New (RS)-benzoxazepin-purines with antitumour activity: The chiral switch from (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1- benzoxazepin-3-yl]-9H-purine

Article abstract of DOI:10.1016/j.ejmech.2010.11.011

Completing an SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H or 9H-purines has been prepared under microwave-assisted conditions. Their antiproliferative activities on MCF-7 and MDA-MB-231 cancerous cell l

Full text of DOI:10.1016/j.ejmech.2010.11.011

European Journal of Medicinal Chemistry 46 (2011) 249e258
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New (RS)-benzoxazepin-purines with antitumour activity: The chiral switch from
(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,
1-benzoxazepin-3-yl]-9H-purine
Luisa C. López-Cara a, Ana Conejo-García a, Juan A. Marchal b, Giuseppe Macchione a, Olga Cruz-López a,
,
Houria Boulaiz b, María A. García b c, Fernando Rodríguez-Serrano b, Alberto Ramírez b, Carlos Cativiela d,
Ana I. Jiménez d, Juan M. García-Ruiz e, Duane Choquesillo-Lazarte e, Antonia Aránega b,
,
Joaquín M. Campos a
*
a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, c/ Campus de Cartuja s/n, 18071 Granada, Spain
b Instituto de Biopatología y Medicina Regenerativa (IBIMER), Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Avenida de Madrid s/n,
18071 Granada, Spain
c Unidad de Investigación, Hospital Universitario Virgen de las Nieves, Granada, Spain
d Departamento de Química Orgánica y Química Física, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain
e Laboratorio de Estudios Cristalográficos, IACT, CSIC-Universidad de Granada, Av. del Conocimiento s/n, P.T. Ciencias de la Salud, 18100 Armilla, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Completing an SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-
3-yl)-7H or 9H-purines has been prepared under microwave-assisted conditions. Their antiproliferative
activities on MCF-7 and MDA-MB-231 cancerous cell lines are presented, being the majority of the IC50
Received 14 September 2010
Received in revised form
27 October 2010
Accepted 8 November 2010
Available online 3 December 2010
values below 1
m
M. The most active compound (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-
M against the human
tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine (14) presents an IC50 of 0.166
m
cancerous cell line MDA-MB-231. Compound 14 was the most selective against the human breast
adenocarcinoma MCF-7 and MDA-MB-231 cancer cell lines (Therapeutic Indexes, TIs ¼ 5.1 and 11.0,
respectively) in relation to the normal one MCF-10A. (RS)-14 was resolved into its enantiomers. Both
enantiomers are equally potent, but more potent than the corresponding racemic mixture. (R)-14 induces
apoptosis against MCF-7 up to 52.50% of cell population after 48 h, being more potent than the clinical-
used drug paclitaxel (43%). (RS)-14 induces no acute toxicity in mice after two weeks of treatment.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Antitumour
Benzoxazepine
2,6-Disubstituted purine
Homochiral
MCF-7
Microwave
Seven-membered ring
1. Introduction
In recent years, many studies have shown an association
between cell-cycle regulation and cancer inasmuch as the cell-cycle
With more than 10 million new cases each year cancer is at
present one of the most devastating diseases worldwide with an
immense affliction burden not only for affected individuals, their
relatives and friends but also representing heavy challenges to
health care systems [1]. In the year 2000, cancer was responsible
for 12% of the nearly 56 million deaths worldwide and in many
countries this percentage is even higher with more than a quarter
of deaths attributable to cancer. Moreover, it is expected that cancer
rates can further increase by 50% to 15 million new cases in the year
2020, mainly due to steadily ageing populations in both developed
and developing countries [2].
inhibitors are being considered as a weapon for the management of
cancer [3]. Ultimately a great level of interest has arisen in the G0/
G1 phase regulatory molecules such as cyclin D1, CdkIs, and p53 as
potential therapeutic targets in diseases where control of inap-
propriate cellular proliferation would be a therapeutic benefit [4].
Apoptosis is an essential physiological process throughout the life
of multi-cellular organisms important in the development and in the
maintenance of tissue homeostasis. Apoptosis is involved in
controlling the cell number and proliferation during embryogenesis,
deletionof activated lymphocytes atthe end of the immune response,
elimination of self-reactive lymphocytes, in controlled destruction of
damaged, aged, infected, transformed, and other harmful cells [5,6].
Zivnyetal. have recently reviewedtheapoptotic pathways, molecules
involved in the cross-talk between individual apoptosis pathways,
* Corresponding author. Tel.: þ34 958 243848; fax: þ34 958 243845.
E-mail address: jmcampos@ugr.es (J.M. Campos).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.011

250
L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
apoptosis regulation as well as mechanisms of action of conventional
anticancer drugs and new promising agents, which trigger directly or
indirectly apoptosis of hematologic cancer cells [7].
active racemic compound was resolved and the antiproliferative
activity of its enantiomers was measured.
Modern drug discovery relies on high speed organic synthesis.
Microwave-assisted organic synthesis [15e21] is proving to be
instrumental for the rapid synthesis of compounds with new and
improved biological activities [22,23]. We previously investigated
the Vorbrüggen condensation in microwave-assisted organic
synthesis [24]. Microwave advantage is chiefly 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.
We have reported the synthesis and anticancer activities of
compounds 1e3, 5e6 [8] and trans-7 [9] (Chart 1). In all the cases,
the linkage between the 5-fluorouracil (5-FU) moiety and the
seven-membered ring was carried out through its N ꢀ 1 atom.
The structural nature of 6 implies that this compound cannot
be considered as a 5-FU prodrug and we suspected that neither
would the remaining compounds (1e3, 5e7) be 5-FU prodrugs
[10]. The effects of the uracil benzo-fused seven-membered
O,N-acetal 4 were investigated in the MCF-7 human breast cancer
cell line. The expression of cell-cycle-related proteins (cyclin D1,
and p53) was also explored to investigate the potential mecha-
nisms of the G0/G1 arrest activity of 4 [10]. In addition, the
expression levels of CDKIs, such as p21Waf1/Cip1 and p27Kip1,
were examined in MCF-7 cells [10].
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of derivatives
Preparation of the O,N-acetals 14e17 was achieved by the
microwave-assisted Vorbrüggen one-pot condensation of the cyclic
acetals 25 and 26 [25] and the commercially available purine bases
6-chloro-, 6-bromo- and 2,6-dichloro-purines, using chloro-
trimethylsilane (TMSCl), 1,1,1,3,3,3-hexamethyldisilazane (HMDS)
and tin(IV) chloride as the Lewis acid in anhydrous acetonitrile. The
reaction mixture was microwave-irradiated at a temperature of
140 ꢂC or 160 ꢂC for 5 min (Scheme 1). Compounds 27 and 28 were
isolated from the reactions and the acyclic O,N-acetal 23 was also
obtained in the synthesis of 14. Traces of the N-70 regioisomer 24
were detected in the synthesis of 15. The following modifications
were carried out on 15: a) selective nucleophilic substitution of the
chorine atom at position 6 of the purine ring using NaI and tri-
fluoroacetic acid (TFA) to yield 18; b) reduction of the nitro group
with SnCl2 to give rise to 19 and 20; and c) the treatment with the
PhSH to produce 21 and 22 (see Experimental Part for details).
