Synthesis and Broad Antiviral Activity of Novel 2-aryl-isoindolin-1-ones towards Diverse Enterovirus A71 Clinical Isolates

Article abstract of DOI:10.3390/molecules24050985

Enterovirus 71 (EV-A71) is the main causative pathogen of childhood hand, foot and mouth disease. Effective medicine is currently unavailable for the treatment of this viral disease. Using the fragment-hopping strategy, a series of 2-aryl-isoindolin-1-one compounds were designed, synthesized and investigated for their in vitro antiviral activity towards multiple EV-A71 clinical isolates (H, BrCr, Shenzhen98, Jiangsu52) in Vero cell culture in this study. The structure–activity relationship (SAR) studies identified 2-phenyl-isoindolin-1-ones as a new potent chemotype with potent antiviral activity against EV-A71. Ten out of the 24 tested compounds showed significant antiviral activity (EC₅₀ < 10 μM) towards four EV-A71 strains. Compounds A3 and A4 exhibited broad and potent antiviral activity with the 50% effective concentration (EC₅₀) values in the range of 1.23–1.76 μM. Moreover, the selectivity indices of A3 and A4 were significantly higher than those of the reference compound, pirodavir. The western blotting experiment indicated that the viral VP1 was significantly decreased at both the protein and RNA level in a dose-dependent manner following treatment with compound A3. Moreover, compound A3 inhibited the viral replication by acting on the virus entry stage. In summary, this study led to the discovery of 2-aryl-isoindolin-1-ones as a promising scaffold with potent anti-EV-A71 activities, which deserves further in-depth studies.

Full text of DOI:10.3390/molecules24050985

molecules
Article
Synthesis and Broad Antiviral Activity of Novel
2-aryl-isoindolin-1-ones towards Diverse Enterovirus
A71 Clinical Isolates
Yixuan Wang 1,†, Huiqiang Wang 1,2,†, Xinbei Jiang 1, Zhi Jiang 1, Tingting Guo 1,2, Xingyue Ji 1,
Yanping Li 1,*, Yuhuan Li 1,2,* and Zhuorong Li 1
1
CAMS Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology, Chinese Academy
of Medical Sciences and Peking Union Medical College, 1 Tiantanxili, Beijing 100050, China;
18645148632@163.com (Y.W.); wanghuiqiang@imb.pumc.edu.cn (H.W.); Jiangxinbei2011@163.com (X.J.);
jiangzhi450@163.com (Z.J.); guott2019@163.com (T.G.); jixingyue@imb.pumc.edu.cn (X.J.);
lizhuorong@imb.pumc.edu.cn (Z.L.)
2
Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of
Medical Sciences and Peking Union Medical College, 1 Tiantanxili, Beijing 100050, China
*
†
Correspondence: liyanping@imb.pumc.edu.cn (Y.L.); liyuhuan@imb.pumc.edu.cn (Y.L.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 February 2019; Accepted: 6 March 2019; Published: 11 March 2019
Abstract: Enterovirus 71 (EV-A71) is the main causative pathogen of childhood hand, foot and
mouth disease. Effective medicine is currently unavailable for the treatment of this viral disease.
Using the fragment-hopping strategy, a series of 2-aryl-isoindolin-1-one compounds were designed,
synthesized and investigated for their in vitro antiviral activity towards multiple EV-A71 clinical
isolates (H, BrCr, Shenzhen98, Jiangsu52) in Vero cell culture in this study. The structure–activity
relationship (SAR) studies identified 2-phenyl-isoindolin-1-ones as a new potent chemotype with
potent antiviral activity against EV-A71. Ten out of the 24 tested compounds showed significant
antiviral activity (EC50 < 10
broad and potent antiviral activity with the 50% effective concentration (EC50) values in the range of
1.23–1.76 M. Moreover, the selectivity indices of A3 and A4 were significantly higher than those of
µM) towards four EV-A71 strains. Compounds A3 and A4 exhibited
µ
the reference compound, pirodavir. The western blotting experiment indicated that the viral VP1 was
significantly decreased at both the protein and RNA level in a dose-dependent manner following
treatment with compound A3. Moreover, compound A3 inhibited the viral replication by acting on
the virus entry stage. In summary, this study led to the discovery of 2-aryl-isoindolin-1-ones as a
promising scaffold with potent anti-EV-A71 activities, which deserves further in-depth studies.
Keywords: 2-aryl-isoindolin-1-ones; enterovirus A71; synthesis; antiviral; structure activity
relationship
1. Introduction
Hand, foot and mouth disease (HFMD) is a seasonal epidemic infectious disease that mainly
affects children under five years old [1,2]. Its common features include symptoms of pyrexia, vomiting,
papulovesicular rash on the palms and soles, and oral ulcers in infected children. Enterovirus 71
(EV-A71), which was first isolated in 1969, is an enterovirus belonging to the Picornaradae family and
is considered to be the main pathogen involved in HFMD [
severe nervous system diseases including encephalitis, myoclonic jerks, neurogenic pulmonary edema
and even death [ ]. EV-A71 infection has been a public health problem in China since the domestic
3,4]. Moreover, EV-A71 infection can cause
2,5
outbreak in 2008, which led to nearly 490,000 reported cases [
6]. More recently, cases of infection with
Molecules 2019, 24, 985; doi:10.3390/molecules24050985
www.mdpi.com/journal/molecules

