Novel substituted heteroaromatic piperazine and piperidine derivatives as inhibitors of human enterovirus 71 and coxsackievirus A16

Article abstract of DOI:10.3390/molecules18055059

A series of substituted heteroaromatic piperazine and piperidine derivatives were found through virtual screening based on the structure of human enterovirus 71 capsid protein VP1. The preliminary biological evaluation revealed that compounds 8e and 9e have potent activity against EV71 and Coxsackievirus A16 with low cytotoxicity.

Full text of DOI:10.3390/molecules18055059

Molecules 2013, 18, 5059-5071; doi:10.3390/molecules18055059
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Substituted Heteroaromatic Piperazine and Piperidine
Derivatives as Inhibitors of Human Enterovirus 71 and
Coxsackievirus A16
Xian Zhang 1,2,†, Hongliang Wang 2,†, Yuhuan Li 3, Ruiyuan Cao 2, Wu Zhong 2, Zhibing Zheng 2,
Gang Wang 1, Junhai Xiao 2,* and Song Li 1,2,
*
1
Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
Liangning, China; E-Mails: zhangxian85@163.com (X.Z.); wg0115@sina.com (G.W.)
National Engineering Research Center for the Strategic Drugs, Beijing Institute of Pharmacology
and Toxicology, Beijing 100850, China; E-Mails: whl83319@126.com (H.W.);
2
21cc@163.com (R.C.); zhongwu@bmi.ac.cn (W.Z.); zzbcaptain@aliyun.com (Z.Z.)
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China; E-Mail: yuhuanlibj@126.com
3
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: xiaojunhai@139.com (J.X.);
lis@bmi.ac.cn (S.L.); Tel.: +86-10-6693-0673-712 (J.X.).
Received: 19 April 2013; in revised form: 22 April 2013 / Accepted: 26 April 2013 /
Published: 29 April 2013
Abstract: A series of substituted heteroaromatic piperazine and piperidine derivatives
were found through virtual screening based on the structure of human enterovirus 71
capsid protein VP1. The preliminary biological evaluation revealed that compounds 8e and
9e have potent activity against EV71 and Coxsackievirus A16 with low cytotoxicity.
Keywords: virtual screening; piperazine derivative; piperidine derivatives; anti-EV71
activity; anti-CVA16 activity

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1. Introduction
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) belong to the genus Enterovirus of
family Picornaviridae [1]. Both viruses are common causes of hand, foot, and mouth disease (HFMD),
which mostly affects young children [2]. The clinical manifestations associated with HFMD caused by
both viruses include fever, sore throat, diarrhea, and papulovesicular rash on the hands, feet, and
oropharyngeal mucosa. Moreover, EV71 infections can damage the central nervous system, leading to
viral meningitis, encephalitis, or severe myocarditis with or without fatal pulmonary edema [3–5].
Many epidemic outbreaks have been reported in Southeast and East Asia because of the high of
morbidity and mortality rates associated with such infections. Between January 2009 and May 2011,
the EV71 epidemic in China has led to 1,000 deaths [6]. Although few inhibitors and vaccines have
been reported, such as compound 1, shown in Supplementary Figure S1 [7–10], no antiviral agent has
been approved by the US Food and Drug Administration for treating HFMD caused by enteroviral
infections. Clinical treatments are directed only toward relieving the most prominent symptoms of
each clinical syndrome. Therefore, we need to develop novel EV71 and CVA16 inhibitors.
In recent years, there are some organic molecules were identified for HFMD therapy [11], and
EV71-related anti-virus activity had been identified by screening traditional Chinese medicines [12,13].
EV71 and CVA16, which are non-enveloped viruses, comprise a positive single-stranded RNA
genome packed within an outer capsid composed of four proteins (VP1–VP4) [1]. Mature EV71, like
other enteroviruses, has a hydrophobic pocket on VP1 that penetrates from the surface deep into the
interior of the VP1 β-barrel, which underlies a canyon-like surface depression [14] and harbors a
natural lipid (possibly sphingosine) called the “pocket factor” [15]. Releasing the pocket factor
contributes to activation because it is required for initiating the uncoating and liberation of the RNA
genome; thus, potential antivirals simulate natural lipids and inhibit uncoating [16], which provides a
theoretical basis for virtual screening.
Our laboratory built an in-house chemical database of nearly 10,000 compounds. Based on the
crystal structure of the EV71 capsid protein [Protein Data Bank (PDB) ID: 3VBH], we screened our
database using DOCK 6.0 and found novel N-containing heterocyclic piperazines and piperidines as
EV71 inhibitors and CVA16 inhibitors. Our findings provide a basis for anti-EV71 and anti-CVA16
drug development.
2. Results and Discussion
2.1. Computer Screening
Identifying a suitable site on the VP1 protein for binding small organic ligands is critical for
successfully implementing the computer screening strategy for developing effective anti-EV71
inhibitors. The binding site is a hydrophobic pocket that penetrates from the surface deep into the
interior of the VP1 β-barrel and harbors a natural lipid (possibly sphingosine) called the “pocket
factor” by Wang [16]. The closure of the VP1 pocket after the pocket factor was expelled initiated
EV71 uncoating with pocket collapse; which switches mature EV71 into expanded particles and
transforms into a conformation with a high-receptor affinity. Therefore; the release of the pocket factor
is required for initiating viral uncoating; and the discovery of potential anti-EV71 inhibitors focuses on

