Regiospecific microwave-assisted synthesis and cytotoxic activity against human breast cancer cells of (RS)-6-substituted-7- or 9-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-7H- or -9H-purines

Article abstract of DOI:10.1016/j.ejmech.2007.10.025

Extended studies on the synthesis and pharmacological evaluation of (RS)-6-substituted-7 or 9-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-7H- or -9H-purines are presented. The microwave-assisted organic synthesis has provided faster access to the target compo

Full text of DOI:10.1016/j.ejmech.2007.10.025

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1742e1748
http://www.elsevier.com/locate/ejmech
Short communication
Regiospecific microwave-assisted synthesis and cytotoxic activity
against human breast cancer cells of (RS )-6-substituted-7- or
9-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-7H- or -9H-purines
a
Ana Conejo-Garcıa , Marıa C. Nunez , Juan A. Marchal , Fernando Rodrıguez-Serrano ,
a
b,c
d
´
Antonia Aranega , Miguel A. Gallo , Antonio Espinosa , Joaquın M. Campos
´
´
´
~
b,c
a
a
a,
*
´
´
a Departamento de Qu´ımica Farmace´utica y Orga´nica, Facultad de Farmacia, c/ Campus de Cartuja s/n, 18071 Granada, Spain
b Instituto de Biopatolog´ıa y Medicina Regenerativa (IBIMER), Spain
c Departamento de Anatom´ıa y Embriolog´ıa Humana, Facultad de Medicina, Avenida de Madrid s/n, 18071 Granada, Spain
d Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Paraje de Las Lagunillas s/n, 23071 Jae´n, Spain
Received 31 August 2007; received in revised form 22 October 2007; accepted 22 October 2007
Available online 25 October 2007
Abstract
Extended studies on the synthesis and pharmacological evaluation of (RS )-6-substituted-7 or 9-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-7H-
or -9H-purines are presented. The microwave-assisted organic synthesis has provided faster access to the target compounds with the advantage of
selective obtaining the N-70 or N-90 regioisomers simplifying their isolation. To test the behaviour of the products (including the purine bases) on
cellular systems, cytotoxic activity against the MCF-7 human breast cancer cell line was determined, and the three most active compounds were
used to study the cell cycle distribution and apoptosis in the MCF-7 cell line.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Antitumour compounds; Seven-membered rings; Microwave; MCF-7; 6-Substituted purines
1. Introduction
family complex with CDKs 4 and 6 during G1 phase, cyclin
E with CDK2 in late G1, cyclin A with CDK2 in S phase,
and cyclin B with CDK1 (also known as cdc2) in late G2/M
[4].
Cyclin-dependent kinases (CDKs) mediate proliferation
and neuronal development, while aberrant CDK activity is
associated with cancer and neurodegeneration [1e5]. Conse-
quently, a large number of ATP-competitive CDK inhibitors
from a variety of chemical classes have been identified [6e
9]. The 2,6,9-trisubstituted purines, which form an ample class
of CDK 1, 2, and 5 inhibitors, were developed to combat these
pathologies [2]. The CDKs control cell cycle progression in
proliferating eukaryotic cells [3]. The activity of CDKs is de-
pendent on the presence of cyclin partners whose levels are se-
quentially regulated to ensure that the phases of the cell cycle
proceed in the correct order. For example, cyclins of the D
The lead guanine-based CDK inhibitor O6-cyclohexylme-
thylguanine (NU2058, 1; Fig. 1) was found to be an inhibitor
of both CDK1 and CDK2. Moreover, 1 showed a mean of
IC50 ¼ 13 ꢀ 7 mM on 57 cell lines of the National Cancer In-
stitute, NCI [10]. On the other hand, the structure of 2 with
a naturally occurring base such as uracil, associated with the
inhibition of MCF-7 cell proliferation through decreasing cy-
clin D1 and CDK1, make it a very attractive agent, opening
a new strategy in cancer chemotherapy using similar com-
pounds endowed with potent antitumour activities and with
null toxicity [11]. Later on, we substituted the pyrimidine
base for the purine one (with several substituents at its position
6), with the objective of increasing both the lipophilicity and
the structural diversity of the target molecules [12].
* Corresponding author. Tel.: þ34 958 243848; fax: þ34 958 243845.
E-mail address: jmcampos@ugr.es (J.M. Campos).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.025

A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
1743
the cyclic acetal 3 [13] and the purine bases 4e10 [15,16] us-
ing trimethylchlorosilane (TCS), 1,1,1,3,3,3-hexamethyldisila-
zane (HMDS) and tin(IV) chloride as the Lewis acid in
anhydrous acetonitrile. When the reaction was carried out at
45 ꢁC for 24e72 h (Scheme 1, method a), the N-90 and the
N-70 cyclic alkylated purine regioisomers (11e13 and 18e
20, respectively) were produced and separated by flash chro-
matography. The isolated single products 11e13 and 18e20
were obtained with an average yield of 20%.
Modern drug discovery relies on high speed organic synthe-
sis. Microwave-assisted organic synthesis [17e23] is proving
to be instrumental for the rapid synthesis of compounds with
new and improved biological activities [24,25]. To investigate
Fig. 1.
Here we propose to extend our studies with the novel
6-substituted-N-90-purines 11e17 and the corresponding
N-70-purines 18e24 (Scheme 1). The use of the cyclohexylme-
thyloxy group as substituent at position 6 of the purine ring is ev-
ident from the previous findings. Moreover other bulky groups
such as the phenoxy, phenylthio, p-fluorophenylthio, 2,4-di-
chlorophenylthio ones, and smaller groups such as the allyloxy
or the methylthio ones at the 6 position were synthesized in order
to demonstrate the importance of the size of the substituent of
the purine ring on the biological activity. As we have previously
reported, the presence of an amino group on the purine ring is
detrimental to the antiproliferative activity [13] and accordingly
it was decided to use 6-substituted purines as bases instead of 2-
amino-6-substituted purines such as 1. To test the behaviour of
the products (including the purine bases 4e10) on cellular sys-
tems, cytotoxic activity against the MCF-7 human breast cancer
cell line was determined, and the three most active compounds
were used to study the cell cycle distribution and apoptosis in
the MCF-7 cell line.
