Synthesis and antitumor activity of selenium-containing quinone-based triazoles possessing two redox centres, and their mechanistic insights

Article abstract of DOI:10.1016/j.ejmech.2016.06.019

Selenium-containing quinone-based 1,2,3-triazoles were synthesized using click chemistry, the copper catalyzed azide-alkyne 1,3-dipolar cycloaddition, and evaluated against six types of cancer cell lines: HL-60 (human promyelocytic leukemia cells), HCT-11

Full text of DOI:10.1016/j.ejmech.2016.06.019

Accepted Manuscript
Synthesis and antitumor activity of selenium-containing quinone-based triazoles
possessing two redox centres, and their mechanistic insights
Eduardo H.G. da Cruz, Molly A. Silvers, Guilherme A.M. Jardim, Jarbas M. Resende,
Bruno C. Cavalcanti, Igor S. Bomfim, Claudia Pessoa, Carlos A. de Simone,
Giancarlo V. Botteselle, Antonio L. Braga, Divya K. Nair, Irishi N.N. Namboothiri,
David A. Boothman, Eufrânio N. da Silva Júnior
PII:
S0223-5234(16)30497-4
10.1016/j.ejmech.2016.06.019
EJMECH 8680
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 November 2015
Revised Date: 2 June 2016
Accepted Date: 11 June 2016
Please cite this article as: E.H.G. da Cruz, M.A. Silvers, G.A.M. Jardim, J.M. Resende, B.C. Cavalcanti,
I.S. Bomfim, C. Pessoa, C.A. de Simone, G.V. Botteselle, A.L. Braga, D.K. Nair, I.N.N. Namboothiri,
D.A. Boothman, E.N. da Silva Júnior, Synthesis and antitumor activity of selenium-containing quinone-
based triazoles possessing two redox centres, and their mechanistic insights, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.019.
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
Graphical abstract
Selenium-containing quinones were designed and synthesized by click chemistry
reaction and evaluated against several human cancer cell lines showing, in some cases,
IC50 values below 0.3 µM.
New class of compounds
possessing two redox centres
O
O
SePh
N
O
O
SePh
N
N
O
O
O
O
First Redox Centre
H
N
N
N
N
O
O
Br
O
O
O
O
O
N
O
O
O
O
N
N
SePh
O
O
O
O
HN
n
Insertion of a second
redox centre
N
N
O
O
SePh
N
n = 1 or 2
n
Potent activity against 6 cancer cell lines
n = 1 or 2
25 compounds evaluated
against cancer cell lines
Mechanistic role for NQO1 elucidated for
selected compounds

ACCEPTED MANUSCRIPT
Synthesis and Antitumor Activity of Selenium-Containing Quinone-based Triazoles
Possessing Two Redox Centres, and their Mechanistic Insights
Eduardo H. G. da Cruz,a Molly A. Silvers,b Guilherme A. M. Jardim,a Jarbas M. Resende,a
Bruno C. Cavalcanti,c Igor S. Bomfim,c Claudia Pessoa,c,d Carlos A. de Simone,e Giancarlo
V. Botteselle,f Antonio L. Braga,f Divya K. Nair,g Irishi N. N. Namboothiri,g David A.
Boothman,b and Eufrânio N. da Silva Júniora*
aInstitute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais,
CEP 31270-901, Belo Horizonte, MG, Brazil; bDepartments of Pharmacology and
Radiation Oncology, Simmons Comprehensive Cancer Center, University of Texas
Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-8807, USA;
cNational Laboratory of Experimental Oncology, Department of Physiology and
Pharmacology, Federal University of Ceará, CEP 60180-900, Fortaleza, CE, Brazil;
dFiocruz-Ceará, CEP 60180-900, Fortaleza, CE, Brazil; eInstitute of Physics, University of
São Paulo, 13560-160, São Carlos, SP, Brazil; fDepartment of Chemistry, Federal
University of Santa Catarina, 88040-900, Florianópolis, SC, Brazil; gDepartment of
Chemistry, Indian Institute of Technology Bombay, Mumbai 400 076, India.
Corresponding author: E.N. da Silva Júnior: E-mail: eufranio@ufmg.br; Fax: +55 31
34095700; Tel.: +55 31 34095720.
Abstract: Selenium-containing quinone-based 1,2,3-triazoles were synthesized using click
chemistry, the copper catalyzed azide-alkyne 1,3-dipolar cycloaddition, and evaluated
against six types of cancer cell lines: HL-60 (human promyelocytic leukemia cells), HCT-
116 (human colon carcinoma cells), PC3 (human prostate cells), SF295 (human
glioblastoma cells), MDA-MB-435 (melanoma cells) and OVCAR-8 (human ovarian
carcinoma cells). Some compounds showed IC50 values < 0.3 µM. The cytotoxic potential
of the quinones evaluated was also assayed using non-tumor cells, exemplified by
peripheral blood mononuclear (PBMC), V79 and L929 cells. Mechanistic role for
NAD(P)H:Quinone Oxidoreductase 1 (NQO1) was also elucidated. These compounds could
provide promising new lead derivatives for more potent anticancer drug development and
delivery, and represent one of the most active classes of lapachones reported.
Keywords: β-Lapachone; Quinone; Click chemistry, Chalcogens, Selenium
1

ACCEPTED MANUSCRIPT
1. Introduction
Development of diverse therapeutics is of paramount importance in the fight
against different types of cancer [1,2]. Quinones are considered as privileged structures
and are among the most important drugs used against cancer [3]. Although single-target
drugs successfully inhibit or activate a specific target [4], drugs that are able to act
simultaneously on diverse biological targets are more attractive in the design of new
effective drugs [5]. In this context, quinoidal structures represent an essential multi-
target class of compounds [6].
Naturally occurring naphthoquinones such as lapachol and β-lapachone (β-lap),
isolated from the heartwood of Tabebuia, are among the most studied for their potential
anti-tumor activity [7]. Docampo et al. [8] found significant activity for β-lap against
Sarcoma 180 ascites tumor cells (S-180 cells) in vitro, and in mice bearing S-180
tumors. Although the antitumor activity of β-lap against Yoshida sarcoma and Walker
256 carcinoma cells in culture has been investigated [8,9], the exact mechanism of
action was not known until recently [10].
β-Lapachone specifically destroys cancer cells with elevated endogenous levels
of NAD(P)H:quinone oxidoreductase 1 (NQO1) [11] regardless of p53, caspase, or cell
cycle status [12]. While in clinical trials, β-lap (i.e., ARQ 501) has been inaccurately
touted as a cell cycle checkpoint activator [13], the major determinant of cell death is
through NQO1 expression [11,12a,14]. The drug is not a substrate for known multidrug
resistance or drug pumps [15,16] and β-lap cell death is not affected by changes in cell
cycle position, oncogenic drivers, or pro- or anti-apoptotic factors [11,12a]. Finally, the
drug targets (i.e., is ‘bioactivated’ by) NQO1, a Phase II, carcinogen-inducible enzyme
that is also induced by ionizing radiation (IR) in some cancer, but not normal, cells
[17,18].
β-Lap’s use as a chemotherapeutic agent is curtailed by its high hydrophobicity
which causes methemoglobinemia in patients [19]. When mixed with the carrier
hydroxypropyl-β-cyclodextrin, the carrier itself can contribute to hemolysis [20].
Recently, Ohayon and coworkers [21] shed light on the hypothesis of β-lap being able
to act nonreversibly for inhibition of deubiquitinases. These authors suggested that the
therapeutic effect of β-lap could be also related to ubiquitin specific peptidase 2 (USP2)
oxidation, which is likely a downstream effect of reactive oxygen species (ROS)
generation via NQO1 futile cycle metabolism of β-lap. NQO1 is the unique gene, that
2

ACCEPTED MANUSCRIPT
when deleted, leads to resistance to β-lap and other NQO1 bioactivatable drugs [22,23],
strongly suggesting that most downstream effects cited by others are a result of
upregulation of NQO1-derived ROS and Ca2+ intracellular increases, as well as rapid
and dramatic losses in NAD+/ATP caused by PARP1 hyperactivation ending in NAD+-
Keresis [24].
In the last few years, our group has been intensively dedicated to the synthesis
and evaluation of lapachones against cancer cell lines [25,26]. We discovered a series of
lapachones with modified C-rings (Scheme 1a) with potent activity against cancer
lineages [27]. Lately, interest in preparing quinone-based triazoles has been stimulated
by our discovery of bioactive compounds endowed with unique subunits in their
chemical structures [28]. Recently, Perumal et al. [29] prepared amino-1,4-
naphthoquinone-appended triazoles with antimycobacterial activity designed by the
same molecular hybridization strategy [30]. β-Lapachone-based 1,2,3-triazoles possess
significant activity with IC50 values below 2 µM for MDA-MB-435 cancer cells. These
compounds promoted cell death by an apparent apoptotic cell death mechanism
associated with significant ROS production [31]. The approach of inserting a triazole
moiety in 1,4-naphthoquinones was also effective, since this unit is known as a potent
pharmacophoric group [32]. Recently, 1,4-naphthoquinone-based 1,2,3-triazoles
(Scheme 1a) were reported as having high activity in the range of 1.4 to 1.9 µM in HL-
60 human promyelocytic leukemia cells [33].
From another perspective, organoselenium compounds show antitumor,
antimicrobial, anti-neurodegenerative and antiviral properties [34]. A series of
selenoproteins are involved in important physiological processes [35]. Jacob and co-
workers [36] demonstrated the potential antitumor activity of selenium-containing
quinones (Scheme 1b) capable of mimicking the enzymatic activity of the human
enzyme, glutathione peroxidase (GPx). GPx targets redox sensitive thiol proteins, while
simultaneously generating reactive oxygen species at a critical threshold. Thus, these
drugs act as ROS-users and ROS-enhancers to affect downsteam targets [37]. This
action would complement the mechanism of action of β-lap, since death caused by this
agent relies upon the hyperactivation of PARP1, which is stimulated by ROS (H2O2)
[24].
In continuation of our program for obtaining novel potent antitumor
naphthoquinones and based on recent findings reported by our group, we discovered
3

