Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)- 9H- or 7H-purines against several human solid tumour cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.02.012

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use.

Full text of DOI:10.1016/j.ejmech.2014.02.012

European Journal of Medicinal Chemistry 76 (2014) 118e124
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-proliferative activity of 2,6-dichloro-9- or
7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human
solid tumour cell lines
,
,c,
Fátima Morales a 1, Alberto Ramírez b 1, Ana Conejo-García a, Cynthia Morata b,
Juan A. Marchal b, Joaquín M. Campos a
,
*
a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, 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 Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales y de la Salud, Paraje de las Lagunillas s/n, 23071 Jaén, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or
purine moieties with notable anti-proliferative activity against human breast, colon and melanoma
cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the
other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives
were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs
are obtained between the anti-proliferative IC50 values for compounds 26e33 and the clog P against the
melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-
9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the
tumour cell lines assayed, showing single-digit micromolar IC50 values. This exemplifies the potential of
our previously reported purine compounds to qualify as lead structures for medicinal chemistry cam-
paigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective
activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for
a future therapeutic use.
Received 1 August 2013
Received in revised form
19 January 2014
Accepted 8 February 2014
Available online 10 February 2014
Keywords:
Anti-tumour compounds
Apoptosis
5-Fluorouracil
Breast cancer
Colon cancer
Melanoma
Purines
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Metastatic melanoma is a disease with limited treatment op-
tions and a poor prognosis. Malignant melanoma is the most
Breast cancer is the first cause of death by neoplasia among
women of industrialized countries and represents nearly 25% of
non-accidental deaths of women between 35 and 54 years of age
[1]. The frequency of breast cancer increases with age up to the
menopause and subsequently continues to rise but more slowly [2].
Conventional cancer chemotherapy blocks cell division but lacks
selectivity for oncogenic cells which can result in serious cytotoxic
side effects for the patient. The key to specificity in cancer
chemotherapy may be found in the pharmacological targeting of
specific molecules avoiding cytotoxicity against normal cells [3].
aggressive form of skin cancer with increasing incidence over the
past years [4]. Metastatic melanoma has a short median survival
and is responsible for most skin cancer deaths [5]. Often mela-
nomas are characterized by resistance to cytotoxic agents because
of the inactivation of apoptotic pathways [6].
Colorectal cancer is a common disease that results in significant
worldwide morbidity and mortality. It is the second leading cause
of global cancer mortality, and accounts for over 600,000 deaths
annually [7].
Having previously reported the synthesis and anticancer activ-
ities of cyclic 5-fluorouracil (5-FU) O,N-acetalic compounds (1), we
changed 5-FU for uracil (2), with the aim of finding an anti-
proliferative agent endowed with a new mechanism of action [8].
Following our ongoing Anticancer Drug Programme we planned
the synthesis of compounds bearing a pyrimidine base, and also the
oxygen atom at position 1 of the seven-membered cycle was
replaced by its isosteric sulfur atom (3, 4) and its oxidized states.
Abbreviations: DCA, Dichloroacetate; FACS, Flow cytometry; 5-FU, 5-
Fluorouracil; MW, Microwave; SRB, Sulforhodamine-B.
* Corresponding author.
E-mail address: jmcampos@ugr.es (J.M. Campos).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.02.012
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

F. Morales et al. / European Journal of Medicinal Chemistry 76 (2014) 118e124
119
Later on, the pyrimidine base was substituted for the purine one
with the objective of increasing both the lipophilicity and the
structural diversity of the target molecules (5e19). Structures were
designed in which both structural entities (such as the benzohe-
terocyclic ring and the purine base) were linked through a meth-
and MDA-MB-231 cancer cell lines (Therapeutic Indexes ¼ 5.1
and 11.0, respectively) in relation to the normal MCF-10A. (RS)-
16 was resolved into its enantiomers. Both homochiral forms are
equally potent, but more so than the corresponding racemic
mixture [10].
ylene linker (20e25).
A
series of (RS)-9-(2,3-dihydro-1,4-
The small molecule sodium dichloroacetate (DCA) inhibits the
growth of C6 glioma tumours in both C6 brain tumour-bearing rats
and C6 tumour-bearing nude mice [11]. Sánchez et al. have re-
ported that the combination of DCA with bortezomib significantly
extends the survival of myeloma-bearing mice over bortezomib
alone and provides preclinical support for the use of this drug
combination against myeloma [12].
benzoxathiin-3-ylmethyl)-9H-purine derivatives (20e22) were
obtained and the anticancer activity for the most active compounds
was correlated with their capability to induce apoptosis. In order to
complete 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
(23e25) was prepared [9] (Chart 1).
