9-Arylpurines as a novel class of enterovirus inhibitors

Article abstract of DOI:10.1021/jm901240p

Here we report on a novel class of enterovirus inhibitors that can be structurally described as 9-arylpurines. These compounds elicit activity against a variety of enteroviruses in the low μM range including Coxsackie virus A16, A21, A24, Coxsackie virus B3, and echovirus 9. Structure-activity relationship (SAR) studies indicate that a chlorine or bromine atomis required at position 6 of the purine ring for antiviral activity. The most selective compounds in this series inhibited Coxsackie virus B3 replication in a dose-dependent manner with EC₅₀ values around 5-8 μM. Notoxicity on different cell lineswas observed at concentrations up to 250 μM. Moreover, no cross-resistance to TBZE-029 and TTP-8307 CVB3 resistant strains was detected.

Full text of DOI:10.1021/jm901240p

316 J. Med. Chem. 2010, 53, 316–324
DOI: 10.1021/jm901240p
9-Arylpurines as a Novel Class of Enterovirus Inhibitors
†
‡
Leire Aguado,† Hendrik Jan Thibaut,‡ Eva-Marı
ꢀ
ꢀ ꢀ
´
Marıa-Jesus Perez-Perez*
´
a Priego,† Marı
a-Luisa Jimeno,§ Marı
a-Jose Camarasa, Johan Neyts, and
ꢀ
´ ´
,†
†
‡
Instituto de Quımica Medica (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain, Rega Institute for Medical Research, Katholieke Universiteit
´
Leuven, B-3000 Leuven, Belgium, and Centro de Quımica Organica Lora-Tamayo (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
ꢀ
§
´
ꢀ
Received August 19, 2009
Here we report on a novel class of enterovirus inhibitors that can be structurally described as 9-arylpurines.
These compounds elicit activity against a variety of enteroviruses in the low μM range including Coxsackie
virus A16, A21, A24, Coxsackie virus B3, and echovirus 9. Structure-activity relationship (SAR) studies
indicate that a chlorine or bromine atom is required at position 6 of the purine ring for antiviral activity. The
most selective compounds in this series inhibited Coxsackie virus B3 replication in a dose-dependent
manner with EC50 valuesaround5-8 μM. No toxicity on different cell lines was observed at concentrations
up to 250 μM. Moreover, no cross-resistance to TBZE-029 and TTP-8307 CVB3 resistant strains was
detected.
Introduction
substrate of adenosine deaminase.12 More recently, Ueno
et al. prepared bis(hydroxymethyl)benzene residues directly
connected to the nucleobases to construct a nucleic acid
analogue.13 The synthesis of these 9-arylpurines has
been carried out following the classical method described by
Greenberg in 1959 that involves reaction of 5-amino-4,
6-dihalopyrimidines with anilines followed by a ring closing
reaction.14 However, this strategy requires prolonged heating
and yields are variable.
More recently, another synthetic approach to 9-arylpurines
has been described that consist in the cross coupling reaction
of the purine base with arylboronic acids catalyzed by copper
salts, initially described by Ding15 and further studied by
Gundersen,16 by adapting the general procedure of Chan,17
Lam,18 and Evans19 and recently reported by other labora-
tories.20,21 Making use of this approach, we synthesized a
number of 9-((3-hydroxymethy)phenyl)purines that were
evaluated against the replication of different DNA and
RNA viruses. Interestingly, compounds with a chlorine atom
at position 6 of the purine base (Chart 1, X = Cl) exerted
significant activity against Coxsackie virus type B3 (CVB3)
replication at nontoxic concentrations. On the basis of their
low molecular weight, their structure that significantly differs
from previously reported anti-CVB3 compounds1 and their
selectivity index, we initiated a program to synthesize different
9-arylpurines. Here we report on the synthesis, antiviral
evaluation, and structure-activity relationships on this new
family of anti CVB3 compounds.
Enteroviruses are responsible of a broad spectrum of acute
and chronic human diseases, including respiratory infections,
meningitis, encephalitis, pancreatitis, myocarditis, and neo-
natal sepsis. Enteroviruses, belonging to the Picornaviridae
family, are small nonenveloped and spherical RNA viruses,
with a diameter of about 30 nm. Despite their high impact on
human health, no drugs have been approved for the treatment
of enterovirus infections.1,2 The genus Enterovirus comprises
more than 60 serotypes of which Coxsackie virus type B3
(CVB3a) can be considered as the prototype for the nonpolio
enterovirus group. CVB3 is an important human pathogen
that may cause acute and chronic viral myocarditis in children
and young adults which eventually can progress to cardio-
myopathy.3,4 In addition to heart infections, CVB3 also
causes chronic inflammatory diseases of the pancreas and
central nervous system.1,5,6 Several molecules have been
reported to be selective inhibitors of enteroviruses, some of
which have entered clinical trials7 but none of them has been
formally approved. Therefore there is an urgent need for the
discovery and development of chemical entities able to selec-
tively inhibit enterovirus replication.
