In Vitro Antiviral Activity of New Oxazoline Derivatives as Potent Poliovirus Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01482

The final stages of polio eradication are proving more difficult than the early phases, and the development of effective drugs and treatments is considered a priority; thus, the research is ongoing. A screening of our in-house chemical library against poliovirus Sabin strains led to the identification of compounds 5 and 6 as hits active at submicromolar concentrations. Derivatives of these compounds were synthesized as a preliminary structure-activity-relationship study. Among them, 7 and 11 were highly active against poliovirus Sabin 1-3. Compound 11 was also very potent against a large panel of wild and vaccine-derived polioviruses. Time-of-addition experiments suggest that 5 and 7 could be active at an early stage of viral replication, whereas 11 was active at same concentration at all stages of viral replication. A ligand-based approach was applied to find the common structural features shared by the new compounds and already-known poliovirus inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01482

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Article
In vitro Antiviral Activity of New Oxazoline
Derivatives as Potent Poliovirus Inhibitors
Valentina Noemi Madia, Antonella Messore, Luca Pescatori, Francesco Saccoliti,
Valeria Tudino, Alessandro De Leo, Luigi Scipione, Lucia Fiore, Eric Rhoden,
Fabrizio Manetti, M. Steven Oberste, Roberto Di Santo, and Roberta Costi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01482 • Publication Date (Web): 04 Dec 2018
Downloaded from http://pubs.acs.org on December 5, 2018
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In vitro Antiviral Activity of New Oxazoline
Derivatives as Potent Poliovirus Inhibitors
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a
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Valentina Noemi Madia, Antonella Messore, Luca Pescatori, Francesco
a
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Saccoliti, Valeria Tudino, Alessandro De Leo, Luigi Scipione, Lucia Fiore,
c
d
c
a*
Eric Rhoden, Fabrizio Manetti, M. Steven Oberste, Roberto Di Santo,
Roberta Costia
aDipartimento di Chimica e Tecnologie del Farmaco, Dipartimento di Eccellenza
2
018-2022, Istituto Pasteur-Fondazione Cenci Bolognetti, “Sapienza” Università
di Roma, p.le Aldo Moro 5, I-00185, Roma, Italy.
bIstituto Superiore di Sanità, CRIVIB, Viale Regina Elena 299, I-00161, Roma,
Italy.
cDivision of Viral Diseases, Centers for Disease Control and Prevention, 1600
Clifton Road, Atlanta, GA 30329, USA.
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dDipartimento di Biotecnologie Chimica e Farmacia, Dipartimento di Eccellenza
2018-2022, Università degli Studi di Siena, via A. Moro 2, I-53100 Siena, Italy,
LDS Lead Discovery Siena Srl, via Fiorentina 1, 53100 Siena, Italy
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KEYWORDS. Poliomyelitis, Poliovirus, Antivirals, Oxazoline, Vaccine-derived
polioviruses.
ABSTRACT: The final stages of polio eradication are proving more difficult than
the early phases and the development of effective drugs and treatments is considered a
priority, thus the research is ongoing. A screening of our in-house chemical library
against poliovirus Sabin strains led to identification of 5 and 6 as hits active at
submicromolar concentration. Derivatives of these compounds were synthesized
as a preliminary structure-activity relationship study. Among them, 7 and 11
were highly active against poliovirus Sabin 1, 2 and 3. 11 was also very potent
against a large panel of wild and vaccine-derived polioviruses. Time of addition
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experiments suggest that 5 and 7 could be active at an early stage of viral
replication, while 11 was active at same concentration at all stages of viral
replication. A ligand-based approach was applied to find the common structural
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Introduction
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Poliovirus (PV), the causative agent of poliomyelitis, is a member of the genus
Enterovirus within the Picornaviridae family, a large family of small non-
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enveloped positive-stranded RNA viruses. Although eliminated in all but two
countries, it still represents a potential risk for humans until polio eradication is
accomplished at global level.
