Novel selenadiazole derivatives as selective antitumor and radical scavenging agents

Article abstract of DOI:10.1016/j.ejmech.2018.07.063

Twenty-seven novel benzo[c][1,2,5]selenadiazole-5-carboxylic acid (BSCA) derivatives were designed and synthesized. Anti-proliferative activity of these structures was tested in vitro against a panel of five human cancer cell lines, including prostate (PC-3), colon (HT-29), leukemia (CCRF-CEM), lung (HTB-54) and breast (MCF-7). Four compounds (5, 6, 7 and 19) showed potent inhibitory activity with GI₅₀ values below 10 μM in at least one of the cancer cell lines. The selectivity of these compounds was further examined in two non-malignant cell lines derived from breast (184B5) and lung (BEAS-2B). Compound 7 exhibited promising anti-proliferative activity (GI₅₀ = 3.7 μM) in MCF-7 cells, together with high selectivity index (SI > 27.1). The induction of cell death by compound 7 was independent of the apoptotic process and it did not affect cell cycle progression either. Likewise, radical scavenging properties of the new selenadiazole derivatives were confirmed by testing their ability to scavenge DPPH radicals. Four compounds (1, 2, 8 and 9) showed potent radical scavenging activity, compound 9 being the most effective. Overall, while compound 7 was identified as the most cell growth inhibitory agent and selectively toxic to cancer cells, compound 9 proved to be the most potent antioxidant among the selenadiazole derivatives synthesized. This series of compounds can serve as an excellent scaffold to achieve new and potent antioxidant compounds useful for several diseases, i.e. cancer, neurodegenerative, heart diseases and leishmaniasis, considering the high radical scavenging activity and low toxicity showed by most of the compounds.

Full text of DOI:10.1016/j.ejmech.2018.07.063

Accepted Manuscript
Novel selenadiazole derivatives as selective antitumor and radical scavenging agents
Ana Carolina Ruberte, Daniel Plano, Ignacio Encío, Carlos Aydillo, Arun K. Sharma,
Carmen Sanmartín
PII:
S0223-5234(18)30628-7
10.1016/j.ejmech.2018.07.063
EJMECH 10598
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2018
Revised Date: 23 July 2018
Accepted Date: 27 July 2018
Please cite this article as: A.C. Ruberte, D. Plano, I. Encío, C. Aydillo, A.K. Sharma, C. Sanmartín, Novel
selenadiazole derivatives as selective antitumor and radical scavenging agents, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.063.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Highlights
•
•
27 novel BSCA derivatives were designed and synthesized
Compound 7 potently inhibited cancer cell viability and showed high
selectivity.
•
•
Compound 7 neither induced apoptosis nor had effect on cell cycle
progression.
Compound 9 showed significant high radical scavenging activity.
1

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Novel selenadiazole derivatives as selective
antitumor and radical scavenging agents
Ana Carolina Ruberte1,2,, Daniel Plano1,2,3, Ignacio Encío4, Carlos
Aydillo1,2, Arun K. Sharma3 and Carmen Sanmartín1,2*
1Department of Organic and Pharmaceutical Chemistry, University of Navarra,
Irunlarrea 1, E-31008 Pamplona, Spain.
2Instituto de Investigación Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008
Pamplona, Spain.
3Department of Pharmacology; Penn State Cancer Institute, CH72;
Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
4Department of Health Sciences, Public University of Navarra, Avda. Barañain s/n, E-
31008 Pamplona, Spain.
* Prof. Carmen Sanmartín
Department of Organic and Pharmaceutical Chemistry
University of Navarra
Irunlarrea, 1, E-31008 Pamplona
SPAIN
+34 948 425 600 (Telephone)
+34 948 425 649 (Fax)
e-mail: sanmartin@unav.es
2