Given our interest in purine nucleosides and analogues,8-10
we focused our attention on 9-((3-hydroxymethyl)phe-
nyl)purines, compounds that have been scarcely explored
and that in some instances have been proposed as nucleoside
surrogates. Chern et al. reported in 1993 the synthesis of the
8-aminoguanine derivative (Chart 1, X=OH, Y=Z=NH2)
that was found inactive against purine nucleoside phosphory-
lase.11 Later on, Brakta et al. described the synthesis of the
adenine derivative (Chart 1, X = NH2, Y = Z = H) as a
Results and Discussion
Chemistry. Initially, the synthesis of 9-(3-(hydroxymethy)-
phenyl)purines was performed employing the conditions set
up by Bakkestuen and Gundersen16 for the regioselective
N9-arylation of purines employing arylboronic acids in the
presence of Cu(II). Thus, the 6-chloropurine (1) (1.0 equiv)
was allowed to react with 3-hydroxymethylphenylboronic acid
*To whom correspondence should be addressed. Phone: 34 91
2587579. Fax: 34 91 5644853. E-mail: mjperez@iqm.csic.es.
a Abbreviations: CVB3, Coxsackie virus type B3; MAOS, micro-
wave-assisted organic synthesis; MTS, 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
r
pubs.acs.org/jmc
Published on Web 11/19/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 317
Chart 1. General Structure of 9-(3-(Hydroxymethy)phenyl)-
purines
Scheme 2. Substitution Reactions at Position 6 in Compound 3a
Scheme 1. Synthesis of 9-(3-(Hydroxymethy)phenyl)purines
from Arylboronic Acidsa
a Reagents and conditions: (a) H2, Pd/C, NaOAc, EtOH, 30 °C, 2 h
(12, 63% yield); (b) MeONa, MeOH, Δ, 2 h, (13, 95% yield); (c) MeNH2,
MeOH, rt, 3 h (14, 95% yield); (d) HCl 1N, 1,4-dioxane, 90 °C, 16 h (15,
42% yield).
a Reagents and conditions: (a) purine base (1,4-7, 1.0 equiv), aryl
boronic acid 2 (2.0 equiv), 1,10-phenanthroline (2.0 equiv), Cu(OAc)2
(2.0 equiv), DMF, rt, 3 days (3, 60% yield; 8, 46% yield; 9, 30% yield; 10,
43% yield; 11, 58% yield).
Table 1. Synthesis of 6-Chloro-9-phenylpurines Differently Substituted
at the Phenyl Ring
(2) (2.0 equiv) in the presence of 1,10- phenanthroline
(2.0 equiv), cupric acetate (1.0 equiv) in dry dichloromethane
and in the presence of activated 4 A molecular sieves for 4 days
at room temperature. Under these conditions, the desired
coupling product 3 was obtained but the yield was quite low
(26%). To improve this yield, different assays were performed
including the employment of other bases such as triethylamine,
pyridine, or other solvents such as 1,4-dioxane or DMF. Also
the impact of the presence or absence of 4 A molecular sieves
was evaluated.22 In our hands, the best conditions were 1.0 equiv
of 6-chloropurine, 2.0 equiv of the aryl boronic acid, 2.0 equiv of
1,10-phenanthroline, 2.0 equiv of cupric acetate in DMF, and
the absence of molecular sieves. Under these modified condi-
tions, compound 3 was isolated in 60% yield (Scheme 1).
Similarly, the arylboronic acid 2 reacted with different purine
bases (4-7) to yield the 9-arylpurines (8-11) (Scheme 1) in
moderate to good yields (30-60%). The 6-chloropurine deri-
vative 3 was further modified at position 6 using standard
conditions in purine chemistry (Scheme 2). Thus, catalytic
hydrogenation of 3 in the presence of Pd(C) and sodium
acetate23 gave the purine derivative 12 in 63% yield. Nucleo-
philic substitution reactions of 3 with sodium methoxide24 and
methylamine9 in methanol yielded the 6-OMe and 6-NHMe
derivatives (13 and 14, respectively) in excellent yields (95%).
Alternatively, reaction of 3 with 1 M HCl in refluxing dioxane25
afforded the hypoxanthine 15 in 42% yield.
The antiviral evaluation of this first series of compounds
revealed that only compounds 3, 9, and 10 showed significant
antiviral activity. Thus, the presence of a chlorine or bromine
atom at position 6 of the purine seemed to be important for
the antiviral effect. Therefore the next series of compounds
kept the 6-chloropurine as the base while different substitu-
ents were introduced at positions 3 and/or 4 of the phenyl
moiety.
Reaction of the arylboronic acids 16-24 with 6-chloro-
purine (1) in the presence of Cu(OAc)2 and 1,10-phenanthro-
line in DMF at room temperature for 3 days afforded the
9-arylpurines 25-33, with the yields specified in Table 1. It is
entry reactive
R1
R2
CH2OH
product yield (%)
1
2
3
4
5
6
7
8
9
16
17
18
19
20
21
22
23
24
H
COCH3
25
26
27
28
29
30
31
32
33
40
62
35
39
36
43
36
31
28
H
COCH3
H
H
COPh
H
COOCH3
OCH3
H
OCH2CH(CH3)2
H
OCH2O
H
OCH2CH(CH3)2
worth mentioning that the reaction conditions are compa-
tible with a variety of functional groups at the aryl moiety
including ketones (entries 2, 3 and 4), esters (entry 5), or
ethers (entries 6-10). Yields in general are moderate, but still
this procedure has the advantage of affording the targeted
compounds in a single reaction step from commercial pre-
cursors. The antiviral testing of this new series of compounds
revealed that the 3-acetyl derivative 26 was more active
against CVB3 than the initial hit 3. Thus the next series of
compounds were designed based on the structure of com-
pound 26, keeping the chlorine atom at position 6 of the
purine base and the 3-acetyl group at the phenyl moiety.
To synthesize 9-arylpurines structurally related to com-
pound 26, a different synthetic approach was employed that
consisted in a microwave assisted (MAOS) protocol starting
from 4,6-dichloro-5-aminopyrimidines that reacted with
anilines, followed by cyclization, as very recently described
by us.26 Reaction of the 4,6-dichloro-5-aminopyrimidines

318 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Aguado et al.
(34 and 35) with anilines (36) or acetamides (37) under
microwave conditions afforded the 4,5-diamino-6-chloro-
pyrimidines (39-42) (Scheme 3).26 Hydrogenation of the
commercially available 4-fluor-3-nitroacetophenone in the
presence of Pt(S)/C27,28 for 2 h at room temperature afforded
the aniline 38 that reacted with the dichloropyrimidine 34 to
yield the diaminopyrimidine 43 in 38% yield for both steps.