We have reported that compounds 8 and 9, with a thymine
linked to a seven-membered ring (Chart 2) exhibited in vitro anti-
proliferative activities (12.74 ꢁ 4.79 and 30.05 ꢁ 0.71
mM, respec-
tively) against the MCF-7 cell line [11].
Moreover, the uracil O,N-acetals 10e13 (Chart 3) derived from
the tetrahydrobenzoxazepine moiety, whose syntheses were
reported [12], show in vitro antiproliferative activities against the
MCF-7 human breast cancer cell line in the micromolar range [13].
Taken together, the antiproliferative activities of the naturally
occurring pyrimidine compounds 8e13 are an outstanding fact that
has not been previously reported in bibliography. Therefore these
compounds may serve as prototypes for the development of even
more potent structures, endowed with a new mechanism of action.
Once the alkylated pyrimidine derivatives had been studied, the
shift was made to the more lipophilic purine ones [14]. Herein we
will report the synthesis and antiproliferative activities of purine
derivatives 14e24 (Chart 4) against the cancerous MCF-7 and MDA-
MB-231 human breast cancer cell lines and the corresponding
normal one (MCF-10A) to define the in vitro TI as a measure of the
selectivity. From a structural point of view, the compounds studied
herein differ from others previously reported [13] by the addition of
an extra halogen or PhS- groups on the purine ring. Finally the most
2.1.2. Other products in the reaction between acetals 25 and 26 and
purines
Compounds 27 and 28 were obtained along with the cyclic and
acyclic O,N-acetals in the reaction of purines with 25 and 26, respec-
tively. Their importance lies in the information that they provide on the
mechanism of the reaction with purines as none of these olefins have
NO2
R6
N
NO2
N
i)
N
14 + 23
15 + 24
16
O S O
N
O S O
+
+
N
H
N
R2
17
OMe
O
O
R2 = H, Cl
R6 = Cl, Br
27 p-NO2
28 o-NO2
25 p-NO2
26 o-NO2
ii)
18
iii)
19
20
15
iv)
21
22
Scheme 1. Reagents and conditions: i) purine, TMSCl, HMDS, SnCl4 (1 M solution in CH2Cl2), 140 or 160 ꢂC, microwave, 5 min; ii) NaI, TFA, butanone, ꢀ15 ꢂC, 6 h; iii) SnCl2$2H2O,
EtOH, reflux, 2 h; iv) PhSH, K2CO3, DMF, rt, 4 h.

L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
251
been isolated in the corresponding reactions with uracil or 5-FU [12].
Although it could have been thought that the formation of the seven-
membered sulfonenamides 27 and28 was simply due to an elimination
of methanol from the cyclic O,O-acetals 25 and 26, our previous results
reported on the acyclic O,N-acetals 6-chloro-7-{2-[N-(2-hydrox-
ymethylphenyl)-2- or 4-nitrobenzenesulfonamide]-1-methoxyethyl}-
7H-purines clearly support a more complicated reaction mechanism
[26].
Compounds 27 and 28 could have been originated in a process
which shared the reaction mechanism with the N-700 / N-900
transformation, via elimination of the purine ring after its activa-
tion as a leaving group by the coordination of the Lewis acid to one
of its electron-rich positions. This elimination would be easier for
the highest energy N-700 regioisomers than for the more stable N-900
ones. The formation of the cyclic olefin would be justified by the
resonance of the final electronic system.
important commercial reason is that as patents on racemic drugs
expire, pharmaceutical companies have the opportunity to extend
patent coverage through development of the chiral switch enan-
tiomers with desired bioactivity [27].
2.1.4. Resolution of (RS)-14 into its two enantiomers
(RS)-9-[1-(p-Nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-ben-
zoxazepin-3-yl]-2,6-dichloro-9H-purine (14) is resolved into its
25
two enantiomers: [(R)-14: [
a
]
¼ ꢀ43.6 (c ¼ 0.22, THF), and (S)-
D
25
14: [
a]
¼ þ41.0 (c ¼ 0.23, THF)] using a semipreparative column
D
CHIRALPAKÒ and a mixture of hexane/t-BuOMe/iPrOH as eluant
[28]. The details for the X-ray data for enantiomer (S)-14 are shown
in the Supporting Information Section.
2.2. Biological activities
According to an elimination mechanism, loss of the purine ring
would lead to the oxocarbenium ions 29 and 30, which could either
attack nucleophile positions of the purine giving rise to the
formation of the N-900 O,N-acetals (14e17), or eliminate one
Table 1 shows the antiproliferative activity (IC50 values) for
14e24 and 27, 28. All the compounds were first assayed as anti-
proliferative agents against the human breast adenocarcinoma cell
line MCF-7 (p53 wild-type and ras mutated). The seven most active
compounds (14, 15, 18e20, and 23e24) were selected to be further
assayed on the human breast cancer cell line MDA-MB-231, which
has high levels of mutant p53, the most commonly mutated gene in
human cancer. Additionally, we used a non-cancerous human
mammary epithelial cell line (MCF-10A), in order to study the
therapeutic index against breast cancer.
It must be pointed out that from the twenty IC50 values against
the two cancerous cell lines, the majority of the IC50 values were
below 1 mM. As shown in Table 1, all compounds were more active
as antiproliferative agents against MDA-MB-231 than against the
MCF-7 human breast cancer cell line, except for the acyclic deriv-
ative 23, whose antiproliferative effect remains the same in both
b
proton in relation to the Oþ atom, with formation of a double
bond to give 27 and 28 (Scheme 2). The progress of the cationic
intermediate in one way or another would depend on the speed of
each process.
2.1.3. Homochiral drugs
The issue of drug chirality is nowa major theme in the design and
development of new drugs, underpinned by a new understanding of
the role of molecular recognition in many pharmacologically rele-
vantevents. In general, threemethods areutilized for the production
of a chiral drug: the chiral pool, separation of racemates, and
asymmetric synthesis. Although the use of chiral drugs predates
modern medicine, only since the 1980’s has there been a significant
increase in the development of chiral pharmaceutical drugs. An
cancer cell lines. The IC50 ¼ 0.166
mM for compound 14 against the
NO2
Stabilization of the oxocarbeniumion
by addition of the purinering
NO2
NO2
O
S
N
O
O
O
S
N
O
O
H
R2
SnCl4
a)
O
S
N
O
O
N
N
N
Cl3Sn
a)
N
R6
N
SnCl4
N
N
b)
N
N
N
R6
Cl
N
R6
R2
N
29 Isomer: p-NO2
Cyclic N-7'' O,N-acetals
R2
30
Isomer: o-NO2
N-9'' Regioisomers
14-17
Stabilization of the oxocarbeniumion
by an elimination of a hydrogenatom
b)
HCl
NO2
27 p-NO2
O
S
N
O
28
o-NO2
O
Scheme 2. Mechanism proposed for the formation of 1,5-dihydro-4,1-benzoxazepines 27 and 28 from cyclic N-700 O,N-acetals.

252
L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
Table 1
Table 2
Antiproliferative activitiesa for compounds 14e24 against the cancerous cell lines
Therapeutic indexes for the most representative compounds.
MCF-7 and MDA-MB-231, and the non-cancerous cell line MCF-10A.