Outbreaks of poliomyelitis caused by vaccine-derived polioviruses (VDPVs) that
reverted to wild phenotype, unexpectedly occurred in 2000 in Hispaniola2
followed by subsequent detections of independent emergences in other
countries. Immunodeficient long-term PV excretors constitute a significant reservoir for PV
post-eradication and are a major concern for the WHO. The occurrence of immunodeficiency-
related VDPV (iVDPV) must be considered a potential source for outbreaks and
for reemergence of polio after eradication.
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At present, the only means to prevent poliomyelitis are the two type of
vaccines: the Sabin live attenuated oral polio vaccines (OPVs) and the Salk
inactivated polio vaccine (IPV) given to young children routinely or during
supplemental immunization activities (SIAs).
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Antiviral drugs would offer a promising complementary or alternative approach
to the use of vaccine to control PV infections, especially for chronic persistent
infections in immunodeficient individuals during the final stages of eradication
and in the post-eradication era. For this reason, the WHO has supported the
development of antiviral drugs against PV.
However, no antiviral agents are currently available in the market to treat
infections by PV and other enteroviruses, and only the protease inhibitor AG-
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404 (1) and the uncoating inhibitor pocapavir (V-073, 2) are in clinical trials
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(Figure 1). Among them, the latter is the most advanced and was recently
demonstrated to be active in an oral vaccine human challenge model. Additional
candidates acting as uncoating inhibitors such as H1PVAT (3) and pirodavir (4)
continue to be profiled and evaluated with the aim to have different antivirals
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available for a combined use and to overcome the problem of possible
emergence of resistant variants.5
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Figure 1. Structures of the anti-PV agents in clinical trials or under evaluation:
the protease inhibitor 1 and the uncoating inhibitors 2-4.
Recently, we reported some 3(2H)-isoflavenes as weak anti-PV agents that
prevented viral entry with a mechanism similar to that of 2, by interfering with
receptor binding or by preventing conformational changes needed for viral
capsid uncoating.6,7 Mutations that interfered with the antiviral activity of these
compounds appeared during in vitro replication of type 2 polio vaccine strains
in the presence of these compounds. Some mutations acted directly by
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affecting binding while others exerted their effect indirectly by altering viral
capsid stability.6,8 However, our attempts to improve the activity of these
compounds or their drug-like properties were unsuccessful.
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Thus, as a further attempt to discover new anti-polio agents characterized by
scaffolds different from both the drugs in clinical trials and from our isoflavenes,
we decided to perform a screening of our in-house library of compounds
including various heterocyclic derivatives and related intermediates, previously
designed as potential antitumor or antimicrobial drugs. The library includes
about 1000 molecular entities but a selection has been performed picking up
compounds characterized by different chemical scaffolds and excluding
isoflavene derivatives. The resulting 40 compounds were tested at fixed
concentration (10 M) against Sabin 1, Sabin 2, and Sabin 3 polio strains. As
5 showed a significant activity, we decided to test also 6, an analogue included
in the original library. Both compounds were further assayed at different
concentrations and showed activity at concentration ranging from about 0.2 to
91 M (Table 1).
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Results and Discussion
Compounds and
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are
5-(4-(4,5-dihydrooxazol-2-yl)phenoxy)-1-(5-
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methylthiophen-2-yl)pentan-1-one derivatives (Table 1) and can be structurally
described as two aryl portions (one thienyl and one phenyl) separated by an
oxyalkylcarbonyl linker. Thus, to some extent, these scaffolds could resemble
those of compounds 2-4.
To increase the potency of these hits we decided to perform some preliminary
structure-activity relationship (SAR) studies. First, the aromatic portions (5-
methylthiophen-2-yl and (4-(4,5-dihydrooxazol-2-yl)phenyl moieties) of the 5 and
6 were kept unchanged while the spacer X was modified (Figure 2). In
particular, we designed and synthesized new derivatives 7-13 in which the
oxygen atom of 5 and 6 was replaced by sulfur atom, sulfinyl or sulfonyl
functions, or methylene, ethylene, and ethine groups. We took care to retain the
distance between the phenyl ring and carbonyl group to possibly increase the
lipophilicity of the new compounds. Finally, we designed two further molecules
characterized by a phenyl spacer, partially mimicking the one of 2 (Figure 1
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and Figure 2), resulting in compounds 14 and 15 (oxygen and sulfur
derivatives, respectively) as conformationally restrained derivatives of 5 and 7.