the Vorbruggen condensation in microwave-assisted organic
¨
synthesis, the reaction mixture was microwave-irradiated at
a temperature of 130 ꢁC for 5 min. No starting material was
left after the reaction time. The most important feature is
that only the N-90 isomer was isolated in the reaction [method
(b), 14e17]. The main advantage of the microwave is the
quick access to the target molecules as well as the better yield
obtained in the only isomer formed making the purification
processes much easier. The average yield in the synthesis of
15e17 is 37% but the allyloxy-substituted purine derivative
14 was isolated in only 10% yield. It was envisioned that
the high reactivity of the allyloxy group would lead to lower
yield of the reaction. Trying to improve the yield in the synthe-
sis of 14 we decided to repeat the reaction at a lower temper-
ature. Surprisingly and interestingly, when the microwave
reaction was carried out at 100 ꢁC, only the N-70 isomer 21
was isolated (Scheme 1, method c). We then validated method
(c) by synthesizing the thio-substituted analogues 22e24
[Scheme 1, method (c)] and the N-70 isomers were isolated
with an average yield of 26%.
2. Results and discussion
2.1. Chemistry
In short we can confirm the regiospecificity of this method-
ology. Depending on the temperature by microwave activation
only one of the isomers was obtained in better yield than the
2.1.1. Synthesis of derivatives
The preparation of the target molecules 11e24 was
achieved by the Vorbruggen [14] one-pot condensation of
¨
Scheme 1. Reagents and conditions. Purine base (4e10), TCS, HMDS, SnCl4, anhydrous MeCN. Method (a), (b) or (c): (a) 45 ꢁC, 24e72 ꢁC; (b) microwave,
130 ꢁC, 5 min; (c) microwave, 100 ꢁC, 5 min.

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A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
Table 2
one produced by conventional heating making the purification
process easier. The isolated regioisomers N-70 or N-90 might be
prepared in this simple manner. It might be argued that this ap-
parent selectivity was due to the differences in the balance of
side products and the ease of isolation of the products, since
the isolated yields are below 50%. However, NMR spectra
of the crudes supported the selectivity of the processes because
neither the N-70 nor the N-90 isomers were detected following
methods (b) and (a), respectively.
Antiproliferative activities against the MCF-7 cell line for 5-FU, for the purine
bases (4e10), and for the seven-membered alkylated purine derivatives (N-90
isomers: 11e17; N-70 isomers: 18e24)
Compound IC50 (mM) Compound IC50 (mM) Compound IC50 (mM)
5-FU [26] 4.32 ꢀ 0.02
9
21.4 ꢀ 0.42 17
23.6 ꢀ 4.11 18
11.6 ꢀ 0.93 19
24.7 ꢀ 3.82 20
12.0 ꢀ 0.59 21
5.04 ꢀ 1.68 22
7.12 ꢀ 0.46 23
11.2 ꢀ 1.32 24
24.0 ꢀ 1.95
13.4 ꢀ 1.94
21.1 ꢀ 2.93
8.4 ꢀ 0.91
1
2 [12]
24.2 ꢀ 0.67 10
5.00 ꢀ 0.05 11
32.3 ꢀ 1.42 12
28.4 ꢀ 0.45 13
44.7 ꢀ 0.74 14
31.0 ꢀ 0.16 15
31.6 ꢀ 0.54 16
4
5
6
7
8
20.9 ꢀ 1.24
15.6 ꢀ 3.74
14.1 ꢀ 0.67
31.8 ꢀ 5.46
2.1.2. Spectroscopic characteristics of the N-90 and N-70
alkylated purines
As we previously reported, assignments of N-90 versus N-70
isomers can be readily made from the 13C NMR signal of the
C-40 peaks (CDCl3) [12,26]: the signal circa d 151 ppm is
characteristic of the C-40 atom for the N-90 regioisomers,
whilst the signal circa d 160 ppm is characteristic of the C-
40 atom for the N-70 regioisomers. Besides examining the C-
40 chemical shift trend in 11e24 (Table 1) it can be noticed
that the presence of the thio substituent in 13, 15e17, 20,
22e24 as compared to 11, 12, 14, 18, 19 and 21 causes a small
downfield shift of 2 ppm (d 148 versus 151 ppm in the N-90
isomers and d 159 versus 162 ppm in the N-70 isomers).
The influence of the substituent on the chemical shift of C-
50 is more marked; the oxy-substituted purine derivatives were
N-90 regioisomers are not significant with the exception of
the allyloxy derivatives 14 and 21. The biological effect is de-
pendent on the substituent present in position 6 of the purine
ring although a clear structureeactivity relationship between
the size of this moiety and the antiproliferative effect of the
MCF-7 human breast cancer cell line is not observed. The
most active compound (14), that presents an allyloxy group
as substituent at position 6 of the purine ring, shows an
IC50 ¼ 5.04 ꢀ 1.68 mM nearly equipotent as 5-FU. The follow-
ing two more active compounds, 15 and 20, present bulky sub-
stituents as the phenylthio and 2,4-diclorophenylthio ones,
respectively. The CDK1 and CDK2 inhibitor NU2058 (1)
has a low activity as an antiproliferative agent in the MCF-7
human breast cancer cell line.
To study the mechanisms of the antitumour and antiproli-
ferative activities of the most active compounds (14, 15 and
20), the effects on the cell cycle distribution were analyzed
by flow cytometry (Table 3). DMSO-treated cell cultures con-
tained a 58.62 ꢀ 0.74 of the G0/G1-phase cells, a 33.82 ꢀ 0.72
of the S-phase cells and a 7.55 ꢀ 1.34 of the G2/M-phase cells.
In contrast, MCF-7 cells treated during 48 h with the IC50 con-
centrations of 14, 15 and 20 showed important differences in
cell cycle progression compared with DMSO-treated control
cells. The cell cycle regulatory activities can be divided into
the following two groups: (a) the breast cancer cells for 14
showed an accumulation in the S phase, up to 37.00 ꢀ 2.00
of the cells, mainly at the expense of the G0/G1-phase popula-
tion that decreased to a percentage of 55.63 ꢀ 1.57 of the cells;
(b) compounds 15 and 20 accumulated the cancerous cells in
the G2/M-phase (11.08 ꢀ 1.01 and 19.16 ꢀ 0.56, respectively)
at the expense of the S-phase cells (26.82 ꢀ 1.26 and
22.73 ꢀ 0.37, respectively) (Table 1).
at lower ppm (dC-5 121 ppm for N-90 regioisomers 11, 12 and
0
14, and 112 ppm for the N-70 regioisomers 18, 19 and 21)
when compared with the thio-substituted purine analogues
(dC-5 130 ppm for N-90 regioisomers 13 and 15e17 and
0
122 ppm for the N-70 regioisomers 20 and 22e24) which
means a downfield shift of 10 ppm for this signal.
