Discovery of Potent EV71 Capsid Inhibitors for Treatment of HFMD

Article abstract of DOI:10.1021/acsmedchemlett.7b00188

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which can spread its infections to the central nervous and other systems with severe consequences. The viral caspid protein VP1 is a well-known target for antiviral efficacy because its occupancy by suitable compounds could stabilize the virus capsid, thus preventing uncoating of virus for RNA release. In this Letter, design, synthesis, and biological evaluation of novel anti-EV71 agents (aminopyridyl 1,2,5-thiadiazolidine 1,1-dioxides) are described. One of the most promising compounds (14) showed excellent antiviral activity against EV71 (EC₅₀ = 4 nM) and exhibited excellent in vivo efficacy in the EV71 infected mouse model.

Full text of DOI:10.1021/acsmedchemlett.7b00188

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Letter
Discovery of Potent EV71 Capsid Inhibitors for Treatment of HFMD
Peng Li, Jun Yu, Fei Hao, Haiying He, Xuyang Shi, Jiao Hu, Li Wang, Chunyan Du, Xiao Zhang, Ya
Sun, Fusen Lin, Zhengxian Gu, Deming Xu, Xinsheng Chen, Liang Shen, Guoping Hu, Jian Li, Shuhui
Chen, Wei Xiao, Zhenzhong Wang, Qingming Guo, Xiujuan Chang, Xuyang Tian, and Tianwei Lin
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00188 • Publication Date (Web): 10 Jul 2017
Downloaded from http://pubs.acs.org on July 17, 2017
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ACS Medicinal Chemistry Letters
Discovery of Potent EV71 Capsid Inhibitors for Treatment of HFMD
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† ⊥ §
† §
†
†
†
†
†
†
,
Peng Li , , Jun Yu , , Fei Hao , Haiying He*, , Xuyang Shi , Jiao Hu , Li Wang , Chunyan Du , Xiao
Zhang†, Ya Sun†, Fusen Lin†, Zhengxian Gu†, Deming Xu†, Xinsheng Chen†, Liang Shen†, Guoping Hu†,
Jian Li†, Shuhui Chen†, Wei Xiao‡, Zhenzhong Wang‡, Qingming Guo‡, Xiujuan Chang‡, Xuyang
Tian⊥, Tianwei Lin⊥
† WuXi AppTec (Shanghai) Co., Ltd., 288 FuTe Zhong Road, Shanghai 200131, People’s Republic of China.
‡ Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichangnan Road, Lianyungang, 222001, People’s Republic of China.
⊥ State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102,
People’s Republic of China.
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KEYWORDS
HFMD, Enterovirus 71, Capsid inhibitor
ABSTRACT: Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), which can spread its
infections to the central nervous and other systems with severe consequences. The viral caspid proteinꢀVP1 is a wellꢀknown target
for antiviral efficacy because its occupancy by suitable compounds could stabilize the virus capsid, thus prevent uncoating of virus
for RNA release. In this paper, design, synthesis and biological evaluation of novel antiꢀEV71 agents (aminopyridyl 1,2,5ꢀ
thiadiazolidine 1,1ꢀdioxides) are described. One of the most promising compounds 14 showed excellent antiviral activity against
EV71 (EC50 = 4 nM) and exhibited excellent in vivo efficacy in the EV71 infected mouse model.
Introduction
from SterlingꢀWinthrop is a prominent series of capsidꢀ
binding compounds. Pleconaril in this series is a typical small
molecule inhibitor which has been shown to have a broad
spectrum of activities against enteroviruses and rhinoviruses
and has been tested in clinical trials [7]. According to what
Shia and coworkers reported, Pleconaril showed no antiviral
activity against EV71. Nevertheless, using skeletons of WIN
compounds as templates, they identified a novel series of imꢀ
idazolidinone derivatives with significant antiviral activity
against enterovirus 71. Among this series of compounds,
DBPR103 was identified as a very potent enterovirus 71 inhibꢀ
itor (EC50 = 54 nM) with no apparent cytotoxic effect toward
RD (rhabdomyosarcoma) cell lines (CC50 > 25 ꢁM) [8ꢀ9].
