Identification, synthesis and pharmacological evaluation of novel anti-EV71 agents via cyclophilin A inhibition

Article abstract of DOI:10.1016/j.bmcl.2015.11.002

In this work, the relationship between cyclophilin A (CypA) and EV71 prompted us to screen a series of small molecular CypA inhibitors which were previously reported by our group. Among them, compounds 1 and 2 were discovered as non-immunosuppressive anti-EV71 agents with an EC₅₀ values of 1.07 ± 0.17 μM and 3.36 ± 0.45 μM in virus assay, respectively, which were desirably for the further study. The subsequent chemical modifications derived a novel class of molecules, among which compound 11 demonstrated the most potent anti-EV71 activity in virus assay (EC₅₀ = 0.37 ± 0.17 μM), and low cytotoxicity (CC₅₀ > 25 μM). The following CypA enzyme inhibition studies indicated that there was not only the enzyme inhibition activity, undoubtedly important, functioning in the antiviral process, but also some unknown mechanisms worked in combination, and the further study is underway in our laboratory. Nevertheless, to the best of our knowledge, compound 11 was probably the most potent small molecular anti-EV71 agent via CypA inhibitory mechanism to date. Consequently, our study provided a new potential small molecule for curing EV71 infection.

Full text of DOI:10.1016/j.bmcl.2015.11.002

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5682–5686
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification, synthesis and pharmacological evaluation of novel
anti-EV71 agents via cyclophilin A inhibition
Wenzhong Yan a,y, Jie Qing b,c,y, Hanbing Mei a, Junxiu Nong c, Jin Huang a, Jin Zhu a, Hualiang Jiang a,
Lei Liu b, , Linqi Zhang c, , Jian Li a,
⇑
⇑
⇑
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
b Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry,
Tsinghua University, Beijing 100084, China
c School of Medicine, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2015
Revised 17 October 2015
Accepted 2 November 2015
Available online 2 November 2015
In this work, the relationship between cyclophilin A (CypA) and EV71 prompted us to screen a series of
small molecular CypA inhibitors which were previously reported by our group. Among them, compounds
1
and
1.07 0.17
study. The subsequent chemical modifications derived a novel class of molecules, among which com-
pound 11 demonstrated the most potent anti-EV71 activity in virus assay (EC50 = 0.37 0.17 M), and
low cytotoxicity (CC50 > 25 M). The following CypA enzyme inhibition studies indicated that there
2
were discovered as non-immunosuppressive anti-EV71 agents with an EC50 values of
l
M and 3.36 0.45 M in virus assay, respectively, which were desirably for the further
l
l
Keywords:
Cyclophilin A
EV71
HFMD
Small molecule inhibitor
l
was not only the enzyme inhibition activity, undoubtedly important, functioning in the antiviral process,
but also some unknown mechanisms worked in combination, and the further study is underway in our
laboratory. Nevertheless, to the best of our knowledge, compound 11 was probably the most potent small
molecular anti-EV71 agent via CypA inhibitory mechanism to date. Consequently, our study provided a
new potential small molecule for curing EV71 infection.
Ó 2015 Elsevier Ltd. All rights reserved.
O
Enterovirus 71 (EV71) is one of the major causative agent of the
large outbreaks of hand, foot and mouth disease (HFMD) in chil-
dren, which have occurred in recent years in the Asia–Pacific
region, including China.1–3 According to the data from the Chinese
Center for Disease Control and Prevention (CDC), there were more
than 1,600,000 cases of EV71 infection, with more than 500 deaths
in mainland China in 2011.4 However, there are currently no vac-
cines or antiviral agents for preventing of EV71 infection and also
no specific drugs reported in clinical study.4 Some anti-EV71 inhi-
bitors have been reported in pre-clinical research,4 such as rupin-
NH
O
O
O
N
H
N
H
N
O
O
O
F
rupintrivir (AG7088)
Anti-EV71 EC50 = 0.014 µM5
Figure 1. Chemical structure and anti-EV71 activity of rupintrivir.