2.2. Biological activities
The antitumour potential of the target molecules is reported
against the MCF-7 human breast cancer cell line including 5-
FU as reference drug (Table 2). The purine O,N-acetals 11e24
are more active than their corresponding purine bases 4e10.
The differences in the antiproliferative effect of the N-70 and
Table 1
Quaternary carbon signals of the purine rings
Compound
Isomer
R
C-40
C-50
C-60
11
12
14
13
15
16
17
18
19
21
20
22
23
24
N-90
N-90
N-90
N-90
N-90
N-90
N-90
N-70
N-70
N-70
N-70
N-70
N-70
N-70
OCH2eC6H11
OC6H5
151.64
152.46
151.72
148.90
148.54
148.57
147.86
161.55
162.81
161.93
159.62
159.39
159.03
158.13
121.53
121.63
121.43
130.95
130.79
130.74
130.62
111.95
112.04
112.10
122.30
122.24
121.74
122.65
161.41
160.52
160.70
159.53
161.44
162.69
162.39
157.05
156.18
156.51
151.36
Missing
162.48
154.00
In response to 15 (and 20), the percentage of apoptotic
cells increased, from 0.22 ꢀ 0.31 in control cells to a maxi-
mum of 73.37 ꢀ 0.12 (and 65.28 ꢀ 1.92) apoptotic cells at
a concentration equal to their IC50 against the MCF-7 cell
line. This is a remarkable property because the demonstration
of apoptosis in MCF-7 breast cancer cells by known apopto-
sis-inducing agents has proved to be difficult. These com-
pounds are more potent as apoptosis inductors against the
MCF-7 human breast cancer cells than paclitaxel (TaxolÒ),
which induced programmed cell death up to 43% of cell
population [27].
OCH2CH]CH2
SC6H5eCl2-(2,4)
SC6H5
SC6H5eF-( p)
SMe
OCH2eC6H11
OC6H5
OCH2CH]CH2
SC6H5eCl2-(2,4)
SC6H5
SC6H5eF-( p)
SMe

A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
1745
Table 3
Cell cycle distribution and apoptosis induction in the MCF-7 human breast
cancer cell line after treatment for 48 h for the three most active compounds
Initiator 2.0 single-mode microwave instrument producing
controlled irradiation at 2.450 GHz (Biotage AB, Uppsala).
Reaction time refers to hold time at 130 ꢁC, 100 ꢁC or
60 ꢁC, not to total irradiation time. These parameters were es-
tablished following the basic principles of microwave-assisted
organic synthesis. The temperature was measured with an IR
sensor outside the reaction vessel. Anhydrous acetonitrile
was purchased from VWR International Eurolab. 6-
Substituted purines 4e8 were synthesized according to litera-
ture procedures [15,16]. 6-Methylthiopurine and NU2058
were purchased from Aldrich.
Compound
Cell cyclea
Apoptosisb
G0/G1
S
G2/M
Control
58.62 ꢀ 0.74
55.63 ꢀ 1.57
59.10 ꢀ 1.28
58.10 ꢀ 0.19
33.82 ꢀ 0.72
37.00 ꢀ 2.00
26.82 ꢀ 1.26
22.73 ꢀ 0.37
7.55 ꢀ 1.34
7.37 ꢀ 0.43
11.08 ꢀ 0.01
19.16 ꢀ 0.56
0.22 ꢀ 0.16
44.47 ꢀ 2.98
73.37 ꢀ 0.12
65.28 ꢀ 1.92
14
15
20
a
Determined by flow cytometry [29].
Apoptosis was determined using an Annexin V-based assay [30]. The data
b
indicate the percentage of cells undergoing apoptosis in each sample. All ex-
periments were conducted in duplicates and gave similar results. The data are
means ꢀ SEM of three independent determinations.
4.1.1. 6-(2,4-Dichlorophenylthio)purine 9
A mixture of 6-chloropurine (0.8 g, 5.17 mmol), 2,4-chlor-
obenzenethiol (1 g, 5.58 mmol) and triethylamine (2.5 mL,
15.5 mmol) in 20 mL of n-butanol was refluxed for 24 h.
The reaction mixture was cooled to room temperature and
the solvent was removed in vacuo. The remaining oil was pu-
rified by flash column chromatography on silica gel using
CH2Cl2/MeOH (9.5/0.5) as eluent to afford 9 as an off-white
3. Conclusion
In conclusion, a series of (RS )-6-substituted-7 or 9-(2,3-di-
hydro-5H-1,4-benzodioxepin-3-yl)-7H- or -9H-purines were
synthesized. The microwave-assisted organic synthesis has
provided faster access to the target compounds with the advan-
tage of the selective preparation of the N-70 or N-90 re-
gioisomers, simplifying their isolation. The antitumour effect
of the target molecules against the MCF-7 human breast can-
cer cell line indicates that the biological effect is dependent on
the substituent at position 6 of the purine ring. The superiority
of chlorine substituents [12] over the substituents used in this
study can be highlighted. At present, studies are being carried
out to determine the mechanism of action at the molecular
level of the most active compounds.
1
solid (1.18 g, 77%); mp 262e264 ꢁC. H NMR (DMSO-d6,
300 MHz): d (ppm) 8.55 (s, 1H), 8.52 (s, 1H), 7.87 (d,
J ¼ 2.3 Hz, 1H), 7.82 (d, J ¼ 8.3 Hz, 1H), 7.55 (dd, J ¼ 2.3,
8.3 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz): d (ppm)
157.05, 152.29, 150.77, 144.66, 144.53, 140.58, 139.91,
136.50, 130.50, 128.91, 126.38. HR (LSIMS) calcd for
C11H6Cl2N4S (M þ H)þ 296.9768, found 296.9768. Anal.
for C11H6Cl2N4S (C, H, N, S).
