Pyrazolopyrimidines: Potent Inhibitors Targeting the Capsid of Rhino- and Enteroviruses

Article abstract of DOI:10.1002/cmdc.201500304

There are currently no drugs available for the treatment of enterovirus (EV)-induced acute and chronic diseases such as the common cold, meningitis, encephalitis, pneumonia, and myocarditis with or without consecutive dilated cardiomyopathy. Here, we report the discovery and characterization of pyrazolopyrimidines, a well-tolerated and potent class of novel EV inhibitors. The compounds inhibit the replication of a broad spectrum of EV in vitro with IC₅₀ values between 0.04 and 0.64 μM for viruses resistant to pleconaril, a known capsid-binding inhibitor, without affecting cytochrome P450 enzyme activity. Using virological and genetics methods, the viral capsid was identified as the target of the most promising, orally bioavailable compound 3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340). Its prophylactic as well as therapeutic application was proved for coxsackievirus B3-induced chronic myocarditis in mice. The favorable pharmacokinetic, toxicological, and pharmacodynamics profile in mice renders OBR-5-340 a highly promising drug candidate, and the regulatory nonclinical program is ongoing. Curing the common cold! A cluster of pyrazolopyrimidines with potent broad-spectrum activity against enteroviruses was discovered. Extensive structure-property relationship analyses led to the identification of 3-(4-trifluoromethyl-phenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine, shown to be a blocker of the viral capsid protein, as a lead compound for drug development with favorable physicochemical, pharmacokinetic, and toxicological properties.

Full text of DOI:10.1002/cmdc.201500304

DOI: 10.1002/cmdc.201500304
Communications
Pyrazolopyrimidines: Potent Inhibitors Targeting the
Capsid of Rhino- and Enteroviruses
Vadim A. Makarov,[b] Heike Braun,[a] Martina Richter,[a] Olga B. Riabova,[b]
Johannes Kirchmair,[c] Elena S. Kazakova,[b] Nora Seidel,[a] Peter Wutzler,[a] and
Michaela Schmidtke*[a]
There are currently no drugs available for the treatment of en-
terovirus (EV)-induced acute and chronic diseases such as the
common cold, meningitis, encephalitis, pneumonia, and myo-
carditis with or without consecutive dilated cardiomyopathy.
Here, we report the discovery and characterization of pyrazolo-
pyrimidines, a well-tolerated and potent class of novel EV in-
hibitors. The compounds inhibit the replication of a broad
spectrum of EV in vitro with IC50 values between 0.04 and
0.64 mm for viruses resistant to pleconaril, a known capsid-
binding inhibitor, without affecting cytochrome P450 enzyme
activity. Using virological and genetics methods, the viral
capsid was identified as the target of the most promising,
orally bioavailable compound 3-(4-trifluoromethylphenyl)ami-
no-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340). Its
prophylactic as well as therapeutic application was proved for
coxsackievirus B3-induced chronic myocarditis in mice. The fa-
vorable pharmacokinetic, toxicological, and pharmacodynamics
profile in mice renders OBR-5-340 a highly promising drug can-
didate, and the regulatory nonclinical program is ongoing.