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Abstract
Twenty-seven novel benzo[c][1,2,5]selenadiazole-5-carboxylic acid (BSCA) derivatives
were designed and synthesized. Anti-proliferative activity of these structures was tested
in vitro against a panel of five human cancer cell lines, including prostate (PC-3), colon
(HT-29), leukemia (CCRF-CEM), lung (HTB-54) and breast (MCF-7). Four
compounds (5, 6, 7 and 19) showed potent inhibitory activity with GI50 values below 10
µM in at least one of the cancer cell lines. The selectivity of these compounds was
further examined in two non-malignant cell lines derived from breast (184B5) and lung
(BEAS-2B). Compound 7 exhibited promising anti-proliferative activity (GI50 = 3.7
µM) in MCF-7 cells, together with high selectivity index (SI>27.1).
The induction of cell death by compound 7 was independent of the apoptotic process
and it did not affect cell cycle progression either. Likewise, radical scavenging
properties of the new selenadiazole derivatives were confirmed by testing their ability to
scavenge DPPH radicals. Four compounds (1, 2, 8 and 9) showed potent radical
scavenging activity, compound 9 being the most effective. Overall, while compound 7
was identified as the most cell growth inhibitory agent and selectively toxic to cancer
cells, compound 9 proved to be the most potent antioxidant among the selenadiazole
derivatives synthesized. This series of compounds can serve as an excellent scaffold to
achieve new and potent antioxidant compounds useful for several diseases, i.e. cancer,
neurodegenerative, heart diseases and leishmaniasis, considering the high radical
scavenging activity and low toxicity showed by most of the compounds.
Keywords: Anti-proliferative activity, radical scavenging, selenadiazole, selenium.
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1. Introduction
Cancer is a major public health problem worldwide due to its high incidence, morbidity
and mortality [1]. According to the American Cancer Society, 1,735,350 new cancer
cases are projected to occur in the United States in 2018 [2], the most prevalent being
prostate, breast, lung, and colorectal cancers [3].
There are many different types of cancer treatments, including surgery, radiation
therapy, and/or systemic therapy (e.g., chemotherapy, immunotherapy). Chemotherapy
is an effective treatment against cancer but is often associated with undesired side
effects, making the seeking of new chemotherapies a priority. Over the last decade,
selenium (Se) compounds have been demonstrated to have inhibitory effects on cancer
cell growth and proliferation. Many mechanisms of action have been identified,
including the induction of apoptosis, the modulation of the activity of some kinases,
antioxidant effects mainly through selenoproteins [4], or a combination of these and
other mechanisms [5, 6]. Several chemical entities have been identified: methylseleno
derivatives such as methylseleninic acid and selenomethionine, isoselenocyanates,
selenosemicarbazones, selenourea scaffolds, selenocyanates and diselenides, or sugar-
conjugated Se analogues [7-9] as attractive scaffolds for the development of new
anticancer agents.
Recently, fused selenoheteroaryl derivatives have received great attention due to their
possible usefulness as therapeutic drugs for cancer treatment. Some representative
examples include 2-Phenyl-1,2-benzisoselenazol-3[2H]-one (ebselen, EBS) [10], 2,2'-
(1,2-Ethanediyl)bis(1,2-benzoselenazol-3[2H]-one) (ethaselen, BBSKE) [11] and 1,2,5-
selenadiazole derivatives [12] (Figure 1). EBS shows high antioxidant capacity through
the catalysis of several essential reactions for the protection of cellular components
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from oxidative and free radical damage, acting as a mimetic of glutathione peroxidase
[13]. Besides, it is noteworthy for its excellent pharmacological profile as well as for its
therapeutic safety as demonstrated by outcomes of Phase II clinical trials for prevention
of noise-induced hearing loss [14].
On the other hand, BBSKE has demonstrated antioxidant and anti-inflammatory
activities, as well as important antitumoral effects in lung [11] and colon cancer models,
alone or in combination with cisplatin or sunitinib, through apoptosis induction [15, 16].
Furthermore, it inhibited the proliferation of PC-3 and DU-145 prostate cancer cell lines
by cell cycle arrest at the S phase and the induction of apoptosis [17]. In addition, it
presents an excellent pharmacological profile, as shown in Phase I clinical trials [18].
Other well documented seleno heteroaryl derivatives are 1,2,5-selenadiazole derivatives
(Figure 1), that have been identified as novel agents with anti-proliferative effect
against human cancer cells through the induction of apoptosis or cell cycle arrest with
the involvement of oxidative stress. Among them, 4-(benzo[c][1,2,5]selenadiazol-6-yl)-
benzene-1,2-diamine (SD-1 in Figure 1) is a potent apoptosis inducer in glioma acting
through the mitochondrial pathway [19]. Moreover, these 1,2,5-selenadiazole
compounds have shown to be less toxic to non-tumoral cells [12, 20, 21]. There have
been many reports related to structural modifications in this chemical scaffold,
particularly at position “5”. So, Xie et al. [22] described novel 5-susbstituted
selenadiazoles (SD-2 in Figure 1) as apoptosis inducers in A375 human melanoma
cells, with some of them (SD-3 in Figure 1) acting as intracellular redox balance
disruptors [20]. In addition, the introduction of simple groups, i.e. 5-methyl or 5-nitro
(SD-4 in Figure 1), enhanced the anticancer activity alleviating the toxicity to the main
organs [21]. On the other hand, some of these 5-functionalized selenadiazole derivatives
(SD-3 in Figure 1) have demonstrated better anti-proliferative activity and higher
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stability than the reference drug mitomycin, a clinically used anti-bladder cancer drug
[12].
Figure 1. Structures of EBS, BBSKE and 5-substituted 1,2,5-selenadiazole.
Based on the above facts and as a continuation of our previous work [23], we optimized
the bioactive entity benzo[c][1,2,5]selenadiazol-5-carboxylic acid (BSCA) by
manipulating the “5” position with a wide variety of moieties (compounds 1-27, Figure
2) using an amide linkage. The amide linkage was chosen because of it plays a major
role in biological systems, including peptides and proteins [24]. Likewise, this
functionality is widely used in medicinal chemistry and it is present in numerous
anticancer drugs such as flutamide, bicalutamide and sorafenib [25, 26].
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Figure 2. General structure of the novel selenadiazole compounds.
All the synthesized compounds were tested in vitro against a panel of five human tumor
cell lines and two non-malignant cell lines in order to determine their anti-proliferative
activity and their selectivity. Likewise, the capability of the synthesized compounds to
interact with the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was examined.
The most active and selective compound (compound 7) was further evaluated for its
apoptotic effects and cell cycle distribution. The structure of this compound was
confirmed by X-ray diffraction.
2. Results and discussion
2.1. Design
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The novel BSCA analogs were designed by optimizing the parent scaffold by
manipulating the “5” position with a variety of moieties using the following criteria:
a) Several electron-withdrawing or electron-donating groups were substituted in
the phenyl ring in order to modulate the electronic distribution. Moreover,
several chemical Se forms, with proven antitumoral effects, e. g. selenol [27]
and methylseleno [28] were also incorporated in “para” position of the phenyl
ring.
b) Modulation of the length of the linker between the amide and R groups, from 0
to 4 methylenes (carbon atoms), with the aim of finding the optimal chain length
required for activity.
c) Several heterocycles (mono- and bi-cyclic) with proven cytotoxic effects, such
as 2-aminothiazole [29, 30], aminoquinoline [31-33] and 2-aminobenzothiazole
[34, 35], which also have antioxidant capacity [36].
d) Alkyl, cycloalkyl and hydroxyalkyl chains in order to modulate the lipophilicity
along with the entropy of the molecules.
e) Finally, a symmetric bi-functionalized amine was used to obtain compound 27,
in order to evaluate whether the presence of a second selenadiazole ring would
impact on the antitumor activity.
2.2. Chemistry
The synthetic procedures for the preparation of the targeted compounds (1-27) are
summarized in Schemes 1 and 2. The benzo[c][1,2,5]selenadiazole-5-carboxylic acid
(BSCA) was obtained as previously reported by us [23]. Chlorination of this acid with
thionyl chloride, followed by reaction with the corresponding amines at room
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temperature in dry chloroform and 1 equivalent of triethylamine have been used to
prepare compounds 1-27. The starting amines that are not commercially available, such
as 4-(methylselanyl)aniline (7i) and 4-aminobenzeneselenol (8i), were synthesized as
shown in Scheme 2. The amine 7i was synthesized by treating a methanolic solution of
4-Aminophenylselenocyanate and sodium hydroxide (0.4 N) with iodomethane in a
1:1.5 molar ratio at room temperature for 40 min. 4-Aminophenylselenocyanate was
prepared following a previously described synthetic procedure [23]. The synthetic route
for amine 8i was based on the reaction between 4-bromoaniline and selenourea in a
1:1.1 molar ratio at reflux in absolute ethanol as solvent for 2 h.
Scheme 1. General schematic for the synthesis of novel BSCA derivatives (1-27).
Scheme 2. Schematic for the synthesis of amines 7i and 8i.
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The structures of all the compounds were confirmed using spectroscopic methods (IR,
1H-NMR, 13C-NMR) and elemental analyses, as described in the Experimental Section.
In general, the IR spectra of the compounds displayed absorption bands around 3395-
3093 cm-1 originated from the N-H stretching vibration. The strong bands between 1689
1
and 1620 cm-1 corresponded to carbonyl bonds stretching. Inspection of H-NMR
spectra revealed one sharp peak in the range of 12.96-8.18 ppm due to the presence of
the secondary amide proton. The presence of methylene as linker between the amide
group and phenyl ring led to a decrease in the chemical shift of this sharp peak from
10.56 ppm (compound 1) to 9.37, 8.90, 8.82 and 8.78 ppm (compounds 20, 22, 23 and
25, respectively). The proton signals for the heteroaryl ring containing Se appear as a
singlet between 8.69 and 5.64 ppm, and two multiplets from 7.36 to 8.72 ppm. The
chemical shift of the methyl group attached to the chalcogen elements (oxygen, sulfur
or Se) appear at 3.76 (compounds 5), 2.48 (compound 6) and 2.34 ppm (compound 7),
respectively. For the methylene linker (-CH2-) directly attached to oxygen and nitrogen
atoms, the proton signals show in the range of 3.51-3.29 ppm, and the rest of protons for
13
other methylene linkers appear at higher fields (1.76-1.48 ppm). Similarly, in the C-
NMR spectra, the chemical shifts appeared between 122.72 and 37.71 ppm while
methylene groups signals were observed in 33.16-26.59 ppm region.
2.3.
Biological evaluation
2.3.1. Anti-proliferative activity
All the newly synthesized compounds were screened for their anti-proliferative activity
against a panel of five human tumor cell lines: lymphocytic leukemia (CCRF-CEM),
lung carcinoma (HTB-54), colon carcinoma (HT-29), prostate adenocarcinoma (PC-3)
and breast adenocarcinoma (MCF-7). The in vitro inhibition of cell viability was
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analyzed by a colorimetric microassay based on the reduction of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) according to a method previously
described [37]. The five cell lines were treated with each compound for 72 h at two
concentrations (100 µM and 10 µM). Compound 21 was not tested at 100 µM due to
solubility problems. The data are expressed as percentage of cell growth ± SEM at least
3 independent experiments performed in quadruplicates (Table S1). Results at 10 µM
concentration are summarized in Figure 3.
A
B
125
100
75
50
25
0
125
100
75
50
25
0
Cancer Cell lines
Cancer Cell lines
C
D
125
100
75
50
25
0
125
100
75
50
25
0
Cancer Cell lines
Cancer Cell lines
Figure 3. Anti-proliferative effect of synthesized compounds 1 – 6 (A), 7 – 13 (B), 14 –
20 (C) and 21 – 27 (D) on five human cancer cell lines after 72 h of treatment with a
dose of 10 µM for each compound.
As shown in Figure 3, some compounds were active under our experimental conditions,
MCF-7 cells being the most sensitive cells. These results allowed us to determine some
preliminary structure-activity relationships, since:
a) The length of the linker between the phenyl ring and the amide group seems
not to be important for inhibition of cell viability, as no significant
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differences were observed with the increase of the linker length (compounds
1, 20, 22, 23 and 25).
b) Among the different substituents on the phenyl ring, methylchalcogen
(methoxy, methylthio, methylseleno) groups seemed to be the most active
ones (compounds 5, 6 and 7 respectively). Nevertheless, the presence of
three methoxy groups leads to a lower anti-proliferative effect (compound
10).
c) Great differences in the cell viability can be observed among mono- and bi-
cyclic heterocycles with proven cytotoxic effects. Thus, compound 19, with
a benzothiazole substituent, inhibited viability of MCF-7 and HTB-54 cells,
while compound 16, with a thiazole group, showed negligible effect against
all tested cell lines.
d) The incorporation of nitrogen atom into a six-member cycle (piperidyl or
piperazinyl in compounds 13 and 27, respectively) in the “5” position of the
selenadiazolic core seems to decrease the inhibitory effect of these
derivatives as compared to the analog carbocyclic substituent (cyclohexyl in
compound 12).
As shown in Figure 3, compounds 5, 6, 7 and 19 were found to be the most active, with
reduction of the cell growth by >50%, after 72 h of treatment at 10 µM, in at least two
cell lines. They were further tested at four different concentrations (1, 10, 50 and 100
µM), in order to establish their dose-response curve. As a guide with regard to
selectivity, these compounds were further examined for toxicity in two non-malignant
cell lines, one established from normal breast tissue (184B5) and another established
from normal bronchial epithelium (BEAS-2B). Results are expressed as GI50, TGI,
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LC50, and selectivity index (SI) that was calculated as the ratio of the GI50 values
determined for the non-malignant and the tumoral cells (GI50 (184B5)/ GI50 (MCF-7)
and GI50 (BEAS-2B) / GI50 (HTB-54)). The obtained results are shown in Tables 1 and
2. EBS and BBSKE were used as reference drugs, given that both are seleno
heterocyclic compounds that are currently under clinical trials. BBSKE has been
synthesized according to a previously reported synthetic route [38], with minor
modifications described in the supplementary material. Furthermore, doxorubicin [39]
and etoposide [40, 41] were used as positive controls due to the promising results
shown by these compounds in vitro and in vivo against different tumor types.
Table 1. Anti-proliferative activity (average GI50, TGI and LC50 values
expressed in µM) of selenadiazoles 5, 6, 7 and 19 and EBS, BBSKE, doxorubicin and
etoposide.
Cell lines
HT-29
PC-3
CCRF-CEM
a
c
a
c
a
c
Comp.
5
GI50
TGIb LC50
GI50
9.8
TGIb LC50
GI50
TGIb
LC50
40.3
53.4
>100
>100
>100
>100
>100
>100
>100
>100
83.9
62.4
85.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6
7
7.4
8.8
8.8
22.3
>100
20.3
0.1
>100
>100
33.2
4.0
>100
>100
46.0
10.9
>100
24.8
>100
>100
39.1
1.2
>100
>100
61.0
6.6
>100
57.1
5.0
>100
73.8
>100
90.5
25.4
0.3
19
EBS
8.5
BBSKE
doxorubicin
etoposided
25.1
1x10-2
3x10-2
7x10-2
31.6
>100
>100
0.6
4.0
79.4
1.6
50.5
89.9
a GI50: concentration that reduces by 50% the growth of treated cells with respect
to untreated controls.
b TGI: concentration that completely inhibits the cell growth.
c LC50: concentration that kills 50% of the initial cells.
d NCI data (http://dtp.nci.nih.gov).
e n.d., not determined.
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Table 2. Anti-proliferative activity (average GI50, TGI and LC50 values
expressed in µM) and selectivity (calculated SI values) of selenadiazoles 5, 6, 7 and 19
and EBS, BBSKE, doxorubicin and etoposide.
Cell lines
MCF-7
184B5
HTB-54
BEAS-2B
a
c
a
c
a
c
a
c
Comp.
GI50
TGIb LC50
GI50
TGIb LC50
GI50
TGIb LC50
GI50
TGIb LC50
77.1
>100 >100
>100 >100
>100 >100
82.2 >100
>100
>100
18.7
18.0
>100 >100 >100 >100 >100
>100 >100 96.4 >100 >100
5
6
6.2
86.9
72.7
>100
>100
>100 >100
>100
>100
>100 >100 >100 >100 >100
>100 >100 >100 >100 >100
7
3.7
6.3
6.5
13.7
95.6
>100
19
58.8
24.2
>100 >100
30.1
19.2
1.2
>100
31.5
4.6
>100
43.8
8.1
68.6
19.7
83.7
30.3
1.3
98.7
40.9
3.5
49.5
20.6
0.1
76.9
30.3
1.5
>100
40.0
1.8
EBS
40.7
0.3
72.8
7.8
0.79
574
BBSKE
2x10-3
<1x10-2
doxorubicin
etoposidee
19.9
>100 >100
n.d.f
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a
GI50: concentration that reduces by 50% the growth of treated cells with respect to
untreated controls.
b TGI: concentration that completely inhibits the cell growth.
c LC50: concentration that kills 50% of the initial cells.
d SI: Selectivity index was calculated as the ratio of the GI50 values determined for the
non-malignant and the tumoral cells (GI50 (184B5)/ GI50 (MCF-7) and GI50 (BEAS-2B)
/ GI50 (HTB-54)).
e NCI data (http://dtp.nci.nih.gov).
f n.d.: not determined.
As shown in Tables 1 and 2, the selected cancer cell lines present different sensitivity
profiles to the action of these derivatives. Thus, three compounds (6, 7 and 19)
effectively inhibited cell viability with GI50 values ranging from 3.7 µM to 18 µM on
solid tumors (MCF-7, HTB-54 and PC-3 cell lines). However, these compounds were
inactive against liquid tumors (CCRF-CEM cell line).
Interestingly, these selenadiazoles exhibited greater anti-proliferative activity than EBS
in solid tumors, along with higher selectivity indexes (Tables 1 and 2). For instance,
compounds 6 (GI50 = 6.2 µM) and 7 (GI50 = 3.7 µM) in MCF-7 cells were 9.5 and 15.9
times more active, respectively, than standard EBS (GI50 = 58.8 µM) and they also
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showed at least 25.9 and 53.1 times higher selectivity index, respectively. Besides, we
have improved significantly the selectivity indexes of BBSKE and the potency of
starting compound, BSCA [23].
Moreover, compound 7, with two Se atoms in the structure and having GI50 value of 3.7
µM and SI > 27.1 in MCF-7 cell line, emerged as the most active and selective
derivative. Taking into account the effects on the cell viability in the four cancer cell
lines tested, together with the selectivity (Tables 1 and 2), derivative 7 was selected as
the lead compound for further biological evaluation.
2.3.2. Evaluation of cell cycle progression and apoptosis induction
Various scientific pieces of evidence show that Se compounds are potent antitumor
agents owing to the induction of apoptosis and the inhibition of cell proliferation [6,
42]. Compound 7 presented notable inhibitory activity in MCF-7 cells. Therefore, we
decided to evaluate whether this activity was related to its ability to induce apoptosis
and / or its effect on the cell cycle progression in MCF-7 cells. This study was
conducted using the Apo DirectTM kit (BD Pharmigen) [43] based on the TUNEL
technique with 50 µM of the compound and 72 h of treatment. Camptothecin was
employed as a positive control.
As shown in Figure 4, the obtained results demonstrated that the inhibition of cell
growth was independent both of the apoptotic process and effect on cell cycle
progression.
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A
A
B
B
A
A
72 h
72 h
112200
72 h
72 h
110000
110000
80
80
80
80
60
60
60
60
40
40
40
40
20
20
20
20
0
0
0
0
7 (50 µM)
DMSO
DMSO
Camptothecin (3 µM)
7 (50 µM)
7 (50 µM)
DMSO
DMSO
7 (50 µM) Camptothecin (3 µM)
Figure 4. The activity of compound 7 was independent of apoptotic process and it did
not modulate cell cycle progression. (A) Bar chart representing the distribution of cells
in different phases of the cell cycle. (B) Percentage of apoptotic cells were calculated.
Results are expressed as mean ± SD of at least three independent experiments
performed in duplicate. *p< 0.05, **p<0.01 and ***p<0.001 with respect the control.
2.4. X-ray
crystallography
of
N-(4-
(methylselanyl)phenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (compound
7)
The structure of the lead compound 7 was further confirmed by X-ray diffraction after
developing a single crystal. Structure and crystal packing of the compound are
presented in Figures 5 and 6, respectively, and selected interatomic distances and
angles for this structure are collected in Table S2 and S3 in the Supplementary material.
The crystal data and structure refinement are listed in Table S4. Hydrogen bonding
interactions in the crystal structure are given in Table S4.
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Figure 5. ORTEP diagram of compound 7 with displacement ellipsoids drawn at 50%
probability level.
Figure 6. Crystal packing of compound 7; some hydrogen atoms were omitted for
clarity.
2.5. DPPH free radical-scavenging activity.
Se compounds, including EBS, show potential chemopreventive activity, which has
been related to their strong antioxidant activity and their ability to interact with redox
status of the cell [44]. We decided to evaluate the antioxidant capacity of the
selenadiazole derivatives using the DPPH method [45].
Determinations were performed at five different concentrations of all the synthesized
compounds ranging from 3.13 x 10-5 to 0.25 mg/mL and were recorded at different time
17

