Synthesis and antiproliferative activity of novel symmetrical alkylthio- and alkylseleno-imidocarbamates

Article abstract of DOI:10.1016/j.ejmech.2010.11.013

The study described here concerns the synthesis of a series of thirty new symmetrically substituted imidothiocarbamate and imidoselenocarbamate derivatives and their evaluation for antitumoral activity in vitro against a panel of five human tumor cell lines: breast adenocarcinoma (MCF-7), colon carcinoma (HT-29), lymphocytic leukemia (K-562), hepatocarcinoma (Hep-G2), prostate cancer (PC-3) and one non-malignant mammary gland-derived cell line (MCF-10A). The GI₅₀ values for eighteen of the compounds were below 10 μM in at least one cell line. Two cancer cells (MCF-7 and HT-29) proved to be the most sensitive to five compounds (1b, 2b, 3b, 4b and 5b), with growth inhibition in the nanomolar range, and compounds 1b, 3b, 7b, 8b and 9b gave values of less than 1 μM. In addition, all of the aforementioned compounds exhibited lower GI₅₀ values than some of the standard chemotherapeutic drugs used as references. The results also reveal that the nature of the aliphatic chain (methyl is better than benzyl) at the selenium position and the nature of the heteroatom (Se better than S) have a marked influence on the antiproliferative activity of the compounds. These findings reinforce our earlier hypothesis concerning the determinant role of the selenomethyl group as a scaffold for the biological activity of this type of compound. Considering both the cytotoxic parameters and the selectivity index (which was compared in MCF-7 and MCF-10A cells), compounds 2b and 8b (with a selenomethyl moiety) displayed the best profiles, with GI₅₀ values ranging from 0.34 nM to 6.07 μM in the five cell lines tested. Therefore, compounds 2b and 8b were evaluated by flow cytometric analysis for their effects on cell cycle distribution and apoptosis in MCF-7 cells. 2b was the most active, with an apoptogenic effect similar to camptothecin, which was used as a positive control. Both of them provoked cell cycle arrest leading to the accumulation of cells in either G₂/M and S phase. These two compounds can therefore be considered as the most promising candidates for the development of novel generations of antitumor agents.

Full text of DOI:10.1016/j.ejmech.2010.11.013

European Journal of Medicinal Chemistry 46 (2011) 265e274
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of novel symmetrical
alkylthio- and alkylseleno-imidocarbamates
,
Elena Ibáñez a, Daniel Plano a, María Font b, Alfonso Calvo c, Celia Prior c, Juan Antonio Palop a
,
*
Carmen Sanmartín a
a Synthesis Section, Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
b Molecular Modeling Section, Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
c Oncology Division, Center for Applied Medical Research, CIMA, University of Navarra, Pío XII, 53, E-31008 Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2010
Received in revised form
18 October 2010
Accepted 6 November 2010
Available online 27 November 2010
The study described here concerns the synthesis of a series of thirty new symmetrically substituted
imidothiocarbamate and imidoselenocarbamate derivatives and their evaluation for antitumoral activity
in vitro against a panel of five human tumor cell lines: breast adenocarcinoma (MCF-7), colon carcinoma
(HT-29), lymphocytic leukemia (K-562), hepatocarcinoma (Hep-G2), prostate cancer (PC-3) and one non-
malignant mammary gland-derived cell line (MCF-10A). The GI50 values for eighteen of the compounds
were below 10
sensitive to five compounds (1b, 2b, 3b, 4b and 5b), with growth inhibition in the nanomolar range, and
compounds 1b, 3b, 7b, 8b and 9b gave values of less than 1 M. In addition, all of the aforementioned
mM in at least one cell line. Two cancer cells (MCF-7 and HT-29) proved to be the most
Keywords:
Selenium
m
compounds exhibited lower GI50 values than some of the standard chemotherapeutic drugs used as
references. The results also reveal that the nature of the aliphatic chain (methyl is better than benzyl) at
the selenium position and the nature of the heteroatom (Se better than S) have a marked influence on
the antiproliferative activity of the compounds. These findings reinforce our earlier hypothesis con-
cerning the determinant role of the selenomethyl group as a scaffold for the biological activity of this
type of compound. Considering both the cytotoxic parameters and the selectivity index (which was
compared in MCF-7 and MCF-10A cells), compounds 2b and 8b (with a selenomethyl moiety) displayed
Antiproliferatives
Thioimidocarbamates
Selenoimidocarbamates
the best profiles, with GI50 values ranging from 0.34 nM to 6.07 mM in the five cell lines tested.
Therefore, compounds 2b and 8b were evaluated by flow cytometric analysis for their effects on cell
cycle distribution and apoptosis in MCF-7 cells. 2b was the most active, with an apoptogenic effect
similar to camptothecin, which was used as a positive control. Both of them provoked cell cycle arrest
leading to the accumulation of cells in either G2/M and S phase.
These two compounds can therefore be considered as the most promising candidates for the devel-
opment of novel generations of antitumor agents.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
generated growing interest [3e13]. At present, our main focus in
the laboratory is the synthesis and evaluation of the cytotoxic
Cancer is still a major health problem and it represents the main
cause of death in many countries. Although major advances have
been made in the chemotherapeutic management of some patients,
the continued commitment to the laborious task of discovering
new anticancer agents remains critically important [1,2]. In the last
three years, among the wide range of compounds tested as
potential anticancer agents, several structurally diverse derivatives
that contain a selenium template have been reported and have
activity of selenium-containing compounds. Taking into account
the results obtained in previous studies [14e18], we present here
twenty new derivatives that contain this trace element. In addition,
based on a rational bioisosteric approach, the differences in the
anticarcinogenic activity between two similar chemical elements,
selenium and sulfur, were compared. The previous hypothesis that
isosteric replacement of sulfur by selenium would result in more
effective anticancer agents [14], is reinforced by the higher activity
shown by selenium derivatives as compared to the activity of the
sulfur analogs [19e22]. With the aim of finding further support for
the starting hypothesis, ten methylthio analogs were synthesized
and screened. It is well known that certain alkylthio groups have
* Corresponding author. Tel.: þ34 948 425 600; fax: þ34 948 425 649.
E-mail address: jpalop@unav.es (J.A. Palop).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.013

266
E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
proven to be key structural components for antitumoral agents
[23e28].
O
H2N
SeCH3
COOH
Considering the information given above, as well as the chem-
ical structural patterns previously described by our group [14,15]
and the structures of chemotherapeutic drugs with recognized
efficacy in cancer treatment, we propose a general structure (Fig. 1)
for the new compounds described here. The following character-
istics were taken into consideration: (1) Molecular symmetry, as
a broad concept, because this is a structural property that is
frequently present in many cytotoxic drugs [29] and some reported
sulfur [24e28] and selenium derivatives have this property
[6,9,13]. (2) A central scaffold consisting of a methyl imidothiocar-
bamate (methylisothiourea) or methyl (methylselenourea) and
benzyl (benzylselenourea) imidoselenocarbamates connected by
a carbonyl group onto two identical lateral heteroaromatic rings.
The methyl group was selected on the basis of data in the literature
that show this moiety is present (Fig. 2) in selenoderivatives
with anticancer activity [30,31]. There is increasing evidence
that anticarcinogenic effects for some seleno compounds have been
consistently associated with methylselenol, an active metabolite
of methylseleno derivatives. The benzyl template is present
(Fig. 2) in 1,4-phenylenebis(methylene)selenocyanate (p-XSC)
and in p-xylylbis(methylselenide) (p-XMS), two organoselenium
compounds with relevant biological properties since they act as
regulators of cell cycle, growth factor signaling, angiogenesis and
cell invasion and metastasis [32].
Se
CH3
OH
Methylseleninic acid
CH3Se
Methylselenocysteine
NCSe
H2N
SeCH3
COOH
SeCH3
p-XMS
SeCN
p-XSC
Selenomethionine
Fig. 2. Some selenoderivatives with anticancer activity.
obtained in yields ranging from 2 to 56%. The purity of each
compound was assessed by TLC and elemental analysis and the
structures were identified by spectroscopic methods such as IR, 1H
NMR and MS. In the IR spectra, the compounds showed absorption
The choice of lateral rings was based on data that support the
antitumor activity of mono-, bi- or tri-cyclic heteroaromatic
systems following a model based on a fragmental approach. Among
the many moieties identified, the following are included in our
series of chemical structures: furyl [33,34], thienyl [35,36], iso-
xazole [37,38], benzothiophene [39], benzodioxole [40], quinoline
[41,42], phenylquinoline [43,44] and acridine [45,46].
Finally, in the design of the new series of compounds we
considered the potential adjustment to the Lipinski rules, which is
taken as an estimation of the potential bioavailability. According to
the aforementioned rules, an orally active drug must not violate
more than one of the following criteria: ꢀ5 hydrogen donors
(nOHNH), ꢀ10 hydrogen acceptors (nON), MW ꢀ 500, clogP ꢀ 5.
In view of the rational outline above and our previous work
related to finding new structures with potential chemotherapeutic
activities, we report here the synthesis of thirty previously unre-
ported alkylthio- and alkylseleno-imidocarbamate derivatives and
their biological evaluation as potential antitumoral agents.
2. Results and discussion
2.1. Chemistry
The synthesis of the bisacylimidocarbamates 1e10a, 1e10b and
1e10c was carried out according to Scheme 1, starting from the
appropriate S-methyl imidothiocarbamate (a), Se-methyl imidose-
lenocarbamate (b) or Se-benzyl imidoselenocarbamate (c) as
hydrohalides, and the corresponding heteroaryl acyl chloride
(1e10) in a 1:2 M ratio in chloroform in the presence of pyridine as
a catalyst at room temperature for 48 h. The compounds were
R
O
X
O
Heteroaryl
Heteroaryl
NH
N
Scheme 1. Synthesis of bisacylimidocarbamates 1e10a, 1e10b and 1e10c.
