Synthesis and antiproliferative activity of novel methylselenocarbamates

Article abstract of DOI:10.1016/j.ejmech.2014.06.076

A series of new aliphatic, aromatic and heteroaromatic carbamate derivatives containing a methylseleno moiety were synthesized and evaluated in vitro for their cytotoxic activity against a panel of human cell lines including CCRF-CEM (lymphoblastic leukaemia), K-562 (lymphocytic leukaemia), HT-29 (colon carcinoma), HTB-54 (lung carcinoma), PC-3 (prostate carcinoma), MCF-7 (breast adenocarcinoma), 184B5 (non-malignant, mammary gland derived) and BEAS-2B (non-malignant, derived from bronchial epithelium). Most of the compounds are highly cytotoxic with GI₅₀ values below 10 μM in every tested tumour cell line. Based on its cytotoxic parameters, selectivity index and ADME profile, the biological activity of compound 2, the propyl derivative, was further analysed in CCRF-CEM and HTB-54 cells. Results showed that this compound is able to induce apoptosis in a time- and dose-dependent manner. Involvement of caspases in cell death induction by 2 was detected. Besides, compound 2 was also able to induce cell cycle arrest at G₀/G₁ in CCRF-CEM cells and at G₂/M in HTB-54 cells.

Full text of DOI:10.1016/j.ejmech.2014.06.076

European Journal of Medicinal Chemistry 83 (2014) 674e684
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
methylselenocarbamates
,
*
Beatriz Romano a, María Font a, Ignacio Encío b, Juan Antonio Palop a
,
Carmen Sanmartín a
a
ꢀ
ꢀ
Departamento de Química Organica y Farmaceutica, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain
b
~
Departamento de Ciencias de la Salud, Universidad Pública de Navarra, Avda. Baranain s/n, 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:
A series of new aliphatic, aromatic and heteroaromatic carbamate derivatives containing a methylseleno
moiety were synthesized and evaluated in vitro for their cytotoxic activity against a panel of human cell
lines including CCRF-CEM (lymphoblastic leukaemia), K-562 (lymphocytic leukaemia), HT-29 (colon
carcinoma), HTB-54 (lung carcinoma), PC-3 (prostate carcinoma), MCF-7 (breast adenocarcinoma), 184B5
(non-malignant, mammary gland derived) and BEAS-2B (non-malignant, derived from bronchial
Received 2 May 2014
Received in revised form
11 June 2014
Accepted 30 June 2014
Available online 1 July 2014
epithelium). Most of the compounds are highly cytotoxic with GI50 values below 10 mM in every tested
tumour cell line. Based on its cytotoxic parameters, selectivity index and ADME profile, the biological
activity of compound 2, the propyl derivative, was further analysed in CCRF-CEM and HTB-54 cells.
Results showed that this compound is able to induce apoptosis in a time- and dose-dependent manner.
Involvement of caspases in cell death induction by 2 was detected. Besides, compound 2 was also able to
induce cell cycle arrest at G0/G1 in CCRF-CEM cells and at G2/M in HTB-54 cells.
Keywords:
Carbamate
Methylseleno
Cytotoxicity
Apoptosis
Cell cycle arrest
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
activity through induction of apoptosis, cell cycle arrest and/or
modulation of different kinases [14e23]. Among these compounds,
Cancer is a serious clinical problem: it affects millions of patients
worldwide, reduces life quality and is one of the leading causes of
death [1]. At present, there are many antitumour drugs available for
clinical use. Despite this fact, treatment of cancer still presents
many obstacles and both, lack of selectivity with the consequent
side-effects and occurrence of intrinsic or acquired resistance of
tumours to chemotherapy point out the necessity to develop new
anticancer therapies [2]. In this context, selenium compounds have
attracted considerable attention during the last decade since they
have been shown to inhibit tumour development and growth in a
variety of cancer [3e9]. In particular, converging lines of evidence
support the hypothesis that methylselenol is a key intermediate
metabolite for effective cancer prevention and treatment by
methyl-selenium compounds [10e13].
we described some methylseleno derivatives whose cytotoxic ac-
tivity depends on the release of methylselenol [10,14,18,22,24].
Upon this, and considering the chemical structural patterns pre-
viously described by us, we designed a general structure for the
new compounds presented here. This structure (Fig. 1) keeps a
central scaffold of Se-methylselenourea, a group whose effective-
ness to release methylselenol has been proven. Since carbamates
are considered privileged scaffolds in cancer therapy [25e28],
keeping a general standard of molecular symmetry [18] and based
on a hybrid design approach, here, we try the presence of two
carbamate functions attached to this group. Carbamate functions
have several roles: first, they act as a link between central and
ending scaffolds enabling their chemical accessibility; second, they
slightly increase polarity in this area as compared to the structures
that we have previously described [14,16,18,21,23]; and third, they
theoretically enable the hydrolysis of the compounds towards
anionic species that could act as prodrugs. Besides, to explore the
influence of the ending pieces of the symmetric molecule on its
activity we introduced either an aliphatic or an aromatic structure
in these zones.
Over the last years we have been involved in the development,
design and synthesis of structurally modified selenium derivatives,
some of which exhibited significant cytotoxic and antiproliferative
* Corresponding author. Department of Organic and Pharmaceutical Chemistry,
University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain.
E-mail address: jpalop@unav.es (J.A. Palop).
The use of fatty acids as adjuvants in cancer treatment is well
established. In vitro studies have displayed the capacity of these
http://dx.doi.org/10.1016/j.ejmech.2014.06.076
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
675
Fig. 1. General structure of new pharmacophoric hybrids.
acids, categorized by the number of carbon atoms in the aliphatic
chain, to increase the activity of standard chemotherapeutic agents
in a range of cell lines [29e33]. Thus, to juggle with the carbamate
group and to imitate the structure of short chain fatty acids, we
performed a bioisosteric replacement of a methylene group by an
oxygen atom of ether, which causes a slight increase in polarity
while keeping a similar bond angle and chain flexibility. Following
this approach, here we tested short chain fatty acids (4e7 atoms in
length). On the other hand, we selected a series of aromatic rings
[14] bearing either one electron-donating group or one electron-
withdrawing group to test aromatic endings. These rings were
either directly linked to the carbamate, or separated from it by a
methylene group. In two cases, the ring was a polycyclic system
such as Fmoc [34] or a heteroaromatic unit such as the benzo[b]
thiophene-1,1-dioxide [35].
Additionally, the cytotoxic activity of every synthesised com-
pound was tested against a panel of six human tumour cell lines as
a representative selection of solid, liquid and hormone-dependent
tumours, including breast adenocarcinoma (MCF-7), colon carci-
noma (HT-29), lymphocytic leukaemia (K-562), lymphoblastic
leukaemia (CCRF-CEM), prostate carcinoma (PC-3) and lung carci-
noma (HTB-54). As a guide with regard to selectivity, two cell lines
derived from non-malignant cells, one from mammary gland
(184B5) and the other from bronchial epithelium (BEAS-2B), were
also tested. Moreover, to further analyse the mechanism of action of
the new compounds, the ability to induce apoptosis and cell cycle
arrest of derivative 2, one of the most active and selective com-
pounds, was also tested.
Scheme 1. Synthesis of compounds 1e16. (A) Preparation of the corresponding
chloroformate. (B) Preparation of Se-methylselenourea. (C) Synthesis of compounds
15e16. (D) Synthesis of compounds 1e14.
methylimidoselenocarbamate hydroiodide in different conditions
(temperature, solvents, catalyst) always yielded 4-nitrophenol.
Hence, to test the influence of the nitro group with regards bio-
logical activity, we decided to replace it with a nitrobenzyl group
(13).
The same traditional synthetic procedure was used for the
preparation of the bis derivative of 1,1-dioxobenzo[b]thiophenyl.
Unfortunately, this procedure failed to afford the expected product
and the monomer derivative (15) was isolated instead (78% yield).