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points (30 ́, 90´, 180´). The ascorbic acid was used as the positive control and the EBS
was used as a reference compound.
The results (Figure 7) demonstrated that only four compounds (1, 2, 8 and 9) were able
to scavenge the DPPH activity, with values above 20% of inhibition at 0.25 mg/mL.
Compound 9 presented DPPH scavenging values greater than 40% at 0.25 mg/mL,
displaying greater radical scavenging than EBS. Finally, as shown in Figure 7, these
compounds exhibited concentration-dependent but not time-dependent radical
scavenging activity in vitro.
Time (minutes)
120
30´
90´
180´
100
80
60
40
20
0
Ascorbic acid
Comp.
Comp.
Comp.
Comp.
EBS
3,13E-05
3.13E-05
33.1,133EE-0-044
9
33,1.133EE-0-022
1.25E-01
1,25E-01
2,520.E5-0E1-01
1
3,
3.13E-05
33,1.133EE-0-044
8
3.13E-02
3,13E-02
1.25E-01
1,25E-01
22,5.500EE-0-011
2
3,13E-05
3.13E-05
3.13E-04
3,
3.13E-02
3,13E-02
1.25E-01
1,25E-01
2.50E-01
2,50E-01
Concentration (mg /mL)
Figure 7. Analysis of DPPH radical scavenging activity for compounds 1, 2, 8 and 9
at different concentrations and different time points.
3. Conclusion
In this study a series of twenty-seven selenadiazole derivatives were synthesized and
their anti-proliferative effect against five human tumor cell lines by standard MTT
assay, and antioxidant activity using DPPH test were evaluated. Results demonstrated
18

