Novel seleno- and thio-urea derivatives with potent in vitro activities against several cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2016.02.042

A series of novel selenourea derivatives and corresponding thiourea analogs were synthesized and tested against a panel of six human cancer cell lines: melanoma (1205Lu), lung carcinoma (A549), prostatic carcinoma (DU145), colorectal carcinoma (HCT116), p

Full text of DOI:10.1016/j.ejmech.2016.02.042

Accepted Manuscript
Novel seleno- and thio-urea derivatives with potent in vitro activities against several
cancer cell lines
Verónica Alcolea, Daniel Plano, Deepkamal N. Karelia, Juan Antonio Palop, Shantu
Amin, Carmen Sanmartín, Arun K. Sharma
PII:
S0223-5234(16)30121-0
10.1016/j.ejmech.2016.02.042
EJMECH 8393
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 December 2015
Revised Date: 15 February 2016
Accepted Date: 16 February 2016
Please cite this article as: V. Alcolea, D. Plano, D.N. Karelia, J.A. Palop, S. Amin, C. Sanmartín, A.K.
Sharma, Novel seleno- and thio-urea derivatives with potent in vitro activities against several cancer cell
lines, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel seleno- and thio-urea derivatives with
potent in vitro activities against several
cancer cell lines
Verónica Alcolea1,2,3, Daniel Plano1,2,3, Deepkamal N. Karelia1, Juan
Antonio Palop2,3, Shantu Amin1, Carmen Sanmartín2,3 and
Arun K. Sharma1,
1Department of Pharmacology; Penn State Hershey Cancer Institute, CH72;
Penn State College of Medicine, 500 University Drive, Hershey, PA 17033.
2Department of Organic and Pharmaceutical Chemistry, University of Navarra,
Irunlarrea 1, E-31008 Pamplona, Spain.
3Instituto de Investigación Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008
Pamplona, Spain.
Author to whom correspondence should be addressed
Arun K. Sharma, Ph.D.
Department of Pharmacology
Penn State College of Medicine
Penn State Hershey Cancer Institute, CH72
Penn State Milton S. Hershey Medical Center
500 University Drive
Hershey, PA 17033
Phone: 717-531-4563
Fax:
717-531-0244
E-mail: aks14@psu.edu

ACCEPTED MANUSCRIPT
Abstract
A series of novel selenourea derivatives and corresponding thiourea analogs were
synthesized and tested against a panel of six human cancer cell lines: melanoma
(1205Lu), lung carcinoma (A549), prostatic carcinoma (DU145), colorectal carcinoma
(HCT116), pancreatic epithelioid carcinoma (PANC-1) and pancreatic adenocarcinoma
(BxPC3). In general, we found that the selenium-containing derivatives were more
potent than their isosteric sulfur analogs. Four selenourea derivatives (1e, 1f, 1g and 1i)
showed IC50 values below 10 µM in all of tested cell lines at 72 h. On the basis of its
potent activity, compound 1g was selected for further biological evaluation in different
colon cancer cell lines. Our results indicated that compound 1g induced apoptosis by
caspase activation, along with inhibition of anti-apoptotic proteins.
Keywords: selenium, selenourea, cytotoxicity, apoptosis

ACCEPTED MANUSCRIPT
1. Introduction
Cancer is known to be one of the leading causes of death in the world [1]. A
considerable amount of progress has been made in the field of cancer treatment.
However, the current chemotherapeutic agents are associated with unwanted side effects
and poor quality of life. Moreover, drug resistance is also a rising issue with current
therapies. Therefore, there is an unmet need to develop novel cancer therapeutic agents
which are effective against the disease and can improve quality of life [2].
In the last decade, a wide range of novel agents have emerged as potential anti-cancer
agents. Among them, selenium (Se) containing molecules have generated a growing
interest for their anti-cancer activity and safety [3-6]. The chemical form in which
selenium is used and the dose are the most critical factors in its activity [7].
Guanidine group has been emphasized over the years for its anti-cancer properties. The
anti-cancer activity of this framework is generally associated with its ability to bind to
various substituents on the DNA helix via the three nitrogen atoms present in its
structure [8,9]. Besides, guanidines can act as urokinase [10] or cyclin kinase inhibitors
[11]. Recently, some guanidine compounds have been reported to sensitize prostate
cancer cells towards known chemotherapeutic agents such as staurosporine [12].
Moreover, compounds including the isothiourea group, for example the compound S,S-
(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea (PBIT), have been described as potent
and selective inhibitors of the inducible nitric oxidase synthase (iNOS) [13]. This kind
of inhibitors has been shown in several studies as potential chemopreventive agents
[14,15]. The isosteric selenium analog (PBISe) was found superior to PBIT as apoptosis
inductor, iNOS inhibitor and cell growth inhibitor in several cancer cell lines [16-18].

ACCEPTED MANUSCRIPT
On the basis of these results, we have designed new hybrid compounds by introducing
selenium in the form of isoselenourea, into different carbo- and hetero-cyclic scaffolds
which have previously been shown to possess antitumor properties. These scaffolds
include nitrofuran [19,20], isoxazole [21-23], 4-phenylpyrrole [24], imidazolidone [25-
27], quinoline [28,29], quinoxaline [30-32], coumarin [33,34], acridine [35,36],
anthraquinone [37] and benzodioxole [38,39]. We have selected variable mono, bi or tri-
cyclic carbo- and hetero-cycles in order to confer different volume, rigidity, and
electronic and lipophilic characteristics to the target molecules.
In addition, to evaluate the differences in the anti-cancer activities between two similar
chemical elements, selenium and sulfur, when attached to above referred
carbo/heterocyclic scaffolds, the isosteric isothiourea analogs were also synthesized.
Literature reports, both from our group [40-42] and others [43], have demonstrated the
enhanced cytotoxicity of selenium compounds as compared to corresponding sulfur
isosteres. In spite of these two elements are in the same column of the periodic table,
selenium is a larger atom with more labile electrons that likely contributes to the
observed differences in activity. This study examines the effect of newly synthesized
hybrid molecules on various cancer cell lines and begins to evaluate the underlying
mechanism of action.
2. Results and discussion
2.1. Synthesis of seleno- and thio-urea derivatives
The twenty novel compounds synthesized are summarized in Figure 1. The compounds
were prepared according to previously published procedures [13] with some

ACCEPTED MANUSCRIPT
modifications (Scheme 1), starting from the commercially available alkyl halides and
selenourea (compounds 1a-j) or thiourea (compounds 2a-j). The reactions were carried
out under different conditions, depending on the alkyl halide used (more information in
experimental section), in a 1:1.1 (alkyl halide:selenourea) molar ratio using absolute
ethanol as solvent. After isolation and purification of the compounds, a yield of 15-92
% range was achieved. This variability in yield cannot be explained by chemical
properties of the reagents. No correlation was found between yields and chemical
features as nucleophilic activity of seleno-/thio-urea or the ligand, kind of halogen or
ligand volume. Therefore, the differences might be ascribed to other physical factors.
The unalike reaction times (0.5-6 h) and temperature conditions (0 ºC, r.t., reflux), along
with the varying isolation and purification procedures (wash, recrystallization,
precipitation) could be proposed as the main causes of the diverse yield values obtained.
Figure 1. General structures of compounds. The wavy bond indicates the position
where the seleno- or thio-urea group is attached.