R5
R6
X
X
7
X
1
N
9
N
N
R2
N
N
O
O
O
NH
O
N
N
N
N
R2
O
R6
1
2
3
4
X = O; R5 = F
X = O; R5 = H
X = S; R5 = F
X = S; R5 = H
5
10 X = O; R2 = H; R6 = Cl
11
12 X = O; R2 = R6 = Cl
X = O; R2 = H; R6 = Cl
6 X = O; R2 = H; R6 = l
7 X = O; R2 = R6 = Cl
8
X = O; R2 = H; R6 = Cl
13 X = S; R2 = H; R6 = Cl
X = S; R2 = H; R6 = Cl
14
9 X = SO2; R2 = H; R6 = Cl
X = SO2; R2 = H; R6 = Cl
R1
N
R1
N
7
N
9
N
N
N
O
R6
O
N
N
R6
N
N
R2
1 = SO2-C6H4-pNO2; R2 = H; R6 = Cl
18
15
16
R
1 = SO2-C6H4-pNO2, R6 = Cl
R
R1 = SO2-C6H4-pNO2; R2 = R6 = Cl
19 R1 = Fmoc, R6 = Cl
17 R1 = Fmoc, R6 = Cl
9
N
O
S
R2
N
N
R6
N
O
N
R6
N
N
N
9
R2
S
20
R2 = H, R6 = Cl
23 R2 = H, R6 = Cl
24 R2 = H; R6 = Br
21 R2 = H; R6 = Br
22 R2 = Cl; R6 = Cl
25
R2 = R6 = Cl
Chart 1. Benzo-fused seven-membered linked to pyrimidines (1e4), to purines (5e19), and benzo-fused six-membered rings linked to purines (20e25).
Compound 16 presents an IC50 of 0.166
man cancerous cell line MDA-MB-231. Compound 16 was the
most selective against the human breast adenocarcinoma MCF-7
m
M against the hu-
We took the benzo-fused seven- and six-membered scaffolds
linked to the pyrimidine or purine moieties 1e25 (Chart 1) as
leader compounds, and decided to maintain the double-ringed
H
O
H
H
R2
6
4
1
3
3
EtO
F
EtO
F
9H
H
6
7
NH
4
NH
N
R2
5
5
5
N
2
2
N
N 1
2
O
O
6
4
O
N
O
H
N
O
N1
3
8
5
8
2
H
H
4
H
H
1
H
N
7
H
N
N
R1
6
EtO
EtO
3
9
R1
31
28 R1= H R2= Cl
29 R1= H R2= Br
30 R1=R2= Cl
R1= H R2= Cl
O
O
32 R1= H R2= Br
33 R1=R2= Cl
27
26
Fig. 1. The HMBC interactions that discriminate N-9 (28e30) and N-7 (31e33) purine
Fig. 2. The HMBC interactions that discriminate N-1 (26) and N-3 (27) 5-FU
regioisomers.
derivatives.

120
F. Morales et al. / European Journal of Medicinal Chemistry 76 (2014) 118e124
nitrogenous bases and, as a first approach, connect the other
components to the ethyl acetate (drastic molecular pruning) rest,
which is related to DCA. Such a very simple reasoning has guided us
in spite of a remote resemblance to the model structures. Should
compounds 26e33 (Chart 2) show notorious anti-proliferative ac-
tivity, the simplicity of their preparation and the use of ordinary
chemistry militate in their favour, although is not always accepted
with enthusiasm by organic chemists. In this communication we
report the synthesis and anti-proliferative activity against the hu-
man breast cancer cell line MCF-7, the human colon carcinoma cell
line HCT-116, and two human melanoma cell lines such as A-375
and G-361 of two 5-FU derivatives (26, 27), and six purine scaffolds
(28e33) (Chart 2).
Chinese journal, in which its melting point is not provided
[15].
