1,2,3-Triazole-, arylamino- and thio-substituted 1,4-naphthoquinones: Potent antitumor activity, electrochemical aspects, and bioisosteric replacement of C-ring-modified lapachones

Article abstract of DOI:10.1016/j.bmc.2014.01.033

1,2,3-Triazole-, arylamino- and thio-substituted naphthoquinones (24, 8, and 2 representatives, respectively) were synthesized in moderate yields and evaluated against several human cancer cell lines (blood, ovarian, breast, central nervous system, colon, and prostate cancers and melanoma), showing, for some of them, IC₅₀ values below 2 μM. The cytotoxic potential of the tested naphthoquinones was also assayed on non-tumor cells such as human peripheral blood mononucluear cells (PBMC) and two murine fibroblast lines (L929 and V79 cells). α-Lapachone- and nor-α-lapachone-based 1,2,3-triazoles and arylamino-substituted naphthoquinones showed potent cytotoxicity against different cancer cell lines. The compounds may represent promising new lead derivatives for anticancer drug development. The electrochemical properties of selected compounds were evaluated in an attempt to correlate them with antitumor activity.

Full text of DOI:10.1016/j.bmc.2014.01.033

Bioorganic & Medicinal Chemistry 22 (2014) 1608–1619
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2,3-Triazole-, arylamino- and thio-substituted
1,4-naphthoquinones: Potent antitumor activity, electrochemical
aspects, and bioisosteric replacement of C-ring-modified lapachones
Eduardo H. G. da Cruz a, Caio M. B. Hussene a, Gleiston G. Dias a, Emilay B. T. Diogo a,
Isadora M. M. de Melo a, Bernardo L. Rodrigues a, Mauro G. da Silva b, Wagner O. Valença c,
Celso A. Camara c, Ronaldo N. de Oliveira c, Yen G. de Paiva d, Marilia O. F. Goulart d
Bruno C. Cavalcanti e, Claudia Pessoa e, Eufrânio N. da Silva Júnior a,
⇑
a Instituto de Ciências Exatas, Departamento de Química, UFMG, 31270-901 Belo Horizonte, MG, Brazil
b Departamento de Química Fundamental, UFPE, 50670-901 Recife, PE, Brazil
c Departamento de Ciências Moleculares, UFRPE, 52171-900 Recife, PE, Brazil
d Instituto de Química e Biotecnologia, UFAL, Tabuleiro do Martins, 57072-970 Maceió, AL, Brazil
e Laboratório Nacional de Oncologia Experimental, Departamento de Fisiologia e Farmacologia, UFC, 60430-270 Fortaleza, CE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
1,2,3-Triazole-, arylamino- and thio-substituted naphthoquinones (24, 8, and 2 representatives, respec-
tively) were synthesized in moderate yields and evaluated against several human cancer cell lines (blood,
ovarian, breast, central nervous system, colon, and prostate cancers and melanoma), showing, for some of
Received 16 December 2013
Revised 13 January 2014
Accepted 20 January 2014
Available online 31 January 2014
them, IC50 values below 2
non-tumor cells such as human peripheral blood mononucluear cells (PBMC) and two murine fibroblast
lines (L929 and V79 cells). -Lapachone- and nor- -lapachone-based 1,2,3-triazoles and arylamino-
lM. The cytotoxic potential of the tested naphthoquinones was also assayed on
a
a
Keywords:
1,4-Naphthoquinone
Lapachol
1,2,3-Triazoles
Click chemistry
Cancer
substituted naphthoquinones showed potent cytotoxicity against different cancer cell lines. The com-
pounds may represent promising new lead derivatives for anticancer drug development. The electro-
chemical properties of selected compounds were evaluated in an attempt to correlate them with
antitumor activity.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Compounds that are able to modulate the redox balance in can-
cer cells are potential candidates for the development of anticancer
Reactive oxygen species (ROS) are important participants in
regulating normal cellular processes. A deregulated redox balance
may contribute to the development of several human diseases,
including cancers, as recently reported by Nogueira and Hay.1 Sev-
eral types of cancer cells exhibit disturbed intracellular redox bal-
ance, differentiating them from their non-cancerous counterparts.2
ROS may act directly with DNA, lipids, and proteins to induce cell
damage.3 The alkylation of crucial proteins and nucleic acids can
also cause cell damage.4 Compared to non-cancerous cells, the
reactive oxygen species (ROS) levels are considerably closer to
the critical redox threshold at which cell death is induced.2 These
biochemical differences between healthy and malignant tissues
are significant and may be exploited in the design of selective
drugs.2
drugs.5–7 In general, these compounds catalyze the oxidation of re-
dox-sensitive, thiol-containing proteins and enzymes and/or sig-
nificantly increase intracellular ROS levels. These features relate
to subsequent processes that lead to apoptosis.7 Quinones belong
to this class of compounds, as they are capable of increasing intra-
cellular ROS levels over a critical threshold and therefore may in-
duce apoptosis in cancer cells.