ACCEPTED MANUSCRIPT
Scheme 1. General schematic for synthesis of selenoureas (series 1) and thioureas
(series 2)
Mechanistically, these reactions follow is a second-order nucleophilic substitution. The
lone electron pair of Se or S atom attacks the methylene group and simultaneously the
halogen atom leaves. Subsequently, the C=Se bond recovers the pi electron pair and one
of the amino groups loses a proton, leading to the imine group formation.
The purity of all the products was determined by thin layer chromatography (TLC) and
elemental analysis. Purity of all final compounds was 95 % or higher and their
structures were confirmed by IR, 1H and 13C NMR, mass spectrometry (MS) and
elemental analysis.
2.2. Biology
2.2.1. Effect on cancer cell viability
The effects of the novel compounds on cancer cell viability were evaluated in a panel of
six human cancer cell lines: 1205Lu (melanoma), A549 (lung), DU145 (prostate),
HCT116 (colon), PANC-1 (pancreas) and BxPC3 (pancreas). MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was performed in order

ACCEPTED MANUSCRIPT
to measure cancer cell viability as previously described [45]. Results are expressed as
half maximal inhibitory concentration (IC50), the concentration that produces 50 %
growth inhibition. The anti-cancer effect of each agent was tested at seven different
concentrations between 0.5 and 50 µM and three different time points (24, 48 and 72 h).
The results are summarized in Tables 1A and 1B.
Table 1A. Screening of novel seleno- and thio-urea derivatives on cancer cell viability
in melanoma (1205Lu), lung (A549) and prostate (DU145) cancer cell lines
IC50 (µM)
Cell lines
1205Lu
48 h
A549
48 h
DU145
48 h
Comp.
24 h
72 h
24 h
72 h
24 h
72 h
13.0 ± 4.0 16.7 ± 1.7 11.6 ± 1.2 34.4 ± 4.1 14.9 ± 1.8 6.2 ± 0.7 31.8 ± 4.0 14.9 ± 2.9 12.9 ± 1.5
1a
1b
1c
1d
1e
1f
1g
1h
1i
>50.0
19.1 ± 2.3 15.7 ± 2.1 13.0 ± 1.0 22.9 ± 0.9 10.2 ± 0.9 9.0 ± 1.0
17.4 ± 3.1 14.5 ± 2.4 11.3 ± 1.5
14.4 ± 1.7 9.9 ± 1.2 7.1 ± 0.3 39.6 ± 16.2 15.1 ± 0.7 8.1 ± 0.4 19.6 ± 1.2 11.6 ± 0.8
21.0 ± 2.5 4.4 ± 0.8 1.9 ± 0.3 15.7 ± 1.6 5.2 ± 0.6 4.5 ± 0.3 11.4 ± 1.9 8.4 ± 0.6
>50.0 21.8 ± 3.0
>50.0
>50.0 36.3 ± 1.4
>50.0
>50.0
>50.0
>50.0 15.6 ± 4.7
>50.0
8.9 ± 1.2 2.2 ± 0.5 1.9 ± 0.4 21.4 ± 10.2 6.2 ± 1.6
8.2 ± 2.3
7.3 ± 0.5
6.8 ± 0.3
1.0 ± 0.3
6.9 ± 0.4
7.4 ± 1.4
>50.0
3.0 ± 0.8
3.3 ± 0.5 2.8 ± 0.3 2.2 ± 0.1
2.0 ± 0.4 1.0 ± 0.2
2.3 ± 0.9 2.8 ± 0.3
19.0 ± 3.7 13.2 ± 1.2 9.3 ± 1.2 11.2 ± 1.5 7.8 ± 0.8 5.5 ± 0.2 18.8 ± 2.5 14.0 ± 1.5
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 3.4 ± 1.3
>50.0 2.8 ± 1.0 1.2 ± 0.3
>50.0 22.0 ± 8.4
>50.0
>50.0
>50.0
>50.0
>50.0 25.3 ± 6.4 16.3 ± 3.0 12.8 ± 1.9
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
1j
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
2a
2b
2c
2d
2e
2f
2g
2h
2i
9.4 ± 0.8 8.5 ± 0.8 6.3 ± 0.5
9.7 ± 1.3 8.7 ± 0.6 7.6 ± 0.5 29.8 ± 23.5 22.9 ± 4.7 31.2 ± 5.4
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 39.2 ± 1.4
>50.0 37.4 ± 7.6
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 31.6 ± 11.5
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 31.7 ± 3.8
2j

ACCEPTED MANUSCRIPT
Table 1B. Screening of novel seleno- and thio-urea compounds in colon (HCT116) and
pancreatic (PANC-1 and BxPC3) cancer cell lines
IC50 (µM)
Cell lines
HCT116
48 h
PANC-1
48 h
BxPC3
48 h
Comp.
24 h
72 h
24 h
72 h
24 h
72 h
31.8 ± 3.0 16.7 ± 2.1 14.4 ± 2.6 12.1 ± 1.9 11.6 ± 1.3
9.8 ± 1.1 20.4 ± 2.7 10.5 ± 0.7 8.2 ± 0.5
>50.0 24.7 ± 3.8 16.5 ± 2.7 >50.0 >50.0 36.9 ± 5.8
>50.0 15.6 ± 1.6 13.6 ± 1.8 42.9 ± 4.6 39.4 ± 3.8 15.6 ± 2.2
>50.0 >50.0 31.4 ± 7.2 >50.0 >50.0 >50.0
9.6 ± 1.2 36.6 ± 2.3 17.0 ± 3.4 7.8 ± 0.7
6.6 ± 1.4 20.6 ± 2.5 19.4 ± 1.5 10.0 ± 1.5
>50.0 >50.0 6.6 ± 2.1
0.7 ± 0.4 6.6 ± 3.1 2.9 ± 1.1 2.0 ± 0.5 3.6 ± 1.4 2.9 ± 0.5 2.2 ± 0.2
1a
1b
1c
1d
1e
1f
1g
1h
1i
>50.0
14.6 ± 3.3
>50.0
31.8 ± 3.8 23.9 ± 3.4
3.7 ± 1.1
3.8 ± 0.4
5.9 ± 1.4
12.6 ± 3.9
29.7 ± 6.4 19.6 ± 2.2 10.6 ± 0.9 18.0 ± 3.3 15.9 ± 3.8
26.7 ± 5.6
2.9 ± 2.1
8.9 ± 1.9
0.7 ± 0.6
29.2 ± 2.7 17.9 ± 2.4 15.6 ± 1.7 23.5 ± 4.2 15.3 ± 2.2 12.6 ± 1.0 19.8 ± 3.0 19.1 ± 4.2 18.8 ± 2.5
>50.0
>50.0
>50.0
45.0 ± 1.0
4.6 ± 1.1
>50.0 37.4 ± 10.1 13.6 ± 3.5
>50.0 6.8 ± 1.5 2.4 ± 0.6
14.7 ± 4.8 8.3 ± 2.1
>50.0 46.3 ± 1.8
>50.0 44.3 ± 6.2
1j
>50.0
>50.0 45.1 ± 24.3
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
2a
2b
2c
2d
2e
2f
2g
2h
2i
>50.0 34.8 ± 7.6 22.1 ± 1.7
>50.0 30.8 ± 9.2 7.0 ± 7.7
20.2 ± 2.5 10.7 ± 3.6 10.3 ± 2.8 7.6 ± 0.6
>50.0 40.5 ± 5.3 34.3 ± 6.0
>50.0
>50.0
7.4 ± 0.7
>50.0
>50.0
>50.0
>50.0
6.8 ± 0.5 4.1 ± 0.7 9.0 ± 1.5 7.9 ± 1.1
>50.0 28.5 ± 6.6 22.9 ± 3.5
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 37.3 ± 2.1 25.7 ± 1.7
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0 47.2 ± 2.6
>50.0 24.6 ± 2.6
>50.0
>50.0
>50.0
>50.0 31.5 ± 3.8
>50.0 40.6 ± 17.8
2j
As shown in Tables 1A and 1B, the isosteric replacement of sulfur by selenium was
correlated with a significant decrease in cancer cell viability, with the exception of the
anthracene-9,10-dione scaffold (compound 2d) in 1205Lu, HCT116, PANC-1 and
BxPC3 cell lines. In fact, the activity of sulfur-containing derivatives remained over the
maximum studied concentration (50 µM) in almost all cases. A more in-depth analysis
of the cell viability effects showed that seven out of the ten selenium-containing
compounds possessed IC50 values below 20 µM after 72 h of treatment in all of the
tested cell lines. On the contrary, only one of the sulfur-containing derivatives
(compound 2d) showed IC50 values below 32 µM in all cell lines screened at the same
time-point. Taken together, these results suggest that the presence of the selenium atom
is crucial for the biological effects of these derivatives.