ACCEPTED MANUSCRIPT
potent chalcogen-containing β-lapachones (Scheme 1b) [38]. Here, we describe fifteen
novel selenium-containing quinones and our strategy was based on inserting this
pharmacophoric group, generating 1,2,3-triazole selenium-containing lapachones
(Scheme 1c). Selected naphthoquinones with a structural framework with recognized
activity against several types of cancer cell lines were used in the preparation of the new
compounds. The structures were designed as multi-target ligands potentially giving rise
to NQO1 cell death mechanisms of action.
Scheme 1.
2. Results and Discussion
2.1 Chemistry
The first class of compounds prepared possessing two redox centres was
selenium-containing dihydropyran naphthoquinones obtained from lapachol (1)
(Scheme 2). α-Lapachone 2 was prepared by acid catalyzed cyclization from 1, and then
two selenium-containing derivatives, 6 and 7, were synthesized from 2. Compound 6,
was prepared in moderate yield (75%) by copper(I) catalyzed click reaction [39]
between compound 4 and (azidomethyl)(phenyl)selane. The intermediate compound 4
was obtained by the reaction of 3-ethynylaniline and the bromo derivative 3. As
previously reported [40], 4-azide-α-lapachone (5) was easily synthesized from the
reaction of 3 with sodium azide in dichloromethane. The reaction of 5 and phenyl
propargyl selenide affords selenium-containing α-lapachone 1,2,3-triazole 7. Finally,
from the azide derivative 9, prepared as reported by us [41], β-lapachone-based 1,2,3-
triazole 10 containing the chalcogen was obtained as a red solid. Compounds 3-10 are
racemic. However, compounds 9 and 10 are single diastereomers, the relative
stereochemistry is trans. The trans-stereochemistry was confirmed by comparison with
previously reported data [41,47a].
Scheme 2.
We began the synthesis of selenium-containing dihydrofuran naphthoquinones,
the second class of compounds, initially by synthesizing nor-α-lapachone derivatives 15
and 16 (Scheme 3). Since the synthesis of arylamino substituted lapachones and
4

ACCEPTED MANUSCRIPT
azidoquinones are well established in our group [25,40,42], compounds 13 and 14 were
prepared as shown in Scheme 3. Following click methodology, compounds 13 and 14
were reacted with selenium-containing azide and alkyne, respectively, to furnish the
naphthoquinones 15 and 16 in 70% and 80% yield, respectively.
Scheme 3.
From nor-lapachol (17), the bromo intermediate 18 was synthesized following
the methodology described by Pinto and co-workers (Scheme 4) [26,43]. Synthesis of
various antitumor compounds from 18 was reported, as for instance, arylamino and
alkoxy substituted nor-β-lapachone [26], lapachones in the presence of 1,2,3-triazole
moiety [44] and hybrids with chalcones [45]. The unpublished arylamino substituted
lapachone 19 bearing a terminal alkyne group was prepared based on the previously
described compounds possessing activity against cancer cell lines [26]. The formation
of the selenium-containing 1,2,3-triazole 21 from 19 herein described, allowed us to
access the product designed with two redox centres. Using the same strategy discussed
above, compound 22 was obtained from the azide derivative 20, previously reported by
our group [46] (Scheme 4).
Scheme 4.
At this juncture, we described the synthesis of lapachones obtained from
lapachol (1) and nor-lapachol (17), their inferior homologue. Recently, we reported the
synthesis of a new class of naphthoquinone compounds, containing a pendant 1,2,3-
triazole motif from C-allyl lawsone (23) [47]. The iodination of 23 affords compounds
24 and 27 in 68% yield and 1:1 ratio (Scheme 5), which were easily separated by
column chromatography. With these compounds in hand, the respective azide
derivatives, compunds 25 and 28, were synthesized by reaction of sodium azide in
dimethylformamide. The respective selenium derivatives, compounds 26 and 29, were
prepared by Cu-catalyzed azide-alkyne cycloaddition (Scheme 5).
Scheme 5.
5

ACCEPTED MANUSCRIPT
1,4-naphthoquinone coupled to selenium-containing 1,2,3-triazole was also a
subject of our study. From compounds 33-35 and 39, the respective triazolic derivatives,
compounds 36-38 and 40, were prepared using methodology discussed previously
(Scheme 6). Suitable crystals of compounds 35 and 38 were obtained, and the structures
were solved by crystallographic methods.
Scheme 6.
Recently, we reported a straightforward approach for the obtention of enantio-
enriched α-lapachone derivatives [48]. In order to identify new enantio-enriched
antitumor compounds, lawsone (41) was used to prepare the nitro-derivatives 42 and 43
via organocatalysis with a chiral squaramide (Scheme 7). The nitro group of the
.
quinones was reduced to the amino group using NiCl2 6H2O/NaBH4 to afford
aminoquinones 44 and 45. These compounds were used immediately after synthesis,
due to their instability, for the preparation of azidoquinones 46 and 47, after a
diazotransfer reaction. In the last step, we prepared the chalcogenium-containing 1,2,3-
triazoles 48 and 49 for reaction with phenyl propargyl selenide by using a classical click
procedure via catalysis with sodium ascorbate and copper(II) sulfate in a 1:1 mixture of
dichloromethane and water.
Scheme 7.
Structures of the novel compounds 4, 6, 7, 10, 13, 15, 16, 19, 21, 22, 26, 29, 36,
1
37, 38, 40, 48 and 49 were determined by H and 13C NMR and 2D NMR spectra
(COSY, HMBC and HSQC). Electrospray ionization mass spectra were also obtained to
confirm compound identities.
2.2. Biological Studies
All of the selenium-containing quinone-based 1,2,3-triazoles described
(Schemes 2-7) and their synthetic precursors were evaluated in vitro using the MTT
assay against six cancer cell lines: HL-60 (human promyelocytic leukemia cells, NQO1-
), HCT-116 (human colon carcinoma cells, NQO1), PC3 (human prostate cells,
6

ACCEPTED MANUSCRIPT
NQO1+), SF295 (human glioblastoma cells, NQO1+), MDA-MB-435 (melanoma cells,
NQO1+) and OVCAR-8 (human ovarian carcinoma cells, NQO1+). β-Lapachone and
doxorubicin were used as positive controls (Table 1). NQO1- normal cells, human
peripheral blood mononucluear (PBMC), and murine fibroblast immortalized cell lines
(V79 and L929) were used to evaluate the selectivity of the compounds. Mechanistic
aspects of selected compounds were also studied for NQO1-dependency using the fairly
specific NQO1 inhibitor, dicoumarol. As previously described [49], the compounds
were classified according to their activity as highly active (IC50 < 2 ꢀM), moderately
active (2 ꢀM < IC50 < 10 ꢀM), or inactive (IC50 >10 ꢀM).
The results showed most of compounds were highly active against all cancer cell
lines evaluated, with IC50 values < 2 ꢀM. In general terms, ortho-quinoidal compounds
were more active than para-quinones. However, α-lapachone derivatives with potent
antitumor activities were also identified.
Naphthopyranquinones 5-7, 9 and 10 presented high to moderate activities (IC50
in the range of 0.92 to 5.46 µM) and the non-active compound 4 was the exception of
this class. For the selenium-containing quinones 6 and 10, the strategy of insertion of a
second redox centre was a success and these derivatives were more active than their
naphthoquinoidal precursors. Recently, we reported the synthesis and antitumor
activities of several α-lapachone-based 1,2,3-triazoles [33]. It is important to highlight
that the selenium-containing-based 1,2,3-triazole 7 displayed better activity than the
compounds without the chalcogen.
Naphthofuranquinones were the second class of compounds evaluated. Para-
naphthoquinones 15 and 16 were active against all cancer cell lines studied. In the last
few years, we described nor-α-lapachone-based 1,2,3-triazoles obtained from lapachol
(1) with IC50 values > 2 µM [33]. The strategy herein used to prepare compounds 15 and
16 with the presence of selenium improved the activities of nor-α-lapachone-based
1,2,3-triazoles, and these derivatives presented IC50 values ranging from 0.68-1.71 µM
for 15, and 1.59-2.95 µM for 16.
Nor-β-lapachone and derivatives are among the most potent compounds from
the lapachol group [25,26]. Recently, we demonstrated [50] the cytotoxicity and genetic
toxicity of nor-β-lapachone in human lymphocytes, HL-60 leukaemia, and immortal
normal murine V79 fibroblasts ranging from 2.5 and 5 ꢀM. This compound failed to
induce DNA damage in nontumor cells, but at the highest concentrations, it induced
DNA single and double strand breaks and increased the frequency of chromosomal
7

ACCEPTED MANUSCRIPT
aberrations. The biological effects of nor-β-lapachone is related to its ability to deplete
reduced glutathione (GSH), which leads to a GSSG-dominant pro-oxidant cellular status
that contributes to its antiproliferative properties.
In this context, we described the potent antitumor activities of nor-β-lapachones
[26,28]. Importantly, C-ring modified, nor-β-lapachone with arylamino groups were the
most active lapachones described [26]. These compounds present significant
antiproliferative effects in human myeloid leukaemia cell lines and induce oxidative
DNA damage by ROS generation. They also somehow impair DNA repair activity,
while triggering cell death, which may be apoptosis [51]. Compound 21 was designed
based on prior experience with these bioactive lapachones. This compound contains the
structural framework of the 3-arylamino-nor-β-lapachone derivatives reported before,
but with a second redox chalcogen centre inserted by click chemistry reaction. This
substance was highly active against all cancer cell lines evaluated, with IC50 values
ranging from 0.07 to 0.38 µM. Moreover, compound 21 exhibited a high selectivity
index (SI represented by the ratio of cytotoxicities between normal cells and different
lines of cancer cells). For instance, PBMC vs HL-60 = 19.8. By comparison,
doxorubicin, a standard clinically used drug against various types of cancers, contained
a selectivity index value of 10.6. In the same way, compound 22 (IC50 in the range of
1.06 to 2.56 µM) was more active than nor-β-lapachone-based 1,2,3-triazole without the
chalcogen atom. Herein, two important examples of successful preparation of potent
antitumor quinones with two redox centres are reported. Compound 26, another nor-
lapachone derivative obtained from C-allyl lawsone (23), also exhibited potent
antitumor activity. This drug was considered highly active with IC50 values ranging
from 0.07 to 0.29 µM, suggesting a highly active structure. Compounds 21 and 26
presented similar antitumor activities, showing the importance of the ortho-
naphthofuranquinone moiety and the triazole selenium-containing group that potentially
works together in the same two redox centre structures.
1,4-Naphthoquinones 36-40 were also evaluated and the compounds were
considered moderately active with exception of compound 40, which was inactive
against all cancer cells examined. The last compounds evaluated were asymmetric α-
lapachone 48 and 49 and these substances were inactive. As recently described by us
[45], the asymmetric α-lapachone derivatives are inactive against several cancer cell
lines evaluated. Herein, we tried to improve their activities using the approach of
8