This prompted us to seek alternative routes to prepare the corre-
sponding 1,1-dioxobenzo[b]thiophenylbiscarbamate. To our sur-
prise, modification of the reaction conditions (temperature,
solvents, molar ratio, catalyst…) resulted in decomposition of both,
reagents and monomer yielding complex reaction mixtures.
Despite some of the derivatives (7, 10 and 13) were isolated in a
poor yield because their purification was troublesome, all newly
synthesized compounds are pure and stable and their structures
were confirmed by spectroscopic (IR, 1H NMR and 13C NMR), mass
spectrometry (MS) and elemental analysis.
2. Results and discussion
2.1. Chemistry
2.2. Biological evaluation
The general synthetic route used to obtain the desired bisimi-
doselenocarbamates was straightforward and it is outlined in
Scheme 1. The synthesis was performed from Se-methylselenourea
hydroiodide, obtained by a method previously developed in our
laboratory [18], and the corresponding chloroformates, synthesized
according to standard procedures [36,37] or purchased commer-
cially. The reaction was carried out stirring at room temperature for
24ꢀ72 h in a 1:2 molar ratio using dried chloroform and in the
presence of pyridine. When necessary, dimethylaminopyridine
(DMAP) was also included as a basic catalyst. After isolation and
purification of the compounds, this procedure gave yields within
the range of 5e78%.
Since chloroformates bearing different groups on the aryl ring
behave differently, this reaction outcome might be due to the in-
fluence of these substituents. For instance, replacement of electron-
donating groups on the aromatic ring (8 and 9) by electron-
withdrawing groups (10 and 11) offered a noticeable decrease in
yield from 41% to 5%. Moreover, all our attempts to generate the
corresponding 4-nitrophenylbiscarbamate were unsuccessful since
2.2.1. Cytotoxicity
Every synthesised compound was screened for its cytotoxic and
antiproliferative activity against CCRF-CEM, K-562, MCF-7, HT-29,
PC-3 and HTB-54 cells. Cytotoxicity assays were performed by the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide] method as previously described [38]. The cytotoxic effect of
each substance was tested at five different concentrations between
0.01 and 100 mM. Obtained results are shown in Table 1 and are
expressed as GI50, i.e. the concentration that reduces by 50% the
growth of treated cells compared to untreated controls, TGI, the
concentration that completely inhibits cell growth, and LC50, the
concentration that kills 50% of the cells.
As shown in Table 1, CCRF-CEM and HTB-54 cells were generally
more sensitive and K-562 more resistant to the cytotoxic effect. In
fact, most of the target compounds were active as cytostatic agents
against HTB-54, PC-3, CCRF-CEM and MCF-7 cells, with GI50 values
below 5.5 mM. Moreover, compounds 1, 2, 3, 6, 7, 10, 12 and 15 in
HTB-54 cells, 1, 5, 7, and 10 in PC-3 cells, 1, 2, 5, 7, 9, 10, 11, 12, 13, 14,
15 and 16 in CCRF-CEM cells, 1, 7, 10 and 11 in HT-29 cells and 15 in
the
reaction
of
4-nitrophenylchloroformate
with

676
B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
Table 1
Cytotoxic activities (GI50, TGI and LC50
mM) of the compounds in human tumour cell lines.
Ref.
R
HTB-54a
GI50
PC-3b
GI50
CCRF-CEMc
GI50 TGI
2.97 5.44
K-562d
GI50
HT-29e
GI50
MCF-7f
GI50
TGI
LC50
TGI
LC50
LC50
TGI
LC50
TGI
LC50
9.37
TGI
LC50
1
2
3
4
5
6
7
8
Ethyl
Propyl
Butyl
Hexyl
Isobutyl
Allyl
Phenyl
4-Methoxy-Ph 3.42
4-Methyl-Ph
4-Cl-Ph
4-F-Ph
2.29
2.17
2.74
6.05
5.67
5.81
9.81
9.16
8.88
2.91
5.30
5.30
5.64 8.37
7.91
7.78
6.77 39.76 82.10
2.55 6.18 9.82
48.01 11.65 43.92 76.19
2.92 6.15
5.30 9.04
4.93 8.02
4.78 8.51
5.91 27.02 67.44
7.17 27.77 66.15
5.17 8.30
4.10 7.97
7.39 26.92 73.12
5.42 8.44
5.62 9.27
4.93 8.70
5.80 9.80
60.17
36.47
45.66
8.91 50.08 2.37 5.07
9.35 53.67 4.38 8.77
9.74 45.44 82.04
5.13 17.57 65.14
6.96 38.53 74.05
3.60 8.24
4.43 8.01
2.42 5.41
3.03 7.32
4.49 8.13
2.92 6.24
2.27 5.99
22.40 51.96 81.51
5.43 11.24 60.15 7.11 28.41 65.04 21.25 48.62 76.08
3.75
6.68 22.58 63.35 6.95 32.18 66.53
3.51
4.00
6.28 14.14 60.87 2.25 4.99
3.14
4.12
20.53 46.53 72.53 1.85 4.70
7.39 33.38 68.72 2.39 5.09
5.95 18.13 60.13 0.70 3.35
5.84 11.85 56.72 0.49 2.45
5.48
0.63
2.24
2.41
2.16
6.73
6.08
5.66
7.88
6.55
6.09
5.77
5.90
33.11
9.76
9.15
51.18
21.54
9.63
12.80
8.84
6.25 8.75
3.25 5.97
8.70
5.29 34.46 69.33
5.06 29.00 65.61
4.68 19.16 81.59
49.73
43.30
8.39
36.97
44.37
9.56
43.08
45.31
5.98 8.54
2.79 5.26
7.15 17.87 3.96 7.57
7.74
32.54 10.86 48.62 86.39
9
2.56
2.55
1.41
2.96
0.98
0.91
1.09
2.19
n.d.i
9.64
7.73
7.98
8.14
7.55
7.79
7.00
6.47
7.86
5.88 29.93 65.24
7.53 36.99 71.57
21.80 52.51 83.22
47.95
59.43
58.24
53.84
10
11
12
13
14
15
16
6.06 8.98
3.17 5.57
7.79 34.33 3.24 5.69
9.72
Benzyl
4.82 10.72 56.54 11.58 40.41 69.25
4-Nitrobenzyl
Fmoc
36.88 70.71
14.81 43.83 72.85
6.08 34.37 69.08
4.99 28.60 64.70
4.51 28.57 69.31
6.81 34.19 68.71 11.89 42.49 73.09
4.17 8.01
5.50 14.63 56.25
6.28 24.87 65.48
9.43
5.20
6.50
n.d.
54.42
9.30
54.78
n.d.
39.50
5.23 8.27
3.10 5.68
4.63 8.22
37.08
8.26
77.10
Benzodioxog
4-Methyl-Phg
Etoposideh
Cisplatinh
9.59 60.29 2.00 4.93
3.98 79.43 1.58 50.47 89.95 12.59 >100 >100 31.62 >100 >100 19.95 >100 >100
5.01 >100 >100 7.95 >100 >100 3.16 >100 >100
32.73 50.00
5.01 50.12 >100 1.72 >100 >100
a
Lung carcinoma.
b
c
d
e
f
Prostate carcinoma.
Lymphoblastic leukaemia.
Lymphocytic leukaemia.
Colon carcinoma.
Breast adenocarcinoma.
Monosubstituted derivatives.
g
h
i
NCI data (http://dtp.nci.nih.gov/).
No data.
MCF-7 cells exhibited a strong cytotoxic activity with LC50 below
10 M.
Regarding the influence of the side chain, it can be observed that
2B)/TGI (HTB-54) and LC50 (BEAS-2B)/LC50 (HTB-54) for lung cells.