Compounds 40-43 were subjected to microwave irradiation
with trimethylorthoformate in acetic anhydride at 120 °C for
1 h26 to afford the purines 44-47. Moreover, the 4,5-
diaminopyrimidines 39 and 40 are versatile intermediates
that have been employed to access to the 8-methylpurines
(48 and 49) and the 8-oxo compounds (50-51) as described
(Scheme 4).26 When these diaminopyrimidines 39 and 40
reacted with NaNO2 in CH2Cl2 in the presence of HCl at
room temperature for 30 min,29 the triazolo derivatives 52
and 53 were obtained in 77 and 75% yield, respectively. Two
of the most potent compounds (44 and 51) were transformed
into the tertiary alcohols (54 and 55, Scheme 5) in 53 and
45% yield, respectively, by reaction with CH3MgI. Finally,
to increase the distance between the purine base and the
aromatic substituent, the benzyl derivative 6-chloro-9-(3-
methoxybenzyl)purine (56) together with its N7-isomer
(57) (Figure 1) were synthesized following the described
procedure30 and both compounds were included for antiviral
evaluation.
Compounds 50 and 51 may exist as a tautomeric equili-
brium between the keto and the enol form (Figure 2).
Detailed 15N NMR studies performed on 8-hydroxyadeno-
sine had led to the conclusion that this nucleoside exi-
sts predominantly as the keto form in solution.31 These
authors even proposed to use the prefix “8-oxo” instead of
Scheme 3. Synthesis of 9-(3-Acetylphenyl)-6-chloropurines
through MAOS
Scheme 5. Synthesis of 3-(2-Hydroxypropyl)phenyl Deriva-
tives 54 and 55a
a Reagents and conditions: (a) isobutyl alcohol, HCl (cat.), MW,
150 °C, 10 min-1 h; (b) HC(OCH3)3, Ac2O, MW, 120 °C, 1 h. The
synthesis of 39-42 and 44-46 has been described in ref 26.
a Reagents and conditions: (a) CH3MgI, THF, 0 °C, 90 min.
Scheme 4. Cyclization Reactions Starting from 4,5-Diaminopyrimidinesa
a Reagents and conditions: (a)NaNO2, HCl, CH2Cl2, rt, 30 min. The synthesis of 48-51 has been described in ref 26.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 319
Table 2. Antiviral Evaluation against CVB3 of the 9-Arylpurines in
Vero Cellsa
compd
EC50 (μM)b
EC90 (μM)c
30 ( 9.2
CC50 (μM)d
3
14.2 ( 3.9
>8.8
>100
8.8 ( 1.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
44 ( 0.35
44 ( 0.21
>100
>100
>100
>100
>100
>100
8
9
17 ( 3
22 ( 0
>100
>100
25
29 ( 0.0
10
11
12
13
14
15
25
26
27
28
29
30
31
32
33
39
40
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Figure 1. Structural formulas of the benzylic derivatives 56 and 57.
>100
>100
>100
11 ( 3
20 ( 4.6
9.1 ( 3.1
51 ( 7.9
85 ( 13
22 ( 11
22 ( 7.4
>100
7.3 ( 2.3
35.3 ( 0.9
67 ( 9
11.1 ( 3.3
12.0 ( 5.3
29 ( 2
Figure 2. Keto-enol tautomerism for compound 50.
>100
>100
>100
>100
“8-hydroxy” to name this class of compounds. However, in
more recent examples even involving biologically active
compounds, the designation as “8-hydroxy”32 or “8-oxo”33
is found in the literature. To determine which of the two
forms is the predominant for the here described purines 50
and 51, [1H-15N] NMR correlation experiments have been
carried out for compound 50 in DMSO-d6. In the [1H-15N]
gHSQC spectrum, a correlation signal was observed bet-
ween a proton at 12.40 ppm and the signal of a nitrogen at
-250 ppm. Moreover, in the [1H-15N] gHMBC experi-
ments, a correlation peak was detected between this proton
and a signal around -220 ppm. Thus, the signal at -250 ppm
was assigned to N7 of the purine, while the signal at
-220 ppm corresponds to N9. The other two signals in the
[1H-15N] gHMBC spectrum around -90 and -120 ppm,
with correlations peaks with a proton at 8.42 ppm, corre-
spond to N1 and N3 of the purine ring. The complete
assignment of the 1H and 13C NMR spectra was performed
based on 1H-13C correlation experiments (gHSQC and
gHMBC) (see Supporting Information). From these experi-
ments, it is noticeable that the signal corresponding to the
C-8 of the purine appears at 151.9 ppm. These experiments
point to the keto form as the predominant one under these
experimental conditions because all the correlations are in
agreement with this form. If the enol form should have
been the predominant, the signal corresponding to N7
should appear approximately at -160 ppm. Thus for these
6-chloropurines, as previously shown for adenines, the pre-
dominant form in DMSO-d6 is the keto form.
8.7 ( 3.1
>57
>100
14 ( 0.5
9.3 ( 0.5
12.2 ( 1.1
11 ( 0.0
9.3 ( 2.7
6.7 ( 2.1
>44
15 ( 3.8
22 ( 4.5
15 ( 0.1
18 ( 5.9
10 ( 2.1
>44
8.0 ( 3.4
22 ( 0.1
53 ( 18
75 ( 3
1.2 ( 0.4
0.7 ( 0.3
7.4 ( 2.1
29 ( 0.5
70 ( 23
>100
58 (TBZE-029)
59 (enviroxime)
a All data are mean values ( standard deviation for at least three
independent experiments. b 50% effective concentration or concentra-
tion required to protect 50% of the cells against the cytopatic effect of
the virus. c 90% effective concentration or concentration required to
protect 90% of the cells against the cytopatic efect of the virus. d 50%
cytotoxic concentration or concentration cytotoxic to 50% of the cells.