During our research, Rao and coworkers reported a paper usꢀ
ing structural analysis of the complete virus to guide the deꢀ
sign of EV71 inhibitors. Using pyridyl imidazolidinones as a
template, they discovered a potent EV71 capsid inhibitorꢀ
NLD, which was able to protect cells from EV71 infection at a
concentration of 25 pM [10ꢀ11]. Based on these studies, here
we report the chemical synthesis and biological evaluation of
novel aminopyridyl 1,2,5ꢀthiadiazolidine 1,1ꢀdioxides as
EV71 caspid inhibitors to treat HMFD.
Hand, foot, and mouth disease (HFMD) is a common infecꢀ
tious disease mainly occurring in young children. Clinical
symptoms of HMFD are characterized by fever, sore throat
and vesicular rash on the hands and feet as well as exanthema
on oral mucosa and tongue. For several decades, HFMD has
been a very common infection in children all over the world,
especially in the AsiaꢀPacific region. HFMD is caused by a
spectrum of pathogens in the enterovirus (EV) family, enteroꢀ
virus 71 (EV71) and coxsackievirus A16 (Cox A16) are reꢀ
garded as the major causative agents. In particular, EV71 is
more often associated with neurological diseases such as menꢀ
ingitis, encephalitis, monoplegia, acute flaccid paralysis and
even death, especially among young children (≤ 5 years old)
[1ꢀ2]. So far, no marketed antiꢀenterovirus drugs are available
for the treatment of EV71ꢀrelated disease, though the first
EV71 vaccine is now approved by cFDA to prevent this disꢀ
ease [3]. Supportive therapy is still the primary treatment for
severe cases of EV71 infection. Ribavirin was used for treatꢀ
ing mild HFMD in clinic. But Ribavirin showed no antiꢀEV71
activity with EC50 > 100 ꢁM in the cytopathogenic effect inhiꢀ
bition assay (CPE) in house. Considering the danger of severe
EV71 infection in children, there is an urgent need to develop
effective antiviral treatment to prevent or treat EV71ꢀrelated
disease before next EV71 outbreak.
EV71 has a nonꢀenveloped, positiveꢀsense, singleꢀstranded
RNA encoding capsid proteins (VP1, VP2, VP3, and VP4) and
nonstructural proteins (2A, 2B, 2C, 3A, VPg, 3C, and 3D) that
are involved in the life cycle of viruses including absorption,
invasion, shelling, and replication. All the functional protein
involved in the life cycle of viruses can be targets for the deꢀ
velopment of antiviral drugs [4]. We have previously disꢀ
closed the discovery of a series of peptidomimetic 3Cpro inꢀ
hibitors [5].
Scheme 1 Chemical structures of Pleconaril, DBPRꢀ103
and NLD
The viral proteinꢀVP1 is a wellꢀknown target for antiviral deꢀ
sign, because its hydrophobic pocket can be occupied by suitꢀ
able compounds, which lead to stabilize the virus capsid and
prevent uncoating of virus for RNA release [6]. WIN series
Result and Discussion
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Pleconaril and DBPRꢀ103 were tested in the cytopathogenic
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effect inhibition assay (CPE) inꢀhouse. Pleconaril showed no
antiꢀEV71 activity with EC50 > 100 ꢁM and DBPRꢀ103 was a
potent EV71 inhibitor with EC50 = 91 nM. Data were conꢀ
sistent with those reported in literatures [8ꢀ9]. Our research
was focused on structure modifications of pyridyl imidazoliꢀ
done compound DBPRꢀ103. Therefore, the primary task of the
project is to identify a novel skeleton based on pyridyl imidazꢀ
olidone series that could maintain potent antiviral activity. As
reported by Chern, the five carbon alkyl linker and terminal
pyridine play a very important role in influencing antiꢀEV71
activity [9]. We keep these 2 positions unchanged.