Ratios between 3 and 6 refer to moderate selectivity; ratios greater
than 6 indicate high selectivity toward the corresponding cell line,
while compounds not meeting either of these criteria rated non-
selective [39]. Based upon these parameters, no selectivity was
m
elongation of alkyl chains from ethyl (1) to hexyl (4) led to a
noticeable decrease in cytotoxicity in HTB-54, PC-3, CCR-CEM and
HT-29 cells. Moreover, since 5 was cytotoxic for PC-3 and CCRF-
CEM cells and cytostatic for HTB-54 cells, while 3 was cytotoxic
for HTB-54 cells and cytostatic for PC-3 and CCRF-CEM cells,
comparison between butyl (3) and isobutyl (5) derivatives revealed
that activity profiles are also dependent on the presence or absence
of a ramification in the alkyl chain. Besides, comparison between
propyl (2) and allyl (6) derivatives revealed that attachment of an
unsaturated unit instead of a saturated one resulted in a decrease in
cytotoxic activity in PC-3, K-562, CCRF-CEM and MCF-7 cells.
As for compounds bearing aromatic rings, the anticancer activity
was unaffected by the nature (electron-donating or electron-
withdrawing) of the substituent in the phenyl ring. For instance,
replacement of the hydrogen atom (7) by a methyl group (9) or by a
Table 2
Cytotoxic activities (GI50, TGI and LC50 mM) of the compounds in 184B5 and BEAS-2B
cells.
Compound
BEAS-2Ba
GI50
184B5b
GI50
TGI
6.22
48.42
28.21
50.56
6.36
6.52
7.00
7.00
6.30
5.75
5.98
40.60
34.35
5.69
LC50
8.11
73.68
65.34
75.60
8.39
9.00
9.22
9.22
9.09
TGI
6.01
LC50
8.15
1
2
3
4
5
6
7
8
3.67
23.16
7.22
25.51
4.32
4.05
4.79
4.79
3.52
3.53
3.95
13.50
3.54
3.45
4.96
6.20
3.86
3.01
4.15
5.69
4.15
3.22
4.39
4.39
3.82
4.31
4.21
6.12
5.14
4.71
1.32
3.81
5.55
6.41
8.99
6.17
5.78
6.47
6.47
6.21
6.31
6.18
11.87
8.51
7.41
4.28
6.24
8.09
8.67
47.23
8.19
8.34
8.55
8.55
8.59
8.23
8.16
55.24
42.68
23.69
7.23
8.66
chlorine atom (10) yielded GI50 values of 4.10, 5.42 and 5.62
MCF-7 cells, 2.79, 2.25 and 3.17 M in CCRF-CEM cells, 2.42, 4.49
and 2.92 M in HT-29 cells and 2.16, 2.56 and 2.55 M in HTB-
mM in
m
mM
m
m
54 cells. Similarly, the presence (12) or absence (7) of a methylene
group as spacer between the carbamic moiety and the phenyl ring
had little or no influence on the activity in any of the tumour cell
lines.
In order to determine the selectivity index (SI) of the com-
pounds, cytotoxicity of the methylselenocarbamate derivatives was
also evaluated against 184B5 and BEAS-2B, two cell lines derived
9
10
11
12
13
14
15
16
7.97
8.01
67.70
65.63
7.94
44.58
58.05
from non-malignant cells (Table 2). The SI was calculated from GI50
,
8.92
22.55
TGI and LC50 values according to the formula SI ¼ GI50 (184B5)/GI50
(MCF-7), TGI (184B5)/TGI (MCF-7) and LC50 (184B5)/LC50 (MCF-7)
for breast cells, and SI ¼ GI50 (BEAS-2B)/GI50 (HTB-54), TGI (BEAS-
a
Bronchial epithelium cell culture derived from non-malignant cells.
Mammary gland cell culture derived from non-malignant cells.
b

B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
677
found with breast cell lines. Compounds 2, 3, 12 and 15 however
exhibited a good SI in lung cell lines (Table 3).
value was also calculated. Results show that compound 2 possess
drug like molecule (DLM) features.
To compare activity among compounds, GI50 MID (average
sensitivity of a cell line towards the tested compounds) was
calculated and differences against MID for every compound were
represented as a mean graph midpoint (MG-MID) (Fig. 2). Most
active compounds in a particular cell line of the screen are char-
acterized by a high negative difference to the MID value. Positive
values denote smaller activities. Thus, though most of the com-
pounds show up similar activity profiles among cell lines, com-
pounds 2, 4 and 12 showed a less toxic effect in BEAS-2B cells.
Besides, compound 2 (SI 11, 9 and 8 for GI50, TGI and LC50 in BEAS-
2B) displays the highest negative difference in K-562, the less
sensitive cell line to the compounds.
In addition, under our experimental conditions, most of the
compounds displayed comparable or higher in vitro cytotoxicity
values than the traditional anticancer agents cisplatin and etopo-
side, which were used here as a reference. For example, in HT-29
and MCF-7 cells, the whole series was more active than etopo-
side. A similar behaviour was observed in HTB-54 cells with the
exception of compound 4 (Table 1 and Fig. 2). It is remarkable that
in K-562, the least sensitive cell line, compound 2 is five-fold better
8 (in terms of cytotoxicity) than etoposide and two-fold than
cisplatin.
Considering its selectivity in lung cells as indicative of a superior
therapeutic index and its cytotoxicity on a broad-spectrum of the
tested cell lines, compound 2 was selected to further analyse the
molecular mechanisms involved in the antiproliferative activity of
the new methylselenocarbamates. Curves with the original data
from which the GI50, TGI and LC50 values for compound 2 were
calculated are shown in Fig. 3.
As a filter for drug-like properties, Lipinski descriptors for
bioavailability estimation were calculated for compound 2 using
the freely accessible program MIPC (Molinspiration Property
Calculator) [40]. Obtained results are shown in Table 4. Lipinski
parameters describe important molecular properties for drug
pharmacokinetics in the human body, especially their oral ab-
sorption. According to them, an oral active drug must not violate
more than one of the following criteria: <5 hydrogen donors
(nOHNH), <10 hydrogen acceptors (nON), MW <500, log Pcalc <5.
Moreover, TPSA a predictive indicator of membrane penetration
2.2.2. Determination of apoptosis and effects on cell cycle
progression
Apoptosis and cell cycle arrest are two major targets of many
anticancer drugs such as doxorubicin and camptothecin. In addi-
tion, it is well established that they are mediators of selenium
compounds anticancer activity [11,41,42]. To analyse whether the
cytotoxic effect caused by compound 2 could be mediated by these
events, we evaluated the apoptotic status of the cells and the cell
cycle phase distribution in both CCRF-CEM and HTB-54 cell cultures
by flow cytometry. With this purpose, Annexin V-FITC and propi-
dium iodide staining for CCRF-CEM cells and the TUNEL technique
for HTB-54 cells were used for apoptosis determination and cell
cycle evaluation. Cell cultures were treated with 0.5e20
mM of
compound 2, DMSO (vehicle) or 6 M of Camptothecin, used as a
m
positive control, for 4e48 h. Obtained results are shown in Fig. 4ꢀ7.
As can be observed in Figs. 4 and 5, compound 2 was able to
induce apoptosis in a time- and dose-dependent manner in both
cell lines. For instance, after treatment of CCRF-CEM cell cultures
with different amounts of 2 for 24 h, the number of Annexin V
positive cells in the cultures rose from 5.2% at 0.5 and 1
at 2 M, 19.8% at 5 M, 37.8% at 10 M and 67.3% at 20
On the other hand, when the cultures were exposed to 10
m
M to 9.6%
M (Fig. 4B).
M of 2
m
m
m
m
m
the incidence of apoptotic cells at the culture rose from 8.0% after
4 h to 9.8 after 8 h, 37.8 after 24 h and 58.8% after 48 h (Fig. 4C).
Similar results were obtained with HTB-54 cells (Fig. 5B and C).