Electrochemistry is the standard method for studying redox
systems. Electrochemical techniques applied to biology are contin-
uously described in the literature and provide both kinetic and
thermodynamic information.8 Electrochemical methods are useful
in the characterization and ultimately the design of redox-modu-
lating natural products and drugs, including potential antioxidants
and anticancer agents.9 Among these techniques, cyclic voltamme-
try can rapidly evaluate the redox properties of some of those com-
pounds, including quinones. The usual parameters normally
obtained and employed, especially in cyclic voltammetry, are the
⇑
Corresponding author. Tel.: +55 31 34095720; fax: +55 31 34095700.
E-mail address: eufranio@ufmg.br (E.N. da Silva Júnior).
http://dx.doi.org/10.1016/j.bmc.2014.01.033
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
1609
potentials of the oxidation (Epa) and reduction (Epc) peaks or Ere-
dox (Epc + Epa)/2 (for reversible systems) or Epc ꢀ Epc/2 (for irre-
versible ones). The potential Eredox or similar parameters give a
quantitative measure of the ease of reduction of an oxidant or elec-
tron acceptor, A, since the more positive the value of the potential
of the couple E (A/Aꢀ), the more powerful the oxidant. Similarly, the
more negative the value of E (Aꢀ/A2ꢀ), the more powerful the reduc-
tant.9 The Epc or the Eredox of the first peak or system, respec-
tively, are the ones to be used in correlation of electrochemistry
and biological activities.8a With this approach these data may even
be useful in the rational design of such compounds as part of a spe-
cial chemical structure–electrochemical potential–biological activ-
ity relationship. Nonetheless, one must bear in mind that
electrochemical parameters such Epa and/or Epc are only one mea-
sure and other data, like abundance, distribution, accessibility are
also decisive factors in vitro or cell environments.8b,10
The second group, 1,4-naphthoquinone-based 1,2,3-triazoles
7–19, was previously described (Fig. 2).21 Compounds 20–24 are
herein described for the first time.
para-Naphthoquinones 7–19 were synthesized by clicking a
series of N-phthalimidoalkylazides with the respective naphtho-
quinones. Compounds 18 and 19 were prepared by the same meth-
odology using 1-azido-3-nitrobenzene (Fig. 2).
Unpublished compounds 20–24 were obtained by reaction of
azide-substituted carbohydrate or naphthoquinone and the al-
kyne-substituted quinone. Compounds 20–24 were obtained in
high yields using copper iodide(I) in acetonitrile as the reaction
medium (Scheme 1).
The unpublished arylamino-substituted
a-lapachones 28–35,
were prepared from lapachol (25), as shown in Scheme 2. The
first step consisted of the simple cyclization of lapachol (25) to
a-lapachone (26) using HCl/HOAc. Compound 27 reacted with
The mechanism of action of quinonoid anti-tumor agents has
been thoroughly investigated.10 Under aerobic conditions, that is,
in organs with sufficient blood supply, a one-electron reduction
predominates, resulting in free-radical intermediates that undergo
back oxidation in the presence of oxygen, releasing reactive oxygen
species (ROS).8,11 This may cause damage to the DNA of the tumor
cell, but normal cells are also negatively affected. A two-electron
reduction of the quinone function followed by its inactivation
through subsequent glucuronidation and/or sulfation or by the
conversion of the hydroquinone into an alkylating intermediate
is an alternative pathway.11 This mechanism is believed to pre-
dominate under anaerobic conditions.12
various anilines containing electron donating and electron with-
drawing groups to prepare 28–35. The compounds were obtained
in high yields, and their structures were determined by IR, 1H
and 13C NMR and by comparing their spectroscopic data with those
of already reported similar compounds.22 Electrospray ionization
mass spectra and combustion analysis data were also obtained
for unpublished compounds.