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First, imidazolidone modification was designed and evaluated
by the CPE assay. Among this series, 2ꢀketopiperazines 1 and
1,2,5ꢀthiadiazolidine 1,1ꢀdioxide 5 [12] kept antiviral activity
with EC50 as 180 nM and 110 nM, respectively. The rest modiꢀ
fications all resulted into loss of antiviral activity. Compound
1 (CC50 = 3.2 ꢁM) has moderate cytotoxicity while compound
5 (CC50 > 100 ꢁM) has no such issue.
Another structure modification strategy was the cyclization of
phenyl alkyl ether. According to this strategy, several benꢀ
zoheterocycle compounds were designed and evaluated, as
showed in table 2. Most of these compounds lost activity, exꢀ
cept the benzodihydropyran 6 (EC50 = 48 nM), which is 2 folds
more potent than DBPRꢀ103. Compound 6 also showed modꢀ
erate cytotoxicity (CC50 = 3 ꢁM) like 1.
a The compound concentration required to reduce the virusꢀinduced cell death by 50%
was defined as EC50.
b The compound concentration required to reduce cell viability to 50% of the tested
control culture was defined as CC50.
Table 2 Structure−Activity Relationship of phenyl alkyl
ether cyclization.
Table 1 Structure−Activity Relationship of imidazolidone
modification.
a The compound concentration required to reduce the virusꢀinduced cell death by 50%
was defined as EC50.
b The compound concentration required to reduce cell viability to 50% of the tested
control culture was defined as CC50.
The three potent compounds 1, 5 and 6 were further evaluated
and compared with DBPR103 through in vitro ADME and in
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vivo PK profiles. The mouse liver microsomal stability of 5 (< total, compound 11 exhibited 100 folds improvement in activiꢀ
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10 uL/min/mg) indicated slower metabolism than the other 3
compounds (40 ꢀ 50 uL/min/mg). The PK data with 2 mg/kg
IV and 5 mg/kg IP are summarized in Table 3. Following
IV/IP administration, the elimination halfꢀlife (T1/2) of 1, 5 and
6 ranged from 5 to 6 h, which is better than DBPR103 and
would be suitable for once daily dosing. Compound 6 exhibitꢀ
ed the lowest clearance and best exposure in these compounds.
Compound 5 also showed good exposure which was 2 folds
than that of DBPR103 both in intraperitoneal and intravenous
injection. However, 1 and 6 showed hERG inhibition issues
(IC50 = 0.37 ꢁM and 1.75 ꢁM, respectively), compared with 5
(IC50 > 30 ꢁM). Compound 5 was selected to further test in
oral PK. The oral exposure of 5 indicated it is also suitable for
oral dosing. Taking all factors into consideration, compound 5
is believed to be more suitable as a lead compound.
ty compared to 5 with these 2 steps modification. Furthermore,
10 and 11 have no cytotoxicity (CC50 > 100 ꢁM).
Scheme 2 Chemical structures of 5, 10 and 11
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To the best of our knowledge, no aldoxime ether structures
were tested in clinical therapies. While ketoxime ether strucꢀ
tures were widely used in pharmaceutical molecules in the
market, like fluvoxamine, ceftriaxone sodium, cefetamet
pivoxil and cefuroxime (Scheme 3). This might be because
aldoximes ether is more easily metabolized to aldehydes,
which are potential genotoxic agents. In this respect, we specꢀ
ulate it will be more drugꢀlike and safer to replace aldoxime
ether of 11 to ketoxime ether. So we designed several comꢀ
pounds on the basis of 11, including acetophenone oxime ether
(12), propiophenone oxime ether (13), benzocyclopentanone
oxime ether (14), benzocyclohexanone oxime ether (15) and
some heterocyclicꢀfused ring as bioisosteres of oxime ether
(16, 17, and 18).