Since caspases play a vital and active role at the initiation and
execution of the apoptotic process [43], to assess whether caspases
were involved or not in the process of cell death induced by 2, the
apoptotic status of the cells in CCRF-CEM cell cultures treated for
1 h with 50 mM z-VAD-fmk followed by 24 h of exposure to 10 mM of
2 was determined. As shown in Fig. 4D pretreatment with z-VAD-
fmk decreased the percentage of apoptotic cells from 36.5% to 14%
thus confirming the involvement of caspases in cell death induction
by 2.
Cell cycle distribution of CCRF-CEM cell cultures treated with 2
is shown in Fig. 6. As shown, an increase of the hypodiploid subG1
population could be detected after 24 h of exposure to 5 mM 2 or
higher, a result that confirms its ability to induce apoptosis (Fig. 6B).
At the same time, a significant increase in G0/G1 cell population, as
well as a reduction of the S and G2/M cell populations were
detected, thus suggesting cell cycle arrest at G0/G1. Time course
analysis of cell cycle distribution in CCRF-CEM cell cultures upon
Table 3
Selectivity index (SI) of the compounds in lung cell lines.
treatment with 10 mM of compound 2 confirmed a cell cycle arrest
at G0/G1 at 24 and 48 h (Fig. 6C).
A parallel study carried out in HTB-54 cells also displayed that 2
is able to induce disturbances in cell cycle in a time and dose
dependent manner (Fig. 7). Thus, while low concentrations of 2
(1e2
10 M treatment with 2 increased the percentage of S and G2/M
cells whilst decreasing the G0/G1 cell population, indicating
blockage of the cycle at G2/M. Additionally, both exposure to 20
of 2 and longer incubation times led to a strong increase in the
subG1 cell population associated with a reduction in the size of the
other phases. These data suggest that cells were not able to recover
cell damage.
mM) failed to block cell cycle progression after 24 h, 5 and
Compound
GI50
TGI
LC50
m
1
2
3
4
5
6
7
8
2
11
3
1
2
2
2
1
3
1
3
5
4
4
5
1
1
9
5
1
1
1
1
1
3
1
1
7
1
1
2
1
1
8
7
1
<1
1
1
<1
1
1
1
8
1
<1
5
<1
mM
9
10
11
12
13
14
15
16
3. Conclusions
Our recent studies on the research and development of new
antitumoural agents led us to identify the methylseleno entity as
determinant for the biological activity. In this paper, we show that a
number of new methylselenocarbamates hybrids (1e16) display

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B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
high cytotoxicity against a broad spectrum of human cancer cell
lines with GI50 values below 10 M for many of them. Compound 2,
m
with a good selectivity index in lung cells was selected for addi-
tional pharmacological studies. Results showed that 2 induced a
caspase-mediated process of cell death that is time and dose
dependent. Besides, compound 2 was also able to induce cell cycle
arrest at G0/G1 in CCRF-CEM cells and at G2/M in HTB-54 cells. The
potent anticancer activity, synthetic accessibility and potential
selectivity of these compounds strongly encourage us to continue
the development and testing of novel methylselenocarbamate hy-
brids aimed at investigating their structureeactivity relationship
and pharmacological mechanism(s) of action.
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Mettler FP82 þ FP80
apparatus (Greifense, Switzerland) and have not been corrected.
The 1H and 13C NMR spectra were recorded on a Bruker 400
Ultrashield™ spectrometer (Rheinstetten, Germany) using TMS as
the internal standard. The IR spectra were obtained on a Thermo
Nicolet FT-IR Nexus spectrophotometer with KBr or NaCl pellets.
Elemental microanalyses were carried out on vacuum-dried sam-
ples using a LECO CHN-900 Elemental Analyzer. Silica gel 60
(0.040e0.063 mm) 1.09385.2500 (Merck KGaA, 64271 Darmstadt,
Germany) was used for Column Chromatography and Alugram® SIL
G/UV254 (Layer: 0.2 mm) (MachereyeNagel GmbH & Co. KG. Post-
fach 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., Barcelona, Spain),
Panreac Química S.A. (Montcada i Reixac, Barcelona, Spain), Sig-
maeAldrich Química, S.A. (Alcobendas, Madrid, Spain), Acros Or-
ganics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgium) and
Lancaster (Bischheim-Strasbourg, France).
4.1.1. General procedure for compounds 1e6
A solution of the corresponding chloroformate (8.08 mmol) in
dry chloroform (25 mL) was slowly added dropwise to a stirred
Fig. 2. MG-MID for compounds 1e16 in tumour (A) and non-malignant cell lines (B).
Compounds are depicted at the vertical axis. Horizontal bars represent the deviation
respect Gl50 MG-MID value. Compounds with a horizontal bar pointing to the right
(positive values) have a Gl50 that is bigger than the mean, whereas horizontal bars
pointing to the left (negative values) indicate Gl50 values smaller than the mean.
Fig. 3. Cytotoxicity of compound 2 in human cell lines. Cytotoxicity was determined by
a colorimetric microassay based on the use of MTT as described in the Experimental
protocols Section. Data are expressed as the percentage of growth SEM of at least
3 independent experiments performed in quadruplicate.

B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
679
1653 (C]N). 1H NMR (400 MHz, CDCl3)
d
: 1.35 (t, 6H, 2CH3,
Table 4
Theoretical structural properties of compound 2.
JCH ꢀCH ¼ 7:1 Hz); 2.3 (s, 3H, SeCH3); 4.26 (c, 4H, 2CH2,
2
3
JCH ꢀCH ¼ 7:1 Hz), 11.95 (s, 1H, NH) 13C NMR (100 MHz, CDCl3)
d:
2
3
8.4 (1C, SeCH3); 14.9 (2C, 2CH3); 62.6 (2C, 2CH2); 148.9 (1C, CONH);
153.0 (1C, CON); 162.6 (1C, CSe). MS (m/z % abundance): 282 (Mþ
,
ꢃ
14), 191 (19), 177 (100), 159 (52), 149 (19), 132 (71), 115 (44), 105
(43), 88 (40), 69 (38), 57 (33). Elemental Analysis for C8H14N2O4Se,
Calcd/Found (%): C: 34.16/34.74; H: 4.98/5.09; N: 9.96/10.11.
Comp. log P MW TPSA
2.56 309 77.00
n-OH acceptors n-OHNH donors Volume
244.61
2
6
1
4.1.3. Methyl N,N0-bis(propoxycarbonyl)imidoselenocarbamate (2)
From propyl chloroformate. Yellow oil. Yield: 29%. IR (NaCl)
cmꢀ1: 3167 (NeH), 2969 (CeHalif), 1747 (C]O), 1651 (C]N). 1H
solution of Se-methylselenourea (3.94 mmol) in dry chloroform
(25 mL) and pyridine (2.5 mL). The mixture was stirred for 24e72 h
at room temperature. Water was added to the reaction and the
mixture was extracted with dichloromethane (3 ꢁ 20 mL). The
organic layer was washed with water (3 ꢁ 20 mL), dried with
Na2SO4 and the solvents were removed under vacuum by rotatory
evaporation and the residue was treated with ethyl ether (100 mL).
NMR (400 MHz, CDCl3)
d
: 0.96 (t, 6H, 2 CH3, JCH ꢀCH ¼ 7:0 Hz);
3
2
1.72 (dd, 4H, 2
3H, SeCH3); 4.14 (t, 4H, 2
b
CH2 J
¼ 7:0 Hz, J
¼ 14:0 Hz); 2.28 (s,
CH2ꢀCH2
CH3ꢀCH2
a
CH2, J
¼ 6:0 Hz); 11.90 (s, 1H, NH).