Compound
Therapeutic index (TI)
MCF-7
Compound
IC50 MCF-7 (
mM)
IC50 MDA-MB-231
M)
IC50 MCF-10A (mM)
MDA-MB-231
(
m
14
15
18
19
20
23
24
5.14
4.00
0.57
1.85
0.80
5.25
1.27
11.0
5.50
1.37
3.25
2.53
4.55
4.00
14
15
16
17
18
19
20
21
22
0.355 ꢁ 0.011
0.383 ꢁ 0.027
1.226 ꢁ 0.348
3.618 ꢁ 0.273
0.610 ꢁ 0.043
0.820 ꢁ 0.050
1.530 ꢁ 0.040
9.710 ꢁ 0.380
13.85 ꢁ 1.790
0.355 ꢁ 0.122
0.990 ꢁ 0.090
4.32 ꢁ 0.020
0.166 ꢁ 0.063
0.280 ꢁ 0.006
N.D.b
1.825 ꢁ 0.503
1.530 ꢁ 0.198
N.D. b
N.D.b
N.D.b
0.256 ꢁ 0.002
0.467 ꢁ 0.017
0.487 ꢁ 0.006
N.D.b
0.351 ꢁ 0.020
1.520 ꢁ 0.498
1.233 ꢁ 0.217
N.D.b
N.D.b
N.D.b
23
0.409 ꢁ 0.074
0.318 ꢁ 0.066
N.D.b
1.863 ꢁ 0.050
1.265 ꢁ 0.163
N.D.b
racemic compound showing the greatest differences against MCF-7
cells. (S)-14 shows higher antitumour activity, up to twice that of
(R)-14 in the MCF-7 cell line. Studies with other compounds
showed similar results with more potency in cytotoxicity in an
enantiomer in comparison with the racemate. This enantioselective
cytotoxicity indicates that the enantiomers of some chiral drugs
may differ both quantitatively and qualitatively in their biological
activity [30,31]. Moreover, enantiomers can posses minimal in vitro
but a dramatic in vivo chiral dependency in their antitumour
activities [32,33] (Table 3).
24
5-FUc
a
All experiments were conducted in duplicate and gave similar results. The data
are means ꢁ SEM of three independent determinations. The treatment time was
48 h.
b
N.D. ¼ Not determined.
c
Taken from Ref. [14].
human cancerous cell line MDA-MB-231 stands out over the rest of
the values (Fig. 1).
A comparison between the cancerous cell lines (MCF-7 and
MDA-MB-231) and the corresponding normal one (MCF-10A) was
established in an intent to define the in vitro therapeutic index as
a measure of the selectivity. The in vitro therapeutic index (TI) of
a drug is defined as the ratio of the toxic dose to the therapeutic
dose (In vitro TI ¼ IC50 non-tumour cell line/IC50 tumour cell line)
[29]. TI was better for compounds 14, 15 and 23 against both cancer
cell lines with values up to 11.0, 5.50 and 4.55, respectively against
MDA-MB-231 cell line. 2,6-Dichloro derivatives 14 and 23 were the
most selective compounds against the human breast adenocarci-
noma MCF-7 cancer cell line (TIs ¼ 5.1 and 5.2, respectively) in
relation to the normal one. The iodine derivative 18 showed the
most toxic effect against the non-tumour MCF-10A human
mammary epithelial cell line (Table 2, Fig. 1).
2.2.2. Selection of compounds to study the cell cycle
Once the antitumour activity of compounds was determined
against the different breast cell lines, we carried out a selection
between those that showed a great cytotoxic effect against MCF-7,
including (R)-14 and (S)-14, in order to determine their influence on
the several cell-cycle phases. In this study we have included drugs
used in clinic against breast cancer, such 5-FU and paclitaxel, with
a known mechanism of action at the level of cell cycle.
2.2.3. Cell-cycle analysis
In order to analyze if the antitumour effects of the drugs
involve changes in cell-cycle distribution, the non-tumour cell line
MCF-10A and the breast cancer cell lines MCF-7 and MDA-MB-231
were treated with the compounds during 48 h and then analyzed
by flow cytometry (Supporting Information Tables 1e3). The non-
accumulation in a specific phase was detected during treatment
with the drugs in most of the cell lines analyzed in comparison
with control-DMSO-treated cells. Only the (R)-14 enantiomer was
able to induce in MDA-MB-231 cells an accumulation in both G0/
G1 and G2/M phases with the consequently significant decreased
in the S phase. Also an accumulation in the phase G2/M was
detected in MCF-7-18 treated cells. Treatment with 5-FU and
paclitaxel, as have been described previously [34], induced accu-
mulation in the S or G2/M phases depending on the cell line
analyzed. Similar data were obtained when cell lines were treated
for 24 h with 0.5 mM mimosine to synchronize the cells in the G1/
S phase (data not shown). These results indicate that compounds
inhibited all phases of the cell cycle, probably through the inhi-
bition of protein synthesis as it has been proved with other anti-
tumour drugs [35].
2.2.1. In vitro cytotoxic activities
When the homochiral forms were analyzed we found differ-
ences in the IC50 values between (S)-14 and (R)-14 enantiomers,
although no differences in activity were found between the two
enantiomers against the MDA-MB-231 cell line. However both
enantiomers present higher antiproliferative activity than the
2,5
2
1,5
MCF-10A
MCF-7
MDA-MB-231
1
Table 3
Antiproliferative activities of (RS)-14 and its enantiomers against the cancerous cell
lines MCF-7 and MDA-MB-231.
0,5
0
Compound
MCF-7 (
m
M)
MDA-MB-231 (mM)
14
(R)-14
(S)-14
0.355 ꢁ 0.011
0.19 ꢁ 0.001
0.10 ꢁ 0.001
0.166 ꢁ 0.063
0.11 ꢁ 0.001
0.11 ꢁ 0.001
14
15
18
19
20
23
24
Fig. 1. Comparison between antiproliferative activities for the most representative
compounds against the cancerous cell lines MCF-7 and MDA-MB-231, and the non-
cancerous cell line MCF-10A.
All experiments were conducted in duplicate and gave similar results. The data are
means ꢁ SEM of three independent determinations.

L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
253
2.2.4. Apoptosis assay
arrest since cells exposed to specific agents typically enter
apoptosis from a given phase of the cell cycle [10,36,38,39].
Differences in cytotoxicity, cell-cycle analysis or apoptotic levels
between (R)-14 and (S)-14 suggest distinct signalling pathways as
has been shown with other antitumour enantiomers [40]. More-
over, the amount of cells undergoing apoptosis in response to the
compounds may likely have been higher than these values, due to
the fact that only adherent cells were stained and counted.
Representative results for the MCF-7 cells at 48 h of treatments
are shown in Fig. 2A.
The effects of compounds on the pattern of cell death were also
confirmed by confocal microscopy after staining with FITC-conju-
gated annexin V and the nuclear non-vital stain PI. MCF-7 cells
treated with compounds showed several staining patterns (Fig. 2B).
Some cells displayed an intense FITC staining located at the plasma
membrane and a nucleus with intensely PI-labeled marginated
chromatin, suggesting that they were in the course of apoptosis.