4.1.2. General procedures for the reaction between (RS )-
3-methoxy-2,3-dihydro-5H-1,4-benzodioxepine 3 and the
purine bases 4e10
4. Experimental protocols
4.1.2.1. Method (a). A suspension of 3 (200 mg, 1.11 mmol),
TCS (142 mL, 1.11 mmol), HMDS (232 mL, 1.11 mmol) and
the corresponding purine derivative 4, 5 or 9 (1.22 mmol) in
anhydrous acetonitrile (6 mL) was prepared under argon atmo-
sphere and kept at room temperature for 20 min. The reaction
mixture was then cooled at ꢂ25 ꢁC and a 1.0 M solution of
SnCl4 (1.22 mL, 1.22 mmol) in CH2Cl2 was added dropwise.
After 48e72 h of stirring at 45 ꢁC, the reaction was quenched
by the addition of a concentrated aqueous solution of Na2CO3
and extracted with CH2Cl2 (3 ꢃ 20 mL). The combined or-
ganic layers were washed with brine, dried (Na2SO4), filtered
and the solvent removed under vacuum. The residue was puri-
fied by flash chromatography under gradient elution conditions
using mixtures of hexane/ethyl acetate (1/1 to 1/4) or CH2Cl2/
MeOH (100/1 to 100/5) to afford 11 and 18, 12 and 19 and 13
and 20. Traces of the acyclic alkylated analogues [26] were
detected in the reactions.
4.1. Chemistry
Melting points were taken in open capillaries on an Electro-
thermal melting point apparatus and are uncorrected. Nuclear
1
magnetic resonance spectra were recorded on a 400 MHz H
and 100 MHz 13C NMR Bruker ARX 400 or 300 MHz H
1
and 75 MHz 13C NMR Bruker AMX-300 spectrometers or
1
400 MHz H and 100 MHz 13C NMR Varian NMR-System-
TM 400 or 300 MHz H and 75 MHz 13C NMR Varian In-
1
ova-TM spectrometers at ambient temperature. Chemical
shifts (d) are quoted in parts per million (ppm) and are refer-
enced to the residual solvent peak. Signals are designated as
follows: s, singlet; d, doublet; dd, doublet of doublets; ddd,
double doublet of doublets; dt, doublet of triplets; t, triplet;
m, multiplet. The HMBC spectra were measured using a pulse
sequence optimized for 10 Hz (inter-pulse delay for the evolu-
tion of long-range couplings: 50 ms) and a 5/3/4 gradient com-
bination. In this way, direct responses (1J couplings) were not
completely removed. High-resolution liquid secondary ion
mass spectra (HR LSIMS) were carried out on a VG AutoSpec
Q high-resolution mass spectrometer (Fisons Instruments). All
products had satisfactory C, H and N analyses (within ꢀ0.4%).
Small scale microwave-assisted synthesis was carried out in an
4.1.2.1.1. (RS )-6-Cyclohexylmethyloxy-9-(2,3-dihydro-5H-
1,4-benzodioxepin-3-yl)-9H-purine 11. White solid (10%);
1
mp 156e158 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.53
(s, 1H), 8.20 (s, 1H), 7.29 (t, J ¼ 7.6 Hz, 1H), 7.20 (dd,
J ¼ 2.0, 7.4 Hz, 1H), 7.13e7.07 (m, 2H), 6.28 (dd, J ¼ 2.5,
6.5 Hz, 1H), 4.90 (d, J ¼ 14.1 Hz, 1H), 4.85 (d, J ¼ 14.1 Hz,
1H), 4.55 (dd, J ¼ 2.3, 12.9 Hz, 1H), 4.43 (dd, J ¼ 6.7,

1746
A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
12.9 Hz, 1H), 4.39 (d, J ¼ 6.7 Hz, 2H), 1.96e1.65 (m, 6H),
1.33e1.07 (m, 5H). 13C NMR (CDCl3, 100 MHz): d (ppm)
161.41, 158.88, 152.59, 151.64, 140.62, 130.57, 129.95,
129.33, 124.22, 121.53, 120.76, 83.93, 73.80, 72.54, 68.95,
37.31, 29.8 (ꢃ2), 26.47, 25.76 (ꢃ2). HR (LSIMS) calcd for
C21H24N4O3Na (M þ Na)þ 403.1746, found 403.1748. Anal.
for C21H24N4O3 (C, H, N).
calcd for C20H14N4O2NaSCl2 (M þ Na)þ 467.0112, found
467.0112. Anal. for C20H14Cl2N4O2S (C, H, N, S).
4.1.2.1.6. (RS )-6-(2,4-Dichlorophenylthio)-7-(2,3-dihydro-
5H-1,4-benzodioxepin-3-yl)-7H-purine
20. White
solid
(28%); mp 176.7e179.3 ꢁC. 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.70 (s, 1H), 8.57 (s, 1H), 7.60 (d, J ¼ 8.4 Hz, 1H),
7.53 (d, J ¼ 2.2 Hz, 1H), 7.32e7.19 (m, 3H), 7.12e7.05 (m,
2H), 6.49 (dd, J ¼ 2.3, 5.0 Hz, 1H), 4.79 (d, J ¼ 13.9 Hz, 1H),
4.73 (d, J ¼ 13.9 Hz, 1H), 4.64 (dd, J ¼ 2.4, 13.2 Hz, 1H),
4.49 (dd, J ¼ 5.0, 13.1 Hz, 1H). 13C NMR (CDCl3, 75 MHz):
d (ppm) 159.62, 158.84, 153,16, 151.36, 146.29, 140.32,
138.54, 137.33, 130.70, 130.59, 130.27, 129.54, 128.15,
125.26, 124.73, 122.30, 120.85, 85.41, 73.96, 67.98. HR
(LSIMS) calcd for C20H14N4O2NaSCl2 (M þ Na)þ 467.0112,
found 467.0112. Anal. for C20H14Cl2N4O2S (C, H, N, S).