trivir (AG7088),
a human rhinovirus (HRV) NS3C protease
inhibitor which was in phase II clinical trials for anti-HRV, was
found possessed the ability for inhibiting EV71 replication with
an EC50 value of 0.014 l
M (Fig. 1).5 Thus, the development of effec-
EV71 is
a kind of positive-sense, non-enveloped, single-
stranded RNA virus in the Enterovirus genus of the Picornaviridae
family6–9 which can cause serious aseptic meningitis, myocarditis,
encephalitis, and acute flaccid paralysis, hence leading to high
mortality.10 In the virus lifecycle, the single-stranded RNA can
encode a polypeptide with a molecular weight of approximate
250 kDa,11 which is initially processed into one structural (P1)
and two non-structural (P2 and P3) regions. Among these three
regions, P1 is further proteolyzed into VP1–VP4 to generate the
tive anti-EV71 agent is quite urgent and possesses important
significance.
⇑
Corresponding authors.
E-mail addresses: lliu@mail.tsinghua.edu.cn (L. Liu), zhanglinqi@biomed.
tsinghua.edu.cn (L. Zhang), jianli@ecust.edu.cn (J. Li).
y
These authors made equal contributions to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.11.002
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

W. Yan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5682–5686
5683
Table 1
viral capsid, while P2 and P3 are ultimately resulted in replicase
Chemical structures and anti-EV71 assay results of compounds 1–3a
proteins. Targeting these regions can derive series of direct-acting
antivirals (DAA) such as capsid proteins, NS2C protein, NS3A pro-
tein and NS3C protease inhibitors currently in preclinical study,
which were the vast majority of the reported anti-EV71 agents at
present.4 However, RNA virus (such as EV71) is notoriously pos-
sessed high mutation rate, only a single mutation in the viral target
may hinder the binding of an inhibitor, thus making it ineffective.
Taking hepatitis C virus (HCV) as an example, currently there are
more than 20 DAAs targeting the NS5A protein, polymerase and
viral protease in advanced clinical trials, whereas these DAAs had
to be utilized in combination in order to avoid resistance.12–15
Thus, a strategy targeting host factors which are essential for viral
replication such as cyclophilin A (CypA) may possess extensive
prospect and may bring light to the cure of EV71 infection.
CypA, the most critical member of the cyclophilin (Cyp) protein
family from vertebrates and other organisms, which is proved to be
identical to peptidyl–prolyl cis–trans isomerase (PPIase), has been
researched extensively.16 Via its PPIase activity, CypA plays a crit-
ical role in a lot of cellular processes, including protein secretion,
transcriptional regulation, mitochondrial function, and immune
response.17,18 Besides the functions mentioned above, CypA also
takes a crucial part in the replication of diverse kinds of viruses,
such as HCV, human immunodeficiency virus type 1 (HIV-1), vesic-
ular stomatitis virus (VSV), influenza virus, human papillomavirus
(HPV) and vaccinia virus.19,20 Inspiringly, the essential role of CypA
in the infection of EV71 was just demonstrated by Qing and his
coworkers that CypA assists the entrance step of EV71 as a capsid
uncoating regular.10
O
O
NH NH
R1
Compd R1
CypA enzyme inhibition
assay IC50
M) SDb
Anti-EV71 assay
M) SDb CC50
(lM)
(
l
EC50
(l
1
2-F
0.159 0.007
0.032 0.002
0.263 0.024
0.023 0.00039
1.07 0.17
3.36 0.45
5.85 0.34
3.66 0.51
>20
>20
>20
>10
2
3
2,6-di-OH
2,6-di-F
CsA
a
The synthesis and HPLC analysis data of 1–3 was according to Ref. 21.
Each value indicates the mean SD of three independent experiments.
b
Table 2
The spleen cell proliferation inhibition activities of compounds 1–2 and CsA
Compd
T spleen cells IC50
(
l
M) SDa
B spleen cells IC50 (l
M) SDa
1
2
CsA
>100
>100
33.03 5.72
0.01 0.005
51.93 8.68
0.04 0.008
a
Each value indicates the mean SD of three experiments using one mouse with
triplicate sets in each assay. Half-maximal inhibitory concentrations (IC50 values)
are obtained by log-probit analysis of inhibitory curves using SPSS, a windows
statistical software package.