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EV-A71 have been increasing worldwide [
7–
9]. Although EV-A71 vaccines have been successfully
developed and were approved in China in 2015, a protective immunological barrier has not been
built due to low levels of vaccination in the population [10]. In addition, anti-EV-A71 drugs are
under-developed and no specific antiviral agent is available on the market to treat EV-A71 infections.
N-aryl benzamide was identified as a new broad antiviral chemotype in our previous research.
The representative compound 3-amino-N-(4-bromophenyl)-4-methoxybenzamide (Figure 1) exerts
moderate anti-EV-A71 activity against multiple virus strains [11]. However, the amide bond in N-aryl
benzamide is liable to hydrolysis which may impose on the stability and the in vivo efficacy of the
target compound. In order to mitigate this limitation, the incorporation of a cyclization constraint
within this structure afforded a new series of 2-aryl-isoindolin-1-one compounds (Figure 1). The
antiviral activity was evaluated in Vero cells infected with different EV-A71 clinical isolates. Although
2-Aryl isoindolin-1-ones with a variety of pharmacological effects, such as tumor necrosis factor
(TNF)-
α
regulation, Alzheimer’s diagnostic probe, anti-diabetes effects and the promotion of bone
14], antiviral studies based on this type of scaffold have been less
growth have been reported [12
–
frequent. Herein, we describe preliminary SAR studies toward N-aryl isoindolin-1-one as a novel
potent anti-EV-A71 chemotype. Several derivatives with favorable potency were obtained. Compounds
A3 and A4 exhibited significant antiviral activity against four tested EV-A71 clinical isolates at a low
micromole concentration. In addition, the preliminary mechanism of action studies revealed that
compound A3 played an antiviral role in the virus entry stage.
Figure 1. Structure modification strategy of anti-enterovirus 71 (EV71) compounds.
2. Results and Discussion
2.1. Chemistry
As shown in Schemes 1 and 2, different substituted iodobenzenes were chosen to react with
commercially available isoindolin-1-one (
Ullmann coupling reaction. Amides A11
in the presence of 1-Hydroxybenzotriazole (HOBt) and N,N0-diisopropylcarbodiimide (DIC) in
N,N-dimethylformide (DMF) (Scheme 2). As shown in Scheme 3, intermediates were obtained
by a similar method to that used for the synthesis of A1 A10 with or as the starting materials.
Then, and were alkylated with excessive iodopropane in the presence of anhydrous potassium
carbonate in acetonitrile to afford compounds B1 B4. Compounds and were also acylated
with propionyl chloride to quantitatively yield the target compounds C1 and C2, respectively.
1
) to yield 2-aryl isoindolin-1-ones A1–A10 by a modified
–
A13 were prepared by condensing A10 with amines
4–6
–
2
3
4
5
–
4
5
Also, compound C3 was synthesized by the propionylation of
6.
Furthermore, starting from
methyl 5-bromo-2-methylbenzoate ( ) in Scheme 4, intermediate
7
9 was subsequently obtained
through bromination and a microwave-aided cyclization. Eventually, a phenyl, phenylamino or
methylpiperazin group was introduced at the C6-position of isoindolinon by a Buchwarld or Suzuki
coupling reaction to afford the final compounds D1–D3 with a yield of less than 40%.
9

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Scheme 1. Preparation of N-substitutedphenyl-isoindolin-1-ones.
Scheme 2. Preparation of N-(4-carbamoyl)phenyl-isoindolin-1-ones.
Scheme 3. Preparation of 5 or 6-substituted N-phenyl-isoindolin-1-ones.
Scheme 4. Preparation of 6-substituted N-(4-chlorophenyl)isoindolin-1-ones.
2.2. In Vitro Antiviral Activity of N-aryl-isoindolin-1-ones against Multiple EV-A71 Clinical Isolates
An in-vitro antiviral assay was established using the Africa monkey kidney cell line Vero which
was infected with different EV-A71 clinical isolates (BrCr, H, Shenzhen98 and Jiangsu52). A total of 24
synthetic compounds were initially screened in such an assay to determine their inhibitory effect on
virus replication at 3-fold diluted concentrations serial (400~0.18
µM). The 50% effective concentration
(EC50) of each compound was calculated according to its inhibitory rate values with the Reed–Muench

Molecules 2019, 24, 985
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method. The 50% cytotoxic concentration (CC50) was defined as the concentration that inhibited 50%
cellular growth in comparison with the controls. The selectivity index (SI) was calculated as CC50/EC50.
As shown in Table 1, the reference compound pirodavir, a potent picornavirus inhibitor, displayed
potent inhibitory activity against four virus strains with EC50 values in the submicromolar range (EC50
= 0.38~0.76
compounds, 10 compounds (A1
10 M) and acceptable SI values towards four EV-A71 strains. Moreover, all EV-A71 strains showed
µM) as well as high SI values (between 88 and 44) in this test model. Among the 24 tested
–
A5 A7 B1 and D3) showed significant antiviral activity (EC50 <
,
,
4
,
6
,
µ
similar sensitivity to the tested compounds. Importantly, compounds A3 and A4 possessed superior
antiviral profiles (SI = 98–199) derived from their significantly lower cytotoxicity, although their EC50
values were about 2–4 fold higher (EC50 = 1.23~1.76 µM) than those of the reference compound,
pirodavir. Generally, the EV-A71 H strain was less sensitive to most of the active compounds as
compared to the other virus strains.
Table 1. Antiviral activity of tested compounds towards four enterovirus A71 (EV-A71) clinical isolates
in vitro.
BrCr
H
Shenzhen98
Jiangsu52
Compd.
CC50 (µM)
SI
47
67
199
140
54
/
44
/
/
/
/
SI
13
49
168
98
38
/
34
/
/
/
/
SI
47
49
199
98
49
/
44
/
/
/
/
SI
47
58
199
131
54
/
40
/
/
/
/
EC50 (µM)
EC50 (µM)
EC50 (µM)
EC50 (µM)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
4
370.14 ± 39.81
218.06 ± 22.31
257.43 ± 35.33
172.64 ± 34.05
68.39 ± 5.72
218.06 ± 22.31
318.23 ± 54.51
76.50 ± 13.10
96.47 ± 9.34
46.22
7.87 ± 0.91
3.23 ± 0.25
1.29 ± 0.24
1.23 ± 0.28
1.27 ± 0.01
NA *
7.27 ± 0.86
12.62 ± 2.22
17.00 ± 2.92
12.92 ± 2.24
NA
28.71 ± 4.07
4.43 ± 0.88
1.53 ± 0.16
1.76 ± 0.19
1.81 ± 0.21
NA
7.87 ± 0.91
4.43 ± 0.68
1.29 ± 0.24
1.76 ± 0.15
1.39 ± 0.17
NA
7.27 ± 0.86
12.95 ± 2.00
17.00 ± 2.92
14.61 ± 2.24
NA
7.87 ± 0.91
3.74 ± 0.80
1.29 ± 0.24
1.32 ± 0.22
1.27 ± 0.01
NA
7.92 ± 0.86
12.62 ± 2.22
17.00 ± 2.92
14.61 ± 2.24
NA
9.35 ± 1.44
NA
NA
13.65 ± 5.71
115.47
NA
179.56 ± 27.26
26.99 ± 3.38
/
/
24.44 ± 9.37
>22.22
/
/
30.26 ± 2.38
/
/
30.26 ± 2.38
/
/
38.49
>22.22
>22.22
>22.22
230.94
5.27 ± 0.44
9.12 ± 0.76
1.89 ± 0.32
1.59 ± 0.35
34.63 ± 6.54
18.42 ± 2.41
NA
44
17
91
43
/
13
/
26
/
/
/
/
16
88
11.66 ± 2.10
16.24 ± 1.90
2.11 ± 0.23
5.41 ± 0.63
NA
19
9
82
13
/
/
/
/
/
/
/
/
20
44
5.27 ± 0.44
9.12 ± 0.76
2.00 ± 0.25
1.59 ± 0.35
34.63 ± 6.54
18.42 ± 2.41
NA
43
17
86
43
/
13
/
26
/
/
/
/
11
67
5.74 ± 0.54
9.70 ± 0.76
1.89 ± 0.32
1.59 ± 0.43
40.28 ± 5.54
20.61 ± 1.01
NA
40
16
91
43
/
11
/
25
/
/
2
/
32
54
5
6
158.98 ± 17.10
172.64 ± 34.05
68.39 ± 5.72
257.43 ± 35.33
230.94 ± 0.00
123.89 ± 12.59
151.55 ± 27.01
230.94 ± 0.00
113.42 ± 17.24
115.47
B1
B2
B3
B4
C1
C2
C3
D1
D2
D3
NA
NA
>44.44
NA
NA
66.67
5.85 ± 0.60
5.85 ± 0.60
5.91 ± 0.65
NA
NA
NA
NA
NA
NA
NA
19.81 ± 3.40
3.73 ± 0.78
0.50 ± 0.06
NA
56.45 ± 14.45
15.12 ± 3.23
1.29 ± 0.33
0.62 ± 0.10
115.47
42.35 ± 5.47
19.56 ± 3.77
2.71 ± 0.35
0.38 ± 0.04
19.56 ± 3.77
2.09 ± 0.54
0.76 ± 0.09
pirodavir 33.33
*
NA means no antiviral activity was observed at maximum nontoxic concentration; / represents selectivity index
(SI) value of less than 10.
2.3. SAR Analysis
Most of the active compounds were 2-substitutedphenyl isoindolin-1-ones, whereas only a
minority of the active compounds contained an amino group on the isoindolin-1-one moiety.
Specifically, the replacement of phenyl (A1) with 4-halophenyl (A2–A4) increased the antiviral activity,
whereas 4-methoxyphenyl substitution (A5) did not influence much of the antiviral activity against
the four tested virus strains. Furthermore, 4-bromophenyl substitution (A4) provided comparable
activity to 4-chlorophenyl substitution (A3), both of which were more potent than 4-fluorophenyl A2
Therefore, it can be concluded that lipophilicity is more important for potency than the electronic effect.
Meanwhile, a halogen bond may exist within the interaction between compound A3 or A4 and their
target. Although 3,4-dichlorophenyl substitution (A5) showed a similar potency as 4-chlorophenyl
.
(A3), the former has a lower SI due to its increased cytotoxicity. The unfavorable meta-position
substitution was further evidenced by the increased cytotoxicity of A8 with multiple substitution as