EVs such as echoviruses 9, 11, 30 (ECHO-9, -11, and -30) and
coxsackieviruses B1-6 (CVB1-6) can cause severe conditions, in-
cluding aseptic meningitis, encephalitis, and acute and chronic
myocarditis with or without consecutive dilated cardiomyopa-
thy.[2a]
Broad-spectrum anti-enteroviral drugs are urgently needed,
but despite intensive research no specific drugs have been ap-
proved for the treatment of EV infections so far.[3] Drug candi-
dates include the RNA synthesis inhibitor enviroxime and the
protease 2C inhibitor AG 7088.[3a,b] Pleconaril, a capsid inhibitor
with broad-spectrum activity against EVs,[4] failed approval by
the US Food and Drug Administration for the oral treatment of
the common cold due to a lack of efficacy and safety concerns
related to the induction of certain cytochrome P450 enzymes.[5]
Results from a phase II clinical trial (NCT00394914) with an in-
tranasal formulation of pleconaril have yet to be published.[6]
1,2-Benzisoxazoles represent a second class of potential capsid
inhibitors, with drug candidate vapendavir currently under de-
velopment.[7] Its binding mode and activity spectrum closely
resemble those of pleconaril,[4a,b,7–8] which may be a factor in
the observed cross-resistance with pleconaril.[7b]
The genus enterovirus (EV) of the family picornaviridae includes
more than 100 rhinovirus (RV) serotypes and about 70 EV sero-
types, each of them classified into species A–D.[1] These viruses
are capable of causing a wide range of acute and chronic dis-
eases.[2] Each year, millions of people suffer from the common
cold, asthma, chronic obstructive pulmonary disease exacerba-
tion, otitis or pneumonia after infection with rhinoviruses.[2b]
High-level resistance to capsid inhibitors resulting from
single amino acid substitution of residues forming the hydro-
phobic inhibitor binding pocket of viral capsid protein 1 (VP1)
[a] Dr. H. Braun,+ M. Richter,+ Dr. N. Seidel, Prof. Dr. P. Wutzler,
Dr. M. Schmidtke+
Virology & Antiviral Therapy, Jena University Hospital
Hans-Knçll-Str. 2, 07745 Jena (Germany)
E-mail: michaela.schmidtke@med.uni-jena.de
[b] Dr. V. A. Makarov,+ Dr. O. B. Riabova,+ E. S. Kazakova
A. N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, build. 2, Moscow 119071 (Russia)
[c] Prof. Dr. J. Kirchmair
Center for Bioinformatics, University of Hamburg
Bundesstrasse 43, 20146 Hamburg (Germany)
[+] These authors contributed equally to this work.
have been observed.[9] It has been suggested that substituted
amino acids block the integration of the inhibitors into the
pore. Such single amino acid substitutions in VP1 were also
shown to confer high-level pleconaril resistance to EVs,[4a,c,8,10]
whereby I1092L substitution, situated in proximity of the cen-
tral, methylated phenoxy group of pleconaril in VP1, was most
commonly observed for CVB3.[4c,8] We demonstrated the crucial
role of the central ring of capsid inhibitors in anti-enteroviral
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500304.
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
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tutions of the aniline are possible. Most active compounds
comprise at least one halogen or trifluoromethyl group (e.g.,
compounds 24, 25, 32, 36, and 41).
Compound 36 (MW: 370.3; logP=2.85; Scheme 1) showed
particularly favorable physicochemical, antiviral, and pharmaco-
kinetic properties (Figure 1 and Table 1; see also, Tables S2
and S4 in the Supporting Information). It was synthesized in
four steps with high yield,[12b] as summarized in Scheme 1.
Compound 25, an analogue with a 4-fluoroaniline moiety,
was also highly active in vitro but almost inactive in vivo (re-
sults not shown). The compound is hydroxylated in vivo to
form the 100- to 5000-fold weaker metabolite 15. This renders
25 unsuitable for oral administration. By introducing an elec-
tron-withdrawing trifluoromethyl group into 36, its pharmaco-
kinetic and metabolic properties were improved (Tables S4
and S5 in the Supporting Information).
Scheme 1. Synthetic route to compound 36.[12] Reagents and conditions:
a) toluene, reflux, 3 h, 95%; b) NaH, CH2(CN)2, DMF, 208C, 5 h, 95%;
c) NH2NH2 H2O, EtOH, reflux, 2.5 h, 67%; d) PhC(NH)NH2, NaAc, butanol,
melt at 170–1808C, 25 min, 25%.
activity and showed that select-
ed ring decorations can break
drug resistance.[11] These and fur-
ther considerations led to the
exploration of a series of pyrazo-
lo[3,4-d]pyrimidines as novel
drug candidates for the treat-
ment of EV infections (Scheme 1;
see also, Table S1 in the Support-
ing Information).[12] Herein, we
report their synthesis, physico-
chemical characteristics, struc-
ture–activity relationships and
unique anti-enteroviral activities.