Antiviral Evaluation. The compounds synthesized were
submitted to an antiviral screening against different DNA
and RNA viruses. Significant antiviral activity was observed
against Coxsackie virus type B3 (CVB3) replication in Vero
cells, as shown in Table 2. Two of the most potent described
CVB3 inhibitors, 58 (TBZE-029)34 and 59 (enviroxime),35
(Figure 3), were included as reference compounds (Table 2).
The antiviral activity is expressed as the 50% effective
concentration (EC50) and the 90% effective concentration
(EC90). Cytotoxicity to noninfected cells is expressed as
CC50. The evaluation of the first series of compounds (3,
8-15) revealed that a chlorine or bromine atom was required
at position 6 of the purine in order to get antiviral activity, as
shown for compounds 3, 9, and 10. The nonsubstituted
compound (12) or those with NHMe, OMe, or carbonyl at
Figure 3. Structural formulas of well established anti-CVB3 com-
pounds.
position 6 of the purine (compounds 13, 14, and 15, re-
spectively) were antivirally inactive.
Keeping the 6-chloropurine as the base, different substit-
uents were introduced at the positions 3 and/or 4 of the
aryl ring (compounds 25-31). In particular, the 4-CH2OH

320 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Aguado et al.
derivative (25) was similar in antiviral activity to its 3-isomer
(3). However, when comparing the 3- and 4-isomers with
an acetyl substituent (26 and 27), it became clear that the
3-isomer (26) was 5-fold more potent than the 4-isomer. A
similar trend was observed for the isobutoxy derivatives 31
and 32, showing that the 3-isomer (31) is moderately active
while the 4-isomer (32) is inactive at the highest concentra-
tion tested (100 μM). It is interesting to notice that different
substituents at position 3 of the aryl moiety are compatible
with a significant antiviral activity including a hydroxy-
methyl (3), an acetyl (26), a methylester (29), or a methoxy
group (30). However, a double substitution at positions 3
and 4, as shown for the 1,3-dioxolane derivative 33, renders
an inactive compound. Thus, compound 26 with a 3-acetyl
substituent at the phenyl ring was even more potent that the
initial hit, the hydroxymethyl derivative 3, and was chosen as
the prototype for further modifications.
The structure-activity relationship studies of the latest
series of compounds (39, 40, 44-57) indicated that the purine
ring can incorporate a methyl group at positions 2, 8, or
both, as shown for compounds 44, 48, and 49, respectively,
without compromising the antiviral activity. However,
the opening of the imidazole ring of the purine, as in the
4,5-diaminopyriminides 39 and 40, or the incorporation of a
nitrogen at position 8 as in the triazolo derivatives 52 and 53,
provide antivirally inactive compounds. Moreover, the tri-
azolo derivatives show a certain degree of cytotoxicity at
44 μM. If a keto group is introduced at position 8 of the
purine (compounds 50 and 51), the antiviral activity is
maintained or even slightly increased compared to the
8-unsubstituted analogues (26 and 44, respectively) based
on the EC50 and EC90 values. Concerning the aryl moiety,
incorporation of a methoxy group at position 6 of the phenyl
ring (compounds 45 and 46), annihilates the antiviral effect.
However, introduction of a fluor atom at position 4 (47)
keeps the antiviral activity compared to 26, in agreement
with the well documented bioisosterism fluor/hydrogen.36
Furthermore, replacement of the acetyl group at position 3
of the phenyl ring in compounds 44 and 51 by a 2-hydroxy-
propyl (54 and 55, respectively) keeps the antiviral activity in
particular for compound 54. Finally the N9-benzylic deriva-
tive 56, where a methylene unit has been introduced between
the aryl moiety and the purine ring, proved to be much less
potent than the corresponding N9-aryl analogue (30), point-
ing to the importance of the direct attachment of the phenyl
ring to the purine in order to obtain a significant antiviral
effect.
Table 3. Cellular Toxicity of Compounds 3, 26, 51, and 54, Determined
in Three Different Cell Lines, As Assessed with the MTS Method and the
CellTiter-Glo Luminescent Cell Viability Assaya
MTS
CC50 (μM)b
luminescence
CC50 (μM)b
compd
3
26
51
54
Vero cells
158 ( 23
206 ( 14
>250
HeLa cells
175 ( 7
144 ( 14
>250
MRC-5 cells
229 ( 16
238 ( 4
>250
Vero cells
200 ( 78
>250
>250
>250
>250
>250
>250
a Data are mean values ( SD for at least three independent experi-
ments. b 50% cytotoxic concentration.
Table 4. Antiviral Evaluation of Compound 26 against a Selected Panel
of Enterovirusesa
species
virus (strain)
EC50 (μM)b
enterovirus A
Coxsackie virus A16 (G-10)c
enterovirus 71 (BrCr)d
Coxsackie virus A9 (Bozek)e
Coxsackie virus B3 (Nancy)f
echovirus 9 (Hill)c
2.7 ( 0.76
>100
>100
enterovirus B
7.3 ( 2.3
3.3 ( 1.2
>100
echovirus 11 (Gregory)c
polio virus 1 (Sabin)f
enterovirus C
rhinovirus
>100
Coxsackie virus A21 (Coe)c
Coxsackie virus A24 (clinical)c
rhinovirus 2g
4.5 ( 1.6
2.7 ( 0.87
>100
rhinovirus 14g
>100
a Data are mean values ( SD for at least three independent experi-
ments. b 50% effective concentration. c Cultured in MRC-5 cells at
37 °C. d Cultured in RD cells at 37 °C. e Cultured in Hela R at 37 °C.
f Cultured in Vero cells at 37 °C. g Cultured in Hela R at 35 °C.
was found against CVB3 (Nancy) and echovirus 9. Coxsack-
ie virus A21 (CVA21) and Coxsackie virus A24 (CVA24)
were also inhibited by compound 26. However, this com-
pound was inactive against 15 selected rhinoviruses, polio-
virus 1, enterovirus 71, and echovirus 11.
