Efficient synthesis and anti-enteroviral activity of 9-arylpurines

Article abstract of DOI:10.1016/j.ejmech.2012.01.022

To further explore the anti-enteroviral activity of 9-aryl-6-chloropurines, three different series of compounds with a dialkylamino, (alkyl)amido, or oxazolidinone substituent at the aryl ring have been synthesized, in most cases with the aid of microwave

Full text of DOI:10.1016/j.ejmech.2012.01.022

European Journal of Medicinal Chemistry 49 (2012) 279e288
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient synthesis and anti-enteroviral activity of 9-arylpurines
Leire Aguado a, María-Dolores Canela a, Hendrik Jan Thibaut b, Eva-María Priego a, María-José Camarasa a,
,
Pieter Leyssen b, Johan Neyts b, María-Jesús Pérez-Pérez b
*
a Instituto de Química Médica (IQM-CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
To further explore the anti-enteroviral activity of 9-aryl-6-chloropurines, three different series of
compounds with a dialkylamino, (alkyl)amido, or oxazolidinone substituent at the aryl ring have been
synthesized, in most cases with the aid of microwave-assisted synthesis. The resulting compounds
Received 20 December 2011
Received in revised form
10 January 2012
Accepted 11 January 2012
Available online 17 January 2012
efficiently inhibit Coxsackie virus type B3 (CVB3) replication with EC50 values varying from 3 to 15 mM,
and with no significant toxicity in Vero cells. The most potent compounds also selectively inhibit the
replication of other enteroviruses including Coxsackie virus B4 and Echo virus 11. The cross-resistance
studies performed with different 9-aryl-6-chloropurines indicate that they all belong to the same
pharmacological family and differ from other CVB3 drugs such as enviroxime.
Keywords:
Purines
Microwave-assisted synthesis
Oxazolidinone
Enterovirus
Ó 2012 Elsevier Masson SAS. All rights reserved.
Coxsackie virus B3
1. Introduction
target. TBZE-029 has been proposed to interact with the 2C protein
[7], a well-conserved protein that is essential for viral replication
Enteroviruses, which belong to the Picornaviridae family, are
small, non-enveloped, eicosaheadral, positive-sense single-
stranded RNA viruses. They are responsible for a spectrum of
diseases in humans, i.e respiratory infections, meningitis, enceph-
alitis, pancreatitis, myocarditis and neonatal sepsis [1]. Among
enteroviruses, Coxsackievirus B3 (CVB3) is considered to be
a pathogen of significant importance as it is the most common
cause of acute and chronic viral myocarditis, mostly in children and
young adults [2,3]. In addition, CVB3 is also associated with several
inflammatory diseases of the pancreas and of the central nervous
system, or can cause acute episodes of hand-foot and mouth
disease [4e6]. Despite the impact of CVB3 infections on human
health, there is currently no drug approved for the treatment of
CVB3 or other enteroviral infections.
and which is hypothesized to have ATPase/helicase activity. On the
other hand, the non-structural protein 3A, a key partner in the viral
replication complex, seems to be involved in the mechanism of
action of TTP-8307 [9] and enviroxime [10].
We recently described the anti-enteroviral activity of a novel
family of compounds that structurally can be described as
9-arylpurines, and of which compound 4 is the prototype (Fig. 1)
[11]. These compounds can easily be synthesized in two steps and
have an attractive low molecular weight. Moreover, they efficiently
and selectively inhibit enteroviral replication without apparent
adverse effects on several host lines i.e. Vero, HeLa, and MRC-5
cells. Interestingly, compound 4 did not show cross resistance
when evaluated for selective antiviral activity on the replication of
TTP-8307- or TBZE029-resistant CVB3, suggesting a different
mechanism of action [11]. For RNA viruses, such as HIV and HCV
[12], it was shown to be essential to combine antiviral drugs with
a different mechanism of action to prevent rapid emergence of
resistance during treatment, a strategy which recently has been
proposed for enteroviruses as well [13].
In recent years, a number of compounds have been described as
efficient and selective inhibitors of CVB3 replication in vitro,
including TBZE-029 (1) [7], enviroxime (2) [8], and TTP-8307 (3) [9]
(Fig. 1). Mechanism-of-action studies performed with these
compounds point towards non-structural proteins as their main
Structureeactivity relationship studies performed with a first
series of compounds revealed that a chloro or bromo residue on
position 6 of the purine is essential for the compounds to selec-
tively inhibit CVB3 replication [11]. The introduction of a carbonyl
at position 8 and/or a methyl at position 2 of the purine base, as
in compound 5 (Fig. 1), led to a compound with similar antiviral
Abbreviations: CVB3, coxsackie virus type B3; MAOS, microwave-assisted
organic synthesis.
* Corresponding author. Tel.: þ34 91 258 75 79; fax: þ34 91 5644853.
E-mail address: mjperez@iqm.csic.es (M.-J. Pérez-Pérez).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.022

280
L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
2. Results and discussion
2.1. Chemistry
Our previously described 9-aryl-6-chloropurines have been
prepared by either of the following two synthetic approaches: i)
Copper-mediated coupling of the corresponding aryl boronic acids
with 6-chloropurine leading exclusively to the N-9 substituted
compounds [11]; ii)
a microwave-assisted two-step protocol
involving the reaction of anilines with 4,6-dichloro-5-
aminopyrimidines, followed by cyclization [11,15]. This second
strategy has been employed for the synthesis of the compounds
described in this study. Thus, reaction of the diamines 7, 8 or 9 with
the dichloropyrimidines 10 or 11 in isobutanol at 150 ꢀC for 1 h in
the presence of HCl yielded the 4,5-diamino-6-chloropyrimidines
12e17 in good to excellent yields (Scheme 1). In most cases, these
diaminopyrimidines were isolated from the reaction mixture by
filtration and used as such in the next step. Then, reaction of 12e17
with trimethyl orthoformate in acetic anhydride at 120 ꢀC for 1 h led
to the 9-arylpurines 18e23. Alternatively, as shown in Scheme 2,
the diaminopyrimidines 14 and 15 reacted with triphosgene in THF
under microwave irradiation at 120 ꢀC for 20 min [15] to prepare
the corresponding 8-oxopurines 24 and 25. According to our
previous studies, the keto form should be the predominant one in
DMSO-d6 [11]. Moreover, reaction of 25 with MeI in the presence of
K2CO3 [16] afforded the N-7 methyl derivative 26 in 44% yield. On
the other hand, reaction of 25 with ethyl or phenyl chloroformate
in CH2Cl2 at 0 ꢀC in the presence of DIPEA, as described for
8-oxoadenines [16], efficiently afforded the 8-substituted purines
27 and 28 in 74 and 75% yield, respectively.
The next series of compounds were aimed to contain an
acetanilide as the aryl substituent. Thus, reaction of 30-amino-
acetanilide (29) with 5-amino-4,6-dichloropyrimidine (10) or its
2-methyl analogue (11) in isobutanol in the presence of acetic acid
under MAOS at 150 ꢀC for 15 min afforded the acetamides 30 and
31 in good yields (Scheme 3). It should be emphasized that the
employment of acetic acid instead of HCl was crucial to perform
the reaction at the NH2 without affecting the acetamide. Cycli-
zation of 30 and 31 with trimethyl ortoformate was performed in
the presence of ethanosulphonic acid [17] using MAOS to obtain
the purines 32 and 33 in 72 and 80% yield, respectively. The
presence of the free NH at the acetamide allowed the possibility
for further functionalization. Indeed, the acetamides 32 or 33
Fig. 1. Reference compounds with selective anti-CVB3 activity including 9-arylpurines
(4-6).
activity [11]. Direct attachment of the aryl ring to the 9-position of
the purine produced better inhibitory activity compared to
a benzylic substituent [11]. Finally, a variety of substituents could be
introduced at the aryl moiety without compromising the antiviral
activity. In general, substitutions at the meta position resulted in
better EC50 values that when the same substituent was introduced
at the para position. Interestingly, the nature of the substituent
seems not to be a limiting factor as both a hydrogen-bond acceptor,
as in 4, or a hydrogen bond donor, as in 6, resulted in similar
antiviral activities. Recently other 6-chloropurines with a bicyclic
scaffold at position 9 have also been reported as inhibitors of CVB3
replication [14].