4.1.2.1.2. (RS )-6-Cyclohexylmethyloxy-7-(2,3-dihydro-5H-
1,4-benzodioxepin-3-yl)-7H-purine 18. White solid (21%);
1
mp 101e104 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.64
(s, 1H), 8.39 (s, 1H), 7.33e7.29 (m, 1H), 7.23 (dd, J ¼ 1.5,
7.5 Hz, 1H), 7.13e7.09 (m, 2H), 6.30 (dd, J ¼ 2.5, 6.5 Hz,
1H), 4.81 (s, 2H), 4.58 (dd, J ¼ 2.2, 12.6 Hz, 1H), 4.44e4.36
(m, 2H), 4.28 (dd, J ¼ 6.5, 12.6 Hz, 1H), 1.83e1.76 (m, 6H),
1.26e1.10 (m, 5H). 13C NMR (CDCl3, 100 MHz): d (ppm)
161.55, 158.88, 157.04, 152.59, 143.97, 130.74, 130.11,
129.38, 124.42, 120.84, 111.95, 86.49, 74.64, 72.51, 68.99,
37.28, 29.86 (ꢃ2), 26.40, 25.73 (ꢃ2). HR (LSIMS) calcd for
C21H24N4O3Na (M þ Na)þ 403.1746, found 403.1746. Anal.
for C21H24N4O3 (C, H, N).
4.1.2.2. Method (b). In a microwave vial, a suspension of 3
(100 mg, 0.55 mmol), TCS (71 mL, 0.55 mmol), HMDS
(116 mL, 0.55 mmol) and the corresponding purine derivative
(6e8 and 10, 0.61 mmol) in anhydrous acetonitrile (3 mL)
was prepared under an argon atmosphere and kept at room
temperature for 20 min. The reaction mixture was then cooled
at ꢂ25 ꢁC and a 1.0 M solution of SnCl4 (0.61 mL,
0.61 mmol) in CH2Cl2 was added dropwise. The vial was
then sealed and after 5 min of microwave irradiation at
130 ꢁC, the reaction was quenched by the addition of a concen-
trated aqueous solution of Na2CO3 and extracted with CH2Cl2
(3 ꢃ 20 mL). The combined organic layers were washed with
brine, dried (Na2SO4), filtered and the solvent removed under
vacuum. The crude product was loaded onto a silica column
and purified by flash column chromatography using hexane/
ethyl acetate (1/1 to 1/4) as eluent to afford 14e17.
4.1.2.1.3.
(RS )-6-Phenoxy-9-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-9H-purine 12. White solid (21%); mp 182e
1
184 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.56 (s, 1H),
8.37 (s, 1H), 7.49e7.46 (m, 2H), 7.35e7.23 (m, 5H), 7.16e
7.11 (m, 2H), 6.34 (dd, J ¼ 2.3, 6.3 Hz, 1H), 4.94 (d,
J ¼ 14.3 Hz, 1H), 4.90 (d, J ¼ 14.3 Hz, 1H), 4.63 (dd,
J ¼ 2.3, 13.0 Hz, 1H), 4.50 (dd, J ¼ 6.3, 13.0 Hz, 1H). 13C
NMR (CDCl3, 100 MHz): d (ppm) 160.52, 158.87, 152.62,
152.46, 141.76, 130.42, 130.00, 129.71 (ꢃ2), 129.34,
125.93, 124.25, 121.91 (ꢃ2), 121.63, 120.75, 84.01, 73.72,
69.02, one carbon missing. HR (LSIMS) calcd for
C20H17N4O3 (M þ H)þ 361.1301, found 361.1300. Anal. for
C20H16N4O3 (C, H, N).
4.1.2.2.1.
(RS )-6-Allyloxy-9-(2,3-dihydro-5H-1,4-benzo-
4.1.2.1.4.
(RS )-6-Phenoxy-7-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-9H-purine 14. White solid (7%); mp 117e
1
dioxepin-3-yl)-7H-purine 19. White solid (25%); mp 192e
119 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.55 (s, 1H),
1
193 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.67 (s, 1H),
8.23 (s, 1H), 7.33e7.27 (m, 1H), 7.22 (dd, J ¼ 1.5, 7.5 Hz,
1H), 7.13e7.07 (m, 2H), 6.28 (dd, J ¼ 2.5, 6.5 Hz, 1H), 6.15
(ddd, J ¼ 5.6, 10.5, 17.3 Hz, 1H), 5.48 (dd, J ¼ 1.4, 17.3 Hz,
1H), 5.32 (dd, J ¼ 1.3, 10.4 Hz, 1H), 5.12 (dt, J ¼ 1.3, 1.4,
5.6 Hz, 2H), 4.88 (s, 2H), 4.59 (dd, J ¼ 2.5, 13.0 Hz, 1H),
4.45 (dd, J ¼ 6.5, 13.0 Hz, 1H). 13C NMR (CDCl3,
100 MHz): d (ppm) 160.70, 158.90, 152.52, 151.72, 140.85,
132.42, 130.52, 129.96, 129.32, 124.21, 121.53, 120.75,
118.67, 83.91, 73.77, 68.97, 67.79. HR (LSIMS) calcd for
C17H17N4O3 (M þ H)þ 325.1301, found 325.1301. Anal. for
C17H16N4O3 (C, H, N).
8.57 (s, 1H), 7.53e7.47 (m, 2H), 7.38e7.23 (m, 5H),
7.18e7.10 (m, 2H), 6.51 (dd, J ¼ 2.2, 6.0 Hz, 1H), 4.91 (d,
J ¼ 14.0 Hz, 1H), 4.85 (d, J ¼ 14.1 Hz, 1H), 4.71 (dd,
J ¼ 2.2, 12.9 Hz, 1H), 4.39 (dd, J ¼ 6.0, 12.9 Hz, 1H). 13C
NMR (CDCl3, 100 MHz): d (ppm) 162.79, 158.79, 152.68,
151.87, 145.15, 130.52, 130.12, 129.90 (ꢃ2), 129.70,
129.37, 126.27, 124.45, 121.91, 121.77 (ꢃ2), 120.77,
86.25, 74.46, 68.75. HR (LSIMS) calcd for C20H16N4O3Na
(M þ Na)þ 383.1120, found 383.1121. Anal. for
C20H16N4O3 (C, H, N).