Other cells showed a peculiar staining pattern, because they
exhibited nuclei with the same features observed in true apoptotic
cells and, at the same time, cytoplasms homogeneously stained for
annexin V. In fact, the FITC staining was located not only at the cell
surface, but also within the cytoplasm (Fig. 2B). Therefore, these
cells were considered as aponecrotic cells as previously has been
established [41]. In addition, patches of localised partially
condensed chromatin were found in other cells abutted along the
inner part of the nuclear membrane (Fig. 2B). In the control
cultures, most of the cells turned out to be negative for both
staining except for some dying cells with the staining features of
apoptosis (data not shown). The present data support the effect of
the compounds in some of the series of steps of the apoptotic
process where a wide range of intermediate morphological and
biochemical types of cell death occur [38,42].
Finally, to determine if the observed growth inhibition was due
to apoptosis, both flow cytometry and confocal microscopy
studies were carried out. The cells were treated with the IC50
values of compounds and stained using Annexin V and propidium
iodide (PI) at 24 and 48 h post-drug treatment. Apoptosis assays
were accomplished in the MCF-7 human breast cancer cell line,
where the demonstration of programmed cell death by known
apoptosis-inducing agents has proven difficult and only few
cytotoxic agents act preferentially through an apoptotic mecha-
nism in human breast cancer cells [36,37]. Paclitaxel (TAXOLÒ)
induced programmed cell death of up to 43% of the cell pop-
ulation. Simultaneous staining with annexin V-FITC and the PI
non-vital dye made it possible to distinguish between early
apoptosis (stained positive for annexin V-FITC and negative for PI),
and late apoptosis or cell death (stained positive for both annexin
V-FITC and PI). In MCF-7 control-DMSO cultures neither early nor
late apoptosis were detected after 24 h or 48 h. Similarly,
compounds did not induce apoptosis after 24 h of treatment
(Supporting Information, Table 4). In contrast, MCF-7 cells treated
during 48 h with the novel compounds showed a significant
increase of early apoptotic cells in relation to the control culture
with percentages varying from 13.93% in cells treated with 24 to
43.30% and 41.99% after treatment with 23 and (R)-14, respec-
tively. The percentage of late apoptotic cells also increased in
MCF-7 cells treated with the drugs in comparison with control-
DMSO-treated cells (Table 5 Supporting Information). It should be
noted that levels of early apoptosis induced by (R)-14 were almost
double in comparison with the corresponding racemic compound
14, which may explain the enantioselective antiproliferative
activity shown by this enantiomer. These high apoptotic
percentages shown by (R)-14 are consistent with the G1 and G2/M
Fig. 2. Apoptosis induction in the MCF-7 human breast cancer cell line after treatment for 48 h with the compounds. (A) Representative panels of cytometry analysis for control
cells treated with 0.5% DMSO alone, 23 and (R)-14. In each panel, lower left quadrant (LL) shows viable cells which are negative for both annexin V-FITC and PI, lower right (LR)
shows annexin V positive cells which are in the early stage of apoptosis, upper left (UL) shows only PI positive cells which are dead, and upper right (UR) shows both annexin V and
PI positive, which are in the stage of late apoptosis. (B) Confocal microscopy analysis using simultaneous staining with annexin V-FITC (green) and PI (red) in MCF-7 after treatment
with 23. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

254
L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
NO2
R9
X
F
O
O
N
O
O
NH
NH
O
R7
N
O S O
N
10 ortho isomer; IC50: 45.17+ 0.48
12 para isomer; IC50: 39.78+2.60
M
M
O
O
1
2
3
4
R7 = R9 = H, X = F
N
O
O
R7 = OMe; R9 = H; X = F
R7 = H; R9 = OMe; X = F
R7 = R9 = H; X = H
NH
NH
O
5
NO2
OH
O
F
O
O
11 ortho isomer; IC50: 50.90+3.87
13 para isomer; IC50: 44.28+ 4.65
M
M
S
N
O
O
N
O
O
O
HN
N
O
O
O
N
O
N
NH
O
O
O
F
HN
F
Chart 3.
O
6
7
carried out to determine the mechanism of action at the molecular
level of the most active compounds.
Chart 1.
4. Experimental protocols
2.2.5. In vivo toxicity
Toxicity was determined selecting (RS)-14, which was the most
in vitro cytotoxic compound against MCF-7 cells. We examined the
acute-toxicity profile of (RS)-14 in BALB/c mice when it was
administered in a single i.p. bolus injection (n ¼ 25) at dose levels of
50, 75, 100, 150 and 200 mg/kg or via gavage (n ¼ 25) in a single p.o.
bolus at dose levels of 0.05, 0.5, 5 and 50 mg/kg. (RS)-14 was
nontoxic to BALB/c mice even at the highest i.p. bolus dose of
200 mg/kg and p.o. bolus dose of 50 mg/kg after 2 weeks. Control
mice (n ¼ 10; 5 mice for the i.p. group and 5 mice for the p.o. group)
were treated with vehicle alone. All 50 (RS)-14-treated mice
remained healthy and gained weight throughout the 15-day
observation period, with no evidence of morbidity.
4.1. Chemistry
Melting points were taken in open capillaries on an Electro-
thermal melting point apparatus and are uncorrected. Nuclear
magnetic resonance spectra were recorded on a 400 MHz 1H and
100 MHz 13C NMR Varian NMR-System-TM 400 or 300 MHz 1H and
75 MHz 13C NMR Varian Inova-TM spectrometers at ambient
temperature. Chemical shifts (d) are quoted in parts per million
(ppm) and are referenced 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; pt, pseudotriplet; m, multiplet. High-resolution liquid
secondary ion mass spectra (HR LSIMS) were carried out on a VG
AutoSpec Q high-resolution mass spectrometer (Fisons Instru-
ments). Small scale microwave-assisted synthesis was carried out
in an Initiator 2.0 single-mode microwave instrument producing
controlled irradiation at 2.450 GHz (Biotage AB, Uppsala). Reaction
time refers to hold time at 160 ꢂC or 140 ꢂC, not to total irradiation
time. The temperature was measured with an IR sensor on the
outside of the reaction vessel. Anhydrous acetonitrile was
purchased from VWR International Eurolab. 6-Chloropurine, 6-
bromopurine and 2,6-dichloropurine were purchased from Aldrich.
Analyses indicated by the symbols of the elements or functions
were within ꢁ0.4% of the theoretical values.
3. Conclusion
The anticarcinogenic potency of the target molecules is reported
against one non-tumour cell line and two cancerous ones. The most
active compound (14) presents an IC50 of 166 nM, being the most
potent structure so far reported. These results provide promising
information for further development of potent antiproliferative
agents. Compound 14 induces no acute toxicity in mice after 2
weeks of treatment. Compound 14 and its enantiomers associated
with the inhibition of cancer cell proliferation caused in MCF-7 and
MDA-MB-231 cells would make them very attractive agents,
opening a new strategy in cancer chemotherapy using similar
compounds endowed with potent antitumour activities with future
clinical application in breast cancer. At present, studies are being
4.1.1. General procedures for the preparation of the substituted
purine O,N-acetals
A suspension of 25 or 26 and the corresponding purine base in
anhydrous acetonitrile (5 mL/mmol) was prepared under argon
atmosphere and cooled to 0 ꢂC. At this temperature, TMSCl, HMDS
and a 1.0 M solution of SnCl4 in CH2Cl2 were added subsequently.