In the present study, we describe the synthesis, antiviral eval-
uation, and exploration of the structureeactivity relationship (SAR)
of novel 9-arylpurines incorporating N-attached substituents on
the aryl ring, that is, dialkylamino, (alkyl)amido or oxazolidinones
functionalities versus the C-attached substituents in our first series
(exemplified by compounds 4e6). Besides this, additional substi-
tutions at position 7 or 8 of the purine have been explored.
Scheme 1. Reagents and conditions: (a) Isobutyl alcohol, HCl (cat), MW, 150 ꢀC, 10-20 min; (b) HC(OCH3)3, Ac2O, MW, 120 ꢀC, 1h. The synthesis of 12, 13 and 18, 19 has been
described in ref. 15.

L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
281
a
Scheme 2. Reagents and conditions: (a) triphosgene, THF, MW, 120 ꢀC 1h; (b) CH3I, K2CO3, DMF, rt, overnight; (c) EtOCOCl or PhOCOCl, CH2Cl2, DIPEA, 0 ꢀC, 3 h.
were alkylated by treatment with NaH in the presence of
molecular sieves at ꢁ30 ꢀC, followed by the addition of alkyl
halides. In this way, compounds with a methyl (34, 35), ethyl (36),
or methylcyclopropyl (37) substituent at the NH of the acetamide
were obtained. These compounds, particularly those with
a methyl or an ethyl substituent, retained selective antiviral
activity against CVB3. Based on these SAR data, it was considered
to develop a third series of compounds containing an oxazolidin-
2-one at the aryl ring as a possible ring-closed analogue of an N-
alkyl amide.
a
Scheme 3. Reagents and conditions: (a) Isobutyl alcohol, AcOH, MW, 150 ꢀC, 15-30 min; (b) HC(OCH3)3, Ac2O or EtSO3H, MW, 120 ꢀC, 1h; (c) NaH, 4 Å molecular sieves, DMF,
ꢁ30 ꢀC, CH3I (for 34 and 35), EtI (for 36) or methylcyclopropylbromide (for 37).

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L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
For the synthesis of the oxazolidin-2-ones (Scheme 4), the
starting material was the 30-aminoacetanilide 29. A solution of 29
in acetonitrile was treated with 2-chloroethyl chloroformate in the
presence of Cs2CO3 and DIPEA and microwaved-irradiated for 2 h at
120 ꢀC to obtain the oxazolidinone 38 in 81% yield. The employ-
ment of MAOS allowed the reaction to be performed in just 2 h
compared to 12 h refluxing as reported for a similar example [18].
Reaction of 38 with the dichloropyrimidines 10 and 11 afforded the
diaminopyrimidines 39 and 40 in 41 and 51% yield, respectively.
Treatment of 39 and 40 with trimethyl orthoformate in the pres-
ence of ethanosulfonic acid allowed the synthesis of the purines 41
and 42 in 84 and 86% yield, respectively. Alternatively, reaction of
39 and 40 with triphosgene led to the 8-oxopurines 43 an 44 in 85
and 84% yield, respectively.
a
Scheme 5. Reagents and conditions: (a) 12.5 N NaOH, EtOH, MW, 50 ꢀC, 15 min.
ring by performing the reaction in other solvents (dioxane, pyri-
dine, .) failed, and the starting material remained intact.
On the other hand, while reaction of the oxazolidinone 38 with
KCN in the presence of 18-crown-6 [20] led to the expected
3-aminopropinionitrile (data not shown), all attempts to perform
the same reaction on the purine derivative 41 failed, leading to
extensive decomposition.
Besides their interest as ring-closed analogues of N-alkyl
amides, oxazolidin-2-ones can also be considered as interesting
intermediates towards the synthesis of
-aminonitriles. In this sense, the oxaxolidin-2-one 41 was treated
with NaOH in EtOH [19] to obtain the corresponding -amino
alcohol. However, the isolated reaction product was identified as
compound 45 (Scheme 5) whereas the presence of -amino alcohol
b-amino alcohols or
b
b
2.2. Antiviral evaluation
b
is accompanied by the concomitant substitution at position 6 of the
chloro by an OEt. Attempts to selectively open the oxazolidinone
The compounds were evaluated for selective antiviral activity on
the replication of Coxsackie virus type B3 (CVB3), Nancy strain,
in Vero cells (Table 1). The previously-identified inhibitors,
compounds 4, 5 and 6, are included for comparison. TBZE-029 and
enviroxime, two other selective inhibitors of CVB3 replication
(Fig. 1), were included as reference compounds. The antiviral
Table 1
Antiviral activity, expressed as EC50
ievirus B3, Nancy strain, in Vero cells.
(m
M), of the 9-arylpurines against Coxsack-
M)b M)c
Compound ID
EC50
(
m
M)a
EC90
(
m
CC50 (m
4 (TP219)
5
6
18
19
20
21
22
23
24
25
26
27
28
32
33
34
35
36
37
41
42
43
44
7.3 ꢂ 2.3d
6.7 ꢂ 2.1d
8.0 ꢂ 3.4d
15 ꢂ 2d
9.1 ꢂ 3.1
10 ꢂ 2.1
7.4 ꢂ 2.1
23 ꢂ 2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>200
131.3 ꢂ 10.0
75.9 ꢂ 1.3
>174
>200
>100
10 ꢂ 1d
15 ꢂ 3
3.8 ꢂ 0.9d
12 ꢂ 1d
5 ꢂ 1
28 ꢂ 1
6.3 ꢂ 1.2d
11 ꢂ 4.3d
16 ꢂ 4d
ND
ND
26 ꢂ 8
10 ꢂ 2d
15 ꢂ 1
11.3 ꢂ 0.6d
12.6 ꢂ 0.3d
13.6 ꢂ 6.2
18.5 ꢂ 7.3
11.8 ꢂ 1.3d
7.8 ꢂ 1.3d
6.1 ꢂ 1.0d
6.1 ꢂ 2.7d
34.5 ꢂ 3.5d
33.7 ꢂ 2.5d
10.6 ꢂ 0.5d
8.9 ꢂ 2.7d
18.2 ꢂ 0.5
19.9 ꢂ 0.3
14.8
24.2 ꢂ 9.7
17.5 ꢂ 2.7
ND
8.3 ꢂ 1.4
10.9 ꢂ 4.5
52.3 ꢂ 6.8
47.5 ꢂ 4.3
15.6 ꢂ 1.4
15.3 ꢂ 2.0
33.5 ꢂ 16.1
>200
307 ꢂ 17
>200
>200
>200
>200
>200
>200
>200
>100
>100
25.4 ꢂ 12.3d
>200
45
58(TBZE-029)
59(Enviroxime)
1.2 ꢂ 0.4d
ND
ND
0.7 ꢂ 0.3d
a
EC50 ¼ concentration of compound, calculated from the doseeresponse curves,
that reduces virus-induced cell death by 50%.
b
EC90 ¼ concentration of compound, calculated from the doseeresponse curves,
that reduces virus-induced cell death by 90%.
c
CC50 ¼ concentration of compound, calculated for the doseeresponse curves,
that elicits 50% anti-metabolic effect on uninfected host cells.
d
when checked microscopically, at least one concentration of compound
completely protects the cells from virus-induced cytopathic effects without causing
any visible morphological alterations to the cells compared to the uninfected,
untreated control condition; all data are mean values ꢂ standard deviation for at
least three independent experiments. ND: Not determined.
a
Scheme 4. Reagents and conditions: (a) ClCH2CH2COCl, Cs2CO3, DIPEA, CH3CN, MW,
120 ꢀC, 2h; (b) 10 or 11, isobutyl alcohol, HCl (cat), MW, 150 ꢀC, 15-30 min; (c)
HC(OCH3)3, EtSO3H, MW, 120 ꢀC, 30-60 min; (d) triphosgene, THF, MW, 120 ꢀC, 1h.