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Figure 2. Structures of hit compounds 5 and 6 and the newly designed
oxazolynes 7-15.
Chemistry. The alkyl oxazoline derivatives 5 and 6 were synthesized as
reported in Scheme 1. From 2-methylthiophene and 5-chlorovaleroyl chloride in
the presence of AlCl , we synthesized the desired compound 16 that was
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reacted with derivatives 17 or 18 in N,N-dimethylformamide (DMF) in the
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chromatographic purification, 5 and 6, respectively. Oxazolines 17 and 18 were
synthesized from commercially available 4-hydroxybenzonitrile that reacted with
the proper ethanolamine in anhydrous chlorobenzene in the presence of
anhydrous ZnCl2.
Scheme 1. Synthetic pathway for 5 and 6 derivativesa
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ethanolamine, dry ZnCl , dry chlorobenzene, 160 °C, 24 h; (iii) (R)-(-)-2-amino-
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The newly designed compounds 7-10 were synthesized following a multistep
approach as described in Scheme 2. Compound 16 was reacted with 4-
mercaptobenzonitrile in DMF in the presence of K CO as a base to obtain the
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desired thioether 19. The last was reacted in the presence of opportune
ethanolamine and Si-propylsulfonic acid to obtain, after chromatographic
purification, the desired oxazolines 7 and 8. At the same time, 19 was oxidized
in two different ways (with m-chloroperbenzoic acid, mCPBA, or H O /POCl ) to
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obtain the respective sulfonyl nitrile 20 and sulfinyl nitrile 21 that were
successively reacted with ethanolamine and Si-propylsulfonic acid to obtain the
oxazolines 9 and 10, respectively.
The synthesis of 11 was performed following the procedure described in
Scheme 3. Compound 22 was synthesized though Heck reaction form 4-bromo-
benzonitrile and 5-hexenoic acid, then reacted with oxalyl chloride in
dichloromethane (DCM) in the presence of catalytic amount of DMF to obtain
the acyl chloride 23, that was rapidly reacted with 2-methylthiophene to obtain
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the intermediate 24. The last compound was reacted with ethanolamine and Si-
propylsulfonic acid to obtain 11.
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Scheme 2. Synthesis of the alkyl oxazolines 7-10a
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ii) ethanolamine, Si-propylsulfonic acid, 120 °C, 4 h; (iii) (R)-(-)-2-amino-1-
propanol, Si-propylsulfonic acid, 120 °C, 4 h; (iv) mCPBA, CHCl , room temp,
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0 min; (v) H O , POCl , CHCl /EtOH, room temp, 1 h.
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Scheme 3. Synthesis of the alkyl oxazoline 11a
NC
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2
3
4
5
6
7
8
9
0
i
ii
iii
iv
+
HO
CN
O
Cl
HO
S
24
O
O
O
S
O
22
23
1
1
a
Reagents and conditions: (i) TBACl, Pd(OAc) , PPh , KOAc, dry DMF, room
2
3
temp, 7 d; (ii) oxalyl chloride, dry CH Cl , dry DMF (cat.), N , room temp, 2 h;
2
2
2
(iii) 2-methylthiophene, AlCl , dry CH Cl , N , room temp, 2 h; ethanolamine, Si-
3
2
2
2
propylsulfonic acid, 120 °C, 4 h.