The here described compounds are significantly different
in their structure to previously reported anti-CVB3 com-
pounds. Therefore, it was of interest to determine the anti-
viral activity of the prototype compound 26 against CVB3
strains that are resistant to the picornavirus 2C targeting
compound 58 (TBZE-029)37 or to the picornavirus 3A
targeting compound 60 (TTP-8307).38 The replication of
wild type CVB3 and these two mutant variants in the
presence of the three compounds (58, 60, and the arylpurine
26) is represented in Figure 4. As shown in Figure 4c,
compound 26 retained wild-type activity against these two
drug resistant variants of CVB3, suggesting a different mode
of action or a different molecular interaction with these two
viral proteins 2C and 3A.
Most of the compounds had no or little adverse effect on
Vero cells according to the CC50 values shown in Table 2.
The antiproliferative activity of selected compounds (3, 26,
51, and 54) was further determined in three different cell lines
(Vero, HeLa, and MRC-5 cells) by the MTS (3-(4,5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium) method (Promega, Leiden, The
Netherlands). Additionally, Vero cells survival was deter-
mined using the CellTiter-Glo luminescent cell viability
assay (Promega). Compounds 51 and 54 did not result in
any detectable toxicity at the highest concentration tested
(Table 3).
The most potent congener, compound 26, was selected for
the evaluation against a panel of other enteroviruses
(Table 4). Compound 26 proved to be active against Cox-
sackie virus A16 (CVA16) among enterovirus A. Also, in the
group of enterovirus B, antiviral activity in the low μM range
Conclusions
The present paper describes the discovery of a novel class of
enterovirus replication inhibitors, particularly against
Coxsackie virus type B3, with a scaffold of 9-arylpurines.
The here described compounds are easily accessible by two
different synthetic pathways: the coupling of purines bases
with aryl boronic acids catalyzed by copper salts, and a
microwave-assisted protocol from 4,6-dichloro-5-aminopyr-
imidines that reacted with anilines or acetamides, followed by
heterocyclization. Several of the synthesized structures are
selective inhibitors of in vitro CVB3 replication. Substituents
at the purine moiety are crucial for the antiviral effect,
particularly at position 6, where a chlorine or bromine atom

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 321
Figure 4. Replication of wild-type CVB3 and mutant viruses in the presence of 58 (TBZE-029) (graph A), 60 (TTP-8307) (graph B), and
compound 26 (graph C). Wild-type replication (WT) is represented as black bars. Mutant 1 [TTP8307r CVB3] (in white bars) is a resistant strain
to TTP8307 with an I54F substitution in 3A. Mutant 2 [TBZE029r CVB3] (in gray bars), resistant to TBZE029, is characterized by three
mutations affecting viral protein 2C (A224 V þ I227 V þ A229 V). Each graph represents the number of plaque forming units, produced by
wild-type virus or by the recombinant viruses at a given compound concentration.
is required. The direct linkage of the aryl moiety to the purine
ring is also important for the antiviral activity. However, the
aryl ring can incorporate different substituents in particular at
position 3. Thus, the most active compounds (26, 44, 51, and
54) inhibit CVB3, with EC50 values of 5-10 μM. Moreover,
compound 26 remains active against CVB3 strains that are
resistant to compounds such as 58 and 60, which targets the
viral 2C and the 3A proteins, respectively. The mechanism of
action of this novel class of compounds is currently being
studied and will be the subject of further investigation. An
effect on the binding of the virus to or the entry in the host cell
has already been excluded. Interestingly, compound 26 has
also no effect on viral RNA synthesis but yet efficiently
prevents the production of infectious progeny virus. Therefore,
these 9-arylpurines can be considered as a novel class of entero-
virus inhibitors characterized by their simplicity in structure, low
molecular weight, and selective inhibitory activity against entero-
virus replication that deserve further exploration.
Experimental Section
Chemistry Procedures. Melting points were obtained on a
Reichert-Jung Kofler apparatus and are uncorrected. The
elemental analysis was performed with a Heraeus CHN-O-
RAPID instrument. The elemental compositions of the com-
pounds agreed to within (0.4% of the calculated values. For all
the tested compounds, satisfactory elemental analysis was ob-
tained supporting >95% purity. Electrospray mass spectra
were measured on a quadrupole mass spectrometer equipped
with an electrospray source (Hewlett-Packard, LC/MS HP
1
1100). H and 13C NMR spectra were recorded on a Varian
INNOVA 300 operating at 299 MHz (1H) and 75 MHz (13C),
respectively, and Varian INNOVA-400 operating at 399 MHz
(1H) and 99 MHz (13C), respectively.

322 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Aguado et al.
Analytical TLC was performed on silica gel 60 F254 (Merck)
precoated plates (0.2 mm). Spots were detected under UV light
(254 nm) and/or charring with ninhydrin. Separations on silica
gel were performed by preparative centrifugal circular thin-layer
chromatography (CCTLC) on a Chromatotron (Kiesegel 60
PF254 gipshaltig (Merck)), with layer thickness of 1 and 2 mm
and flow rate of 4 or 8 mL/min, respectively. Flash column
chromatography was performed with silica gel 60 (230-
400 mesh) (Merck).