L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
283
Table 2
Some of the most potent inhibitors of CVB3 replication were also
evaluated for selective antiviral activity on the replication of other
enteroviruses as showninTable 2. Compounds 20, 32 and 35 efficiently
and selectively inhibited the replication of echo virus 11 and Coxsackie
virus type B4, but no significant antiviral activity was observed on the
replication of enterovirus 71, poliovirus 1, or rhinovirus 14.
Subsequently, it was assessed whether or not the compounds
described in this study belong to the same pharmacological class as
our previously reported arylpurines, exemplified by the methyl-
ketone 4 (TP219). To this end, CVB3 resistant to the antiviral effect
of compound 4 was evaluated for susceptibility to inhibition by
treatment with compounds containing either an amino, amido, or
oxazolidinone substitution. The EC50 values obtained were as
Antiviral activity, expressed as EC50
enteroviruses.
(mM), of selected compounds against different
Compound PV1a
EV71b
Echo11c
CVB4d
hRV14e
(BGM cells) (RD cells) (BGM cells) (Vero cells) (Hela cells)
4 (TP219)
>100
>100
>174
>158
>151
>100
>100
>174
>158
>151
21.6 ꢂ 12.0 28.8 ꢂ 5.5f >100
20
32
35
43
12.5 ꢂ 2.5
20.8 ꢂ 8.9f
20.0 ꢂ 1.7f
6.9 ꢂ 0.6f >100
12.1 ꢂ 2.0f >100
13.0 ꢂ 7.5f >100
86.9 ꢂ 20.0 24.1 ꢂ 3.7f >100
a
PV1 ¼ poliovirus type 1, Sabin strain.
EV71 ¼ enterovirus 71, BRCR strain.
b
c
d
e
f
ECHO11 ¼ echovirus 11, Gregory strain.
CVB4 ¼ Coxsackievirus B4, E2 Edwards strain.
hRV14 ¼ human rhinovirus type 14.
follows: 4 (TP219) EC50 > 100
EC50 > 50 M, and 35: EC50 > 50
The inability of the novel compounds to inhibit the replication of
compound 4-resistant virus confirms that the different 6-chloro-9-
arylpurines belong to the same pharmacological family.
m
M; 20: EC50 > 100
m
M, 32:
when checked microscopically, at least one concentration of compound
m
m
M; enviroxime: EC50 1.3 ꢂ 0.1
mM.
completely protects the cells from virus-induced cytopathic effects without causing
any visible morphological alterations to the cells compared to the uninfected,
untreated control condition; the EC50 is derived from the doseeresponse curves
and represents the concentration of compound that is calculated to inhibit virus-
induced cell death by 50%.
activity is expressed as the 50% effective concentration (EC50) and
the 90% effective concentration (EC90). The adverse effect of the
2.3. Maps of electrostatic potential
compounds on non-infected cells is expressed as CC50
As shown in Table 1, most of the newly-synthesized compounds
have selective antiviral activity on the replication of CVB3 with EC50
.
At this moment, the target(s) of the 6-chloro-9-arylpurines is
still unknown and the exact mechanism of action still has to be
unravelled. To obtain some more information on the effect of the
substituents on the aryl ring, molecular electrostatic potential
(MEP) maps were calculated for selected dialkylamino (18 and 20),
(alkyl)amido (32 and 34) and oxazolidinone (41) compounds. The
prototype compound 4 was also included in this study.
The geometry of the compounds was first optimized by means of
the ab initio program Gaussian 03, and MEP maps were calculated
based on the optimized structures using the program DelPhi. The
results are shown in Fig. 2. As the same purine ring is present in all of
values ranging from 3 to 10
where observed when uninfected Vero cells were treated with
concentrations up to 100e200 M, with the exception of
mM. No significant adverse effects
m
compound 28. Interestingly, when the substituent at position 6 is
not a chlorine, as in compound 45, the anti-CVB3 activity is lost.
These data are consistent with our previously-reported SAR that
indicated that a chlorine or bromine residue at position 6 is
required to obtain compounds with selective antiviral activity [11].
Fig. 2. MEP maps of compounds 4, 18, 20, 32, 34 and 41. Positive and negative isopotential surface are coloured in cyan and pink, respectively.

284
L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
the studied structures, it is logical that the MEP maps corresponding
to the base show a common pattern. Concerning the aryl moiety,
overall it may be considered that there are no big differences among
this class of compounds: in all cases it is visible the presence of
localized negative and positive potential regions affecting respec-
tively the left and right faces of the aryl moiety. A more careful
analysis shows that the negative isosurface for the oxazolidinone
derivative 41 is the one that better resembles the MEP maps of the
prototype 4. However, this does not translate into a similar activity
for compounds 4 and 41. Of course, other factors including intra-
cellular delivery, metabolic stability etc. may account for the
different EC50 values. On the other hand, the dimethylamino deriv-
ative 18 with a higher contribution of the positive isosurface at the
left hand side is equipotent with the prototype compound 4. In
conclusion, MEP maps do not show a strict pattern among the active
compounds (i.e compounds 4 and 20), suggesting that this part of
the molecule can incorporate quite different substituents in terms of
electrostatic properties without compromising antiviral activity.
Microwave reactions were performed using the Biotage Initiator
2.0 single-mode cavity instrument from Biotage (Uppsala). Exper-
iments 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.
4.1.1. General procedure for the reaction of 4,6-dichloropyrimidines
with arylamines
A microwave vial was charged with the corresponding 4,6-
dichloropyrimidine (1.0 mmol), the appropriate arylamine
(1.0 mmol), 4 mL of isobutanol and 50 mL of 37% aqueous HCl. The
reaction vessel was sealed and heated in a microwave reactor at
150 ꢀC for 10 min. After cooling, the reaction mixture was worked
up as indicated in each case.
The synthesis of 12 and 13 was performed as previously
described [15].
4.1.1.1. 6-Chloro-N4-[30e(N0,N0-dimethylamino)phenyl]pyrimidine-
4,5-diamine (14). Following the general procedure, a microwave
vial was charged with 5-amino-4,6-dichloropyrimidine (164 mg,
1.0 mmol), N1,N1-dimethyl-1,3-phenylenediamine hydrochloride
3. Conclusions
Novel 9-aryl-6-chloropurines have been synthesized and eval-
uated for selective antiviral activity on CVB3 replication. From
a synthetic point of view, most of the reactions have been per-
formed through microwave-assisted synthesis (MAOS), signifi-
cantly reducing the reaction times compared to previous reported
examples (i.e the cyclization of the diaminopyrimidines to the
purines in the presence of ethanesulphonic acid was performed in
1 h instead of the three days reported in a similar example [17]).
Moreover, MAOS is compatible with a selective functionalization of
the molecules as shown for the acetamides or the oxazolidinones.
From a biological point of view, it should be emphasized that
most of the synthesized compounds keep selective antiviral activity
on the replication of CVB3. Moreover, the most potent compounds
(209 mg, 1.0 mmol), isobutanol (4 mL) and 37% aqueous HCl (50 mL).
After cooling, the product was isolated by filtration, dried and used
as such for the next step. Yield: 187 mg (71%). EM (ES, positive
mode): m/z 264 (M þ H)þ with a Cl isotopic pattern. 1H NMR
(DMSO-d6, 300 MHz) d: 3.32 (s, 6H, N(CH3)2), 4.73 (bs, 2H, NH2),
7.22e7.40 (m, 4H, Ar), 8.43 (s, 1H, H-2), 8.61 (s, 1H, NH).
4.1.1.2. 6-Chloro-N4-[30e(N0,N0-dimethylamino)phenyl]-2-methyl-
pyrimidine-4,5-diamine (15). Following the general procedure,
a
microwave vial was charged with 5-amino-4,6-dichloro-2-
methylpyrimidine (178 mg, 1.0 mmol), N1,N1-dimethyl-1,3-
phenylenediamine hydrochloride (209 mg, 1.0 mmol), isobutanol
(i.e 20 or 36) are characterized by EC90 values in the low
mM range.