ACCEPTED MANUSCRIPT
insertion of the chalcogen in these quinones, but the strategy failed. Selectivity index for
the most active compounds was summarized in Table 2.
Table 1.
Table 2.
Previous studies from our laboratory revealed that compounds 50-53 (Figure 1)
can be considered as prototypes possessing potent antitumor activities against diverse
cancer cells [25,26]. To deepen our knowledge about the mechanism of action of
compounds 50-53 and compare the previously reported structures with selenium-
containing lapachone-based 1,2,3-triazoles, we examined their potential NQO1-
dependent cytotoxicities using a set two-hour exposure, with or without the NQO1
inhibitor, dicoumarol. Such exposures take advantage of elevated NQO1 levels
specifically in most solid cancers compared to associated normal tissue [11]. Within the
class of selenium-containing quinones, we selected compounds 21 and 22 to evaluate
their characteristics by an NQO1-dependent mechanism.
Figure 1.
NQO1-dependency assessments. Within the drug concentration range tested,
the compounds showed activity against human A549 non-small cell lung
adenocarcinoma, an alveolar basal epithelial cell line. These cells express high levels of
NQO1 (3000 + 300 enzymatic units). Cell death for many of the compounds tested were
NQO1-specific, since addition of dicoumarol (DIC, an NQO1 inhibitor) spared their
lethality. Based on the survival curves (Figure 2), previously reported arylamine
substituted nor-β-lapachones have predicted IC50 as follows, compounds: 50 = 2.6 µM,
51 = 1.8 µM, 52 = 2.4 µM and 53 = 1.3 µM. Compound 53 showed the most dramatic
lethality within a narrow therapeutic window, going from 93% viability at 0.8 µM to
11% viability at 1.6 µM. Overall, compounds 50-53 were NQO1-specific drugs
exhibiting similar or lower IC50 values than β-lapachone. Compounds 21 and 22,
selenium-containing quinones, with IC50 values = 0.64 and 1.2 µM, respectively, were
the most active of this series and were NQO1-dependent (Table 3). They showed
9

ACCEPTED MANUSCRIPT
tremendous therapeutic windows using DIC treatment as a surrogate for responses to
NQO1- cells, such as that found for nearly all human normal tissue [12a]. These
responses are indicative of NQO1-dependent futile redox cycling of these drugs that
create massive ROS, specifically H2O2, that ultimately cause PARP1 hyperactivation
and programmed necrosis [23,24,52].
Figure 2.
Table 3.
The externalization of phosphatidylserine is considered an important marker in
the apoptotic process. After treatments, selected compounds 21 and 22 induced a
significant increase on populations of PC3 cells with phosphatidylserine expressed on
the cell surface (Figure 3). On the other hand, phosphatidylserine externalization was
not observed in cultures pre-treated with NAC before 21 and 22 exposure or co-treated
with dicoumarol (Figure 3). Our data show that cytotoxic mechanisms of tested
compounds may involve drug bioreduction by quinone reductase NQO1 as well
emphasizing the ROS contribution on the cytotoxicity suggesting that tested
compounds-induced apoptosis is associated with ROS production. Finally,
corroborating these studies, we observed that a short exposure (1 h) to compound 21 led
to the generation of intracellular ROS. In other hand, in cultures pre-exposed with NAC,
compound 21 was not able to generate ROS, which may be explained due the
antioxidant protection exercised by NAC (See Figure S1 in the Supporting
Information).
Figure 3.
3. Conclusions
By using the strategy of conjoining two redox centres, a quinoidal moiety and
the atom of selenium, we prepared and evaluated the activities of novel and diverse
selenium-containing quinone-based 1,2,3-triazole compounds against cancer versus
normal cell lines. We assessed these drugs for lethality overall (Table 1) as well as for
specific role of NQO1. In general, our approach was efficient and we identified
10

ACCEPTED MANUSCRIPT
compounds with IC50 values below 0.3 µM that were more potent than β-lapachone or
doxorubicin, a standard clinically used agent against several types of cancers. We also
studied the efficacy of two more active compounds 21 and 22 and found that these are
specifically bioactivated by NQO1 with higher potency than nor-β-lapachone,
previously published by us. Interestingly, we found that several of these drugs contained
glutathione peroxidase (GPx)-like activities with selected selenium-containing nor-β-
lapachone-based 1,2,3-triazoles being the most potent, and functionally acting on these
two targets. Annexin V cytometry assay was also used to visualize the cell population in
viable, early and late apoptosis stage for compounds 21 and 22. The cytotoxic
mechanisms of 21 and 22 are intrinsically related with ROS contribution on the
cytotoxicity suggesting that apoptosis is associated with ROS production. In order, we
have described different classes of quinones, ortho- and para-quinoidal systems with
potent antitumor activity. For example, compound 29 (para-quinone) has IC50 in the
range of 0.62 to 2.42 µM in the cancer cell lines evaluated. Ortho-quinones, exemplified
for compounds 10, 21, 22 and 26, presented IC50 among 0.07 to 2.52 µM. Finally, we
have described potent antitumor naphthoquinone compounds that emerge as promising
molecules for the therapeutic use of cancers overexpressing NQO1.
4. Experimental Section
4.1. Chemistry
4.1.1. General procedures
Lawsone were acquired from Sigma-Aldrich (St. Louis, MO, USA). Lapachol
(1) (2-hydroxy-3-(3’-methyl-2’-butenyl)-1,4-naphthoquinone) was extracted from the
heartwood of Tabebuia sp. (Tecoma). C-allyl lawsone (23) was prepared from lawsone
as previously reported [53]. A saturated aqueous sodium carbonate solution was added
to the sawdust of ipe tree. Upon observing rapid formation of lapachol sodium salt,
hydrochloric acid was added, allowing the precipitation of lapachol. Then, the solution
was filtered and a yellow solid was obtained. This solid was purified by
recrystallizations with hexane. All chemicals were obtained from commercial sources
and used without further purification. Solvents were distilled and when required were
dried by distillation according to standard procedure [54].
11

ACCEPTED MANUSCRIPT
For the synthesis of (azidomethyl)(phenyl)selane (PhSeCH2N3): Initially
(chloromethyl)(phenyl)selane was prepared by the reaction of a solution of diphenyl
diselenide (3.0 mmol) in THF (6.0 mL) with NaBH4 (2 eq.) in EtOH (6.0 mL) and
CH2Cl2 (30 mL). The mixture was kept under reflux in inert atmosphere and stirred for
12 h. After this period and subsequently extraction with H2O, the organic phase were
combined, dried over MgSO4, and concentrated under vacuum. The residue was purified
by flash chromatography on silica gel using hexane as the eluent [55]. To a solution of
(chloromethyl)(phenyl)selane (PhSeCH2Cl) (3.0 mmol) in CH3CN (5.0 mL), sodium
azide (4.5 mmol) and 18-crown-6 (0.60 mmol) were added at room temperature. Then
the reaction mixture was stirred for 48 h under nitrogen atmosphere. After this time, 30
mL of H2O was added and the organic phase was extracted with CH2Cl2. The organic
layers were combined, dried over MgSO4, and concentrated under vacuum. The residue
was purified by flash chromatography on silica gel using hexane as the eluent. The
product was obtained in 90% yield [56].
For the synthesis of phenyl propargyl selenide: To a solution of diphenyl
diselenide (1.0 mmol) in THF (8.0 mL) with NaBH4 (2 eq.) in EtOH (4 mL). The
mixture was kept under agitation at temperatue of 0 ºC under inert atmosphere and,
then, propargyl bromide (2.0 mmol) in THF (4 mL) was added. After 10 minutes, 30
mL of H2O was added and the organic phase was extracted with ethyl acetate. The
organic phases were combined, dried over MgSO4, and concentrated under vacuum. The
residue was purified by flash chromatography on silica gel using hexane as the eluent.
The product was obtained in 85% yield [57].
Melting points were obtained on a Thomas Hoover and are uncorrected. Column
chromatography was performed on silica gel (SiliaFlash G60 UltraPure 60-200 µm, 60
Å). Infrared spectra were recorded on an FTIR Spectrometer IR Prestige-21-Shimadzu.
1H and 13C NMR were recorded at room temperature using a Bruker AVANCE
DRX200 and DRX400 MHz, in the solvents indicated, with tetramethylsilane (TMS) as
internal reference. Chemical shifts (δ) are given in parts per million (ppm) and coupling
constants (J) in Hertz (Hz). The mass spectrometer was operated in the positive ion
mode. A standard atmospheric pressure photoionization (APPI) source was used to
generate the ions. The sample was injected using a constant flow (3 µL/min). The
solvent was an acetonitrile/methanol mixture. The APPI-Q-TOF MS instrument was
calibrated in the mass range of 50-3000 m/z using an internal calibration standard (low
concentration tuning mix solution) supplied by Agilent Technologies. Data were
12

ACCEPTED MANUSCRIPT
processed employing Bruker Data Analysis software version 4.0. Compounds were
named following IUPAC rules as applied by ChemBioDraw Ultra (version 12.0).
4.1.2. Procedures to prepare arylamino lapachones 4, 13 and 19
To prepare 4 and 13: Compounds 3 and 12 (1.0 mmol) were dissolved in 25
mL of CH2Cl2 and an excess of 3-ethynylaniline (117 mg, 1.2 mmol) was added. The
mixture was left under stirring overnight, followed by the addition of 50 mL of water.
The organic phase was extracted with CH2Cl2, washed with 10% HCl (3 x 50 mL),
dried over sodium sulfate, and filtered. The solvent from the crude was evaporated
under reduced pressure and it was purified by column chromatography on silica-gel,
using eluents with an increasing polarity gradient mixture of hexane and ethyl acetate
(9/1 to 7/3).
To prepare 19: To a solution of nor-lapachol (17) (228 mg, 1.0 mmol) in 25 mL
of chloroform, 2 mL of bromine was added. The bromo intermediate 18 precipitated
immediately as an orange solid. After removal of bromine, by adding dichloromethane
and then removing the organic solvent with dissolved bromine by rotary evaporator, an
excess of 3-ethynylaniline (117 mg, 1.2 mmol) was added in CH2Cl2 and the mixture
was stirred overnight. The crude reaction mixture was poured into 50 mL of water. The
organic phase was separated and washed with 10% HCl (3 x 50 mL), dried over sodium
sulfate, filtered, and evaporated under reduced pressure. The product 19 was obtained
after purification by column chromatography in silica-gel, eluted with an increasing
polarity gradient mixture of hexane and ethyl acetate (9/1 to 7/3).
4.1.3. General procedures to prepare the azide derivatives
The previously published azido derivatives 5, 9, 14, 20, 25, 39, 46 were prepared
as described in the literature [40-42,46-48]. Compound 28 (1.0 mmol) was prepared
from 27 in the presence of sodium azide (120 mg, 1.85 mmol) in 2 mL of
dimethylformamide (DMF). The mixture was stirred at room temperature until product
formation was complete as determined by thin layer chromatography. After extraction
with CH2Cl2, the residue was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Compound 28 was obtained after purification by column
chromatography on silica gel eluting with a gradient mixture of hexane:ethyl acetate
13