ACCEPTED MANUSCRIPT
that “5” position is a favorable site to carry out modifications, given that many analogs
formed by decorating several substituent over this position through an amide linkage
displayed better anti-proliferative effects than the parent structure (BSCA). Compounds
5, 6, 7 and 19 were the most potent in inhibiting viability of at least one cancer cell line
with GI50 values ranging from 3.7 to 9.8 µM. These activities were 10-fold more potent
than the reference EBS and the selectivity indexes were greater than EBS and BBSKE.
Furthermore, the radical scavenging capacity of 1, 2, 8 and 9 was greater than EBS.
Taking together all the results, compound 7 emerged as the most promising and suggest
the importance of methylphenylselane group for BSCA scaffold. It is interesting to note
that contrary to the parent compound (BSCA) and EBS, compound 7 induced cell
growth inhibition independent of the apoptotic process and without affecting the cell
cycle progression. This behavior is quite unique and warrants further investigation of
the in-depth mechanism by which this compound is potently exerting inhibition of
viability of breast cancer cells while sparing the non-tumoral 184B5 cells. It is clear that
compound 7, with GI50 greater than 100 µM in normal cells and promising anti-
proliferative activity in MCF-7 cells (GI50 = 3.7 µM) is highly promising as a lead for
developing effective new chemotherapeutics, particularly for breast cancer.
Compound 9 on the other hand having better radical scavenging activity than EBS may
prove to be potent antioxidant. These data suggest that appropriate manipulation of EBS
structure can lead to a set of promising small drug-like anti-cancer molecules with
diverse mechanisms of action. Given the greater selectivity and moderate antioxidant
activity, these compounds can serve as excellent scaffolds to achieve new and potent
antioxidant compounds useful for several diseases such as cancer, neurodegenerative,
heart and leishmaniasis diseases.
19