Molecules 2013, 18
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compounds that are structurally similar to natural lipids to occupy the hydrophobic pocket; the binding
site. Some amino acids are important in EV71 VP1 and the natural lipid complex crystal structure
(PDB ID: 3VBH). The hydroxyl group of natural lipids was 2.88 Å away from the N atom of Ile113. It
indicates the formation of H-bond. Morever the natural lipid and residues Ile111; Asp112; Thr114;
Val192; Met230; and Phe233 interact through molecular electrostatic potential; and Van der Waals force.
The identification of a VP1 capsid-binding pocket facilitated the computational screening of 10,000
organic compounds using our in-house chemical database for potential ligands for this pocket. This
screening selected 60 compounds for cell culture cytopathic effect (CPE) assays. Most of the 60
compounds were known as HRV inhibitors before. It is not surprising since the homology of EV71
VP1 coat protein (Sequence ID: Q91PB1) with human rhinovirus (HRV) 2 coat protein (PDB ID:
1FPN) and HRV16 coat protein (PDB ID: 1AYM) from UniProtKB were up to 40% and 38%,
respectively. Among the 60 cmpounds identified, 8e is novel and 9e is known to inhibit HRV and their
docking models in the hydrophobic pocket are shown in Figure 1 as compared to compound 1
developed by Shih et al. [10] shown in Supplementary Figures S2–S3. Ile111, Val192, Met230, and
Phe233 formed hydrophobic interactions with different moieties of compounds 1, 8e, and 9e. The
carbonyl group of imidazolidinone in compound 1 was 3.49 Å away from the N atom of Ile113,
indicating formation of H-bond. It was also observed that the formation of π-π effects between the
compound 1 and residues Phe155 and Trp203 existed. Besides, Phe137, Phe155 and 8e interacted
through π-π effect, whereas Tyr201 and Asn228 performed σ-π effect with 9e. It seems that those
compounds have more potential for EV71 VP1 inhibition. Fifteen of these compounds displayed
significant inhibitory effect (median inhibitory concentration [IC50]: 1 μM to 100 μM), with the
structure of the 15 organic inhibitors shown in Table 1. This finding also indicates further structural
optimization directions for our research.
Figure 1. (a) Structures of compounds 8e, and 9e. (b) The natural lipid (pink) was docked
in the hydrophobic pocket on the VP1 of EV71. The hydroxy group of the natural lipid was
2.88 Å from the N of Ile113 (green), which suggests a potential H-bond (red). (c) The
structure of 8e (blue) docked in the pocket with important interactions shown by orange
lines. (d) The structure of 9e (orange) docked in the pocket with important interactions
shown by orange lines.
(a)

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Figure 1. Cont.
(b)
(c)
(d)