All synthesized compounds
(~80, plus pleconaril as a refer-
ence) were initially subjected to
cytotoxicity (see the Supporting
Information) and antiviral testing
(inhibition of the cytopathic
effect, CPE) in HeLa cell cultures.
Most of them were well tolerat-
ed (Table S2 in the Supporting
Information). Analogues derived
from a 3-phenylamino-6-phenyl-
pyrazolo[3,4-d]pyrimidine-4-
amine scaffold were particularly
promising, exhibiting strong ac-
tivity against CVB3, RV2, 5, 8, 42,
and 48 (RV5, RV42, and RV48 are
known[4a] and confirmed by our
data as high-level pleconaril-re-
sistant; activity measured as de-
scribed in Table S2 in the Sup-
porting Information). Amino
Figure 1. Anti-enteroviral activity of 36. A) Dose-dependent inhibition of CVB3 Nancy- and CVB3 97927-induced
cytopathic effect in HeLa cells by compound 36 in comparison to pleconaril. Data represent the meanÆSD (error
bars) of at least three independent determinations. B) Distribution of mean IC50 values of compound 36 among
groups at positions 3 and 4 of
the central ring are critical for
antiviral activity. Different substi- the studied enterovirus strains. Mean and/or SD values were calculated from the results of at least three assays.
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with CVB3 Nancy in HeLa cells (Figure 2A). Pretreatment of
cells with 36 prior to virus inoculation had no effect on virus-
induced plaque formation. In contrast, incubation of 36 with
cell-free virus resulted in strong loss of virus infectivity as
shown by high percentage of plaque reduction in Figure 2A.
Here, high concentrations of CVB3 Nancy were incubated with
or without the inhibitor in test medium for one hour at 378C.
Then the mixture was diluted 1:106 to obtain noneffective in-
hibitor concentrations and added to confluent cell monolayers
for virus adsorption.
Table 1. Impact of amino acid substitutions detected in compound 36-
resistant CVB3 Nancy variants on plaque inhibition in HeLa cells.
CVB3
Nancy
Amino acid
substitution
IC50 [mm][a]
Compd 36
Pleconaril[b]
Compd 25
wildtype
none
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
0.16Æ0.03
35.03Æ0.01
n.a.
0.25Æ0.06
3.43Æ0.97
n.a.
36-resistant
36-resistant
36-resistant
36-resistant
36-resistant
P1094S
S1190T
I1207N
I1207m
S2209C
n.a.
n.a.
n.a.
n.a.
31.53Æ7.89
32.47Æ4.59
Acute (>3 gkgÀ1) and chronic (12.5, 50 and 200 mgkgÀ1
doses, 30 days treatment in male BALB/c mice) toxicological
studies in mice showed no indications of adverse anatomical,
behavioral or physiological effects (see Table S3 in the Support-
ing Information). In vitro ADMET profiling of 36 (Table S4 in
the Supporting Information) included analysis of plasma pro-
tein binding and recovery (96.7% and 100%, respectively),
plasma stability (100%), mutagenicity (none), and hERG chan-
nel inhibition (low). Compound 36 is neither an inducer nor an
inhibitor of relevant cytochrome P450 isoforms. A 100% meta-
bolic stability of 36 in human liver microsomes was observed
(see the Supporting Information, Table S4) explaining in vitro
its efficacy after oral administration in mice. The in vitro data
were confirmed by pharmacokinetics studies in mice during
a 100 mgkgÀ1 per os administration. The in vivo results show
that 36 has druglike pharmacokinetic properties, as reported
in the Supporting Information (Table S5), and in part summar-
[a] Data represent the meanÆSD of at least three independent determi-
nations in plaque reduction assays performed in duplicate; [b] not active
(n.a.) due to naturally occurring pleconaril resistance (L1092).