(4 mL) and 37% aqueous HCl (50 mL). The product was isolated by
Furthermore, significant and selective antiviral activity was
observed on the replication of CVB4 and echovirus 11, and appears
to be independent of host cell type. Currently, the mechanism of
action of these 9-arylpurines is being elucidated. Our preliminary
results indicate that these compounds impede correct assembly of
infectious virus particles [21]. Additional studies are ongoing to
confirm these results and to unravel the precise mechanism.
filtration, dried and used as such for the next step. Yield: 271 mg
(98%). EM (ES, positive mode): m/z 278 (M þ H)þ with a Cl isotopic
pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 2.38 (s, 3H, CH3), 3.09 (s,
6H, N(CH3)2), 4.80 (s, 2H, NH2), 6.81 (m, 1H, Ar), 7.25e7.69 (m, 3H,
Ar), 9.33 (s, 1H, NH).
4.1.1.3. 6-Chloro-N4-[40e(N0,N0-dimethylamino)phenyl]pyrimidine-
4,5-diamine (16). According to the general procedure, a microwave
vial was charged with 5-amino-4,6-dichloropyrimidine (123 mg,
0.75 mmol), N1,N1-dimethyl-1,4-phenylenediamine (102 mg,
4. Experimental
4.1. Synthesis
0.75 mmol), isobutanol (3 mL) and 37% aqueous HCl (38 mL). After
cooling, ethyl acetate was added (15 mL) and the crude reaction
mixture was subsequently washed with saturated aqueous NaHCO3
(15 mL) and brine (15 mL). The organic layer was dried over Na2SO4,
filtered and evaporated to dryness. Treatment with diethyl ether
afforded 132 mg (67%) of 16 as a brown solid, used as such for the
next step. EM (ES, positive mode): m/z 264 (M þ H)þ with a Cl
Melting points were obtained on
a Reichert-Jung Kofler
apparatus and are uncorrected. The elemental analysis was per-
formed with a Heraeus CHN-O-RAPID instrument. The elemental
compositions of the compounds agreed to within ꢂ0.4% of the
calculated values. Electrospray mass spectra were measured on
a quadrupole mass spectrometer equipped with an electrospray
source (HewlettePackard, LC/MS HP 1100). 1H 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.
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 ChromatotronR (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 in a Biotage Horizon instrument.
isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 2.86 (s, 6H,
N(CH3)2), 5.28 (s, 2H, NH2), 6.72 (d, 1H, J ¼ 8.4 Hz, Ar), 7.43 (d, 1H,
J ¼ 8.2 Hz, Ar), 7.76 (s, 1H, H-2), 8.36 (s, 1H, NH).
4.1.1.4. 6-Chloro-N4-[40e(N0,N0-dimethylamino)phenyl]-2-methyl-
pyrimidine-4,5-diamine (17). According to the general procedure,
a
microwave vial was charged with 5-amino-4,6-dichloro-2-
methylpyrimidine (133 mg, 0.75 mmol), N1,N1-dimethyl-1,4-
phenylenediamine (102 mg, 0.75 mmol), isobutanol (3 mL) and
37% aqueous HCl (38 mL). After cooling, ethyl acetate was added
(15 mL) and the crude reaction mixture was subsequently washed
with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The
organic layer was dried over Na2SO4, filtered and evaporated to

L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
285
dryness. Treatment with diethyl ether afforded 168 mg (81%) of 17
as a brown solid, used as such for the next step. EM (ES, positive
mode): m/z 278 (M þ H)þ with a Cl isotopic pattern. 1H NMR
a white solid. Mp 186e187 ꢀC. EM (ES, positive mode): m/z 288
(M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d:
2.49 (s, 3H, CH3), 2.66 (s, 6H, N(CH3)2), 6.88 (d, 2H, J ¼ 8.3 Hz, Ar), 7.57
(d, 2H, J ¼ 8.4 Hz, Ar), 8.81 (s,1H, H-8). Anal. calc. for (C14H14ClN5): C,
58.44; H, 4.90; N, 24.34. Found C, 58.21; 4.73; N, 24.12.
(DMSO-d6, 300 MHz) d: 2.24 (s, 3H, CH3), 2.86 (s, 6H, N(CH3)2), 5.04
(s, 2H, NH2), 6.73 (d, 1H, J ¼ 8.4 Hz, Ar), 7.49 (d, 1H, J ¼ 8.3 Hz, Ar),
8.27 (s, 1H, NH).
4.1.3. 6-Chloro-9-[3-(N,N-dimethylamino)phenyl]purin-8-one (24)
A microwave vial was charged with compound 14 (100 mg,
0.38 mmol), anhydrous THF (1.8 mL) and triphosgene (374 mg,
1.30 mmol). The reaction vessel was sealed and heated in a micro-
wave reactor at 120 ꢀC for 20 min. The reaction mixture was cooled
to 0 ꢀC, quenched with water and extracted with ethyl acetate
(20 mL). The organic layer was dried over Na2SO4, filtered, evapo-
rated to dryness in vacuo and purified by CCTLC (dichlor-
omethane:methanol, 30:1) to yield 63 mg (57%) of 24 as a whitþe
solid. Mp 221e223 ꢀC. EM (ES, positive mode): m/z 290 (M þ H)
4.1.2. General procedure for the synthesis of N-9-arylpurines
starting from 4,5-diaminopyrimidines
A
solution of the corresponding 4,5-diaminopyrimidine
(1.0 mmol) in a 1:1 mixture (5 mL) of trimethyl orthoformate and
acetic anhydride in a microwave vessel was irradiated at 120 ꢀC for
1 h. After cooling, volatiles were evaporated and the residue was
dissolved in CHCl3 (15 mL) and subsequently washed with a satu-
rated aqueous NaHCO3 solution (15 mL) and brine (15 mL). The
organic layer was dried over Na2SO4, filtered and evaporated to
dryness in vacuo. The resulting residue was purified as specified.
The synthesis of 18 and 19 was performed as previously
described [15].
with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 2.92 (s,
6H, N(CH3)2), 6.80e6.90 (m, 3H, Ar), 7.30 (t, 1H, J ¼ 7.8 Hz, Ar), 8.40
(s,1H, H-2),12.28 (s,1H, NH). Anal. calc. for (C13H12ClN5O): C, 53.89;
H, 4.17; N, 24.17. Found: C, 53.67; H, 4.22; N, 24.24.
4.1.2.1. 6-Chloro-9-[3e(N,N-dimethylamino)phenyl]purine
(20).
According to the general procedure, compound 14 (130 mg,
0.49 mmol) reacted with trimethyl orthoformate (1.5 mL) in
acetic anhydride (1.5 mL) at 120 ꢀC for 1 h in a microwave reactor.
The residue was worked up and purified by CCTLC in the Chro-
matothron (dichloromethane:ethyl acetate, 2:1) to yield 68 mg
(51%) of 20 as a white solid. Mp 160e162 ꢀC. EM (ES, positive
mode): m/z 274 (M þ H)þ with a Cl isotopic pattern. 1H NMR
4.1.4. 6-Chloro-2-methyl-9-[3-(N,N-dimethylamino)phenyl]purin-
8-one (25)
As described for the synthesis of compound 24, a microwave vial
was charged with compound 15 (100 mg, 0.36 mmol), anhydrous
THF (1.6 mL) and triphosgene (356 mg, 1.20 mmol) and heated in
a microwave reactor at 120 ꢀC for 20 min. After worked up, the
residue was purified by CCTLC (dichloromethane:methanol, 30:1) to
yield 65 mg (60%) of 25 as a white solid. Mp 185e187 ꢀC. EM (ES,
positive mode): m/z 304 (M þ H)þ with a Cl isotopic pattern.1H NMR
(DMSO-d6, 300 MHz) d: 3.04 (s, 6H, N(CH3)2), 6.82e6.98 (m, 3H,
Ar), 7.42 (t, 1H, J ¼ 8.0 Hz, Ar), 8.39 (s, 1H, H-8), 8.81 (s, 1H, H-2).