4.1.2.1.5. (RS )-6-(2,4-Dichlorophenylthio)-9-(2,3-dihydro-
4.1.2.2.2. (RS )-6-Phenylthio-9-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-9H-purine 15. White solid (31%); mp 186e
5H-1,4-benzodioxepin-3-yl)-9H-purine
13. White
solid
(21%); mp 72e73 ꢁC melts, 154 ꢁC liquefies. 1H NMR
(CDCl3, 300 MHz): d (ppm) 8.64 (s, 1H), 8.33 (s, 1H), 7.68
(d, J ¼ 8.50 Hz, 1H), 7.58 (d, J ¼ 2.0 Hz, 1H), 7.36e7.21 (m,
3H), 7.14e7.09 (m, 2H), 6.29 (dd, J ¼ 1.8, 6.2 Hz, 1H), 4.89
(s, 2H), 4.61 (dd, J ¼ 1.8, 12.9 Hz, 1H), 4.44 (dd, J ¼ 6.2,
12.9 Hz, 1H). 13C NMR (CDCl3, 75 MHz): d (ppm) 159.53,
158.99, 152.68, 148.90, 142.20, 141.02, 138.75, 137.19,
130.95, 130.60, 130.20, 129.52, 128.08, 125.56, 124.46,
120.94, 84.07, 73.84, 69.21, one carbon missing. HR (LSIMS)
188 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.63 (s, 1H),
1
8.31 (s, 1H), 7.67e7.65 (m, 2H), 7.48e7.46 (m, 3H), 7.33e
7.28 (d, J ¼ 6.0 Hz, 1H), 7.13e7.09 (m, 2H), 6.28 (dd,
J ¼ 2.0, 6.5 Hz, 1H), 4.91 (d, J ¼ 14.1 Hz, 1H), 4.87 (d,
J ¼ 14.1 Hz, 1H), 4.53 (dd, J ¼ 2.5, 13.0 Hz, 1H), 4.44 (dd, J ¼
6.5, 13.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d (ppm)
161.45, 158.99, 152.85, 148.54, 141.89, 135.85 (ꢃ2), 130.79,
130.58, 130.15, 129.86, 129.55 (ꢃ2), 129.49, 127.28, 124.41,
120.91, 84.00, 73.86, 69.17. HR (LSIMS) calcd for

A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
1747
C20H16N4O2NaS (M þ Na)þ 399.0892, found 399.0892. Anal.
for C20H16N4O2S (C, H, N).
86.41, 74.69, 68.95, 67.88. HR (LSIMS) calcd for
C17H16N4O3Na (M þ Na)þ 347.1120, found 347.1120. Anal.
for C17H16N4O3 (C, H, N).
4.1.2.2.3. (RS )-6-p-Fluorophenylthio-9-(2,3-dihydro-5H-
1,4-benzodioxepin-3-yl)-9H-purine 16. White solid (35%);
4.1.2.3.2. (RS )-6-Phenylthio-7-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-7H-purine 22. White solid (19% at 60 ꢁC and
17% at 100 ꢁC); mp 148e151 ꢁC. 1H NMR (CDCl3,
300 MHz): d (ppm) 8.71 (s, 1H), 8.52 (s, 1H), 7.56 (m, 2H),
7.41 (m, 4H), 7.30e7.04 (m, 3H), 6.53 (dd, J ¼ 2.2, 5.3 Hz,
1H), 4.81 (d, J ¼ 14.1 Hz, 1H), 4.75 (d, J ¼ 14.1 Hz, 1H), 4.64
(dd, J ¼ 2.6, 13.2 Hz, 1H), 4.41 (dd, J ¼ 5.3, 12.8 Hz, 1H).
13C NMR (CDCl3, 75 MHz): d (ppm) 159.39, 159.00, 153.40,
146.15, 135.66, 130.84, 130.38, 130.13, 129.78, 129.68,
127.01, 124.82, 122.24, 121.01, 85.70, 74.31, 68.35, one carbon
missing. HR (LSIMS) calcd for C20H16N4O2NaS (M þ Na)þ
399.0892, found 399.0891. Anal. for C20H16N4O2S (C, H, N, S).
4.1.2.3.3. (RS )-6-p-Fluorophenylthio-9-(2,3-dihydro-5H-
1,4-benzodioxepin-3-yl)-7H-purine 23. White solid (35% at
1
mp 197e199 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.63
(s, 1H), 8.31 (s, 1H), 7.65e7.61 (m, 2H), 7.32e7.29 (m, 1H),
7.22e7.09 (m, 5H), 6.28 (dd, J ¼ 2.5, 6.5 Hz, 1H), 4.89 (s,
2H), 4.59 (dd, J ¼ 2.5, 13.0 Hz, 1H), 4.44 (dd, J ¼ 6.5,
13.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d (ppm) 165.18,
162.69, 158.97, 152.79, 148.57, 141.99, 138.07, 137.98,
130.10, 130.74, 130.53, 130.17, 129.49, 124.42, 120.91,
116.96, 116.74, 84.02, 73.84, 69.18. HR (LSIMS) calcd for
C20H16FN4O2S (M þ H)þ 395.0978, found 395.0979. Anal.
for C20H15FN4O2S (C, H, N).
4.1.2.2.4. (RS )-6-Methylthio-9-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-9H-purine 17. White solid (45%); mp 109e
1
111 ꢁC. H NMR (CDCl3, 400 MHz): d (ppm) 8.75 (s, 1H),
1
8.26 (s, 1H), 7.35e7.27 (m, 1H), 7.21 (dd, J ¼ 1.5, 7.5 Hz,
1H), 7.06e7.15 (m, 2H), 6.28 (dd, J ¼ 2.3, 6.7 Hz, 1H), 4.91
(d, J ¼ 14.1 Hz, 1H), 4.86 (d, J ¼ 14.1 Hz, 1H), 4.60 (dd,
J ¼ 2.4 and 12.9 Hz, 1H), 4.44 (dd, J ¼ 6.7 and 12.9 Hz, 1H),
2.73 (s, 3H). 13C NMR (CDCl3, 100 MHz): d (ppm) 162.36,
158.99, 152.50, 147.86, 141.38, 131.40, 130.62, 130.13,
129.49, 124.40, 120.91, 83.93, 73.89, 69.14, 12.04. HR
(LSIMS) calcd for C15H14N4O2NaS (M þ Na)þ 337.0735,
found 337.0735. Anal. for C15H14N4O2S (C, H, N).