Molecules 2019, 24, 985
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compared to A7, which has a single substituent at the para-position. The importance of lipophilicity at
the C4-position of the 2-phenyl group for antiviral activity was further confirmed by the comparison
between the halogen group (A2
trend was also observed for and
A10 was further transformed into corresponding amide derivatives (A11
–
A5) and isoquinoline (A9) or the carboxyl group (A10). The same
. The potency was decreased when the carboxylic acid group of
A13). Moreover, the potency
5
6
–
was significantly compromised and even lost when the 2-phenyl group was replaced with a bulkier
group such as biphenyl (A6). These results suggested that a steric limitation exists on the substituents
at the pare-position of the 2-phenyl ring.
Compounds of the B, C and D series as well as compounds
substitution effect on the isoindolin-1-one fragment. Firstly, the introduction of an amino group at
the C6-position of isoindolin-1-one ( ) slightly increased antiviral activity. On the contrary, the same
substitution at the C5-position ( ) decreased the activity as compared to A7. The presence of alkylamino
4–6 were synthesized to probe the
4
5
group at the C6-postion (B1) led to a dramatically increased potency as compared to B3 with the
same substitution at the C5-position. The same trend was observed for the dialkylamino substituted
compounds B2 and B4. Of note, compounds with dialkylamino substitution on the isoindolin-1-one
fragment consistently displayed weaker or even no activity compared to those with alkylamino group
substitution. In addition, the higher potency of B1 over A7 illustrated the role of NH at the C6-position
of isoindolin-1-one in antiviral activity. However, the acylation of C6-amino (C1) induced a several-fold
decrease in potency in comparison with amino alkylation (B1). Among compounds C1
–C3, only the
C6-amide compound C1 showed moderate activity, while the C5-amide compounds (C2 and C3
)
completely lost their antiviral potency. These results reiterated the importance of the substitution
position on isoindolin-1-one fragments. Encouraged by the good tolerance of 6-alkylamino substitution
for anti-EV-A71 activity, we decided to further explore modification at that position to facilitate the SAR
study. Unfortunately, the introduction of 6-phenyl (D1) resulted in a substantial loss in potency relative
to that of A3. Similarly, 6-phenylamino substitution (D2) resulted in a 10-fold loss in activity against
four in vitro models. Interestingly, 6-(4-methylpiperazin)-2-(4-chlorophenyl)-isoindolin-1-one (D3
showed comparable antiviral activity to that of A3. This is in contradiction to the former speculation
about the importance of having the NH group at the C6-postion as a hydrogen-bond donor. A possible
explanation for this disparity is that steric limitation at the C6-position plays a more crucial role in
maintaining antiviral potency than the hydrogen bond.
)
Altogether, it appears that 2-substitutedphenyl-isoindolin-1-one is an important pharmacophore
for anti-EV-A71 activity. A lipophilic group with size restriction was shown to be more favorable at the
C4-position of the phenyl ring. A small amino group at the C6-position of the isoindolin-1-one moiety
was also well tolerated.
2.4. Compound A3 Inhibits EV-A71 Replication at both the RNA and Protein Levels
To further confirm the anti-EV-A71 activity, the expression levels of VP1 RNA and protein were
determined. Vero cells were infected with the H strain of EV-A71 for 1 h followed by treatment with
A3 of various concentrations for another 24 h. As shown in Figure 2A, A3 treatment decreased the
levels of viral VP1 protein in a dose-dependent manner in vitro. Moreover, compound A3 significantly
reduced VP1 RNA expression level in a dose-dependent way in the reverse transcription-quantitative
polymerase chain reaction (RT-qPCR) assay (Figure 2B). Meanwhile, the antiviral efficacy of A3 was
also tested using viral titer reduction assays. We observed a dose-dependent reduction in viral titers
when the cells were treated with A3 after EV-A71 infection (Figure 2C). Those results convincingly
demonstrated that A3 inhibited EV-A71 replication in vitro.