Fig. 1. General structure of compounds.

Table 1
Average GI50, TGI and LC50 values (
m
M) for compounds for compounds 1e10a, 1e10b and 1e10c.
MCF-7a
HT-29b
K-562c
GI50
Hep-G2d
GI50
PC-3e
GI50
MCF-10Af
GI50
Comp.
GI50
TGIh
LC50
GI50
TGI
LC50
TGI
LC50
TGI
LC50
TGI
LC50
TGI
LC50
g
i
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
1c
2c
3c
4c
5c
6c
7c
8c
9c
10c
56.08
44.49
10.28
44.65
77.83
9.51
24.55
23.90
43.08
27.68
0.03
>100
88.96
57.10
78.07
>100
43.40
59.69
>100
87.70
>100
88.92
5.64
31.18
28.14
57.52
32.66
52.77
8.03
>100
>100
>100
>100
>100
78.48
94.83
>100
>100
>100
>100
>100
>100
>100
>100
80.64
>100
60.70
>100
>100
>100
>100
>100
83.76
>100
87.90
>100
>100
97.04
>100
19.95
>100
>100
>100
>100
>100
77.14
77.92
35.58
51.82
>100
16.76
46.42
>100
23.69
69.17
5 ꢁ 10ꢂ5
3.4 ꢁ 10ꢂ4
0.0049
6.3 ꢁ 10ꢂ4
7.9 ꢁ 10ꢂ4
5.62
0.53
0.20
0.46
7.63
7.19
55.07
6.51
9.25
6.82
>100
>100
76.52
91.96
>100
52.46
>100
>100
>100
>100
>100
>100
38.80
>100
>100
40.14
>100
>100
32.59
63.95
38.08
>100
>100
54.65
>100
53.40
>100
>100
>100
>100
6.31
>100
>100
>100
>100
>100
88.16
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
90.69
>100
89.11
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.40
31.62
>100
79.43
>100
>100
64.84
72.59
36.95
56.85
>100
34.66
67.99
65.75
34.39
45.02
8.78
6.07
4.81
4.98
0.87
32.43
9.23
3.80
5.80
27.16
9.53
>100
>100
78.89
98.68
>100
66.63
>100
>100
>100
75.98
>100
>100
28.46
>100
45.63
60.99
46.30
29.92
30.58
60.98
44.18
58.48
95.82
55.84
57.34
42.79
>100
72.61
47.39
44.53
5.01
>100
>100
>100
>100
>100
98.60
>100
>100
>100
>100
>100
>100
90.03
>100
>100
89.55
85.20
74.75
73.05
94.79
80.59
87.42
>100
88.20
94.72
70.63
>100
>100
88.86
80.65
19.95
>100
>100
>100
>100
>100
>100
>100
65.25
63.41
>100
15.81
61.42
57.57
6.24
90.25
0.76
0.63
0.69
0.62
0.51
9.59
5.84
4.16
4.54
22.30
7.65
34.71
4.42
34.08
9.12
21.01
>100
8.54
25.81
30.42
n.d.k
n.d.
n.d.
n.d.
n.d.
n.d.
>100
>100
>100
>100
>100
52.99
>100
>100
48.15
>100
8.66
>100
>100
>100
>100
>100
75.84
>100
>100
>100
>100
>100
12.51
8.67
10.04
>100
73.29
73.14
87.77
65.42
82.46
76.51
81.46
>100
91.59
>100
76.99
>100
90.28
78.94
98.51
n.d.
56.18
39.02
24.68
41.48
99.24
8.58
28.71
76.30
7.48
51.08
0.36
1.34
0.97
0.10
0.09
37.16
4.24
2.33
3.97
41.28
30.48
49.86
52.70
35.91
53.12
30.53
>100
34.84
39.26
42.30
0.25
>100
98.25
57.38
82.45
>100
39.36
54.54
>100
>100
87.26
56.67
63.03
7.03
>100
>100
90.08
>100
>100
72.55
80.38
>100
>100
>100
>100
>100
>100
>100
>100
94.02
>100
>100
>100
>100
84.78
>100
>100
>100
>100
86.31
>100
>100
>100
>100
19.95
>100
79.43
>100
>100
>100
8.22
19.70
0.09
1.55
16.85
0.02
1.01
0.50
0.04
1.00
5.94
9.26
3.16
1.95
2.89
0.49
5.58
20.34
0.71
18.25
8.80
72.52
3.68
24.44
7.80
37.94
55.34
23.55
8.46
53.69
n.d.
46.52
55.10
23.72
21.35
>100
5.60
5.03
42.17
0.54
>100
40.16
41.14
5.49
85.69
90.50
64.11
75.14
>100
54.56
9.05
82.19
48.81
>100
98.07
73.51
7.82
0.0034
0.30
1.50
4.02
1.55
9.35
7.6 ꢁ 10ꢂ4
9 ꢁ 10ꢂ5
5.07
7.09
4.74
7.53
39.32
65.59
36.79
43.64
40.22
80.12
57.63
>100
>100
69.19
>100
58.42
>100
>100
97.07
77.38
3.16
15.41
34.65
45.97
52.10
20.05
53.28
57.98
>100
28.33
54.10
>100
62.92
>100
>100
45.56
88.14
n.d.
63.94
73.21
>100
83.86
76.90
88.31
>100
>100
>100
83.76
>100
87.90
>100
>100
97.04
>100
n.d.
41.01
23.62
23.68
10.13
52.38
39.45
58.09
45.46
62.84
58.92
49.00
>100
48.85
52.37
64.46
n.d.
0.99
0.66
0.49
73.92
8.80
72.52
3.68
24.44
7.80
37.94
55.34
23.55
8.46
53.69
0.02
7.52
>100
57.98
>100
28.33
54.10
>100
62.92
>100
>100
45.56
88.14
5.01
29.54
7.63
23.48
19.97
14.94
97.84
29.34
9.06
9.60
0.10
0.06
6.51
57.27
26.12
6.07
26.92
0.20
Doxorubicinj
Camptothecinj
Etoposidej
Taxolj
0.01
0.16
0.05
0.79
>100
0.20
>100
>100
50.12
>100
15.85
>100
>100
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.06
0.63
0.004
5.01
0.1
>100
3.98
12.59
50.12
>100
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
19.95
0.003
3.16
>100
>100
>100
>100
31.62
0.003
7.95
12.59
0.004
5.01
Cisplatinj
Methotrexatej
0.06
0.04
0.03
n.d.
n.d.
n.d.
n.d.
n.d.
a
Breast adenocarcinoma.
Colon carcinoma.
Leukemia.
Liver carcinoma.
Prostate carcinoma.
b
c
d
e
f
Nontumorigenic breast epithelial cells.
g
h
i
Concentration that inhibits 50% of cell growth.
Concentration that inhibits 100% of cell growth.
Concentration that kills 50% of cells.
NCI data (http://dtp.nci.nih.gov).
No data.
j
k

268
E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
Fig. 3. Cytotoxic effect of compounds 2b and 8b on MCF-7, HT-29, K-562, Hep-G2, PC-3 and MCF-10A cells. Data are expressed as the percentage of growth ꢃ SEM of at least 3
independent experiments performed in quadruplicate.
bands around 3400e3450 cmꢂ1 originated from the NeH stretch-
ing vibration. The strong bands between 1646 and 1776 cmꢂ1
corresponded to carbonyl vibrations. In most of the 1H NMR
spectra, one sharp peak from 13.77 to 15.11 ppm was observed due
to the presence of the proton in secondary amide NH. The protons
on heteroaryl rings were split into multiple peaks from 6.08 to
9.86 ppm. The chemical shift of methyl group attached to the
controls. In order to assess the selectivity of the compounds for
tumor cells, cytotoxicity was tested in MCF-10A normal cells and
the results were compared to those of MCF-7 cancer cells (Table 1).
The analyses were all carried out with a minimum of three inde-
pendent experiments and values were calculated after 72 h
exposure.
The methylthio derivatives (1e10a) exhibited comparable and
weak antiproliferative activities in all cancer cells tested, as shown
by the results in Table 1. Compound 6a in MCF-7 and in PC-3 and
compound 9a in Hep-G2 and in PC-3 had GI50 values lower than
heteroatom sulfur or selenium appeared at
2.44e2.67 respectively.
d 2.59e2.82 and
d
10
methylseleno compounds (1e10b) presented GI50 values below
10 M in all the cell lines tested, with the exception of 6b in PC-3
(GI50 value of 37.16 M) and K-562 (32.43 M), and 10b in all cell
lines apart from HT-29. Among the cancer cell lines tested, MCF-7
and HT-29 were the most susceptible ones, with GI50 values in the
nanomolar ranges for 2b, 4b and 5b in MCF-7 and 1b, 2b, 3b, 4b and
5b in HT-29. Moreover, GI50 values for 1b, 3b, 7b, 8b and 9b in MCF-
mM. However, when sulfur was replaced by selenium all of the
2.2. Biological evaluation
2.2.1. Cytotoxicity
m
m
m
All thirty of the synthesized compounds were screened for
their cytotoxic and antiproliferative activities against a panel of
five human tumor cell lines: breast adenocarcinoma (MCF-7),
colon carcinoma (HT-29), lymphocytic leukemia (K-562), hep-
atocarcinoma (Hep-G2) and prostate cancer (PC-3) as well as the
non-malignant mammary gland cell line MCF-10A. These cell lines
represent common cancer tumor types. Regardless of the type of
tumor cell, several common features were observed in the anti-
proliferative activities of the compounds. Cytotoxicity assays were
based on the reactivity of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide] according to a method previously
7, and the values for 7b, 8b and 9b in HT-29, were below 1 mM.