60 ꢁC); mp 82e84 ꢁC melts, 135e137 ꢁC liquefies. H NMR
(CDCl3, 400 MHz): d (ppm) 8.81 (s, 1H), 8.70 (s, 1H), 7.69e
7.63 (m, 2H), 7.41e7.35 (m, 1H), 7.31e7.16 (m, 5H), 6.60
(dd, J ¼ 2.2, 4.8 Hz, 1H), 4.91 (d, J ¼ 14.1 Hz, 1H), 4.84 (d,
J ¼ 14.1 Hz, 1H), 4.74 (dd, J ¼ 2.2, 13.2 Hz, 1H), 4.44 (dd,
J ¼ 5.2, 13.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d (ppm)
165.81, 162.48, 159.03, 158.99, 153.20, 146.27, 138.15,
138.04, 130.77, 130.45, 129.70, 124.90, 121.74, 121.02,
117.28, 116.99, 104.99, 85.69, 73.23, 68.27. HR (LSIMS) calcd
for C20H15FN4O2NaS (M þ Na)þ 417.0797, found 417.0799.
Anal. for C20H15FN4O2S (C, H, N, S).
4.1.2.3. Method (c). In a microwave vial, a suspension of 3
(100 mg, 0.55 mmol), TCS (71 mL, 0.55 mmol), HMDS
(116 mL, 0.55 mmol) and the corresponding purine derivative
(6e8 and 10, 0.61 mmol) in anhydrous acetonitrile (3 mL)
was prepared under argon atmosphere and kept at room tem-
perature for 20 min. The reaction mixture was then cooled at
ꢂ25 ꢁC and a 1.0 M solution of SnCl4 (0.61 mL, 0.61 mmol)
in CH2Cl2 was added dropwise. The vial was then sealed
and after 5 min of microwave irradiation at 100 ꢁC the reac-
tion was quenched by the addition of a concentrated aqueous
solution of Na2CO3 and extracted with CH2Cl2 (3 ꢃ 20 mL).
The combined organic layers were washed with brine, dried
(Na2SO4), filtered and the solvent removed under vacuum.
The crude product was loaded onto a silica column and puri-
fied by flash column chromatography using hexane/ethyl ace-
tate (1/1 to 1/4) as eluent to afford 21e24. Traces of the
acyclic alkylated analogues [26] were detected in the synthesis
of 22e24. The use of 60 ꢁC as the reaction temperature did not
prevent the formation of these acyclic derivatives.
4.1.2.3.4. (RS )-6-Methylthio-9-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-7H-purine 24. White solid (20% at 100 ꢁC,
25% at 60 ꢁC); mp 69 ꢁC melts, 95 ꢁC foam, 124 ꢁC liquefies.
1H NMR (CDCl3, 400 MHz): d (ppm) 8.95 (s, 1H), 8.58 (s,
1H), 7.39e7.34 (m, 1H), 7.29e7.26 (m, 1H), 7.20e7.14 (m,
2H), 6.49 (dd, J ¼ 2.4, 5.5 Hz, 1H), 4.84 (s, 2H), 4.68 (dd,
J ¼ 2.4, 13.0 Hz, 1H), 4.46 (dd, J ¼ 5.5, 13.0 Hz, 1H), 2.82 (s,
3H, SMe). 13C NMR (CDCl3, 100 MHz): d (ppm) 158.98,
158.13, 154.51, 152.91, 145.49, 130.81, 130.38, 129.67,
124.80, 122.65, 121.00, 85.70, 74.43, 68.38, 12.83. HR
(LSIMS) calcd for C15H14N4O2NaS (M þ Na)þ 337.0735,
found 337.0736. Anal. for C15H14N4O2S (C, H, N, S).
4.2. Biology
4.2.1. Cell culture and drug treatments
The human breast cancer MCF-7 cells were grown at 37 ꢁC
in an atmosphere containing 5% CO2, with Dulbecco’s modi-
fied Eagle’s medium (DMEM) (Gibco, Grand Island, NY) sup-
plemented with 10% heat-inactivated fetal bovine serum
(FBS) (Gibco), 2% L-glutamine, 2.7% sodium bicarbonate,
1% Hepes buffer, 40 mg/L gentamicin and 500 mg/L ampicil-
lin. After the synthesis and purification of the compounds
stock solutions were prepared as previously described [28].
Briefly, the drugs were dissolved in DMSO and stored at
ꢂ20 ꢁC. For each experiment, the stock solutions were further
diluted in medium to obtain the desired concentrations. The fi-
nal solvent concentration in cell culture was ꢄ0.5% v/v of
DMSO, a concentration without effect on cell replication.
4.1.2.3.1.
(RS )-6-Allyloxy-9-(2,3-dihydro-5H-1,4-benzo-
dioxepin-3-yl)-7H-purine 21. Viscous oil (30% at 100 ꢁC). 1H
NMR (CDCl3, 300 MHz): d (ppm) 8.68 (s, 1H), 8.45 (s, 1H),
7.33 (t, J ¼ 7.6 Hz, 1H), 7.24 (d, J ¼ 7.0 Hz, 1H), 7.16e7.11
(m, 2H), 6.37 (d, J ¼ 4.5 Hz, 1H), 6.08 (ddd, J ¼ 5.1, 10.5,
17.0 Hz, 1H), 5.48 (d, J ¼ 17.0 Hz, 1H), 5.32 (d, J ¼ 10.5 Hz,
1H), 5.12 (d, J ¼ 5.1 Hz, 2H), 4.87 (d, J ¼ 14.1 Hz, 1H), 4.82
(d, J ¼ 14.1 Hz, 1H), 4.62 (dd, J ¼ 2.0, 13.0 Hz, 1H), 4.33
(dd, J ¼ 6.5, 13.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
d (ppm) 161.93, 159.00, 156.51, 152.74, 144.41, 132.14,
130.90, 130.27, 129.56, 124.60, 120.99, 119.05, 112.10,

1748
A. Conejo-Garc´ıa et al. / European Journal of Medicinal Chemistry 43 (2008) 1742e1748
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J.A. Endicott, A.E. Gibson, B.T. Golding, R.J. Griffin, P. Jewsbury,
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[8] T.M. Sielecki, J.F. Boylan, P.A. Benfield, G.L. Trainor, J. Med. Chem. 43
(2000) 1e18.