The temperature was allowed to rise to 10 ꢂC before microwave
irradiating at 140 ꢂC or 160 ꢂC for 5 min. The reactions were
quenched by cooling (ice/water bath) and by addition of distilled
water. The pH was fixed to 7e8 with a saturated NaHCO3 solution
and the aqueous phase extracted with CH2Cl2 and EtOAc. The
combined organic layers were dried (Na2SO4), filtered and evapo-
rated. The residues were purified by flash chromatography under
O
O
O
Me
Me
S
S
N
O
O
N
O
O
NH
O
NH
O
9
8
Chart 2.

L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
255
NO2
R
R2
R2
R2
O S O
N
O S O
N
H
N
N
N
N
N
N
N
R6
R6
R6
N
N
N
O
O
O
N
N
N
21 R2 = R6 = SPh
22 R2 = Cl, R6 = SPh
14 Isomer: p-NO2, R2 = R6 = Cl
15 Isomer: o-NO2, R2 = R6 = Cl
16 Isomer: p-NO2, R2 = H, R6 = Br
17 Isomer: o-NO2, R2 = H, R6 = Br
18 R = NO2, R2 = Cl, R6 = l
19 R = NH2, R2 = R6 = Cl
20 R = NHOH, R2 = R6 = Cl
NO2
NO2
Cl
O S O
Cl
Cl
N
O S O
N
N
N
N
OMe
N
N
Cl
N
O
N
OH
N
24
23
Chart 4.
gradient elution using mixtures of hexane/ethyl acetate (4/1 /1/1)
or CH2Cl2/MeOH (100/1 /100/5) to afford 14e17. Compounds 27
and 28 were also isolated in the reactions. Compound 23 was iso-
lated in the synthesis of 14. Traces of the N-7 regioisomer analogue
was detected in the synthesis of 15.
Three different conditions were investigated; method a): purine
(2.5 equiv), TMSCl (4.0 equiv), HMDS (4.0 equiv), SnCl4 (1 M solu-
tion in CH2Cl2, 4.0 equiv), 160 ꢂC, microwave, 5 min; method b):
purine (2.5 equiv), TMSCl (4.0 equiv), HMDS (4.0 equiv), SnCl4 (1 M
solution in CH2Cl2, 4.0 equiv), 140 ꢂC, microwave, 5 min; method c):
purine (1.5 equiv), TMSCl (1.5 equiv), HMDS (1.5 equiv), SnCl4 (1 M
solution in CH2Cl2, 1.5 equiv), 140 ꢂC, microwave, 5 min.
(C-5). HR LSIMS (m/z) calcd. for
355.0365, found 355.0365. Anal. C15H12N2O5S (C, H, N, S).
C
15H12N2O5NaS (M þ Na)þ
4.1.1.3. (RS)-2,6-Dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tet-
rahydro-4,1-benzoxazepin-3-yl]-9H-purine (14). Yellowish solid
[method b): 20%], mp: 173e175 ꢂC. 1H NMR (CDCl3, 300 MHz):
d
(ppm) 8.44 (d, J ¼ 8.8 Hz, 2H), 8.17e8.11 (m, 3H), 7.43e7.31 (m,
4H), 6.00 (d, J ¼ 8.8 Hz,1H), 4.78 (d, J ¼ 13.8 Hz,1H), 4.74 (dd, J ¼ 1.2,
14.7 Hz, 1H), 4.60 (d, J ¼ 13.8 Hz, 1H), 3.65 (dd, J ¼ 10.0, 14.7 Hz, 1H).
13C NMR (CDCl3, 75 MHz):
d (ppm) 153.72, 152.59, 152.16, 150.79,
146.29, 143.41, 138.78, 136.64, 131.01, 130.52, 130.40, 129.54, 129.10
(ꢃ2), 129.05, 125.18 (ꢃ2), 84.78, 72.05, 54.56. HR LSIMS (m/z) calcd.
for C20H14N6O5NaSCl2 (M þ Na)þ 543.0021, found 543.0024. Anal.
C20H14Cl2N6O5S (C, H, N, S).
4.1.1.1. 1-[(p-Nitrophenyl)sulfonyl]-1,5-dihydro-4,1-benzoxazepin
(27). Yellow solid, [method b): 20%], mp: 178e180 ꢂC. 1H NMR
(CDCl3, 300 MHz):
d
(ppm) 8.33 (d, J ¼ 8.8 Hz, 2H, H-3,5pnitro), 7.80
4.1.1.4. (RS)-9-{2-[N-(2-Hydroxymethylphenyl)-p-nitro-
(d, J ¼ 9.1 Hz, 2H, H-2,6pnitro), 7.60 (dd, J ¼ 0.9, 7.9 Hz, 1H, H-9), 7.44
(dt, J ¼ 1.5, 7.7 ꢃ 2 Hz, 1H, H-8), 7.31 (dt, J ¼ 1.5, 7.5 ꢃ 2 Hz, 1H, H-
7), 7.14 (dd, J ¼ 1.6, 7.5 Hz, 1H, H-6), 5.94 (d, J ¼ 5.9 Hz, 1H, H-3),
5.78 (d, J ¼ 5.9 Hz, 1H, H-2), 4.01 (s, 2H, H-5). 13C NMR (CDCl3,
benzenesulfonamide]-1-metoxyethyl}-2,6-dichloro-9H-purine
(23). White solid [method b): 12%]: mp: 172e174 ꢂC. [conformer A
(58%), conformer B (42%)] 1H NMR (CDCl3, 300 MHz):
d (ppm)
8.36e8.28 (m, 5H), 8.12 (s, 1H), 7.80 (d, J ¼ 9.2 Hz, 2H), 7.72 (d,
J ¼ 8.8 Hz, 2H), 7.63 (d, J ¼ 7.7 Hz, 1H), 7.61 (d, J ¼ 7.7 Hz, 1H),
7.44e7.39 (m, 2H), 7.30e7.25 (m, 2H), 7.18e7.08 (m, 2H), 6.42 (dd,
J ¼ 0.9, 7.9 Hz, 1H), 6.38 (d, J ¼ 0.9, 7.9 Hz, 1H), 5.86 (pt, J ¼ 6.1 Hz,
1H), 5.69 (dd, J ¼ 4.8, 7.5 Hz, 1H), 4.58e4.53 (m, 3H), 4.44e4.36 (m,
2H), 4.27 (d, J ¼ 12.7 Hz, 1H), 4.19 (dd, J ¼ 7.5, 14.5 Hz, 1H), 3.96 (dd,
75 MHz):
d (ppm) 150.49 (C-4pnitro), 142.93 (C-1pnitro), 141.37 (C-
9a), 138.91 (C-3), 132.26 (C-5a), 130.16, 130.08, 130.00, 128.99 (C-6,
7, 8, 9), 128.90 (C-2pnitro and C-6pnitro), 124.55 (C-3pnitro and C-
5
pnitro), 104.27 (C-2), 70.24 (C-5). HR LSIMS (m/z) calcd. for
C15H12N2O5NaS (M þ Na)þ 355.0365, found 355.0364. Anal.
C15H12N2O5S (C, H, N, S).