As showed in table 4, most of these compounds lost antiviral
activities in CPE assay. Fortunately, benzocyclopentanone
oxime ether (14) kept excellent antiꢀEV71 activity (EC50 = 4
nM).
Scheme 3 Marketed drugs contain ketoxime ether strucꢀ
ture
Table3 Single Dose Mouse in vivo PK Properties.
(A) Data reported are the means from the dosing cohorts (Male
CDꢀ1 mouse, n = 3/dose). (B) Dosed at 2 mg/kg iv (20% solutol
in water), 5 mg/kg ip (20% solutol in water) and 5 mg/kg po (20%
solutol in water). (C) Cl is clearance. V
d
is volume of distribution.
C
max represents the highest observed concentration. Tmax is the
time taken to reach the maximum concentration.T1/2 is the halfꢀlife
of the compounds exposure in plasma. AUC is the area under the
curve. %F is percent bioavailability.
Since the PK profiles of 5 is good, the next goal of the project
is to further increase activity of compounds based on 5 (EC50
=
110 nM). According to Chern reported, antiviral activity of
DBPR compounds could be improved by replacing chlorobenꢀ
zene with oxime ether [13ꢀ14]. So, we planned to introduce
these moieties into 1,2,5ꢀthiadiazolidine 1,1ꢀdioxides to imꢀ
prove the activity. Coincidently, Rao and coworkers reported
a series of EV71 capsid inhibitors (NLD as a representative)
during our research process. As shown in NLD structure
(scheme 1), the methyl group in the linker and oxime ether has
been confirmed to make contribution to activity. The main
difference between NLD and DBPR series is the amine group
in the 2 position of pyridine. With such slight alteration, NLD
was able to protect cells from EV71 infection at a concentraꢀ
tion of 25 pM, which is an amazing improvement in activity.
To confirm this, NLD was synthesized and tested in CPE asꢀ
say. NLD exhibited excellent antiviral activity (EC50 = 0.03
nM), as previously reported [10]. But NLD shows moderate
cytotoxicity (CC50 = 5 ꢁM), and also suffers from severe
hERG inhibition (0.8 ꢁM). We planned to introduce amine
group into our 1,2,5ꢀthiadiazolidine 1,1ꢀdioxide serials to
check whether this modification can improve activity.
The amine group was added to compound 5 to generate 10
[12], which showed 10 folds more potent than 5. This indicatꢀ
ed the amine made a great contribution to the activity. Comꢀ
pound 11 (EC50 = 0.9 nM), which replaced the chlorobenzene
of 10 with oxime ether, is 10 folds more potent than 10. In
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Table 4 Modifications of compound 11.
of NLD. The phenoxyl group sandwiched between Phe135
O
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2
3
4
5
6
7
8
O
and Phe155. And the binding mode of tail oxime group of
compound 14 could overlay well with that of NLD in the coꢀ
crystal structure (pdb code: 4CEY). The aminopyridine group
of compound 12 could overlay with NLD, but its phenoxyl
group could not overlay well with NLD (see SI figure S2).
Both the aminopyridine group and phenoxyl group of comꢀ
pound 16 could not overlay well with NLD (see SI figure S3).
The binding results are consistent with the cellꢀbased bioactivꢀ
ities of 12, 14 and 16. This implies that the inhibition mechaꢀ
nism of compound 14 is targeting on EV71 capsid and blockꢀ
ing viral uncoating as NLD does.
Since NLD suffers from severe hERG inhibition (0.8 ꢁM), we
tested the hERG inhibition of two potent compound 11 and 14.
The hERG activities of 11 (9 ꢁM) and 14 (6 ꢁM) are much
better than NLD. Compounds 11, 14 and NLD were further
tested and compared by PK characterization (table 5). Pharꢀ
macokinetic studies were conducted in mouse to assess the
intraperitoneal and intravenous exposure for 11, 14 and NLD.
