Anti-coxsackievirus B3 activity of 2-amino-3-nitropyrazolo[1,5-a] pyrimidines and their analogs

Article abstract of DOI:10.1016/j.bmcl.2004.10.043

The synthesis of the 2-amino-3-nitropyrazolo[1,5-a]pyrimidines with anti-coxsackievirus B3 activity is described. A novel class of 2-amino-4-nitropyrazolo[1,5-a]pyrimidines has been identified as potent inhibitors of coxsackievirus B3 replication. The syn

Full text of DOI:10.1016/j.bmcl.2004.10.043

Bioorganic & Medicinal Chemistry Letters 15 (2005) 37–39
Anti-coxsackievirus B3 activity of 2-amino-3-nitropyrazolo
[
1,5-a]pyrimidines and their analogs
a
Vadim A. Makarov, Olga B. Riabova, Vladimir G. Granik, Hans-Martin Dahse,
a
a
b
c
Axel Stelzner, Peter Wutzler and Michaela Schmidtke
c
c,
*
a
Department of Medicinal Chemistry, Research Center for Antibiotics, 3a Nagatinskaya str., Moscow 117105, Russia
b
Hans-Knoell-Institute for Natural Products Research, 11a Beutenberg Str., Jena D-07745, Germany
c
Institute of Virology and Antiviral Therapy, Medical Center of the Friedrich Schiller University,
2
Hans-Knoell-Str., Jena D-07745, Germany
Received 19 August 2004; revised 7 October 2004; accepted 13 October 2004
Available online 2 November 2004
Abstract—A novel class of 2-amino-4-nitropyrazolo[1,5-a]pyrimidines has been identified as potent inhibitors of coxsackievirus B3
replication. The synthesis of these compounds is based on the regioselective reaction of 3,5-diamino-5-nitropyrazole with unsymmet-
rical b-diketones at catalysis by hydrochloric acid leading to 2-amino-4-nitropyrazolo[1,5-a]pyrimidines as key steps.
ꢀ
2004 Elsevier Ltd. All rights reserved.
Picornaviruses, in particular enteroviruses and rhinovi-
ruses, are responsible for the majority of human respira-
The 3,5-diamino-4-nitropyrazole was prepared via con-
8
densation of 1-dimethylamino-2-nitroenamine 1 with
1
tory diseases. Moreover, enteroviruses may cause
2
aseptic meningitis, encephalitis, febrile illness, hand-
hydrazine hydrate in ethanol under reflux conditions
and recrystallization of pyrazole 2 from propanol-2.
The condensations of pyrazole 2 with commercially
available b-dicarbonyl compounds in methanol give rise
to the 2-amino-3-nitropyrazolo[1,5-a]pyrimidines 3,4.
Generally, this reaction may result in two regioisomers
(5-R-7-oxo and 7-oxo-5-R-pyrazolo[1,5-a]pyrimidine).
However, the reactions selectively result in 2-amino-3-
nitro-5-R-7-oxopyrazolo[1,5-a]pyrimidines under condi-
tion of catalysis by the methanol solution of hydrochlo-
3
4
5
foot-mouth disease, and myocarditis. The enterovirus,
coxsackievirus B3 (CVB3) is considered to be the most
important etiological agent of virus-induced acute and
6
,7
chronic myocarditis. Because more than 65 enterovi-
rus serotypes exist, it was not possible to generate a vac-
cine until now and considerable interest exists to develop
antiviral compounds. So far, these virus infections are
treated symptomatically because there are no virus-spe-
cific prophylactics and drugs available for clinical use.
9
ric acid or sodium hydroxide. The subsequent reaction
with exchange of chlorine atom in pyrazolopyrimidine 4
with different nucleophilic agents in presence of triethyl-
amine under reflux condition results in a series of final
compounds (5a–m). The reaction was controlled by
TLC (hexane/acetone: 3/1).
In this paper, we discuss the synthesis of 2-amine-3-
nitropyrazolo[1,5-a]pyrimidine derivatives and the re-
sults of investigations on their cytotoxic, antiprolifera-
tive, and antiviral activity. Based on these data the
relationship between the chemical structure and cytotoxi-
city, cell growth (proliferation) inhibition, and anti-
CVB3 activity was analyzed. A general synthetic route
for the synthesis of the pyrazolopyrimidines is described
in Scheme 1.
1
0
The compounds were examined in regard of cytotoxicity
in human cervix carcinoma cells (HeLa), growth inhibi-
tion of mouse fibroblasts (L-929) as well as human
chronic myeloid leukemic cells (K-562) and anti-CVB3
activity in HeLa cells. The results are presented in Table
1
and demonstrate a good compatibility of the test com-
pounds for confluent as well as proliferating cells. None
of the pyrazolo[1,5-a]pyrimidine derivatives inhibited
the influenza virus A or herpes simplex virus type 1-in-
duced cytopathic effect in Madin Darby canine kidney
Keywords: Nitropyrazolo[1,5-a]pyrimidines; Antiviral; Coxsackievirus.
*
Corresponding author. Tel.: +49 3641 65 7222; fax: +49 3641 65
202; e-mail: michaela.schmidtke@med.uni-jena.de
7
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.043