ACCEPTED MANUSCRIPT
with increasing polarity. The unpublished azide derivative 47 was prepared following
the same procedure previously described [48].
4.1.4. General procedures for the preparation of 1,2,3-triazole derivatives
The azidoquinones (1.0 mmol) or quinone with terminal alkynes (1.0 mmol)
.
were reacted with CuSO4 5H2O (0.04 mmol) and sodium ascorbate (0.11 mmol) and the
phenyl propargyl selenide (195 mg, 1.0 mmol) or (azidomethyl)(phenyl)selane (212 mg,
1.0 mmol) in a mixture of CH2Cl2:H2O (12 mL, 1:1, v/v). The mixture was stirred at
room temperature until product formation was complete as determined by thin layer
chromatography. The aqueous phase was extracted with CH2Cl2, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel eluting with a gradient mixture of hexane:ethyl acetate
with increasing polarity.
4.1.5. 4-((3-ethynylphenyl)amino)-2,2-dimethyl-3,4-dihydro-2H-benzo[g]chromene-
5,10-dione (4). Yield: 70%; mp 196-197 ºC; Brown solid. 1H NMR (200 MHz, CDCl3)
δ 8.11-8.03 (m, 2H), 7.75-7.68 (m, 2H), 7.16 (t, J = 7.5 Hz, 1H), 6.95-6.85 (m, 2H),
6.71 (d, J = 8.0 Hz, 1H), 4.68 (t, J = 3.7 Hz, 1H), 3.03 (s, 1H), 2.27 (dd, J = 3.5 and
13
14.4 Hz, 1H), 2.02 (dd, J = 5.5 and 14.4 Hz, 1H), 1.54 (s, 6H); C NMR (CDCl3, 50
MHz) δ 183.7, 180.0, 155.5, 146.3, 134.6, 133.3, 132.1, 131.0, 129.4, 126.5, 126.3,
123.0, 122.8, 118.4, 117.1, 115.2, 84.1, 79.2, 43.7, 37.4, 28.7, 26.3; HRMS (ES+)
calculated for C23H20NO3 [M+H]+: 358.1443; found: 358.1488.
4.1.6.
2,2-dimethyl-4-((3-(1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-
yl)phenyl)amino)-3,4-dihydro-2H-benzo[g]chromene-5,10-dione (6). Yield: 75%;
mp 101-102 ºC; Red solid. 1H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 7.1 and 1.7 Hz,
H9), 8.07 (dd, J = 7.1 and 1.6 Hz, H6), 7.72 (td, J = 7.1, 7.1 and 1.6 Hz, H8), 7.68 (td, J
= 7.1, 7.1 and 1.7 Hz, H7), 7.62 (s, H5-triazole), 7.50 (d, J = 7.4 Hz, H2’’/6’’), 7.33-7.39
(m, H4’’), 7.26-7.28 (m, H2’), 7.28-7.33 (m, H3’’/5’’), 7.20-7.26 (m, H5’), 7.05 (d, J = 7.7
Hz, H4’), 6.69 (dd, J = 8.0 and 1.8 Hz, H6’), 5.71 (s, H12), 4.78 (dd, J = 5.6 and 3.4 Hz,
H4), 2.32 (dd, J = 14.3 and 3.4 Hz, H3a or H3b), 2.06 (dd, J = 14.3 and 5.6 Hz, H3a or
H3b), 1.55 (s, H11’ or H11’’), 1.54 (s, H11’ or H11’’); 13C NMR (CDCl3, 100 MHz) δ 183.5
(C5), 180.0 (C10), 155.3 (C10a), 148.5 (C4-triazole), 147.4 (C1’), 134.8 (C2’’/6’’), 134.3
14

ACCEPTED MANUSCRIPT
(C8), 133.1 (C7), 132.1 (C9a), 131.3 (C3’), 130.9 (C5a), 129.8 (C5’), 129.6 (C3’’/5’’), 129.0
(C4’’), 127.4 (C1’’), 126.4 (C9), 126.2 (C6), 119.4 (C5-triazole), 118.9 (C4a), 115.7 (C4’),
113.6 (C6’), 110.7 (C2’), 79.1 (C2), 44.8 (C12), 43.2 (C4), 37.6 (C3), 29.0 (C11’ or C11’’),
26.1 (C11’ or C11’’); HRMS (ES+) calculated for C30H27N4O3Se [M+H]+: 571.1248;
found: 571.1234.
4.1.7. 2,2-dimethyl-4-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-3,4-dihydro-
1
2H-benzo[g]chromene-5,10-dione (7). Yield: 70%; mp 168-169 ºC; Yellow solid. H
NMR (200 MHz, CDCl3) δ 8.18-8.09 (m, 1H), 8.04-7.96 (m, 1H), 7.80-7.68 (m, 2H),
7.50-7.39 (m, 2H), 7.24-7.10 (m, 4H), 5.71 (t, J = 6.3 Hz, 1H), 4.13 (s, 2H), 2.73 (dd, J
= 5.5 and 14.4 Hz, 1H), 2.29 (dd, J = 6.3 and 14.4 Hz, 1H), 1.50 (s, 3H), 1.21 (s, 3H);
13C NMR (CDCl3, 50 MHz) δ 182.7, 179.5, 156.3, 145.3, 134.7, 133.7, 131.8, 131.1,
129.7, 129.1, 127.5, 126.8, 126.6, 122.0, 115.0, 79.4, 49.6, 39.0, 27.0, 26.5, 20.8;
HRMS (ES+) calculated for C24H21N3O3SeNa [M+Na]+: 502.0646; found: 502.0643.
4.1.8.
3-bromo-2,2-dimethyl-4-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-
3,4-dihydro-2H-benzo[h]chromene-5,6-dione (10). Yield: 90%; mp 105-106 ºC;
Orange solid. 1H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 7.6 and 1.4 Hz, H7), 7.90 (dd,
J = 7.6 and 1.2 Hz, H10), 7.73 (td, J = 7.6, 7.6 and 1.2 Hz, H8), 7.63 (td, J = 7.6, 7.6 and
1.4 Hz, H9), 7.48-7.52 (m, H2’/6’), 7.48 (s, H5-triazole), 7.22-7.25 (m, H3’/5’), 7.22-7.25
(m, H4’), 5.55 (d, J = 8.9 Hz, H4), 4.93 (d, J = 8.9 Hz, H3), 4.17 (d, J = 13.3 Hz, H12a),
4.13 (d, J = 13.3 Hz, H12b), 1.71 (s, H11’ or H11’’), 1.64 (s, H11’ or H11’’); 13C NMR
(CDCl3, 100 MHz) δ 177.8 (C6), 176.4 (C5), 162.7 (C10b), 144.5 (C4-triazole), 135.2
(C8), 134.1 (C2’/6’), 132.3 (C9), 130.6 (C10a), 130.5 (C6a), 129.4 (C1’), 129.2 (C7), 129.1
(C3’/5’), 127.6 (C4’), 125.3 (C10), 125.0 (C5-triazole), 110.3 (C4a), 83.4 (C2), 54.4 (C3),
58.9 (C4), 20.7 (C11’ or C11’’), 27.4 (C11’ or C11’’), 20.7 (C12); HRMS (ES+) calculated
for C24H21BrN3O3Se [M+H]+: 557.9931; found: 557.9923.
4.1.9. 3-((3-ethynylphenyl)amino)-2,2-dimethyl-2,3-dihydronaphtho[2,3-b]furan-
4,9-dione (13). Yield: 65%; mp 165-167 ºC; Brown solid. 1H NMR (200 MHz, CDCl3)
δ 8.14-8.04 (m, 2H), 7.78-7.66 (m, 2H), 7.15 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 7.5 Hz,
1H), 6.74 (s, 1H), 6.62 (dd, J = 2.1 and 8.0 Hz, 1H), 4.87 (s, 1H), 3.03 (s, 1H), 2.27 (s,
1H), 1.65 (s, 3H), 1.54 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 181.7, 178.7, 159.9,
146.8, 134.6, 133.2, 133.1, 131.5, 129.4, 126.5, 126.3, 123.0, 122.4, 122.1, 116.3,
15

ACCEPTED MANUSCRIPT
114.0, 94.9, 83.9, 62.2, 29.5, 27.1, 21.5; HRMS (ES+) calculated for C22H17NO3Na
[M+Na]+: 366.1106; found: 366.1257.
4.1.10.
2,2-dimethyl-3-((3-(1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-
yl)phenyl)amino)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione (15). Yield: 70%; mp
120-122 ºC; Red solid. 1H NMR (400 MHz, CDCl3) δ 8.10 (dd, J = 7.4 and 1.4 Hz, H8),
8.07 (dd, J = 7.4 and 1.6 Hz, H5), 7.73 (td, J = 7.4, 7.4 and 1.6 Hz, H7), 7.68 (td, J =
7.4, 7.4 and 1.4 Hz, H6), 7.60 (s, H5-triazole), 7.50 (d, J = 7.5 Hz, H2’’/6’’), 7.27-7.39 (m,
H3’’/5’’), 7.27-7.39 (m, H4’’), 7.22 (t, J = 7.8 Hz, H5’), 7.20 (s, H2’), 7.04 (d, J = 7.8 Hz,
H4’), 6.61 (dd, J = 7.8 and 1.8 Hz, H6’), 5.71 (s, H11), 4.96 (s, H3), 4.05 (sl, NH), 1.67 (s,
13
H10’ or H10’’), 1.57 (s, H10’ or H10’’); C NMR (CDCl3, 100 MHz) δ 181.8 (C4), 178.6
(C9), 159.8 (C9a), 148.3 (C4-triazole), 147.5 (C1’), 134.8 (C2’’/6’’), 134.5 (C7), 133.2
(C8a), 133.1 (C6), 131.9 (C3’), 131.6 (C4a), 129.9 (C5’), 129.6 (C3’’/5’’), 129.0 (C4’’), 127.4
(C1’’), 126.5 (C8), 126.2 (C5), 122.3 (C3a), 119.4 (C5-triazole), 115.9 (C4’), 113.1 (C6’),
110.5 (C2’), 95.5 (C2), 62.3 (C3), 44.7 (C11), 27.2 (C10’ or C10’’), 21.6 (C10’ or C10’’);
HRMS (ES+) calculated for C29H25N4O3Se [M+H]+: 557.1092; found: 557.1101.
4.1.11.
2,2-dimethyl-3-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[2,3-b]furan-4,9-dione (16). Yield: 80%; mp 172-174 ºC; Yellow
solid. 1H NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 7.4 and 1.6 Hz, H8), 8.08 (dd, J = 7.4
and 1.6 Hz, H5), 7.80 (td, J = 7.4, 7.4 and 1.6 Hz, H7), 7.76 (td, J = 7.4, 7.4 and 1.6 Hz,
H6), 7.40 (dd, J = 7.9 and 1.4 Hz, H2’/6’), 7.08-7.19 (m, H3’/5’), 7.08-7.19 (m, H4’), 6.98
(s, H5-triazole), 5.92 (s, H3), 4.14 (d, J = 13.5 Hz, H11), 4.09 (d, J = 13.5 Hz, H11), 1.67
(s, H10’ or H10’’), 1.02 (s, H10’ or H10’’); 13C NMR (CDCl3, 100 MHz) δ 180.5 (C4), 177.8
(C9), 151.1 (C9a), 145.8 (C4-triazole), 134.9 (C7), 133.9 (C2’/6’), 133.6 (C6), 132.7 (C8a),
131.6 (C4a), 129.13 (C1’), 129.06 (C3’/5’), 127.5 (C4’), 126.8 (C8), 126.5 (C5), 121.2 (C5-
triazole), 118.3 (C3a), 94.5 (C2), 67.3 (C3), 27.4 (C10’ or C10’’), 20.7 (C10’ or C10’’), 20.5
(C11); HRMS (ES+) calculated for C23H19N3O3SeNa [M+Na]+: 488.0489; found:
488.0486.
4.1.12. 3-((3-ethynylphenyl)amino)-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-
1
4,5-dione (19). Yield: 70%; mp 205-206 ºC; Red solid. H NMR (200 MHz, CDCl3) δ
8.10 (dd, J = 2.0 and 8.0 Hz, 1H), 7.76-7.58 (m, 3H), 7.12 (t, J = 7.8 Hz, 1H), 6.88 (d, J
= 7.8 Hz, 1H), 6.69 (s, 1H), 6.58 (dd, J = 2.0 and 8.0 Hz, 1H), 4.79 (s, 1H), 3.02 (s, 1H),
16