Parallel cultures of MCF-7 cells in medium with DMSO were
used as controls.
4.2.2. Cytotoxicity assays in vitro
The effect of anticancer drugs on cell viability was assessed
using the sulforhodamine-B (SRB) colorimetric assay. Aliquots
of MCF-7 cell suspension (30 ꢃ 103 cells/well) were seeded
onto 24-well plates and incubated for 24 h. The cells were
then treated with different concentrations of drugs in the culture
medium. After three days the wells were aspirated, fresh me-
dium and further drug were added, and cells were maintained
for 3 more days. Thereafter, the cells were processed as de-
scribed previously [29], using a Titertek Multiscan apparatus
(Flow, Irvine, California) at 492 nm. We evaluated the linearity
of the SRB assay with cell number for each MCF-7 cell stock
before each cell growth experiment. The IC50 values were cal-
culated from semilogarithmic doseeresponse curves by linear
interpolation. All of the experiments were plated in triplicate
wells and were carried out at least twice.
[9] M. Knockaert, P. Greegard, L. Meijer, Trends Pharmacol. Sci. 23 (2002)
417e425.
[10] C.E. Arris, T. Boyle, H. Calvert, N.J. Curtin, J.A. Endicott, E.F. Garman,
A.E. Gibson, B.T. Golding, S. Grant, R.J. Griffin, P. Jewsbury,
L.N. Johnson, A.M. Lawrie, D.R. Newell, M.E.M. Noble,
E.A. Sausville, R. Schultz, W. Yu, J. Med. Chem. 43 (2000) 2797e2804.
´~
[11] J.A. Marchal, M.C. Nunez, I. Suarez, M. Dıaz-Gavilan, J.A. Gomez-
´
´
´
´
´
Vidal, H. Boulaiz, F. Rodrıguez-Serrano, A. Aranega, M.A. Gallo,
´
A. Espinosa, J.M. Campos, Breast Cancer Res. 105 (2007) 237e246.
´~
´
´
´
´
[12] M.C. Nunez, M.G. Pavani, M. Dıaz-Gavilan, F. Rodrıguez-Serrano,
J.A. Gomez-Vidal, J.A. Marchal, A. Aranega, M.A. Gallo,
´
A. Espinosa, J.M. Campos, Tetrahedron 62 (2006) 11724e11733.
[13] E. Saniger, J.M. Campos, A. Entrena, J.A. Marchal, I. Suarez,
´
A. Aranega, D. Choquesillo, J. Niclos, M.A. Gallo, A. Espinosa, Tetrahe-
´
dron 59 (2003) 5457e5467.
´
4.2.3. Cell cycle distribution analysis
[14] H. Vorbruggen, B. Bennua, Chem. Ber. 114 (1981) 1279e1286.
¨
Cells at70%confluenceweretreatedwitheitherDMSOalone
or with concentrations of the compounds determined by their
IC50 values. Fluorescence-activated cell sorting (FACS) analysis
was performed at 48 h of treatment as described [30]. All exper-
iments were performed in duplicates and yielded similar results.
[15] S.A. Laufer, D.M. Domeyer, T.R.F. Scior, W. Albrecht, D.R.J. Hauser,
J. Med. Chem. 48 (2005) 710e722.
[16] R.J. Griffin, A.H. Calvert, N.J. Curtin, D.R. Newell, B.T. Golding, J.A.
Endicott, M.E. Noble, F.T. Boyle, P.J. Jewsbury, G.B. Patent
9,902,162, 1998; Chem. Abstr. 130 (1999) 139576.
[17] C.O. Kappe, Angew. Chem., Int. Ed. 43 (2004) 6250e6284.
[18] B.L. Hayes, Aldrichimica Acta 37 (2004) 66e77.
´
[19] A. De la Hoz, A. Dıaz-Ortiz, A. Moreno, Chem. Soc. Rev. 34 (2005)
4.2.4. Apoptosis detection by staining with annexin V-FITC
and propidium iodide
The Annexin V-FITC Apoptosis Detection kit I (Pharmin-
gen, San Diego, CA, USA) was used to detect apoptosis by
flow cytometry according to Ref. [30]. All experiments were
performed in duplicates and yielded similar results.
164e178.
[20] C.O. Kappe, A. Stadler, Microwaves in Organic and Medicinal Chemis-
try, Wiley-VCH, Weinheim, 2005.
[21] P. Lidstrom, J.P. Tierney (Eds.), Microwave-assisted Organic Synthesis,
¨
Blackwell, Oxford, 2005.
[22] A. Loupy, Microwaves in Organic Synthesis, Wiley-VCH, Weinheim,
2002.
Acknowledgments
[23] B.L. Hayes, Microwave Synthesis: Chemistry at the Speed of Light,
CEM, Matthews, NC, 2002.
[24] F. Al-Obeidi, R.E. Austin, J.F. Okonya, D.R.S. Bond, Mini Rev. Med.
Chem. 3 (2003) 449e460.
[25] C.O. Kappe, D. Dallinger, Nat. Rev. Drug Discov. 5 (2006) 51e63.
We thank the European Commission (A.C.-G. Marie Curie
Programme MERG-CT-2005-030616), the Instituto de Salud
´
Carlos III (Fondo de Investigacion Sanitaria Project No.
PI041206 and PI070227), and the Consejerıa de Innovacion,
´~
´
´
[26] M.C. Nunez, F. Rodrıguez-Serrano, J.A. Marchal, O. Caba, A. Aranega,
M.A. Gallo, A. Espinosa, J.M. Campos, Tetrahedron 63 (2007) 183e190.
[27] D.E. Saunders, W.D. Lawrence, C. Christensen, N.L. Wappler, H. Ruan,
G. Deppe, Int. J. Cancer 70 (1997) 214e220.
´
´
´
Ciencia y Empresa of the Junta de Andalucıa (Excellence Re-
search Project No. 00636 and M.C.N. research contract) for fi-
nancial support.
´
[28] J.A. Marchal, H. Boulaiz, I. Suarez, E. Saniger, J. Campos, E. Carrillo,
´
J. Prados, M.A. Gallo, A. Espinosa, A. Aranega, Invest. New Drugs 22
(2004) 379e389.
[29] M. Villalobos, N. Olea, J.A. Brotons, M.F. Olea-Serrano, J.M. Ruiz de
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