Comparison of the results with the cytotoxicity values for the
standard drugs showed that three (2b, 4b, 5b) of the compounds
had GI50 values lower than those found for doxorubicin, nine lower
than those obtained for etoposide (1b, 2b, 3b, 4b, 5b, 6b, 7b, 8b and
9b) and two (4b and 5b) lower than those observed for taxol in
MCF-7 cells. The results reveal that the selenomethyl derivatives
are more antiproliferative than the corresponding benzyl deriva-
tives. In addition, the results indicate that the replacement of the
methyl group by benzyl had less effect than the replacement of
sulfur by selenium. For example, GI50 values in MCF-7 for
compounds 2b, 4b and 5b were at least four orders of magnitude
lower than those determined for 2c, 4c and 5c in the same cell line.
Similar effects were observed in HT-29 and, to a lesser extent, in K-
562, Hep-G2 and PC-3. The likely explanation for this finding is that
compounds 1e10b, with a selenomethyl moiety, act as methyl-
selenol precursors. It has been suggested that methylselenol is
a critical selenium metabolite for anticancer activity [48e50].
In spite of this, compounds 1c, 3c, 5c and 9c showed
described by Denizot and Lang [47]. Results are expressed as GI50
:
the concentration that reduces by 50% the growth of treated cells
with respect to untreated controls; TGI: the concentration that
completely inhibits cell growth, and LC50: the concentration that
kills 50% of the cells. The cytotoxic effect of each substance was
tested at five different concentrations between 0.01 and 100 mM, or
at lower levels when the GI50 was less than 10 nM. Mean GI50, TGI
and LC50 values are summarized in Table 1. Doxorubicin, campto-
thecin, etoposide, taxol, cisplatin and methotrexate were used as
Table 2
Average GI50 values (mM) for compounds 1e5b, 8b and 9b in PC-3 and HT-29 cell
lines using CV method.
pronounced inhibitory effects (GI50 < 10
mM) on at least three
PC-3a
HT-29b
GI50
c
Comp.
GI50
Table 3
1b
2b
3b
4b
5b
8b
9b
0.16
0.63
0.24
0.11
0.03
1.42
3.72
1.0 ꢁ 10ꢂ4
9.0 ꢁ 10ꢂ4
0.0059
Effects of 2b and 8b on apoptosis in MCF-7 cells at a concentration of 15
mM for 24
and 48 h using the Apo-Direct kit (BD Pharmingen) based on the TUNEL technique.
Compound
24 h
48 h
7.1 ꢁ 10ꢂ4
4.9 ꢁ 10ꢂ4
0.46
Control
2b
8b
9.77 ꢃ 2.34
19.61 ꢃ 0.75
29.35 ꢃ 3.19**
7.70 ꢃ 1.63
71.08 ꢃ 1.88**
27.19 ꢃ 1.89**
71.45 ꢃ 7.80**
0.85
Camptothecin
21.25 ꢃ 6.16
a
Prostate carcinoma.
Colon carcinoma.
Concentration that inhibits 50% of cell growth.
b
Results are expressed as percent (%) of apoptotic cells and data are representative of
three experiments (mean ꢃ SEM). **p < 0.01 with respect to the control.
c

E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
269
non-selective [51]. If we consider the results as a whole, the most
promising candidates are 2b and 8b. These compounds were highly
selective for MCF-7 cell growth inhibition compared to normal
mammary cells, as evidenced by the ratios of their GI50 (GI50 MCF-
10A/GI50 MCF-7 ranged from 31 for 2b to 2723 for 8b) and TGI (TGI
MCF-10A/TGI MCF-7 ranged from 6.5 for 2b to 7.3 for 8b). Fig. 3 shows
exemplary curves with the original data from which the GI50, TGI
and LC50 values for compounds 2b and 8b were calculated.
Although MTT test is the most usual method for evaluating the
cytotoxic effects of chemicals in cultured cells, this assay could be
questioned when testing selenium-containing compounds. Sele-
nium could have an influence on these results due to modification of
redox processes in cells. In order to validate the GI50 values obtained
fromthe MTTassay, crystalviolet (CV) assayswereperformed for the
seven most active compounds (1e5b, 8b and 9b) in two represen-
tative cell lines (PC-3 and HT-29). The GI50 values obtained from the
CV method (Table 2) validated those obtained from the MTT assay.
Therefore, we consider the MTT assay as a valid cytotoxicity method
for the organoselenium compounds studied in this paper.
Analysis of the Lipinski descriptors for bioavailability estimation
[52,53] using the freely accessible program OSIRIS Explorer Prop-
erties was also carried out. Compounds 2b (clogP ¼ 3.15; MW ¼ 357;
nOHNH ¼ 1; nON ¼ 4) and 8b (clogP ¼ 4.09; MW ¼ 447; nOHNH ¼ 1;
nON ¼ 6) met the Lipinski criteria. However, the reference drugs for
pharmacological testing, doxorubicin, etoposide and taxol, which
were also analyzed with this program, did not fulfill the Lipinski
criteria. For example, doxorubicin (clogP ¼ 0.48; MW ¼ 543;
nOHNH ¼ 7; nON ¼ 12) did notfulfill three out of fourconditions and
etoposide (clogP ¼ 0.53; MW ¼ 588; nOHNH ¼ 3; nON ¼ 13) and
taxol (clogP ¼ 3.64; MW ¼ 853; nOHNH ¼ 4; nON ¼ 15) did not fulfill
two of them.
Fig. 4. Compounds 2b and 8b induce an apoptotic effect on MCF-7 cells at 48 h. MCF-7
cells were incubated either with 15 M of the indicated compound or vehicle (control
m
cells) for 24 and 48 h. The results are presented as the mean ꢃ SEM of three inde-
pendent experiments (duplicate wells). **p < 0.01 with respect to the control.
tumoral cell lines. In addition, these chemical structures were more
active than the positive etoposide against HT-29 cell growth (with
the exception of 2c and 7c). Once again, the test compounds were
more active against MCF-7 and HT-29 cell growth.
In the present study a precise structureeactivity relationship
cannot be defined, although some general trends related to the
heteroaromatic rings can be highlighted. When the size of the
heteroaryl group was increased (from five members to a fused-ring
system) a loss of antiproliferative activity was observed. This
suggests that the bulk of the ring is critical for the antitumor
activity. Compounds 1e5b showed GI50 values between
2.2.2. Apoptosis and effects on cell cycle progression
5 ꢁ 10ꢂ5 mM in HT-29 and 8.78
m
M in K-562, whereas compounds
Mounting evidence indicates that apoptosis is a critical mech-
anism for cancer prevention by Se compounds [15,54e56]. In
addition, cell cycle arrest is one of the targets of many anticancer
drugs, including doxorubicin and camptothecin. To gain further
insight into the mechanisms of action of these new active
compounds (2b and 8b) and as a preliminary study, we decided to
assess whether this activity was related to their ability to induce
apoptosis and affect cell cycle progression in MCF-7 cells, due to the
remarkable cytotoxic activity of the compounds 2b and 8b in this
6e10b gave values that ranged from 0.20 in HT-29 to 73.92
m
M in
MCF-7. A relationship between the presence of substituents with
strongly deactivating electron-withdrawing groups (e.g., nitro) in
the thienyl ring and increased activity in seleno compounds seems
to exist. For example, the unsubstituted derivatives 2b and 2c are
less active than the nitro-derivatives 4b and 4c, mainly in MCF-7,
HT-29 and PC-3 cell lines.
The tested compounds also affected the proliferation of the
normal mammary gland cell line MCF-10A, although GI50 and TGI
values were usually higher than those obtained for cancer cell lines.
The ratio between the cytotoxic parameters found in MCF-10A and
those observed in MCF-7 can be considered as a measurement of
compound selectivity. Ratios between 3 and 6 refer to moderate
selectivity, ratios greater than six indicate high selectivity, while
compounds that do not fulfill either of these criteria are rated
cell line. The cells were treated with 15
mM of the corresponding
compound for 24 and 48 h and the apoptotic status (Table 3 and
Fig. 4) and cell cycle progression (Figs. 5 and 6) were determined by
flow cytometry analysis [57]. The apoptotic status of the cells after
24 and 48 h of treatment with 15
mM of the corresponding
compound was determined using the Apo-Direct kit (BD Pharmin-
gen) [57] based on the TUNEL technique. Camptothecin was used as
Fig. 5. Effects of 2b and 8b on cell cycle distribution in MCF-7 cells. Exponentially growing cells were treated with 15 mM of the indicated compound or vehicle (control) for 24 h.
The changes of cell cycle phase distribution were assessed by DNA flow cytometric analysis. Results are expressed as percentages of total cell counts. Camptothecin was used as
a positive control. Results are presented as the mean ꢃ SEM of three independent experiments (duplicate wells). * 0.05 > p > 0.01 with respect the control.

270
E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
Fig. 6. Effects of 2b and 8b on cell cycle distribution in MCF-7 cells. Exponentially growing cells were treated with 15 mM of the indicated compound or vehicle (control) for 48 h.
The changes of cell cycle phase distribution were assessed by DNA flow cytometric analysis. Results are expressed as percentages of total cell counts. Camptothecin was used as
a positive control. Results are presented as the mean ꢃ SEM of three independent experiments (duplicate wells). * 0.05 > p > 0.01 with respect the control.
a positive control. The results obtained are shown in Table 3 and
Figs. 4e6. As can be seen, 2b led to a marked increase in the
proportion of apoptotic cells in the cultures accompanied of accu-
mulation in S and G2/M phases and a diminution of cell proportion
in G0/G1 phase at both times. On the other hand, 8b only exhibited
a light apoptotic effect at 48 h and had a similar behaviour on cell
cycle distribution.