(30 mL) was stirred at room temperature for 3 h. Volatiles were
removed, and the residue was purified by CCTLC in the
Chromatotron (CH2Cl2:MeOH, 20:1) to yield 65 mg (95%) of
14 as a white solid; mp 197-199 °C. MS (ES, positive mode): m/z
256 (M þ H)þ. 1H NMR (DMSO-d6) δ 3.04 (s, 3H, CH3), 4.66
(d, 2H, J=5.6 Hz, CH2O), 5.45 (t, 1H, J=5.7 Hz, OH), 7.45
(d, 1H, J=7.6 Hz, Ar), 7.60 (t, 1H, J=7.8 Hz, Ar), 7.79 (d, 1H,
J=8.0 Hz, Ar), 7.92 (s, 1H, Ar), 7.93 (br s, 1H, NH), 8.36, 8.62 (s,
2H, H-2, H-8). Anal. (C13H13N5O) C, H, N.
Microwave reactions were performed using the Biotage Initi-
ator 2.0 single-mode cavity instrument from Biotage (Uppsala).
Experiments were carried out in sealed microwave process vials
utilizing the standard absorbance level (400 W maximum
power). The temperature was measured with an IR sensor on
the outside of the reaction vessel.
General Procedure for the Synthesis of N9-Arylpurines. A
mixture containing the corresponding arylboronic acid
(1.0 mmol), the purine base (0.5 mmol), 1,10-phenanthroline
(180 mg, 1.0 mmol), and cupric acetate (181 mg, 1.0 mmol) in
DMF (5 mL) was stirred at room temperature for 3 days.
Volatiles were removed and the residue was taken up in EtOAc
(50 mL) and washed with a solution of EDTA (400 mg in 50 mL
of water). The aqueous phase was further extracted with EtOAc
(2 ꢀ 20 mL). The combined organic phases were dried on
anhydrous Na2SO4, filtered, and evaporated. The residue was
then purified as indicated for each compound.
9-[3-(Hydroxymethyl)phenyl]hypoxanthine (15). A solution of
3 (53 mg, 0.2 mmol) in 1,4-dioxane (5 mL) was treated with 1N
HCl (5 mL) and heated at 90 °C overnight. After cooling to
room temperature, the mixture was neutralized by the addition
of Amberlite IRA400 (OH- form), filtered and evaporated. The
residue was then purified by CCTLC in the Chromatotron
(CH2Cl2:MeOH, 10:1) to yield 20 mg (42%) of 15 as a white
solid; mp 120-122 °C. MS (ES, positive mode): m/z 243 (M þ
1
H)þ. H NMR (DMSO-d6) δ 4.58 (d, 2H, J=5.4 Hz, CH2O),
5.37 (t, 1H, J=5.4 Hz, OH), 7.41-7.70 (m, 4H, Ar), 8.08 (s, 1H,
H-2), 8.43 (s, 1H, H-8), 12.41 (br s, 1H, NH). Anal.
(C12H10N4O2) C, H, N.
The synthesis of compounds 25-33 was performed following
the general procedure for the synthesis of N9-arylpurines and all
details and analytical and spectroscopic data are included in the
Supporting Information.
N4-(30-Acetyl-40-fluorophenyl)-6-chloropyrimidine-4,5-diamine
(43). A solution of 4-fluor-3-nitroacetophenone (150 mg, 0.82
mmol) in EtOH (6 mL) was hydrogenated in the presence of
Pt(S)/C (20 mg) at 30 psi at room temperature for 2 h. The
mixture was then filtered through celite, washed with EtOH and
MeOH, and the filtrate was evaporated. The crude, which
contained 38, was allowed to react with 5-amino-4,6-dichloro-
pyrimidine (34, 123 mg, 0.75 mmol) in the presence HCl (37% v/
v) (50 μL) in isobutyl alcohol (4 mL) at 120 °C for 1 h in a
microwave reactor. Volatiles were removed and the residue was
taken up in EtOAc (20 mL) and washed with a saturated, cold
NaHCO3 solution (15 mL) and brine (15 mL). The organic phase
was dried on anhydrous Na2SO4, filtered, and evaporated. The
residue was purified by flash column chromatography (hexane:
EtOAc, 2:1) to afford 80 mg (38% for the two steps) of 43 as a
yellow solid. MS (ES, positive mode): m/z 281 (M þ H)þ showing
6-Chloro-9-[3-(hydroxymethyl)phenyl]purine (3). Following
the general procedure for the synthesis of N9-arylpurines,
reaction of 6-chloropurine (1, 77 mg, 0.5 mmol) and 3-(hydroxy-
methyl)phenylboronic acid (2, 152 mg, 1.0 mmol) afforded a
residue that was purified by CCTLC in the Chromatotron
(CH2Cl2:MeOH, 50:1) to yield 77 mg (60%) of 3 as a white
solid; mp 197-199 °C. MS (ES, positive mode): m/z 261 (M þ
H)þ showing the isotopic Cl pattern. 1H NMR (DMSO-d6)
δ 4.74 (d, 2H, J=6.0 Hz, CH2O), 5.54 (t, 1H, J=5.9 Hz, OH),
7.58-7.96 (m, 4H, Ar), 8.97 (s, 1H, H-8), 9.22 (s, 1H, H-2). 13
C
NMR (DMSO-d6, 75 MHz) δ: 62.5 (CH2O), 122.0, 122.6, 127.0,
130.0, 134.0, 146.6 (Ar), 131.6, (C-5), 144.4 (C-8), 150.0 (C-4),
151.7 (C-6), 152.5 (C-2). Anal. (C12H9ClN4O) C, H, N.
The synthesis of compounds 8-11 was performed following a
similar procedure and all details, and analytical and spectro-
scopic data are included in the Supporting Information.
9-[3-(Hydroxymethyl)phenyl)purine (12). To a solution of 3
(120 mg, 0.5 mmol) in EtOH (10 mL), Pd(C) (10%) (20 mg) and
sodium acetate (113 mg, 1.4 mmol) were added. The reaction
mixture was then hydrogenated at 35 psi at 30 °C overnight.