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Table 1. Anti-EV71 activity, anti-CVA16 activity, and cytotoxicity of compounds 8a–f and compounds 9a–h.
EV71
IC50
(μM)
CVA16
TC50
(μM)
TC0
(μM)
Cpd
Ar
X
Y n
R
IC50
(μM)
>7.7
SI
SI
1
-
-
-
-
-
78 ± 5.3
7.7 ± 0.5
0.6 ± 0.0
137
-
N N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
9a
9b
9c
9d
N
N
N
C
C 2
C 2
C 2
C 2
C4H9
19.2 ± 0.7
7.7 ± 0.3
1.2 ± 0.0
21.6 ± 1.4
>31.5
16.1
8.7
_
1.4 ± 0.1
>29.7
13.8
-
N N
N N
N N
189 ± 17.3
62.9 ± 2.4
193 ± 19.3
29.7 ± 2.2
31.5 ± 2.1
30.5 ± 2.0
OC2H5
10.8 ± 0.3
22.1 ± 1.1
5.8
8.7
CO2CH3
>31.5
_
9e
9f
C
C
C 2
N 2
CO2C2H5
CO2C2H5
>513
>512
>513
1.0 ± 0.2
513
12.7 ± 0.4
16.0 ± 0.9
>40.3
>32.0
128 ± 9.5
25.4 ± 1.7
>20.2
9g
9h
N
N
C 3
C 3
CO2CH3
CO2C2H5
256 ± 22.7
359 ± 21.5
128 ± 3.8
31 ± 4.3
16.0 ± 0.6
4.9 ± 0.2
16.0
73.7
50.8 ± 4.1
4.9 ± 0.2
5.0
73.7

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Table 1. Cont.
EV71
IC50
(μM)
CVA16
TC50
(μM)
TC0
(μM)
Cpd
8a
Ar
X
N
N
N
N
N
N
Y n
C 5
C 5
C 6
C 6
C 6
C 6
R
IC50
(μM)
SI
SI
OCH3
277 ± 2.2
66.6 ± 1.0
54.1 ± 8.1
56.0 ± 3.3
43.1 ± 4.7
15.6 ± 0.3
138 ± 9.6
33.3 ± 3.9
6.8 ± 0.1
28.0±2.5
6.8 ± 0.8
7.8 ± 0.4
100 ± 9.0
2.8
69.2 ± 5.5
5.3 ± 0.3
6.8 ± 0.4
14.0 ± 0.5
1.2 ± 0.5
7.8 ± 0.6
4.0
8b
8c
OC2H5
OC2H5
C2H5
21 ± 1.2
4.3 ± 0.1
4.4 ± 0.3
4.3 ± 0.0
3.9 ± 0.1
3.2
12.7
12.7
10.1
4.0
12.7
8.0
8d
8e
4.0
CH(CH3)2
C(CH3)3
34.8
2.0
8f

Molecules 2013, 18
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2.2. Chemistry
Compounds 9a–h (Table 1) were previously reported as HRV inhibitors [17,18]. Because
compounds 8a–f have never been reported, we describe their synthesis in this paper (Scheme 1). To
prepare piperazine-1-yl-alkyl alcohol 4a–b, ethyl piperazine-1-carboxylate 2 was coupled with
compounds 3a–b in the presence of K2CO3 in acetonitrile and exposed the ethoxycarbonyl group in the
presence of aq NaOH in ethanol. The 2-bromo group of 2-bromothiazole (5) was substituted with
compounds 4a–b in the presence of Na2CO3 in dimethylformamide (DMF) to yield compounds 6a–b.
Finally, compounds 8a–f were synthesized through the Mitsunobu reaction by coupling compounds
6a–b and 4-substituted phenols 7a–e.
Scheme 1. Synthesis of compounds 8a–f.
Reagents and conditions: (i) K2CO3, MeCN, reflux; (ii) 10% NaOH (aq), EtOH, reflux; and
(iii) Na2CO3, DMF, 80 °C.
2.3. Biology
The anti-EV71 activity, anti-CVA16 activity, and cytotoxicity of the compounds on the EV71 test
strain (Shenzhen 98, shzh01-8) and CVA16 (laboratory viral culture) were evaluated by using cell
culture cytopathic effect assays. The results are listed in Table 1.
2.3.1. Results for Anti-EV71 Activity
In the series of imidazolidinone compounds reported by Shia [10], the length of the alkyl linker
does not contribute significantly to antiviral activity. However, we found such length to be important,