The last class of compounds was obtained using similar meth-
odology. The respective bromo derivatives were prepared and were
reacted with thiophenol as shown in Scheme 3. Compounds 36 and
39 were isolated in moderate yields as yellow solids.
Recently, our research group has described the synthesis of re-
dox modulating quinone compounds. To access substances with
different redox behaviors, we have appended groups such as aryl-
amines,13 1,2,3-triazoles,14 azides,14 among others, onto the qui-
none moiety.15
2.2. Electrochemistry
Several studies have investigated and reported on the electro-
chemical behavior of quinones. Some correlation has been ob-
served between the electrochemical parameters (Epc1: potential
of the first reduction peak) and biological activities.23
The single electron reduction of quinones by enzymes such as
NADH-cytochrome P450 oxidoreductase initiates redox cycling
and oxidative stress and then the relative one-electron reduction
potentials of quinones control the position of the equilibrium
defining the electron transfer between the semiquinone and oxy-
gen.8b Also, the first reduction step of quinones can be correlated
with the unoccupied molecular orbital of lower energy (LUMO).23b
In the context of cancer, there is a considerable number of po-
sitive correlations.24
Through our efforts to design molecular scaffolds using hybrid-
ization protocols, we report herein the extension of our approach,
which includes modification of 1,4-naphthoquinones with tria-
zoles (1–19) and sugars (20–21) to enhance the bioavailability of
the compounds, the insertion of a second quinone group (22–24)
to potentially increase ROS release and through the use of phenyl-
thiolated compounds to connect chalcogens16 to the main struc-
ture. Additionally, we have synthesized compounds 28–35,
isomers of substituted arylamino-nor-b-lapachones,13 which are
widely recognized as highly active cytotoxic compounds, in order
to compare the effect of the para-quinone moiety on their activity
and electrochemical behavior. Among the thirty-four compounds
assayed, sixteen are new and are fully characterized. Eleven of
the cell lines used are cancerous, and three are non-cancerous.
The quinone/semiquinone/hydroquinone (Q/SQꢀꢀ/H2Q) triad is
an important component of many redox systems in biology. It is
a vital link for the transfer of electrons through cells and tissues
(Eqs. 1–3).25
2. Results and discussion
2.1. Chemistry
2-Bromo-1,4-naphthoquinone and 2-chloro-1,4-naphthoqui-
none were acquired from Sigma-Aldrich (St. Louis, MO, USA).
Lapachol (25) (2-hydroxy-3-(30-methyl-20-butenyl)-1,4-naphtho-
quinone) was extracted from the heartwood of Tabebuia sp.
(Tecoma) and purified by a series of recrystallizations.17 Nor-lapachol
(38) (2-hydroxy-3-(20-methyl-propenyl)-1,4-naphthoquinone)
was obtained as a crystalline orange solid by Hooker oxidation of
quinone 25.18
In typical measurements, cyclic voltammograms (CV) were re-
corded in aprotic medium (DMF + TBAP, 0.1 mol Lꢀ1), at a scan rate
of 100 mV sꢀ1. This enabled determination of the electrochemical
reduction behavior of each compound. Cathodic and anodic peaks
for each compound are listed in Table 1.
It was not feasible to study all of the quinones in the present
work. Representative compounds 20 and 21 (Scheme 1), 28 and
34 (Scheme 2) and 39 (Scheme 3) were selected to encompass each
of the structural classes. The present electrochemical study com-
plements recently published results.21 In Table 1, the previously re-
ported electrochemical data of compounds 2, 3 and 10 are
presented in a more complete form to allow comparison and ratio-
nalization of the electrochemical behavior. It is out of the scope of
As previously described, the first group of compounds was pre-
pared using click chemistry reaction19 with the respective azide
derivatives.20 4-Azido-
a-lapachone and 3-azido-nor-a-lapachone
were synthesized and used to prepare the 1,2,3-triazole analogues
1–6 in moderate yields, as shown in Figure 1.