The elimination halfꢀlife (T1/2) of 11, 14 are 1.6h and 1.5h,
respectively. NLD showed high clearance and its halfꢀlife
(T1/2) is only 0.3h. The IV/IP exposure of 14 is better than 11
and NLD. Also considering the poor drugꢀlike property of
aldoxime ether in compound 11, compound 14 was selected
for further exploration in in vivo efficacy model.
R3
S
N
O
N
N
H2N
EC50 (nM)a
CC50
(
)
b
Compd No
R3
µ M
H
O
N
> 100
11
0.9
9
O
N
600
10
11
12
13
14
15
16
17
18
19
20
21
22
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12
13
> 100
O
N
554
> 100
O
N
4
42
14
15
O
N
309
> 100
N
Table 5 Single Dose Mouse in vivo PK Properties
> 1000
> 100
16
17
Cpd.
11
14
NLD
iv@1 mpk
Cl (mL/min/kg)
T1/2 (h)
N
N
N
> 100
> 100
> 1000
352
34
1.5
2.8
821
105
0.3
36
1.6
3.2
592
2.4
Vd (L/kg)
18
371
AUC (nM*h)
O
a The compound concentration required to reduce the virusꢀinduced cell death by 50%
was defined as EC50.
b The compound concentration required to reduce cell viability to 50% of the tested
control culture was defined as CC50.
ip@5 mpk
Cmax (nM)
Tmax (h)
2152
0.7
1558
0.3
1443
0.5
3868
88
1711
92
AUC (nM*h)
%F
2449
79
(A) Data reported are the means from the dosing cohorts (Male
CDꢀ1 mouse, n = 3/dose). (B) Dosed at 2 mg/kg iv (20% solutol
in water) and 5 mg/kg ip (20% solutol in water). (C) Cl is clearꢀ
ance. V is volume of distribution. Cmax represents the highest
d
observed concentration. Tmax is the time taken to reach the maxiꢀ
mum concentration.T1/2 is the halfꢀlife of the compounds exposure
in plasma. Tlast is the time taken to reach the last measurable conꢀ
centration. AUC is the area under the curve. %F is percent bioaꢀ
vailability.
ICR mouse model (3 days old) with lethalꢀdose (1 x 106 PFU)
EV71 strain 695F infection was used to imitate the severe
complications of EV71 infected children. Lycorine, as reportꢀ
ed in an EV71 infected model, was used as a positive control
[15ꢀ16]. Ribavirin is a broadꢀspectrum antiviral agent and was
used for treating mild HFMD in clinic. Compound 14 was
dosed 10 mg/kg/d via intraperitoneal injection route at differꢀ
ent time. One is dosing before EV71 infection (group 3), the
other is dosing after EV71 infection (group 4). The vehicle,
lycorine and ribavirin groups were dosed before EV71 infecꢀ
tion. The results showed that mice in vehicle group all died at
day 8. Ribavirin group mice all died at day 5 (100 mg/kg) and
day 6 (50 mg/kg), due to toxicity of Ribavirin. Lycorine group
mice protection was only 30% after 14 days dosing. Comꢀ
pound 14 showed excellent protection on EV71 infected
mouse, which was 100% in both two groups (Figure 2). The
Figure 1. Predicted binding mode of compound 14 on EV71
capsid. Xꢀray structure of NLD (PDB code 4CEY) was used
as docking template. Pink dash stands for hydrogen bonding.
The figure was generated with PyMOL Molecular Graphics
System, version 1.5.0.4 Schrödinger, LLC.
The molecular modeling method was used to illustrate the
binding mode of compound 12, 14 and 16. The results indicatꢀ
ed that the overall binding mode of compound 14 was similar
to that of the NLD on EV71 capsid. The aminoꢀpyridine group
could form hydrogen bonding with Asp112. The sulphonyl
group could form hydrogenꢀbonding to the backbone NH of
residue Ile113 as that of the carbonyl oxygen of the imidazole
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body weight and clinical score also showed 14 was much betꢀ met medical need to develop effective antiviral treatment. The
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ter than lycorine.