38
V. A. Makarov et al. / Bioorg. Med. Chem. Lett. 15 (2005) 37–39
H N
NO2
NH
2
NC
NO2
H
2
N
NO
2
N
O
b
a
N
N
H N
NMe2
N
H
NH
2
2
2
R
1
3
c
H N
NO2
NH
H N
NO2
NH
2
2
d
N
O
N
O
N
N
Cl
R
4
5
3
a R = 2,4-Cl-5-NO2 5a R = NHNH2
5h R = NHC(NH )SCH Ph
2
2
3
3
3
b R = H
5b R = NHNH-C H -F-4
5i R = NHC(NH )SEt
6
4
2
c R = 2-F
d R = 4-F
5
5
5
5
5
c R = NHNH-C H -OMe-4
d R = NHNH-C H -SO H -4 5k R = N(CH CH ) NCONEt
e R = NHNHCH CH OH
5j R = N(CH CH ) NCH CH OH
6
4
2
2
2
2
2
2
6
4
3
2
2 2
5l R = N(CH CH ) NCHPh
2
2
2 2 2 2
f R = NHCH CH OH
5mR = SC(S)NEt2
2
2
g R = NHNHC(S)NH2
Scheme 1. Synthesis of 2-amino-3-nitropyrazolo[1,5-a]pyrimidine derivatives. Reagents and conditions: (a) NH
ethyl R-benzoylacetic acid ester, MeOH, HCl/MeOH or NaOH, reflux, 90%; (c) ethyl 4-chloro-3-oxobutanoate, HCl/MeOH, MeOH, reflux, 83%; (d)
nucleophylic agent, Et N, EtOH, reflux, 50–80%.
2 2 2
NH H O, EtOH, reflux, 86%; (b)
3
11
12
Table 1. 50% cytotoxic concentration (CC50), 50% growth inhibitory concentration (GI50), 50% inhibitory concentration determined with
1
3
13
coxsackievirus B3 in HeLa cells (IC50), therapeutic index (TI), and logP of 2-amino-4-nitropyrazolo[1,5-a]pyrimidine
15
Compound
CC50 (lg/ml) in HeLa cells
GI50 (lg/ml) in
L-929 cells K-562 cells
IC50 (lg/ml) CVB3
TI (CC50:IC50
)
LogP
3
3
3
3
5
5
5
4
5
5
a
>100
>100
66.5
>100
42.6
>100
66.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
3.94
29.60
20.35
50.29
66.45
5.81
>25.6
>3.4
2.77 ± 0.27
2.54 ± 0.09
2.34 ± 0.16
2.34 ± 0.16
0.42 ± 0.09
2.65 ± 0.22
2.46 ± 0.27
b
c
3.2
>2.0
d
a
35.2
41.4
>50
>50
19.9
>50
26.5
Not active
>17.2
3.8
b
19.7
5.1
c
, 5d–k
17.53
>50
>50
>50
Not active
Not active
Not active
l
14.8
>50
9.0
33.1
3.73 ± 0.16
3.06 ± 0.12
m
>50
or green monkey kidney cells, respectively (results not
shown). Six 2-amino-3-nitro-7-hydroxypyrazolo[1,5-a]-
pyrimidine derivatives with a benzene moiety or hydra-
zinomethylene substitutes in position 5 of the pyrimidine
ring (compounds 3a–d, 5b, c) exhibited a specific inhib-
itory effect against CVB3 (Fig. 1).
3c
3a
1
00
3
5
3
b
c
d
8
6
4
2
0
0
0
0
0
SARS studies revealed that only compounds containing
a free 2-amino group and 3-nitro- and 7-oxo- substitutes
showed antiviral activity. A complete loss of antiviral
activity was observed if compounds with other than
these substitutes were synthesized (structures not
shown). Interestingly, the anti-CVB3 activity of another
nitrocontaining compound, the 2-(3,4-dichlorophen-
oxy)-5-nitrobenzonitrile, was published during the prep-
5b
1
4
aration of this manuscript. It exhibits broad-spectrum
anti-picornavirus activity and inhibits CVB3 replication
in primary human myocardial fibroblasts. Furthermore,
the anti-CVB3 activity was completely lost after substi-
tution of the phenyl group to piperasino, thiosemicarb-
asido, dithiocarbamato-groups or other moiety
10
100
concentration (µg/ml)
Figure 1. Mean anti-coxsackievirus B3 activity and standard deviation
of pyrazolopyrimidines 3a–d, 5b, and 5c determined in three inde-
13
(
compounds 4, 5a,g–m).
pendently performed CPE inhibitory assays.