1610
E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
O
O
O
O
N
O
1
- R = (CH2)4CH3
4 - R =
5 - R =
6 - R =
(CH2)5CH3
2
- R =
Ph
Cy
OH
3 - R = Cy
N
O
N
N
N
N
R
R
Figure 1. Nor-a-lapachone and a-lapachone-based 1,2,3-triazoles 1–6.
7
- R = H, n = 1
8 - R = H, n = 2
9 - R = H, n = 3
O
10
- R = H, n = 4
11 - R = Cl, n = 1
12 - R = Cl, n = 2
N
N
N
N
N
n
O
O
N
O
O
H
N
H
N
O
N
13
- R = Cl, n = 3
14 - R = Cl, n = 4
15 - R = Br, n = 1
NO2
R
R
18
19
- R = H
- R = Br
16
- R = Br, n = 2
17 - R = Br, n = 3
Figure 2. 1,4-Naphthoquinone-based 1,2,3-triazoles 7–19.
O
Cl
Cl
O
H2N
CH3CN
OAc
OAc
O
N
N
O
OAc
O
O
O
O
H
N
H
N
R'
R
AcO
N3
OAc
OAc
N
H2N
AcO
OAc
R
R
O
CH3CN
O
CuI, CH3CN
20
- R = Br
R = Br, Cl or H
R' = Br
21 - R = H
R = Br or H
O
N3
O
N
N
O
H
22 - R = H
N
O
N
23
24
- R = Cl
- R = Br
CuI, CH3CN
O
R
O
Scheme 1. 1,4-Naphthoquinone-based 1,2,3-triazoles 20–24.
the present paper, the rationalization of the electrochemical
behavior.
As has been noted, the compounds described here can be di-
vided among four structural classes: dihydropyran-para-naphtho-
quinones (2 and 3), para-naphthoquinone derivatives (10, 20 and
The major electrochemical parameters for each compound are
listed in Table 1. Epc1 values are most commonly used to compare
biological activities. The ease of reduction is as follows:
39 > 2 ꢁ 3 > 20 > 28 > 34 > 21 > 10
Compounds 28 and 34 display electrochemical behavior similar
to m-nitroaniline-substituted nor-b-lapachone.26 As observed in
Figure 3, four and three cathodic peaks are displayed for 28 (para
derivative) and 34 (meta derivative), respectively. The first peak of
the CV of 28 has a cathodic peak potential (Epc1) of ꢀ0.674 V, while
for 34, the value is ꢀ0.684 V. These cathodic peaks display two cor-
responding backward peaks, revealing quasi-reversible behavior.
For 34, the first pair of peaks observed is consistent with an ini-
tial one-electron reduction of the para-quinone to form the semi-
quinone radical according to Eq. 4:
21), nitroaniline-substituted
a-lapachones (28 and 34) and a thio-
lated nor- -lapachone (39). Figure 3 shows the cyclic voltammo-
a
grams of the compounds 20, 21, 28, 34 and 39.
The first two quinones (20 and 21), similar to compounds 2 and
3,21 exhibited quasi-reversible reduction behavior (Fig. 3), which is
typical of simple and well-behaved quinones (Eqs. 1 and 2). The CV
profiles of the other four compounds (10,21 28, 34 and 39) are
much more complex due to the presence of other electroactive
groups.

E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
1611
O
O
O
OH
HCl/HOAc
O
25
O
26
NBS
CCl4
In.
28 - R1 = H; R2 = NO2
O
O
29
- R1 = H; R2 = Br
H2N
R1
R2
O
30 - R1 = H; R2 = I
31 - R1 = H; R2 = Cl
O
32
- R1 = H; R2 = OMe
33 - R1 = F; R2 = H
CH2Cl2
O
HN
R1
R2
O
27
Br
34 - R1 = NO2; R2 = H
35
- R1 = Br; R2 = H
Scheme 2. Arylamino-substituted
a-lapachones 28–35.
O
O
O
O
NBS
1.
O
OH
HCl
CH3COOH
O
CCl4
benzoyl per.
2.
SH
CH2Cl2
O
O
S
26
25
36
Hooker
oxidation
NBS
CCl4
benzoyl per.
O
O
O
1.
HCl
CH3COOH
OH
O
O
2.