CH2ꢀCH2
13C NMR (100 MHz, CDCl3)
(2C, 2 CH2); 70.5 (2C, 2
d
: 10.6 (1C, SeCH3); 13.2 (2C, 2CH3); 24.5
b
aCH2); 155.3 (1C, CONH); 162.3 (1C, CON);
164.6 (1C, CSe). MS (m/z % abundance): 311 (Mþꢃ, 30), 251 (16), 215
(7), 129 (75), 46 (87), 69 (19), 43 (100), 27 (16). Elemental Analysis
for C10H18N2O4Se, Calcd/Found (%): C: 38.84/38.96; H: 5.87/5.51; N:
9.06/9.31.
4.1.2. Methyl N,N0-bis(ethoxycarbonyl)imidoselenocarbamate (1)
From ethyl chloroformate. Yellow solid; mp: 67e70 ꢂC. Yield:
49%. IR (KBr) cmꢀ1: 3228 (NeH), 2983e2931 (CeHalif), 1802 (C]O),
Fig. 4. Analysis of cell death induction in CCRF-CEM cells by compound 2. Apoptosis was determined by Annexin V-FITC staining as described in Experimental protocols. (A) Flow
cytometry analysis of a representative experiment after 24 h incubation of the cells in the presence or absence (control) of 2. (B) Percentage of apoptotic cells in the cultures after
incubation of the cells either with 2 (0.5e20
induction by the pan-caspase inhibitor z-VAD-fmk. Cells were pre-incubated for 1 h either with or without 50
used as a positive control. Results are presented as the mean SEM of three independent experiments performed in duplicate. *p < 0.05 and **p < 0.01 respect to control cells.
mM) or vehicle (control) for 24 h. (C) Time course analysis of cell death induction by compound 2 (10
mM). (D) Prevention of cell death
m
M z-VAD-fmk and then with 10 M of 2 for 24 h. Camptothecin was
m

680
B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
Fig. 5. Analysis of cell death induction in HTB-54 cells by compound 2. Apoptosis was determined by TUNEL as described in Experimental protocols. (A) Flow cytometry analysis of a
representative experiment after 24 h incubation of the cells in the presence or absence (control) of 2. (B) Percentage of subdiploid cells in the cultures after incubation of the cells
either with 2 (1e10
mM) or vehicle (control) for 24 h. (C) Time course analysis of cell death induction by compound 2 (10 mM). Camptothecin was used as a positive control. Results
are presented as the mean SEM of three independent experiments performed in duplicate. *p < 0.05 respect to control cells.
4.1.4. Methyl N,N0-bis(butoxycarbonyl)imidoselenocarbamate (3)
(m, 2H, 2CH); 2.28 (s, 3H, SeCH3); 4.32 (d, 4H, 2CH2,
From butyl chloroformate. Yellow oil. Yield: 74%. IR (NaCl) cmꢀ1
:
JCH ꢀCH ¼ 6:7 Hz), 11.51 (s, 1H, NH). 13C NMR (100 MHz, CDCl3)
d:
2
3167 (NeH), 2961 (CeHalif), 1748 (C]O), 1652 (C]N). 1H NMR
8.4 (1C, SeCH3); 19.6 (4C, 4CH3); 28.0 (2C, 2CH); 72.6 (2C, 2CH2);
153.1 (1C, CONH); 160.1 (1C, CON); 162.5 (1C, CSe). MS (m/z %
abundance): 337 (Mþꢃ, 2), 279 (25), 253 (27), 205 (14), 179 (36), 153
(27), 69 (15), 57 (100). Elemental Analysis for C12H22N2O4Se, Calcd/
Found (%): C: 42.73/42.77; H: 6.53/6.53; N: 8.31/8.36.
(400 MHz, CDCl3)
d
: 0.96 (t, 6H, 2 CH3, J
¼ 7:0 Hz); 1.42 (dq,
CH3ꢀCH2
2
4H, 2
g
CH2, J
¼ 7:0 Hz, JgCH ꢀbCH ¼ 14 Hz); 1.70 (dt, 4H,
CH3ꢀCH2
2
2
bCH2, J
¼ 6:0 Hz, JbCH ꢀgCH ¼ 14:0 Hz); 2.30 (s, 3H,
bCH2ꢀaCH2
2
2
SeCH3); 4.20 (t, 4H, 2
13C NMR (100 MHz, CDCl3)
(2C, 2 CH2); 31.0 (2C, 2
a
CH2, J
¼ 6:0 Hz); 11.79 (s, 1H, NH).
aCH2ꢀbCH2
d
: 8.4 (1C, SeCH3); 14.4 (2C, 2CH3); 19.3
g
bCH2); 66.2 (2C, 2
a
CH2); 153.1 (1C, CONH);
4.1.7. Methyl N,N0-bis(allyloxycarbonyl)imidoselenocarbamate (6)
160.1 (1C, CON); 162.6 (1C, CSe). MS (m/z % abundance): 338 (Mþ
,
ꢃ
From allyl chloroformate. Yellow oil. Yield: 21%. IR (NaCl) cmꢀ1
:
3), 306 (14), 279 (17), 253 (15), 206 (31), 191 (12), 179 (27), 153 (20),
126 (15), 111 (15), 69 (36), 57 (100). Elemental Analysis for
3168 (NeH), 2937 (C-Halif), 1749 (C]O), 1652 (C]N). 1H NMR
(400 MHz, CDCl3) : 2.31 (s, 3H, SeCH3); 4.69 (d, 4H, 2 CH2,
d
C
12H22N2O4Se, Calcd/Found (%): C: 42.73/42.66; H: 6.53/6.63; N:
JCH ]CH ¼ 6:0 Hz); 5.31 (d, 2H, 2 CH ¼ , JCHgemeCHcis ¼ 10.0 Hz); 5.37
2
8.31/8.05.
(d, 4H, 2
(100 MHz, CDCl3)
a
CH2, J
¼ 6:0 Hz); 11.94 (s, 1H, NH). 13C NMR
CH2ꢀCH
d
: 10.7 (1C, SeCH3); 69.5 (2C, 2CH2); 121.3 (2C,
4.1.5. Methyl N,N0-bis(hexyloxycarbonyl)imidoselenocarbamate (4)
2CH2]); 135.2 (2C, 2CH]); 154.9 (1C, CONH); 161.7 (1C, CON);
164.1 (1C, CSe). MS (m/z % abundance): 307 (Mþꢃ, 15), 291 (8), 249
(10), 211 (6), 167 (14), 138 (8), 123 (15), 90 (21), 81 (24), 69 (19), 41
(100). Elemental Analysis for C10H14N2O4Se, Calcd/Found (%): C:
39.35/39.80; H: 4.62/4.36; N: 9.18/9.31.
From hexyl chloroformate. Yellow oil. Yield: 74%. IR (NaCl) cmꢀ1
:
3163 (NeH), 2956e2930 (CeHalif), 1748 (C]O), 1653 (C]N). 1H
NMR (400 MHz, CDCl3)
1.36 (m, 8H, 2
d
: 0.90 (t, 6H, 2 CH3, JCH ꢀCH ¼ 7:0 Hz);
3
2
d
þ 2εCH2); 1.38 (m, 4H, 2
gCH2); 1.71 (t, 6H, 2bCH2,
JbCH ꢀgCH ¼ JbCH ꢀaCH ¼ 7:0 Hz); 2.30 (s, 3H, SeCH3); 4.18 (t, 4H,
2
2
2
2
2
a
CH2, J
CDCl3)
¼ 7:0 Hz); 11.79 (s, 1H, NH). 13C NMR (100 MHz,
aCH2ꢀbCH2
4.1.8. General procedure for compounds 7e14 and 16
d: 6.9 (1C, SeCH3); 13.2 (2C, 2CH3); 21.5 (2C, 2εCH2); 27.5 (2C,
A solution of the corresponding chloroformate (8.08 mmol) in
dry chloroform (25 mL) was slowly added dropwise to a stirred
solution of Se-methylselenourea (3.94 mmol) in dry chloroform
(25 mL) and pyridine (2.5 mL). For compounds 9, 12 and 16 dime-
thylaminopyridine (8.08 mmol) was added. The mixture was stir-
red for 24e72 h at room temperature. Solvents were removed
under vacuum by rotatory evaporation and the residue was treated
and purified.