Compound 36 strongly inhibited the cytopathic effect of the
pleconaril-resistant CVB3 Nancy (L1092[4c]) as well as pleconaril-
susceptible CVB3 97927 (I1092) with IC50 values of 0.16 and
1.05 mm, respectively (Figure 1A; see also the Supporting Infor-
mation).
We postulate that inhibitor binding to viral capsid proteins
leads to the prevention of virus adsorption and might also be
responsible for the observed antiviral effect during and after
adsorption. The detection of amino acid substitutions in VP1
(2”P1094S, 2”S1190T, 1”I1207N, 1”I1207M) and VP2 (2”
S2209C) of high-level 36-resistant CVB3 Nancy variants sup-
ports this (Table 1). In agreement with pleconaril studies,[8]
a concentration of 1 mgmLÀ1 (2.7 mm) of 36 was used for their
isolation. In ten independently prepared pools of wild-type
CVB3 Nancy, the mean frequency of resistance to 36 was ~7”
105, corresponding well with the data published for pleconar-
il.[8] Based on these phenotypic and genotypic data, we expect
that the lipophilic 36 binds inside the hydrophobic pocket of
VP1 in a similar fashion to pleconaril and vapendavir.[7b,15] The
potential binding mode of 36 as derived by automated ligand
docking with GOLD[16] corroborates this mechanism of action
(Figure 2B,C). The model suggests that the observed mutations
causing high-level resistance induce larger conformational rear-
rangements leading to the disruption of hydrophobic interac-
tions formed by the trifluorophenyl moiety of 36 (which is
postulated to correspond to the methylisoxazole moiety in ple-
conaril).
ized
here:
Cmax
:
1.25 mgmLÀ1
;
tmax
:
3 h;
AUC0Àt
:
6418.3 ngmLhÀ1; AUCoÀ1: 9598.53 ngmLhÀ1; t1/2: 3.5 h; Cmax
/
AUC0Àt: 0.2 hÀ1; MRT: 3.54 h; Cl: 10.42 mLhkgÀ1; Kel: 0.18 hÀ1
;
Vd: 52.72 mLkgÀ1.
Effective compound concentrations were reached by oral ad-
ministration of 100 mgkgÀ1 of 36 twice daily, for 7 days. When
treatment was started 3 h prior to (prophylactically) or 1 and 3
days p. i. (therapeutically), 36 exerted a strong antiviral effect
in a model of CVB3-induced chronic myocarditis in NMRI mice
(Figure 3; see also, Tables S6 and S7 in the Supporting Informa-
tion). Placebo-treated, CVB3-infected mice markedly lost body
weight (Figure 3A) and became ill (Figure 3B) on day 3 p. i.
Prophylactic and therapeutic treatment significantly improved
both study parameters (Figure 3). As a result of viral replica-
tion, pronounced inflammation as well as necrosis and fibrosis
of myocardial tissue were observed in hematoxylin-eosin and
sirius red-stained heart specimens, respectively, in placebo-
treated animals on day 28 p. i. (Figure 3C). CVB3 also com-
pletely destroyed the exocrine pancreas tissue (Figure 3C).
Compound 36 significantly decreased tissue damage as well as
inflammation in the heart and pancreas when treatment was
started 3 h before, or 1 or 3 days p. i. No therapeutic effect
was observed when treatment started 7 days p. i. (results not
shown).
A panel of 46 RV serotypes comprising viruses from species
A (n=33) and B (n=13), two EV species A, nine EV species B,
one EV species C, and two EV species D were assessed regard-
ing their sensitivity to 36 in CPE inhibition assays in HeLa
cells.[13] Compound 36 inhibited 52 of the 60 strains tested
(Figure 1B). Strong antiviral activity was detected against
ECHO11 and EV68, which are known to cause outbreaks of
aseptic meningitis and acute lower respiratory tract infections,
respectively.[14] The highest activities were measured against
high-level pleconaril-resistant viruses including RV5, RV42,
RV44, RV48, and RV69.[4a,c,10] The IC50 values for these specific
EVs ranged from 0.04 to 0.64 mm (Table S2 in the Supporting
Information).