2.2. Spectroscopic analysis
Compounds 26e33 were identified by NMR-spectroscopy and
high resolution mass spectroscopy. HSQC and HMBC studies were
employed for the unequivocally identification of each atom of H
and C. The discrimination between the N-9 and N-7 substituted
purine derivatives (28e33) relies on the observation of the 1,3-
relationship between the hydrogen atoms of the carbon linked to
the purine and the quaternary carbons of the purine moiety: C4pur
and C5pur in the HMBC spectrum (Figs. S5eS16). While these
O
F
F
NH
NH
R6
EtO
EtO
7
9
N
N
R2
O
N
O
N
O
N
N
O
O
1
3
N
EtO
EtO
N
N
N
R2
R6
O
O
27
26
28 R2 = H; R6 = Cl
29 R2 = H; R6 = Br
31 R2 = H; R6 = Cl
32
33 R2 = R6 = Cl
R
2 = H; R6 = Br
30
R
2 = R6 = Cl
Chart 2. Target molecules under study in this paper: N-1 (26) and N-3 (27) 5-FU derivatives, N-9 (28e30) and N-7 (31e33) purine compounds.
2. Results and discussion
hydrogen atoms are correlated with C4pur in the N-9 regioisomers,
they are correlated with C5pur in the N-7 regioisomers (Fig. 1).
In the 5-FU derivatives, the identification of the N-1 isomer (26)
relies on the observation of the 1,3-connection between the
hydrogen atoms of the carbon linked to the 5-FU and the sole ter-
tiary carbon of the 5-FU moiety (C65FU) and the opposite connec-
tion: H65FU and the carbon linked to the 5-FU (Fig. 2) in the HMBC
spectrum (Figs. S1eS4). These correlations do not exist in its isomer
N-3 (27). The correlation in 27 appears between the hydrogen
2.1. Chemistry
Compounds 26e33 were obtained as shown in Scheme 1.
Nucleophilic substitution assisted by microwave (MW) irradi-
ation of ethyl chloroacetate using water as solvent and Et3N as
a base afforded compounds 26e33. This rapid, convenient and
green protocol was previously described for the synthesis of 28
and 30 [13]. Whereas the authors reported the isolation of the
N-9 regioisomers (28 and 30), we obtained the N-9 and the N-
7 isomers in each reaction (28 and 31, 30 and 33, respectively
using the same conditions. We also conducted the synthesis of
the bromopurine and 5-FU derivatives obtaining the N-9 and
N-7 bromopurine isomers (29 and 32) and the N-1 and N-3 5-
FU regioisomers (26 and 27). Compound 26 was previously
reported [14], although in a non-easily accessible journal. The
synthesis of its N-3 5-FU isomer (27) is described in another
atoms of the carbon linked to the 5-FU and C45FU
.
2.3. Biological activities
Table 1 shows the anti-proliferative activities against the MCF-7,
HCT-116, A-375 and G-361 cancerous cell lines for the target
compounds. As a rule of thumb, the following can be stated: (a) The
2,6-dichloropurine derivatives (30, 33) are the most active com-
pounds showing single-digit micromolar IC50 values against all the
Scheme 1. Reagents and conditions: a) Et3N, H2O, 105 ꢀC, MW, 8 min (13% for 26, 6% for 27, 24% for 28, 28% for 29, 40% for 30, 12% for 31, 9% for 32, 15% for 33).

F. Morales et al. / European Journal of Medicinal Chemistry 76 (2014) 118e124
121
Table 1
Eq. 2 shows the anti-proliferative activity of the targeted com-
pounds against the G-361 cell line:
Anti-proliferative activitiesa for compounds 26e33 against the cancerous cell lines
MCF-7, HCT-116, A-375 and G-361.
