1 of non-toxic 19, which generated compound 20, increased
the antiviral activity against the influenza A and B viruses by 2–3-fold. However, latent
toxicity was still detected at the highest concentration, resulting in approximately 30% cell
death at 300
µM (Figure 3A,B). It confirmed that being linked with a chemical entity at the
20 position of R2, the methoxy group at R7 could be attributed to cytotoxicity.
Table 2. Antiviral activity of dimethoxyphenylisoquinolone derivatives against influenza viruses and toxicity in MDCK cells.
EC50 (µM) b against Influenza Virus (S.I. c
)
Substitution
R5
CC50 (µM) a against
Compound
MDCK Cells
R1
R2
R6
R7
PR8 d
HK e
Lee f
20-Et,
1
Me
H
OMe
OMe
39.0
40.3
0.2 (195.0)
0.3 (130.0)
0.6 (65.0)
1.1 (36.6)
40+50-OCH2O
20-CHCH2,
18
19
Me
Me
H
H
OMe
OMe
OMe
OMe
2.8 (14.4)
1.4 (28.8)
40+50-OCH2O
20-C2H4OH,
>300.0
59.9 (>5.0)
46.2 (>6.5)
22.2 (>13.5)
40+50-OCH2O
20-C2H4OH,
20
21
H
H
H
OMe
OMe
OMe
H
>300.0
>300.0
29.8 (>10.1)
18.5 (>16.2)
11.9 (>25.2)
10.9 (>27.5)
15.6 (>19.2)
9.9 (>30.3)
40+50-OCH2O
H
OMe

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Table 2. Cont.
EC50 (µM) b against Influenza Virus (S.I. c
)
Substitution
R5
CC50 (µM) a against
Compound
R1
MDCK Cells
R2
R6
R7
PR8 d
HK e
Lee f
>300.0 (n.d.
22
H
H
20-OH
30-Me
OMe
OMe
OMe
OMe
H
H
>300.0
>300.0 (n.d.)
>300.0 (n.d.)
g
)
23
>300.0
>300.0
>300.0
>300.0 (n.d.)
>300.0 (n.d.)
25.0 (>16.2)
>300.0 (n.d.)
1.6 (>187.5)
43.8 (>19.7)
>300.0 (n.d.)
>300.0 (n.d.)
48.4 (>30.6)
AMT h
RBV i
0.03
OSV-C j
>300.0
b
0.05 (>6000)
0.37 (>810.8)
(>10,000)
a
50% cytotoxic concentration, concentration at which cell viability was reduced by 50%; 50% effective concentration, concentra-
c
tion required to improve the viability of influenza virus-infected MDCK cells by 50%; Selectivity index, the ratio of CC50/EC50
;
A/Puerto Rico/8/34 (H1N1); e A/Hong Kong/8/68 (H3N2); f B/Lee/40; g not determined; h amantadine hydrochloride; i ribavirin;
oseltamivir carboxylate.
d
j
Figure 3. Antiviral activity and cytotoxicity of compounds 20 and 21. (A,C) Chemical structure of
compounds 20 and 21. ( ) Antiviral activity and cytotoxicity of compounds 20 and 21. MDCK
B,D
cells were mock-infected (no virus) or infected individually with influenza A viruses (PR8 and HK)
or influenza B virus (Lee) in the presence of increasing concentrations of each compound. On day
2, percentage cell viability was measured using fluorescein diacetate; the fluorescence intensity of
mock-infected DMSO-treated cells and virus-infected, DMSO-treated cells was set as 100% and 0%,
respectively. (
MDCK cells were infected with PR8 at an MOI of 3 in the presence of 0.2% DMSO or compound
21 (30 or 100
M) at 33 ◦C for 5 h. Viral NP was visualized using an anti-NP antibody and Alexa
E) Confocal microscopy showing reduction of viral NP amounts by compound 21.
µ
488-conjugated goat anti-mouse IgG (green), while cellular nuclei were counterstained with DAPI
(blue). Original magnification, ×40.