SH
CH2Cl2
S
O
O
O
37
39
38
Scheme 3. Thio derivatives 36 and 39.
Table 1
Major electrochemical parameters of the quinones (c = 1 ꢃ 10ꢀ3 mol Lꢀ1), in DMF/TBAP, 0.1 mol Lꢀ1, versus Ag/AgCl, Clꢀ1
,
m
= 100 mV sꢀ1
Epa2 (V)
Compounds
Epc1 (V)
Epc2 (V)
Epc3 (V)
Epc4 (V)
Epa1 (V)
Epa3 (V)
Epa4 (V)
2a
ꢀ0.632
ꢀ0.630
ꢀ0.807
ꢀ0.642
ꢀ0.797
ꢀ0.674
ꢀ0.684
ꢀ0.556
ꢀ1.116
ꢀ1.147
ꢀ1.348
ꢀ1.281
ꢀ1.378
ꢀ1.177
ꢀ1.169
ꢀ1.132
—
—
—
—
ꢀ0.940
ꢀ0.967
ꢀ1.151
ꢀ1.098
—
ꢀ1.333
ꢀ1.050
ꢀ1.385
ꢀ0.540
ꢀ0.547
ꢀ0.779
ꢀ0.544
ꢀ0.709
ꢀ1.045
ꢀ0.811
ꢀ1.139
—
—
—
—
—
—
—
—
—
—
—
3a
10a
20
21
28
34
39
ꢀ2.085
ꢀ2.471
—
—
—
—
—
ꢀ1.392
ꢀ1.842
ꢀ1.500
ꢀ2.114
ꢀ0.596
ꢀ0.607
ꢀ0.479
—
—
ꢀ0.030
Epc1: potential of the first cathodic peak; Epc2: potential of the second cathodic peak; etc; Epa1: potential of the first anodic peak; etc.
a
Ref. 21.
½Qꢂ-PhNO2 þ eꢀ¡½Qꢀꢀꢂ-PhNO2:
ð4Þ
For compound 28, the two waves are separated
(Epc2 = ꢀ1.177 V and EpIIIc = ꢀ1.392 V) and until now, it has not
been possible to determine their nature. Studies on this subject
are under way.
For compound 39, the most readily reduced in the series and, to
our knowledge, a previously unreported compound, a different
electrochemical profile is observed. The first two redox systems
correspond to Eqs. 1 and 2, and the third one, which has an irre-
versible nature, suggests the occurrence of a dissociative electron
transfer with cleavage of the C–S bond. Studies to elucidate the
electrochemical behavior are ongoing.
According to a previous article reporting the reduction of a sim-
ilar compound (nor-b-lapachone derivative) on mercury,27 the sec-
ond cathodic pair of peaks, twice as intense as the first peak and
appearing at ꢀ1.169 V, should correspond to the subsequent one-
electron reduction of the electrogenerated semiquinone and to
the formation of the nitroanion radical (Eq. 5), leading to a dianion
quinone-nitro radical, which is further reduced, in the third peak,
in a conventional nitroaromatic reduction:10
½Qꢀꢀꢂ-PhNO2 þ eꢀ¡½Q2ꢀꢂ-PhNOꢀ2ꢀ
:
ð5Þ

1612
E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
15
10
5
15
Epa2
Epa2
10
5
0
scan 1
scan 2
Epa1
Epa1
0
-5
-5
-10
-15
-10
Epc2
Epc2
Epc1
Epc1
-15
-1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
-1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
(a)
(b)
12
6
12
6
Epa2
Epa2
scan 1
scan 2
Epa1
Epa1
0
0
21
-6
-12
-6
-12
Epc1
Epc2
Epc1
Epc2
-1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
-1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
(c)
(d)
Epa3
Epa3
Epa2
10
0
10
0
Scan 1
Epa2
Scan 2
Epa1
Epa1
-10
-20
-30
-40
-50
-10
-20
-30
-40
-50
Epc1
Epc2
Epc1
Epc2
Epc3
Epc3
Epc4
-2.0
Epc4
-2.0
-3.0
-2.5
-1.5
-1.0
-0.5
0.0
-3.0
-2.5
-1.5
-1.0
-0.5
0.0
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
(e)
(f)
Figure 3. Cyclic voltammograms of naphthoquinones 20, 21, 28, 34, and 39 (c = 1 ꢃ 10ꢀ3 mol Lꢀ1). DMF/TBAP (0.1 mol Lꢀ1), glassy carbon electrode, versus Ag/AgCl, Clꢀ1
,
cathodic direction,
v .