ACCEPTED MANUSCRIPT
1.68 (s, 3H), 1.57 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 175.4, 169.7, 147.1, 134.7,
132.7, 131.2, 129.6, 129.4, 128.6, 125.2, 122.9, 122.2, 116.1, 115.0, 114.1, 96.8, 84.1,
61.5, 27.4, 21.8; HRMS (ES+) calculated for C22H17NO3Na [M+Na]+: 366.1106; found:
366.1108.
4.1.13.
2,2-dimethyl-3-((3-(1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-
yl)phenyl)amino)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (21). Yield: 50%; mp
135-137 ºC; Red solid. 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 7.4 Hz, H6), 7.70 (td,
J = 7.4, 7.4 and 1.1 Hz, H7), 7.74 (dd, J = 7.4 and 1.1 Hz, H9), 7.64 (td, J = 7.4, 7.4 and
1.6 Hz, H8), 7.59 (s, H5-triazole), 7.50 (d, J = 6.9 Hz, H2’’/6’’), 7.28-7.40 (m, H3’’/5’’),
7.28-7.40 (m, H4’’), 7.14 (s, H2’), 7.20 (dd, J = 8.1 and 7.7 Hz, H5’), 7.02 (d, J = 7.7 Hz,
H4’), 6.56 (dd, J = 8.1 and 2.2 Hz, H6’), 5.51 (s, H11), 4.89 (s, H3), 3.70-4.37 (sl, NH),
1.60 (s, H10’ or H10’’), 1.17 (s, H10’ or H10’’); 13C NMR (CDCl3, 100 MHz) δ 181.0 (C5),
175.4 (C4), 169.6 (C9b), 148.3 (C4-triazole), 147.8 (C1’), 134.8 (C2’’/6’’), 134.6 (C7),
132.5 (C8), 131.3 (C3’), 131.2 (C9a), 129.8 (C5’), 129.6 (C3’’/5’’), 129.5 (C6), 128.9 (C4’’),
127.5 (C5a), 127.4 (C1’’), 125.1 (C9), 119.5 (C5-triazole), 115.5 (C4’), 115.1 (C3a), 113.0
(C6’), 110.2 (C2’), 96.9 (C2), 61.6 (C3), 44.8 (C11), 27.5 (C10’ or C10’’), 21.8 (C10’ or
C10’’); HRMS (ES+) calculated for C29H24N4O3SeNa [M+Na]+: 579.0911; found:
579.0890.
4.1.14.
2,2-dimethyl-3-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (22). Yield: 80%; mp 186-188 ºC; Yellow
1
solid. H NMR (200 MHz, CDCl3) δ 8.19 (d, J = 7.3 Hz, H6), 7.68-7.82 (m, H7), 7.68-
7.82 (m, H8), 7.68-7.82 (m, H9), 7.41 (d, J = 7.1 Hz, H2’/6’), 7.08-7.21 (m, H3’/5’), 7.08-
7.21 (m, H4’), 7.00 (s, H5-triazole), 5.86 (s, H3), 4.05-4.16 (m, H11), 1.70 (s, H10’ or
H10’’), 1.05 (s, H10’ or H10’’); 13C NMR (CDCl3, 50 MHz) δ 180.0 (C5), 174.5 (C4), 171.1
(C9b), 145.5 (C4-triazole), 134.9 (C7), 134.0 (C2’/6’), 133.4 (C8), 131.4 (C9a), 129.9 (C6),
129.2 (C1’), 129.1 (C3’/5’), 127.5 (C4’), 126.6 (C5a), 125.6 (C9), 121.2 (C5-triazole), 111.2
(C3a), 95.9 (C2), 66.7 (C3), 27.6 (C10’ or C10’’), 20.9 (C10’ or C10’’), 20.6 (C11); HRMS
(ES+) calculated for C23H19N3O3SeNa [M+Na]+: 488.0489; found: 488.0482.
4.1.15.
2-((4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2,3-
dihydronaphtho[1,2-b]furan-4,5-dione (26). Yield: 85%; mp 163-164 ºC; Orange
solid. 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 7.4 and 1.5 Hz, H6), 7.59 (td, J = 7.4,
17

ACCEPTED MANUSCRIPT
7.4 and 1.4 Hz, H7), 7.53 (td, J = 7.4, 7.4 and 1.5 Hz, H8), 7.44 (dd, J = 7.4 and 1.1 Hz,
H9), 7.33-7.41 (m, H2’/6’), 7.31 (s, H5-triazole), 7.13-7.18 (m, H3’/5’), 7.13-7.18 (m, H4’),
5.33-5.42 (m, H2), 4.55 (dd, J = 14.6 and 7.3 Hz, H10a), 4.64 (dd, J = 14.6 and 3.9 Hz,
H10b), 4.11 (s, H11), 3.23 (dd, J = 15.8 and 10.2 Hz, H3a), 2.83 (dd, J = 15.8 and 7.0 Hz,
13
H3b); C NMR (CDCl3, 100 MHz) δ 180.4 (C5), 175.2 (C4), 186.6 (C9b), 146.4 (C4-
triazole), 134.7 (C7), 132.9 (C2’/6’), 132.3 (C8), 130.6 (C9a), 129.8 (C6), 129.8 (C1’),
129.2 (C3’/5’), 127.5 (C4’), 126.9 (C5a), 124.4 (C9), 122.7 (C5-triazole), 114.7 (C3a),
84.3 (C2), 53.3 (C10), 29.6 (C3), 20.2 (C11); HRMS (ES+) calculated for
C22H18N3O3Se [M+H]+: 452.0513; found: 452.0896.
4.1.16. 2-(azidomethyl)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione (28). Yield: 90%;
1
mp 116-117 ºC; Yellow solid. H NMR (200 MHz, CDCl3) δ 8.06-7.94 (m, 2H), 7.71-
7.57 (m, 2H), 5.23-5.09 (m, 1H), 3.75 (dd, J = 3.7 and 13.3 Hz, 1H), 3.56 (dd, J = 5.0
and 13.3 Hz, 1H), 3.26 (dd, J = 10.8 and 17.4 Hz, 1H), 3.02 (dd, J = 7.5 and 17.4 Hz,
1H); 13C NMR (CDCl3, 50 MHz) δ 181.8, 177.1, 159.4, 134.1, 133.0, 132.6, 131.2,
126.1, 125.8, 124.0, 83.6, 53.5, 30.0; HRMS (ES+) calculated for C13H10N3O3 [M+H]+:
256.0722; found: 256.0716.
4.1.17.
2-((4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2,3-
dihydronaphtho[2,3-b]furan-4,9-dione (29). Yield: 90%; mp 169-171 ºC; Yellow
solid. 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 7.4 and 1.5 Hz, H8), 7.93 (dd, J = 7.4
and 1.6 Hz, H5), 7.66 (td, J = 7.4, 7.4 and 1.6 Hz, H7), 7.62 (td, J = 7.4, 7.4 and 1.5 Hz,
H6), 7.32-7.37 (m, H5-triazole), 7.32-7.37 (m, H2’/6’), 7.11-7.17 (m, H3’/5’), 7.11-7.17 (m,
H4’), 5.23-5.32 (m, H2), 4.64 (dd, J = 14.7 and 3.9 Hz, H10b), 4.57 (dd, J = 14.7 and 5.6
Hz, H10a), 4.05 (s, H11), 3.26 (dd, J = 17.4 and 10.6 Hz, H3b), 2.95 (dd, J = 17.4 and 8.1
13
Hz, H3b); C NMR (CDCl3, 50 MHz) δ 181.6 (C4), 177.2 (C9), 159.0 (C9a), 146.4 (C4-
triazole), 134.4 (C7), 133.1 (C2’/6’), 132.7 (C8a), 133.2 (C6), 131.3 (C4a), 129.7 (C1’),
129.1 (C3’/5’), 127.4 (C4’), 126.4 (C8), 126.2 (C5), 124.1 (C3a), 123.1 (C5-triazole), 83.0
(C2), 52.9 (C10), 30.1 (C3), 20.3 (C11); HRMS (ES+) calculated for C22H18N3O3Se
[M+H]+: 452.0513; found: 452.0512.
4.1.18.
2-chloro-3-(((1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)amino)-1,4-naphthoquinone (36). Yield: 65%; mp 127-128 ºC; Orange
solid. 1H NMR (200 MHz, CDCl3) δ 8.14 (dd, J = 1.2 and 7.5 Hz, 1H), 8.03 (dd, J = 1.2
18