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We tried to synthesize more primitive phenylisoquinolone derivatives in which all
side chains were removed except for the dimethoxyl groups at R5 and R6. Compound 21
exhibited ideal antiviral activity profiles, including a dose-dependent response, as well
as tolerable cytotoxicity (Table 2; Figure 3C,D). It was active against all three viral strains
tested, with EC50 values ranging from 9.9 to 18.5
polymerase inhibitor, ribavirin (RBV). Most importantly, it was not cytotoxic, even at the
highest concentration (300 M). We evaluated its specific antiviral activity by visualizing
µM, and with higher potency than a
µ
the reduction of viral NP expression in PR8-infected cells using fluorescence microscopy
(Figure 3E). By contrast, hydroxylation (22) or methylation (23) of the phenyl ring resulted
in complete loss of activity (Table 2). This finding was intriguing because the 20-OH of
compound
3
hardly affected antiviral activity when compared with that of compound
2
.
0
Collectively, it stresses again that the 2 position in Ring C is involved in both antiviral activ-
ity and cytotoxicity, depending on the methoxylation stoichiometry of Ring A. In summary,
the chemical modification of cytotoxic antiviral compound
1 identified seven safer (CC50,
>300 M) compounds ( 11, and 19 21) exhibiting a broad spectrum antiviral activity.
µ
2–
4
,
–
Among these, 6,7-dimethoxy-3-phenylisoquinolone (compound 21) was most potent in the
cell culture system, satisfying S.I. values over 15 against all three viral strains tested.
2.6. Anti-Influenza Viral Activity of Compound 21 in Human Cells by Suppressing Viral
Polymerase Activity
A plaque assay confirmed that compound 21 inhibited progeny virus production
into MDCK culture supernatants in a dose-dependent manner (Figure 4A). We wondered
whether its antiviral efficacy can be reproducible in human cells. Human lung epithelial
cells, A549, were infected with PR8 and treated with the compound for one day. Western
blot analysis with the cell lysates revealed an inhibition of NP expression (Figure 4B).
Furthermore, like the primary hit compound
polymerase activity in HeLa cells (Figure 4C). However, our previous time-of-addition
experiment with compound , still showing antiviral activity between 6 and 7 h post-
1 (Figure 2C), it efficiently suppressed viral
1
infection, motivatied us to investigate whether a later step of the virus life cycle can be
influenced. As NA is a representative viral protein playing a pivotal role in the virus
release, its activity was tested with a purified PR8 virus. The result showed that it had no
inhibitory effect at a maximum concentration of 30
µM, while OSV-C efficiently blocked
NA activity with a 50% inhibitory concentration (IC50) of 0.30
±
0.04 nM (Figure 4D). These
data clearly suggested that compound 21 affects viral polymerase activity, and its antiviral
activity is reliable in human cells, ensuring its potential for the development of antivirals
to treat human influenza infections through additional chemical modifications.
Figure 4. Cont.

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Figure 4. Antiviral activity of compound 21 in human cells. (A) Plaque assay. PR8-infected MDCK cells (MOI, 0.01) were
treated with compound 21 or OSV-C, as a control, for one day. Culture supernatants were serially diluted 10-fold and
used to infect fresh MDCK cells. On day 3, the number of plaques was counted by crystal violet staining. ( ) Antiviral
B
activity in A549 cells. Cells were infected with PR8 (MOI, 0.01) and treated with compound 21 or RBV, as a control. After
SDS-PAGE and electrotransfer, viral NP protein was detected with anti-NP antibody and HRP-conjugated goat anti-rabbit
IgG. β-Actin was used as a loading control. (C) Viral polymerase activity assay in HeLa cells. Cells were transfected
with plasmids expressing viral polymerases, including pVP-PB2, -PB1, -PA, or -NP (200 ng in well of a 24-well plate),
together with reporter plasmids pHH21-FLuc (200 ng per well) and phRL-CMV (10 ng per well). At 6 h after transfection,
cells were treated with increasing concentrations of compound
1 or RBV. On day 1, viral polymerase activity (%) was
determined by measuring FLuc normalized to RLuc. (
D
) NA activity assay. PR8 virus (2.8
×
103 PFU) was pre-incubated
◦
with increasing concentrations of compound 21 or OSV-C at 37 C for 20 min in 96-well plates. At 30 min after addition of
a chemiluminescent NA substrate, luminescence was measured and expressed as relative luminescence units (RLU). In
(
A
,
C
,
D
), values are expressed as the mean
±
standard deviation from three different experiments. *, p < 0.05; ***, p < 0.001;