ACCEPTED MANUSCRIPT
4. Experimental
4.1. Chemistry
4.1.1. Material and methods
Melting points (mp) were determined with a Mettler FP82 + FP80 apparatus
(Greifensee, Switzerland). The proton (1H) and carbon (13C) NMR spectra were
recorded on a Bruker 400 Ultrashield™ spectrometer (Rheinstetten, Germany) using
DMSO-d6 as solvent. The IR spectra were obtained on a Thermo Nicolet FT- IR Nexus
spectrophotometer with KBr pellets. Purity of all final compounds was 95% or higher
and was dwtermined by elemental microanalyses carried out on vacuum-dried samples
using a LECO CHN-900 Elemental Analyzer. Furthermore, Column Chromatography
using Silica gel 60 (0.040 - 0.063 mm) (Merck KGaA, Darmstadt, Germany) and Thin
Layer Chromatography (TLC) assays were carried out in Alugram® SIL G/UV254
sheets (Layer: 0.2 mm) (Macherey-Nagel, Düren, Germany). Chemicals were purchased
from E. Merck (Darmstadt, Germany), Panreac Química S.A. (Montcada i Reixac,
Barcelona, Spain), Sigma-Aldrich Química, S.A. (Alcobendas, Madrid, Spain), Acros
Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgium) and Lancaster
(Bischheim-Strasbourg, France).
4.1.2. Benzo[c][1,2,5]selenadiazole-5-carboxylic acid (BSCA)
This compound was prepared according to a previously published procedure [23].
Briefly, a mixture of 3,4-diaminobenzoic acid and SeO2 was heated at 260 ºC. The
crude product was washed with water and recrystallized from dioxane.
4.1.3. General procedure for the synthesis of compounds (1-26)
20

ACCEPTED MANUSCRIPT
A mixture of BSCA (0.5 g, 2 mmol) and thionyl chloride (20 mL) was stirred at reflux
for 2 h. The reaction was monitored by IR spectroscopy. Formation of the acyl chloride
was confirmed following the wavenumber position in infrared spectroscopy peaks:
carbonyl group showed up around: 1682 cm-1 and the simple bond of OH group was
detected around 3448 cm-1 in the starting acid whereas the carbonyl in the acyl chloride
arose around 1750 cm-1. The resulting acyl chloride was isolated by rotatory
evaporation of the thionyl chloride under vacuum and the excess of thionyl chloride was
removed with 3 fractions of toluene (40 mL). The resulting acyl chloride was used
without further purification. A solution of the corresponding amine (2 mmol) in dry
chloroform (15 mL) was added to a mixture of acyl chloride (0.5 g, 2 mmol) and
triethylamine (0.28 mL, 2 mmol) in dry chloroform (40 mL). The mixture was stirred at
room temperature for 12-48 h. The product was isolated by filtration or by rotatory
evaporation of the solvent under vacuum and washed with water (3 x 50 mL). Final
product was purified by washing, recrystallization, or column chromatography.
4.1.3.1.
N-Phenylbenzo[c][1,2,5]selenadiazole-5-carboxamide (1)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and aniline. The resulting product,
N-phenylbenzo[c][1,2,5]selenadiazole-5-carboxamide (1), did not require further
purification. A brown powder was obtained. Yield: 40 %; mp: 245-246 ºC. IR (KBr): ν
̃
1
3276 (N-H); 1650 cm-1 (C=O). H NMR (400 MHz, DMSO-d6) δ (ppm): 10.56 (s, 1H,
NH), 8.52 (s, 1H, H4), 8.01 (dd, J6-7 = 9.3; J6-4 = 1.7 Hz, 1H, H6), 7.96 (dd, J7-4 = 0.7,
1H, H7), 7.82 (dd, 2H, J2´-3´ = J6´-5´ = 8.5; J2´-4´ = J6´-4´ = 0.9 Hz, H2´ + H6´), 7.41 - 7.36
13
(dd, J3´-4´ = J5´-4´ = 7.6 Hz, 2H, H3´+ H5´), 7.14 (tt, 1H, H4´). C NMR (100 MHz,
DMSO-d6) δ (ppm): 165.6 (C=O), 161.2 + 160.0 (C3 + C8), 139.8 + 135.9 (C5 + C1´),
129.6 + 128.9 (C3´ + C6 +C5´), 124.9 + 124.0 + 123.5 + 121.3 (C4 + C7 + C2´ + C4´ +
77
C6´). Se NMR (76 MHz, DMSO-d6) δ (ppm): 1561.1. Elemental analysis calculated
21