Comp IC50
(
m
M)
IC50
HCT-116
(
m
M)
IC50
A-375
(
m
M)
IC50
G-361
(
m
M)
clogPb
pðIC50ÞGꢁ361 ¼ 4:55ðꢂ0:08Þ þ 0:55ðꢂ0:10Þc log P
MCF-7
n ¼ 8; r ¼ 0:920; s ¼ 0:159; F1;5 ¼ 27:12; p < 0:005
26
27
28
31
29
32
30
33
25.2 ꢂ 0.03 24.6 ꢂ 0.01 38.4 ꢂ 0.06 29.0 ꢂ 0.01 ꢁ0.07 ꢂ 0.20
30.5 ꢂ 0.03 22.6 ꢂ 0.01 50.9 ꢂ 0.13 25.4 ꢂ 0.01 ꢁ0.27 ꢂ 0.19
(2)
35.1 ꢂ 0.03 5.27 ꢂ 0.04 18.9 ꢂ 0.09 21.4 ꢂ 0.01
49.8 ꢂ 0.03 68.0 ꢂ 0.04 14.5 ꢂ 0.12 21.4 ꢂ 0.01
14.1 ꢂ 0.06 23.5 ꢂ 0.04 13.4 ꢂ 0.03 15.8 ꢂ 0.01
20.2 ꢂ 0.06 55.7 ꢂ 0.04 26.7 ꢂ 0.01 18.3 ꢂ 0.01
3.93 ꢂ 0.04 6.20 ꢂ 0.05 1.18 ꢂ 0.03 3.06 ꢂ 0.01
5.63 ꢂ 0.03 6.36 ꢂ 0.06 4.98 ꢂ 0.07 5.67 ꢂ 0.01
0.49 ꢂ 0.15
0.49 ꢂ 0.15
0.56 ꢂ 0.14
0.51 ꢂ 0.14
1.34 ꢂ 0.15
1.32 ꢂ 0.15
No correlation is obtained for the anti-proliferative activity of
26e33 and their clog P values (r ¼ 0.631 against MCF-7, and
r ¼ 0.353 against HCT-116).
The use of cell-cycle-specific treatments in cancer therapy has
greatly benefited from the major advances that have been recently
made in the identification of the molecular actors regulating the
cell cycle and from the better understanding of the connections
between cell cycle and apoptosis [17]. To study the mechanisms of
the anti-tumour activity of the most active compounds (30 and 33),
the effects on the cell cycle distribution and apoptosis were ana-
lysed by flow cytometry (FACS) (Tables 2e4). For this purpose we
used the MCF-7, HCT-116 and A-375 cell lines as representatives for
breast, colon and melanoma tumours, respectively. The A-375
melanoma cell line was selected because of its higher metastatic
phenotype in comparison with G-361 [18]. MCF-7 cells treated for
24 h with 30 and 33 did not show significant differences in the cell
cycle progression compared with DMSO-treated control cells. We
found a slight cell cycle arrest in the G2/M and S-phases induced by
30 (54.63 ꢂ 1.18) and 33 (18.27 ꢂ 0.79), respectively (Table 2). In the
HCT-116 and A-375 treated cells, 30 did not modify the cell cycle
profile and 33 provoked a G2/M cell cycle arrest (28.47 ꢂ 0.07) at
the expense of the G0/G1-phase (33.58 ꢂ 1.90) in the colon cancer
cells and accumulated the A-375 melanoma cells in the G0/G1-
phase (70.55 ꢂ 1.47) at the expense of both G2/M and S phases
(20.10 ꢂ 0.75 and 9.34 ꢂ 0.62, respectively) (Tables 3 and 4). Pre-
viously, we have demonstrated that potent anti-tumour drugs did
not modify the cell cycle in comparison with control cells, due to a
translational block and consequently inhibition of the protein
synthesis by the activation and phosphorylation of the initiation
a
All experiments were conducted in duplicate and gave similar results. The data
are means ꢂ SEM of three independent determinations.
b
Pallas 3.8.1.1 CompuDrug Chemistry Ltd. Copyright (c) 1994, 2006.
assayed cell lines, the N-9 derivative (30) eliciting improved ac-
tivities in all cancerous cell lines than its N-7 regioisomer (33); (b)
In general, the N-9 purine derivatives present an augmented ac-
tivity than that of their N-7 regioisomers except in the case of the 6-
chloropurines (28 and 29), in which this tendency is reversed
against A-375, and G-361: both regioisomers are equally potent in
inhibiting both melanoma cancer cell growth; (c) The 5-FU de-
rivatives evidence intermediate potencies (IC50 z 22 and 50 mM),
being the N-1 isomer (26) more active than N-3 (27) against MCF-7
and A-375, except in the case of HCT-116 and G-361 cell lines.
A quick look at the IC50 values suggests that a correlation with
the lipophilicity of the target compounds may exist, and we
therefore decided to find the corresponding QSAR equations. Used
in its logarithmic form (log P) the octanolewater partition coeffi-
cient is the most widely accepted measure of lipophilicity. Frag-
mental methods make it possible to create data banks and to
perform log P calculations by computer.
Correlations 1 and 2 show the anti-proliferative activities of the
targeted compounds against the melanoma cell line A-375 and the
clog P values of 26e33. Such clog P values reported in Table 1 were
calculated using the PALLAS programme [16].
factor eIF2a [10].