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Figure 2. Compound A3 inhibited EV-A71 replication in vitro. Vero cells (9
plated into 6-well culture plates and infected with EV-A71 (multiplicity of infection, MOI = 0.1) for 1 h.
×
105 cells/well) were
The infected cells were then treated with the indicated concentrations of A3 for 24 h. Intracellular viral
VP1 protein (A) and RNA (B) were determined by Western blot and qRT-PCR assays, respectively. (C)
The inhibition of a viral titer by A3. ** p < 0.01 * p < 0.05.
2.5. Time-of-Addition Assay
In order to explore the inhibitory effect of compound A3 on the EV-A71 viral life cycle, EV-A71
viral protein VP1 was measured via Western blot when Vero cells were treated with compound A3
prior to, during, or after EV-A71 viral incubation (Figure 3). Vero cells pretreated with A3 at 24 or 1 h
prior to infection with EV-A71 did not display any resistance to infection by EV-A71. The maximum
inhibitory activity was observed when the chemical was added during the inoculation of the virus.
Meanwhile, compound A3 treatment exerted significant efficacy when it was added at 1 or 2 h after
EV-A71 infection, suggesting that A3 may act on the early stage of viral life cycle. It is likely that
compound A3 inhibits the EV-A71 replication by acting on the virus entry stage.

Molecules 2019, 24, 985
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Figure 3. Time-of-addition analysis of Vero cells with compound A3 treatment prior to, during or
after EV-A71 infection. ( ) Schematic illustration of experiment to determine which stage of EV-A71
viral cycle was inhibited by compound A3 in Vero cells. ( ) EV-A71 VP1 expression in Vero cells was
significantly decreased when compound A3 (4 M) was administered at 0, 1 and 2 h after EV-A71
A
B
µ
infection (MOI = 1.0) but not at the other time periods. For all experiments, the collected cells were
washed with PBS after 10 h post viral infection, and viral VP1 expression was determined via Western
blot assay.
3. Materials and Methods
3.1. Chemistry
All reagents and solvents used are commercially available and were used without further
1
purification. H-NMR spectra were recorded in CDCl3 or DMSO-d6 on a Bruker Avance III 400MHz or
a WNMR-1 500 MHz spectrometer (Wuhan Zhongke-Niujin, Wuhan, China). The chemical shift was
reported in parts per million, relative to tetramethylsilane as the internal standard. High Resolution
Mass Spectrum (HRMS) was recorded on a Triple TOF 5600+LC/MS/MS system (CADM-YQ-086)
with an Electrospray Ionization (ESI) mass selective detector. The reaction was monitored through TLC
Silica gel 60F254 Aluminium plate (Merck, 60F254D, Darmstadt, Germany). Flash chromatography
was performed on a CombiflashRf 200 (Teledyne, Lincoln, NE, USA) with gel silica column.
General Procedure A for the Synthesis of Compounds A1~A12. Isoindolin-1-one (200 mg, 1.50
mmol) was dissolved in super-dry DMSO (4 mL), and Cs2CO3 (1215 mg, 3.75 mmol), CuI (58 mg, 0.30
mmol) and N1,N2-dimethylethane-1,2-diamine (27 mg, 33
µL, 0.30 mmol) were added to the solution.
The resulting mixture was stirred at room temperature for 10 min, after which iodobenzene (2.25
mmol) was added. Then the mixture was heated to 120 ◦C. When TLC showed that isoindolin-1-one
had been fully converted, the reaction was stopped. The mixture was extracted with ethyl acetate (20
mL) and H2O (10 mL). The water phase was re-extracted with ethyl acetate (20 mL). The organic layer
was combined and washed with brine (10 mL). Then the solution was dried over anhydrous MgSO4,
filtered and concentrated, and the crude residue was purified by flash chromatography over silica gel
using CH2Cl2/CH3OH as the gradient elution to afford the title compounds.
22-phenylisoindolin-1-one (A1). Compound A1 was synthesized from isoindolin-1-one and iodobenzene
1
using general procedure A as a white solid. The yield was 50%. H-NMR (500 MHz, Chloroform-d)
δ
7.98 (d, J = 7.4 Hz, 1H), 7.92 (d, J = 7.8 Hz, 2H), 7.64 (t, J = 7.5 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.49 (d, J
= 8.0 Hz, 2H), 7.22 (d, J = 7.3 Hz, 1H), 4.91 (s, 2H). HRMS(ESI+) m/z calcd for C14H12NO [M + H]+
210.0919, found 210.0921.