J ¼ 5.7, 14.5 Hz, 1H), 3.30 (s, 6H). 13C NMR (CDCl3, 75 MHz):
d (ppm)
152.9, 150.1, 143.5, 143.3, 141.8, 141.7, 141.1, 136.5, 135.2, 131.3, 130.8,
129.7, 129.4, 129.1, 128.8, 128.6, 128.2, 126.6, 126.4, 123.9, 85.5, 85.2,
60.2, 60.0, 57.0, 56.7, 54.7, 53.6. HR LSIMS (m/z) calcd. for
C21H18N6O6NaSCl2 (M þ Na)þ 575.0283, found 575.0287. Anal.
C21H18Cl2N6O6S (C, H, N, S).
4.1.1.2. 1-[(o-Nitrophenyl)sulfonyl]-1,5-dihydro-4,1-benzoxazepin
(28). Brown sirup [method a): 36%; method b): 39%; method c):
29%], 1H NMR (CDCl3, 300 MHz):
d
(ppm) 7.87 (dd, J ¼ 1.8, 7.9 Hz,
1H, H-3onitro), 7.75e7.63 (m, 2H, H-4onitro and H-5onitro), 7.59 (dd,
J ¼ 1.3, 7.9 Hz, 1H, H-6onitro), 7.47 (dd, J ¼ 1.3, 7.9 Hz, 1H, H-6), 7.40
(ddd, J ¼ 1.8, 7.70 ꢃ 2 Hz, 1H, H-7), 7.34 (ddd, J ¼ 1.5, 7.4 ꢃ 2 Hz, 1H,
H-8), 7.23 (dd, J ¼ 1.5, 7.3 Hz, 1H, H-9), 5.96 (d, J ¼ 5.7 Hz, 1H, H-3),
5.77 (d, J ¼ 5.7 Hz, 1H, H-2), 4.49 (s, 2H, H-5). 13C NMR (CDCl3,
4.1.1.5. (RS)-2,6-Dichloro-9-[1-(o-nitrobenzenesulfonyl)-1,2,3,5-tetra-
hydro-4,1-benzoxazepin-3-yl]-9H-purine (15). White solid [method
a): 32%, b): 37%, c): 47%], mp: 154e155 ꢂC. 1H NMR (CDCl3,
75 MHz):
d
(ppm) 148.15, 141.88, 138.77, 134.12, 132.40, 131.57,
300 MHz):
d
(ppm) 8.18 (s, 1H), 8.07 (dd, J ¼ 1.8, 7.9 Hz, 1H), 7.80 (m,
131.30, 130.74, 129.65 (ꢃ2), 128.97, 128.58, 123.83, 104.05, 70.06
3H), 7.40 (m, 2H), 7.31 (m, 1H), 7.08 (d, J ¼ 7.5 Hz, 1H), 6.09 (dd,

256
L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
J ¼ 1.8, 10.1 Hz, 1H), 5.05 (d, J ¼ 13.6 Hz, 1H), 4.82 (d, J ¼ 13.6 Hz, 1H),
4.72 (dd, J ¼ 1.8, 13.2 Hz, 1H), 3.84 (dd, J ¼ 10.1, 13.2 Hz, 1H). 13C NMR
C
20H14N6O5NaSClI (M þ Na)þ 634.9377, found 634.9381. Anal.
C20H14ClIN6O5S (C, H, N, S).
(CDCl3, 75 MHz):
d (ppm) 153.68, 152.48, 152.38, 148.03, 143.70,
138.89, 137.75, 134.95, 133.71, 132.60, 132.55, 131.03, 130.56, 130.04,
129.58, 128.82, 125.02, 85.22, 72.12, 54.80. HR LSIMS (m/z) calcd. for
C20H14N6O5NaSCl2 (M þ Na)þ 543.0021, found 543.0020. Anal.
C20H14Cl2N6O5S (C, H, N, S).
4.1.3. Reduction of 15: formation of amino 19 and hydroxylamino
20 derivatives
SnCl2$2H2O (5.0 equiv) was added at room temperature to
a suspension of 15 in EtOH (2.5 mL/mmol) and heated at the reflux
temperature for 2 h. The reaction mixture was then cooled at 0 ꢂC
and the pH was fixed to 7e8 with saturated NaHCO3 solution. The
aqueous layer was extracted with CH2Cl2 and the combined organic
layers were washed with brine, dried (Na2SO4) and evaporated.
Purification of 19 and 20 was carried out by flash chromatography
using CH2Cl2/acetone (9.9/0.1) as eluent.
4.1.1.6. (RS)-2,6-Dichloro-7-[1-(o-nitrobenzenesulfonyl)-1,2,3,5-tetra-
hydro-4,1-benzoxazepin-3-yl]-7H-purine (24). White solid, [method
a): 2%; method b): 1%; method c): 2%], mp: 172e174 ꢂC. 1H NMR
(acetone-d6, 400 MHz): d (ppm) 8.93 (s, 1H), 8.10e8.07 (m, 3H), 7.94
(m, 1H), 7.57 (d, J ¼ 7.5 Hz, 1H), 7.42e7.44 (m, 2H), 7.21 (d, J ¼ 7.8 Hz,
1H), 6.56(dd,J ¼ 2.0,10.2 Hz,1H), 5.01e4.98(m, 3H), 4.16(dd,J ¼ 10.0,
13.7 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz):
d
(ppm) 163.88, 160.85,
4.1.3.1. (RS)-9-[1-(o-Aminobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-
152.18, 144.00, 139.25, 137.93, 136.29, 133.91, 132.67, 131.00, 130.89,
130.32, 129.96, 129.54, 128.63, 125.86, 121.01, 85.38, 70.40, 53.85. HR
LSIMS (m/z) calcd. for C20H14Cl2N6NaO5S (M þ Na)þ 543.0021, found
543.0018. Anal. C20H14Cl2N6O5S (C, H, N, S).
benzoxazepin-3-yl]-2,6-dichloro- 9H-purine (19). White solid (35%);
mp: 225e227 ꢂC. 1H NMR (CDCl3, 400 MHz):
d (ppm) 8.14 (s, 1H),
7.83e7.81 (m, 2H), 7.63 (dd, J ¼ 1.6, 8.2 Hz, 1H), 7.55 (dd, J ¼ 1.2,
7.8 Hz, 1H), 7.44e7.25 (m, 4H), 6.02 (dd, J ¼ 2.0, 10.2 Hz, 1H),
4.70e4.67 (m, 3H), 3.53 (dd, J ¼ 10.2, 14.9 Hz, 1H). 13C NMR (CDCl3,
4.1.1.7. (RS)-6-Bromo-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahy-
75 MHz): d (ppm) 153.52, 152.34, 152.15, 145.42, 143.81, 143.49,
dro-4,1-benzoxazepin-3-yl]-9H-purine (16). Viscous oil [method d):
139.57, 136.83, 135.45, 130.70, 130.07, 129.99, 129.96, 128.76, 121.59,
118.31, 118.25, 84.37, 71.90, 54.23. HR LSIMS (m/z) calcd. for
C20H16N6O3NaSCl2 (M þ Na)þ 513.0279, found 513.0284. Anal.
C20H16Cl2N6O3S (C, H, N, S).