ACCEPTED MANUSCRIPT
(%) for C13H9N3SeO·1/2 H2O: C: 50.17, H: 3.22, N: 13.51; found: C: 50.27, H: 3.34, N:
13.41.
4.1.3.2.
N-(p-Tolyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (2)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and p-toluidine. The resulting
product, N-(p-tolyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (2), did not require
further purification. A brown powder was obtained. Yield: 81ꢀ%; mp: 237-238.5 ºC. IR
(KBr): ν̃
3284 (N-H), 1645 cm-1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.49
(s, 1H, NH), 8.51 (s, 1H, H4), 8.01 (dd, J6-7 = 9.3; J6-4 = 1.7 Hz, 1H, H6), 7.95 (dd, J7-4 =
0.5 Hz, 1H, H7), 7.70 (d, J3´-2´ = J5´-6´ = 8.4 Hz, 2H, H3´ + H5´), 7.18 (d, 2H, H2´ + H6´),
13
2.29 (s, 3H, -CH3). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.4 (C=O), 161.2 +
160.0 (C3 + C8), 137.3 + 136.0 + 133.8 (C1´ + C4´ + C5), 129.9 + 128.9 (C3´ + C5´ + C6),
123.9 +123.4 + 121.3 (C4+ C7 + C2´ + C6´), 21.4 (-CH3). 77Se NMR (76 MHz, DMSO-
d6) δ (ppm): 1560.5. Elemental analysis calculated (%) for C14H11N3OSe·1⁄2H2O: C:
51.70, H: 3.69, N: 12.92; found: C: 52.14, H: 3.98, N: 12.94.
4.1.3.3.
N-(4-Chlorophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (3)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-chloroaniline. The resulting
product, N-(4-chlorophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (3), did not
require further purification. A brown powder was obtained. Yield: 63 %; mp: 233-235
1
ºC. IR (KBr): ν
̃
3273 (N-H), 1648 cm-1 (C=O). H NMR (400 MHz, DMSO-d6) δ
(ppm): 10.69 (s, 1H, NH), 8.53 (s, 1H, H4), 7.99 (dd, J6-7 = 9.3; J6-4 = 1.7 Hz, 1H, H6),
7.97 (dd, J7-4 = 0.7 Hz, 1H, H7), 7.86 (d, J2´-3´ = J6´-5´ = 8.8 Hz, 2H, H2´ + H6´), 7.45 (d,
13
2H, H3´ + H5´). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.7 (C=O), 161.2 + 160.0
(C3 + C8), 138.8 + 135.6 (C1´ + C4´), 129.5 + 128.7 + 128.4 (C5 + C6 + C3´ + C5´), 124.0
+123.6 +122.8 (C4 + C7 + C2´ + C6´). 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 1562.2.
22

ACCEPTED MANUSCRIPT
Elemental analysis calculated (%) for C12H8ClN3OSe: C: 46.37, H: 2.38, N: 12.48;
found: C: 46.07, H: 2.74, N: 12.45.
4.1.3.4.
N-(4-Nitrophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (4)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-nitroaniline. The product was
purified by recrystallization from ethanol. A yellow powder was obtained. Yield: 9 %;
mp: 245-256 ºC. IR (KBr): ν
̃
3395 (N-H), 1665 (C=O), 1334 cm-1 (NO2). 1H NMR (400
MHz, DMSO-d6) δ (ppm): 11.08 (s, 1H, NH), 8.57 (s, 1H, H4), 8.29 (d, J3´-2´ = J5´-6´
=
9.1 Hz, 2H, H3´ + H5´), 8.09 (d, 2H, H2´ + H6´), 8.00 (dd, J6-7 = 9.4; J6-4 = 1.08 Hz, 1H,
H6), 7.97 (d, 1H, H7). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 166.3 (C=O), 161.2 +
159.8 (C3 + C8), 146.0 + 143.5 (C4´ + C1´), 135.0 + 128.6 (C5 + C6), 125.6 + 124.1
77
+120.8 (C4 + C7 + C2´ + C3´ + C5´ + C6´). Se NMR (76 MHz, DMSO-d6) δ (ppm):
1565.2. Elemental analysis calculated (%) for C13H8N4O3Se·H2O: C: 42.74, H: 2.74, N:
15.34; found: C: 42.30, H: 3.06, N: 14.90.
4.1.3.5.
N-(4-Methoxyphenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (5)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and p-anisidine. The resulting
product N-(4-methoxyphenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (5), did not
require further purification. A yellow powder was obtained. Yield: 68 %; mp: 233-234
1
ºC. IR (KBr): ν
̃
3284 (N-H), 1640 cm-1 (C=O). H NMR (400 MHz, DMSO-d6) δ
(ppm): 10.44 (s, 1H, NH), 8.50 (s, 1H, H4), 8.01 (d, J6-7 = 9.3 Hz, 1H, H6), 7.95 (d, 1H,
H7), 7.72 (d, J2´-3´ = J6´-5´ = 8 Hz, 2H, H2´ + H6´), 6.96 (d, 2H, H3´ + H5´), 3.76 (s, 3H,
13
O-CH3). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.1 (C=O), 161.2 + 160.1 (C3 +
C8 ),156.6 (C4´), 136.0 + 132.9 (C5 + C1´), 128.9 (C6), 123.9 + 123.3 + 122.9 (C4 + C7
+ C2´ + C6´), 114.7 (C3´ + C5´), 56.1 (O-CH3). 77Se NMR (76 MHz, DMSO-d6) δ (ppm):
1560.0. Elemental analysis calculated (%) for C14H11N3O2Se·1/2H2O: C: 48.01, H:
23