Molecules 2013, 18
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as evidenced by our compounds 8b and 8c. The p-monosubstituted phenyl ring crucially affects
antiviral activity [10]. Similarly, the steric requirement of 4-electron donating substitution may have
affected the antiviral activity against EV71 via 8d–f. Moreover, the larger the alkoxy group, the higher
the activity, such as with compounds 8a–b and 9g–h. Compared with that of its corresponding
pyridine isomer 9f, the antiviral activity of the benzene ring of 9e was increased 25-fold. This effect
might be due to the strict steric requirement on the phenyl ring or to the increased in basicity of the
pyridine nitrogen. Although chloropyridine derivatives 9a–h have been known as potent human
rhinovirus capsid-binding inhibitors [17,18], it hasn’t been reported that the series of compounds manifests
anti-EV71 activity. We first discovered compounds 9a–h showed a wide range of anti-EV71 activity.
9a–f were the derivatives of pirodavir that was known as the anti-HRV capsid-binding inbititor [19]
(Supplementary Figure S1, IC50 = 1.2 ± 0.2 μM, TC50 = 31 ± 2.2 μM, SI = 25 [20]). However,
compound 9e exhibited high antiviral potency and an excellent selectivity index (IC50 = 1.0 μM,
TC50 > 512.97 μM, SI = 512.8).
2.3.2. Results for anti-CVA16 Activity
The evaluation of the series of compounds 8b–c revealed that the length of the alkyl linker was not
sensitive to anti-CVA16 activity. 9b exhibited moderate anti-EV71 activity, but it was inactive at the
highest concentration (29.74 μM, TC0 = 29.74 μM). The compounds with the alkoxy, ester, or alkyl
group at position 4 of the phenoxyl ring (compounds 8a–f and 9g–h) revealed that the suitable steric
volume, neither too large nor too small, may be significant for anti-CVA16. Compound 9e was similar
to its corresponding pyridine isomer 9f in terms of anti-coxsackie activity. These effects might be due
to distinct differences in the hydrophobicity of the compounds. It was the first time that their anti-CVA16
activity was reported.
3. Experimental
3.1. General
Melting points (mp; °C, uncorrected) were determined in open glass capillaries using a YRT-3
1
13
electrothermal melting point apparatus. The H-NMR and C-NMR were recorded on a Bruker ARX
400 MHz spectrometer (Karlsruhe, Germany). Chemical shifts are expressed in δ (ppm) with reference
to tetramethylsilane. The mass spectra were processed through a Waters Xevo G2 QTof electrospray
ionization (ESI) spectrometer (Denver, CO, USA). All solvents and reagents were purchased
commercially and used without further purification.
3.2. Computer Screening
The high-resolution X-ray structure of EV71 VP1 with the capsid-binding pocket was used as a
receptor for ligand docking. DOCK 6.0 is an automatic computerized method used to screen small
molecule databases for ligands that bind to a given receptor [21]. DOCK 6.0 defined the EV71 VP1
binding site with a set of overlapping spheres, the centers of which became the potential locations for
ligand atoms. The binding of an organic ligand to the VP1 hydrophobic pocket was evaluated based on
shape complementarity and simplified interaction energy (force field energy). Our in-house database