V. A. Makarov et al. / Bioorg. Med. Chem. Lett. 15 (2005) 37–39
39
The comparison of SAR of our 2-amino-3-nitropyra-
zolo[1,5-a]pyrimidine derivatives with that of polysubst-
ituted pyrazolo[3,4-d]pyrimidines described by J. H.
6. Maier, R.; Krebs, P.; Ludewig, B. Clin. Dev. Immunol.
004, 11, 1.
. Gauntt, C.; Huber, S. Front. Biosci. 2003, 8, 23.
8. Makarov, V. A.; Solovieva, N. P.; Granik, V. G. Chem.
Heterocycl. Compd. 1997, 33, 78.
2
7
1
6
Chern et al. in 2004 indicates that the introduction
of phenyl substitutes led to an increased antiviral activ-
ity of both classes of compounds. But, whereas the most
active polysubstituted pyrazolo[3,4-d]pyrimidines con-
tained a hydrophobic diarylmethyl group at the piper-
asine, the same substitute caused a complete loss of
antiviral activity when introduced into pyrazolo[1,5-a]-
pyrimidine (compound 5l).
9
. Makarov, V. A.; Tafeenko, V. A.; Granik, V. G. Chem.
Heterocycl. Compd. 1998, 34, 1676.
1
1
0. Final compounds were characterized by H NMR, mass
spectrometry, and elemental analysis. This work was
reported in part; Schmidtke, M.; Makarov, V.; Riabova
O.; Dahse, H.-M.; Stelzner, A. Antiviral Res. 2002, 53,
A78.
1
1. To determine the 50% cytotoxic concentration (CC50),
confluent HeLa cell monolayer grown in 96-well plates
were incubated with serial dilutions (factor 2, each
concentration in duplicate) of the respective compounds
Interestingly, a strong correlation between antiviral
1
5
activity and the index of lipophilicity of active com-
pounds was found. It was established, that compounds
with logP 2.2–2.8 range were active, whereas more lipo-
philic or hydrophilic compounds were inactive.
for 72h (37ꢁC, 5% CO
2
). Then, the cells were fixed and
stained with a crystal violet formalin solution. Cytotoxi-
city was quantified spectrophotometrically with a plate
reader as described previously Schmidtke, M.; Schnittler,
U.; Jahn, B.; Dahse, H.-M.; Stelzner, A. J. Virol. Methods
In summary, a novel class of potent coxsackievirus B3
inhibitors was synthesized and evaluated. A method of
regioselective condensation of 3,5-diamino-4-nitropyra-
zole 2 with b-diketones was elaborated resulting selecti-
vity in the synthesis of 2-amino-3-nitro-5-R-7-oxo-
pyrazolo[1,5-a]pyrimidines. The synthetic routes are
brief and scalable, allowing of rapid analogs preparation.
2001, 95, 133.
4
12. 1 · 10 K-562 or L-929 cells were added to 10 dilutions
(
dilution factor 2) of test compounds. After an incuba-
tion time of 72h (37ꢁC, 5% CO ) the proliferation of
2
suspension cultures of K-562 cells were analyzed by an
electronic cell analyzer system (CASY) whereas the
growth of the adherent L-929 cells was studied by
determination of optical density (OD) staining with
methylene blue.
Acknowledgements
1
3. The cytopathic effect (CPE) inhibitory assay was per-
1
1
formed as described previously. Briefly, 50ll of drug
solution and 50ll of a constant amount of virus (1 MOI
for CVB3, 0.1MOI for influenzavirus A, and 0.1MOI for
HSV-1 K1) were added to confluent HeLa, MDCK, or
GMK cell monolayers, respectively, grown in 96-well
plates. The inhibition of the virus-induced CPE was scored
spectrophotometrically 24h (CVB3 and influenza virus A)
or 48h (HSV-1 K1) post infection when untreated infected
control cells showed maximum cytopathic effect. The
therapeutic index (TI) was calculated by dividing the mean
We gratefully acknowledge the financial support from
the Federal Ministry of Education and Research of
Germany (FKZ: 01KX9812). V.A.M. was supported
in part by a grant of the Paul-Ehrlich-Gesellschaft f u¨ r
Chemotherapie e.V.
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50
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15. LogP was calculated for all compounds with usage
3
TM
4
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