The synthesis of 12 and 13 was performed following the chemical pathway
reported in Scheme 4. Alkyne 25 was synthesized though Heck reaction from
4
-bromo-benzonitrile and 5-hexinoic acid, then reacted with oxalyl chloride in
DCM in the presence of a catalytic amount of DMF to obtain the acyl chloride
26, that was rapidly reacted with 2-methylthiophene to obtain the intermediate
2
7. The last compound was reacted with ethanolamine and Si-propylsulfonic
1
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acid to obtain 13. The nitrile 27 was reduced with Pd/C under a hydrogen
atmosphere to obtain the saturated compound 28 that was further reacted with
ethanolamine and Si-propylsulfonic acid to obtain 12 after column
chromatography.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Synthesis of the alkyl oxazolines 12 and 13 a
a
Reagents and conditions: (i) TBACl, Pd(OAc) , PPh , KOAc, dry DMF, room
2
3
temp, 7 d; (ii) oxalyl chloride, dry CH Cl , dry DMF (cat.), N , room temp, 2 h;
2
2
2
1
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9
(
iii) 2-methylthiophene, AlCl , dry CH Cl , N , room temp, 2 h; (iv) ethanolamine,
3
2
2
2
Si-propylsulfonic acid, 120 °C, 4 h; (v) Pd/C cat., H , EtOAc, room temp, 30
psi, 2 h.
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis of 14 and 15 was performed as described in Scheme 5. As
the first step, 3-chloromethylbenzoyl chloride and 2-methylthiophene were
reacted in DCM in the presence of AlCl3 to obtain the ketone 29 that
underwent nucleophilic aliphatic substitution on chloromethyl group by compound
1
7 in the presence of K CO to obtain the oxazoline 14. The intermediate
2
3
oxazoline 17 was obtained by reaction of 4-cyanophenol with ethanolamine in
the presence of dry ZnCl2. Intermediate 29 was reacted with 4-
mercaptobenzonitrile in the presence of K CO as a base to obtain the nitrile
2
3
3
0 that was in turn reacted with ethanolamine and Si-propylsulfonic acid to
obtain 15.
1
5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Synthesis of the alkyl oxazolines 14 and 15a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
OH
N
O
ii
O
N
CN
Cl
17
O
iii
Cl
O
O
S
i
Cl
O
S
29
14
iv
N
CN
O
S
v
S
O
O
15
S
30
S
a
Reagents and conditions: (i) 2-methylthiophene, AlCl , dry CH Cl , N , room
3
2
2
2
temp, 2 h; (ii) ethanolamine, dry ZnCl , dry chlorobenzene, 160 °C, 24 h; (iii)
2
K CO , DMF, 80 °C, 4 h; (iv) 4-mercaptobenzonitrile, K CO , dry DMF, 80 °C,
2
3
2
3
4
h; (v) ethanolamine, Si-propylsulfonic acid, 120 °C, 4 h.
1
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Evaluation of Biological Activities. All the newly synthesized derivatives 7-15
were assayed in vitro against the three polio Sabin strains, in parallel with 5
and 6 (Table 1). Compounds 9, 10, 13, and 15 were inactive against all three
Sabin strains, while derivatives 6, 12, and 14 were inactive against Sabin 1 but
active at micromolar concentration against Sabin 2 and Sabin 3. Interestingly,
compounds 5, 7, 8, and 11 were active against all Sabin strains and at
submicromolar concentration, with the exception of 5 against Sabin 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Antiviral activities of hits 5 and 6, newly synthesized compounds 7-15
and reference drugs 1-4 against three polio Sabin serotypes.
Compd Sabin 1
EC50a (µM)
Sabin 2
Sabin 3
EC50 (µM)
EC50 (µM)
5
6
7
8
9
90.800 ± 1.400
>100
0.187 ± 0.059
0.417 ± 0.242
0.137 ± 0.010
0.302 ± 0.014
>10
0.367 ± 0.027
1.323 ± 0.205
0.330 ± 0.008
0.530 ± 0.043
>10
0.118 ± 0.012
0.169 ± 0.018
>10
1
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1
1
1
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2
2
2
2
2
2
3
3
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3
3
3
3
3
3
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4
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4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