Apoptotic defects in cancer cells are the primary obstacle that
limits the therapeutic efficacy of anticancer agents, and hence the
development of novel agents targeting programmed cell death
pathways has become an imperative mission for clinical research
[19,20]. Although both compounds showed different cell cycle
profiles that were dependent upon the cell line studied, however,
30 and 33 at 24 h induced high levels of apoptosis in all cancer cells
in comparison with DMSO-treated cell cultures (Tables 2e4). This
apoptosis was induced even in the MCF-7 breast cancer cells that
have shown deficiency in the caspase-activation mechanisms [21].
Interestingly, 30 was the most apoptotic compound against the A-
375 melanoma cell line (35.37 ꢂ 0.47), where no modification in
the cell cycle were found. This effect could be explained by a
pðIC50ÞAꢁ375 ¼ 4:40ðꢂ0:12Þ þ 0:85ðꢂ0:15Þc log P
n ¼ 8; r ¼ 0:920; s ¼ 0:224; F1;5 ¼ 32:96; p < 0:005
(1)
where p(IC50 A-375
higher the value of p(IC
is the number of compounds, r is the correlation coefficient, s is the
standard deviation, data within parentheses are standard errors of
estimate, and F1,5 is the Fisher test (p < 0.005).
)
¼ ꢁlog (IC50 A-375
)
, bearing in mind that the
)
50 A-375 the more potent is the compound, n
Table 2
Table 3
Cell cycle distribution and apoptosis induction in the human breast MCF-7 cancer
cell line after treatment for 24 h for the two most active compounds 30 and 33 as
anti-proliferative agents.
Cell cycle distribution and apoptosis induction in the HCT-116 colon cancer cell line
after treatment for 24 h for the two most active compounds 30 and 33 as anti-
proliferative agents.
Compound
Cell cyclea,b
G0/g1
Apoptosisa,b,c
Compound
Cell cyclea,b
G0/g1
Apoptosisa,b,c
S
G2/M
S
G2/M
Control
30
33
34.58 ꢂ 0.09
28.17 ꢂ 1.37
34.54 ꢂ 0.08
14.50 ꢂ 1.06
15.39 ꢂ 0.68
18.27 ꢂ 0.79
50.91 ꢂ 1.15
54.63 ꢂ 1.18
47.19 ꢂ 0.89
10.80 ꢂ 0.85
20.53 ꢂ 0.91
24.10 ꢂ 4.37
Control
30
33
44.70 ꢂ 0.38
45.93 ꢂ 1.11
33.58 ꢂ 1.90
38.26 ꢂ 1.40
36.75 ꢂ 0.70
37.93 ꢂ 1.92
17.02 ꢂ 1.40
17.31 ꢂ 0.44
28.47 ꢂ 0.07
7.27 ꢂ 1.57
20.20 ꢂ 3.18
17.03 ꢂ 1.00
a
a
Determined by flow cytometry [17].
All experiments were conducted in triplicate and gave similar results. The data
Determined by flow cytometry [17].
All experiments were conducted in triplicate and gave similar results. The data
b
b
are means ꢂ SEM of three independent determinations.
are means ꢂ SEM of three independent determinations.
c
c
Apoptosis was determined using an Annexin V-based assay [17]. The data
Apoptosis was determined using an Annexin V-based assay [17]. The data
indicate the percentage of cells undergoing apoptosis in each sample.
indicate the percentage of cells undergoing apoptosis in each sample.

122
F. Morales et al. / European Journal of Medicinal Chemistry 76 (2014) 118e124
Table 4
2.450 GHz (Biotage AB, Uppsala). Reaction time refers to hold time
at 105 ꢀC, not to total irradiation time. The temperature was
measured with an IR sensor outside the reaction vessel. Anhydrous
THF was purchased from VWR International Eurolab. Anhydrous
conditions were performed under argon. 5-FU, 6-chloropurine, 6-
bromopurine and 2,6-dichloropurine were purchased from Aldrich.
Cell cycle distribution and apoptosis induction in the A-375 colon cancer cell line
after treatment for 24 h for the two most active compounds 30 and 33 as anti-
proliferative agents.