ACCEPTED MANUSCRIPT
Compounds 1e, 1f, 1g and 1i were found to be the most active, with IC50 values below
10 µM in all of tested cell lines at 72 h. From our screening, compound 1g emerged as
the most potent agent with low IC50 values (0.7-6.6 µM) in all cancer cell lines tested.
The potent activity of compound 1g may be related to the presence of two selenourea
groups.
Taking into account that compound 1g was the only compound that had single digit IC50
values even at 24 h treatment in all the cancer cell lines tested, it was selected for further
biological evaluation. Compound 2d was the only sulfur-containing compound that
showed a decrease on cell viability in all cancer cell lines screened. Therefore,
compound 2d was chosen as the most promising compound from series 2.
2.2.2. Compounds 1g and 2d reduced cell viability in different colon cancer cell
lines
As observed from Tables 1A and 1B, compound 1g was most potent in HCT116, colon
cancer, cell line. Therefore, we focused our experiments to evaluate the activity of 1g in
other human colon cancer cell lines. Compound 2d, the most potent sulfur containing
compound from series 2, was also evaluated since the thiourea analog PBIT has been
shown to be an effective colon cancer chemopreventive agent [15]. As shown in Table
2 and Figure 2, compound 1g was almost equally effective in reducing the viability of
all three colon cancer cell lines (HCT116, HT29 and RKO) with IC50 ranging from 0.7-
5.0 µM at different treatment time points. This observation is unique in the essence that
all the three cell lines harbor different mutations. As expected, compared to 1g, the
thiourea analog 2d was much less effective in reducing cancer cell viability in all three
colon cancer cell lines tested (Figure 2 and Table 2).

ACCEPTED MANUSCRIPT
We also compared the potency of compound 1g with 5-FU, a first line therapeutic agent
for colon cancer. As shown in Figure 2 and Table 2, compound 1g was up to >50
times more potent than 5-FU. Taken together, these results indicated that 1g not only
emerged as the most potent agent from the series of novel agents synthesized, but it was
also superior to the current therapy, 5-FU, for colon cancer. Hence, compound 1g was
narrowed down as the most promising agent for further biological evaluations.
Table 2. Effects of compounds 1g, 2d and 5-FU on cell viability of different colon
cancer cell lines.
IC50 (µM)
Cell lines
HCT116
HT29
RKO
Comp.
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
2.9 ± 2.0 0.7 ± 0.7
0.7 ± 0.4
5.0 ± 1.0 4.1 ± 1.7 2.2 ± 1.9 3.8 ± 2.9
1.9 ± 1.6 2.5 ± 1.9
1g
2d
20.2 ± 2.5 10.7 ± 3.6 10.3 ± 2.8 17.0 ± 3.28 8.1 ± 2.7 7.4 ± 0.6 28.9 ± 7.3 20.4 ± 1.3 21.0 ± 2.4
>50.0 >50.0 27.8 ± 4.8 >50.0 25.0 ± 2.0 6.3 ± 0.7 >50.0 41.1 ± 12.6 6.8 ± 0.8
5-FU

ACCEPTED MANUSCRIPT
Figure 2. Compound 1g and 2d reduced cancer cell viability in three different
colon cancer cell lines. Colon cancer cells were treated with different concentrations of
the compounds for the given time points. Cell viability was evaluated by MTT assay.
Mean values are represented with their respective standard deviation (± SD).
2.2.3. Compound 1g induced cell death in HCT116 cells at 24 h
The MTT assays performed above demonstrated that 1g substantially reduces the
viability of various cancer cell lines. However, to understand whether the reduction in
cell viability is due the cell death and not due to the inhibition of cell growth, HCT116
cells were treated with 1g for 24 h and were subjected to Live and Dead assay as
described in the experimental section. As shown in Figure 3, treatment of HCT116 cells
with compound 1g increased the amount of cells positive for Ethidium Homodimer
staining (dead cells, Upper left quadrant), while reduced the cells with Calcein staining

ACCEPTED MANUSCRIPT
(live cells, Lower right quadrant). Dose of 5 ꢀM showed more than 28 % cell death
compared to vehicle. Hence, the inhibitory effect of compound 1g on cancer cell
viability was primarily due to cancer cell death.
Figure 3. Compound 1g induced cell death. HCT116 cells were treated with
compound 1g (1, 2.5 or 5 µM) or DMSO (control) for 24 h and stained with Calcein
AM and Ethidium Homodimer.
2.2.4. Compound 1g induced apoptosis in colon cancer cells
In order to determine if 1g induces cell death through apoptosis, the MuseTM Annexin V
& Dead Cell assay was carried out. In this assay, Annexin V is employed to detect the
externalization of phosphatidylserine (PS) to the cell surface, a process occurring in
early apoptosis [46]. This kit also included a dead cell marker (7-ADD). Only the cells
which have lost their cell membrane integrity are stained by 7-ADD. Hence, cells
positive for 7-ADD marker only, are indicative of necrosis (Upper left quadrant). Cells

ACCEPTED MANUSCRIPT
positive for both markers (Annexin V and 7-ADD) are indicative of apoptotic cell death
(Upper right quadrant), while cells only positive for Annexin V are considered to be in
early apoptosis (Lower right quadrant). Cells which are negative for both markers are
present in the Lower left quadrant (healthy cells). As shown in Figure 4A, the treatment
with compound 1g increased the number of apoptotic cells even at low concentration (1
µM). When the dose of 1g was increased to 5 µM, population of early apoptotic cells as
well as late apoptotic cells also increased, indicating the dose dependent effect of 1g in
induction of apoptosis in colon cancer cells. These results are in concordance with the
data obtained from the MTT assay and Live & Dead assay.
To confirm these results, the activation of the effector Caspases 3 and 7 was studied
using MuseTM Caspase 3/7 kit. The kit included a reagent with a DNA binding dye
which contains an effector Caspase 3/7 recognizing sequence (DEVD). Upon the
presence of Caspase 3/7 activity, this sequence is cleaved and DNA binding dye is
released from the reagent, hence staining the DNA of cells having positive Caspase 3/7
activity. This kit also included the dead cell marker 7-ADD. The distribution of cells in
quadrants is the same as in the Annexin V assay: viable (both dyes negatives, Lower left
quadrant), early apoptotic (only positive for Caspase 3/7 reagent, Lower right quadrant),
late apoptotic/dead (positive for both markers, Upper right quadrant) and necrotic cells
(only positive for 7-ADD, Upper left quadrant). As shown in Figure 4B, the amount of
cells positive for the Caspase 3/7 reagent increased dramatically after the treatment with
1g. At the dose of 5 µM, more than 50 % of the cells were positive for the apoptosis
marker (Lower right and Upper right quadrants), suggesting that compound 1g is a
potent apoptosis inducer. The above results clearly demonstrate that compound 1g