Considering the important role of CypA in the infection of EV71,
we focused on the discovery and application of the CypA inhibitors
for anti-EV71 persistently. In the previous study, a novel series of
small molecular CypA inhibitors with nanomolar inhibitory poten-
cies which possess the same scaffold of 1-(benzoyl)-3-(9H-fluoren-
9-yl)-urea had been reported by our group.21 In view of the close
relationship between CypA and EV71, we wondered if these com-
pounds could be utilized as a novel kind of anti-EV71 agent as
we expected. Therefore, a series of experiments were performed
in this Letter aiming at realizing our idea.
B
OH
O
O
B
F
O
O
N
H
N
H
C
N
H
N
H
C
OH
A
A
Figure 2. Three structural regions of compounds 1 and 2.
Due to the tight relationship between CypA and EV71, the com-
pounds which possessed potent CypA enzyme activities in our pre-
vious work were selected for the antiviral test and CsA was
selected as the control. As we expected, the active compounds
were achieved and the three most potent compounds (1–3) were
shown in Table 1, from which we can see that all the three com-
pounds showed good anti-EV71 activities and compounds 1 and
2 were equal or better compared with the control (1.07 0.17
Table 3
The chemical structures of compounds 4–9 and their corresponding anti-EV71
activities
and 3.36 0.45
as the leads for further study.
lM vs 3.66 0.51 lM). Thus 1 and 2 were selected
X
O
(R1)
m
N
H
N
H
n
Before the further chemical modification of compounds 1 and 2,
the immunosuppressive ability was evaluated firstly, because an
anti-EV71 agent via CypA inhibition should possess enough low
inhibition activity against proliferation of spleen cells to avoid side
effects. The result was optimistic, the two compounds showed
quite low inhibition activities (more than 10 times) against prolif-
eration of spleen cells compared with its anti-EV71 activity
(Table 2), which could be regarded as non-immunosuppressive
lead compounds for further research.
Compd
R1
X
n
Anti-EV71 activity
EC50
(l
M) SDa
CC50 (lM)
4
5
6
7
8
9
2,6-Di-OH
2-F
3,5-Di-OH
2,4-Di-OH
2-OH,5-F
2-F
H,H
H,H
O
O
O
0
0
0
0
0
1
>10
>25
>25
>25
3.78 0.72
>10
>25
>25
>25
>25
>25
>50
O
The lead compounds 1 and 2 can be divided into three regions
(Fig. 2). Considering of the importance of the acylurea moiety
(region C) for CypA inhibition,21 it was retained in most derivatives
only except compounds 4 and 5 (Table 3), which were designed to
evaluate the influence of the acylurea moiety for the anti-EV71
activity. By changing the other two structural regions A–B, two ser-
ies of derivatives were achieved. Firstly, to evaluate the impact of
region A on the antiviral activity, four compounds (6–9, Table 3)
a
Data are the mean value of three independent experiments.
were obtained by altering the site of the substituent on the ben-
zene ring (2 vs 6, 7 and 8) or replacing the benzene ring with ben-
zyl group (1 vs 9) but remaining the fluorene moiety (region B).
Secondly, keeping region A remained and introducing varies kinds

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W. Yan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5682–5686
of substituents (F, Cl, Me, CF3) to the fluorene moiety (1 vs 16–19, 2
vs 10–15) or replacing the fluorine ring with hydrogenated fluo-
rene or benzfluorene (1 vs 22, 2 vs 20–21), another 13 novel
derivatives were achieved and their chemical structures were
shown in Table 4. All novel derivatives (4–22) were confirmed
P95% purity (Supporting information, Table 3S).
Scheme 1 depicts the general synthetic route for the prepara-
tion of compounds 5, 9 and the benzyl protected precursors 4p,
6p, 7p, 8p of compounds 4, 6, 7, 8, respectively. In general, 9H-flu-
oren-9-ylamine (25a) was selected as the starting material and
was reacted with triphosgene in the presence of triethylamine to
afford the corresponding isocyanate (24a), which was reacted with
intermediates 23c–h directly to afford compounds 5, 9 and inter-
mediates 4p, 6p, 7p, 8p. Finally, the four intermediates were trea-
ted with Pd/C under the circumstance of hydrogen at the
temperature of 40 °C to provided compounds 4, 6, 7, 8. The details
for synthetic procedures and structural characterizations are
described in Supporting information.