Compound
Cell cyclea,b
G0/G1
Apoptosisa,b,c
S
G2/M
Control
30
33
57.97 ꢂ 0.93
58.48 ꢂ 1.03
70.55 ꢂ 1.47
29.60 ꢂ 0.78
28.78 ꢂ 0.26
20.10 ꢂ 0.75
12.42 ꢂ 0.70
12.73 ꢂ 0.77
9.34 ꢂ 0.62
6.83 ꢂ 0.40
35.37 ꢂ 0.47
27.13 ꢂ 3.07
4.1.1. General procedure for the microwave-assisted syntheses of
26e33
a
Ethyl chloroacetate (6 mmol) was added dropwise to a mixture
of Et3N (4 mmol) and the corresponding purine derivative or 5-FU
(2 mmol) in water (5 mL). The microwave vial was sealed and
irradiated at 105 ꢀC for 8 min. After completion of irradiation time
and cooling to room temperature through rapid pressurized air
supply gas-jet, the resulting mixture was extracted with CH2Cl2
(3 ꢃ 50 mL) and the organic phase was dried (MgSO4). The solvent
was evaporated and the residue was purified by flash chromatog-
raphy using CH2Cl2/CH3OH (10/0.1) as eluent.
Determined by flow cytometry [17].
All experiments were conducted in triplicate and gave similar results. The data
b
are means ꢂ SEM of three independent determinations.
c
Apoptosis was determined using an Annexin V-based assay [17]. The data
indicate the percentage of cells undergoing apoptosis in each sample.
preferentially apoptotic mechanism of action. Moreover, the fact
that 33 gathered cells at G2/M and G0/G1 phases respectively in the
colon and melanoma cancer cells accompanied by high levels of
programmed death cell indicates that this compound has different
cytotoxic effects on each tumour cell type.
4.1.1.1. 5-Fluoro-1-(ethoxycarbonylmethyl)-3H-pyrimidine-2,4-dione
(26). White solid (57 mg, 13%); mp: 165e166 ꢀC (lit14 164e165 ꢀC).
1H NMR (300 MHz, CDCl3):
d
¼ 8.83 (s, 1H, H1), 7.21 (d, J ¼ 5.3 Hz,
3. Conclusion
1H, H6), 4.43 (s, 2H, CH2CO), 4.27 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.31 (t,
J ¼ 7.1 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
¼ 167.09 (CO),
The anti-proliferative potential of the target molecules is re-
ported against four human cancerous cell lines. Two QSARs are
obtained between the anti-proliferative IC50 values for compounds
26e33 and the clog P against the melanoma cell lines A-375 and G-
361. Using our purine derivatives as lead structures, we have ob-
tained a simplified analogue with a remarkable bioactivity. The
most active compounds are always 30 and 33 and the results
indicate that the anti-proliferative activity of 33 is correlated with
its ability to induce apoptosis against the human melanoma cell
line A375. The mechanism through which molecules 30 and 33
elicit their effects is currently being elucidated.
156.87 (C4), 149.31 (C2), 141.60 (C5), 128.86 (C6), 62.67 (CH2O),
49.02 (CH2CO), 14.24 (CH3). HRMS m/z [M þ H]þ calcd for
C8H10FN2O4: 217.0546, found: 217.0546. Anal. Calc. for C8H9FN2O4:
C, 44.45; H, 4.20; N, 12.96. Found: C, 44.21; H, 4.39; N, 13.01.
4.1.1.2. 5-Fluoro-3-(ethoxycarbonylmethyl)-1H-pyrimidine-2,4-
dione (27). Light orange solid (26 mg, 6%); mp: 131e132 ꢀC. 1H NMR
(300 MHz, CDCl3):
d
¼ 9.26 (s,1H, H1), 7.29 (pst,1H, H6), 4.68 (s, 2H,
CH2CO), 4.24 (q, J ¼ 7.1 Hz, 2H, CH2O),1.30 (t, J ¼ 7.1 Hz, 3H, CH3). 13
C
NMR (75 MHz, CDCl3):
d
¼ 167.09 (CO), 156.77 (C4), 149.59 (C2),
139.70 (C5), 128.59 (C6), 62.67 (CH2O), 49.07 (CH2CO), 14.24 (CH3).
HRMS m/z [M þ H]þ calcd for C8H10FN2O4: 217.0546, found:
217.0546. Anal. Calc. for C8H9FN2O4: C, 44.45; H, 4.20; N, 12.96.
Found: C, 44.21; H, 4.59; N, 12.63.