****, p < 0.0001.
3. Discussion
Screening a chemical library identified that compound
against both influenza A and B viruses with EC50 values ranging from 0.2 to 0.6
exhibited highly selective antiviral efficacy; nevertheless, it showed severe cytotoxicity
(inducing > 40% cell death at concentrations > 0.41 M on day 2). This was improved by
reducing incubation time for 1 day, which resulted in 80% average viability at a concentra-
tion of 11.11 M. However, the problem of intrinsic cytotoxicity had to be solved to make it
1
had an inhibitory effect
M. It
µ
µ
µ
more druggable. Therefore, we synthesized additional isoquinolone derivatives. Among
the 22 modified compounds, we found that compound 21 did not affect cell viability when
incubated with cells for 2 days at the maximum tested concentration of 300
µM. Although
this modification reduced antiviral efficacy by 16.5–92.5-fold compared with compound
1,
compound 21 sufficiently suppressed influenza viral infection at subtoxic concentrations,
showing a desirable antiviral profile with high S.I. values (>15) against all three viral strains
tested (Figure 3D and Table 2). A time-of-addition study and enzymatic assay revealed
that these isoquinolone compounds target the viral RNA replication step during the virus
life cycle, not HA-mediated viral adsorption onto the cell surface or NA-mediated progeny
viral release. It was pondered when compound 21 is a direct acting polymerase inhibitor,
which viral protein among PB2, PB1 and PA could be a putative target molecule. To make
rational conjecture, an in silico docking study was performed to compare the structural
similarities and docking patterns by using the three polymerase inhibitors including VX787
(PB2 inhibitor), T705 (favipiravir, PB1 inhibitor) and Baloxavir (PA inhibitor) as controls.
Interestingly, the modeling data displayed that compound 21 could interact with PB2 in
a similar way to VX787 (Supplementary Figures S6–S8) [20
–22]. In detail, compound 21
formed stacked interactions with Phe104 and Phe23 of PB2 as observed in the binding
π-π
pattern of VX787 to PB2. It also generated a hydrogen bond to Glu61 and H2O4140, which
could increase its binding affinity to the protein. On the basis of the in silico docking
analysis, it is expected that compound 21 could inhibit the polymerase activity by binding

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to PB2. However, at present, it is not yet experimentally determined whether it directly
binds to one of the vRNP components, i.e., PB2, PB1, PA, or NP, or to cellular factors that
are participated in viral genome amplification. Particularly, in the latter case, to address
any concerns regarding the species-specific antiviral activity of the compounds which
were discovered from MDCK cell-based screening, we evaluated its antiviral efficacy and
polymerase inhibitory effect in human epithelial cells, A549 and HeLa, respectively (Figure
4B,C). The results ensured that even when it recognizes a host factor, it could not be a
matter for antiviral drug development in further extensive chemical modifications.
The structure-activity and structure-cytotoxicity relationship studies of isoquinolone
derivatives had four outcomes: (i) the simplest core skeleton, phenylisoquinolone (
and its 20-substituted derivatives (
and , but not ) are non-toxic but marginally active;
(ii) complete loss of antiviral activity is induced by dimethylamino substitutions at R4 (as
shown for and 16 18),
to 10); (iii) methylation at R1 increases cytotoxicity (as shown for
although this can be moderated by substitution with either formyl (15) or hydroxyethanol
2)
3
4
5
6
1
–
(
19) groups; and (iv) most of the compounds with mono-methoxy at R6 (17) or dimethoxy
at both R6 and R7 (18 to 20) are effective but intrinsically toxic, while R5- and R6-substituted
dimethoxy compounds are not toxic, but their antiviral activity is absolutely dependent on
chemical substituents on R2 (21 to 23). Collectively, the data presented herein show that
strategic chemical optimization of hit compound
1 generated compound 21, which may be
the basis for development of a novel therapeutic agent affecting the viral RNA replication
step of different (sub)types of influenza viruses.
4. Materials and Methods
4.1. Chemical Synthesis
Isoquinolone derivatives were synthesized as described previously [11–19]. The
structure of newly synthesized compounds 7–10 and 21 was confirmed by 300 MHz
(Varian Unity Plus 300 MHz; Varian Inc., Palo Alto, CA, USA) and 400 MHz (Bruker
Ascend 400 MHz; Bruker Daltonik, Bremen, Germany) nuclear magnetic resonance (NMR)
spectrometry. Purity was estimated to be ≥99%.