Anal. calc. for (C13H12ClN5): C, 57.04; H, 4.42; N, 25.59. Found C,
57.33; H, 4.38; N, 25.45.
(DMSO-d6, 300 MHz) d: 2.46 (s, 3H, CH3), 2.92 (s, 6H, N(CH3)2),
6.79e7.32 (m, 4H, Ar),12.09 (s,1H, NH). Anal. calc. for (C14H14ClN5O):
C, 55.36; H, 4.65; N, 23.06. Found: C, 55.29; H, 4.72; N, 23.14.
4.1.2.2. 6-Chloro-9-[3-(N,N-dimethylamino)phenyl]-2-methylpurine
(21). A microwave vial was charged with compound 15 (278 mg,
1.0 mmol), trimethyl ortoformate (5 mL) and hydrochloric acid
4.1.5. 6-Chloro-9-(3-(N,N-dimethylamino)phenyl)-2,7-dimethyl-
7H-purin-8(9H)-one (26)
(90
m
L) and the vessel was microwave-irradiated at 120 ꢀC for 1 h.
To a solution of compound 24 (100 mg, 0.32 mmol) in anhydrous
After cooling, the reaction mixture was worked up according to the
general procedure, and purified by CCTLC in the Chromatotron
(dichloromethane:methanol, 30:1). Yield: 117 mg (41%). Mp
127e129 ꢀC. EM (ES, positive mode): m/z 288 (M þ H)þ with a Cl
DMF (6 mL), K2CO3 (150 mg, 1.09 mmol) and iodomethane (62 mL,
0.98 mmol) were added. The reaction was stirred at room temper-
ature overnight. The reaction mixture was diluted with dichloro-
methane (20 mL) and washed with saturated aqueous NaCl (15 mL).
The organic layer was dried over Na2SO4, filtered and evaporated to
dryness. The residue was purified by CCTLC (hexane:ethyl acetate,
1:1) to yield 44 mg (44%) of 26 as a white solid. Mp: 173e175 ꢀC. EM
(ES, positive mode): m/z 318 (M þ H)þ with a Cl isotopic pattern. 1H
isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 2.60 (s, 3H, CH3),
2.89 (s, 6H, N(CH3)2), 6.83e7.42 (m, 4H, Ar), 8.96 (s, 1H, H-8). Anal.
Calc. for (C15H14ClN5): C, 58.44; H, 4.90; N, 24.34. Found: C, 58.22;
H, 5.12; N, 24.11.
NMR (DMSO-d6, 300 MHz)
3.58 (s, 3H, N-CH3), 6.72e7.36 (m, 4H, Ar); 13C NMR (DMSO-d6,
75 MHz) : 24.83 (CH3), 28.85 (CH3-N), 39.93 (N(CH3)2), 110.79,
d: 2.74 (s, 3H, CH3), 2.90 (s, 6H, N(CH3)2),
4.1.2.3. 6-Chloro-9-[4-(N,N-dimethylamino)phenyl]purine
(22).
According to the general procedure, compound 16 (130 mg,
0.49 mmol) reacted with trimethyl orthoformate (1.5 mL) in
acetic anhydride (1.5 mL) at 120 ꢀC for 1 h in a microwave reactor.
The residue was worked up and purified by CCTLC in the Chro-
matothron (dichloromethane:ethyl acetate, 2:1) to yield 75 mg
(56%) of 22 as a white solid. Mp 196e198 ꢀC. EM (ES, positive
mode): m/z 274 (M þ H)þ with a Cl isotopic pattern. 1H NMR
d
112.26, 114.29, 129.33, 133.15, 150.90 (Ar), 114.29 (C-5), 134.66 (C-6),
150.66 (C-4), 152.11 (CO),159.35 (C-2). Anal. calc. for (C15H16ClN5O):
C, 56.69; H, 5.08; N, 22.04. Found:C, 56.71; H, 5.24; N, 22.18.
4.1.6. 6-Chloro-9-(3-(N,N-dimethylamino)phenyl)-2-methyl-9H-
purin-8-yl ethyl carbonate (27)
(DMSO-d6, 300 MHz)
d
: 2.98 (s, 6H, N(CH3)2), 6.88 (d, 2H,
A solution of compound 25 (67 mg, 0.22 mmol) in CH2Cl2 (3 mL)
J ¼ 8.2 Hz, Ar), 7.60 (d, 2H, J ¼ 8.4 Hz, Ar), 8.80, 8.95 (s, 2H, H-2,
H-8). Anal. calc. for (C13H12ClN5): C, 57.04; H, 4.42; N, 25.59.
Found C, 56.98; H, 4.41; N, 25.39.
and DIPEA (132
chloroformate (64
m
L, 0.76 mmol) was cooled at 0 ꢀC, and ethyl
mL, 0.66 mmol) was added. The reaction was
stirred at 0 ꢀC for 3 h. The reaction mixture was diluted with
dichloromethane (20 mL) and washed with saturated aqueous NaCl
(15 mL). The organic layer was dried over MgSO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (hexane:ethyl acetate) to yield 61 mg (74%) of 27 as
a white solid. Mp: 101e103 ꢀC. EM (ES, positive mode): m/z 376
(M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
4.1.2.4. 6-Chloro-9-[4-(N,N-dimethylamino)phenyl]-2-methylpurine
(23). According to the general procedure, compound 17 (100 mg,
0.36 mmol) reacted with trimethyl orthoformate (1 mL) in acetic
anhydride (1 mL) at 120 ꢀC for 1 h in a microwave reactor. The
residue was worked up and purified by CCTLC in the Chromatothron
(dichloromethane:ethyl acetate, 3:1) to yield 48 mg (47%) of 23 as
d: 1.36 (t, 3H, J ¼ 7.1 Hz, CH3), 2.48 (s, 3H, CH3), 2.89 (s, 6H, N(CH3)2),

286
L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
4.45 (q, 2H, J ¼ 7.1 Hz, CH2), 6.75e6.87 (m, 3H, Ar), 7.33 (t, 1H,
for (C13H10ClN5O): C, 54.27; H, 3.50; N, 24.34. Found: C, 54.11; H,
3.73; N, 24.55.
J ¼ 8.0 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz)
d: 13.92 (CH2CH3),
24.88 (CH3), 39.96 (N(CH3)2), 64.59 (CH2CH3), 111.37, 112.72, 114.81,
129.43, 132.51, 150.98 (Ar), 114.98 (C-5), 139.49 (C-6), 148.82, 152.46
(C-4, C-8), 147.50 (CO), 162.33 (C-2). Anal. calc. for (C17H18ClN5O3):
C, 54.33; H, 4.83; N, 18.64. Found: C, 54.30; H, 4.81; N, 58.53.
4.1.11. N-(3-(6-Chloro-2-methyl-9H-purin-9-yl)phenyl)acetamide (33)
As described for the synthesis of 32, the diaminopyrimidine 31
(213 mg, 0.73 mmol) reacted with trimethyl orthoformate (2 mL) and
ethanesulfonic acid (6 m
L, 0.07 mmol) at 120 ꢀC for 1 h in a microwave
4.1.7. 6-Chloro-9-(3-(N,N-dimethylamino)phenyl)-2-methyl-9H-
purin-8-yl phenyl carbonate (28)
reactor. The residue was worked up and purified by CCTLC in the
Chromatothron (dichloromethane:methanol, 30:1) to yield 216 mg
(98%) of 33 as a white solid. Mp: 269e271 ꢀC. EM (ES, positive mode):
m/z 302 (M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6,
As described for the synthesis of 27, compound 25 (100 mg,
0.32 mmol) was dissolved in CH2Cl2 (5 mL) and DIPEA (192
m
m
L,
L,
1.10 mmol) and cooled at 0 ꢀC. Then, phenyl chloroformate (125
300 MHz)
d
: 2.08 (s, 3H, CH3), 2.68 (s, 3H, CH3), 7.43 (d,1H, J ¼ 8.3 Hz,
0.98 mmol) was added and the reaction was stirred at 0 ꢀC for 1 h.