In summary, considering both the biological activities and the
Lipinski parameters, the two most active and selective compounds
(2b and 8b) should be considered as novel candidates for further
studies to understand the exact mechanism of action in cancer
cells. Work is currently in progress in our laboratory with this aim
and results will be reported in due course.
spectra were obtained on a Thermo Nicolet FT-IR Nexus spectro-
photometer with KBr pellets. Elemental microanalyses were carried
out on vacuum-dried samples using a LECO CHN-900 Elemental
Analyzer. AlugramÒ SIL G/UV254 (Layer: 0.2 mm) (MachereyeNagel
GmbH & Co. KG. Postfach 101352, D-52313 Düren, Germany) was
used for Thin Layer Chromatography. Chemicals were purchased
from E. Merck (Darmstadt, Germany), Scharlau (F.E.R.O.S.A., Barce-
lona, Spain), Panreac Química S.A. (Montcada i Reixac, Barcelona,
Spain), SigmaeAldrich Química S.A. (Alcobendas, Madrid, Spain),
Acros Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, België)
and Lancaster (Bischheim-Strasbourg, France).
4.1.1. General procedure for the synthesis of compounds aec
The appropriate alkyl halide (45.0 mmol) was added dropwise
to a cooled (0 ꢄC), stirred mixture of thiourea (2.47 g, 32.5 mmol) or
selenourea (4.0 g, 32.5 mmol) in dry ethanol (25 mL). The mixture
was heated under reflux for 90 min. The solvent was removed in
vacuo and the product was recrystallized from ethanol.
3. Conclusions
The biological activities of the designed compounds confirm our
hypothesis that molecular symmetry and the presence of a seleno-
methyl moiety is a valid approach to obtain potent new antitumor
agents. Eighteen compounds exhibited inhibitory effects (<10
m
M)
4.1.1.1. Methyl imidothiocarbamate hydroiodide (a). IR (KBr):
on at least one cell line. MCF-7 and HT-29 were the most susceptible
lines, with GI50 values in the nanomolar range for three (2b, 4b and
5b) and five (1b, 2b, 3b, 4b and 5b) compounds, respectively. In
3500e3200, 1634 cmꢂ1 1H NMR (400 MHz, DMSO-d6,
; d): 2.24 (t,
3H, SeCH3); 8.87 (bs, 3H, NH$HI, NH2). Anal. Calcd. for C2H6N2S$HI
(%): C, 11.01; H, 3.21; N, 12.85. Found: C, 11.16; H, 3.12; N, 12.92.
addition,1b, 3b, 7b, 8b and9b showed GI50 values lowerthan 1 mMin
these same cell lines. It is noteworthy that some of these compounds
were more cytotoxic than the reference drugs currently used in the
treatment of cancer patients. As a result of the preliminary SAR, it
was revealed that the nature of the aliphatic chain (methyl > benzyl)
at the selenium site and the nature of the heteroatoms (Se > S) have
a critical influence on the antiproliferative activity of the molecules.
Considering all of the biological results and the predicted values for
Lipinski parameters, compounds 2b and 8b were found to be the
most appropriate as candidates for further testing with the aim of
finding more selective and active anticancer drugs. For these
reasons, they were evaluated by flow cytometric analysis for their
effects on cell cycle distributions and apoptosis induction in MCF-7
cells. Results indicated that both of them effectively induced cell
cycle accumulation at S and G2/M phases and arrest at G0/G1, which
consequently trigger apoptosis.
4.1.1.2. Methyl imidoselenocarbamate hydroiodide (b). IR (KBr):
3319e3102, 1635 cmꢂ1 1H NMR (400 MHz, DMSO-d6,
; d): 2.45 (s,
3H, SeeCH3); 8.90 (bs, 3H, NH$HI, NH2). Anal Calcd. for
C2H6N2Se$HI (%): C, 9.45; H, 2.76; N, 11.03. Found: C, 9.02; H, 2.51;
N, 10.66.
4.1.1.3. Benzyl imidoselenocarbamate hydrobromide (c). IR (KBr):
3219e3086, 1657 cmꢂ1 1H NMR (400 MHz, DMSO-d6,
; d): 4.54 (s,
2H, SeeCH2); 7.28 (t, 1H, J3e4 ¼ J4e5 ¼ 7.5 Hz, H4); 7.35 (t, 2H,
J2e3 ¼ J5e6 ¼ 7.5 Hz, 2H, H3, H5); 7.41 (d, 2H, H2, H6); 9.16 (s, 2H,
NH2); 9.28 (s, 2H, NH$HBr). Anal Calcd. for C8H10N2Se$HBr (%): C,
32.66; H, 3.74; N, 9.53. Found: C, 32.50; H, 3.76; N, 9.36.
4.1.2. General procedure for the synthesis of compounds 1e10
Compounds 1, 2, 3, 4 and 5 were commercially available.
Compounds 6, 7, 8, 9 and 10 were prepared from the corresponding
carboxylic acid(9.18 mmol) and thionyl chloride (30mL) with heating
under reflux for 2 h. The thionyl chloride was removed in vacuo. The
resulting acyl chloride was used without further purification.
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Mettler FP82 þ FP80
apparatus (Greifense, Switzerland) and are uncorrected. The 1H NMR
spectra were recorded on a Bruker 400 UltrashieldÔ spectrometer
(Rheinstetten, Germany) using TMS as the internal standard. The IR
4.1.3. General procedure for the synthesis of compounds 1e10a,
1e10b and 1e10c
A solution of the corresponding acyl chloride 1e10 (9.18 mmol)
in chloroform (25 mL) was slowly added dropwise to a stirred

E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
271
solution of compounds aec (4.59 mmol) in dry chloroform (40 mL)
and pyridine (5 mL). The mixture was stirred for 48 h at room
temperature. Solvents were removed under vacuum by rotatory
evaporation and the residue was treated with water (100 mL) and
purified.
4.1.3.7. Methyl N,N0-di(3,4-methylenedioxybenzoyl)-imidothiocarba-
mate (7a). From methyl imidothiocarbamate hydroiodide a and
3,4-methylenedioxy benzoyl chloride 7. Recrystallized from
ethanol/DMF. Yield: 56%. m.p. 195e196 ꢄC. IR (KBr): 3430,
1694 cmꢂ1; 1H NMR (4000MHz, CDCl3,
d): 2.63 (s, 3H, SCH3); 6.08 (s,
2H, CH2); 6.11 (s, 2H, CH2 ); 6.90 (d, 1H, J2e3 ¼ 8.2 Hz, H3); 6.95 (d,
4.1.3.1. Methyl N,N0-di(fur-2-ylcarbonyl)-imidothiocarbamate (1a)
. From methyl imidothiocarbamate hydroiodide a and 2-furoyl
chloride 1. Recrystallized from ethaꢂn1ol/DMF. Yield: 38%.
1H, J2 e3 ¼ 8.2 Hz, H3 ); 7.51 (d, 1H, J2e6 ¼ 1.7 Hz, H6); 7.61 (dd, 1H,
0
0
0
0
0
0
0
H2); 7.79 (d, 1H, J2 e6 ¼ 1.4 Hz, H6 ); 8.02 (dd, 1H, H2 ); 14.53 (s, 1H,
NH). MS (m/z, % abundance): 386 (Mþ, 8); 149 (100). Anal Calcd. for
C18H14N2O6S (%): C, 55.96; H, 3.63; N, 7.25. Found: C, 55.71; H, 4.03;
N, 7.11.
m.p.163e164 ꢄC. IR (KBr): 3398, 1676 cm
;
1H NMR (400 MHz,
0
CDCl3,
d): 2.63 (s, 3H, SCH3); 7.19e7.21 (m, 2H, H4, H4 ); 7.65 (dd, 1H,
0
0
0
0
0
J5 e3 ¼ 0.9 Hz, J5 e4 ¼ 4.9 Hz, H5 ); 7.72 (dd, 1H, J5e3 ¼ 0.8 Hz,
J5e4 ¼ 5.0 Hz, H5); 7.84 (dd, 1H, J3e4 ¼ 3.8 Hz, H3); 7.98 (dd, 1H,
4.1.3.8. Methyl N,N0-di(quinolin-3-ylcarbonyl)-imidothiocarbamate
(8a). From methyl imidothiocarbamate hydroiodide a and quino-
line-3-carbonyl chloride 8. Recrystallized from ethanol/DMF. Yield:
40%. m.p. 206e207 ꢄC. IR (KBr): 3416,1703 cmꢂ1 1H NMR (400 MHz,
0
0
0
J3 e4 ¼ 3.7 Hz, H3 ); 14.37 (s, 1H, NH). MS (m/z, % abundance): 278
(Mþ, <1); 111 (100). Anal Calcd. for C12H10O4N2S$0.9HCl (%): C,
46.32; H, 3.22; N, 9.00. Found: C, 46.46, H, 3.52; N, 9.03.
CDCl3,
d
): 2.78 (s, 3H, SCH3); 7.66 (t, 1H, J8e7 ¼ J8e9 ¼ 8.0 Hz, H8);
4.1.3.2. Methyl N,N0-di(thien-2-ylcarbonyl)-imidothiocarbamate (2a)
. From methyl imidothiocarbamate hydroiodide a and 2-thio-
phenecarbonyl chloride 2. Recrystallized from ethanol/DMF. Yield:
7.72 (t, 1H, J8 e7 ¼ J8 e9 ¼ 8.4 Hz, H8 ); 7.87 (t, 1H, J7e6 ¼ 8.0 Hz, H7);
0
0
0
0
0
0
0
0
0
7.92 (t,1H, J7 e6 ¼ 8.4 Hz, H7 ); 8.02 (d,1H, H9); 8.10 (d,1H, H9 ); 8.20
0
(d, 1H, H6); 8.23 (d, 1H, H6 ); 8.83 (d, 1H, J4e2 ¼ 2.2 Hz, H4); 9.12 (d,
14%. m.p. 153e154 ꢄC. IR (KBr): 3447, 1673 cmꢂ1
;
1H NMR
1H, J4 e2 ¼ 1.6 Hz, H4 ); 9.56 (d, 1H, H2); 9.83 (d, 1H, H2 ); 14.96 (s,
1H, NH). MS (m/z, % abundance): 400 (Mþ, 1); 57 (100). Anal Calcd.
for C22H16N4O2S (%): C, 66.00; H, 4.00; N, 14.00. Found: C, 65.86; H,
4.16; N, 14.32.