ACCEPTED MANUSCRIPT
and 7.5 Hz, 1H), 7.97-7.57 (m, 2H), 7.51-7.40 (m, 3H), 7.33-7.23 (m, 3H), 6.49 (sl,
1H), 5.68 (s, 2H), 5.10 (d, J = 6.3 Hz, 2H); 13C NMR (CDCl3, 50 MHz) δ 180.0, 176.7,
144.8, 143.6, 134.8, 134.7, 132.5, 132.3, 129.7, 129.5, 129.0, 126.9, 126.8, 126.7,
121.8, 111.6, 43.6, 40.0; HRMS (ES+) calculated for C20H16ClN4O2Se [M+H]+:
459.0127; found: 459.0128.
4.1.19.
2-bromo-3-(((1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)amino)-1,4-naphthoquinone (37). Yield: 60%; mp 124-126 ºC; Orange
solid. 1H NMR (200 MHz, CDCl3) δ 8.13 (dd, J = 1.2 and 8.0 Hz, 1H), 8.01 (dd, J = 1.2
and 8.0 Hz, 1H), 7.77-7.57 (m, 2H), 7.48 (s, 1H), 7.46-7.41 (m, 2H), 7.36-7.20 (m, 3H),
6.50 (sl, 1H), 5.68 (s, 2H), 5.11 (d, J = 6.3 Hz, 2H); 13C NMR (CDCl3, 50 MHz) δ
180.1, 176.7, 146.2, 145.0, 135.0, 132.8, 132.3, 130.0, 129.8, 129.3, 127.2, 127.1,
122.1, 44.6, 40.7; HRMS (ES+) calculated for C20H16BrN4O2Se [M+H]+: 502.9622;
found: 502.9612.
4.1.20.
2-(((1-((phenylselanyl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-1,4-
1
naphthoquinone (38). Yield: 60%; mp 162-164 ºC; Red solid. H NMR (200 MHz,
CDCl3) δ 8.10 (dd, J = 7.6 and 1.3 Hz, H8), 8.06 (dd, J = 7.6 and 1.3 Hz, H5), 7.74 (td, J
= 7.6, 7.6 and 1.3 Hz, H6), 7.64 (td, J = 7.6, 7.6 and 1.3 Hz, H7), 7.46 (dd, J = 7.8 and
1.5 Hz, H2’/6’), 7.39 (s, H5-triazole), 7.27-7.35 (m, H3’/5’), 7.27-7.35 (m, H4’), 6.22-6.35
(m, NH), 5.77 (s, H3), 5.68 (s, H10), 4.45 (d, J = 5.8 Hz, H9); 13C NMR (CDCl3, 50
MHz) δ 183.0 (C1), 181.6 (C4), 147.4 (C4-triazole), 143.2 (C2), 134.82 (C2’/6’), 134.78
(C6), 133.4 (C4a), 132.2 (C7), 130.4 (C8a), 129.7 (C3’/5’), 129.1 (C4’), 127.0 (C1’), 126.3
(C5), 126.2 (C8), 121.7 (C5-triazole), 101.7 (C3), 44.7 (C10), 38.1 (C9); HRMS (ES+)
calculated for C20H17N4O2Se [M+H]+: 425.0517; found: 425.0512.
4.1.21.
2-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-1,4-naphthoquinone
1
(40). Yield: 60%; mp 117-120 ºC; Brown solid. H NMR (200 MHz, CDCl3) δ 8.36 (s,
1H), 8.24-8.12 (m, 2H), 7.88-7.79 (m, 2H), 7.72 (s, 1H), 7.59-7.48 (m, 2H), 7.29-7.26
13
(m, 2H), 4.25 (s, 2H), 1.6 (s, 1H); C NMR (CDCl3, 50 MHz) δ 183.6, 179.1, 146.8,
139.1, 134.9, 134.2, 133.4, 131.3, 130.9, 129.3, 129.1, 127.6, 127.1, 126.4, 126.2,
123.8, 20.2; HRMS (ES+) calculated for C19H14N3O2Se [M+H]+: 396.0251; found:
396.0242.
19

ACCEPTED MANUSCRIPT
4.1.22.
(3R,4S)-3-azido-4-(naphthalen-1-yl)-3,4-dihydro-2H-benzo[g]chromene-
1
5,10-dione (47). Yield: 78%; mp 115-117 ºC; Light yellow solid. H NMR (200 MHz,
CDCl3) δ 8.27 (d, J = 8.3 Hz, 1H), 8.23-8.20 (m, 1H), 8.03-8.00 (m, 1H), 7.93 (d, J =
8.1 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.76-7.68 (m, 3H), 7.59 (t, J = 8.1 Hz, 1H), 7.35
(t, J = 7.3 Hz, 1H), 7.11 (d, J = 7.3 Hz, 1H), 5.14 (dd, J = 2.3 and 1.8 Hz, 1H), 4.46 (dt,
J = 12.5 and 2.3 Hz, 1H), 4.19 (d, J = 12.5 Hz, 1H), 4.13 (d, J = 1.8 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) δ 183.2, 179.1, 155.7, 136.0, 134.6, 133.6, 132.3, 131.3, 130.6,
129.7, 129.0, 127.6, 126.8, 126.7, 126.5, 126.2, 125.4, 122.2, 119.7, 64.6, 56.9, 36.4;
HRMS (ES+) calculated for C23H15N3O3Na [M+Na]+: 404.1006; found: 404.1007.
4.1.23.
(3R,4S)-4-phenyl-3-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-yl)-3,4-
dihydro-2H-benzo[g]chromene-5,10-dione (48). Yield: 70%; mp 187-188 ºC; Yellow
1
solid. H NMR (200 MHz, CDCl3) δ 8.22-8.16 (m, 1H), 8.02-7.98 (m, 1H), 7.78-7.73
(m, 2H), 7.40-7.26 (m, 7H), 7.15 (s, 1H), 7.13-7.02 (m, 3H), 4.98-4.96 (m, 1H), 4.70-
4.65 (m, 2H), 4.50-4.44 (m, 1H), 4.10 (s, 2H); 13C NMR (CDCl3, 50 MHz) δ 182.3,
178.4, 154.6, 145.8, 140.0, 134.5, 133.5, 133.4, 131.7, 130.7, 129.2, 128.9, 128.0,
127.7, 127.4, 126.5, 120.3, 119.7, 64.6, 58.3, 40.7, 20.2; HRMS (ES+) calculated for
C28H21N3O3SeH [M+H]+: 528.0826; found: 528.0821.
4.1.24. (3R,4S)-4-(naphthalen-1-yl)-3-(4-((phenylselanyl)methyl)-1H-1,2,3-triazol-1-
yl)-3,4-dihydro-2H-benzo[g]chromene-5,10-dione (49). Yield: 65%; mp 185-188 ºC;
Yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 8.6 Hz, H9’’), 8.12 (dd, J = 6.9
and 1.9 Hz, H9), 7.89 (dd, J = 6.8 and 2.0 Hz, H6), 7.84 (d, J = 8.1 Hz, H6’’), 7.72 (d, J =
8.2 Hz, H4’’), 7.64-7.70 (m, H7), 7.64-7.70 (m, H8), 7.62 (dd, J = 8.6 and 7.5 Hz, H8’’),
7.50 (dd, J = 8.1 and 7.5 Hz, H7’’), 7.31 (d, J = 7.4 Hz, H2’/6’), 7.25 (dd, J = 8.2 and 7.1
Hz, H3’’), 7.20 (s, H5-triazole), 7.06 (d, J = 7.1 Hz, H2’’), 7.00 (t, J = 7.4 Hz, H3’/5’), 6.92
(t, J = 7.4 Hz, H4’), 5.42 (s, H4), 5.00 (s, H3), 4.54 (d, J = 12.8 Hz, H2a), 4.36 (dl, J =
13
12.8 Hz, H2b), 4.04 (s, H11); C NMR (CDCl3, 100 MHz) δ 182.2 (C5), 178.4 (C10),
155.0 (C10a), 145.9 (C4-triazole), 135.9 (C1’), 134.5 (C8), 134.3 (C5’’), 133.5 (C2’/6’),
133.6 (C7), 131.9 (C9a), 130.9 (C5a), 130.4 (C10’’), 129.3 (C1’), 129.2 (C6’’), 129.1 (C4’’),
128.9 (C3’/5’), 127.8 (C8’’), 127.5 (C4’), 126.7 (C6), 126.6 (C9), 126.5 (C7’’), 125.7 (C2’’),
124.9 (C3’’), 122.7 (C9’’), 120.3 (C5-triazole), 120.0 (C4a), 64.4 (C2), 56.4 (C3), 37.2 (C4),
20.3 (C11); HRMS (ES+) calculated for C32H24N3O3Se [M+H]+: 578.0983; found:
578.0982.
20

ACCEPTED MANUSCRIPT
4.2. Crystallographic data
The structures of the compounds 35 and 38 were determined from X-ray
diffraction on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on κ-
goniostat) using graphite monochromated MoKα radiation (0.71073 Å), at room
temperature. Data collections were carried out using the COLLECT software [58] up to
50° in 2θ. Final unit cell parameters were based on 6482 reflections for compound 35
and 10161 reflections for compound 38. Integration and scaling of the reflections,
correction for Lorentz and polarization effects were performed with the HKL DENZO-
SCALEPACK system of programs [59]. The structures were solved by direct methods
with SHELXS-97 [60]. The models were refined by full-matrix least squares on F2
using SHELXL-97 [61]. The program ORTEP-3 [62]. was used for graphic
representation and the program WINGX [63] to prepare materials for publication. All H
atoms were located by geometric considerations placed (C–H = 0.93-0.97 Å; N-H =
0.86 Å) and refined as riding with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other). An
Ortep-3 diagram of compounds 35 and 38 are shown in Scheme 6 and the main
crystallographic data are listed as following:
For compound 35: Empirical formula: C13H7BrNO2; Formula weight: 289.11;
Temperature: 293(2) K; Wavelength: 0.71073 Å; Crystal system: triclinic; Space group:
P-1; Unit cell dimensions: a = 7.2940(3) Å; b = 7.9110(3) Å; c = 9.7940(4) Å; α=
3;
77.133(2)°; β= 89.477(2)°; γ = 84.157(2)°; Volume: 548.03(4) Å Z: 2; Density
3
-1
(calculated): 1.75 Mg/m ; Absorption coefficient: 2.140 mm ; F(000): 286; Crystal
3
size: 0.32 x 0.22 x 0.10 mm ; Theta range for data collection: 3.5 to 27.5°; Index
ranges: -9≤h≤9, -9≤k≤10, -12≤l≤12; Reflections collected: 8533; Independent
2
reflections: 2498 [R(int) = 0.092]; Refinement method: Full-matrix least-squares on F ;
Data / restraints / parameters: 2190 / 0 / 154; Goodness-of-fit on F2: 1.06; Final R
indices [I>2sigma(I)]: R1 = 0.042, wR2 = 0.109; R indices (all data): R1 = 0.048, wR2
= 0.114.
For compound 38: Empirical formula: C20H16N4O2Se; Formula weight: 419.3;
Temperature: 293(2) K; Wavelength: 0.71073 Å; Crystal system: monoclinic; Space
group: C2/c Unit cell dimensions: a = 34.0429(8) Å; b = 4.7831(2) Å; c = 23.3921(7) Å;
3;
α = 90°; β = 111.3°; γ = 90° ; Volume: 3548.6(2) Å Z: 8; Density (calculated):1.57
3;
-1;
Mg/m Absorption coefficient: 2.140 mm F(000): 1680; Crystal size: 0.28 x 0.11 x
21