1
the isotopic Cl pattern. H NMR (DMSO-d6) δ 2.57 (s, 3H,
CH3), 5.44 (br s, 2H, NH2), 7.43 (m, 1H, Ar), 7.80 (s, 1H, H-2),
7.83 (m, 1H, Ar), 8.19 (m, 1H, Ar), 8.65 (s, 1H, NH).
9-(3-Acetyl-4-fluorophenyl)-6-chloropurine (47). A solution of
43 (75 mg, 0.27 mmol) and trimethyl orthoformate (0.75 mL) in
acetic anhydride (0.75 mL) was placed in a microwave vessel.
The reaction was irradiated at 120 °C for 1 h. After cooling,
volatiles were evaporated and the residue was dissolved in
CHCl3 (15 mL) and washed with a saturated NaHCO3 solution
(15 mL) and brine (15 mL). The organic phase was dried on
anhydrous Na2SO4, filtered, and evaporated. The residue was
purified by CCTLC in the Chromatotron (CH2Cl2:EtOAc, 2:1).
The UV-positive fractions were combined, evaporated, and
further purified by CCTLC in Chromatotron (CH2Cl2:MeOH,
20:1) to afford 30 mg (39%) of 47 as a yellow solid; mp 162-
164 °C. MS (ES, positive mode): m/z 291 (M þ H)þ showing the
isotopic Cl pattern. 1H NMR (DMSO-d6) δ 2.64 (s, 3H, CH3),
7.77 (dd, 1H, J=9.9, 1.0 Hz, Ar), 8.25 (m, 1H, Ar), 8.43 (dd, 1H,
J = 7.2, 2.2 Hz, Ar), 8.85, 9.02 (s, 1H, H-2, H-8). Anal.
(C13H8ClFN4O) C, H, N.
3-(3-Acetylphenyl)-7-chlorotriazolo[4,5-d]pyrimidine (52). To
a suspension of 3926 (150 mg, 0.57 mmol) in CH2Cl2 (2 mL) and
1 M HCl (2 mL), NaNO2 (41 mg, 0.6 mL) was added in small
portions. The mixture was stirred at room temperature for 30
min. Then, CH2Cl2 (10 mL) was added and the organic layer was
washed with brine, dried on anhydrous Na2SO4, filtered, and
evaporated. The final residue was purified by flash column
chromatography (CH2Cl2:EtOAc, 2:1) to yield 120 mg (77%)
of 52 as a brown solid; mp 127-129 °C. MS (ES, positive mode):
Na2SO4 10H2O was then added and the mixture was stirred for
3
2 h, filtered through celite, and the filtrate was evaporated. The
residue was purified by CCTLC in the Chromatotron (CH2Cl2:
MeOH, 30:1) to yield 65 mg (63%) of 12 as a white solid; mp
1
130-132 °C. MS (ES, positive mode): m/z 227 (M þ H)þ. H
NMR (DMSO-d6) δ 4.62 (d, 2H, J=3.6 Hz, CH2O), 5.41 (t, 1H,
J=4.8 Hz, OH), 7.45 (d, 1H, J=7.7 Hz, Ar), 7.59 (t, 1H, J=7.8
Hz, Ar), 7.77 (d, 1H, J=7.9 Hz, Ar), 7.88 (s, 1H, Ar), 9.02, 9.03,
9.29 (s, 3H, H-2, H-6, H-8). Anal. (C12H10N4O) C, H, N.
9-[3-(Hydroxymethyl)phenyl]-6-methoxypurine (13). To a stir-
red solution of 3 (90 mg, 0.3 mmol) in MeOH (5 mL), MeONa
(56 mg, 1.0 mmol) was added and the reaction mixture was
refluxed for 2 h. After cooling to room temperature, the mixture
was neutralized with AcOH and volatiles were removed. The
residue was treated with CH3CN, and the precipitate was
filtered and washed with CH3CN. The filtrate was then evapo-
rated to obtain 82 mg (95%) of 13 as a white solid; mp 154-156
°C. MS (ES, positive mode): m/z 257 (M þ H)þ. 1H NMR
(DMSO-d6) δ 4.12 (s, 3H, CH3), 4.60 (s, 2H, CH2), 5.41 (br s,
1H, OH), 7.42 (d, 1H, J=7.6 Hz, Ar), 7.55 (t, 1H, J=7.8 Hz, Ar),
7.72 (d, 1H, J=7.7 Hz, Ar), 7.83 (s, 1H, Ar), 8.60, 8.78 (s, 2H,
H-2, H-8). Anal. (C13H12N4O2) C, H, N.
6-(Methylamino)-9-[3-(hydroxymethyl)phenyl]purine (14). A
solution of 3 (70 mg, 0.2 mmol) in MeNH2 in MeOH (33%)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 323
1
m/z 274 (M þ H)þ showing the isotopic Cl pattern. H NMR
(CDCl3) δ 2.73 (s, 3H, CH3), 7.77 (t, 1H, J=7.9 Hz, Ar), 8.12 (d,
1H, J=7.8 Hz, Ar), 8.49 (d, 1H, J=8.0 Hz, Ar), 8.88 (s, 1H, Ar),
9.07 (s, 1H, H-2). Anal. (C12H8ClN5O) C, H, N.
of compound that inhibited virus-induced cytopathic effect
formation by 50% and was calculated using linear interpolation.
Each experiment was repeated at least three times.
Cytotoxic Assays. Cytotoxic evaluation of the selected com-
pounds was performed using the MTS-method or CellTiter-Glo
luminescent cell viability assay (Promega) and the 50% cyto-
toxic concentration (CC50) was calculated as the concentration
of compound that inhibited cell proliferation by 50% using
linear interpolation. Briefly, the same experimental setup was
used as for the antiviral assay, but for cytotoxicity determina-
tion, uninfected cultures were incubated with serial dilution of
compound for three days at 37 °C. Each experiment was
repeated at least three times.