4. Experimental protocols
4.1. Chemistry
4.1.1.3. 6-Chloro-9-(ethoxycarbonylmethyl)-9H-purine
White solid (117 mg, 24%); mp: 97e98 ꢀC (lit 96e98 ꢀC) [22]. 1H
NMR (300 MHz, CDCl3):
¼ 8.74 (s, 1H, H2), 8.20 (s, 1H, H8), 5.05 (s,
2H, CH2CO), 4.27 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.29 (t, J ¼ 7.1 Hz, 3H,
CH3). 13C NMR (75 MHz, CDCl3):
(C4), 151.39 (C6), 145.58 (C8), 131.33 (C5), 62.82 (CH2O), 44.69
(CH2CO), 14.20 (CH3). HRMS m/z [M þ H]þ calcd for C9H10ClN4O2:
241.0414, found: 241.0488. Anal. Calc. for C9H9ClN4O2: C, 44.92; H,
3.77; N, 23.28. Found: C, 44.99; H, 3.59; S, 23.39.
(28).
Melting points were taken in open capillaries on an Electro-
thermal melting point apparatus and are uncorrected. Analytical
thin layer chromatography was performed using Merck Kieselgel
60 F254 aluminum sheets, the spots being developed with UV light
d
d
¼ 166.56 (CO), 152.37 (C2), 152.03
(l
¼ 254 nm). All evaporation was carried out in vacuo with a Büchi
rotary evaporator and the pressure controlled by a Vacuubrand
CVCII apparatus. For flash chromatography, Merck silica gel 60 with
a particle size of 0.040e0.063 mm (230e400 mesh ASTM) was
used. Elemental analyses were within ꢂ0.4% of the theoretical
values. Nuclear magnetic resonance spectra have been carried out
at the Centro de Instrumentación Científica/Universidad de Gran-
ada, and recorded on a 300 MHz 1H and 75 MHz 13C NMR Varian
Inova-TM spectrometers at ambient temperature. Chemical shifts
4.1.1.4. 6-Chloro-7-(ethoxycarbonylmethyl)-7H-purine
(31).
White solid (59 mg, 12%); mp: 122e123 ꢀC. 1H NMR (300 MHz,
CDCl3):
d
¼ 8.87 (s, 1H, H2), 8.28 (s, 1H, H8), 5.23 (s, 2H, CH2CO),
4.27 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.27 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR
(d
) are quoted in parts per million (ppm) and are referenced to the
(75 MHz, CDCl3):
d
¼ 166.76 (CO), 162.00 (C4), 152.81 (C2), 149.72
residual solvent peak. Signals are designated as follows: s, singlet;
d, doublet; t, triplet; pst, pseudo-triplet; q, quartet. The HMBC
spectra were measured using a pulse sequence optimized for 10 Hz
(inter-pulse delay for the evolution of long-range couplings: 50 ms)
and a 5/3/4 gradient combination. In this way, direct responses (J
couplings) were not completely removed. High-resolution Nano-
Assisted Laser Desorption/Ionization or Electrospray Ionization
mass spectra were carried out on a Bruker Autoflex or a Waters LCT
Premier Mass Spectrometer, respectively. Small scale microwave-
assisted synthesis was carried out in an Initiator 2.0 single-mode
microwave instrument producing controlled irradiation at
(C6), 143.16 (C8), 122.80 (C5), 62.90 (CH2O), 48.19 (CH2CO), 14.17
(CH3). HRMS m/z [M þ H]þ calcd for C9H10ClN4O2: 241.0414, found:
241.0487. Anal. Calc. For C9H9ClN4O2: C, 44.92; H, 3.77; N, 23.28.
Found: C, 45.21; H, 3.59; N, 23.42.
4.1.1.5. 6-Bromo-9-(ethoxycarbonylmethyl)-9H-purine
(29).
White solid (157 mg, 28%); mp: 104e105 ꢀC. 1H NMR (300 MHz,
CDCl3):
d
¼ 8.71 (s, 1H, H2), 8.23 (s, 1H, H8), 5.05 (s, 2H, CH2CO),
4.28 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.30 (t, J ¼ 7.2 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3):
(C8), 143.46 (C6), 133.89 (C5), 62.85 (CH2O), 44.75 (CH2CO), 14.21
d
¼ 166.52 (CO), 152.36 (C2), 150.80 (C4), 145.53

F. Morales et al. / European Journal of Medicinal Chemistry 76 (2014) 118e124
123
(CH3). HRMS m/z [M þ H]þ calcd for C9H10BrN4O2: 284.9909, found:
284.9910. Anal. Calc. for C9H9BrN4O2: C, 37.92; H, 3.18; N, 19.65.
Found: C, 37.72; H, 3.49; N, 19.38.
doseeresponse curves by linear interpolation. All of the experi-
ments were plated in triplicate wells and were carried out twice.