After worked up, the residue was purified by flash chromatography
(hexane:ethyl acetate) to yield 102 mg (75%) of 28 as a white solid.
Mp: 162e164 ꢀC. EM (ES, positive mode): m/z 424 (M þ H)þ with
Ar), 7.53 (t, 1H, J ¼ 8.1 Hz, Ar), 7.69 (d, 1H, J ¼ 8.2 Hz, Ar), 8.09 (s, 1H,
Ar), 8.90 (s,1H, H-8),10.24 (bs,1H, NH). Anal. calc. for (C14H12ClN5O):
C, 55.73; H, 4.01; N, 23.21. Found: C, 55.46; H, 4.04; N, 22.98.
a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d
: 2.51 (s, 3H,
4.1.12. N-(3-(6-Chloro-9H-purin-9-yl)phenyl)-N-methylacetamide (34)
A mixture containing 32 (80 mg, 0.28 mmol), NaH (60%, 35 mg,
0.84 mmol) and activated 4 Å molecular sieves in anhydrous DMF
(1 mL) under an argon atmosphere was cooled at ꢁ30 ꢀC, then
CH3), 2.90 (s, 6H, N(CH3)2), 6.77e6.86 (m, 3H, Ar), 7.34e7.41 (m, 4H,
Ar), 7.51e7.56 (m, 2H, Ar). Anal. calc. for (C21H18ClN5O3): C, 59.51; H,
4.28; N, 16.52. Found: C, 59.28; H, 4.30; N, 16.46.
iodomethane (35 mL, 0.58 mmol) was added. The mixture was stir-
4.1.8. N-(3-((5-Amino-6-chloropyrimidin-4-yl)amino)phenyl)
acetamide (30)
red for 1 h at ꢁ30 ꢀC. A saturated aqueous solution of NH4Cl (0.5 mL)
was added. The reaction mixture was filtered through celite, diluted
with dichloromethane (10 mL) and washed with water (5 mL) and
brine (5 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by CCTLC in the
Chromatothron (dichloromethane:ethyl acetate,1:1) to yield 60 mg
(71%) of 34 as a white solid. Mp: 232e234 ꢀC. EM (ES, positive
mode): m/z 302 (M þ H)þ with a Cl isotopic pattern.1H NMR (DMSO-
A microwave vial was charged with 30-aminoacetanilide 29
(300 mg, 2.0 mmol), 5-amino-4,6-dichloropyrimidine (10) (328 mg,
2.0 mmol), acetic acid (100 mL) and isobutanol (10 mL) and was
irradiated at 150 ꢀC for 30 min. After cooling, dichloromethane was
added (20 mL) and the crude reaction mixture was washed with
saturated aqueous NaHCO3 (20 mL). The organic layer was dried over
Na2SO4, filtered and evaporated to dryness. The residue was purified
by flash chromatography (dichloromethane:methanol).Yield:
300 mg (54%). Mp 200e202 ꢀC. EM (ES, positive mode): m/z 278
d6, 300 MHz) d: 1.92 (s, 3H, COCH3), 3.25 (s, 3H, NCH3), 7.52 (d, 1H,
J ¼ 8.2 Hz, Ar), 7.71 (t,1H, J ¼ 8.0 Hz, Ar), 7.94 (m, 2H, Ar), 8.88, 9.17 (s,
2H, H-2, H-8). Anal. calc. for (C14H12ClN5O): C, 55.73; H, 4.01; N,
23.21. Found: C, 55.64; H, 4.25; N, 23.04.
(M þ H)þ with a Cl isotopic pattern; 1H NMR (DMSO-d6, 300 MHz)
d:
2.04 (s, 3H, CH3), 5.47 (br s, 2H, NH2), 7.14e7.38 (m, 3H, Ar), 7.83, 7.95
(s, 2H, Ar, H-2), 8.59 (br s, 1H, NH), 9.93 (br s, 1H, NHCO).
4.1.13. N-(3-(6-Chloro-2-methyl-9H-purin-9-yl)phenyl)-N-
methylacetamide (35)
4.1.9. N-(3-((5-Amino-6-chloro-2-methylpyrimidin-4-yl)amino)
phenyl)acetamide (31)
As shown for compound 34, a mixture containing 33 (61 mg,
0.20 mmol), NaH (60%, 25 mg, 0.60 mmol) and activated 4 Å
molecular sieves in anhydrous DMF (1 mL) reacted with iodo-
As described for the synthesis of 30, a microwave vial was
charged with 5-amino-4,6-dichloro-2-methylpyrimidine (11)
(365 mg, 2.0 mmol), 30-aminoacetanilide (29) (300 mg,2.0 mmol),
methane (25
m
L, 0.40 mmol) for 1 h at ꢁ30 ꢀC. After worked up, the
residue was purified by CCTLC in the Chromatothron (dichlor-
omethane:methanol, 10:1) to yield 50 mg (78%) of 35 as a whitþe
solid. Mp 184e186 ꢀC. EM (ES, positive mode): m/z 316 (M þ H)
isobutanol (10 mL) and acetic acid (100 m
L) and irradiated at 150 ꢀC
for 15 min. After worked up, the residue was purified by flash
chromatography (dichloromethane:methanol). Yield: 362 mg
(62%). Pale brown solid. Mp: 201e203 ꢀC. EM (ES, positive mode):
m/z 292 (M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6,
with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 1.93 (s,
3H, CH3), 2.70 (s, 3H, COCH3), 3.24 (s, 3H, NCH3), 7.49 (d, 1H,
J ¼ 8.3 Hz, Ar), 7.70 (t,1H, J ¼ 8.0 Hz, Ar), 7.89 (m, 2H, Ar), 9.04 (s, 2H,
H-8). Anal. calc. for (C15H14ClN5O): C, 57.06; H, 4.47; N, 22.18.
Found: C, 56.84; H, 4.54; N, 22.02.
300 MHz) d: 2.06 (s, 3H, CH3), 2.33 (s, 3H, CH3), 5.22 (br s, 2H, NH2),
7.12e7.24 (m, 2H, Ar), 7.50 (d,1H, J ¼ 8.2 Hz, Ar), 8.10 (s, 1H, Ar), 8.51
(br s, 1H, NH), 9.90 (br s, 1H, NHCO).
4.1.14. N-(3-(6-Chloro-2-methyl-9H-purin-9-yl)phenyl)-N-
ethylacetamide (36)
As described for 35, a mixture containing 33 (50 mg, 0.16 mmol),
NaH (60%, 20 mg, 0.49 mmol) and activated 4 Å molecular sieves in
anhydrous DMF (1 mL) under an argon atmosphere was cooled
4.1.10. N-(3-(6-Chloro-9H-purin-9-yl)phenyl)acetamide (32)
Compound 30 (194 mg, 0.70 mmol) reacted with trimethyl
orthoformate (2 mL) and ethanesulfonic acid (6 mL, 0.07 mmol) at
120 ꢀC for 1 h in a microwave reactor. After cooling, dichloro-
methane was added (20 mL) and the crude reaction mixture was
washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL).
The organic layer was dried over Na2SO4, filtered and evaporated to
dryness. The residue was purified by CCTLC in the Chromatothron
(dichloromethane:methanol, 30:1) to yield 60 mg (72%) of 32 as
a white solid. Mp 215e217 ꢀC. EM (ES, positive mode): m/z 288
at ꢁ30 ꢀC, then iodoethane (26
mL, 0.48 mmol) was added. After
stirring for 1 h at ꢁ30 ꢀC, the reaction was worked up. The
residue was purified by CCTLC in the Chromatothron (dichlor-
omethane:methanol, 20:1) to yield 21 mg of 36 (40%) as a whitþe
solid. Mp: 176e178 ꢀC. EM (ES, positive mode): m/z 330 (M þ H)
with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 1.07 (t,
(M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d
:
3H, J ¼ 7 Hz, CH3), 1.86 (s, 3H, CH3), 2.70 (s, 3H, CH3), 3.72 (q, 2H,
J ¼ 7.0 Hz, CH2), 7.46 (d, 1H, J ¼ 8.1 Hz, Ar), 7.72 (t, 1H, J ¼ 7.9 Hz, Ar),
7.87 (s, 1H, Ar), 7.94 (d, 1H, J ¼ 7.8 Hz, Ar), 9.06 (s, 1H, H-8). Anal.
calc. for (C16H16ClN5O): C, 58.27; H, 4.89; N, 21.24. Found C, 58.14; H,
4.95; N, 21.35.