0
0
0
0
0
(400 MHz, CDCl3,
d
): 2.62 (s, 3H, SCH3); 7.19e7.22 (m, 2H, H4, H4 );
0
0
0
0
0
7.65 (dd, 1H, J5 e3 ¼ 1.2 Hz, J5 e4 ¼ 4.9 Hz, H5 ); 7.72 (dd, 1H,
J5e3 ¼ 1.1 Hz, J5e4 ¼ 4.9 Hz, H5); 7.84 (dd, 1H, J3e4 ¼ 3.8 Hz, H3); 7.98
0
0
0
(dd, 1H, J3 e4 ¼ 3.8 Hz, H3 ); 14.37 (s, 1H, NH). MS (m/z, % abun-
dance): 310 (Mþ, 17); 111 (100). Anal Calcd. for C12H10O2N2S3 (%): C,
46.45; H, 3.23; N, 9.03. Found: C, 46.70; H, 3.31; N, 9.39.
4.1.3.9. Methyl N,N0-di(2-phenylquinolin-3-ylcarbonyl)-imidothio-
carbamate (9a). From methyl imidothiocarbamate hydroiodide
a and 2-phenylquinoline-3-carbonyl chloride 9. Recrystallized from
ethanol/DMF. Yield: 31%. m.p. 201e202 ꢄC. IR (KBr): 3430,
4.1.3.3. Methyl N,N0-di(3-chlorothien-2-ylcarbonyl)-imidothiocarba-
mate (3a). From methyl imidothiocarbamate hydroiodide a and 3-
chloro-2-thiophenecarbonyl chloride 3. Recrystallized from
ethanol/DMF. Yield: 18%. m.p.165e166 ꢄC. IR (KBr): 3426,
1701 cmꢂ1 1H NMR (400 MHz, CDCl3,
d): 2.74 (s, 3H, SCH3);
0
0
0
0
7.57e7.59 (m, 7H, H13, H14, H15, H8 , H13 , H14 , H15 ); 7.71 (t, 1H, H8);
7.79 (t, 1H, H7); 7.87 (t, 1H, H7 ); 8.29e8.31 (m, 6H, H6, H12, H16, H6 ,
0
0
1662 cmꢂ1; 1H NMR (400 MHz, CDCl3,
d): 2.59 (s, 3H, SCH3); 7.06 (d,
H12 , H16 ); 8.33 (s, 1H, H3); 8.50 (d, 1H, H9); 8.76 (s, 1H, H3 ); 8.89 (d,
0
0
0
0
0
0
0
1H, J4 e5 ¼ 5.3 Hz, H4 ); 7.08 (d, 1H, J4e5 ¼ 5.3 Hz, H4); 7.51 (d, 1H,
1H, H9 ); 14.34 (s, 1H, NH). MS (m/z, % abundance): 65 (100). Anal
0
H5 ); 7.63 (d, 1H, H5); 13.77 (s, 1H, NH). MS (m/z, % abundance): 379
Calcd. for C34H24N4O2S (%): C, 73.91; H, 4.35; N, 10.14. Found: C,
74.01; H, 4.55; N, 10.30.
(Mþ, 1); 145 (100). Anal Calcd. for C12H8N2O2Cl2S3 (%): C, 37.99; H,
2.11; N, 7.39. Found: C, 37.84; H, 2.37; N, 7.17.
4.1.3.10. Methyl N,N0-di(acridin-9-ylcarbonyl)-imidothiocarbamate
(10a). From methyl imidothiocarbamate hydroiodide a and 9-
acridinecarbonyl chloride 10. Recrystallized from ethanol/DMF.
Yield: 6%. m.p. >300 ꢄC. IR (KBr): 3423, 1776 cmꢂ1 1H NMR
4.1.3.4. Methyl N,N0-di(5-nitrothien-3-ylcarbonyl)-imidothiocarba-
mate (4a). From methyl imidothiocarbamate hydroiodide a and 5-
nitro-3-thiophenecarbonyl chloride 4. Recrystallized from ethanol/
DMF. Yield: 27%. m.p. 225e226 ꢄC. IR (KBr): 3430, 1702 cmꢂ1
;
1H
(400 MHz, CDCl3,
d): 2.82 (s, 3H, SCH3); 6.30 (s, 1H, NH); 6.71 (d, 2H,
0
0
0
0
NMR (400 MHz, CDCl3,
d
): 2.66 (s, 3H, SCH3); 8.33 (s, 1H, H5 ); 8.37
H6, H10); 6.96 (t, 2H, H5, H11); 7.08 (d, 2H, H6 , H10 ); 7.20 (t, 2H, H5 ,
0
0
0
0
(s, 1H, H5); 8.45 (s, 1H, H3); 8.48 (s, 1H, H3 ); 14.51 (s, 1H, NH). MS
(m/z, % abundance): 110 (100). Anal Calcd. for C12H8N4O6S3 (%): C,
36.00; H, 2.00; N, 14.00. Found: C, 36.24; H, 2.26; N, 13.80.
H11 ); 7.50 (t, 2H, H4, H12); 7.73 (t, 2H, H4 , H12 ); 7.78 (d, 2H,þH3, H13);
0
0
8.24 (bs, 2H, H3 , H13 ). MS (m/z, % abundance): 500 (M , 1); 57
(100). Anal Calcd. for C30H20N4O2S$0.1HCl (%): C, 71.48; H, 3.99; N,
11.12. Found: C, 71.29; H, 4.11; N, 11.46.
4.1.3.5. Methyl N,N0-di(5-isoxazol-5-ylcarbonyl)-imidothiocarbamate
(5a). From methyl imidothiocarbamate hydroiodide a and iso-
xazole-5-carbonyl chloride 5. Purified by washed with water aꢂn1d
4.1.3.11. Methyl
N,N0-di(fur-2-ylcarbonyl)-imidoselenocarbamate
(1b). From methyl imidoselenocarbamate hydroiodide b and 2-
furoyl chloride 1. Recrystallized from ethanol. Yield: 48%. m.p.
diethylether. Yield: 50%. m.p. 177e178 ꢄC. IR (KBr): 3387, 1706 cm
;
1H NMR (400 MHz, CDCl3,
d
): 2.67 (s, 3H, SCH3); 7.09 (s, 1H, H4 ); 7.18
131e132 ꢄC. IR (KBr): 3436, 1684 cmꢂ1 1H NMR (400 MHz, CDCl3,
d
):
0
0
0
0
0
0
0
(s, 1H, H4); 8.41 (s, 1H, H3); 8.47 (s, 1H, H3 ); 14.14 (s, 1H, NH). MS (m/
z, % abundance): 96 (100). Anal Calcd. for C10H8O4N4S (%): C, 42.86;
H, 2.86; N, 20.00. Found: C, 42.90; H, 2.92; N, 20.30.
2.44 (s, 3H, SeCH3); 6.58 (dd, 1H, J4 e3 ¼ 1.7 Hz J4 e5 ¼ 3.5 Hz, H4 );
6.62 (dd, 1H, J4e3 ¼ 1.7 Hz, J4e5 ¼ 3.5 Hz, H4); 7.40 (dd, 2H,
0
0
0
0
J5e3 ¼ J5 e3 ¼ 0.8 Hz H5, H5 ); 7.66 (dd, 1H, H3); 7.69 (dd, 1H, H3 );
14.23 (s, 1H, NH). MS (m/z, % abundance): 326 (Mþ, 5); 231 (100).
Anal Calcd. for C12H10N2O4Se (%): C, 44.31; H, 3.08; N, 8.61. Found:
C, 44.44; H, 3.15; N, 8.71.
4.1.3.6. Methyl N,N0-di(thienaphthen-2-ylcarbonyl)-imidothiocarba-
mate (6a). From methyl imidothiocarbamate hydroiodide a and
thianaphthene-2-carbonyl chloride 6. Recrystallized from ethanol/
DMF. Yield: 43%. M.p. 226e227 ꢄC. IR (KBr): 3436, 1685 cmꢂ1
;
1H
4.1.3.12. Methyl N,N0-di(thien-2-ylcarbonyl)-imidoselenocarbamate
(2b). From methyl imidoselenocarbamate hydroiodide b and 2-
thiophenecarbonyl chloride 2. Recrystallized from ethanol/DMF.
Yield: 47%. m.p. 161e162 ꢄC. IR (KBr): 3442, 1668 cmꢂ1 1H NMR
NMR (400 MHz, CDCl3, d): 2.72 (s, 3H, SCH3); 7.49e7.51 (m, 4H, H6,
0
0
0
0
H7, H6 , H7 ); 7.93e7.95 (m, 4H, H5, H8, H5 , H8 ); 8.11 (s, 1H, H3); 8.27
þ
0
(s, 1H, H3 ); 14.68 (s, 1H, NH). MS (m/z, % abundance): 410 (M , 9);
0
0
161 (100). Anal Calcd. for C20H14N2O2S3$0.5HCl (%): C, 56.04; H,
3.39; N, 6.54. Found: C, 56.16; H, 3.40; N, 6.43.