Molecules 2019, 24, 985
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2-(4-fluorophenyl)isoindolin-1-one (A2). Compound A2 was synthesized from isoindolin-1-one and
1
1-fluoro-4-iodobenzene using general procedure A as a yellow solid. The yield was 32%. H-NMR
(500 MHz, DMSO-d6)
δ 7.98 (dd, J = 8.8, 4.8 Hz, 2H), 7.84 (d, J = 7.5 Hz, 1H), 7.73 (s, 2H), 7.61 (d, J = 7.4
Hz, 1H), 7.35 (t, J = 8.7 Hz, 2H), 5.08 (s, 2H). HRMS(ESI+) m/z calcd for C14H11FNO [M + H]+ 228.0825,
found 228.0827.
2-(4-chlorophenyl)isoindolin-1-one (A3). Compound A3 was synthesized from isolindolin-1-one and
1
1-chloro-4-iodobenzene using general procedure A as a white solid. The yield was 49%. H-NMR (500
MHz, Chloroform-d)
δ 7.97 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.57 (d, J
= 7.1 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 4.89 (s, 2H). HRMS(ESI+) m/z calcd for C14H11NOCl [M + H]+
244.0529, found 244.0522.
2-(4-bromophenyl)isoindolin-1-one (A4). Compound A4 was synthesized from isoindolin-1-one and
1
1-bromo-4-iodobenzene using general procedure A as a brown solid. The yield was 44%. H-NMR
(500 MHz, DMSO-d6)
δ 7.93 (d, J = 8.6 Hz, 2H), 7.83 (d, J = 7.6 Hz, 1H), 7.75–7.62 (m, 4H), 7.58 (t, J = 7.3
Hz, 1H), 5.06 (s, 2H). HRMS(ESI+) m/z calcd for C14H11BrNO [M + H]+ 288.0024, found 288.0026.
2-(2,4-dichlorophenyl)isoindolin-1-one (A5). Compound A5 was synthesized from isolindolin-1-one and
1
2,4-dichloro-1-iodobenzene using general procedure A as a white solid. The yield was 48%. H-NMR
(500 MHz, Chloroform-d) δ 8.01 (d, J = 7.6 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.58 (dd, J = 12.0, 5.9 Hz,
3H), 7.43 (t, J = 6.5 Hz, 2H), 4.85 (s, 2H). HRMS(ESI+) m/z calcd for C14H10NOCl2 [M + H]+ 278.0139,
found 278.0132.
2-([1,1’-biphenyl]-4-yl)isoindolin-1-one (A6). Compound A6 was synthesized from isoindolin-1-one and
1
4-iodo-1,1’-biphenyl using general procedure A as a yellow solid. The yield was 26%. H-NMR (500
MHz, DMSO-d6) δ 8.05 (d, J = 8.3 Hz, 2H), 7.84 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.73 (t, J = 5.6
Hz, 4H), 7.60 (d, J = 7.1 Hz, 1H), 7.51 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.7 Hz, 1H), 5.12 (s, 2H). HRMS(ESI+)
m/z calcd for C20H16NO [M + H]+ 286.1232, found 286.1220.
2-(4-methoxyphenyl)isoindolin-1-one (A7). Compound A7 was synthesized from isolindolin-1-one and
1
1-iodo-4-methoxybenzene using general procedure A as a white solid. The yield was 53%. H-NMR
(500 MHz, Chloroform-d)
δ 7.96 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.54
(s, 2H), 7.01 (d, J = 8.5 Hz, 2H), 4.85 (s, 2H), 3.87 (s, 3H). HRMS(ESI+) m/z calcd for C15H14NO2 [M +
H]+ 240.1025, found 240.1020.
2-(3,4,5-trimethoxyphenyl)isoindolin-1-one (A8). Compound A8 was synthesized from isoindolin-1-one
and 3,4,5-trimethoxy-1-iodobenzene using general procedure A as a yellow solid. The yield was 37%.
1H-NMR (500 MHz, DMSO-d6)
δ 7.77 (d, J = 7.6 Hz, 1H), 7.69 (dd, J = 6.7, 1.0 Hz, 2H), 7.60–7.51 (m,
1H), 7.28 (s, 2H), 5.05 (s, 2H), 3.83 (s, 6H), 3.67 (s, 3H). HRMS(ESI+) m/z calcd for C17H18NO4 [M + H]+
300.1236, found 300.1222.
2-(isoquinolin-7-yl)isoindolin-1-one (A9). Compound A9 was synthesized from isoindolin-1-one and
1
7-iodoisoquinoline using general procedure A as a yellow solid. The yield was 48%. H-NMR (500
MHz, Chloroform-d)
δ 9.34 (s, 1H), 8.56 (d, J = 9.0 Hz, 2H), 8.32 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.94 (d,
J = 9.1 Hz, 1H), 7.69 (s, 2H), 7.66–7.50 (m, 2H), 5.05 (s, 2H). HRMS(ESI+) m/z calcd for C17H13N2O [M
+ H]+ 261.1028, found 261.1034.
4-(1-oxoisoindolin-2-yl)benzoic acid (A10). Compound A10 was synthesized from isoindolin-1-one and
1
4-iodobenzoic acid using general procedure A as a white solid. The yield was 31%. H-NMR (500
MHz, DMSO-d6)
δ 12.87 (s, 1H), 8.11–8.05 (m, 1H), 8.02 (dd, J = 9.1, 2.2 Hz, 1H), 7.83 (d, J = 7.6 Hz,
1H), 7.75–7.67 (m, 1H), 7.59–7.54 (m, 0H), 5.09 (s, 1H). HRMS(ESI+) m/z calcd for C15H12NO3 [M + H]+
254.0817, found 254.0805
General Procedure for the Synthesis of Compounds A11
mmol) was dissolved in DMF (3 mL). HOBt (69 mg, 0.4 mmol) and DIC (80
–
A13. Compound A10 (100 mg, 0.3
L, 0.4 mmol) were added
µ