Molecules 2013, 18
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was chosen as the small-molecule database because it included approximately 10,000 available small
organic compounds. The molecular structures were generated using the CONCORD heuristic
algorithm (developed by R. Pearlman at the University of Texas). The parameters used for docking
were set to the defaults. For example, the maximum orientation was 1500, and the bump_max was 3.
Following four rounds of inflexible and flexible virtual screens, 50 molecules with the best shape
complementarity scores and 50 with the best force field scores were selected from the DOCK 6.0
screening. The resulting 60 compounds were then chosen for the cell culture CPE assay. Of these 60
compounds, 37 were from the shape list, 23 were from the force field list, and 9 were on both lists.
3.3. Chemical Synthesis
3.3.1. Synthesis of Compounds 4a,b, and 6a,b
5-(4-(Thiazol-2-yl)piperazin-1-yl)pentan-1-ol (6a). Ethyl piperazine-1-carboxylate 2 (25.5 g, 161.39 mmol),
5-bromopentan-1-ol (22.43 g, 161.39 mmol), and K2CO3 (55.68 g, 403.48 mmol) were refluxed
overnight in 200 mL of acetonitrile. Upon cooling, the reaction mixture was filtered to remove
insoluble solids, and the filtrate was evaporated in vacuo to remove the acetonitrile to produce an oily
residue. The crude product was purified via column chromatography (CH2Cl2/MeOH/Et3N 100:1:0.5)
to afford intermediate ethyl 4-(5-hydroxypentyl)piperazine-1-carboxylate as a yellow oil (20.48 g,
52.0%). Ethyl 4-(5-hydroxypentyl)piperazine-1-carboxylate (15.9 g, 65.09 mmol) and 10% aq NaOH
(150 mL) were then refluxed overnight in 150 mL of EtOH. The solvent was removed under reduced
pressure, and the crude residue was diluted with 200 mL of brine and extracted with dichlormethane
(5 × 200 mL). The organic layer was dried over MgSO4, filtered, and concentrated. The resulting
yellow oil 4a (9.69 g, 86.5%) was used for the next step without further purification. 4a (8.6 g, 50 mmol) in
DMF (10 mL) was added to a stirred suspension of 2-bromothiazole (12.30 g, 75 mmol) and Na2CO3
(5.3 g, 50 mmol) in DMF (40 mL) at 0 °C in a three-necked flask. The resulting mixture was stirred
overnight at 80 °C and removed in vacuo to yield a yellow solid. The residue was washed with ether
(3 × 200 mL) and recrystallized with petroleum ether to yield a white solid 6a (5.95 g, 46.5%).
6-(4-(thiazol-2-yl)piperazin-1-yl)hexan-1-ol (6b). Compound 6b was obtained as a white solid
(6.84 g, 21.4%) from 6-bromohexane-1-ol according to the same procedure.
3.3.2. Preparation of Compounds 8a–f
2-(4-(5-(4-Methoxyphenoxy)pentyl)piperazin-1-yl)thiazole (8a). Diethyl azodicarboxylate (0.35 g, 2 mmol)
was added to a solution of 6a (0.51 g, 2 mmol), 4-methoxyphenol (0.22 g, 2 mmol), and
triphenylphosphine (0.52 g, 2 mmol) in dry tetrahydrofuran (10 mL) at 0 °C in a three-necked bottle.
The resulting mixture was stirred overnight at ambient temperature. The solvent was then removed
under reduced pressure to yield a crude product, which was then purified via column chromatography
(petroleum ether/acetone 15:1) to provide a white solid of 8a (0.15 g, 20.7%): mp = 83 °C to 85 °C;
1H-NMR (CDCl3): δ7.20 (d, 1H, J = 3.6 Hz), 6.83 (s, 4H), 6.58 (d, 1H, J = 3.2 Hz), 3.92 (t, 2H, J = 6.4 Hz),
3.77 (s, 3H), 3.52 (br, 3H), 2.57 (br, 3H), 2.42 (br, 1H), 1.80 (m, 2H), and 1.50–1.60 (m, 4H); 13C-NMR
(dimethyl sulfoxide [DMSO]): δ171.37, 153.20, 152.67, 139.49, 115.25, 114.57, 108.08, 67.75, 57.72,