ACCEPTED MANUSCRIPT
induced cell death through apoptosis, as indicated by presence of PS on outer leaflet of
cell membrane and presence of Caspase 3/7 activity.
To evaluate the mechanism by which 1g induces apoptosis, we investigated its effect on
anti-apoptotic proteins, XIAP and Bcl-2, by Western blot analysis. As shown in Figure
4C, the levels of both XIAP and Bcl-2 were reduced in a dose dependent manner.
Further, we evaluated the effect of 1g at the protein levels of pro-Caspase 3 and full-
length PARP (Figure 4D). At doses similar to at which the reduction in anti-apoptotic
proteins (Bcl-2 and XIAP) was observed, there was also a decrease in the pro-Caspase 3
and whole PARP levels. The full-length Caspase 3 (pro-Caspase 3) is inactive, whereas
its cleaved form is active. The active form of Caspase 3 is able to cleave other substrates
as PARP [46]. Therefore, the reduction of both proteins in this experiment indicated the
activation of Caspase 3. The expression of both pro-Caspase 3 and whole PARP
decreased significantly at 2.5 µM dose of 1g and were undetectable after treatment with
5 µM dose. These results are in concordance with the data obtained in the Annexin V
and Caspase 3/7 assays.

ACCEPTED MANUSCRIPT
Figure 4. Compound 1g induced apoptosis and inhibited anti-apoptotic proteins.
After 24 h of treatment with the indicated amounts of compound 1g, HCT116 cells were
analyzed with a MuseTM automated cell analyzer using the MuseTM Annexin V & Dead
Cell Kit according to manufacturer’s instructions (A). Analogous independent
experiments were analyzed with MuseTM Caspase 3/7 Kit to confirm the results (B). The
effects of compound 1g on the expression of two anti-apoptotic proteins, Bcl-2 and
XIAP (C), as well as on the protein levels of pro-Caspase 3 and full-length PARP (D)
were evaluated by Western blot analysis after 24 h of treatment with increasing
concentrations of 1g. β-Actin was used as loading control.
3. Conclusion
In this work, a novel series of selenourea derivatives and its corresponding thiourea
analogs were designed and synthesized. The MTT assay confirmed that the selenium-

ACCEPTED MANUSCRIPT
containing derivatives were much more potent than their corresponding sulfur analogs.
Therefore, the replacement of sulfur by selenium seems to be a valid approach to obtain
new agents with more potent anti-cancer activity. After screening in a panel of six
human cancer cell lines, 1g was identified as the most promising compound. This
compound showed low IC50 values in all cancer cell lines and time points tested and it
was found to be up to >50 times more potent than 5-FU in three different colon cancer
cell lines. Live and Dead, Annexin V, Caspase 3/7 and Western blot assays
demonstrated that compound 1g induced apoptosis by caspases activation, along with
inhibition of the anti-apoptotic proteins Bcl-2 and XIAP. Hence, 1g has the potential to
be developed as a chemotherapeutic agent for colon cancer treatment and warrants
further in depth in vitro and in vivo studies in colon cancer models.
4. Experimental
4.1. Chemistry
4.1.1. Material and methods
Melting points (mp) were determined with a Mettler FP82+FP80 apparatus (Greifense,
Switzerland). Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker 400
UltrashieldTM spectrometer (Rheinstetten, Germany) using DMSO-d6 as solvent.
Chemical shifts are given in parts per million (ppm) (δ relative to residual solvent peak
for 1H and 13C). IR spectra were recorded on a Thermo Nicolet FT-IR Nexus
spectrophotometer. Elemental analysis was performed on a LECO CHN-900 Elemental
Analyzer. Purity of all final compounds was 95 % or higher. Chemicals were purchased
from E. Merck (Darmstadt, Germany), Panreac Química S.A. (Montcada i Reixac,
Barcelona, Spain), Sigma-Aldrich Quimica, S.A. (Alcobendas, Madrid, Spain) and
Acros Organics (Janssen Pharmaceuticalaan, Geel, Belgium).

ACCEPTED MANUSCRIPT
4.1.2. General procedure of synthesis of compounds
The corresponding alkyl halide reagent (1 mmol) was added to a mixture of selenourea
(1.1 mmol) (compounds 1a-j) or thiourea (compounds 2a-j) in absolute ethanol (20
mL). The mixture was stirred at reflux, room temperature or 0 ºC for 0.5-6 h. The
product was isolated by filtration or by rotatory evaporation of the solvent under
vacuum and purified by recrystallization or washing.
4.1.3. Acridin-9-ylmethyl carbamimidoselenoate hydrobromide (1a)
Conditions: 1 h at 0 ºC. The precipitate was washed with dichloromethane and hexane
(25 mL of each). A pink powder was obtained; Yield: 15 %; mp: 222 ºC (direct
combustion). 1H NMR (400 MHz, DMSO-d6): δ 3.58 (s, 2H, -CH2-), 7.01-7.04 (m, 4H,
H2 + H3 + H6 + H7), 7.31 (t, 2H, J= 7.6 Hz, H4 + H5), 7.39 (d, 2H, J= 7.8 Hz, H1 +
H8), 9.32 + 9.63 + 9.71 (bs + bs + bs, 3H, NH + NH2), 11.06 ppm (bs, 1H, HBr). 13C
NMR (100 MHz, DMSO-d6): δ 44.1 (-CH2-), 69.1 (C9), 115.4 (C8a + C9a), 120.7 (C1 +
C8), 121.2 (C2 + C7), 126.3 (C4 + C5), 130.0 (C3 + C6), 138.2 (C4a + C10a), 171.5
ppm (Se-C-(NH)(NH2). IR (KBr): ν̃ 3293-3198 (s; N-H, N-H2), 3052 (s; C-Harom), 1636
(s; C=N), 1578-1484 cm-1 (m; C-Carom). MS (m/z (% abundance)): 194(100). Elemental
analysis calculated (%) for C15H13N3SeꢁHBrꢁH2O: C: 43.58, H: 3.39, N: 10.17; found:
C: 43.22, H: 3.25, N: 9.85
4.1.4. (6-Bromobenzo[d][1,3]dioxol-5-yl)methyl carbamimidoselenoate
hydrobromide (1b)
Conditions: 6 h at reflux. The precipitate was filtered and purified by recrystallization
from ethanol. A pink powder was obtained. Yield: 49 %; mp: 182-183 ºC. 1H NMR (400