Viral Plaque Assays. For determination of viral plaques,
Vero cells, grown to confluence in six-well plates, were infected
with CVB3 at 37 °C. After 2 h, the virus was removed, the cells
were washed twice with phosphate-buffered saline (PBS), and
the growth medium was replaced with agar (final concentra-
tion, 0.5%) in the presence or absence of compound. After 3 to
4 days, plaques were visualized. Briefly, cells were fixed with
2 mL of a solution containing 4% formaldehyde, after which
the agar was removed. A 2% Giemsa solution was used to stain
the cells.
3-(3-Acetylphenyl)-7-chloro-5-methyltriazolo[4,5-d]pyrimidine
(53). Following a procedure analogous to that described for the
synthesis of 52, a solution of 4026 (120 mg, 0.43 mmol) in CH2Cl2
(1.5 mL) and 1 M HCl (1.5 mL) was allowed to react with
NaNO2 (32 mg, 0.46 mmol) to afford 92 mg (75%) of 53 as a
brown solid; mp 109-111 °C. MS (ES, positive mode): m/z 288
(M þ H)þ showing the isotopic Cl pattern. H NMR (CDCl3)
1
δ 2.72(s, 3H, CH3), 2.93(s, 3H, CH3), 7.75 (t, 1H, J=7.9 Hz, Ar),
8.10 (m, 1H, Ar), 8.47 (m, 1H, Ar), 8.86 (s, 1H, Ar). Anal.
(C13H10ClN5O) C, H, N.
6-Chloro-2-methyl-9-[3-(hydroxyprop-2-yl)phenyl]purine (54).
A solution of 4426 (100 mg, 0.35 mmol) in anhydrous THF (9
mL) at 0 °C was treated with methylmagnesium iodide (175 μL)
for 90 min. The reaction mixture was quenched by the addition
of a saturated NH4Cl solution (2 mL). Ethyl acetate (10 mL) and
water (10 mL) were added. The organic phase was dried on
anhydrous Na2SO4, filtered, and evaporated. The final residue
was purified by CCTLC in the Chromatotron (CH2Cl2:EtOAc,
2:1) to yield 56 mg (53%) of 54 as a white solid; mp 154-156 °C.
MS (ES, positive mode): m/z 303 (M þ H)þ showing the isotopic
1
Cl pattern. H NMR (DMSO-d6) δ 1.49 (s, 6H, (CH3)2), 2.69
Acknowledgment. L.A. thanks the Spanish Ministerio
ꢀ
(s, 3H, 2-CH3), 5.21 (s, 1H, OH), 7.53-7.69 (m, 3H, Ar), 7.90
(t, 1H, J=7.8 Hz, Ar), 8.97 (s, 1H, H-8). Anal. (C15H15ClN4O)
C, H, N.
de Educacion y Ciencia for a FPU predoctoral fellowship.
E-M.P. has a CSIC contract from the I3P programme
financed by the Fondo Social Europeo (F.S.E.). We thank
Marıa Nares for excellent technical assistance. This work has
´
been supported by a grant of the Spanish CICYT (SAF2006-
12713-C02-01) and has received the FAES FARMA SA
award for young researchers in the XIV call sponsored by
the Spanish Society of Medicinal Chemistry (SEQT). This
work was also supported by the VIZIER EU FP7-Integrated
Project (LSHG-CT-2004-51196).
6-Chloro-9-(3-hydroxyprop-2-yl)-2-methylpurin-8-one (55).
Following a procedure analogous to that described for the
synthesis of 54, a solution of 5126 (42 mg, 0.14 mmol) in
anhydrous THF (3.5 mL) was treated with CH3MgI (73 μL,
0.21 mmol) at 0 °C for 3 h. The final residue was purified by
CCTLC in the Chromatotron (CH2Cl2:EtOAc, 1:1) to afford
20 mg (45%) of 55 as a white solid; mp 170-172 °C. MS (ES,
positive mode): m/z 319 (M þ H)þ showing the isotopic Cl
1
pattern. H NMR (DMSO-d6) δ 1.46 (s, 6H, (CH3)2), 2.47 (s,
3H, 2-CH3), 7.36-7.67 (m, 4H, Ar), 12.13 (br s, 1H, NH). Anal.
Supporting Information Available: Synthesis and spectro-
scopic data of compounds 8-11 and 25-33. Elemental analysis
of compounds 3, 8-15, 25-33, 47, 52-55 is included. Figure S1
(C15H15ClN4O2) C, H, N.
NMR Experiments Performed for Compound 50. 1H, 13C and
15N NMR spectra were recorded at 303 K, using DMSO-d6 as
the solvent, on a Varian SYSTEM 500 NMR spectrometer
1
represents the H and 13C chemical shifts from compound 50
based on 1H-13C NMR correlation experiments. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
equipped with a 5 mm HCN cold probe. H and 13C chemical
shifts are reported in ppm from tetramethylsilane and 15N
chemical shifts from nitromethane. All assignments have been
performed on the basis of heteronuclear multiple quantum
coherence experiments (gHMQC and gHMBC). Two-dimen-
sional [1H-13C] NMR experiments (gHSQC and gHMBC) were
obtained using a 1H spectral window of 4808 Hz, a 13C spectral
windows of 30 000 Hz, 1 s of relaxation delay, 1024 data points,
and 200 time increments, with a linear prediction to 512. The
data were zero-filled to 4096 ꢀ 4096 real points. Typical
numbers of transients per increment were 4 and 16, respectively.
Two-dimensional [1H-15N] gHSQC and [1H-15N] gHMBC
experiments was carried out with the same conditions, using
16 and 64 transients per increment, respectively, and a 15N
spectral window of 15200 Hz.
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