1
1
1
1
0
1
2
3
>10
>10
>10
0.338 ± 0.106
>10
0.027 ± 0.002
9.273 ± 0.101
9.087 ± 0.834
>10
0.053 ± 0.016
9.070 ± 0.130
9.160 ± 0.036
>10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>10
14
>10
1
5
>10
>10
>10
1b
2c
3d
4e
0.173 ± 0.137
0.017 ± 0.000
0.010 ± 0.001e
0.0045
0.305 ± 0.148
0.016 ± 0.012
0.026 ± 0.014
0.137 ± 0.007
0.020 ± 0.007
0.218 ± 0.162
0.008
0.005
a
e
Effective concentration 50% (µM). Reference 3. c Reference 4. d Reference
9. Reference 10.
b
f
The most interesting hits found in the random screening, (5, 7 and 11) that
showed the best activity in the assays against Sabin strains, have been further
assayed against a large panel of wild, iVDPV and circulating VDPV (cVDPV)
(Table 2). The prototype 5 was inactive against 7 isolates, active at micromolar
concentrations (2.10-67.47 M) against 15 isolates and at submicromolar
1
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9
concentrations (0.02-0.99 M) against 22 isolates. In particular, it was very
potent against 9 isolates showing nanomolar activity (Table 2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. In vitro antiviral activity of 5, 7 and 11 in HeLa cells against PV
Sabin, wild and VDPV polio strains.
EV
Entry Code
No.
5
7
11
Serotyp
e
Country-Year
Origin
EC50a ± SD EC50a ± SD EC50a ± SD
1
10688 Taiwan-2003
10689 Taiwan-2003
PV1
PV1
iVDPV 0.04 ± 0.00 2.80 ± 1.06 0.040 ± 0.01
iVDPV 0.06 ± 0.00 2.03 ± 0.56 0.034 ± 0.01
cVDPV 48.7 ± 15.2 2.00 ± 0.36 1.26 ± 0.19
cVDPV 67.5 ± 19.5 0.56 ± 0.11 0.98 ± 0.14
cVDPV >100 ± 0.00 >10 ± 0.00 0.78 ± 0.12
cVDPV 24.2 ± 7.72 >10 ± 0.00 0.98 ± 0.66
iVDPV 7.22 ± 1.15 6.23 ± 1.76 2.30 ± 1.09
iVDPV 8.41 ± 1.58 7.43 ± 0.61 0.58 ± 0.31
iVDPV 7.89 ± 1.79 4.90 ± 1.31 1.13 ± 0.32
iVDPV 0.07 ± 0.01 3.73 ± 1.31 0.02 ± 0.01
iVDPV >100 ± 0.00 >10 ± 0.00 1.11 ± 0.16
2
3
10690 Phillipines-2004 PV1
10691 Phillipines-2004 PV1
10692 Phillipines-2004 PV1
10693 Phillipines-2004 PV1
10694 USA-MN-2005 PV1
10695 USA-MN-2005 PV1
10222 USA-MD-1981 PV1
4
5
6
7
8
9
10
10223 USA-IN-1982
PV1
1
1
10224 USA-NY-1987 PV1
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
1
2
10225 USA-NC-1990 PV1
iVDPV 35.7 ± 3.97 >10 ± 0.00 0.81 ± 0.17
cVDPV 4.61 ± 2.88 8.47 ± 0.65 0.40 ± 0.16
Dominican
Republic-2000
13
10235
10236
PV1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Dominican
Republic-2000
14
15
PV1
PV1
PV1
cVDPV 2.72 ± 1.58 0.72 ± 0.16 0.72 ± 0.12
cVDPV 5.75 ± 5.66 6.47 ± 1.78 0.28 ± 0.05
cVDPV 2.10 ± 1.50 7.47 ± 0.67 0.19 ± 0.05
10237 Haiti-2000
Dominican
10238
1
1
6
7
Republic-2000
10239 Haiti-2001
10240 Haiti-2001
10241 Haiti-2001
10242 Haiti-2001
PV1
PV1
PV1
PV1
cVDPV 0.94 ± 0.21 5.63 ± 1.50 0.22 ± 0.07
cVDPV 3.62 ± 1.35 8.47 ± 1.25 0.27 ± 0.04
cVDPV 3.17 ± 3.04 8.07 ± 0.35 0.37 ± 0.04