4%]. 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.79 (s, 1H), 8.41 (d,
J ¼ 8.8 Hz, 2H), 8.21 (s, 1H), 8.07 (d, J ¼ 8.8 Hz, 2H), 7.42e7.35 (m,
3H), 7.29e7.27 (m, 1H), 6.14(dd, J ¼ 1.8, 10.0 Hz, 1H), 4.80e4.76 (m,
2H), 4.60 (d, J ¼ 13.8 Hz, 1H), 3.75 (dd, J ¼ 10.0, 14.7 Hz, 1H). 13C
NMR (CDCl3, 75 MHz):
d
(ppm) 152.74, 151.92, 150.73, 146.47,
4.1.3.2. (RS)-2,6-Dichloro-9-[1-(o-hydroxylaminobenzene-sulfonyl)-
142.94, 142.80, 138.76, 137.05, 131.91, 130.53, 130.36, 129.58, 128.93
(ꢃ2), 128.72, 125.06 (ꢃ2), 84.98, 71.84, 54.76. HR LSIMS (m/z) calcd.
for C20H15N6O5NaSBr (M þ Na)þ 552.9906, found 552.9906. Anal.
C20H15BrN6O5S (C, H, N, S).
1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine (20). Viscous
oil (35%). 1H NMR (CDCl3, 400 MHz):
d (ppm) 8.13 (s, 1H), 7.42 (ddd,
J ¼ 1.2, 8.2, 16.6 Hz, 1H), 7.60 (ddd, J ¼ 1.6, 7.4 ꢃ 2 Hz, 1H), 7.47 (ddd,
J ¼ 1.6, 7.8 Hz, 1H), 7.36 (ddd, J ¼ 1.2, 7.4 Hz, 1H), 7.29 (dd, J ¼ 1.4,
8.0 Hz, 1H), 7.26 (m, 1H), 7.20 (dd, J ¼ 1.6, 7.4 Hz, 1H), 6.84 (ddd,
J ¼ 1.2, 7.8 Hz, 1H), 6.66 (dd, J ¼ 2.1, 10.0 Hz, 1H), 4.77 (dd, J ¼ 2.0,
14.9 Hz, 1H), 4.38 (d, J ¼ 13.7 Hz, 1H), 3.67 (d, J ¼ 13.3 Hz, 1H), 3.53
4.1.1.8. (RS)-6-Bromo-9-[1-(o-nitrobenzenesulfonyl)-1,2,3,5-tetrahy-
dro-4,1-benzoxazepin-3-yl]-9H-purine (17). Viscous oil, [method d):
4%], 1H NMR (CD3OD, 400 MHz):
d
(ppm) 8.71 (s, 1H), 8.67 (s, 1H),
(dd, J ¼ 10.3,15.1 Hz,1H). 13C NMR (CDCl3, 75 MHz):
d (ppm) 153.64,
8.02 (d, J ¼ 8.0 Hz, 1H), 7.91 (m, 2H), 7.81 (m, 1H), 7.47 (d, J ¼ 8.0 Hz,
1H), 7.40 (t, 1H), 7.33 (m, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 6.25 (dd,
J ¼ 2.0, 10.2 Hz, 1H), 5.02 (d, J ¼ 13.3 Hz, 1H), 4.84 (d, J ¼ 13.3 Hz,
1H), 4.72 (dd, J ¼ 2.0, 14.9 Hz, 1H), 4.12 (dd, J ¼ 10.2, 14.9 Hz, 1H). 13C
153.24, 151.86, 147.03, 143.82, 138.29, 136.97, 135.41, 131.70, 131.12,
130.15, 129.75, 129.71, 129.20, 123.84, 120.71, 115.20, 83.23, 71.35,
54.70. HR LSIMS (m/z) calcd. for C20H17Cl2N6O4S (M þ H)þ
507.0404, found 507.0441. Anal. C20H16Cl2N6O4S (C, H, N, S).
NMR (CD3OD, 75 MHz):
d (ppm) 153.33, 151.27, 149.32, 146.06,
143.62, 140.32, 139.60, 136.30, 135.08, 134.26, 133.64, 132.67, 131.25,
130.69, 130.27, 129.60, 126.72, 86.41, 72.41, 55.14. HR LSIMS (m/z)
calcd. for C20H15N6O5NaSBr (M þ Na)þ 552.9906, found 552.9899.
Anal. C20H15BrN6O5S (C, H, N, S).
4.1.4. Substitutions on 15: formation of 2,6-dithiophenyl 21 and 6-
thiophenyl 22 derivatives
4.1.4.1. (RS)-2,6-Dithiophenyl-9-[1,2,3,5-tetrahydro-4,1-benzox-
azepin-3-yl]-9H-purine (21). K2CO3 (3.0 equiv) and PhSH (1.1 equiv)
were added at room temperature to a solution of 15 (1.0 equiv) in
DMF (5 mL/mmol). The mixture was stirred at room temperature
for 4 h. The solvent was evaporated and water was added to the
residue. The aqueous layer was extracted with CH2Cl2 and the
combined organic layers were washed with brine, dried (Na2SO4)
and evaporated. Purification of 21 was carried out by flash chro-
matography using EtOAc/hexane 1/4 as eluent. White solid, (42%),
4.1.2. Substitutions on 15: formation of 6-iodo derivative 18
To a solution of 15 (1.0 equiv) in butanone (20 mL/mmol), NaI
(20.0 equiv) and TFA (5.0 equiv) were added subsequently at ꢀ15 ꢂC
and the reaction mixture was stirred at this temperature for 6 h. The
solvent was evaporated and water was added to the residue. The
aqueous layer was extracted (CH2Cl2) and the combined organic
layers were washed (NaHSO4 and brine), dried (Na2SO4) and evap-
orated. Purification of 18 was carried out by flash chromatography
using EtOAc/hexane 1/2 as eluent.
mp: 82e86 ꢂC. 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.17 (s, 1H), 7.6