In conclusion, pyrazolopyrimidines represent a novel class of
compounds targeting the capsid of clinical important RVs and
EVs. Notably, lead compound 36 is orally available, inhibits
a broad-spectrum of EVs and RVs, helps to overcome the re-
sistance of known capsid inhibitors, and does not exert an
effect on relevant cytochrome isoforms. The favorable pharma-
The effect of 0.5 and 1 mm of 36 on different steps of the
viral replication cycle was studied in plaque reduction assays
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Figure 2. Mechanism of action of compound 36. A) 0.5 mm (&) or 1.0 mm (&) of compound 36 were added to cells or only to cell-free virus before infection
and at different time points during infection in a plaque reduction assay with CVB3 Nancy in HeLa cells. B) Viral capsid protein (VP1: orange; VP2-4: grey)
with the highest-ranked docking pose obtained for 36 (carbon atoms in green; ChemPLP score[17] =90.6). C) Detail of this docking pose. Carbon atoms of ple-
conaril are turquoise, and that of 36 are green. Nitrogen, oxygen, sulfur and fluorine atoms are colored blue, red, yellow and light blue, respectively. The mo-
lecular shapes of 36 and pleconaril as positioned within the hydrophobic pocket show similarities. Compound 36 shows excellent shape complementarity
with the hydrophobic pocket.
cokinetic, toxicological, and pharmacodynamics profile in mice
toxicity and antiviral activity (CPE inhibition assay) of pyrazolopyri-
midines in HeLa cells, mechanism of action studies (modified
plaque-reduction assay), isolation[8] and phenotypic (plaque-reduc-
tion assay), as well as genetic characterization of 36-resistant var-
iants. Information on the cells, virus strains, growth conditions, and
virus titer determination are also described.
renders 36 a highly promising drug candidate.
Experimental Section
Chemistry: The synthesis of compound 36 as shown in Scheme 1
is described in detail in the Supporting Information together with
synthetic protocols for access to control compounds (guanidine
hydrochloride and pleconaril).
In vivo models: The in vivo studies on the prophylactic and thera-
peutic antiviral effect, the pharmacokinetic, and the toxicity of 36
were performed in mice. The experimental design was reviewed
and approved by local government (ThüringerLandesamt für Leb-
ensmittelsicherheit und Verbraucherschutz); the registration
number is 02-001/07 (antiviral studies), and by the State Scientific
Center for Antibiotics, Moscow; the registration number is 267/
2010 (pharmacokinetic and toxicity). For details, see the Support-
ing Information.
Computational methods: Information on the homology model
used to predict the binding mode and ligand docking methods is
also given in the Supporting Information.
In vitro assays: Also presented in the Supporting Information are
details of the in vitro studies performed, including analysis of cyto-
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Figure 3. Prevention of CVB3-induced chronic myocarditis in NMRI mice. A) Influence of treatment with 36 on body weight, B) on symptoms shown as clinical
score, and C) on histopathological changes in heart and pancreas tissue. Statistical analysis of histopathological and clinical score was performed using the
Mann-Whitney U test to compare 36-treated, and placebo-treated infected mice. After confirming normal distribution of data with the Kolmogorov-Smirnov
test, a t-test was applied for statistical analysis of body weight change of untreated, 36-treated mice in comparison to untreated, placebo-treated infected
mice. *p ꢀ0.05; **p ꢀ0.01; ***p ꢀ0.001.
Keywords: antivirus agents · drug discovery · structure–
Acknowledgements
property relationships · synthetic drugs · virology
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Received: July 14, 2015
Published online on August 10, 2015
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