ACCEPTED MANUSCRIPT
MHz, DMSO-d6): δ 4.51 (s, 2H, -CH2-Se), 6.09 (s, 2H, O-CH2-O), 7.19 (s, 1H, H4),
7.28 (s, 1H, H7), 9.26 + 9.34 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR (100
MHz, DMSO-d6): δ 32.4 (-CH2-Se), 103.2 (O-CH2-O), 111.4 (C4), 113.7 (C6), 115.7
(C7), 129.2 (C5), 148.2 (C7a), 149.1 (C3a), 166.8 ppm (Se-C-(NH)(NH2)). IR (KBr): ν
3233-3078 (s; N-H, N-H2), 2889 (w; C-Haliph), 1644 cm-1 (s; C=N). MS (m/z (%
abundance)): 294(11), 213(100), 157(11), 124(16), 75(21). Elemental analysis
calculated (%) for C9H9N2BrO2 SeꢁHBr: C: 25.90, H: 2.40, N: 6.72; found: C: 26.02, H:
2.37, N: 6.75.
̃
4.1.5 (5-Nitrofuran-2-yl)methyl carbamimidoselenoate hydrobromide (1c)
Conditions: 1h at reflux. The precipitate was washed with ethyl ether (50 mL). A white
powder was obtained. Yield: 80 %; mp: 223 (direct combustion). 1H NMR (400 MHz,
DMSO-d6): δ 4.67 (s, 2H, -CH2-), 6.81 (d, 1H, J= 3.7 Hz, H3), 7.69 (d, 1H, J= 3.8 Hz,
H4), 9.25 + 9.35 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR (100MHz, DMSO-d6):
δ 22.4 (-CH2-), 113.6 (C3), 115.2 (C4), 152.0 (C5), 156.4 (C2), 166.1 ppm (1C, -Se-C-
(NH)(NH2)). IR (KBr): ν̃
3241-3093 (s; N-H, N-H2), 1661 cm-1 (s; C=N). MS (m/z (%
abundance)): 207(72), 160(39), 126(80), 80(79), 52(100). Elemental analysis calculated
(%) for C6H7N3O3 SeꢁHBr: C: 21.90, H: 2.45, N: 12.77; found: C: 21.64, H: 2.48, N:
12.69.
4.1.6. (9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl carbamimidoselenoate
hydrochloride (1d)
Conditions: 4 h at reflux. The precipitate was washed with dichloromethane and ether
(25 mL of each). A green powder was obtained; Yield: 75 %; mp: 221-223 ºC. 1H NMR
(400 MHz, DMSO-d6): δ 4.73 (s, 2H, -CH2 -), 7.92-7.96 (m, 3H, H3 + H5 + H8), 8.20-

ACCEPTED MANUSCRIPT
8.21 (m, 3H, H1 + H6 + H7), 8.28 (bs, 1H, H4), 9.37 + 9.45 ppm (bs + bs, 4H, NH +
NH2 + HCl). 13C NMR (100 MHz, DMSO-d6): δ 30.1 (-CH2-), 127.5 (C5 + C8), 127.9
(C4), 128.1 (C1), 132.8 (C4a), 133.8 (C6 + C7), 134.0 (C3), 135.3 (C8a + C10a), 135.6
(C9a), 146.1 (C2), 166.7 (Se-C-(NH)(NH2)), 182.8 (C10), 183.1 ppm (C9). IR (KBr): ν
̃
3192-3075 (s; N-H, N-H2), 1661 (s; C=O), 1539 cm-1 (s; C=N). MS (m/z (%
abundance)): 302(14), 221(100), 193(49), 165(29), 139(7), 82(7). Elemental analysis
calculated (%) for C16H12N2O2Se ꢁHCl: C: 50.61, H: 3.45, N: 7.38; found: C: 50.15, H:
3.46, N: 7.40.
4.1.7. Quinolin-2-ylmethyl carbamimidoselenoate hydrochloride (1e)
The commercially available 2-(chloromethyl)quinoline hydrochloride was treated with
basic water (50 mL) in order to obtain the free base, which was used for the synthesis of
1e. The reaction was carried out at 0 ºC for 30 minutes. Then, the mixture was filtered
and the solvent was removed under vacuum by rotatory evaporation. The residue was
precipitated with ether (10 mL) and washed with acetone, dichloromethane and hexane
(25 mL of each). A dark brown powder was obtained. Yield: 48 %; mp: 113-114 ºC. 1H
NMR (400 MHz, DMSO-d6): δ 4.77 (s, 2H, -CH2 -), 7.65 (d, 2H, J=8.35 Hz, H3 + H8),
7.80 (t, 1H, J= 7.6 Hz, H6), 7.98-8.04 (m, 2H, J= 7.6 Hz, H5 + H7), 8.46 (d, 1H, J= 8.2
Hz, H4), 9.54 + 9.89 ppm (bs + bs, 4H, NH + NH2 + HCl). 13C NMR (100 MHz,
DMSO-d6): δ 21.4 (-CH2-), 121.3 (C3), 127.5 (C6 + C4a), 128.9 (C5 + C8), 131.4 (C7),
138.8 (C4), 145.7 (C8a), 158.8 (C2), 168.9 ppm (Se-C-(NH)(NH2)). IR (KBr): ν̃ 3254-
3105 (s; N-H, N-H2), 1645 cm-1 (s; C=N). MS (m/z (% abundance)): 223(6), 143(100),
128(16), 115(21). Elemental analysis calculated (%) for C11H11N3S ꢁHClꢁ1/2H2O: C:
42.63, H: 4.20, N: 13.56; found: C: 42.36, H: 3.94, N: 13.95.

ACCEPTED MANUSCRIPT
4.1.8. (6,7-Dimethoxy-2-oxo-2H-chromen-4-yl)methyl carbamimidoselenoate
hydrobromide (1f)
Conditions: 3 h at reflux. The precipitate was washed with ethyl ether (50 mL). A
yellow powder was obtained. Yield: 79 %; mp: 221-222 ºC. 1H NMR (400 MHz,
DMSO-d6): δ 3.85 (s, 6H, (-O-CH3) ×2), 4.68 (s, 2H, -CH2-), 6.33 (s, 1H, H3), 7.11 (s,
1H, H5), 7.42 (s, 1H, H8), 9.26 + 9.36 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR
(100 MHz, DMSO-d6): δ 27.6 (-CH2-), 57.6 + 57.1 ((-OCH3) ×2), 101.4 (C8), 107.6
(C5), 110.6 (C3), 112.7 (C4a), 146.8 (C6), 150.3 (C8a), 152.6 (C7), 153.7 (C4), 160.9
(C2), 165.7 ppm (Se-C-(NH)(NH2)). IR (KBr): ν̃ 3302-3220 (m; N-H, N-H2), 2977 (s;
C-Haliph), 1687 (s; C=O), 1648 (s, C=N), 1611-1467 cm-1 (m; C-Carom). MS (m/z (%
abundance)): 300(47), 220(98), 205(22), 191(100), 177(26), 147(29), 124(27), 80(37).
Elemental analysis calculated (%) for C13H14N2 O4Se ꢁHBr: C: 36.99, H: 3.58, N: 6.64;
found: C: 36.56, H: 3.53, N: 6.47.
4.1.9. Quinoxaline-2,3-diylbis(methylene)dicarbamimidoselenoate dihydrobromide
(1g)
Conditions: 2 h at room temperature. The precipitate was washed with ethyl ether (50
mL). A brown powder was obtained. Yield: 82 %; mp: 175 ºC (direct combustion). 1H
NMR (400 MHz, DMSO-d6): δ 5.03 (s, 4H, ((-CH2-) ×2), 7.89-7.90 (m, 2H, H6 + H7),
8.03-8.05 (m, 2H, H5 + H8), 9.24 + 9.38 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C
NMR (100 MHz, DMSO-d6): δ 23.4 ((-CH2-) ×2), 128.4 (C5 + C8), 131.7 (C6 + C8),
140.5 (C4a + C8a), 152.1 (C2 + C3), 167.7 ppm ((-Se-C-(NH)(NH2)) ×2). IR (KBr): ν
̃
3360-3257 (s; N-H, N-H2), 2991 (s; C-Caliph), 1632 (s; C=N), 1550-1487 cm-1 (m; C-
Harom). MS (m/z (% abundance)): 258(100), 230(8), 156(66), 129(22), 103(29), 76(31).