Molecules 2019, 24, 985
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to the solution and the resulting solution was stirred for 20 min at room temperature. Amine was
added into the resulting mixture and stirred at room temperature overnight. When TLC showed
that the reaction was complete, the mixture was extracted with EA and H2O. The water phase was
re-extracted with EA. The organic layer was combined and washed with brine (10 mL). Then the
solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude residue was
purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient elution to
afford the title compounds.
2-(4-(4-methylpiperazine-1-carbonyl)phenyl)isoindolin-1-one (A11). Compound A11 was prepared from
1
A10 and 1-methypiperazine using the above procedure as a white solid. The yield was 61%. H-NMR
(500 MHz, DMSO-d6)
δ 8.03–7.98 (m, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.74–7.67 (m, 2H), 7.59–7.54 (m, 1H),
7.52–7.47 (m, 2H), 5.07 (s, 2H), 3.45 (dd, J = 59.5, 52.5 Hz, 4H), 2.35 (d, J = 16.8 Hz, 4H), 2.20 (s, 3H).
HRMS(ESI+) m/z calcd for C20H22N3O2 [M + H]+ 336.1712, found 336.1697.
2-(4-(morpholin-4-carbonyl)phenyl)isoindolin-1-one (A12). Compound A12 was prepared from A10 and
morpholine using the above procedure as a white solid. The yield was 65%. 1H-NMR (500 MHz,
DMSO-d6)
δ 8.01 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.76–7.65 (m, 2H), 7.61–7.47 (m, 3H), 5.08
(s, 2H), 3.62 (s, 8H). HRMS(ESI+) m/z calcd for C19H19N2O3 [M + H]+ 323.1396, found 323.1383.
N-(3-(dimethylamino)propyl)-N-methyl-4-(1-oxoisoindolin-2-yl)benzamide (A13). Compound A13 was
prepared from A10 and 3-dimethylamino-propylamine using the above procedure as a shallow yellow
1
solid. The yield was 40%. H-NMR (500 MHz, DMSO-d6)
δ 8.54 (t, J = 5.5 Hz, 1H), 8.02 (d, J = 8.9 Hz,
2H), 7.93 (d, J = 8.9 Hz, 2H), 7.81 (d, J = 7.6 Hz, 1H), 7.76–7.65 (m, 2H), 7.61–7.52 (m, 1H), 5.08 (s, 2H),
3.30 (dd, J = 12.8, 6.8 Hz, 3H), 2.41 (t, J = 7.1 Hz, 2H), 2.25 (s, 6H), 1.75–1.63 (m, 2H). HRMS(ESI+) m/z
calcd for C20H24N3O2 [M + H]+ 338.1869, found 338.1852.
6-amino-2-(4-methoxyphenyl)isoindolin-1-one (4). Compound 4 was synthesized from 6-aminoisoindolin-
1-one and 1-iodo-4-methoxybenzene using general procedure A as a white solid. The yield was
2
1
56.97%. H-NMR (400 MHz, DMSO-d6)
δ 7.80–7.73 (m, 2H), 7.26 (d, J = 8.1 Hz, 1H), 7.03–6.95 (m,
2H), 6.93–6.82 (m, 2H), 5.38 (s, 2H), 4.77 (s, 2H), 3.77 (s, 3H), 2.55 (s, 7H). HRMS(ESI+) m/z calcd for
C15H15N2O2 [M + H]+ 255.1134, found 255.1140.
5-amino-2-(4-methoxyphenyl)isoindolin-1-one (5). Compound 5 was synthesized from 5-aminoisolindolin-
1-one and 1-iodo-4-methoxybenzene using general procedure A as a yellow solid. The yield was
3
1
51.54%. H-NMR (500 MHz, DMSO-d6)
δ 7.77 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H), 6.99 (d, J
= 8.5 Hz, 2H), 6.87–6.50 (m, 2H), 5.92 (s, 2H), 4.78 (s, 2H), 3.78 (s, 3H). HRMS(ESI+) m/z calcd for
C15H15N2O2 [M + H]+ 255.1134, found 255.1124.
5-amino-2-(p-tolyl)isoindolin-1-one (
and 4-iodotoluene using general procedure A as a yellow solid. The yield was 45.69%. H-NMR (400
6
). Compound
6
was synthesized from 5-aminoisoindolin-1-one
3
1
MHz, DMSO-d6)
δ 7.74 (dd, J = 8.9, 2.4 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.26–7.12 (m, 2H), 6.65 (dd, J =
10.2, 2.1 Hz, 2H), 5.93 (s, 2H), 4.77 (s, 2H), 2.29 (s, 3H). HRMS(ESI+) m/z calcd for C15H15N2O [M +
H]+ 239.1184, found 239.1185.
General Procedure B for the Synthesis of Compounds B1
isoindolin-1-one (300 mg, 1.18 mmol) was dissolved in superdry CH3CN (5 mL), and
K2CO3 (449 mg, 3.54 mmol) was added to the solution. The resulting mixture was stirred
at room temperature, after which 1-iodopropane (2.25 mmol) was added. When TLC
–B4. 6-amino-2-(4-methoxyphenyl)
4
showed that 6-amino-2-(4-methoxyphenyl)isoindolin-1-one was fully converted, the reaction was
stopped. The mixture was filtered and the filtrate was evaporated in vacuo. The residue
was dissolved in CH2Cl2 and then washed with H2O. The water phase was re-extracted
with CH2Cl2. The organic layer was combined and washed with brine (10 mL). Then
the solution was dried over anhydrous MgSO4, filtered and concentrated, and the crude
residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as

Molecules 2019, 24, 985
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the gradient elution to afford 2-(4-methoxyphenyl)-6-(propylamino)isoindolin-1-one (B1) and
6-(dipropylamino)-2-(4-methoxyphenyl)isoindolin-1-one (B2) as a yellow solid with yields of 32% and
26% for B1 and B2, respectively.
Compound B1 1H-NMR (500 MHz, Chloroform-d)
δ 7.75 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 7.4 Hz, 2H),
7.22 (s, 1H), 7.00 (d, J = 8.5 Hz, 2H), 4.77 (s, 2H), 3.88 (s, 3H), 3.25 (t, J = 7.7 Hz, 2H), 1.81 (q, J = 7.6
Hz, 2H), 1.07 (t, J = 7.4 Hz, 3H). HRMS(ESI+) m/z calcd for C18H21N2O2 [M + H]+ 297.1603, found
297.1611.
Compound B2 1H-NMR (500 MHz, Chloroform-d)
δ 7.77 (d, J = 8.5 Hz, 2H), 7.16 (s, 1H), 7.01 (d, J = 8.6
Hz, 2H), 6.90 (s, 1H), 4.87–4.66 (m, 2H), 3.87 (s, 3H), 3.35 (s, 4H), 1.68 (s, 4H), 0.98 (t, J = 7.4 Hz, 6H).
HRMS(ESI+) m/z calcd for C21H27N2O2 [M + H]+ 339.2073, found 339.2055.
2-(4-methoxyphenyl)-5-(propylamino)isoindolin-1-one (B3) and 5-(dipropylamino)-2-(4-methoxyphenyl)
isoindolin-1-one (B4) were synthesized from 5-amino-2-(4-methoxyphenyl)isoindolin-1-one (5) and
1-iodopropane using general procedure B, which were both yellow solids. The yields were 34% and
28% for B3 and B4, respectively.
Compound B3 1H-NMR (500 MHz, Chloroform-d)
δ 7.74 (dd, J = 14.5, 8.5 Hz, 3H), 6.99 (d, J = 8.6 Hz,
2H), 6.71 (d, J = 24.0 Hz, 2H), 4.75 (s, 2H), 3.87 (s, 3H), 3.21 (t, J = 7.1 Hz, 2H), 1.75 (q, J = 7.1 Hz, 2H),
1.07 (t, J = 7.4 Hz, 3H).
Compound B4 1H-NMR (500 MHz, Chloroform-d)
δ 7.75 (dd, J = 13.3, 8.8 Hz, 3H), 6.99 (d, J = 8.8 Hz,
2H), 6.75 (d, J = 8.6 Hz, 1H), 6.66 (s, 1H), 4.76 (s, 2H), 3.87 (s, 3H), 3.37 (t, J = 7.8 Hz, 4H), 1.71 (h, J = 7.5
Hz, 4H), 1.01 (t, J = 7.4 Hz, 6H). HRMS(ESI+) m/z calcd for C21H27N2O2 [M + H]+ 339.2073, found
339.2076.
General Procedure C for the Synthesis of Compounds C1
–
C3. 5-amino-2-(p-tolyl)isoindolin-1-one
6
(70 mg, 0.29 mmol) was dissolved in CH2Cl2 (3 mL), and DIPEA (154
µL, 0.87 mmol) was added to the
solution. Propionyl chloride was dissolved in CH2Cl2 (1 mL) and transferred to a drip funnel, and
dropped into the flask, at a rate of about 1–2 drop(s) per second. After completion of the addition, the
mixture was stirred at room temperature. When TLC showed that 6-amino-2-(p-tolyl)isoindolin-1-one
had been fully converted, the reaction was stopped. The mixture was extracted with H2O (3 mL). The
water phase was re-extracted with CH2Cl2 (10 mL). The organic layer was combined and washed
with brine (10 mL). Then the solution was dried over anhydrous MgSO4, filtered and concentrated,
and the crude residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH as
the gradient elution to afford compound N-(1-oxo-2-(p-tolyl)isoindolin-5-yl)propionamide (C3) as a
1
yellow solid. The yield was 46%. H-NMR (500 MHz, DMSO-d6)
δ 10.27 (s, 1H), 8.07 (s, 1H), 7.80 (d, J
= 7.9 Hz, 2H), 7.71 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 4.97 (s, 2H), 2.41
(q, J = 7.5 Hz, 2H), 2.33 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H). HRMS(ESI+) m/z calcd for C18H19N2O2 [M +
H]+ 295.1447, found 295.1433.
N-(2-(4-methoxyphenyl)-3-oxoisoindolin-5-yl)propionamide (C1). Compound C1 was synthesized from
6-amino-2-(4-methoxyphenyl)isoindolin-1-one (
as a yellow solid. The yield was 49%. H-NMR (500 MHz, Chloroform-d)
4
) and propionyl chloride using general procedure C
1
δ
8.22 (s, 1H), 7.85 (d, J = 8.1
Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.63 (s, 1H), 7.00 (d, J = 8.5 Hz, 2H), 4.83 (s, 2H), 3.87 (s, 3H), 2.77–2.30
(m, 2H), 1.31 (t, J = 7.5 Hz, 3H). HRMS(ESI+) calcd for C18H19N2O3 [M + H]+ 311.1396, found 311.1386.
N-(2-(4-methoxyphenyl)-1-oxoisoindolin-5-yl)propionamide (C2). Compound C2 was synthesized from
5-amino-2-(4-methoxyphenyl)isoindolin-1-one (
as a yellow solid. The yield was 50%. H-NMR (400 MHz, DMSO-d6)
5
) and propionyl chloride using general procedure C
1
δ
10.24 (s, 1H), 8.04 (d, J = 1.6 Hz,
1H), 7.82–7.75 (m, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.61 (dd, J = 8.3, 1.7 Hz, 1H), 7.04–6.97 (m, 2H), 4.94 (s,
2H), 3.77 (s, 3H), 2.39 (q, J = 7.5 Hz, 2H), 1.11 (t, J = 7.5 Hz, 3H). HRMS(ESI+) calcd for C18H19N2O3 [M
+ H]+ 311.1396, found 311.1382.