R2 NH2
23c-h
NH2
O
NCO
a
R2
N
H
N
H
b
25a
R2 = substituted benzoyl, phenylacetyl or benzyl
24a
5, 9, 4p, 6p,
7p, 8p
Scheme 1. Reagents and conditions: (a) (i) triphosgene, 1,2-dichloroethane, 0 °C,
(ii) Et3N, reflux; (b) R2NH2, toluene or acetone, reflux or rt.
O
3
R
NH
2
O
O
3
R
1
R
a
23a-b
b
N
H
N
H
NH
2
NCO
1
1
R
R
Compounds 16–19 and the precursors 10p–15p, 20p–22p of
compounds 10–15, 20–22 were synthesized using the approach
outlined in Scheme 2. Generally, intermediates 25b–i were reacted
with triphosgene in the presence of triethylamine to afford the
corresponding isocyanates (24b–i), which were reacted with
intermediates 23a–b directly to afford 16–19 and the precursors
10p–15p, 20p–22p. Finally, the nine precursors were treated with
Pd/C under the circumstance of hydrogen at 40 °C to provided
compounds 10–15, 20–22, except compound 12 which was
obtained by treating 12p with trifluoroacetic acid (TFA) at room
temperature. It should be noted that compounds 21 and 22 are
racemates. The details for synthetic procedures and structural
characterizations are described in Supporting information.
In the virus assay, compounds 4–9 were tested firstly to inves-
tigate the impact of the benzoylurea group (regions A and C) on the
antiviral activity. The results were summarized in Table 3. Specifi-
cally, the EC50 values were reduced remarkably (EC50 values
25b-i
24b-i
16-19, 10p-15p, 20p-22p
1
3
R
= Substituted-9H-fluoren-9-yl or
R
= 2, 6-di-OH or 2-F
Scheme 2. Reagents and conditions: (a) (i) triphosgene, 1,2-dichloroethane, 0 °C,
(ii) Et3N, reflux; (b) 23a or 23b, toluene, reflux.
B). The results were summarized in Table 4 and showed that ether
disrupting its planarity (1 vs 22, 2 vs 21) or enlarging its volume (2
vs 20) were not obviously beneficial for the antiviral property. The
results of compounds 10–19 which were introduced substituent
groups to the fragment B were interesting. All the compounds
exhibited more potent antiviral activities than the control CsA,
except compound 15. When comes to the derivatives from lead
compound 2 (10–15), the results showed that the substitution at
30 site of the fluorene ring could remarkably increase the anti-
EV71 activity than at the 10 site (11 vs 10 and 2). When comes to
the 30 position of the fluorene, four kinds of substituent groups
were measured and the fluoro-substituted compound 11 showed
the most potent anti-EV71 activity (compared with 12, 13 and
15). However, no significant improvement of the activity was
achieved among the derivatives from lead compound 1 (16–19).
>10 lM) no matter by removing the oxygen of the benzoyl group
(1 vs 5, 2 vs 4) or by inserting a carbon between regions A and C
(1 vs 9). And when changing the positions of the hydroxyl groups,
the anti-EV71 activity was completely lost (EC50 values >25 lM, 2
vs 6, 7).
Directed by this consequence, the benzoylurea groups were
retained in the following compounds for this text, and the whole
structural modifications were focused on the fluorene ring (region
Table 4
The chemical structures of compounds 10–22 and their corresponding anti-EV71 activities
4'
1'
F
O
O
OH
O
O
OH
O
O
O
O
N
H
N
H
R2
N
H
N
H
N
H
N
H
N
H
N
H
R1
OH
OH
7'
22
21
20
Compd
R1
R2
Anti-EV71 activity
M) SDa
CC50
Compd
R1
R2
Anti-EV71 activity
M) SDa
CC50 (lM)
EC50
(l
(lM)
EC50
(l
10
11
12
13
14
15
16
2,6-di-OH
2,6-di-OH
2,6-di-OH
2,6-di-OH
2,6-di-OH
2,6-di-OH
2-F
10-F
30-F
1.13 0.29
0.37 0.17
0.46 0.13
2.72 0.77
1.08 0.28
>10
>25
>25
>25
>25
>25
>25
>25
17
18
19
20
21
22
2-F
2-F
2-F
30-F
2.11 0.58
3.34 0.14
0.92 0.20
1.92 0.37
4.31 0.53
5.25 0.79
>10
>25
>10
>25
>25
>25
30-Cl
30-Cl
30-CH3
30-CH3
20,60-di-F
30-CF3
10-F
0.66 0.21
a
Data are the mean value of three independent experiments.