Molecules 2013, 18
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55.34, 51.92, 48.30, 28.72, 26.01, and 23.53; ESI-HRMS: m/z [M+H]+ calculated for C19H27N3O2S:
362.1902; found: 362.1902.
2-(4-(5-(4-Ethoxyphenoxy)pentyl)piperazin-1-yl)thiazole (8b). Compound 8b was obtained as a white
solid (0.25 g, 33.3%) from compound 6a according to the same procedure: mp = 79 °C to 81 °C;
1H-NMR (CDCl3): δ7.20(d, 1H, J = 3.6 Hz), 6.82 (s, 4H) , 6.57 (d, 1H, J = 3.2 Hz), 3.90–4.00 (m, 4H),
3.52 (br, 4H), 2.57 (br, 4H), 2.43 (br, 2H), 1.79 (m, 2H), 1.49–1.60 (m, 4H), and 1.39 (t, 3H); 13C-NMR
(DMSO): δ171.36, 152.60, 152.43, 139.46, 115.22, 115.15, 108.05, 67.72, 63.26, 57.71, 51.91, 48.29,
28.71, 26.00, 23.52, and 14.78; ESI-HRMS: m/z [M+H]+ calculated for C20H29N3O2S: 376.2059; found:
376.2061.
2-(4-(6-(4-Ethoxyphenoxy)hexyl)piperazin-1-yl)thiazole hydrochloride (8c). Diethyl azodicarboxylate
(0.35 g, 2 mmol) was added to a solution of 6b (0.54g, 2 mmol), 4-ethoxyphenol (0.28g, 2 mmol), and
triphenylphosphine (0.52 g, 2 mmol) in dry tetrahydrofuran (10 mL) at 0 °C in a three-necked bottle.
The resulting mixture was stirred overnight at ambient temperature. The solvent was then removed
under reduced pressure to yield a crude product, which was then purified via column chromatography
(petroleum ether/acetone 15:1) to provide a white solid of 8c (0.32 g). The white solid was added to
acetone (10 mL) to form a transparent solution. A saturated solution of hydrogen chloride in ether was
added dropwise to this solution until the pH was adjusted to about 5. The resultant precipitate was
1
filtered and dried to yield a white solid 8c (0.38 g, yield 41.1%): mp = 222 °C to 224 °C; H-NMR
(DMSO): δ10.99 (s, 1H), 7.26 (d, 1H, J = 3.6 Hz), 7.00 (d, 1H, J = 4.0), 6.83 (s, 4H), 3.88–3.95 (m, 6H),
13
3.49–3.57 (m, 4H), 3.09–3.14 (m, 4H), 1.68–1.73 (m, 4H), and 1.29–1.44 (m, 3H); C-NMR (DMSO):
δ170.54, 153.05, 152.96, 135.37, 115.71, 115.67, 110.35, 68.12, 63.77, 55.76, 49.87, 46.13, 29.02,
26.32, 25.59, 23.36, and 15.29; ESI-HRMS: m/z [M+H]+ calculated for C21H33Cl2N3O2S: 390.2213;
found: 390.2215.
2-(4-(6-(4-Ethylphenoxy)hexyl)piperazin-1-yl)thiazole hydrochloride (8d). 2-(4-(6-(4-Ethylphenoxy)-
hexyl)piperazin-1-yl)thiazole hydrochloride was obtained as a white solid (0.12 g, yield 13.4%) from
compound 6b according to the same procedure: mp = 240 °C to 242 °C; 1H-NMR (DMSO): δ 11.32 (s,
1H), 7.27 (d, 1H, J = 3.6 Hz), 7.06 (d, 2H, J = 8.4), 7.01 (d, 1H, J = 3.6), 6.79 (d, 2H, J = 8.4), 3.86–4.03
(m, 4H), 3.52–3.60 (m, 4H), 3.04–3.09 (m, 4H), 1.67–1.72 (m, 4H), 1.32–1.40 (m, 4H), and 1.09 (t, 3H,
J = 7.6 Hz); 13C-NMR (DMSO): 170.26, 156.70, 137.08, 135.57, 128.65, 114.25, 109.86, 67.17, 55.29,
49.55, 45.37, 28.48, 27.31, 25.81, 25.10, 22.93, and 16.03; ESI-HRMS: m/z [M+H]+ calculated for
C21H33Cl2N3OS: 374.2266; found: 374.2275.
2-(4-(6-(4-Isopropylphenoxy)hexyl)piperazin-1-yl)thiazole
hydrochloride
(8e).
2-(4-(6-(4-
isopropylphenoxy)hexyl)piperazin-1-yl)thiazole hydrochloride was obtained as a white solid (0.33 g,
yield 35.8%) from compound 6b according to the same procedure.: mp = 135 °C to 137 °C; 1H-NMR
(DMSO): δ10.93 (s, 1H), 7.23 (d, 1H, J = 3.6 Hz), 7.13 (d, 2H, J = 8.4 Hz), 6.98 (d, 1H, J = 4.0), 6.83
(d, 2H, J = 8.4), 3.91–4.00 (m, 4H), 3.45–3.57 (m, 4H), 3.08–3.13 (m, 4H), 2.82 (m, 3H), 1.68–1.74
(m, 4H), 1.35–1.47 (m, 4H), and 1.16 (d, 6H, J = 6.8 Hz); 13C-NMR (DMSO): 170.44, 156.75, 140.25,
139.55, 127.14, 114.19, 109.84, 67.16, 55.32, 49.75, 45.10, 32.59, 28.47, 25.80, 25.12, 24.20, and
22.98; ESI-HRMS: m/z [M+H]+ calculated for C22H35Cl2N3OS: 388.2423; found: 388.2429.