2.08 (s, 3H, CH3), 7.47e8.16 (m, 4H, Ar), 8.84, 9.06 (s, 2H, H-2, H-8),
10.28 (br s,1H, NHCO); 13C NMR (DMSO-d6,100 MHz)
d: 24.01 (CH3),
114.25,118.28,118.81,129.94,134.18,140.34 (Ar),131.46 (C-5),146.34
(C-8), 149.60 (C-4), 151.46 (C-6), 152.16 (C-2), 168.73 (CO). Anal. calc.

L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
287
4.1.15. N-(3-(6-Chloro-2-methyl-9H-purin-9-yl)phenyl)-N-
(cyclopropylmethyl)acetamide (37)
As described for 35, a mixture containing 33 (50 mg, 0.16 mmol),
NaH (60%, 20 mg, 0.49 mmol) and activated 4 Å molecular sieves in
anhydrous DMF (1 mL) under an argon atmosphere was cooled
4.1.19. 3-(3-(6-Chloro-9H-purin-9-yl)phenyl)oxazolidin-2-one (41)
A mixture containing 39 (86 mg, 0.28 mmol), trimethyl ortho-
formate (3.8 mL) and ethanesulfonic acid (2.3 L, 0.03 mmol) was
m
irradiated at 120 ꢀC for 1 h in a microwave reactor. After cooling,
dichloromethane was added (20 mL) and the crude reaction
mixture was washed with saturated aqueous NaHCO3 (10 mL) and
brine (10 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (dichloromethane:methanol). Yield: 74 mg þ(84%). Mp
244e246 ꢀC. EM (ES, positive mode): m/z 316 (M þ H) with a Cl
at ꢁ30 ꢀC, then (bromomethyl)cyclopropane (31
mL, 0.32 mmol)
was added and the reaction mixture stirred for 4 h at ꢁ30 ꢀC. After
worked up, the residue was purified by flash chromatography
(dichloromethane/methanol) to yield 29 mg (17%) of 37 as a whitþe
solid. Mp: 138e140 ꢀC. EM (ES, positive mode): m/z 356 (M þ H)
with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d
: 0.11 (q,
isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 4.14 (m, 2H, CH2),
2H, J ¼ 4.9 Hz, CH2), 0.40 (q, 2H, J ¼ 5.3 Hz, CH2), 0.96 (m, 2H, CH2),
1.86 (s, 3H, CH3), 2.69 (s, 3H, CH3), 3.58 (d, 2H, J ¼ 7.0 Hz, CH2), 7.49
(d,1H, J ¼ 8.0 Hz, Ar), 7.71 (t,1H, J ¼ 7.9 Hz, Ar), 7.93 (m, 2H, Ar), 9.07
(s, 1H, H-8). Anal. calc. for (C18H18ClN5O): C, 60.76; H, 5.10; N, 19.68.
Found C, 61.03; H, 5.17; N, 19.45.
4.50 (m, 2H, CH2), 7.72e8.85 (m, 4H, Ar), 8.85 (s, 1H, H-2), 9.11 (s,
1H, H-8). Anal. calc. for (C14H10ClN5O2): C, 53.26; H, 3.19; N, 22.18.
Found: C, 52.98; H, 3.24; N, 22.01.
4.1.20. 3-(3-(6-Chloro-2-methyl-9H-purin-9-yl)phenyl)oxazolidin-
2-one (42)
4.1.16. N-(3-(2-Oxooxazolidin-3-yl)phenyl)acetamide (38)
Compound 40 (50 mg, 0.15 mmol) reacted with trimethyl
orthoformate (1 mL) in the presence of ethanesulfonic acid (2 mL,
A microwave vial was charged with 30-aminoacetanilide 29
(150 mg, 1.0 mmol), 2-chloroethyl chloroformate (124
m
L,
0.02 mmol) at 120 ꢀC for 30 min in a microwave reactor. After
cooling, dichloromethane was added (20 mL) and the crude reac-
tion mixture was subsequently washed with saturated aqueous
NaHCO3 (10 mL) and brine (10 mL). The organic layer was dried
over Na2SO4, filtered and evaporated to dryness. The residue was
worked up and purified by CCTLC in the Chromatothron (dichlor-
omethane:methanol, 30:1) to yield 43 mg (86%) of 42 as a whitþe
solid. Mp: 249e251 ꢀC. EM (ES, positive mode): m/z 330 (M þ H)
1.2 mmol), Cs2CO3 (977 mg, 3.0 mmol) and DIPEA (350 L, 2 mmol)
m
in acetonitrile (5 mL) and was irradiated at 120 ꢀC for 2 h. After
cooling, ethyl acetate was added (20 mL) and the crude reaction
mixture was subsequently washed with saturated aqueous NH4Cl
(20 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (dichloromethane:methanol). Yield: 178 mg (81%). Yeþllo1w
solid Mp 177e179 ꢀC. EM (ES, positive mode): m/z 221 (M þ H) . H
with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 2.69 (s,
NMR (DMSO-d6, 300 MHz)
d: 2.04 (s, 3H, CH3), 4.03 (m, 2H, CH2),
3H, CH3), 4.14 (dd, 2H, J ¼ 8.9, 7.0 Hz, CH2), 4.50 (dd, 2H, J ¼ 9.1,
6.9 Hz, CH2), 7.59e7.68 (m, 2H, Ar), 7.76 (m, 1H, Ar), 8.08 (s, 1H, Ar),
8.97 (s, 1H, H-8). Anal. calc. for (C15H12ClN5O2): C, 62.68; H, 4.51; N,
20.88. Found: C, 61.09; H, 4.45; N, 20.99.
4.43 (m, 2H, CH2), 7.20e7.42 (m, 3H, Ar), 7.83 (s, 1H, Ar), 10.02 (s,
1H, NH).
4.1.17. 3-(3-((5-Amino-6-chloropyrimidin-4-yl)amino)phenyl)
oxazolidin-2-one (39)
4.1.21. 3-(3-(6-Chloro-8-oxo-7H-purin-9(8H)-yl)phenyl)
A
microwave vial was charged with 5-amino-4,6-
oxazolidin-2-one (43)
dichloropyrimidine (10) (112 mg, 0.68 mmol), the oxazolidinone
38 (150 mg, 0.68 mmol), isobutanol (2 mL) and 37% aqueous HCl
(20
A microwave vial was charged with 39 (70 mg, 0.23 mmol) in
anhydrous THF (1 mL) and triphosgene (217 mg, 0.73 mmol) was
added. The reaction was microwaved irradiated at 120 ꢀC for
20 min. After cooling at 0 ꢀC, water was added (20 mL) and the
crude reaction mixture was extracted with isobutanol. The organic
layer was dried over Na2SO4, filtered and evaporated to dryness.
The residue was purified by CCTLC in the Chromatothron
(dichloromethane:methanol, 20:1) to yield 65 mg (85%þ) of 43. Mp
273e275 ꢀC. EM (ES, positive mode): m/z 332 (M þ H) with a Cl
m
L) and irradiated at 120 ꢀC for 30 min. After cooling,
dichloromethane was added (20 mL) and the crude reaction
mixture was washed with saturated aqueous NaHCO3 (10 mL) and
brine (10 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (dichloromethane:methanol) to yield 86 mg (41%) of 39 as
an amorphous solid. EM (ES,positive mode): m/z 306 (M þ H)þ with
a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 4.01 (m, 2H,
isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d: 4.08 (m, 2H, CH2),
CH2), 4.42 (m, 2H, CH2), 5.43 (br s, 2H, NH2), 5.75 (br s, 1H, NH),
7.07e7.95 (m, 4H, Ar), 8.41 (s, 1H, H-2).