cVDPV 37.9 ± 6.68 8.20 ± 1.40 0.02 ± 0.01
18
1
9
20
Madagascar-
2
1
10219
2002
PV2
PV2
cVDPV 0.03 ± 0.01 1.20 ± 0.20 0.03 ± 0.01
cVDPV 0.04 ± 0.02 1.00 ± 0.61 0.02 ± 0.01
Madagascar-
2
2
2
3
10220
2002
10221 USA-CA-1999 PV2
10226 USA-PA-1991 PV2
10227 USA-PA-1991 PV2
10228 USA-NY-1992 PV2
iVDPV 0.02 ± 0.02 2.37 ± 1.25 0.02 ± 0.01
iVDPV >100 ± 0.00 0.13 ± 0.03 0.02 ± 0.01
iVDPV 0.03 ± 0.01 4.33 ± 2.00 0.04 ± 0.01
iVDPV 10.1 ± 1.49 9.10 ± 0.25 0.50 ± 0.17
cVDPV 0.55 ± 0.25 2.77 ± 0.40 0.51 ± 0.18
cVDPV 0.10 ± 0.00 5.33 ± 1.21 0.03 ± 0.00
cVDPV 0.05 ± 0.01 5.33 ± 1.00 0.03 ± 0.00
24
2
2
2
2
2
5
6
7
8
9
10229 Egypt-1993
10230 Egypt-1993
10231 Egypt-1998
PV2
PV2
PV2
2
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10232 Egypt-1999
10233 Egypt-1999
10234 Egypt-1999
10243 Nigeria-2002
10804 Egypt-2007
10805 Iran-2007
PV2
cVDPV 0.54 ± 0.29 6.33 ± 0.86 0.023 ± 0.01
cVDPV 0.45 ± 0.36 8.47± 0.93 0.19 ± 0.17
cVDPV 0.03 ± 0.01 2.47 ± 0.49 0.02 ± 0.02
31
PV2
PV2
PV2
PV3
PV3
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33
c
0.04 ± 0.00 1.77 ± 0.67 0.03 ± 0.01
3
4
iVDPV >100 ± 0.00 0.44 ± 0.25 0.02 ± 0.00
iVDPV 0.27 ± 0.10 1.50 ± 0.44 0.03 ± 0.00
35
Brunhild
USA-1939
e
3
6
PV1
Wild
Wild
0.99 ± 0.09 0.47 ± 0.21 0.07 ± 0.01
>100 ± 0.00 >10 ± 0.00 0.07 ± 0.02
Mahone
USA-1942
y
3
3
7
8
PV1
PV2
Lansing USA-1937
MEF-1 Egypt-1942
Wild
Wild
Wild
0.59 ± 0.07 2.27 ± 0.59 0.06 ± 0.01
>100 ± 0.00 1.26 ± 0.53 0.03 ± 0.00
>100 ± 0.00 2.93 ± 3.01 0.24 ± 0.04
0.35 ± 0.03 1.67 ± 0.02 0.04 ± 0.01
0.17 ± 0.12 1.11 ± 0.29 0.95 ± 0.12
39
PV2
PV2
4
4
4
4
4
4
0
1
2
3
4
P712
Saukett USA-1962
Leon USA-1937
USA-1954
PV3 Wild
PV3 Wild
Sabin 1 Vaccine strain PV1
Sabin 2 Vaccine strain PV2
Sabin 3 Vaccine strain PV3
Effective concentration 50% (µM).
Vaccine 12.5 ± 0.78 0.70 ± 0.36 0.34 ± 0.11
Vaccine 0.03 ± 0.00 0.16 ± 0.01 0.03 ± 0.01
Vaccine 0.32 ± 0.04 0.33 ± 0.01 0.05 ± 0.02
5
a
2
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2
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4
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4
4
4
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4
5
5
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5
5
5
5
5
5
5
6
Compound 7 was inactive against 5 isolates, active at low micromolar
concentrations (1.00-9.10 M) against 32 isolates and at submicromolar
concentrations (0.13-0.72 M) against 8 isolates (Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
More interestingly, 11 showed very potent activity on a wide number of
strains. It was active at micromolar concentration (1.109-2.300 M) on 3 strains
and at submicromolar or nanomolar concentrations against the remaining 41
strains. In particular, it was active at nanomolar concentration against 20 strains
(
20-69 nM, Table 2).