ACCEPTED MANUSCRIPT
Elemental analysis calculated (%) for C12H14N6Se2ꢁ2HBr: C: 25.64 H: 2.87 N: 14.95;
found: C: 25.60 H: 2.68 N: 15.14.
4.1.10. (1,3-Dioxoisoindolin-2-yl)methyl carbamimidoselenoate hydrobromide (1h)
Conditions: 45 min at 0 ºC. The precipitate was washed with acetone and ether (25 mL
of each). A pink powder was obtained. Yield: 50 %; mp: 202-203 ºC. 1H NMR (400
MHz, DMSO-d6): δ 5.44 (s, 2H, -CH2-), 7.89-7.91 (m, 2H, H4 + H7), 7.94-7.96 (m, 2H,
H5 + H6), 9.32 + 9.42 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR (100 MHz,
DMSO-d6): δ 34.2 (-CH2-), 124.4 (C4 + C7), 132.4 (C3a + C7a), 135.9 (C5 + C6),
165.1 (Se-C-(NH)(NH2)), 167.4 ppm (C1 + C3). IR (KBr): ν̃ 3314-3172 (s; N-H, N-H2),
3020 (s; C-Harom), 1774-1717 (s; C=O), 1648 cm-1 (s; C=N). MS (m/z (% abundance)):
241(6), 160(100), 133(10), 104(13), 76(14). Elemental analysis calculated (%) for
C10H9N3O2Se ꢁHBrꢁ1/2H2O: C: 32.26, H: 2.78, N: 11.57; found: C: 31.91, H: 2.59, N:
11.31.
4.1.11. (3,5-Dimethylisoxazol-4-yl)methyl carbamimidoselenoate hydrochloride (1i)
Conditions: 2.5h at reflux. After this time, the mixture was filtered and the solvent was
removed under vacuum by rotatory evaporation. The brown powder was washed with
acetone (25 mL). Yield: 76 %; mp: 168-170 ºC. 1H NMR (400 MHz, DMSO-d6): δ 2.23
(s, 3H, C3-CH3), 2.40 (s, 3H, C5-CH3), 4.41 (s, 2H, -CH2-), 9.50 ppm (bs, 4H, NH2 +
NH + HCl). 13C NMR (100 MHz, DMSO-d6): δ 10.7 ((C5)-CH3), 11.8 ((C3)-CH3), 19.5
(-CH2-), 110.6 (C4), 160.1 (C5), 166.5 (C3), 167.7 ppm (Se-C-(NH)(NH2)). IR (KBr): ν
̃
3220-3100 (s; N-H, N-H2), 1655 cm-1 (s, C=N). MS (m/z (% abundance)): 228(13),
213(22), 190(24), 156(43), 110(98), 68(100), 43(100). Elemental analysis calculated

ACCEPTED MANUSCRIPT
(%) for C7H11N3OSe ꢁHCl: C: 31.30, H: 4.50, N: 15.64; found: C: 30.94 H: 4.59, N:
15.53.
4.1.12. 4-(1H-Pyrrol-1-yl)benzyl carbamimidoselenoate hydrobromide (1j)
Conditions: 2 h at reflux. Then, the mixture was filtered and the solvent was removed
under vacuum by rotatory evaporation. The pink powder was washed with acetone and
1
ether (25 mL of each). Yield: 78 %; mp: 176-177 ºC. H NMR (400 MHz, DMSO-d6): δ
4.55 (s, 2H, -CH2-), 6.26 (t, 2H, J= 2.2 Hz, H3’ + H4’), 7.37 (t, 2H, J= 2.2 Hz, H2’ +
H5’), 7.48 (d, 2H, J=8.0 Hz, H2 + H6), 7.57 (d, 2H, J=8.0 Hz, H3 + H5), 9.17 + 9.28
ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR (100 MHz, DMSO-d6): δ 22.9 (-CH2-),
110.8 (C3’ + C4’), 120.4 (C2’ + C5’), 122.7 (C3 + C5), 129.6 (C2 + C6), 135.8 (C1),
137.4 (C4), 168.1 ppm (Se-C-(NH)(NH2)). IR (KBr): ν̃ 3213-3139 (s; N-H, N-H2),
1654 cm-1 (s, C=N). MS (m/z (% abundance)): 156(100), 124(27), 80(29), 43(45).
Elemental analysis calculated (%) for C12H13N3 SeꢁHBr: C: 40.13, H: 3.93, N: 11.70;
found C: 39.57, H: 3.93, N: 11.45.
4.1.13. Acridin-9-ylmethyl carbamimidothioate hydrobromide (2a)
Conditions: 2h at reflux. The precipitate was recrystallized from ethanol. An orange
powder was obtained. Yield: 16 %; mp: 180-181 ºC. 1H NMR (400 MHz, DMSO-d6): δ
3.62 (s, 2H, -CH2-), 6.99-7.04 (m, 4H, H2 + H3 + H6 + H7), 7.32 (t, 2H, J= 7.6 Hz, H4
+ H5), 7.39 (d, 2H, J= 7.8 Hz, H1 + H8 ), 9.39 + 9.70 + 9.82 (bs +bs + bs, 3H, NH2 +
NH), 11.1 ppm (s, 1H, HBr). 13C NMR (100 MHz, DMSO-d6): δ 48.6 (-CH2-), 68.6
(C9), 115.4 (C8a + C9a), 120.3 (C1 + C8), 121.2 (C2 + C7), 130.2 (C3 + C6), 126.5
(C4 + C5), 138.3 (C4a + C10a), 171.8 ppm (Se-C-(NH)(NH2)). IR (KBr): ν
̃ 3289-3200
(s; N-H, N-H2), 3047 (s; C-Harom), 1639 cm-1 (s; C=N). MS (m/z (% abundance)):