ACCEPTED MANUSCRIPT
3.70, N: 12.00; found: C: 48.09, H: 3.63, N: 11.74.
4.1.3.6.
(6)
N-(4-(Methylthio)phenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-(methylthio)aniline. The
product was purified by recrystallization from ethanol. A yellow powder was obtained.
Yield: 21 %; mp: 260-261 ºC. IR (KBr): ν̃
3093 (N-H), 1647 cm-1 (C=O). 1H NMR (400
MHz, DMSO-d6) δ (ppm): 10.55 (s, 1H, NH), 8.51 (s, 1H, H4), 8.00 (d, J6-7 = 8 Hz, 1H,
H6), 7.95 (d, 1H, H7), 7.78 (d, J2´-3´ = J6´-5´ = 8 Hz, 2H, H2´ + H6´), 7.30 (d, 2H, H3´ +
13
H5´), 2.48 (s, 3H, S-CH3). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.4 (C=O),
161.2 + 160.0 (C3 + C8), 137.2 + 135.7 + 133.6 (C5 + C1´ + C4´), 128.8 + 127.7 (C6 +
C3´ + C5´), 124.0 + 123.5 + 121.9 (C4 + C7 + C2´ + C6´), 16.2 (S-CH3). 77Se NMR (76
MHz, DMSO-d6) δ (ppm): 1561.3. Elemental analysis calculated (%) for
C14H11N3O2SSe: C: 48.27, H: 3.16, N: 12.07; found: C: 47.77, H: 3.64, N: 11.85.
4.1.3.7.
4-(Methylselenyl)aniline (7i)
1.5 mmol of the iodomethane was added to a methanolic solution of 1 mmol of 4-
Aminophenylselenocyanate and 43 mL of sodium hydroxide (0.4 N) and stirred at room
temperature for 40 min. The reaction medium was then poured into water and the
product was isolated by filtration and extraction with dichloromethane (3 x 50 mL). The
product was purified by column chromatography on silica gel using dichloromethane as
eluent. 4-Aminophenylselenocyanate was prepared following the synthetic procedure
previously described [23]. Yield: 22 %. IR (KBr): ν̃ 3447-3355 (N-H2), 3213 (=C-H),
1
2924 (Se-CH3), 1619-1492 cm-1 (C=C). H NMR (400 MHz, CDCl3) δ (ppm): 7.32 (d,
J3-2 = J5-6 = 8.0 Hz, 2H, H3 + H5), 6.63 (d, 2H, H2 + H6), 2.29 (s, 3H, Se-CH3).
4.1.3.8.
N-(4-(Methylselanyl)phenyl)benzo[c][1,2,5]selenadiazole-5-
24

ACCEPTED MANUSCRIPT
carboxamide (7)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-(methylselenyl)aniline (7i).
The crude product was purified by washing with chloroform (3 x 15 mL). A yellow
powder was obtained. Yield: 38 %; mp: 258-260 ºC. IR (KBr): ν̃
3296 (N-H), 1647 cm-
1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.56 (s, 1H, NH), 8.51 (s, 1H, H4),
8.00 (d, J6-7 = 8.9 Hz, 1H, H6), 7.94 (d, 1H, H7), 7.76 (d, J2´-3´ = J6´-5´ = 7.6 Hz, 2H, H2´
+ H6´), 7.42 (d, 2H, H3´ + H5´), 2.34 (s, 3H, Se-CH3). 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 165.9 (C=O), 161.2 + 160.0 (C3 + C8), 138.1 + 135.7 (C5 + C1´), 131.1 + 128.8
+ 126.8 (C6 + C3´ + C4´+ C5´), 124.0 + 123.5 + 122.0 (C4 + C7 + C2´ + C6´), 7.8 (Se-
CH3). 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 1561.5, 189.3. Elemental analysis
calculated (%) for C14H11N3O2Se2·1/2HCl: C: 40.65, H: 2.66, N: 10.16; found: C:
41.06, H: 3.09, N: 10.45.
4.1.3.9.
4-Aminobenzeneselenol (8i)
12 mmol of the 4-bromoaniline was added to a solution of 13.2 mmol of selenourea in
15 mL of absolute ethanol and stirred at reflux for 2 h. The product was isolated by
filtration and washed with water (50 mL). The resulting product, 4-
aminobenzeneselenol (8i), did not require further purification. Yield: 20 %. IR (KBr): ν
̃
1
3428-3322 (N-H2), 3232 (=CH-), 2681 (SeH), 1618-1486 cm-1 (C=C). H NMR (400
MHz, DMSO-d6) δ (ppm): 7.11 (d, J3-2 = J5-6 = 8 Hz, 2H, H3 + H5), 6.50 (d, 2H, H2 +
H6), 5.22 (s, 2H, -NH2), 3.85 (s, 1H, -SeH). Elemental analysis calculated (%) for
C6H7NSe·1/2H2O: C: 39.79, H: 4.42, N: 7.74; found: C: 40.81, H: 3.89, N: 7.94.
4.1.3.10.
(8)
N-(4-Hydroselenophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-aminobenzeneselenol (8i).
25

ACCEPTED MANUSCRIPT
The resulting product, N-(4-hydroselenophenyl)benzo[c][1,2,5]selenadiazole-5-
carboxamide (8), did not require further purification. A brown powder was obtained.
Yield: 80 %; mp: 253.4-255.8 ºC. IR (KBr): ν̃
3273 (N-H), 1647 cm-1 (C=O). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 10.66 (s, 1H, NH), 8.52 (s, 1H, H4), 7.99 (dd, J6-7 = 9.3;
J6-4 = 1.5 Hz, 1H, H6), 7.97 (dd, J7-6 = 0.7 Hz, 1H, H7), 7.81 (d, J2´-3´ = J6´-5´ = 8 Hz, 2H,
13
H2´ + H6´), 7.57 (d, 2H, H3´ + H5´). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.7
(C=O), 161.2 + 160.0 (C3 + C8), 139.2 +135.6 (C1´ + C5), 132.4 + 128.7 (C6 + C3´ +
77
C5´), 124.0 +123.6 + 123.1 + 116.5 (C7 + C4 + C2´ + C4´ + C6´). Se NMR (76 MHz,
DMSO-d6)
δ
(ppm): 1562.4. Elemental analysis calculated (%) for
C13H9N3OSe2·1/2H2O: C: 40.01, H: 2.56, N: 10.77; found: C: 39.82, H: 2.29, N: 10.97.
4.1.3.11.
N-(p-Mercaptophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide (9)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-aminothiophenol. The
resulting product, N-(p-mercaptophenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide
(9), did not require further purification. An orange powder was obtained. Yield: 51 %;
1
mp: 189.0-188.6 ºC. IR (KBr): ν
̃
3399 (NH), 1654 cm-1 (C=O). H NMR (400 MHz,
DMSO-d6) δ (ppm): 8.49 (s, 1H, H4), 7.99 (d, J7-6 = 9.3 Hz, 1H, H7), 7.91 (dd, J6-4
=
1.7 Hz, 1H, H6), 7.15 (d, J2´-3´ = J6´-5´ = 8.5 Hz, 2H, H2` + H6´), 6.66 (d, 2H, H3` + H5´),
13
5.64 (s, 1H, SH). C NMR (100 MHz, DMSO-d6) δ (ppm): 191.9 (C=O), 161.6 +
159.7 (C3 + C8), 151.5 (C1´) 137.1 + 136.9 (C3´ +C4´ + C5´), 126.6 + 125.0 + 124.0 (C5
77
+ C6 + C7) 115.3 + 110.1 (C4 + C2´ + C6´). Se NMR (76 MHz, DMSO-d6) δ (ppm):
1573.7. Elemental analysis calculated (%) for C13H9N3OSSe: C: 46.71, H: 2.71, N:
12.57; found: C: 46.51, H: 3.07, N:12.40.
4.1.3.12.
(10)
N-(3,4,5-Trimethoxyphenyl)benzo[c][1,2,5]selenadiazole-5-carboxamide
26