ACCEPTED MANUSCRIPT
3;
0.10 mm Theta range for data collection: 2.6 to 27.54°; Index ranges: -43≤h≤44, -
5≤k≤6, -30≤l≤30; Reflections collected: 16701; Independent reflections: 4026 [R(int) =
2;
0.063]; Refinement method: Full-matrix least-squares on F Data / restraints /
parameters: 2822 / 0 / 244; Goodness-of-fit on F2: 1.12; Final R índices [I>2sigma(I)]:
R1 = 0.047, wR2 = 0.128; R indices (all data): R1 = 0.073, wR2 = 0.144.
Crystallographic data for the structures were deposited in the Cambridge
Crystallographic Data Centre, with numbers CCDC 1063533 and 1063534.
4.3. Antitumor activity
Compounds were tested for antitumor activity in cell culture in vitro using
several human cancer cell lines obtained from the National Cancer Institute, NCI
(Bethesda, MD). The L929 cells (mouse fibroblast L cells NCTC clone 929) were
obtained from the American Type Culture Collection (Manassas, VA), MDCK cells
were purchased from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil), and the
Chinese hamster lung fibroblasts (V79 cells) were kindly provided by Dr. JAP
Henriques (UFRGS, Brazil). Peripheral blood mononuclear cells (PBMC) were isolated
from heparinized blood from healthy, non-smoker donors who had not taken any
medication at least 15 days prior to sampling by a standard method of density-gradient
centrifugation on Histopaque-1077 (Sigma Aldrich Co. - St. Louis, MO, USA). All
cancer cell lines and PBMC were maintained in RPMI 1640 medium. The L929,
MDCK and V79 cells were cultivated under standard conditions in DMEM with Earle’s
salts. All culture media were supplemented with 20% (PBMC) or 10% (cancer, L929,
MDCK and V79 cells) fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin,
100 µg/mL streptomycin at 37 °C with 5% CO2. PBMC cultures were also
supplemented with 2% phytohaemagglutinin. In cytotoxicity experiments, cells were
plated in 96-well plates (0.1 x 106 cells/well for leukaemia cells, 0.7 x 105 cells/well for
solid tumor as well V79, L929 and MDCK cells, and 1 x 106 cells/well for PBMC). All
tested compounds were dissolved with DMSO. The final concentration of DMSO in the
culture medium was kept constant (0.1%, v/v). Doxorubicin (0.001-1.10 µM) was used
as the positive control, and negative control groups received the same amount of vehicle
(DMSO). The cell viability was determined by reduction of the yellow dye 3-(4,5-
dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan
22

ACCEPTED MANUSCRIPT
product as described by Mosmann [64]. At the end of the incubation time (72 h), the
plates were centrifuged and the medium was replaced by fresh medium (200 µL)
containing 0.5 mg/mL MTT. Three hours later, the MTT formazan product was
dissolved in DMSO (150 µL) and the absorbance was measured using a multiplate
reader (Spectra Count, Packard, Ontario, Canada). Drug effect was quantified as the
percentage of control absorbance of the reduced dye at 550 nm. All cell treatments were
performed with three replicates. All cells were mycoplasma-free.
4.4. DNA Survival Assays
A549 cells were plated into a 48-well plate with 10,000 cells/well in 500 µL of
DMEM containing 10% FBS. The cells were allowed to attach and grow overnight. A
stock of 5 mM of compounds or 10 mM β-lapachone, and 5 mM dicoumarol were made
for the experiment. The 8 drug concentrations (0-3.2 µM) were prepared separately in
15 mL conical tubes with 7 mL of media each. The untreated control is DMSO. The
media was removed from each well and 500 µL of each drug concentration was added
to 6 wells (to produce sextuplet replicates for each concentration). After aliquoting the
drug-containing media, 40 µL of dicoumarol was added to each remaining 15 mL
conical tube (there remains 4 mL left of each drug concentration) to give a final
concentration of 50 µM dicoumarol. The media was removed from a 2nd 48-well plate
and 500 µL of the remaining drug + DIC media was aliquoted/well. The plates were
gently shaken to mix and placed in the incubator for 2 h. After 2 h, all media was
aspirated from the wells and 1 mL of fresh media was aliquoted into each well. The
plates were then left in the incubator for 7 days, or until there was 100% confluency for
the untreated control. Once the control was confluent, the media was discarded and 500
µL/well of 1X PBS was added to wash the wells. The PBS was discarded and 250 µL of
dH2O/well was added. The plates were then put in the -80 °C freezer overnight. The
next day, the plates were thawed completely and 500 µL of Hoechst staining buffer (50
µL of Hoechst 33258 in 50 mL of 1X TNE buffer) was added to each well. The plates
were incubated in the dark at RT for 2 h. After two hours, the plates were read on a
PerkinElmer Victor X3 plate reader and the readings were plotted as the treated/control
(T/C) ± SEM.
23

ACCEPTED MANUSCRIPT
4.5. Annexin V/PI detection
The Annexin V cytometry assay was used to detect cell population in viable,
early and late apoptosis stage as described by Cavalcanti and coworkers [65]. After
short exposure time (6 h) with compounds 21 and 22 at 5 µM, PC3 cells were stained
with fluorescein isothiocyanate (FITC) conjugated Annexin V (Guava Nexin kit, Guava
Technologies, Inc., Hayward, CA, USA) and PI (necrotic-cell indicator), and then they
were subjected to flow cytometry (Guava EasyCyte Mini). Cells undergoing early and
late apoptosis were detected by the emission of the fluorescence from only FITC and,
both FITC and PI, respectively. To determine whether ROS are involved with tested
compounds-induced cytotoxicity, cultures were pre-exposed (24 h) to 5 mM N-
acetylcysteine (NAC), a widely used thiol-containing antioxidant that is a precursor of
GSH which protects against oxidative stress-induced cell death. Also, cultures were co-
treated with dicoumarol (50 µM) in order to evaluate the role of NQO1 on compounds
bioactivation. A total of 10,000 events was evaluated per experiment (n = 3) and
cellular debris was omitted from analysis.
Supporting Information
1H and 13C NMR spectra for all the unpublished compounds and 2D NMR spectra
(COSY, HMBC and HSQC) for compounds 6, 10, 15, 16, 21, 22, 26, 29, 38 and 49.
This material is available free of charge via the Internet at http://pubs.acs.org.
Notes
The authors declare no competing financial interest.
Acknowledgements
This research was funded by grants from the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), numbers: 480719/2012-8, PVE 401193/2014-4,
449348/2014-8, 474797/2013-9 and 454171/2014-5. We would also like to thank
FAPEMIG (APQ-02478-14), FUNCAP, FAPESP, FAPESC, CAPES, INCT-catálise
and also the Physics Institute of USP (São Carlos) for kindly allowing the use of the
24

ACCEPTED MANUSCRIPT
KappaCCD diffractometer. Finally, these studies were also supported by NIH/NCI
grant# CA102792 to DAB.
References
[1] M. Ljungman, Chem. Rev. 109 (2009) 2929.
[2] G.M. Cragg, P.G. Grothaus, D.J. Newman, Chem. Rev. 109 (2009) 3012.
[3] (a) E.A. Hillard, F.C. de Abreu, D.C.M. Ferreira, G. Jaouen, M.O.F. Goulart, C.
Amatore, Chem. Commun. 23 (2008) 2612; (b) K.W. Wellington, RSC Adv. 5
(2015) 20309. (c) S. Bannwitz, D. Krane, S. Vortherms, T. Kalin, C. Lindenschmidt,
N.Z. Golpayegani, J. Tentrop, H. Prinz, K. Müller, J. Med. Chem. 57 (2014) 6226.
[4] J.J. Lu, W. Pan, Y.J. Hu, Y.T. Wang, PLoS One 29 (2012) 40262.
[5] K.S.T. Dias, C. Viegas Júnior, Curr. Neuropharmacol. 12 (2014) 239.
[6] (a) F. Prati, C. Bergamini, M.T. Molina, F. Falchi, A. Cavalli, M. Kaiser, R. Brun,
R. Fato, M.L. Bolognesi, J. Med. Chem. 58 (2015) 6422; (b) F. Prati, M. Bartolini, E.
Simoni, A. de Simone, A.V. Pinto, V. Andrisano, M.L. Bolognesi, Bioorg. Med.
Chem. Lett. 23 (2013) 6254; (c) F. Prati, E. Uliassi, M.L. Bolognesi, Med. Chem.
Commun. 5 (2014) 853.
[7] (a) M. Arnaudon, Comptes Rendus Hebdomadares Des Sianes ‘L‘Acord. Des
Science 46 (1858) 1152; (b) R.H. Thomson, Naturally Occurring Quinones,
Academic Press: London, New York, 1971; (c) S. Fiorito, F. Epifano, C. Bruyère, V.
Mathieu, R. Kiss, S. Genovese, Bioorg. Med. Chem. Lett. 24 (2014) 454; E.J. Park,
K.J. Min, T.J. Lee, Y.H. Yoo, Y.S. Kim, T.K. Kwon, Cell Death Dis. 5 (2014)
e1230.
[8] R. Docampo, F.S. Cruz, A. Boveris, R.P. Muniz, D.M. Esquivel, Biochem.
Pharmacol. 28 (1979) 723.
[9] K. Schaffner-Sabba, K.H. Schmidt-Ruppin, W. Wehrli, A.R. Schuerch, J.W.
Wasley, J. Med. Chem. 27 (1984) 990.
[10] (a) J.J Pink, S.M. Planchon, C. Magliarino, M.E. Varnes, D. Siegel, D. Boothman,
J. Biol. Chem. 275 (2000) 5416; (b) J.J. Pink, S. Wuerzberger-Davis, C. Tagliarino,
S.M. Planchon, X. Yang, C.J. Froelich, D.A. Boothman, Exp. Cell Res. 255 (2000)
144; (c) S.M. Planchon, J.J. Pink, C. Tagliarino, W.G. Bornmann, M.E. Varnes,
D.A. Boothman, Exp. Cell Res. 267 (2001) 95.
25