Compounds 5, 7, and 11 were further tested in time of addition experiments
against Sabin 2 to determine at what stage of the viral cycle the compounds
acted (Figure 3). Interestingly, compounds 5 and 7 showed a decrease of
activity after 1 h post-infection (PI). During the six hours needed for the
complete PV life cycle the lost of activity is increased for 5 and stabilized for 7.
These results suggest that these compounds are active at an early stage of
viral replication. Surprisingly, the derivative 11 acts at any time of viral replication,
probably inhibiting or binding a target that is always present during the replication cycle.
2
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compound 5
compound 7
compound 11
no compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
1
0
1
2
4
6
8
Time of Compound Addition (h)
Figure 3. Effect of compounds in the time of addition assay. Time of addition
experiments on 5, 7, and 11 performed on Sabin 2 virus. Titers indicate the
presence of 1 M 5, 7 or 11 and 100 CCID50 of poliovirus. Titration results
11 12
were obtained using the virus titration assay. , Data represent mean values
from three independent experiments.
Cytotoxicity assays on HeLa cells were also performed on the most active
compounds (5, 7, and 11) to determine the selectivity of these prototypes.
While 7 was slightly cytotoxic at a concentration of 20 µM, 5 and 11 were not
2
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5
6
cytotoxic at concentrations up to 50 µM and they had selectivity indexes (SI)
>1667 and >1852, respectively (Table 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Cytotoxicity in vitro of most active compounds 5, 7 and 11.
Comp
d
EC50a
(µM)
Cytotoxicity
(CC50 , µM)
Therapeutic ratio
(CC /EC )
Virus
SD
Date
b
50
50
5
7
Sabin 2 0.03
Sabin 2 0.137
Sabin 2 0.027
0.010
0.010
0.002
>50
20
07/09/13 >1667
27/02/15 146.0
27/02/15 >1852
1
1
>50
a
b
Effective concentration 50% (µM).
Cytotoxic concentration 50% on Hela
cells (µM).
Preliminary SAR considerations
Some preliminary SAR can be described considering the activity of 5-15
against PVs. Since the inhibitors designed in this work have been tested
against a wide number of PV strains, only the activities against Sabin 2
serotype reported in Table 1 will be considered in this paragraph.
2
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A classic bivalent bioisosteric replacement of the oxygen atom of the ether
function of 5 with a methylene group led to 12 that showed a 50-fold decrease
in antiviral activity (0.187 vs 9.273 M, respectively). However, a partial
rigidification of the alkyl spacer with the trans-styryl moiety found in 11 restored
a submicromolar activity (0.027 M) better than that of 5. These results clearly
suggested that the presence of atoms or chemical groups able to interact with
the phenyl  system of these compounds caused an improvement in activity in
comparison to the linear alkyl spacer.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
4
4
4
4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
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7
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9
0
1
2
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0
1
2
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0
1
2
3
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5
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0
1
2
3
4
5
6
7
8
9
0
The SAR of the new oxazolines could be explained on the basis of the
different preferred conformations adopted by the phenoxy, styryl, and benzyl
moieties found in 5, 11, and 12, respectively. In fact, the Ph-O-CH - and styryl
2
moieties are characterized by a planar arrangement consequent to a significant
rehybridization and the formation of an extended  system between the phenyl
ring and the oxygen lone pair of 5, as well as between the phenyl ring and the
3
olefinic  system in 11. On the contrary, the sp character of the corresponding
alkyl carbon atom of 12 forced the overall alkyl chain to be out of the plane of
2
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1
1
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1
1
1
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1
1
2
2
2
2
2
2
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2
2
2
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5
5
6
the phenyl ring and to adopt a shape significantly different from those found for
5 and 11.
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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2
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8
9
0
A similar trend of SAR considerations, with phenoxy and styryl compounds
more active than the corresponding benzyl analogues, was previously reported
for a series of phenyl oxazole inhibitors of blood platelet aggregation.13,14
Moreover, the additional bioisostere of 5 that contained a thiophenol moiety
(
7) also retained a submicromolar activity (0.137 M), while its oxidation
products 9 and 10 (the corresponding sulfone and sulfoxide derivatives,
respectively), that differed widely in polarity and hybridization, were inactive.