ACCEPTED MANUSCRIPT
267(27), 220(100), 205(15), 193(16), 179(45), 152(16), 110(15). Elemental analysis
calculated (%) for C15H13N3SꢁHBr: C: 51.72, H: 4.02, N: 12.07; found: C: 52.12, H:
4.22, N: 11.97.
4.1.14. (6-Bromobenzo[d][1,3]dioxol-5-yl)methylcarbamimidothioate
hydrobromide (2b)
Conditions: 6 h at reflux. The solvent was removed under vacuum by rotatory
evaporation and the residue was recrystallized from ethanol. The obtained yellow oil
was precipitated with ether (10 mL). A white powder was obtained. Yield: 79 %; mp:
180-181 ºC. 1H NMR (400 MHz, DMSO-d6): δ 4.52 (s, 2H, -CH2-S), 6.13 (s, 2H, O-
CH2-O), 7.22 (s, 1H, H4), 7.31 (s, 1H, H7), 9.28 ppm (bs, 4H, NH2 + NH + HBr). 13C
NMR (100 MHz, DMSO-d6): δ 36.5 (-CH2-S), 103.3 (O-CH2-O), 111.7 (C4), 113.7
(C6), 115.9 (C7), 127.2 (C5), 148.2 (C7a), 149.4 (C3a), 169.9 ppm (S-C-(NH)(NH2)).
IR (KBr): ν̃
3222-3088 (s; N-H, N-H2), 2889 (m; C-Haliph), 1648 cm-1 (s; C=N). MS (m/z
(% abundance)): 294(8), 213(100), 209(32), 192(10), 166(9), 157(14), 76(44).
Elemental analysis calculated (%) for C9H9N2BrO2SꢁHBr: C: 29.19, H: 2.70, N: 7.57;
found C: 29.27 H: 2.95 N: 7.74.
4.1.15. (5-Nitrofuran-2-yl)methyl carbamimidothioate hydrobromide (2c)
Conditions: 1 h at reflux. The precipitate was filtered and washed with ether (25 mL). A
pink powder was obtained. Yield: 41 %; mp: 171-172 ºC. 1H NMR (400 MHz, DMSO-
d6): δ 4.78 (s, 2H, -CH2-), 6.86 (s, 1H, H3), 7.77 (s, 1H, H4), 9.17 + 9.35 ppm (bs + bs,
4H, NH2 + NH + HBr). 13C NMR (100 MHz, DMSO-d6): δ 27.8 (-CH2-), 114.1 (C3),
114.9 (C4), 152.2 (C5), 154.2 (C2), 168.8 ppm (-S-C-(NH)(NH2)). IR (KBr): ν̃ 3219-
3078 (s; N-H, N-H2), 1655 cm-1 (s; C=N). MS (m/z (% abundance)): 205(1), 170(15),
159(13), 154(58), 140(19), 126(75), 114(33), 80(100), 60(31), 52(30). Elemental

ACCEPTED MANUSCRIPT
analysis calculated (%) for C6H7N3O3SꢁHBr: C: 25.53, H 2.83, N: 14.89; found: C:
25.61, H: 2.80, N: 14.96.
4.1.16. (9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl carbamimidothioate
hydrochloride (2d)
Conditions: 3 h at reflux. The precipitate was filtered and washed with dichloromethane
and ether (25 mL of each). A yellow powder was obtained. Yield: 57 %; mp: 233-234
ºC. 1H NMR (400 MHz, DMSO-d6): δ 4.77 (s, 2H, -CH2-), 7.92-7.95 (m, 2H, H5 + H8),
7.99 (d, 1H, J=8.0Hz, H3), 8.19-8.23 (m, 3H, H1 + H6 + H7), 8.29-8.30 (m, 1H, H4),
9.29 + 9.44 ppm (bs, 4H, NH + NH2 + HCl). 13C NMR (100 MHz, DMSO-d6): δ 34.2 (-
CH2-), 127.6 (C5 + C8), 127.8 (C4), 128.2 (C1), 133.2 (C3), 133.8 (C6 + C7), 134.0
(C4a), 135.4 (C8a + C10a), 135.6 (C9a), 143.8 (C2), 169.5 (S-C-(NH)(NH2)), 182.8
(C10), 183.1 ppm (C9). IR (KBr): ν̃ 3274-3195 (m; N-H, N-H2), 3025 (s; C-Harom),
1668(s; C=O), 1650 cm-1 (s; C=N). MS (m/z (% abundance)): 296(4), 254(57),
221(100), 193(55), 165(23), 152(8), 139(6), 82(10). Elemental analysis calculated (%)
for C16H12N2O2S ꢁHCl: C: 57.74, H: 3.94, N: 8.42; found: C: 57.60, H: 4.02, N: 8.17.
4.1.17. Quinolin-2-ylmethyl carbamimidothioate hydrochloride (2e)
The commercially available 2-(chloromethyl)quinoline hydrochloride was treated with
basic water (50 mL) in order to obtain the free base, which was used for the synthesis of
2e. The reaction was carried out at reflux for 3 hours. The solvent was removed under
vacuum by rotatory evaporation and the residue was recrystallized from ethanol. The
precipitate was filtered and washed with dichloromethane and hexane (25 mL of each).
1
A brown powder was obtained. Yield: 34 %; mp: 178-179 ºC. H NMR (400 MHz,
DMSO-d6): δ 4.83 (s, 2H, -CH2-), 7.64-7.67 (m, 2H, H3 + H8), 7.82 (t, 1H, J=8.5 Hz,
H6), 8.02 (t, 2H, J=8.4 Hz, H5 + H7), 8.46 (d, 1H, J=8.4 Hz, H4), 9.50 ppm (bs, 3H,

ACCEPTED MANUSCRIPT
NH + NH2). 13C NMR (100 MHz, DMSO-d6): δ 37.3 (-CH2-), 122.2 (C3), 127.8 (C6 +
C4a), 128.9 (C5 + C8), 131.2 (C7), 138.7 (C4), 147.4 (C8a), 157.1 (C2), 171.1 ppm (S-
C-(NH)(NH2)). IR (KBr): ν̃
3382 (s; N-H, N-H2), 3035 (s; C-Harom), 1676-1641 cm-1 (s;
C=N). MS (m/z (% abundance)): 221(30), 193(100), 165(33), 142(36), 73(44), 55(54).
Elemental analysis calculated (%) for C11H11N3SꢁHCl: C: 52.07, H: 4.77, N: 16.56;
found: C: 51.59, H: 4.80, N: 16.47
4.1.18. (6,7-Dimethoxy-2-oxo-2H-chromen-4-yl)methyl carbamimidothioate
hydrobromide (2f)
Conditions: 3 h at reflux. The precipitate was filtered and washed with ether (25 mL). A
yellow powder was obtained. Yield: 92 %; mp: 253-254 ºC. 1H NMR (400 MHz,
DMSO-d6): δ 3.88 (s, 6H, (-OCH3) ×2), 4.78 (s, 2H, -CH2-), 6.39 (s, 1H, H3), 7.13 (s,
1H, H5), 7.34 (s, 1H, H8), 9.14 + 9.33 ppm (bs + bs, 4H, NH2 + NH + HBr), 13C NMR
(100 MHz, DMSO-d6): δ 31.9 (-CH2-), 57.2 + 57.4 ((-OCH3) ×2), 101.3 (C8), 107.3
(C5), 110.6 (C3), 112.7 (C4a), 146.8 (C6), 150.1 (C8a), 150.8 (C7), 153.7 (C4), 160.9
(C2), 168.9 ppm (Se-C-(NH)(NH2)). IR (KBr): ν̃ 3310-3214 (s; N-H, N-H2), 2975(s; C-
Haliph), 1692 (s; C=O), 1651 (s; C=N). MS (m/z (% abundance)): 300(21), 294(26),
277(29), 252(100), 237(42), 219(26), 209(16), 191(94), 177(9), 147(19), 76(30).
Elemental analysis calculated (%) for C13H14N2O4SꢁHBr: C: 41.61 H: 4.03 N: 7.47;
found: C: 41.36, H: 3.79, N: 7.24.
4.1.19. Quinoxaline-2,3-diylbis(methylene) dicarbamimidothioate dihydrobromide
(2g)
Conditions: 3.5 h at reflux. The precipitate was filtered and washed with ether (25 mL).
A gray powder was obtained. Yield: 16 %; mp: 221 ºC (direct combustion). 1H NMR