W. Yan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5682–5686
5685
Table 5
potent CypA inhibitory activities (CypA enzyme inhibition:
IC50 = 2.76 0.13 M and 1.22 0.39 M, respectively) than the
The CypA enzyme inhibition activities of compounds 4–22
l
l
other compounds except 10. Although some anti-EV71 active com-
pounds (8, 19–22) showed low CypA inhibitory activities, these
compounds (4–7, 9, 15) without anti-EV71 activities were all not
Compd Enzyme inhibition assay IR
Compd Enzyme inhibition assay IR
a
a
(10
lM) or IC50
(l
M)
(10
lM) or IC50 (lM)
4
9.25% (10
lM)
14
15
16
17
18
19
20
21
22
10.33 1.07
5
17.82% (10
l
M)
20.32% (10
10.43 1.34
10.26 0.39
10.8 0.47
lM)
CypA inhibitors (IR value at 10 lM < 25%).
6
Inactive
Considering the important role of CypA for EV71 infection, we
were enlightened and prompted to screen a series of compounds
previously reported as CypA inhibitors. Eventually, two small
molecular CypA inhibitors 1 and 2 with good anti-EV71 activities
were discovered. The following assessment showed that these
two compounds can be regarded as non-immunosuppressive lead
compounds for further research. Hence a series of structural
modification had been performed and then another 19 derivatives
were achieved. It was notable that most of the derivatives (13/19)
were different from the compounds21 previously reported by our
group in which the fluorene moiety was not modified. Among
these compounds, 11 exhibited the most potent antiviral activity
7
8
9
10
11
12
13
21.40% (10
27.72% (10
16.89% (10
0.94 0.33
2.76 0.13
1.22 0.39
lM)
lM)
lM)
28.71% (10
39.12% (10
27.65% (10
33.54% (10
l
M)
M)
M)
M)
l
l
l
11.62 1.12
a
Data are the mean value of five independent experiments.
Consequently, the most potent compound 11 was achieved with
the EC50 value of 0.37 0.17 M that was 9.0-fold and 9.9-fold
l
in virus assays (EC50 = 0.37 0.17
(CC50 > 25 M).
lM) and low cytotoxicity
more potent than 2 and CsA, respectively. Furthermore, all the
three most potent compounds (11–12, 16) showed low cytotoxic-
l
The structure–activity relationship of these derivatives in the
virus assays demonstrated that the 2,6-dihydroxy-benzoylurea
moiety and the planar fluorene ring were crucial for the anti-
EV71 ability. Furthermore, the former seemed to be better than
2-fluoro-benzoylurea moiety. The results of the modification on
the fluorene ring indicated that the substitution at 30 site of the flu-
orene ring was better than 10 site and fluorine was the best sub-
stituent. However, due to the difficulty of the synthesis and
separation of the substituted fluoren-9-ones which were the most
important intermediates for the synthesis of substituted fluorene
derivatives,22 the substitution at 20 and 40 sites of fluorene ring
were not investigated regrettably. But by making significant
efforts, compound 14 was obtained eventually, which could certify
ity (CC50 > 25 lM), indicating that the inhibition of EV71 was not
due to the death of host cells.