(400 MHz, CDCl3,
d
): 2.47 (s, 3H, SeCH3); 7.17 (dd, 1H, J4 e3 ¼ 3.8 Hz,
0
0
0
J4 e5 ¼ 4.9 Hz,1H, H4 ); 7.20 (dd,1H, J4e3 ¼ 3.8 Hz, J4e5 ¼ 4.9 Hz, H4);

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E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
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0
0
7.65 (dd, 1H, J5 e3 ¼ 1.2 Hz, H5 ); 7.73 (dd, 1H, J5e3 ¼ 1.1 Hz, H5); 7.84
C22H16N4O2Se(%): C, 59.06; H, 3.58; N, 12.53. Found: C, 58.87; H,
3.71; N, 12.59.
0
(dd, 1H, H3); 7.98 (dd, 1H, H3 ); 14.49 (s, 1H, NH). MS (m/z, %
abundance): 358 (Mþ, 1); 111 (100). Anal Calcd. for C12H10N2O2S2Se
(%): C, 40.34; H, 2.80; N, 7.84. Found: C, 40.38; H, 2.96; N, 7.88.
4.1.3.19. Methyl N,N0-di(2-phenylquinolin-3-ylcarbonyl)-imidoseleno-
carbamate (9b). From methyl imidoselenocarbamate hydroiodide
b and 2-phenylquinoline-3-carbonyl chloride 9. Recrystallized froꢂm1
ethanol/DMF. Yield: 24%. m.p. 179e182 ꢄC. IR (KBr): 3449, 1694 cm
4.1.3.13. Methyl N,N0-di(3-chlorothien-2-ylcarbonyl)-imidoselenocar-
bamate (3b). From methyl imidoselenocarbamate hydroiodide b and
3-chloro-2-thiophenecarbonyl chloride 3. Recrystallized froꢂm1
ethanol/DMF. Yield: 4%. m.p. 174e175 ꢄC. IR (KBr): 3426, 1654 cm
1H NMR (400 MHz, CDCl3,
d
): 2.59 (s, 3H, SeCH3); 7.58e7.60 (m, 7H,
0
0
0
0
H13, H14, H15, H8 , H13 , H14 , H15 ); 7.71 (t, 1H, H8); 7.79 (t, 1H, H7); 7.87
1H NMR (400 MHz, CDCl3,
d
): 2.44 (s, 3H, SeCH3); 7.06 (d, 1H,
0 0 0 0
(t, 1H, H7 ); 8.29e8.31 (m, 6H, H6, H12, H16, H6 , H12 , H16 ); 8.33 (s, 1H,
0
0
0
0
0
0
J4 e5 ¼ 5.3 Hz, H4 ); 7.09 (d, 1H, J4e5 ¼ 5.3 Hz, H4); 7.53 (d, 1H, H5 );
7.66 (d, 1H, H5); 13.96 (s, 1H, NH). MS (m/z, % abundance): 426 (Mþ,
1); 161 (100). Anal Calcd. for C12H8N2O2Cl2S2Se (%): C, 33.80; H, 1.88;
N, 6.57. Found: C, 34.14; H, 1.85; N, 6.50.
H3); 8.51 (d, 1H, H9); 8.81 (s, 1H, H3 ); 8.91 (d, 1H, H9 ); 14.46 (s, 1H,
NH). MS (m/z, % abundance): 128 (100). Anal Calcd. for C34H24O2N4Se
(%): C, 68.10; H, 4.01; N, 9.36. Found: C, 68.20; H, 4.22; N, 9.09.
4.1.3.20. Methyl N,N0-di(acridin-9-ylcarbonyl)-imidoselenocarbama-
te (10b). From methyl imidoselenocarbamate hydroiodide b and
9-acridinecarbonyl chloride 10. Recrystallized from ethanol/DMF.
Yield: 18%. m.p. 232e233 ꢄC. IR (KBr): 3398, 1692 cmꢂ1 1H NMR
4.1.3.14. Methyl N,N0-di(5-nitrothien-3-ylcarbonyl)-imidoselenocar-
bamate (4b). From methyl imidoselenocarbamate hydroiodide
b and 5-nitro-3-thiophenecarbonyl chloride 4. Purified by washed
with water and diethylether. Yield: 18%. m.p. 199e200 ꢄC. IR (KBr):
(400 MHz, CDCl3,
d
0
): 2.67 (s, 3H, SeCH3); 6.32 (s, 1H, NH); 6.73 (d,
3430, 1690 cmꢂ1 1H NMR (400 MHz, CDCl3,
d): 2.51 (s, 3H, SeCH3);
2H, J5 e6
¼
J10 e11
¼
7.4 Hz, H6 , H10 ); 6.97 (t, 2H,
0
0
0
0
0
0
0
8.34 (s, 1H, H5 ); 8.37 (s, 1H, H5); 8.46 (s, 1H, H3); 8.50 (s, 1H, H3 );
14.64 (s, 1H, NH). MS (m/z, % abundance): 448 (Mþ, 1); 156 (100).
Anal Calcd. for C12H8N4O6S2Se (%): C, 32.21; H, 1.79; N, 12.53.
Found: C, 32.58; H, 1.91; N, 12.47.
J4e5 ¼ J5e6 ¼ J10e11 ¼ J11e12 ¼ 7.0 Hz, H5, H11); 7.08 (d, 2H, H6, H10);
0
0
0
0
0
0
7.20 (t, 2H, J4 e5 ¼ J11 e12 ¼ 7.4 Hz, H5 , H11 ); 7.52 (t, 2H,
0
0
J3e4 ¼ J12e13 ¼ 7.0 Hz, H4, H12); 7.74e7.76 (m, 4H, H3, H13, H4 , H12 );
0
0
0
0
0
0
8.34 (d, 2H, J3 e4 ¼ J12 e13 ¼ 8.8 Hz, H3 , H13 ). MS (m/z, % abun-
dance): 57 (100). Anal Calcd. for C30H20N4O2Se$0.1HCl (%): C, 65.38;
H, 3.65; N, 10.17. Found: C, 65.55; H, 4.02; N, 9.61.
4.1.3.15. Methyl N,N0-di(5-isoxazol-5-ylcarbonyl)-imidoselenocarba-
mate (5b). From methyl imidoselenocarbamate hydroiodide b and
isoxazole-5-carbonyl chloride 5. Recrystallized from ethanol. Yield:
14%. m.p. 162e163 ꢄC. IR (KBr): 3416, 1703 cmꢂ1 1H NMR (400 MHz,
0
4.1.3.21. Benzyl N,N0-di(fur-2-ylcarbonyl)-imidoselenocarbamate (1-
c). From benzyl imidoselenocarbamate hydrobromide c and 2-
furoyl chloride 1. Recrystallized from ethanol. Yield: 56%. m.p.
CDCl3,
d
): 2.54 (s, 3H, SeCH3); 7.20 (bs, 2H, H4, H4 ); 8.45 (bs, 2H, H3,
þ
0
H3 ); 14.25 (s, 1H, NH). MS (m/z, % abundance): 327 (M , 1); 96
(100). Anal Calcd. for C10H8O4N4Se (%): C, 36.70; H, 2.45; N, 17.12.
Found: C, 36.64; H, 2.36; N, 17.24.
146e147 ꢄC. IR (KBr): 3447,1684 cmꢂ1. 1H NMR (400 MHz, CDCl3,
d):
0
4.51 (s, 2H, SeCH2); 6.58 (bs, 1H, H4 ); 6.62 (bs, 1H, H4); 7.26 (t, 1H,
00
00
00
00
00
00
00
00
00
00
J3 e4 ¼ J4 e5 ¼ 7.3 Hz, H4 ); 7.32 (t, 2H, J2 e3 ¼ J5 e6 ¼ 7.3 Hz, H3
,
00
0
00
00
H5 ); 7.40e7.42 (m, 4H, H5, H5 , H2 , H6 ); 7.66 (bs, 1H, H3); þ7.71 (bs,
4.1.3.16. Methyl
N,N0-di(thienaphthen-2-ylcarbonyl)-imidoseleno-
1H, H3 ); 14.26 (s, 1H, NH). MS (m/z, % abundance): 401 (M , 1); 95
0
carbamate (6b). From methyl imidoselenocarbamate hydroiodide
b and thianaphthene-2-carbonyl chloride 6. Recrystallized froꢂm1
ethanol/DMF. Yield: 2%. m.p. 203e204 ꢄC. IR (KBr): 3430,1678 cm
(100). Anal Calcd. for C18H14O4N2Se (%): C, 53.86; H, 3.49; N, 6.98.
Found: C, 54.06; H, 3.26; N, 6.97.
1H NMR (400 MHz, CDCl3,
d): 2.57 (s, 3H, SeCH3); 7.49e7.51 (m, 4H,
4.1.3.22. Benzyl N,N0-di(thienyl-2-carbonyl)-imidoselenocarbamate
(2c). From benzyl imidoselenocarbamate hydrobromide c and 2-
thiophenecarbonyl chloride 2. Recrystallized from ethanol. Yield:
44%. m.p. 183e184 ꢄC. IR (KBr): 3423, 1676 cmꢂ1 1H NMR (400 MHz,
0
0
0
0
H6, H7, H6 , H7 ); 7.94e7.96 (m, 4H, H5, H8, H5 , H8 ); 7.98 (s, 1H, H3);
0
8.11 (s,1H, H3 ); 14.68 (s,1H, NH). MS (m/z, % abundance): 161 (100).
Anal Calcd. for C20H14N2O2S2Se (%): C, 52.52; H, 3.06; N, 6.13.
Found: C, 52.72; H, 3.14; N, 6.12.