ACCEPTED MANUSCRIPT
(400 MHz, DMSO-d6): δ 5.07 (s, 4H, (-CH2-) ×2), 7.89-7.91 (m, 2H, H6 + H7), 8.04-
8.07 (m, 2H, H5+ H8), 9.01 + 9.26 ppm (bs + bs, 4H, NH2 + NH + HBr). 13C NMR
(100 MHz, DMSO-d6): δ 35.9 ((-CH2-) ×2), 129.1 (C5 + C8), 131.6 (C6 + C8), 140.5
(C4a + C8a), 150.1 (C2 + C3), 170.6 ppm ((-Se-C-(NH)(NH2)) x2). IR (KBr): ν̃ 3378-
3243 (s; N-H, N-H2), 3091 (s; C-Harom), 2992 (s; C-Haliph), 1631 cm-1 (s; C=N). MS (m/z
(% abundance)): 272(2), 220(9), 187(31), 156(9), 102(9), 76(100), 60(27). Elemental
analysis calculated (%) for C12H14N6S2 ꢁ2HBr: C: 30.78, H: 3.44, N: 17.95; found: C:
30.43, H: 3.26, N: 18.27.
4.1.20. (1,3-Dioxoisoindolin-2-yl)methyl carbamimidothioate hydrobromide (2h)
Conditions: 2 h at reflux. The precipitate was washed with ethyl ether (50 mL). A white
powder was obtained. Yield: 77 %; mp: 217-219 ºC. 1H NMR (400 MHz, DMSO-d6): δ
5.40 (s, 2H, -CH2-), 7.90-7.92 (m, 2H, H4 + H7), 7.95-7.97 (m, 2H, H5 + H6), 9.30
ppm (bs, 4H, NH2 + NH + HBr). 13C NMR (100 MHz, DMSO-d6): δ 39.4 (-CH2-),
124.5 (C4 + C7), 132.2 (C3a + C7a), 135.9 (C5 + C6), 167.5 (C1 + C3), 168.1 ppm (S-
C-(NH)(NH2)). IR (KBr): ν̃ 3322-3129 (s; N-H, N-H2), 3009 (s; C-Harom), 1774-1716 (s;
C=O), 1655 cm-1 (s; C=N). MS (m/z (% abundance)): 233(40), 191(36), 163(91),
110(49), 75(54), 57(85), 43(100). Elemental analysis calculated (%) for C10H9N3O2S
·HBr: C: 37.99, H: 3.19, N: 13.29; found: C: 37.87, H: 3.22, N: 13.36.
4.1.21. (3,5-Dimethylisoxazol-4-yl)methyl carbamimidothioate hydrochloride (2i)
Conditions: 2.5 h at reflux. The solvent was removed under vacuum by rotatory
evaporation. The yellow solid was washed with acetone and ether (25 mL of each).
Yield: 91 %; mp: 179-181 ºC. 1H NMR (400 MHz, DMSO-d6): δ 2.24 (s, 3H, C3-CH3),
2.40 (s, 3H, C5-CH3), 4.45 (s, 2H, -CH2-), 9.42 ppm (bs, 3H, NH2 + NH). 13C NMR

ACCEPTED MANUSCRIPT
(100 MHz, DMSO-d6): δ 9.7 ((C5)-CH3), 10.8 ((C3)-CH3), 23.6 (-CH2-), 108.0 (C4),
159.2 (C5), 167.5 (C3), 168.8 ppm (-S-C(NH)(NH2)). IR (KBr): ν̃ 3228 (s; N-H, N-H2),
3066 (s; C-Harom), 1652 cm-1 (s; C=N). MS (m/z (% abundance)): 160(16), 144(56),
110(58), 76(53), 68(95), 43(100). Elemental analysis calculated (%) for C7H11N3OS
ꢁHCl: C: 37.92, H: 5.46, N: 18.95; found: C: 37.67, H: 5.62, N: 19.52.
4.1.22. 4-(1H-Pyrrol-1-yl)benzyl carbamimidothioate hydrobromide (2j)
Conditions: reflux for 2 h. The precipitate was filtered and the solvent was evaporated
under vacuum by rotatory evaporation. The residue was precipitated with hexane (10
mL) and washed with acetone and ether (25 mL of each). A brown powder was
obtained. Yield: 75 %; mp: 195-196 ºC. 1H NMR (400 MHz, DMSO-d6): δ 4.52 (s, 2H,
-CH2-), 6.27 (t, 2H, J=2.0 Hz, H3’ + H4’), 7.38 (t, 2H, J=2.0 Hz, H2’ + H5’), 7.50 (d,
1H, J=8.5Hz, H2 + H6), 7.60 (d, 1H, J=8.5 Hz, H3 + H5), 9.01 + 9.20 ppm (bs + bs,
4H, NH2 + NH + HBr). 13C NMR (100 MHz, DMSO-d6): δ 33.6 (-CH2-), 110.6 (C3’ +
C4’), 118.9 (C2’ + C5’), 119.4 (C3 + C5), 130.3 (C2 + C6), 131.7 (C1), 139.4 (C4),
168.9 ppm (S-C-(NH2)(NH)). IR (KBr): ν̃ 3453-3233 (s; N-H + N-H2), 3088 (s; C-
Harom), 1639 cm-1 (s; C=N). MS (m/z (% abundance)): 231(59), 156(100), 128(27),
43(44). Elemental analysis calculated (%) for C12H13N3SꢁHBr: C: 46.16, H: 4.52, N:
13.46; found: C: 45.90, H: 4.76, N: 13.20.
4.2. Biological evaluation
4.2.1. Cell cultures
PANC-1, BxPC3 and 1205Lu cell lines were grown in DMEM medium; DU145 and
A549 cells in RPMI 1640 medium; HT29, HCT116 and RKO cells were grown in
McCoy's 5A medium (Corning # 10-051-CI) at 37 °C and 5 % CO2. All used mediums

ACCEPTED MANUSCRIPT
were supplemented with 10 % fetal bovine serum (FBS) and 100 units/mL of penicillin
and streptomycin (Corning # 30-002-CI). Cell lines were purchased from ATCC.
4.2.2. Reagents and anti-bodies
Dimethyl Sulfoxide (DMSO) was purchased from Fisher (#BP231-100) and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ) from Sigma (#2128).
The antibodies for Caspase 3 (#9668S), PARP (#9542S) and XIAP (#14334s) were
obtained from Cell Signaling and Bcl-2 (#ab7973) from abcam. β-Actin antibody was
purchased from Sigma-Aldrich (A5316).
4.2.3. Cell viability
A total of 3,000 cells/well were treated with either DMSO or increasing concentrations
(0.5-50 µM) of the new seleno- and thio-urea derivatives for 24, 48 or 72 hours. Three
hours prior the termination point, 20 µL of MTT (5 mg/mL stock) was added. Resultant
formazan crystals were dissolved in 50 µL of DMSO and absorbance was measured at
570 nm and 630 nm wavelength. IC50 values were calculated using GraphPad Prism
version 6.01.
4.2.4. Live and Dead assay
HCT116 cells (7 ×104 /well) were seeded in a 6 well plate, grown overnight and treated
with the different concentrations of 1g for 24 h. Then they were trypsinized and stained
with Calcein AM and Ethidium Homodimer-1 (LIVE/DEAD viability/cytotoxicity kit
for mammalian cells, Life Technologies) according to the manufacturer’s instructions.
Data were analyzed by flow cytometry (BD FACSCalibur)