Phenyl chloroformate and 4-methoxyphenyl chloroformate
were prepared from phenol or 4-methoxyphenol respectively, and
trichloromethylchloroformate in toluene as solvent and with DMAP
as catalyst according to the literature [37].
2
dCH2); 39.0 (2C, 2gCH2); 65.5 (2C, 2bCH2); 66.7 (2C, 2aCH2); 151.7
(1C, CONH); 154.2 (1C, CON); 162.0 (1C, CSe). MS (m/z % abun-
dance): 394 (Mþꢃ, 6), 171 (37), 85 (100), 69 (19), 57 (45). Elemental
Analysis for C16H30N2O4Se, Calcd/Found (%): C: 48.45/48.50: H:
7.68/7.84; N: 7.12/6.80.
4.1.6. Methyl N,N0-bis(isobutoxycarbonyl)imidoselenocarbamate
(5)
From isobutyl chloroformate. Yellow oil. Yield: 62%. IR (NaCl)
cmꢀ1: 3167 (NeH), 2963 (CeHalif), 1749 (C]O), 1652 (C]N). 1H
NMR (400 MHz, CDCl3)
d
: 0.95 (d, 12H, 4CH3, J
¼ 6:7 Hz); 2.0
CH3ꢀCH

B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
681
Fig. 6. Effect of compound 2 on cell cycle phase distribution in CCRF-CEM cells. Cells treated with compound 2 were processed for propidium iodide staining and cellular DNA
content determined by flow cytometry. (A) Flow cytometry analysis of a representative experiment performed with cells treated with 1e20 M compound 2 or vehicle (control) for
24 h. (B) Percentage of cells in each phase of the cell cycle after incubation of the cells in the presence or absence (control) of 2 (1e20 M) for 24 h. (C) Time course analysis of cell
cycle phase distribution of CCRF-CEM cell cultures treated with 10 M of 2. Camptothecin was used as a positive control. Results are presented as the mean SEM of three in-
dependent experiments performed in duplicate. *p < 0.05 and **p < 0.01 respect to control cells.
m
m
m
4.1.9. Methyl N,N0-bis(phenoxycarbonyl)imidoselenocarbamate (7)
From phenyl chloroformate. The residue was treated with
toluene (3 ꢁ 20 mL), washed with water (100 mL) and recrystal-
lized from ethanol. A white powder was obtained; mp: 110e112 ꢂC.
Yield: 8%. IR (KBr) cmꢀ1: 3430 (NeH), 1751 (C]O), 1670 (C]N). 1H
(d, 4H, 2H2 þ 2H6, J2e3 ¼ J6e5 ¼ 8.7 Hz); 7.11 (d, 4H, H3 þ H5, H3 þ H5 ,
0
0
J2e3 ¼ J6e5 ¼ 8.7 Hz); 12.18 (s,1H, NeH). 13C NMR (100 MHz, CDCl3)
d:
8.8 (1C, SeCH3); 56.3 (2C, 2OCH3); 115.1 (4C, 2C2 þ 2C6); 123.3 (6C,
2C1 þ 2C3 þ 2C5); 151.9 (1C, CONH); 153.3 (2C, 2C1); 157.8 (1C, CON);
162.9(1C, CSe). MS(m/z% abundance):219(21),191 (64),165(71),124
(100), 109 (62), 81 (31). Elemental Analysis for C18H18N2O6Se, Calcd/
Found (%): C: 49.55/49.43; H: 4.12/4.30; N: 6.41/6.04.
NMR (400 MHz, CDCl3)
d
: 2.33 (s, 3H, SeCH3); 7.20 (d, 4H,
0
2H2 2H6, J2e3 J6e5
þ
¼
¼
7.0 Hz); 7.28 (t, 2H, H4, H4 ,
0
0
J4e3 ¼ J4e5 ¼ 7.0 Hz); 7.44 (m, 4H, H3 þ H5, H3 þ H5 ); 11.97 (s, 1H,
NH). 13C NMR (100 MHz, CDCl3)
d: 11.0 (1C, SeCH3); 116.6 (4C,
4.1.11. Methyl N,N0-bis(4-methylphenoxycarbonyl)
imidoselenocarbamate (9)
2C2 þ 2C6); 127.0 (6C, 2C3 þ 2C4 þ 2C5); 140.4 (2C, 2C1); 153.1 (1C,
CONH); 160.1 (1C, CON); 164.7 (1C, CSe). MS (m/z % abundance):
377 (Mþ), 285 (49), 223 (17), 191 (96), 123 (17), 94 (100), 77 (47), 65
(38). Elemental Analysis for C16H14N2O4Se, Calcd/Found (%): C:
50.93/51.22; H: 3.71/3.72; N: 7.43/7.46.
From 4-methylphenyl chloroformate. The residue was purified
by silica gel column chromatography (toluene/dioxane 90/10)ꢀt1o
give 9 as white powder; mp: 100e102 ꢂC. Yield: 30%. IR (KBr) cm
3426 (NeH), 1756 (C]O), 1637 (C]N). 1H NMR (400 MHz, CDCl3)
:
:
d
2.22 (s, 3H, SeCH3); 2.31 (s, 6H, 2CH3); 7.13 (d, 4H, 2H2 þ 2H6,
4.1.10. Methyl N,N0-bis(4-methoxyphenoxycarbonyl)
imidoselenocarbamate (8)
J2e3 ¼ J6e5 ¼ 8.0 Hz); 7.23 (d, 4H, 2H3 þ 2H5, J2e3 ¼ J6e5 ¼ 8.0 Hz).
13C NMR (100 MHz, CDCl3)
d: 6.9 (1C, SeCH3); 21.2 (2C, 2CH3); 122.3
From 4-methoxyphenyl chloroformate. The residue was treated
with toluene (3 ꢁ 20 mL), washed with water (100 mL) and recrys-
tallizedfromethanol. Awhitepowderwasobtained;mp:100e102 ꢂC.
Yield: 41%. IR (KBr) cmꢀ1: 3426 (NeH), 1756 (C]O), 1663 (C]N). 1H
(2C, C2 þ C6); 130.5 (2C, C3 þ C5); 134.7 (1C, C4) 150.2 (1C, C1); 153.0
(1C, CONH); 159.6 (1C, CON); 161.5 (1C, CSe). MS (m/z % abun-
dance): 299 (9), 203 (44), 191 (100), 123 (15), 107 (75), 91 (22), 77
(27). Elemental Analysis for C18H18N2O4Se$1/2HCl, Calcd/Found (%):
C: 51.03/51.58; H: 4.37/4.52; N: 6.61/7.02.