The outcome of the standard chymotrypsin-coupled assay was
summarized in Table 5, all compounds reduced CypA inhibitory
activities in vitro. Specifically, compounds 4–9 (benzoylurea
moiety were disrupted) and compounds 20–22 (fluorene ring were
replaced) were completely lost the CypA enzyme inhibition
activity (IR value at 10
lM < 40%), compared with 1 and 2 (CypA
enzyme inhibition: IC50 = 0.159 0.007
l
M and 0.032 0.002 M,
l
respectively). Whereas most compounds of 10–19 which
were merely introduced substituent to the fluorene ring could
reserve parts of the enzyme inhibition activities. The two most
potent compounds 11 and 12 in the virus assay exhibited more
Figure 3. (a) Dose-dependent reduction of EV71 VP1 expression. The concentrations of compound 11 in lanes 2–5 were 5, 1, 0.2, and 0.04
lM, respectively. NITD008 (5 lM)
was used as positive control. (b) The effect of adding compound 11 (5 M) to WT-EV71 or S243P-EV71 virus. The data were expressed as the percentage of EV71 RNA from the
l
untreated control cells; the mRNA level of GAPDH was used as an internal control. Each data point represents the average of three replicates. The error bars represent the SEM.
The anti-EV71 effects of the combination of compound 11–NK-1.8K (c) and compound 11–NITD008 (d). Differential surface plot at the 95% confidence level (CI) were
calculated and generated by using MacSynergy II for the drug–drug interaction.

5686
W. Yan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5682–5686
that the introduction of substituent group to the 20 site had no con-
tribution to the improvement of the anti-EV71 activity.
Acknowledgments
Whereas the outcome of the CypA enzyme inhibition assay was
not completely as we expected and indicated that the enzyme inhi-
bition activity only played parts of the role, undoubtedly impor-
tant, in the anti-EV71 mechanism. Therefore, further mechanistic
researches were conducted using compound 11 to verify our spec-
ulation. Results of the western blot analysis which was designed
for testing viral protein expression after drugs treatment showed
that 11 can clearly inhibit the proliferation of EV71 (Fig. 3a). This
inhibition was dose-dependent and the intracellular expression
of EV71 VP1, which is the major component of EV71 capsid, was
obviously attenuated by the treatment of compound 11. In a con-
trast, the intracellular expression of endogenous glyceraldehyde-
3-phosphate dehydrogenase (GADPH) was not affected, being
indicative that the inhibition of compound 11 to EV71 replication
was specific. In this assay, NITD008,23 a nucleoside analog which
was reported to be an anti-EV71 reagent, was selected as positive
control. The results of quantitative RT-PCR based infection assay
revealed that although 11 still be effective to the VP1 mutant virus
S243P-EV71, it show a little resistant comparing to the WT-EV71
virus (3.0 0.6% vs 6.7 0.8%, Fig. 3b), which were in accord with
our previous research.10 These results together demonstrated that
CypA inhibition mechanism indeed functioned in the anti-EV71
process of these compounds, and the unknown effect functioned
together with the former is still necessary to explicitly
demonstrated.
Financial support of this research provided by the National Nat-
ural Science Foundation of China (Grants 21222211, 21372001,
91313303), the Program for New Century Excellent Talents in
University (Grant NCET-12-0853), and the Fundamental Research
Funds for the Central Universities is gratefully acknowledged.
Supplementary data
Supplementary data (synthetic methods, spectroscopic data,
details of the spleen cells proliferation inhibition assay, virus assay
and the CypA enzyme inhibition assay) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2015.11.002.
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Because inhibition of each step of the virus life cycle can affect
viral proliferation, we wondered whether compound 11 could be
combined with other anti-EV71 drugs. Hence NK-1.8K24 (a peptidyl
aldehyde) and NITD008, that both inhibited the proliferation of
EV71, were employed in this study. The results revealed that com-
pound 11–NK-1.8K combination had the maximal percent synergy
extended to the range of 20–30% at a high concentration (0.31–
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l
with the tested doses, as the maximal percent antagonism
extended to the range of ꢀ20% to ꢀ30% (Fig. 3c and d).
In conclusion, our present study provided a new series of potent
anti-EV71 agents. Furthermore, to the best of our knowledge, these
compounds represented the most potent small molecular anti-
EV71 agents via CypA inhibition mechanism to date, and the fur-
ther pre-clinical study of these compounds are currently under
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