4.2. Cells, Viruses, and Antiviral Compounds
Madin-Darby canine kidney (MDCK) cells, human cervical cancer HeLa cells and hu-
man lung epithelial A549 cells were purchased from the American Type Culture Collection
◦
(ATCC, Rockville, MD, USA) and cultured at 37 C/5% CO2 in Minimum Essential Medium
(MEM; Invitrogen, Carlsbad, CA, USA), Dulbecco’s Modified Eagle’s Medium (DMEM;
Invitrogen) or Roswell Park Memorial Institute 1640 Medium (RPMI; Hyclone, Logan, UT,
USA), respectively, supplemented with 10% FBS (Invitrogen). Influenza viruses A/Puerto
Rico/8/34 (PR8), A/Hong Kong/8/68 (HK), and B/Lee/40 (Lee) were obtained from the
ATCC. The two type A influenza strains were amplified in 10-day-old embryonated chicken
eggs, while the type B strain was inoculated into MDCK cells in the presence of 2
TPCK-treated trypsin at 37 C for 3 days. The NA inhibitor, oseltamivir carboxylate (OSV-C;
µ
g/mL
◦
≥
98%), was purchased from United States Biological (Swampscott, MA, USA). The cell
entry blocker (-)-epigallocatechin-3-gallate (EGCG, 95%) and the polymerase inhibitor
≥
ribavirin (RBV) were from Sigma-Aldrich (St. Louis, MO, USA). Stocks were prepared
in water (for EGCG) or DMSO (for OSV-C and RBV); the final concentration of each was
50 mM.
4.3. Cytopathic Effect Assay
The fluorescein diacetate-based cell viability assay was performed as described pre-
viously [23]. Briefly, MDCK cells seeded on 96-well plates (3
×
104 cells per well) were
grown to confluency and then mock-infected or infected with the influenza A or B virus at
a multiplicity of infection (MOI) of 0.0001. After viral absorption for 1 h, cells were washed
with PBS and treated for 3 days at 33 ◦C with 3-fold serial dilutions of each compound
in the presence of 2 µg/mL TPCK-treated trypsin (Sigma-Aldrich). Finally, they were

Pharmaceuticals 2021, 14, 650
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◦
incubated for 15 min at 37 C with 30
µg/mL fluorescein diacetate. Cell viability was
quantified using a Spectra Max M3 plate reader (Molecular Devices, Sunnyvale, CA) at an
excitation wavelength of 485 nm and an emission wavelength of 538 nm. Both CC50 and
EC50 values were determined by comparison with cell viability values from mock-infected
and virus-infected cells, respectively, using GraphPad Prism 8.4.2 (GraphPad Software, La
Jolla, CA, USA).
4.4. Immunoassay
For Western blot analysis, MDCK or A549 cells in 6-well plates (9
were infected with the PR8 virus at an MOI of 0.01 for 1 h and then treated with increasing
concentrations of compound or OSV-C in the presence of TPCK-trypsin (2 g/mL for
MDCK cells at 33 ◦C or 0.1 g/mL for A549 cells at 35 ◦C). On the next day, cell lysates
were subjected to 10% SDS-PAGE (30 g per well), followed by electrotransfer to a PVDF
×
105 cells per well)
1
µ
µ
µ
membrane. Viral proteins NP and HA, and the cellular β-actin protein (a loading control)
were detected using specific antibodies as described previously [24].
For fluorescence microscopy, PR8 virus-infected MDCK cells (MOI, 3) seeded onto 4-
well slides (8.5
×
104 cells per well) were treated with DMSO (delivery vehicle), compound
◦
1, or RBV at 33 C for 5 h. After fixing and permeabilization, viral NP was visualized using
an anti-NP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by an
Alexa 488-conjugated goat anti-mouse IgG (Invitrogen) [10]. Cells were counterstained
with a Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame,
CA, USA). Images were captured under a Zeiss LSM 700 confocal microscope (Carl Zeiss,
Thornwood, NY, USA).
4.5. Plaque Titration
Cell culture supernatants harvested on day 1 post-viral infection in the presence
or absence of compound
1 or OSV-C were centrifuged for 5 min at 3000 g and then
serially diluted (10−1 to 10−6) in MEM. Fresh MDCK cells were seeded into 48-well plates
(
1 × 105 cells per well) and incubated with the diluted samples for 1 h at 33 ◦C. Next, the
◦
cells were washed with PBS and incubated at 33 C in the serum-free overlay medium
[MEM containing 0.6% carboxymethyl cellulose (CMC; Sigma-Aldrich)] supplemented
with 2
µg/mL TPCK-trypsin. On day 3, viral plaques were counted by staining with
crystal violet.