4.2.4. Cell cycle distribution analysis
The cells at 70% confluence were treated with either DMSO
alone or with concentrations of compounds 30 and 33 determined
by their IC50 values. FACS analysis was performed after 24 h of
treatment as described [17]. All experiments were performed in
triplicate and yielded similar results.
4.1.1.6. 6-Bromo-7-(ethoxycarbonylmethyl)-7H-purine
Yellow syrup (50 mg, 9%). 1H NMR (300 MHz, CDCl3):
(32).
¼ 8.90 (s,1H,
d
H2), 8.65 (s, 1H, H8), 5.32 (s, 2H, CH2CO), 4.29 (q, J ¼ 7.1 Hz, 2H,
CH2O), 1.29 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
¼ 166.67 (CO), 159.41 (C4), 152.99 (C2), 149.77 (C8), 133.93 (C6),
122.69 (C5), 63.06 (CH2O), 48.29 (CH2CO), 14.21 (CH3). HRMS m/z
[M þ H]þ calcd for C9H10BrN4O2: 284.9909, found: 284.9991. Anal.
Calc. For C9H9BrN4O2: C, 37.92; H, 3.18; N, 19.65. Found: C, 37.64; H,
3.42; N, 19.33.
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. [17]. Apoptosis induction in the MCF-7,
HCT-116 and A-375 human cancer cell lines after treatment for 24 h
was determined for compounds 30 and 33 at doses of their corre-
sponding IC50. All experiments were performed in triplicate and
yielded similar results.
4.1.1.7. 2,6-Dichloro-9-(ethoxycarbonylmethyl)-9H-purine
(30).
White solid (218 mg, 40%); mp: 120e121 ꢀC (lit13 112e113 ꢀC). 1H
NMR (300 MHz, CDCl3):
d
¼ 8.18 (s, 1H, H8), 5.02 (s, 2H, CH2CO),
4.24 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.28 (t, J ¼ 7.2 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3):
d
¼ 166.25 (CO), 153.30 (C6), 153.26 (C4), 151.95
(C2), 146.33 (C8), 130.39 (C5), 62.90 (CH2O), 44.70 (CH2CO), 14.10
(CH3). HRMS m/z [M þ H]þ calcd for C9H9Cl2N4O2: 275.0024, found:
275.0096. Anal. Calc. for C9H8Cl2N4O2: C, 39.29; H, 2.93; N, 20.37.
Found: C, 39.01; H, 2.63; N, 20.09.
4.2.6. Statistical analyses
All the quantitative data in the present study are reported as
means ꢂ standard derivation from at least three independent ex-
periments. Two-way ANOVA was used for grouped analysis of dif-
ferences followed by Bonferroni post-tests.
4.1.1.8. 2,6-Dichloro-7-(ethoxycarbonylmethyl)-7H-purine
Yellow syrup (82 mg, 15%). 1H NMR (300 MHz, CDCl3):
(33).
¼ 8.29 (s,
d
Acknowledgements
1H, H8), 5.22 (s, 2H, CH2CO), 4.29 (q, J ¼ 7.1 Hz, 2H, CH2O), 1.29 (t,
J ¼ 7.1 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
¼ 166.49 (CO),
We thank the Instituto de Salud Carlos III [Fondo de Inves-
tigación Sanitaria (FIS) through projects no. PI10/00592 and PI10/
02295] and the ERDF (European Regional Development Fund) for
financial supports.
163.61 (C4), 153.53 (C6), 152.00 (C8), 143.90(C2), 122.05 (C5), 63.11
(CH2O), 48.25 (CH2CO), 14.21 (CH3). HRMS m/z [M þ H]þ calcd for
C9H9Cl2N4O2: 275.0024, found: 274.0096. Anal. Calc. for
C9H8Cl2N4O2: C, 39.29; H, 2.93; N, 20.37. Found: C, 38.91; H, 2.64; N,
20.20.
Appendix A. Supplementary data
4.2. Biology
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.012.
4.2.1. Cell culture
MCF-7, HCT-116, A-375 and G-361 cells were grown at 37 ꢀC in
an atmosphere containing 5% CO2, with Dubelcco’s modified Eagle
Medium (Gibco, Grand Island, NY, USA) supplemented with 10%
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