= 100 mV sꢀ1

E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
1613
Epa3
Epa3
15
0
15
0
Epa
Epa1 Epa2
1 Epa2
Scan 1
Scan 2
-15
-30
-45
-15
-30
-45
Epc1
Epc1
Epc2
Epc2
Epc3
Epc3
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
E/ V vs. Ag| AgCl|Cl- (0.1 mol L-1)
E/V vs. Ag|AgCl|Cl- (0.1 mol L-1)
(h)
(g)
15
0
15
0
Scan 1
Scan 2
Epa3
Epa3
Epa4
Epa4
Epa2
Epc2
Epa2
Epa1
Epa1
-15
-30
-15
-30
Epc1
-0.5
Epc1
Epc2
Epc3
Epc3
-1.5
-2.5
-2.0
-1.0
-0.5
0.0
0.5
-2.5
-2.0
-1.5
-1.0
0.0
0.5
E/V vs. Ag|AgCl|Cl- (0.1 mol L-1)
E/V vs. Ag|AgCl|Cl- (0.1 mol L-1)
(i)
(j)
Fig. 3 (continued)
Table 2
Crystal data and structure refinement for compound 36
Empirical formula
Formula weight
C21H17O3S
349.41
Temperature
Wavelength
150(2) K
0.71073 Å
ꢀ
Crystal system, space group
Unit cell dimensions
Triclinic, P1
a = 9.1351(6) Å
b = 9.3814(8) Å
c = 10.4100(8) Å
816.66(11) Å3
2, 1.421 mg/m3
0.216 mmꢀ1
366
a
= 108.220(7)°
b = 100.266(6)°
= 97.646(6)°
c
Volume
Z, calculated density
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Limiting indices
Reflections collected
Independent reflections
Completeness to theta = 26.32°
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
0.16 ꢃ 0.10 ꢃ 0.07 mm3
2.12–29.55°
ꢀ11 6 h 6 12, ꢀ7 6 k 6 12, ꢀ13 6 l 6 14
6145
3797 [R(int) = 0.0274]
100%
Analytical
Full-matrix least-squares on F2
3797/0/228
1.037
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.0424, wR2 = 0.1036
R1 = 0.0553, wR2 = 0.1128
0.350 and ꢀ0.379 e Åꢀ3
Figure 4. ORTEP-3 projection of compound 36, showing atom labeling and
displacement ellipsoids drawn at the 40% probability level.
2.3. X-ray analysis
with one molecule in the asymmetric unit. The bond lengths and
angles are in good agreement with expected values, based on each
atomic type. Two planes may be defined through the carbon skel-
eton of the molecule. Plane 1 is formed by the quinonoid ring
The Ortep-3 diagram of 36 is shown in Figure 4. The crystallo-
graphic data collection and refinement parameters are shown in
Table 2. Compound 36 crystallizes in the triclinic space group P1,
ꢀ

Table 3
Cytotoxic activity expressed by IC50 lg/mL [lM] (95% CI) of compounds 1-24, 28-36 and 39 in cancer and normal cell lines after 72 h exposure, obtained by nonlinear regression for all cell lines from three independent experiments
Compounds
IC50
PC3
l
g/mL [
l
M] (CI 95%)
HL-60
OVCAR-8
MDA-MB435 SF295
HCT-8
HCT-116
DU-145
MCF-7
MX1
HS578t
PBMC
L929
V79
1
0.52 [1.37]
(0.39–0.61)