ACCEPTED MANUSCRIPT
[11] E.A. Bey, M.S. Bentle, K.E. Reinicke, Y. Dong, C.R. Yang, L. Girard, J. Minna,
W.G. Bornmann, J. Gao, D.A. Boothman, Proc. Natl. Acad. Sci. USA 104 (2007)
11832.
[12] (a) E.A. Bey, S.M. Wuerzberger-Davis, J.J. Pink, C.R. Yang, S. Araki, K.E.
Reinicke, M.S. Bentle, Y. Dong, E. Cataldo, T.L. Criswell, M.W. Wagner, L. Li, J.
Gao, D.A. Boothman, J. Cell Physiol. 209 (2006) 604; (b) C. Tagliarino, J.J. Pink,
G.R. Dubyak, A.L. Nieminen, D.A. Boothman, J. Biol. Chem. 276 (2001) 19150; (c)
C. Tagliarino, J.J. Pink, K.E. Reinicke, S.M. Simmers, S.M. Wuerzberger-Davis,
D.A. Boothman, Cancer Biol. Ther. 2 (2003) 141.
[13] C.J. Li, J. Cell Physiol. 209 (2006) 695.
[14] M.S. Bentle, K.E. Reinicke, Y. Dong, E.A. Bey, D.A. Boothman, Cancer Res. 67
(2007) 6936.
[15] G.K. Rekha, N.E. Sladek, Cancer Chemother. Pharmacol. 40 (1997) 215.
[16] M.S. Cunha-Filho, M. Landin, R. Martinez-Pacheco, B. Dacunha-Marinho, Acta
Crystallogr. C 62 (2006) 473.
[17] D.A. Boothman, M. Meyers, N. Fukunaga, S.W. Lee, Proc. Natl. Acad. Sci. USA
90 (1993) 7200.
[18] E.K. Choi, K. Terai, I.M. Ji, Y.H. Kook, K.H. Park, E.T. Oh, R.J. Griffin, B.U.
Lim, J.S. Kim, D.S. Lee, D.A. Boothman, M. Loren, C.W. Song, H.J. Park,
Neoplasia 9 (2007) 634.
[19] L.P. Hartner, L. Rosen, M. Hensley, D. Mendelson, A.P. Staddon, W. Chow, O.
Kovalyov, W. Ruka, K. Skladowski, A. Jagiello-Gruszfeld, M. Byakhov, J. Clin.
Oncol. 25 (2007) 20521.
[20] E. Blanco, E.A. Bey, C. Khemtong, S. Yang, J. Setti-Guthi, H. Chen, C.W.
Kessinger, K.A. Carnevale, W.G. Bornmann, D.A. Boothman, J. Gao, Cancer Res.
70 (2010) 3896.
[21] S. Ohayon, M. Refua, A. Hendler, A. Aharoni, A. Brik, Angew. Chem. Int. Ed. 54
(2015) 599.
[22] L. Cao, L.S. Li, C. Spruell, L. Xiao, G. Chakrabarti, E.A. Bey, K.E. Reinicke, M.C.
Srougi, Z. Moore, Y. Dong, P. Vo, W. Kabbani, C. Yang, X. Wang, F. Fattah, J.C.
Morales, E.A. Motea, W.G. Bornmann, J.S. Yordy, D.A. Boothman, Antioxid Redox
Sign. 21 (2014) 237.
26

ACCEPTED MANUSCRIPT
[23] X. Huang, Y. Dong, E.A. Bey, J.A. Kilgore, J.S. Bair, L.S. Li, M. Patel, E.I.
Parkinson, Y. Wang, N.S. Williams, J. Gao, P.J. Hergenrother, D.A. Boothman,
Cancer Res. 72 (2012) 3038.
[24] Z. Moore, G. Chakrabarti, X. Luo, A. Ali, Z. Hu, F.J. Fattah, R. Vemireddy, R.J.
DeBerardinis, R.A. Brekken, D.A. Boothman, Cell Death Dis. 6 (2015) 1599.
[25] (a) E.N. da Silva Júnior, M.C.B.V. de Souza, A.V. Pinto, M.C.F.R. Pinto, M.O.F.
Goulart, F.W.A. Barros, C. Pessoa, L.V. Costa-Lotufo, R.C. Montenegro, M.O. de
Moraes, V.F. Ferreira, Bioorg. Med. Chem. 15 (2007) 7035; (b) E.N. da Silva Júnior,
M.A.B.F. de Moura, A.V. Pinto, M.C.F.R. Pinto, M.C.B.V. de Souza, A.J. Araújo,
C. Pessoa, L.V. Costa-Lotufo, R.C. Montenegro, M.O. de Moraes, V.F. Ferreira,
M.O.F. Goulart, J. Braz. Chem. Soc. 20 (2009) 635.
[26] E.N. da Silva Júnior, C.F. de Deus, B.C. Cavalcanti, C. Pessoa, L.V. Costa-Lotufo,
R.C. Montenegro, M.O. de Moraes, M.C.F.R. Pinto, C.A. de Simone, V.F. Ferreira,
M.O.F. Goulart, C.K.Z. Andrade, A.V. Pinto, J. Med. Chem. 53 (2010) 504.
[27] S.L. de Castro, F.S. Emery, E.N. da Silva Júnior, Eur. J. Med. Chem. 69 (2013)
678.
[28] E.H.G. da Cruz, P.H.P.R. Carvalho, J.R. Corrêa, D.A.C. Silva, E.B.T. Diogo, J.D.
de Souza Filho, B.C. Cavalcanti, C. Pessoa, H.C.B. de Oliveira, B.C. Guido, D.A. da
Silva Filho, B.A.D. Neto, E.N. da Silva Júnior, New J. Chem. 38 (2014) 2569.
[29] B.D. Bala, S. Muthusaravanan, T.S. Choon, M.A. Ali, S. Perumal, Eur. J. Med.
Chem. 85 (2014) 737.
[30] C. Viegas Júnior, A.C. Danuello, V.S. Bolzani, E.J. Barreiro, C.A.M. Fraga, Curr.
Med. Chem. 14 (2007) 1829.
[31] E.N. da Silva Júnior, B.C. Cavalcanti, T.T. Guimarães, M.C.F.R. Pinto, I.O.
Cabral, C. Pessoa, L.V. Costa-Lotufo, M.O. de Moraes, C.K.Z. de Andrade, M.R.
dos Santos, C.A. de Simone, M.O.F. Goulart, A.V. Pinto, Eur. J. Med. Chem. 46
(2011) 399.
[32] (a) S.G. Agalave, S.R. Maujan, V.S. Pore, Chem. Asian J. 6 (2011) 2696; (b) A.
Massarotti, S. Aprile, V. Mercalli, E.D. Grosso, G. Grosa, G. Sorba, G. Tron,
ChemMedChem 9 (2014) 2497.
[33] E.H.G. da Cruz, C.M.B. Hussene, G.G. Dias, E.B.T. Diogo, I.M.M. de Melo, B.L.
Rodrigues, M.G. da Silva, W.O. Valença, C.A. Camara, R.N. de Oliveira, Y.G. de
27

ACCEPTED MANUSCRIPT
Paiva, M.O.F. Goulart, B.C. Cavalcanti, C. Pessoa, E.N. da Silva Júnior, Bioorg.
Med. Chem. 22 (2014) 1608.
[34] (a) C.W. Nogueira, G. Zeni, J.B.T. Rocha, Chem. Rev. 104 (2004) 6255; (b) A.L.
Braga, J. Rafique, The chemistry of organic selenium and tellurium compounds, ed.
Z. Rappoport, Wiley & Sons, Ltd, Chichester, 2014; Important chapters related to the
use of Se-Modified antioxidants: Synthesis of biologically relevant small molecules
containing selenium. Part A. Antioxidant compounds pg 989-1054; Synthesis of
biologically relevant small molecules containing selenium. Part B. Anti-infective and
anticancer compounds pg 1053-1118; Synthesis of biologically relevant small
molecules containing selenium. Part C. Miscellaneous biological activities 1119-
1174. (c) S. Shaaban, A. Negm, M. A. Sobh, L. A. Wessjohann, Eur. J. Med. Chem.
97 (2015) 190. (d) S. Shaaban, H.E. Gaffer, M. Alshahd, S.S. Elmorsy, Int. J. Res.
Dev. Pharm. L. Sci. 4 (2015) 1654. (e) S. Shaaban, F. Sasse, T. Burkholz, C. Jacob,
Bioorg. Med. Chem. 22 (2014) 3610.
[35] (a) E.E. Alberto, V. Nascimento, A.L. Braga, J. Braz. Chem. Soc. 21 (2010) 2032;
(b) C.T. Santi, C. Scalera, M. Piroddi, F. Galli, Curr. Chem. Bio. 7 (2013) 25; (c) M.
Iwaoka, K. Arai, Curr. Chem. Bio. 7 (2013) 2; (d) E.E. Alberto, D.W. Tondo, D.
Dambrowski, M.R. Detty, F. Nome, A.L. Braga, J. Am. Chem. Soc. 134 (2012) 138.
[36] (a) S. Shabaan, L.A. Ba, M. Abbas, T. Burkholz, A. Denkert, A. Gohr, L.A.
Wessjohann, F. Sasse, W. Weber, C. Jacob, Chem. Commun. 31 (2009) 4702; (b) M.
Doering, L.A. Ba, N. Lilienthal, C. Nicco, C. Scherer, M. Abbas, A.A.P. Zada, R.
Coriat, T. Burkholz, L. Wessjohann, M. Diederich, F. Batteux, M. Herling, C. Jacob,
J. Med. Chem. 53 (2010) 6954; (c) S. Mecklenburg, S. Shaaban, L.A. Ba, T.
Burkholz, T. Schneider, B. Diesel, A.K. Kiemer, A. Röseler, K. Becker, J. Reichrath,
A. Stark, W. Tilgen, M. Abbas, L.A. Wessjohann, F. Sasse, C. Jacob, Org. Biomol.
Chem. 7 (2009) 4753.
[37] S. Shaaban, R. Diestel, B. Hinkelmann, Y. Muthukumar, R.P. Verma, F. Sasse, C.
Jacob, Eur. J. Med. Chem. 58 (2012) 192.
[38] A.A. Vieira, I.R. Brandão, W.O. Valença, C.A. de Simone, B.C. Cavalcanti, C.
Pessoa, T.R. Carneiro, A.L. Braga, E.N. da Silva Júnior, Eur. J. Med. Chem. 101
(2015) 254.
28