Molecules 2013, 18
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2-(4-(6-(4-tert-Butylphenoxy)hexyl)piperazin-1-yl)thiazole (8f). Compound 8f was obtained as a white
solid (0.33 g, 63.0%) from compound 6b according to the same procedure: mp = 30 °C to 32 °C;
1H-NMR (CDCl3): δ7.29 (t, 2H, J = 2.0 Hz), 7.20 (d, 1H, J = 3.6 Hz), 6.83 (d, 2H, J = 8.8 Hz), 6.57 (d,
1H, J = 3.6 Hz), 3.94 (t, 2H, J = 6.4 Hz), 3.52 (br, 4H), 2.57 (br, 4H), 2.41 (br, 2H), 1.78 (m, 2H),
1.37–1.58 (m, 6H), and 1.30 (s, 9H); 13C-NMR (DMSO): 171.37, 156.42, 142.44, 139.46, 126.02,
113.83, 108.04, 67.23, 57.73, 51.92, 48.28, 33.74, 31.38, 26.69, 26.21, and 25.51; ESI-HRMS: m/z
[M+H]+ calculated for C23H35N3OS: 402.2579; found: 402.2563.
3.4. Biological Evaluation
3.4.1. Neutralization Test [22]
This assay measured the ability of the test compounds to inhibit the CPE induced by EV71 and
CVA16 on Vero cells. The 96-well tissue culture plates were seeded with 20,000 Vero cells/mL. The
plates were incubated for 24 h at 37 °C. The virus (100 TCID50) mixed with different concentrations of
the test compounds was added to the cells, which were incubated at 37 °C for 2 h. After adsorption, the
infected cells were overlaid with 50 μL of Dulbecco’s modified Eagle’s medium with 5% fetal bovine
serum, and 2% DMSO. The concentration of the test compound required to reduce the virus-induced
CPE to 4+ relative to the virus control was expressed as IC50 according to the Reed–Muench method.
All assays were performed in triplicate.
3.4.2. Cytotoxicity Assay [23]
The Vero cells treated with the test compounds were incubated at 37 °C for 96 h. After incubation,
the cells were harvested and counted. The concentration of a test compound required to reduce cell
viability to 50% of the tested control culture was expressed as TC50.
4. Conclusions
A computer-based database screening strategy was employed to identify a novel series of aromatic
heterocyclic substituted piperazine and piperidine derivatives as ligands for the EV71 VP1
hydrophobic pocket that exhibits antiviral activity against EV71 and CVA16. Further mechanistic
studies on this new class of anti-EV71 inhibitors are in progress. Our study on the structure–activity
relationships of anti-EV71 and anti-CVA16 indicated that the space volume of the 4-electron donating
group substituent at the phenoxyl ring largely influenced the in vitro anti-EV71 activity of the new
class of potent inhibitors, and the appropriate steric requirement on the p-monosubstituted phenyl ring
may exhibit activity against CVA16. Interestingly, the anti-EV71 activity of this series of compounds
was reduced when the phenoxyl ring was substituted for the corresponding pridine isomer. Moreover,
the length of the alkyl linker influenced the anti-EV71 activity. However, both factors did not affect
the anti-CVA16 activity. The piperidine derivative 9e exhibited the most potent antiviral activity
against EV71 (IC50 = 1 μM), with no apparent cytotoxicity against Vero cells (TC50 > 512.97 μM),
compared with compound 1(IC50 = 1 μM, TC50 = 77.91 μM). Compound 8e exhibited excellent
activity against CVA16 (IC50 = 1.24 μM) and moderate activity against EV71 (IC50 = 4.28 μM), with

Molecules 2013, 18
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weak cytotoxicity on Vero cell lines (TC50 = 43.08 μM). Therefore, these compounds are very
promising candidates for further optimization towards a drug for HFMD treatment.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/5/5059/s1.
Acknowledgments
We acknowledge the financial support of the National High-Technology Research and
Development Program of China (863 Program; Grant No. 2012AA020301) and the National Science
and Technology Major Project of China (Grant No. 2012ZX09103003-001-002).
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8a–f, and 9a–h are available from the authors.
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