(ddd, J ¼ 0.9, 7.5 ꢃ 2 Hz, 1H), 7.53e718 (m, 13H), 6.92 (d, J ¼ 7.9 Hz,
1H), 6.03 (dd, J ¼ 2.4, 7.7 Hz, 1H), 4.95 (d, J ¼ 14.1 Hz, 1H), 4.80 (d,
J ¼ 14.1 Hz, 1H), 3.73 (dd, J ¼ 2.6, 13.6 Hz, 1H), 3.59 (dd, J ¼ 7.5,
4.1.2.1. (RS)-2-Chloro-6-iodo-9-[1-(o-nitrobenzenesulfonyl)-1,2,3,5-
13.6 Hz, 1H). 13C NMR (CDCl3, 75 MHz):
d (ppm) 177.21, 165.50,
tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine (18). Yellow solid
161.25, 149.10, 141.13, 135.39 (ꢃ2), 135.26 (ꢃ2), 130.25, 129.72 (ꢃ2),
129.42, 129.39, 129.26 (ꢃ2), 129.06 (ꢃ2), 129.00, 128.55, 127.02,
122.21, 119.29, 85.22, 70.48, 51.79, one carbon missing. HR LSIMS
(m/z) calcd. for C26H21N5ONaS2 (M þ Na)þ 506.1085, found
506.1084. Anal. C26H21N5OS2 (C, H, N, S).
(66%), mp: 129e132 ꢂC 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.18 (s,
1H), 8.08 (d, J ¼ 8.0 Hz, 1H), 7.81e7.79 (m, 3H), 7.42e7.40 (m, 2H),
7.31e7.32 (m, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 6.06 (dd, J ¼ 8.0, 16.0 Hz,
1H), 5.04 (dd, J ¼ 8.0, 12.0 Hz, 1H), 4.82 (d, J ¼ 12.0 Hz, 1H), 4.73 (dd,
J ¼ 4.0, 16.0 Hz, 1H), 3.82e3.80 (m, 1H). 13C NMR (CDCl3, 75 MHz):
d
(ppm) 153.10, 152.46, 152.34, 148.81, 147.86, 143.64, 138.85,
4.1.4.2. (RS)-2-Chloro-6-phenylthio-9-[1,2,3,5-tetrahydro-4,1-ben-
zoxazepin-3-yl]-9H-purine (22). K2CO3 (1.6 equiv) and PhSH
(0.8 equiv) were added at room temperature to a solution of 15
137.72, 137.68, 134.90, 133.68, 132.57, 132.52, 130.53, 130.00,
128.82, 124.99, 85.18, 72.12, 54.76. HR LSIMS (m/z) calcd. for

L.C. López-Cara et al. / European Journal of Medicinal Chemistry 46 (2011) 249e258
257
(1.0 equiv) in DMF (5 mL/mmol). The mixture was stirred at room
4.2.5. Apoptosis detection by staining with annexin V-FITC and
propidium iodide
The annexin V-FITC apoptosis detection kit I (Pharmingen, San
Diego, CA, USA) was used to detect apoptosis by flow cytometry
according to Marchal et al. [38]. All experiments were performed in
triplicate and yielded similar results.
temperature for 4 h. The solvent was evaporated and water was
added to the residue. The aqueous layer was extracted with CH2Cl2
and the combined organic layers were washed with brine, dried
(Na2SO4) and evaporated. Purification of 22 was carried out by flash
chromatography using EtOAc/hexane 1/2 as eluent. White solid,
(40%), mp: 161e163 ꢂC. 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.27
(s, 1H), 7.95 (ddd, J ¼ 0.4, 7.5 Hz, 1H), 7.64 (m, 2H), 7.46 (m, 3H) 7.19
(m, 2H), 6.87 (d, J ¼ 7.9 Hz, 1H), 6.11 (dd, J ¼ 2.2, 7.4 Hz, 1H), 4.88 (s,
2H), 3.83 (dd, J ¼ 2.2, 13.6 Hz, 1H), 3.40 (dd, J ¼ 7.4, 13.6 Hz, 1H). 13C
4.2.6. Apoptosis detection by confocal microscopy
Cells (5 ꢃ103 cells/well) were seeded onto Labtek chamber-slide
8-well plates. Cells were allowed to adhere for 24 h before dosing
with required concentrations of compounds. Culture medium was
removed and cells were washed with cold 1X PBS. After that, cells
were incubated with both Annexin V-FITC and propidium iodide for
15 min at room temperature in the dark. Cells were then washed
with binding buffer, prepared with mounting medium and cover
slips before confocal microscopic imaging [42]. Cells were imaged
by confocal microscopy using a Leica SP2 Confocal Microscope.
NMR (CDCl3, 75 MHz):
d (ppm) 163.13, 154.06, 149.70, 147.99,
142.22, 135.59 (ꢃ2), 129.97, 129.75, 129.73, 129.56, 129.50 (ꢃ2),
128.46, 126.71, 122.03, 119.19, 85.02, 71.02, 52.85. HR LSIMS (m/z)
calcd. for C20H16N5ONaSCl (M þ Na)þ 432.0662, found 432.0660.
Anal. C20H16ClN5O5S (C, H, N, S).
4.1.4.3. Resolution of (RS)-14. (RS)-9-[1-(p-Nitrobenzenesulfonyl)-
1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-2,6-dichloro-9H-purine
(14) is resolved into its enantiomers: the one that elutes first is (R)-
14 (retention time ¼ 8.5 min), and the one which comes second is
(S)-14 (retention time ¼ 19.8 min), using a column CHIRALPAKÒ IA
(DAICEL CHEMICAL INDUSTRIES, LTD.) semipreparative, and as
eluent a mixture of hexane/t-BuOMe/iPrOH (26/65/9).
4.2.7. Toxicity assays in vivo
Acute toxicity was determined in six-week old BALB/c female
mice (average, 20 g weight) during 2 weeks. (RS)-14 dissolved in
0.25 mL of a mixture of 0.9% sodium chloride and DMSO was
administered in BALB/c in a single i.p. bolus injection (n ¼ 25) at
dose levels of 50, 75, 100, 150 and 200 mg/kg or via gavage (n ¼ 25)
in a single p.o. bolus at dose levels of 0.05, 0.5, 5 and 50 mg/kg.
Control mice were inoculated with the same volume of sodium
chloride (control group) and DMSO alone (DMSO group). Mice were
maintained under standard conditions and for each treatment
schedule, were weighed and assessed twice weekly for systemic
toxicity (listlessness, weight loss) and local toxicity (alopecia, skin
reaction, and leg motility).
4.2. Biology
4.2.1. Cell culture
MCF-7 and MDA-MB-231 cells were grown at 37 ꢂC in an
atmosphere containing 5% CO2, with Dubelcco’s modified Eagle
Medium (DMEM) (Gibco, Grand Island, NY) supplemented 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 ampicillin.
Acknowledgments
This study was supported by the European Commission (A.C-G.
Marie Curie Programme MERG-CT-2005-030616) and the Instituto
de Salud Carlos III (Fondo de Investigación Sanitaria) through
projects no. PI070227 and PI070527. The project “Factoría de Cris-
4.2.2. Drugs and drug treatments
The drugs were dissolved in DMSO or water and stored at ꢀ20 ꢂC.
For each experiment, the stock solutions were further diluted in
medium to obtain the desired concentrations. The final solvent
concentration in cell culture was ꢄ0.1% v/v of DMSO, a concentration
without effect on cell replication. Parallel cultures of cells in medium
with DMSO were used as controls.
talización,
CONSOLIDER
INGENIO-2010”
(CSD2006-00015)
provided X-ray structural facilities for this work. We gratefully
acknowledge the BM16 Spanish beamline at ESRF (Grenoble,
France) for access to synchrotron radiation and for helpful assis-
tance during data collection. D Ch-L thanks CSD2006-00015 for his
research contract.
4.2.3. Cytotoxicity assays in vitro
The effect of anticancer drugs on cell viability was assessed
using the sulforhodamine-B colorimetric assay [38]. Aliquots of
MCF-7 cells suspension (1 ꢃ103 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. Three days
later, the wells were aspirated, fresh medium and treatment were
added, and cells were maintained for 3 additional days. Thereafter,
cells were processed as described previously [38], using a Titertek
Multiscan apparatus (Flow, Irvine, California) at 492 nm. We eval-
uated linearity of the SRB assay with cell number for each cell line
before each cell growth experiment. The IC50 values were calcu-
lated from semilogarithmic doseeresponse curves by linear inter-
polation. All of the experiments were plated in triplicate wells and
were carried out at least twice.
Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2010.11.011.
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