Molecules 2019, 24, 985
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Procedure D for the synthesis of compounds D1
–
D3. Methyl 5-bromo-2-methylbenzoate (2.29
g, 10 mmol) was added to a flask containing 20 mL CCl4, and then N-bromosuccinimide (1.78
g, 10 mmol) and TsOH (172 mg, 1 mmol) were added. The mixture was refluxed until methyl
5-bromo-2-methylbenzoate was fully converted. The solution was washed with saturated NaHCO3
solution, water and brine successively. The organic phase was evaporated under vacuum to afford the
crude product methyl 5-bromo-2-(bromomethyl)benzoate (9).
A 10 mL microwave vial was charged with 4-chloroaniline (638 mg, 5 mmol), methyl
5-bromo-2-(bromomethyl)benzoate (1.57 g, 5.5 mmol), and DMF (4 mL). The vial was sealed and
◦
microwaved at 110 C for 25 min. The reaction mixture was diluted with MeOH (50 mL) and an
off-white precipitate was formed, which was filtered and dried under vacuum to give 10 (1.19 g,
1
73.78%) as a white solid. H-NMR (500 MHz, DMSO-d6)
δ
7.96 (d, J = 8.9 Hz, 3H), 7.90 (d, J = 8.1 Hz,
1H), 7.68 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 5.03 (s, 2H).
A microwave vial was charged with 10 (97 mg, 0.30 mmol), phenylboronic acid (41 mg, 0.33 mmol),
NaOBu-t (58 mg, 0.60 mmol), DMSO (2 mL), and H2O (0.3 mL). The solution was degassed with
nitrogen for 10 min, and Pd(dppf)Cl2 (12 mg, 0.015 mmol) was added. The vial was sealed and
microwaved at 130 ◦C for 2 h. The reaction mixture was poured into EtOAc/H2O and the organic
layer was separated and subsequently passed through a plug of florisil. The obtained organic phase
was concentrated into a residue, which was triturated with MeOH, filtered, and dried under vacuum
1
to afford D1 (43 mg, 44.82%) as a white solid. H-NMR (500 MHz, DMSO-d6)
(d, J = 7.3 Hz, 3H), 7.58–7.50 (m, 4H), 7.45 (t, J = 7.3 Hz, 1H), 5.11 (s, 2H).
δ 8.05–7.98 (m, 4H), 7.80
Compound D2 was synthesized using 10 and aniline using the same method of synthesis as that of D1
D2 was purified by flash chromatography to afford a yellow solid. The yield was 20%. H-NMR (400
.
1
MHz, DMSO-d6)
δ 8.46 (s, 1H), 8.00–7.89 (m, 2H), 7.51 (dd, J = 8.5, 4.1 Hz, 3H), 7.45–7.24 (m, 4H), 7.14
(d, J = 7.9 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 4.93 (s, 2H).
Compound D3 was synthesized using 10 and 4-methylpiperazine using the same method of synthesis
as that of D1
22%. H-NMR (500 MHz, DMSO-d6)
.
D3 was purified by flash chromatography to afford a pale yellow solid. The yield was
7.96 (d, J = 8.9 Hz, 3H), 7.90 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.1
1
δ
Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 5.03 (s, 2H).
3.2. Cells and Viruses
African green monkey kidney (Vero) cells were purchased from the American Type Culture
Collection (ATCC) and were cultured in minimum essential medium (MEM) supplemented with 10%
fetal bovine serum (FBS) (GIBCO) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin)
at 37 ◦C in a 5% CO2 incubator.
EV-A71 strain ShenZhen98 (SZ98) was isolated from the throat swab sample of an HFMD case
which occurred in 1998 in China. This was kindly provided by Dr. Qi Jin, Institute of Pathogen Biology,
Chinese Academy of Medical Science and Peking Union Medical School, Beijing, China. EV-A71 strains
BrCr (VR-1775) and H (VR-1432) were purchased from the ATCC. The EV-A71 strain JS52 was a kind
gift from Dr. Xiangzhong Ye, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. EV-A71 were all
passaged in Vero cells.
3.3. Cytotoxicity Assay
The cytotoxic effects of compounds on Vero cells were detected by the cytopathic effect (CPE)
assay. Briefly, cells (3
×
104 cells/well) were seeded into 96-well culture plates and incubated overnight.
Then, the medium was removed and different concentrations of compounds were applied in triplicate.
After 3 days of incubation, the CC50 was defined as the concentration that inhibited 50% cellular
growth in comparison with the untreated controls, as calculated by the Reed–Muench method.