ACCEPTED MANUSCRIPT
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 3,4,5-trimethoxianiline. The
resulting
product,
N-(3,4,5-trimethoxyphenyl)benzo[c][1,2,5]selenadiazole-5-
carboxamide (10), did not require further purification. A yellow powder was obtained.
3272 (N-H), 1651 cm-1 (C=O). 1H NMR
Yield: 50 %; mp: 207.2-208.3 ºC. IR (KBr): ν
̃
(400 MHz, DMSO-d6) δ (ppm): 10.45 (s, 1H, NH), 8.53 (s, 1H, H4), 8.02 (d, J6-7 = 9.3
Hz, 1H, H6), 7.95 (d, 1H, H7), 7.28 (s, 2H, H2´ + H6´), 3.80 (s, 6H, 2OCH3), 3.66 (s, 3H,
OCH3). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 165.3 (C=O), 161.2 + 160.0 (C3 +
C8), 153.5 (C3´ + C5´), 136.0 + 135.8 + 134.8 + 128.7 (C5 + C6 + C1´ + C4´), 124.0 +
77
123.4 (C7 + C4), 99.0 (C2´ + C6´), 61.0 (OCH3), 56.6 (2OCH3). Se NMR (76 MHz,
DMSO-d6)
δ
(ppm): 1561.6. Elemental analysis calculated (%) for
C16H15N3O4Se·1/2H2O: C: 47.89, H: 3.99, N: 10.47; found: C: 47.25, H: 4.40, N: 10.15.
4.1.3.13.
N-Ethylbenzo[c][1,2,5]selenadiazole-5-carboxamide (11)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and ethylamine solution. The
mixture was filtered and the product was isolated by rotatory evaporation of the solvent
and purified by successive washing with water (50 mL) and hexane (15 mL). A brown
powder was obtained. Yield: 5 %; mp: 161-164 ºC. IR (KBr): ν̃ 3282 (N-H), 2982 (C
sp2), 1637 cm-1 (C=O). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.18 (s, 1H, H4), 7.92 (dd,
J6-7 = 9.3; J6-4 = 1.2 Hz, 1H, H6), 7.90 (d, 1H, H7), 6.34 (s, 1H, NH), 3.62-3.53 (m, 2H,
-CH2-), 1.32 (t, J CH3-CH2= 7.3 Hz, 3H, -CH3). 13C NMR (100 MHz, DMSO-d6) δ (ppm):
166.0 (C=O), 161.1 + 160.2 (C3 + C8), 135.6 + 128.7 (C5 + C6), 123.8 +122.7 (C4 +
C7), 35.2 (-CH2-), 15.5 (-CH3). Elemental “analysis calculated (%) for
C9H9N3OSe·1/2H2O: C: 41.04, H: 3.80, N: 15.96; found: C: 41.50, H: 3.76, N: 15.23.
4.1.3.14.
N-Cyclohexylbenzo[c][1,2,5]selenadiazole-5-carboxamide (12)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and cyclohexylamine. The resulting
27

ACCEPTED MANUSCRIPT
product, N-cyclohexylbenzo[c][1,2,5]selenadiazole-5-carboxamide (12), did not require
further purification. A white powder was obtained. Yield: 63 %; mp: 242.6-246.4 ºC.
1
IR (KBr): ν
̃
3303 (N-H), 2934-2854 (cyclohexane, C sp2), 1631 cm-1 (C=O). H NMR
(400 MHz, TFA) δ (ppm): 11.00 (s, 1H, NH), 7.79 (s, 1H, H4), 7.41 (bs, 2H, H6 + H7),
3.42-3.41 (bs, 1H, H1´), 1.55-1.37 (bs, 2H, H2a´ + H2b´), 1.22-1.21 (bs, 2H, H6a´ + H6b´),
1.14-1.00 (bs, 1H, H4a´), 0.89-0.81 (bs, 4H, H3a´ +H3b´ +H5a´ +H5b´), 0.65-0.61 (bs, 1H,
H4b´). Elemental analysis calculated (%) for C13H14N3OSe·1/2H2O: C: 49.37, H: 4.75,
N: 13.29; found: C: 49.92, H: 5.19, N: 13.27.
4.1.3.15.
N-(Piperidin-1-yl)benzo[c][1,2,5]selenadiazole-5-carboxamide (13)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and piperidine. The resulting
product, N-(piperidin-1-yl)benzo[c][1,2,5]selenadiazole-5-carboxamide (13), did not
require further purification. A brown powder was obtained. Yield: 56 %; mp: 103.3-
1
101.9 ºC. IR (KBr): ν
̃
2935-2855 (C sp2), 1621 cm-1 (C=O). H NMR (400 MHz,
DMSO-d6) δ (ppm): 7.90 (d, J7-6 = 9.2 Hz, 1H, H7), 7.82 (s, 1H, H4), 7.49 (dd, J6-4=1.5
Hz, 1H, H6), 3.61 (bs, 2H, 2H1´), 3.33 (bs, 2H, 2H5´), 1.47 + 1.60 (bs+ bs, 6H, 2H2´ +
13
2H3´ + 2H4´). C NMR (100 MHz, DMSO-d6) δ (ppm): 168.3 (C=O), 160.2 + 159.8
(C3 + C8), 137.8 (C5) + 129.0 (C6), 124.5 + 121.5 (C4 + C7), 48.9 + 43.2 (C1´ + C5´),
26.8 + 26.1 + 24.9 (C2´ + C3´ + C4´). 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 1548.0.
Elemental analysis calculated (%) for C12H13N3OSe: C: 48.99, H: 4.45, N: 14.28; found:
C: 49.29, H: 4.96, N: 14.44.
4.1.3.16.
N-(Pyridin-4-yl)benzo[c][1,2,5]selenadiazole-5-carboxamide (14)
From benzo[c][1,2,5]selenadiazole-5-acyl chloride and 4-aminopyridine. The resulting
product, N-(pyridin-4-yl)benzo[c][1,2,5]selenadiazole-5-carboxamide (14), did not
require further purification. A brown powder was obtained. Yield: 83 %; mp: 277.4-
28