4.47 (m, 2H, CH2), 7.36e7.61 (m, 3H, Ar), 7.90 (t, 1H, J ¼ 1.9 Hz, Ar),
8.42 (s, 1H, H-2), 12.34 (br s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz)
d: 45.18 (CH2N), 61.98 (CH2O), 116.30, 117.95, 121.87, 129.79, 133.22,
4.1.18. 3-(3-((5-Amino-6-chloro-2-methylpyrimidin-4-yl)amino)
phenyl)-oxazolidin-2-one (40)
139.52 (Ar), 120.32 (C-5), 135.35, 150.55 151.52, (C-8, C-6, C-4),
152.32 (C-2), 155.22 (CO). Anal. calc. for (C14H10ClN5O3): C, 50.69; H,
3.04; N, 21.11. Found: C, 50.55; H, 3.12; N, 21.15.
A
mixture
containing
5-amino-4,6-dichloro-2-
methylpyrimidine (11) (240 mg, 1.35 mmol), the oxazolidinone 38
(297 mg, 1.35 mmol), isobutanol (5 mL) and 37% aqueous HCl
(41
4.1.22. 3-(3-(6-Chloro-2-methyl-8-oxo-7H-purin-9(8H)-yl)phenyl)
oxazolidin-2-one (44)
m
L) was irradiated at 150 ꢀC for 30 min. After cooling,
dichloromethane was added (20 mL) and the crude reaction
mixture was washed with saturated aqueous NaHCO3 (20 mL) and
brine (20 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (dichloromethane:methanol) to yield 220 mg (51%) of 40
as a yellow solid. Mp 244e246 ꢀC. EM (ES, positive mode): m/z 320
(M þ H)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
A microwave vial was charged with compound 40 (150 mg,
0.47 mmol), anhydrous THF (2 mL) and triphosgene (445 mg,
1.50 mmol). The reaction vessel was sealed and heated in a micro-
wave reactor at 120 ꢀC for 45 min. The reaction mixture was cooled
to 0 ꢀC, quenched with water and extracted with CH2Cl2 (20 mL).
The organic layer was dried over Na2SO4, filtered, evaporated to
dryness in vacuo and purified by flash chromatography (dichlor-
omethane:methanol) to yield 136 mg (84%) of 44 as a white solid.
Mp 296e298 ꢀC. EM (ES, positive mode): m/z 346 (M þ H)þ with
d
: 2.33 (s, 3H, CH3), 4.07 (dd, 2H, J ¼ 9.0, 7.0 Hz, CH2), 4.45 (dd, 2H,
J ¼ 9.0, 6.9 Hz, CH2), 5.24 (br s, 2H, NH2), 7.19 (d, 2H, J ¼ 8.2 Hz, Ar),
7.32 (t, 1H, J ¼ 8.2 Hz, Ar), 7.55 (d, 1H, J ¼ 8.1 Hz, Ar), 8.16 (s, 1H, Ar),
8.81 (s, 1H, NH).
a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz)
d
: 2.48 (s, 3H,
CH3), 4.08 (m, 2H, CH2), 4.50 (m, 2H, CH2), 7.33e7.86 (m, 4H, Ar),

288
L. Aguado et al. / European Journal of Medicinal Chemistry 49 (2012) 279e288
12.16 (br s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)
d
: 25.42 (CH3),
with a radius of 1.4 Å, defined the solute boundaries, and a minimum
separation of 10 Å was left between any solute atom and the borders
of the box. The interior of the ligands was considered a low-
dielectric medium (ε ¼ 2) whereas the surrounding solvent was
treated as a high-dielectric medium (ε ¼ 80).
55.37 (CH2N), 62.07 (CH2O), 116.69, 117.87, 118.01, 122.13, 129.89,
133.47,135.18, 139.64 (Ar, C-5, C-4),151.58,152.55, (C-6, C-8), 155.30
(CO), 159.93 (C-2). Anal. calc. for (C15H12ClN5O3): C, 52.11; H, 3.50;
N, 20.26. Found C, 51.96; H, 3.55; N, 20.12.
Acknowledgements
4.1.23. 2-((3-(6-Ethoxy-9H-purin-9-yl)phenyl)amino)ethanol (45)
A microwave vial containing compound 41 (50 mg, 0.16 mmol)
and NaOH 12.5N (0.15 mL) in ethanol (0.7 mL) was irradiated at
50 ꢀC for 15 min. Then, ethyl acetate was added (20 mL) and the
crude reaction mixture was washed with brine (20 mL). The organic
layer was decanted, dried and evaporated. The residue was purified
by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1)
to yield 36 mg of 45 (78%). Mp 131e133 ꢀC. EM (ES, positive mode):
L.A. thanks the Spanish Ministerio de Educación y Ciencia (MEC)
for an FPU predoctoral fellowship. M.-C. C. thanks the Fondo Social
Europeo (FSE) and the JAE Predoc Programme for a predoctoral
fellowship. We thank Ms. María Nares for excellent technical
assistance and the support of the Spanish MEC and the Fondo Social
Europeo (FSE). This project has been supported by the Spanish
CICYT (SAF2009-13914-C02-01). We also would like to acknowl-
edge Stijn Delmotte, Tom Bellon, Mieke Flament, and Annelies De
Ceulaer for their excellent technical assistance in the acquisition of
the antiviral data.
m/z 290 (M þ H)þ; 1H NMR (DMSO-d6, 300 MHz)
: 1.44 (t, J ¼ 7.0 Hz,
d
3H, CH3), 3.17 (t, J ¼ 5.8 Hz, 2H, CH2), 3.57 (t, J ¼ 5.8 Hz, 2H, CH2), 4.63
(q, J ¼ 7.0 Hz, 2H, CH2), 5.95 (br s, 1H, NH), 7.01 (m, 4H, Ar), 8.56 (s,
1H, H-8), 8.70 (s, 1H, H-2). Anal. calc. for (C15H17N5O2): C, 60.19; H,
5.72; N, 23.40. Found C, 59.95; H, 5.84; N, 23.57.
Appendix. Supplementary data
4.2. Biological methods
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2012.01.022. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
4.2.1. Antiviral activity
Serial dilutions of the compounds and 100 CCID50 of virus were
added to the appropriate cell line, grown in 96-well plates. After 3
days of incubation at 37 ꢀC (35 ꢀC for rhinovirus 14), a time at which
complete virus-induced cytopathic effect (CPE) was observed in the
infected and untreated virus control conditions (VC), cell viability
was measured using the MTS/PMS method (Promega, Leiden, The
Netherlands).The optical density of each well was measured spec-
trophotometrically at 498 nm using a microplate reader (Safire2,
Tecan, Switzerland). Raw values were converted to percentage of
controls and the 50% effective concentration (EC50) was calculated
by linear interpolation as the concentration of compound that is
supposed to inhibit virus-induced cell death by 50%. Similarly, the
EC90 was calculated. Selective antiviral activity was confirmed by
microscopic quality control i.e. the absence of signs of virus-
induced CPE as well as the absence of compound-induced
adverse effects on the host cell (which is mostly apparent as
alteration of cell morphology as compared to untreated, uninfected
cells). Each experiment was performed at least in triplicate.
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4.2.2. Cytotoxic assays
The same experimental set-up was used as for the antiviral
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was repeated at least three times. The evaluation of the overall anti-
metabolic effect of the selected compounds was performed using
the MTS/PMS method Assay (Promega). Raw values were converted
to percentage of controls and the 50% cytotoxic concentration
(CC50) was calculated using linear interpolation as the concentra-
tion of compound that inhibited overall cell metabolism by 50%.
Microscopic quality control was used to confirm the observations
made in the antiviral assay.
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solutions to the linearized PoissoneBoltzman equation, as imple-
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MEPs. For MEP calculations cubic grids with a resolution of 1.0 Å
were centered on each molecule, and the atomic charges were
distributed onto the grid points. AMBER charges and radii were
used. Solvent-accessible surfaces, calculated with a spherical probe
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