NMR (400 MHz, CDCl3) d: 2.34 (s, 3H, SeCH3); 3.83 (s, 6H, OCH3); 6.93

682
B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
Fig. 7. Effect of compound 2 on cycle phase distribution in HTB-54 cells. Cell cycle analysis of the cells was performed by TUNEL as described in Experimental protocols. (A) Flow
cytometry analysis of a representative experiment performed with cells treated with 1e20 M 2 or vehicle (control) for 24 h. (B) Percentage of cells in each phase of the cell cycle
m
after incubation of the cells in the presence or absence (control) of 2 (1e20
10
m
M) for 24 h. (C) Time course analysis of cell cycle phase distribution of HTB-54 cell cultures treated with
m
M of 2. Camptothecin was used as a positive control. Results are presented as the mean SEM of three independent experiments performed in duplicate. *p < 0.05 and
**p < 0.01 respect to control cells.
4.1.12. Methyl N,N0-bis(4-chlorophenoxycarbonyl)
imidoselenocarbamate (10)
132e134 ꢂC. Yield: 14%. IR (KBr) cmꢀ1: 3430 (NeH), 1761 (C]O),
1664 (C]N). 1H NMR (400 MHz, CDCl3)
d
: 2.34 (s, 3H, SeCH3); 7.15
: 8.9
¼ 24 Hz); 124.4 (d, 4C,
From 4-chlorophenyl chloroformate. The residue was treated
with toluene (3 ꢁ 20 mL), washed with water (100 mL) and
recrystallized from ethanol. A white powder was obtained; mp:
120e122 ꢂC. Yield: 5%. IR (KBr) cmꢀ1: 3430 (NeH), 1761 (C]O),
(m, 8H, Harom); 12.17 (s, 1H, NeH). 13C NMR (100 MHz, CDCl3)
d
(1C, SeCH3); 117.0 (d, 4C, 2C3 þ 2C5, J2
CeF
2C2 þ 2C6, J3
¼ 24 Hz); 130.9 (d, 2C, 2C4, J1
¼ 286 Hz); 147.2
CeF
CeF
(2C, 2C1); 154.1 (1C, CONH); 161.8 (1C, CON); 162.9 (1C, CSe). MS
(m/z % abundance): 303 (49), 191 (100), 112 (72), 95 (61), 57 (24).
Elemental Analysis for C16H12F2N2O4Se, Calcd/Found (%): C: 46.49/
46.10; H: 2.91/2.92; N: 6.78/7.08.
1664 (C]N). 1H NMR (400 MHz, CDCl3)
d: 2.34 (s, 3H, SeCH3); 7.15
(d, 4H, 2H2 þ 2H6, J2e3 ¼ J6e5 ¼ 8.8 Hz); 7.39 (d, 4H, 2H3 þ 2H5,
J2e3 ¼ J6e5 ¼ 8.8 Hz); 12.16 (s, 1H, NeH). 13C NMR (100 MHz, CDCl3)
d
:
8.9 (1C, SeCH3); 117.8 (4C, 2C2
þ
2C6); 124.6 (6C,
2C3 þ 2C4 þ 2C5); 130.3 (2C, 2C1); 153.1 (1C, CONH); 160.1 (1C,
CON); 162.8 (1C, CSe). MS (m/z % abundance): 319 (9),191 (100),128
(81). Elemental Analysis for C16H12Cl2N2O4Se, Calcd/Found (%): C:
43.05/42.80; 2.69/2.83; N: 6.28/6.32.
4.1.14. Methyl N,N0-bis(benzyloxycarbonyl)imidoselenocarbamate
(12)
From benzyl chloroformate. The residue was purified by silica
gel column chromatography (toluene/dioxane 90/10) to give 12 as
white powder; mp: 68e70 ꢂC. Yield: 13%. IR (KBr) cmꢀ1: 3430
4.1.13. Methyl N,N0-bis(4-fluorophenoxycarbonyl)
imidoselenocarbamate (11)
From 4-fluorophenyl chloroformate. The residue was treated
with toluene (3 ꢁ 20 mL), washed with water (100 mL) and
recrystallized from ethanol. A white powder was obtained; mp:
(NeH), 1742 (C]O), 1654 (C]N). 1H NMR (400 MHz, CDCl3)
d
: 2.30
(s, 3H, SeCH3); 5.22 (s, 4H, 2CH2); 7.40 (m, 10H, Harom); 11.98 (s, 1H,
NH). 13C NMR (100 MHz, CDCl3)
: 8.5 (1C, SeCH3); 68.4 (2C, 2CH2);
d
129.1 (10C, 2C2 þ 2C6 þ 2C3 þ 2C4 þ 2C5); 136.4 (2C, 2C1); 153.0 (1C,
CONH); 159.6 (1C, CON); 161.5 (1C, CSe). MS (m/z % abundance):

B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
683
406 (Mþꢃ), 203 (6), 107 (6), 91 (100), 77 (6), 65 (8). Elemental
Analysis for C18H18N2O4Se, Calcd/Found (%): C: 53.33/53.07; H:
4.44/4.48; N: 6.91/6.78.
for C10H12N2O2Se, Calcd/Found (%): C: 44.28/44.22; H: 4.43/3.97; N:
10.33/10.41.
4.2. Biological evaluation
4.1.15. Methyl N,N0-bis(4-nitrobenzyloxycarbonyl)
imidoselenocarbamate (13)
From 4-nitrobenzyl chloroformate. The residue was treated with
toluene (3 ꢁ 20 mL), washed with water (100 mL) and recrystal-
lized from ethanol. A white powder was obtained; mp: 158e159 ꢂC.
Yield: 6%. IR (KBr) cmꢀ1: 3404 (NeH), 1751 (C]O), 1647 (C]N). 1H
4.2.1. Cytotoxic and antiproliferative activities
The cytotoxic effect of each substance was tested at five different
concentrations ranging between 0.01 and 100 mM. Each substance
was initially dissolved in DMSO at a concentration of 0.01 M and
serial dilutions were prepared using culture medium. The plates
with cells from the different lines, to which medium containing the
substance under test was added, were incubated for 72 h at 37 ꢂC in
a humidified atmosphere containing 5% CO2.
Human cell lines were provided by the European Collection of
Cell Cultures (ECACC) or the American Type Culture Collection
(ATCC). Eight cell lines were used: CCRF-CEM (lymphoblastic
leukaemia), K-562 (lymphocytic leukaemia), HT-29 (colon carci-
noma), HTB-54 (lung carcinoma), PC-3 (prostate carcinoma), MCF-7
(breast adenocarcinoma), 184B5 (non-malignant, mammary gland
derived) and BEAS-2B (non-malignant, derived from bronchial
epithelium). CCRF-CEM, HT-29, PC-3, K-562 and HTB-54 cells were
grown in RPMI 1640 medium (Invitrogen) supplemented with 10%
NMR (400 MHz, DMSO) d: 2.15 (s, 3H, SeCH3); 5.29 (s, 4H, 2CH2);
7.68 (d, 4H, 2H2 þ 2H6, J2e3 ¼ J6e5 ¼ 8.0 Hz); 8.25 (d, 4H, 2H3 þ 2H5,
J2e3 ¼ J6e5 ¼ 8.0 Hz); 11.44 (s, 1H, NH). 13C NMR (100 MHz, CDCl3)
d:
8.6 (1C, SeCH3); 67.0 (2C, 2CH2); 124.4 (4C, 2C3 þ 2C5); 129.6 (4C,
2C2 þ 2C6); 144.2 (2C, 2C1); 148.0 (2C, 2C4); 153.0 (1C, CONH); 159.6
(1C, CON); 161.7 (1C, CSe). MS (m/z % abundance): 136 (100), 106
(27), 89 (37), 31 (31). Elemental Analysis for C18H16N4O8Se, Calcd/
Found (%): C: 43.65/43.89; H: 3.16/2.99; N: 11.31/11.25.
4.1.16. Methyl N,N0-bis(9H-fluoren-9-ylmethoxycarbonyl)
imidoselenocarbamate (14)
From fluorenylmethyloxycarbonyl chloride. The residue was
treated with toluene (3 ꢁ 20 mL), washed with water (100 mL) and
recrystallized from ethanol. A white powder was obtained; mp:
foetal calf serum, 2 mM
100
g/mL streptomycin and 10 mM HEPES buffer (pH ¼ 7.4). MCF-
7 cells were grown in EMEM medium (Clonetics) supplemented
with 10% foetal calf serum, 2 mM -glutamine, 100 units/mL peni-
cillin and 100 g/mL streptomycin. 184B5 cells were grown in Hams
L-glutamine, 100 units/mL penicillin,
m
146e147 ꢂC. Yield: 24%. IR (KBr) cmꢀ1
:
3431 (NeH), 3066
(CeHarom), 2931 (CeHali), 1751 (C]O), 1643 (C]N). 1H NMR
(400 MHz, CDCl3) : 1.28 (m, 2H, 2H9); 2.41 (s, 3H, SeCH3); 4.49 (d,
L
m
d
F-12/DMEM (50:50) supplemented is described by Li et al. [44].