In a time-of-addition experiment, the PR8 virus (MOI, 0.01) was loaded onto MDCK
◦
cells cultured in 48-well plates (1.5
×
105 cells per well) for 1 h at 4 C. After washing away
◦
unabsorbed virus, the cells were incubated at 33 C for a further 7 h. In parallel with virus
infection, 10 µM compound 1 or 1 µM EGCG (as a control for entry blocking) was added
to infected cells during adsorption or at different times (0, 1, 2, 4, and 6 h) post-infection.
At 7 h post-infection, all compounds were removed by washing twice with PBS prior to
plaque titration as described above.
4.6. Viral Polymerase Assay
The influenza A viral polymerase activity assay was performed as described pre-
viously, with some modifications. Briefly, HeLa cells were seeded in 24-well plates
(
1 × 105 cells per well). On the next day, the cells were transfected with plasmids ex-
pressing viral polymerases and NP (pVP-PB2, -PB2, -PA, or NP; 200 ng of each), together
with reporter plasmids pHH21-FLuc (200 ng per well) and phRL-CMV (10 ng per well),
which transcribe the negative-sense RNA of the FLuc gene flanked by UTRs from the
influenza viral NS segment from RNA polymerase I and the RLuc mRNA from RNA poly-
merase II, respectively [10]. At 6 h post-transfection, the cells were treated with compound
1
or 21 or RBV. After 1 day, the cell lysates were harvested and tested in a dual luciferase
assay (Promega, Madison, WI, USA).

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4.7. NA Activity Assay
An NA activity assay was performed by using the NA-XTD Influenza Neuraminidase
assay kit (Applied Biosystems, Foster City, CA, USA) [25
(2.8
,
26]. The influenza A virus PR8
◦
×
103 PFU) was mixed with the same volume of compounds at 37 C for 20 min. After
an addition of NA-XTD substrate, the mixture was incubated at room temperature for
30 min. The luminescent signal was maximized by the addition of an NA-XTD accelerator
according to the manufacturer’s instructions and read using a microplate luminometer
(Centro LB960 system; Berthold Technologies, Bad Wildbad, Germany). Half maximum
inhibitory concentrations (IC50s) were determined using GraphPad Prism 8.4.2.
4.8. Statistical Analysis
Statistical analysis was performed using a two-tailed Student’s t-test. p values < 0.05
were considered significant.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/ph14070650/s1, Scheme S1: Synthesis of phenylisoquinolones
Synthesis of phenylisoquinolones 13 15 and 22; Scheme S3: Synthesis of phenylisoquinolone
16; Figure S1: 1H NMR (400 MHz, DMSO-d6) spectrum of ; Figure S2: 1H NMR (400 MHz, DMSO-
d6) spectrum of ; Figure S3: 1H NMR (400 MHz, DMSO-d6) spectrum of ; Figure S4: 1H NMR
(400 MHz, DMSO-d6) spectrum of 10; Figure S5: 1H NMR (400 MHz, DMSO-d6) spectrum of 21
1 and 17–19; Scheme S2:
2,
3,
–
7
8
9
;
Figure S6. Docking pattern comparison of VX787 (A and B) and compound 21 (C and D) to PB2;
Figure S7: Docking pattern comparison of T705 (A and B) and compound 21 (C and D) to PB1;
Figure S8. Docking pattern comparison of Baloxavir (A and B) and compound 21 (C and D) to PA.
Author Contributions: Conceptualization, M.K. and W.-J.C.; methodology, Y.J. and J.H.; formal
analysis, X.L. and H.S.; investigation, Y.J., J.H., X.L. and H.S.; writing—original draft preparation,
M.K. and W.-J.C.; writing—review and editing, H.S. and J.H.; visualization, Y.J.; supervision, M.K.
and W.-J.C.; project administration, M.K. and W.-J.C.; funding acquisition, M.K. and W.-J.C. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grants
funded by the Korea government (MSIT) [NRF-2018M3A9H4089601 to M.K., NRF-
2018M3A9H4089602 to W.-J.C.] and by an intramural fund from KRICT (Grant Number SI2132-
20 to M.K.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Material.
Acknowledgments: We would like to thank the Korea Chemical Bank for providing a chemical
library and the hit compound 1.
Conflicts of Interest: The authors declare no conflict of interest.
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