0.81 [2.12]
(0.69–0.96)
2.53 [6.67]
(2.37–2.70)
1.82 [4.77]
(1.59–2.08)
1.05 [2.77]
(0.94–1.12)
0.80 [2.10]
(0.61–0.93)
1.05 [2.77]
1.69 [4.45]
(1.46–1.73)
1.04 [2.73]
(0.83–1.21)
1.87 [4.93]
1.39 [3.66]
0.94 [2.48]
(0.73–1.16)
1.28 [3.36]
(1.13–1.47)
1.20 [3.16]
(1.08–1.37)
1.19 [3.12]
(1.10–1.28)
0.91 [2.40]
(0.82–0.98)
1.30 [3.41]
(1.18–1.46)
>5
1.17 [3.08]
(1.02–1.30)
1.16 [3.04]
(0.92–1.27)
2.95 [7.77]
(2.66–3.20)
1.82 [4.77]
(1.67–2.19)
1.89 [4.98]
(1.53–2.07)
1.47 [3.85]
(1.13–1.70)
2.12 [5.59]
(1.74–2.41)
1.55 [4.06]
(1.32–1.75)
(0.83–1.25)
1.61 [4.22]
(1.43–1.80)
(1.51–2.06) (1.22–1.48)
1.22 [3.20] 1.17 [3.07]
(1.05–1.42) (1.08–1.24)
2
3
4.08 [10.42] 5.91 [15.10] 4.17 [10.65]
4.42 [11.29] 4.81 [12.29] 4.1 [10.47]
(4.19–4.67) (4.79–4.85) (3.82–4.30) (4.04–4.77)
4.24 [11.17] 2.02 [5.32]
(3.94–4.51)
1.28 [3.44]
(0.93–1.45)
1.2 [3.18]
(1.08–1.37)
>5
4.38 [11.19] 4.57 [11.67] >5
4.25 [10.86] >5
4.89 [12.49] 4.70 [12.00]
(3.79–4.23)
0.72 [1.90]
(0.43–1.02)
1.61 [4.34]
(1.45–1.84)
1.06 [2.70]
(0.85–1.19)
>5
(5.62–6.16)
2.03 [5.35]
(1.91–2.25)
2.68 [7.22]
(2.46–2.81)
2.03 [5.38]
(1.95–2.17)
>5
(3.78–4.39)
1.51 [3.98]
(1.40–1.57)
1.84 [4.95]
(1.69–1.93)
1.04 [2.76]
(0.88–1.17)
>5
(4.29–4.82)
[>12]
[>12]
(4.11–4.48)
3.14 [8.28]
(2.93–3.27)
2.14 [5.76]
(2.09–2.25)
1.87 [4.95]
(1.56–1.91)
>5
[>12]
(4.71–4.94)
3.15 [8.30]
(2.91–3.37)
3.12 [8.40]
(2.71–3.38)
2.50 [6.62]
(2.37–2.71)
>5
(4.61–4.87)
2.74 [7.22]
(2.59–2.93)
2.83 [7.62]
(2.57–3.04)
2.31 [6.12]
(2.16–2.54)
>5
4
1.85 [4.88]
3.86 [10.17] 3.75 [9.88]
3.57 [9.41]
(3.47–3.61)
2.58 [6.95]
(2.33–2.76)
2.18 [5.78]
(2.06–2.31)
>5
2.78 [7.33]
(2.55–2.93)
2.20 [5.92]
(2.07–2.41)
1.59 [4.21]
(1.38–1.74)
>5
3.42 [9.01]
(3.20–3.58)
2.98 [8.02]
(2.74–3.14)
2.78 [7.36]
(2.59–2.94)
>5
(1.82–2.17)
3.37 [9.07]
(3.20–3.48)
2.53 [6.70]
(2.28–2.82)
>5
(1.57–2.17) (3.67–3.95)
3.07 [8.27] 2.16 [5.82]
(2.61–3.47) (1.98–2.31)
2.08 [5.51] 1.62 [4.29]
(1.81–2.20) (1.48–1.85)
(3.62–3.81)
1.74 [4.68]
(1.52–1.90)
2.07 [5.48]
(1.83–2.29)
>5
5
6
7
>5
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
[>12]
>5
8
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
9
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
[>11]
>5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
28
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>13]
>5
[>11]
2.43 [3.66]
(2.25–2.78)
>5
[>8]
2.14 [5.21]
(1.91–2.43)
[>10]
>5
[>11]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>10]
>5
[>9]
>5
[>9]