0
0
CDCl3,
d
): 4.57 (s, 2H, SeCH2); 7.19 (dd, 1H, J4 e3 ¼ 3.8 Hz,
0
0
0
J4 e5 ¼ 4.9 Hz, H4 ); 7.21 (dd, 1H, J4e3 ¼ 3.9 Hz, J4e5 ¼ 4.9 Hz, H4);
4.1.3.17. Methyl N,N0-di(3,4-methylenedioxybenzoyl)-imidoselenoca-
rbamate (7b). From methyl imidoselenocarbamate hydroiodide
b and 3,4-methylenedioxy benzoyl chloride 7. Recrystallized from
ethanol/DMF. Yield: 30%. m.p. 178e179 ꢄC. IR (KBr): 3427,
7.26 (t, 1H, J3 e4
¼
J4 e5
¼
00
00
00
00
00
6.9 Hz, H4 ); 7.33 (ddd, 2H,
00
00
00
00
00
00
0
0
0
J2 e3 ¼ J5 e6 ¼ 7.6 Hz, H3 , H5 ); 7.67 (dd, 1H, J3 e5 ¼ 1.2 Hz, H5 );
0
7.73 (dd, 1H, J3e5 ¼ 1.1 Hz, H5); 7.85 (dd, 1H, H3); 8.02 (dd, 1H, H3 );
14.54 (s, 1H, NH). MS (m/z, % abundance): 434 (Mþ,1); 111 (100).
Anal Calcd. for C18H14N2O2S2Se (%): C, 49.88; H, 3.23; N, 6.47.
Found: C, 49.64; H, 3.33; N, 6.57.
1685 cmꢂ1 1H NMR (400 MHz, CDCl3,
d
): 2.47 (s, 3H, SeCH3); 6.08 (s,
0
2H, CH2); 6.10 (s, 2H, CH2 ); 6.89 (d, 1H, J2e3 ¼ 7.9 Hz, H3); 6.94 (d,
0
0
0
1H, J2 e3 ¼ 8.1 Hz, H3 ); 7.50 (s, 1H, H6); 7.60 (d, 1H, H2); 7.78 (s, 1H,
H6 ); 8.03 (d, 1H, H2 ); 14.65 (s, 1H, NH). MS (m/z, % abundance): 433
(Mþ, 1); 95 (100). Anal Calcd. for C18H14N2O6Se (%): C, 49.88; H,
3.01; N, 6.02. Found: C, 50.01; H, 3.27; N, 6.16.
4.1.3.23. Benzyl N,N0-di(3-chlorothien-2-ylcarbonyl)-imidoselenoca-
rbamate (3c). From benzyl imidoselenocarbamate hydrobromide c
and 3-chloro-2-thiophenecarbonyl chloride 3. Recrystallized from
ethanol/DMF. Yield: 11%. m.p. 175e177 ꢄC. IR (KBr): 3430,
0
0
4.1.3.18. Methyl N,N0-di(quinolin-3-ylcarbonyl)-imidoselenocarbam-
ate (8b). From methyl imidoselenocarbamate hydroiodide b and
quinoline-3-carbonyl chloride 8. Recrystallized from ethanol. Yield:
23%. m.p. 181e182 ꢄC. IR (KBr): 3446, 1686 cmꢂ1 1H NMR (400 MHz,
1646 cmꢂ1 1H NMR (400 MHz, CDCl3,
d
): 4.52 (s, 2H, SeCH2); 7.08 (d,
0
0
0
00
2H, J4e5 ¼ J4 e5 ¼ 5.3 Hz, H4, H4 ); 7.27e7.29 (m,1H, H4 ); 7.33 (t, 2H,
00
00
00
00
00
00
00
00
00
00
00
,
J2 e3 ¼ J3 e4 ¼ J4 e5 ¼ J5 e6 ¼ 7.4 Hz, H3 , H5 ); 7.42 (d, 2H, H2
00
0
H6 ); 7.54 (bs, 1H, H5 ); 7.64 (bs, 1H, H5); 14.00 (s, 1H, NH). MS (m/z,
% abundance): 145 (100). Anal Calcd. for C18H12N2O2Cl2S2Se (%): C,
43.03; H, 2.39; N, 5.58. Found: C, 43.00; H, 2.61; N, 5.40.
CDCl3,
d
): 2.63 (s, 3H, SeCH3); 7.64 (t, 1H, J8e7 ¼ J8e9 ¼ 7.8 Hz, H8);
0
0
0
0
0
7.71 (t, 1H, J8 e7 ¼ J8 e9 ¼ 7.8 Hz, H8 ); 7.86 (t, 1H, J7e6 ¼ 7.8 Hz, H7);
0
0
0
0
7.91 (t, 1H, J7 e6 ¼ 7.8 Hz, H7 ); 8.00 (d, 1H, H9); 8.08 (d, 1H, H9 );
8.23 (d, 1H, H6); 8.25 (d, 1H, H6 ); 8.82 (d, 1H, J4e2 ¼ 2.1 Hz, H4); 9.18
4.1.3.24. Benzyl N,N0-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarb-
amate (4c). From benzyl imidoselenocarbamate hydrobromide c
and 5-nitro-3-thiophenecarbonyl chloride 4. Recrystallized from
0
0
0
0
0
(d,1H, J4 e2 ¼ 1.7 Hz, H4 ); 9.56 (d,1H, H2); 9.84 (d,1H, H2 ); 15.09 (s,
1H, NH). MS (m/z, % abundance): 128 (100). Anal Calcd. for

E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
273
ethanol. Yield: 26%. m.p. 156e157 ꢄC. IR (KBr): 3426, 1692 cmꢂ1 1H
NMR (400 MHz, CDCl3, ): 4.54 (s, 2H, SeCH2); 7.30e7.32 (m, 1H,
H3, H6, H12, H16, H6 , H12 , H16 ); 8.48 (d,1H, H9); 8.75 (s,1H, H3 ); 8.88
0
0
0
0
0
d
(d,1H, H9 ); 14.49 (s,1H, NH). MS (m/z, % abundance): 204 (100). Anal
00
00
00
00
00
00
00
00
00
00
00
H4 ); 7.36 (t, 2H, J2 e3 ¼ J3 e4 ¼ J4 e5 ¼ J5 e6 ¼ 7.3 Hz, H3 , H5 );
Calcd. for C40H28N4O2Se$0.25HCl (%): C, 70.16; H, 4.13; N, 8.19. Found:
C, 70.42; H, 4.17; N, 7.98.
00
00
0
0
7.43 (d, 2H, H2 , H6 ); 8.41e8.43 (m, 4H, H5, H3, H3 , H5 ); 14.65 (s,
1H, NH). MS (m/z, % abundance): 91 (100). Anal Calcd. for
C18H12N4O6S2Se (%): C, 38.60; H, 2.32; N, 10.01. Found: C, 38.09; H,
2.30; N, 9.78.
4.1.3.30. Benzyl N,N0-di(acridin-9-ylcarbonyl)-imidoselenocarbamate
(10c). From benzyl imidoselenocarbamate hydrobromide c and 9-
acridinecarbonyl chloride 10. Recrystallized from ethanol/DMF.
Yield: 29%. m.p. 144e146 ꢄC. IR (KBr): 3606, 1709 cmꢂ1 1H NMR
4.1.3.25. Benzyl N,N0-di(5-isoxazol-5-ylcarbonyl)-imidoselenocarba-
mate (5c). From benzyl imidoselenocarbamate hydrobromide c
and isoxazole-5-carbonyl chloride 5. Recrystallized from ethanol.
Yield: 19%. m.p. 117e119 ꢄC. IR (KBr): 3393, 1706 cmꢂ1 1H NMR
0
(400 MHz, CDCl3,
d
0
): 4.62 (s, 2H, SeCH2); 6.46 (s, 1H, NH); 6.71 (d,
0
0
0
0
0
2H, J5 e6
¼
J10 e11
¼
8.0 Hz, H6 , H10 ); 6.94 (t, 2H,
J4e5 ¼ J5e6 ¼ J10e11 ¼ J11e12 ¼ 7.7 Hz, H5, H11); 7.03 (d, 2H, H6, H10);
0
0
0
0
0
0
00
,
(400 MHz, CDCl3,
(t, 1H, J3 e4
d): 4.58 (s, 2H, SeCH2); 7.14 (bs, 2H, H4, H4 ); 7.28
7.19 (t, 2H, J4 e5 ¼ J11 e12 ¼ 8.0 Hz, H5 , H11 ); 7.36e7.37 (m, 3H, H3
00
00
00
00
00
00
H4
00
00
00
¼
J4 e5
¼
7.5 Hz, H4 ); 7.34 (t, 2H,
,
H5 ); 7.49e7.52 (m, 2H, H2
,
H6 ); 7.56 (t, 2H,
00
00
00
00
00
00
00
00
0
0
J2 e3 ¼ J5 e6 ¼ 7.5 Hz, H3 , H5 ); 7.44 (d, 2H, H2 , H6 ); 8.45þ(bs, 2H,
J3e4 ¼ J12e13 ¼ 7.7 Hz, H4, H12); 7.74e7.77 (m, 4H, H3, H13, H4 , H12 );
0
0
0
0
0
0
0
H3, H3 ); 14.28 (s, 1H, NH). MS (m/z, % abundance): 401 (M , 1); 91
8.33 (d, 2H, J3 e4 ¼ J12 e13 ¼ 8.0 Hz, 2H, H3 , H13 ). MS (m/z, %
abundance): 237 (100). Anal Calcd. for C36H24N4O2Se$0.75HCl (%):
C, 66.42; H, 3.81; N, 8.61. Found: C, 66.78; H, 4.16; N, 8.36.