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3.4. CPE Inhibition Assay for Anti-EV-A71
The anti-EV-A71 activity of compounds was determined by the CPE inhibition method. Briefly,
cells (3
104 cells/well) were plated into 96-well culture plates for incubation for 24 h. The medium
×
was then removed and cells were infected with EV-A71 with 100TCID50 (50% tissue culture infective
doses) in serum-free medium for 1 h at 37 ◦C. Then, the unbound viruses were removed and various
concentrations of compounds were supplemented and incubated for another 48 h. The EC50 was
determined by the Reed–Muench method. The selectivity index was calculated as the ratio of
CC50/EC50 [15].
3.5. Time-of-Addition Assay
The antiviral activity of the tested compound was examined at different time periods prior to,
during or after viral infection. Briefly, Vero cells (9
(MOI = 1.0) at 37 ◦C for 1 h. The media with or without treatment were added during the periods of
24~0 h, 1~0 h, 0~10 h, 1~10 h, 2~10 h, 4~10 h, 6~10 h and 8~10 h. After each incubation period, the
×
105 cells/well) were inoculated with EV-A71
−
−
collected cells were washed with PBS, and then the viral protein was determined by Western blot.
3.6. Western Blot Analysis
Vero cells (9
×
105 cells/well) were plated into 6-well culture plates for incubation for 16
h. The medium was removed and cells were infected with EV-A71 (H, MOI = 0.1). After 1 h,
various concentrations of A3 were supplemented for incubation for another 24 h. The cells were
lysed in the M-PER mammalian protein extraction reagent (Thermo, Rockford, IL, USA) containing
halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined
by the BCA reagents (Thermo). About 15 µg of proteins was denatured and applied to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis products were
transferred to a polyvinyl idenefluoride (PVDF) film and PVDF membranes were then incubated
at room temperature with a specific primary antibody. After a standard washing, membranes were
incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The assay was developed
using a chemiluminescent substrate. The primary antibodies used in this study included antibodies
against β-actin (Cell Signaling Technology), and EV-A71-VP1 (Abnova). Goat anti-mouse HRP-labeled
antibodies were obtained from Cell Signaling Technology [15].
3.7. Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) Quantification
Vero cells (9
×
105cells/well) were plated into 6-well culture plates for incubation for 16 h.
The medium was removed and cells were infected with EV-A71 (H, MOI = 0.1). After 1 h, various
concentrations of A3 were supplemented and incubated for another 24 h. The total RNA of the infected
cells was extracted using the RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions.
The one-step qRT-PCR was performed with SuperScript III Platinum SYBR Green One-step RT PCR
Kit (Invitrogen) using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). The RNA
expression of EV-A71 VP1 was detected with the sense primer 50-GATATCCCACATTCGGTGA-30
and the antisense primer 50-TAGGACACGCTCCATACTCAAG -30 targeting a conserved region of
the VP1 gene.
CTGTGCCCATCTA-30 and the antisense primer 50-CTAGAAGCATTTG CGGTGGACG ATG-30. The
PCR assay was carried out in a 25 L volume and the target fragment amplification was carried out as
follows: reverse transcription at 50 ◦C for 3 min; initial activation of HotStar Taq DNA Polymerase
β
-Actin mRNA was detected using the sense primers 50-TGACGGGGT CACCCACA
µ
◦
◦
◦
at 95 C for 10 min; 40 cycles in two steps: 95 C for 15 s, 60 C for 30 s. The relative amounts of
EV-A71 VP1 mRNA were calculated by the comparative Ct method after normalizing the quantity of
β-actin [15].

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3.8. Statistical Analysis
Data are expressed as the mean
ANOVA using MTLAB software (8.6, MathWorks, 2015, Natick, MA, USA). A threshold of p < 0.05
±
standard error of the mean and were analyzed with one-way
was defined as statistically significant.
4. Conclusions
In this study, twenty-four 2-aryl-isoindolin-1-one analogs were designed and synthesized using
the fragment-hopping strategy. Anti-EV-A71 activity was evaluated in Vero cell cultures infected by
different EV-A71 clinical isolates. Ten of the 2-aryl-isoindolin-1-ones showed broad and significant
antiviral activity at the submicromolar concentration level. 2-(4-Halophenyl)-isoindolin-1-ones
(A3 and A4) exerted more favorable antiviral profiles (SI = 98–199), which is attributed to their
significantly lower cytotoxicity as compared to the reference compound pirodavir in four virus strain
tests. Preliminary SAR studies suggested that 2-substitutedphenyl-isoindolin-1-one is an important
pharmacophore for anti-EV-A71 activity. The small amino group on the isoindolin-1-one moiety
was shown to be acceptable for antiviral potency. Lipophilic substitution with size restriction at the
para-position of the phenyl group might be beneficial for maintaining antiviral activity. Although the
EV-A71 H strain presented a weaker sensitivity to this class of compounds than the other strains, such
as BrCr, Shenzhen98 and Jiangsu52, in this study, A3 treatment significantly decreased the viral VP1
expression at both the protein and RNA levels in a dose-dependent manner. Although the mechanism
of action of this class of compounds is still undetermined, the time-of-addition experiment indicated
that compound A3 played a role in the early stage of EV-A71 viral cycle, and presumably acts on the
virus entry stage. In summary, 2-aryl-isoindolin-1-one is a promising scaffold with broad anti-EV-A71
activity for further study. On-going studies include detailed SAR, the mechanism of action and in vivo
evaluation of compound A3 in an animal model.
Author Contributions: Y.L. (Yanping Li) and Y.L. (Yuhuan Li) designed the study and directed the study’s
optimization strategy. Z.L. summarized the Results and Discussion sections of the text. Y.W., X.J., and Z.J.
prepared the compounds for biological testing and collected the structural data. H.W., T.G. and X.J. conducted the
biological experiments and analyzed the data. All the authors read and approved the final manuscript.
Funding: This study was supported by CAMS Innovation Fund for Medical Sciences (CIFMS, 2017-I2M-3-012),
the Natural Science Foundation of Beijing (7132166) and National Science and Technology Major Projects for
“Major New Drugs Innovation and Development” (2018ZX09711003).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds with the structural data are available from the authors.
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