(A)
novel aminopyridyl 1,2,5ꢀthiadiazolidine 1,1ꢀdioxides were
first synthesized and their antiꢀEV71 activities were evaluated
by CPE assay. In this series, compound 14 was identified to
have excellent antiviral activity against EV71 (EC50 = 4 nM)
and more potent than DBPR103. Furthermore, 14 showed
excellent in vivo efficacy in EV71 infected mouse model.
Thus, 14 is a promising drug candidate for potential applicaꢀ
tion in the clinical therapy of HFMD.
100
90
80
70
60
50
40
30
20
10
0
Group 1 Vehicle
Group 2 Lycorine 0.4 mpk
Group 3 14 10 mpk, before EV71 infection
Group 4 14 10 mpk, after EV71 infection
Group 5 Ribavirin 100 mpk
Group 6 Ribavirin 50 mpk
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Day Post Infection
ASSOCIATED CONTENT
Supporting Information
(B)
6
Group 1 Vehicle
Group 2 Lycorine 0.4 mpk
Group 3 14 10 mpk, before EV71 infection
Group 4 14 10 mpk, after EV71 infection
Group 5 Ribavirin 100 mpk
4
2
0
Synthetic procedures, analytical data, assay protocol are available
free of charge on the ACS Publications website.
Group 6 Ribavirin 50 mpk
AUTHOR INFORMATION
Corresponding Author
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Day Post Infection
* Eꢀmail: he_haiying@wuxiapptec.com
Author Contributions
(C)
10
Group 1 Vehicle
Group 2 Lycorine 0.4 mpk
8
6
4
2
0
Group 3 14 10 mpk, before EV71 infection
Group 4 14 10 mpk, after EV71 infection
Group 5 Ribavirin 100 mpk
Group 6 Ribavirin 50 mpk
§These authors contributed equally.
ABBREVIATIONS
EV71, Enterovirus 71; HFMD, hand, foot and mouth disease; Cox
A16, coxsackievirus A16; CPE, cytopathogenic effect; RD, rhabꢀ
domyosarcoma; ADME, absorption, distribution, metabolism, and
excretion; PK, pharmacokinetics; CYP, Cytochrome P450; hERG,
human etherꢀaꢀgoꢀgo relatedgene; MMS, microsomes metabolic
stability; IV, intravenous; IP, intraperitoneal; PO, per oral; AUC,
area under curve; CL, clearance; T1/2, halfꢀlife; Vd, volume of
distribution; PFU, plaqueforming unit.
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig.2 The iDnayviPvoost aInnfetcitvioinral activity of 14. (A) Survival rate. (B)
Mice body weight. (C) Clinical score. 3ꢀdayꢀold ICR mice are
i.p. inoculated with EV71 and administrated with vehicle
(N=10), lycorine (0.4 mpk, N=10), 14 before EV71 infection
(10 mpk, N=10), 14 after EV71 infection (10 mpk, N=9), Ribꢀ
avirin (100 mpk, N=11, or 50 mpk, N=12).
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The chemical synthesis of 14 is shown in Scheme 4. Starting
from 5ꢀhydroxyꢀ1ꢀindanone 14a, condensation of 14a with
ethoxyamine gave oxime ether 14b. Monosubstitution of 14b
with 1,5ꢀdibromopentane afforded intermediate 14c, which
then reacted with C to give 14d. Final product 14 was obꢀ
tained by the reaction of chloropyridine intermediate 14d with
BocNH2 and Boc deprotection in situ. The synthesis of other
compounds can be found in the supporting information.
Scheme 4 Synthesis of 14
4.
5.
Conclusion
Considering the danger of severe EV71 infection in children
and no marketed antiviral drugs are available, there is an unꢀ
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