BEAS-2B were grown in 25 mL foetal bovine serum (FBS), 5 mL
insulinetransferrinesodium selenite (ITS), 1 mL hydrocortisone,
10 mL sodium pyruvate, 5 mL glutamine, 5 mL penicillin/genta-
4H,
2
CH2, JCH ¼ 7:0 Hz); 7.36 (t, 4H, 2H2
þ
2H7,
2ꢀ9
J1e2 ¼ J2e3 ¼ J6e7 ¼ J7e8 ¼ 7.0 Hz); 7.44 (t, 4H, 2H3 þ 2H6,
J2e3 ¼ J3e4 ¼ J5e6 ¼ J6e7 ¼ 7.0 Hz); 7.65 (d, 4H, 2H1 þ 2H8,
J1e2 ¼ J7e8 ¼ 7.0 Hz); 7.65 (d, 4H, 2H4 þ 2H5, J3e4 ¼ J5e6 ¼ 7.0 Hz);
micin, 10
mL epidermal growth factor (EGF), and 150 mL retinoic acid
12.08 (s, 1H, NeH). 13C NMR (100 MHz, CDCl3)
d: 8.6 (1C, SeCH3);
(1 M). Cytotoxicity was then determined by the MTT method [38].
m
47.1 (2C, 2C9); 68.2 (2C, 2CH2); 121.1 (4C, 2C1 þ 2C8); 26.0 (4C,
2C2 þ 2C7); 128.5 (8C, 2C3 þ 2C4 þ 2C5 þ 2C6); 153.1 (1C, CONH);
159.5 (1C, CON); 161.7 (1C, CSe). MS (m/z % abundance): 368 (45),
191 (100), 163 (48), 135 (24), 97 (22), 57 (56). Elemental Analysis for
Results were obtained from at least 3 independent experiments
performed in quadruplicate and are expressed as GI50, the con-
centration 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.
C
32H26N4O4Se, Calcd/Found (%): C: 66.09/66.42; H: 4.48/4.54; N:
4.82/4.85.
4.1.17. Methyl N-(1,1-dioxidebenzo[b]thiophen-2-yloxycarbonyl)
imidoselenocarbamate (15)
From 1,1-dioxidebenzo[b]thiophen-2-chloroformate. White
powder; mp: 177e178 ꢂC. Yield: 78%. IR (KBr) cmꢀ1: 3419 (NeH),
2932 (CeHali), 1676 (C]O), 1638 (C]N). 1H NMR (400 MHz, DMSO-
4.2.2. Evaluation of cell cycle progression and cell death
For lung carcinoma HTB-54 cells, both 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 ꢀ20 ꢂC for 30 min. After fixation, cells
were recovered by centrifugation, washed, solved in FITC dUTP-
DNA labelling solution and incubated for 1 h at 37 ꢂC. Cells were
then rinsed, solved in PI/RNase staining buffer, incubated in the
dark for 30 min at RT and analysed using a Coulter Epics XL flow
cytometer.
d6) d: 2.21 (s, 3H, SeCH3); 5.01 (s, 2H, CH2); 7.52 (s, 1H, H3); 7.62 (m,
2H, H4 þ H5); 7.70 (t, 1H, H6, J6e7 ¼ 7.4 Hz, J6e5 ¼ 7.4 Hz); 7.87 (d, 1H,
H7, J7e6 ¼ 7.4 Hz); 8.95 (s, 2H, NH2) 13C NMR (100 MHz, DMSO-d6)
d:
6.8 (1C, SeCH3); 57.2 (1C, CH2); 122.1 (1C, C7); 126.9 (1C, C4); 130.7
(1C, C6); 130.9 (1C, Ca); 131.6 (1C, C3); 135.1 (1C, C5); 137.4 (1C, C2);
140.1 (1C, Cb); 161.8 (1C, CO); 172.6 (1C, CSe). MS (m/z % abun-
dance): 274 (17), 196 (18), 179 (87), 131 (100), 115 (64), 103 (28), 77
(26), 69 (21). Elemental Analysis for C12H12N2O4SSe, Calcd/Found
(%): C: 40.11/39.67; H: 3.34/3.51; N: 7.80/7.71.
The apoptotic status of leukaemia CCRF-CEM cells was evaluated
by measuring the exposure of phosphatidylserine on the cell
membranes using the Annexin V-FITC Kit (BD Pharmingen). Briefly,
5 ꢁ 105 cells were pelleted and washed in PBS. Cells were then
stained with Annexin V-FITC and propidium iodide for 15 min at
4.1.18. Methyl N-(4-methylphenoxycarbonyl)
imidoselenocarbamate (16)
Yellow powder; mp: 100e102 ꢂC. Yield: 47%. IR (KBr) cmꢀ1
:
:
3444 (NeH), 1749 (C]O), 1637 (C]N). 1H NMR (400 MHz, CDCl3)
d
2.37 (s, 6H, SeCH3 þ CH3); 7.08 (d, 2H, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.0 Hz);
4
ꢂC in the dark and analysed using a Coulter Epics XL flow cy-
7.22 (d, 2H, H3 þ H5, J2e3 ¼ J6e5 ¼ 8.0 Hz). 13C NMR (100 MHz,
tometer. For cell cycle analysis in CCRF-CEM cells, cells were fixed in
CDCl3)
d
: 6.9 (1C, SeCH3); 21.2 (1C, CH3); 122.3 (2C, C2 þ C6); 130.5
70% ethanol at 4 ꢂC for 20 min and DNA content was measured by
(2C, C3 þ C5); 134.7 (1C, C4) 150.2 (1C, C1); 161.2 (1C, CO); 173.0 (1C,
CSe); MS (m/z % abundance): 272 (Mþꢃ, 2), 203 (33), 191 (88), 165
(26),123 (16),107 (100), 91 (27), 77 (40), 69 (17). Elemental Analysis
staining with propidium iodide (50 mM).
The statistical analysis was carried out using the two-tailed
Student's paired t-test.

684
B. Romano et al. / European Journal of Medicinal Chemistry 83 (2014) 674e684
ꢀ~
[21] E. Ibanez, M. Stoedter, P.J. Hofmann, D. Plano, A. Calvo, P.A. Nguewa, J.A. Palop,
Acknowledgements
C. Sanmartín, L. Schomburg, Structure- and cell-specific effects of imidosele-
nocarbamates on selenoprotein expression and activity in liver cells in cul-
ture, Metallomics 4 (2012) 1297e1307.
The authors wish to express their gratitude to the Ministerio de
ꢀ
ꢀ
[22] E. Domínguez-Alvarez, D. Plano, M. Font, A. Calvo, C. Prior, C. Jacob, J.A. Palop,
Educacion y Ciencia, Spain (SAF 2009-07744) and to CAN Founda-
C. Sanmartín, Synthesis and antiproliferative activity of novel selenoester
derivatives, Eur. J. Med. Chem. 73 (2014) 153e166.
tion (12,566) for financial support for the project. We thank
Nathaniel Edward Bennet Saidu for carefully reading the
manuscript.
[23] I. Lamberto, D. Plano, E. Moreno, M. Font, J.A. Palop, C. Sanmartín, I. Encío,
Bisacylimidoselenocarbamates cause G2/M arrest associated with the modu-
lation of CDK1 and Chk2 in human breast cancer MCF-7 cells, Curr. Med.
Chem. 20 (2013) 1609e1619.
Appendix A. Supplementary data
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[24] M. Font, E. Lizarraga, E. Ibanez, D. Plano, C. Sanmartín, J.A. Palop, Structural
variations on antitumour agents derived from bisacylimidoselenocarbamate.
A
proposal for structure-activity relationships based on the analysis of
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.076.
conformational behavior, Eur. J. Med. Chem. 66 (2013) 489e498.
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