Finally, the introduction of a methyl group on the oxazoline ring of 5 led to 6
that was less active if compared to the parent compound. A similar trend was
found comparing the activity of the thio derivative 7 with that of its methylated
counterparts 8.
Molecular Modelling
A ligand-based approach has been applied to find the common structural
features shared by the new compounds and already known PV inhibitors
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(
namely, 2-4). As a result, a four-feature pharmacophoric model was obtained,
comprised of one hydrogen bond acceptor group (HBA, red sphere, Figure 4),
two aromatic rings (RA1 and RA2, orange rings), and a hydrophobic region
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
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5
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0
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0
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2
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9
0
(
HY, green sphere). Derivative 5, taken as the representative compound of the
new inhibitors, was able to fulfill all the pharmacophoric features (Figure 4, a):
the oxygen atom of the oxazoline ring represented the hydrogen bond acceptor,
the phenyl and thiophene moieties matched the aromatic ring features, and the
methyl group on the thiophene ring was accommodated within the hydrophobic
region of the pharmacophore. As a comparison, 2 was also able to match the
pharmacophore (Figure 4, b). In fact, the oxygen atom of its methoxy group
was the hydrogen bond acceptor, the two terminal phenyl moieties fitted the
aromatic ring features, and one of the two meta chlorine atoms of the m,m-
disubstituted phenyl ring was located within the hydrophobic feature.
Although the three-dimensional coordinates of the VP1 capsid protein of Sabin
2
PV are not available, a rough comparison between the pharmacophoric model
and the structural properties of a Sabin 1 PV homology model could be
2
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6
attempted. In fact, the three-dimensional structure of Sabin 1 was recently
generated by homology modeling, and its complexes with 2-4 have been built
0
1
2
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0
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0
9
by docking calculations. The alignment of the VP1 capsid protein of Sabin 1
(
N0DLC0, 302 amino acids) with VP1 capsid protein of Sabin 2 (N0DQQ2, 301
amino acids) performed with Clustal Omega (UniProt Consortium)15 resulted in
very similar primary sequences, with 234 identical positions (77.5% identity) and
4
9 similar positions. Moreover, 20 (namely, Trp108, Ile110, Thr111, Tyr112,
Lys113, Phe130, Met132, Phe136, Ile157, Tyr159, Pro181, Ser182, Ile183,
Ile194, Val196, Val199, Tyr205, Asp236, Phe237, and Leu240) out of the 22
amino acids surrounding 4 (at a distance lower than 6 Å) in the X-ray three-
dimensional structure with the VP1 capsid protein of type 1 Mahoney strain
(
1PO2 within the Protein Data Bank)16 are conserved in comparison to the
N0DLC0 and N0DQQ2 sequences. This suggested that the corresponding
binding sites that accommodated the small molecule inhibitors could share very
similar structures and interaction patterns with inhibitors.
2
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A visual inspection of the interaction pattern described for the three docked
inhibitors identified several structural elements shared by the pharmacophoric
model and the structure of VP1-inhibitor complexes. In particular, the hydrogen
bond acceptor HBA of the pharmacophore was represented by the methoxy
oxygen of 2 and by the carbonyl group of 4 that made a hydrogen bond
interaction with VP1 Tyr112 (Figure 4, b and c).9 Moreover, RA2 of the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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0
pharmacophore could be accommodated within an aromatic cage constituted by
Phe130 (reported to make  interaction with the methoxyphenyl ring of 2),9
Phe237, Tyr205, and Tyr112, while RA1 within a hydrophobic region that
included Tyr159 (described to make  interaction with the dichlorophenyl ring of
9
2
). Finally, the same hydrophobic cavity, constituted by Leu240, Ile183, Ile194,
and Ile157, also accommodated the hydrophobic feature of the pharmacophore
that corresponded to the methyl group of both the new inhibitors and 4, as well
as to one of the chlorine substituents of 2.
An additional comparison was performed between the pharmacophoric model
and the binding mode of 2 in the complex with the type 2 Lansing strain
2
9
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