(100). Anal Calcd. for C16H12N4O4Se$0.8HCl (%): C, 44.42; H, 2.78; N,
12.96. Found: C, 44.59; H, 2.84; N, 12.92.
4.1.3.26. Benzyl N,N0-di(thienaphthen-2-ylcarbonyl)-imidoselenocar-
bamate (6c). From benzyl imidoselenocarbamate hydrobromide c
and thianaphthene-2-carbonyl chloride 6. Recrystallized from
ethanol/DMF. Yield: 36%. m.p. 192e193 ꢄC. IR (KBr): 3425,
4.2. Cytotoxic and antiproliferative activities
Cell culture materials were obtained from BD Bioscience. Stock
solutions of the different compounds were prepared as follows: All
compounds were dissolved in DMSO at a concentration between
0.01 and 0.005 M. Sterile filtration of the compounds was achieved
1677 cmꢂ1 1H NMR (400 MHz, CDCl3,
d): 4.65 (s, 2H, SeCH2); 7.31 (t,
00
00
00
00
00
00
00
00
00
1H, J3 e4 ¼ J4 e5 ¼ 7.4 Hz, H4 ); 7.38 (t, 2H, J2 e3 ¼ J5 e6 ¼ 7.4 Hz,
00
00
00
00
0
0
H3 , H5 ); 7.49 (m, 6H, H6, H7, H2 , H6 , H6 , H7 ); 7.96e7.99 (m, 4H,
0
0
0
H5, H8, H5 , H8 ); 8.10 (s, 1H, H3); 8.29 (s, 1H, H3 ); 14.71 (s, 1H, NH).
MS (m/z, % abundance): 91 (100). Anal Calcd. for C26H18N2O2S2Se
(%): C, 58.54; H, 3.38; N, 5.25. Found: C, 58.22; H, 3.64; N, 5.17.
using 0.2 mm filter disks. Serial dilutions with supplemented
medium were prepared daily to a final concentration of less than 2%
DMSO in cell culture.
The cytotoxic effect of each substance was tested at five different
concentrations between 0.01 and 100 mM and viability was deter-
mined according to the protocol of Denizot and Lang [47].
4.1.3.27. Benzyl N,N0-di(3,4-methylenedioxybenzoyl)-imidoselenoca-
rbamate (7c). From benzyl imidoselenocarbamate hydrobromide c
and 3,4-methylenedioxy benzoyl chloride 7. Recrystallized from
ethanol/DMF. Yield: 31%. m.p. 203e205 ꢄC. IR (KBr): 3448,
Hep-G2 (liver carcinoma), HT-29 (colon carcinoma), K-562
(leukemia), MCF-7 (breast carcinoma) and PC-3 (prostate carcinoma)
cell lines were routinely maintained in RPMI medium (Lonza/12-702)
supplemented with10% fetal bovineserum and 1% antibioticsintissue
culture flasks at 37 ꢄC and 5% CO2. MCF-10A (nontumorigenic breast
epithelial cells) were routinely maintained in MEBM medium (Lonza/
CC-3150)supplementedwith1%antibioticsandasupplementcocktail
containing human Epithelial Growth factor, Bovine Pituitary Extract,
Insulin, Hydrocortisone and Gentamicin A (Lonza/CC-4136) in tissue
culture flasks at 37 ꢄC and 5% CO2. Culture medium was exchanged at
3-day intervals. MTT-assays were performed with 1 ꢁ 104 Hep-G2
cells/well, 4 ꢁ 103 HT-29 cells/well, 4 ꢁ 103 K-562 cells/well,
1.2 ꢁ104 MCF-7 cells/well, 7 ꢁ 103 MCF-10A cells/well and 2 ꢁ103 PC-
3 cells/well in 96-well plates. Briefly, cells were incubated in the
presence of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide] and analyzed for their ability to generate a purple
formazandye. After incubationfor 72 h, theabsorbancewas measured
at a wavelength of 590 nm and the ratio of viable cells was calculated.
Results are expressed as GI50, the concentration that reduces by 50%
the growth of treated cells with respect to untreated controls, TGI, the
concentration that completely inhibits cell growth, and LC50, the
concentration that kills 50% of the cells. Data were obtained from at
least 3 independent experiments performed in quadruplicate.
1673 cmꢂ1 1H NMR (400 MHz, CDCl3,
d
): 4.55 (s, 2H, SeCH2); 6.08 (s,
0
2H, CH2); 6.11 (s, 2H, CH2 ); 6.90 (d, 1H, J2e3 ¼ 8.2 Hz, H3); 6.95 (d,
0
0
0
00
1H, J2 e3 ¼ 8.1 Hz, H3 ); 7.27e7.29 (m, 1H, H4 ); 7.33 (t, 2H,
00
00
00
00
00
00
00
00
00
00
00
,
J2 e3 ¼ J3 e4 ¼ J4 e5 ¼ J5 e6 ¼ 7.4 Hz, H3 , H5 ); 7.43 (d, 2H, H2
00
0
H6 ); 7.49 (s, 1H, H6); 7.60 (d, 1H, H2); 7.79 (s, 1H, H6 ); 8.05 (d, 1H,
0
H2 ); 14.70 (s, 1H, NH). MS (m/z, % abundance): 149 (100). Anal
Calcd. for C24H18N2O6Se (%): C, 56.58; H, 3.54; N, 5.50. Found: C,
56.53; H, 3.86; N, 5.48.
4.1.3.28. Benzyl N,N0-di(quinolin-3-ylcarbonyl)-imidoselenocarbam-
ate (8c). From benzyl imidoselenocarbamate hydrobromide c and
quinoline-3-carbonyl chloride 8. Recrystallized from ethanol/
DMF. Yield: 19%. m.p. 178e179 ꢄC. IR (KBr): 3417, 1684 cmꢂ1 1H
NMR (400 MHz, CDCl3,
d): 4.70 (s, 2H, SeCH2); 7.31 (t, 1H,
00
00
00
00
00
00
00
00
00
00
J3 e4 ¼ J4 e5 ¼ 7.4 Hz, H4 ); 7.38 (t, 2H, J2 e3 ¼ J5 e6 ¼ 7.4 Hz, H3
,
00
00
00
H5 ); 7.50 (d, 2H, H2 , H6 ); 7.70 (t, 1H, J7e8 ¼ J8e9 ¼ 7.7 Hz, H8); 7.73
0
0
0
0
0
0
(t, 1H, J7 e8 ¼ J8 e9 ¼ 7.7 Hz, H8 ); 7.93e7.96 (m, 2H, H7, H7 ); 8.03 (d,
0
1H, H9); 8.10 (d, 1H, H9 ); 8.24 (d, 1H, J6e7 ¼ 8.5 Hz, H6); 8.32 (d, 1H,
0
0
0
J6 e7 ¼ 8.3 Hz, H6 ); 8.83 (d, 1H, J2e4 ¼ 1.8 Hz, H4); 9.20 (d, 1H,
0
0
0
0
J2 e4 ¼ 1.0 Hz, H4 ); 9.54 (d, 1H, H2); 9.86 (d, 1H, H2 ); 15.11 (s, 1H,
NH). MS (m/z, % abundance): 524 (Mþ, 1); 91 (100). Anal Calcd. for
C28H20N4O2Se (%): C, 64.24; H, 3.82; N, 10.71. Found: C, 63.80; H,
3.72; N, 10.67.
The CV method was performed according to a modification of
the protocol described by Kueng et al. [58]. Each compound was
tested at eight different concentrations ranging from 0.01 to
100 mM. After incubation for 72 h, cells were fixed with 100 mL of
formaldehyde (4%) and were left for at least 20 min. The plates were
stained with 0.1% crystal violet solution for 30 min. After being
4.1.3.29. Benzyl N,N0-di(2-phenylquinolin-3-ylcarbonyl)-imidoselen-
ocarbamate (9c). From benzyl imidoselenocarbamate hydrobromide
c and 2-phenylquinoline-3-carbonyl chloride 9. Recrystallized froꢂm1
ethanol/DMF. Yield: 22%. m.p.180e181 ꢄC. IR (KBr): 3426,1698 cm
washed with water, 200
mL of acetic acid was added and the
1H NMR (400 MHz, CDCl3,
d
): 4.63 (s, 2H, SeCH2); 7.30e7.32 (m, 3H,
absorbance at 590 nm was measured by an automatic microplate
reader. Data were obtained from at least three independent
experiments performed in quadruplicate.
00
00
00
00
00
H3 , H4 , H5 ); 7.44 (d, 2H, H2 , H6 ); 7.60e7.62 (m, 8H, H8, H13, H14
,
0
0
0
0
0
H15, H8 , H13 , H14 , H15 ); 7.70e7.72 (m, 2H, H7, H7 ); 8.23e8.25 (m, 7H,

274
E. Ibáñez et al. / European Journal of Medicinal Chemistry 46 (2011) 265e274
[21] D.
Desai,
S.V.
Madhunapantula,
K.
Gowdahalli,
A.
Sharma,
4.3. Evaluation of cell cycle progression and apoptosis induction
R. Chandagaludoreswamy, K. El-Bayoumi, G.P. Robertson, S. Amin, Bioorg.
Med. Chem. Lett. 20 (2010) 2038e2043.
For breast adenocarcinoma MCF-7 cells, the apoptotic status and
cell cycle analysis of the cells were determined using the Apo-
Direct kit (BD Pharmingen), based on the TUNEL technique, under
the conditions described by the manufacturer. Briefly, for fixation
step cells were suspended in 1% paraformaldehyde in PBS (pH 7.4)
at a concentration of 1 ꢁ 106 cells/mL, incubated on ice for 1 h,
collected by centrifugation, washed, adjusted to 1 ꢁ 106 cells/mL in
70% ice cold ethanol and incubated at ꢂ20 ꢄC for 30 min. After
fixation, cells were recovered by centrifugation, washed, resus-
pended in FITC dUTP-DNA labelling solution and incubated for 1 h
at 37 ꢄC. Cells were then rinsed, resuspended in PI/RNase staining
buffer, incubated in the dark for 30 min at RT and analyzed using
a Coulter Epics XL flow cytometer.
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