>5
[>9]
>5
[>13]
>5
[>11]
0.79 [1.19]
(0.43–1.36)
>5
[>13]
>5
[>11]
1.82 [2.80]
(1.50–2.27)
>5
[>13]
>5
[>11]
1.77 [2.67]
(1.42–2.01)
>5
[>13]
>5
[>11]
1.88 [2.83]
(1.40–2.54)
>5
[>13]
>5
[>11]
1.46 [2.20]
(1.14–1.82)
>5
[>13]
>5
[>11]
2.91 [4.39]
(2.67–3.49) [>7]
>5
[>13]
>5
[>11]
>5
[>13]
>5
[>11]
2.7 [4.07]
(2.28–3.10)
>5
[>13]
>5
[>11]
1.93 [2.91]
(1.24–2.42)
>5
[>13]
>5
[>11]
2.38 [3.59]
(2.07–2.63)
>5
[>13]
>5
[>11]
2.44 [3.68]
(2.16–2.92)
>5
[>13]
>5
[>11]
3.16 [4.76]
(2.90–3.27)
>5
[>8]
2.78 [6.77]
(2.41–3.07)
4.52 [10.16] 3.04 [6.83]
(4.23–4.89)
>5
[>10]
0.76 [2.01]
(0.60–0.97)
[>13]
>5
[>11]
2.38 [3.59]
(1.81–2.67)
>5
>5
[>8]
2.06 [5.02]
(1.58–2.37)
2.41 [5.42]
[>8]
[>8]
[>8]
[>8]
[>8]
[>8]
>5
[>8]
[>8]
[>8]
[>8]
[>8]
0.33 [0.80]
(0.22–0.50)
1.52 [3.42]
(1.15–1.82)
>5
[>10]
1.61 [4.26]
(1.34–1.82)
1.18 [2.86]
(0.97–1.42)
2.7 [6.02]
(2.40–3.03)
>5
[>10]
2.52 [6.66]
(2.18–2.80)
1.27 [3.09]
(0.88–1.52)
1.48 [3.33]
(1.12–1.93)
>5
[>10]
2.05 [5.42]
(1.83–2.17)
1.57 [3.82]
(1.07–2.29)
2.47 [5.55]
(2.14–2.86)
>5
[>10]
2.25 [5.95]
(2.01–2.48)
1.11 [2.70]
(0.74–1.35)
3.15 [7.08]
(2.75–3.62)
>5
[>10]
1.88 [4.97]
(1.52–2.16)
2.19 [5.34]
(1.80–2.34)
2.19 [4.92]
(1.90–2.34)
>5
[>10]
2.32 [6.13]
(1.88–2.85) (2.34–3.29)
1.51 [3.67]
(1.17–1.93)
3.45 [7.76]
(3.28–3.81)
>5
1.52 [3.70]
(1.10–1.83)
1.82 [4.02]
(1.44–2.16)
>5
[>10]
2.09 [5.52]
(1.76–2.45)
2.33 [5.68]
(2.04–2.56)
2.08 [4.68]
(1.73–2.31)
>5
[>10]
2.25 [5.95]
(1.53–2.61)
2.55 [6.21]
(2.24–2.79)
3.16 [7.10]
(2.77–3.45)
>5
[>10]
0.61 [1.61]
(0.47–0.73)
[>12]
2.96 [6.65]
(2.49–3.31) (2.07–2.84)
>5
[>10]
(2.72–3.41)
>5
[>10]
0.52 [1.37]
(0.38–0.61)
>5
[>10]
2.19 [5.79]
[>10]
2.71 [7.16]
2.14 [5.66]
(1.84–2.32) (1.87–2.64)

E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
1615
(atoms C4–C13), and C6 is the most distant atom from the mean
plane of the ring (d(C6-plane equal to 0.524(13) Å). Notably, the
oxygen atom O1 [d(O1-plane) = 0.0333(19) Å] lies in the plane as
well. Atoms O2 [d(O2-plane) = 0.1932(19) Å] and O3 [d(O3-
plane) = 0,1631(21) Å] are close to plane 1. Plane 2 is formed by
the phenyl ring (C14–C19) and includes the sulfur atom, with
d(S1-plane) equal to 0.0866(23) Å. Cremer and Pople ring pucker-
ing parameters28 (Q = 0.4531(19) Å, h = 52.7(2)°,
U = 97.6(3)°) sug-
gest that the pyran ring (C1, C2, C3, O1, C4 and C5) is in
approximately a half-chair conformation.
2.4. Biological activity
All the substances described (Figs. 1 and 2 and Schemes 1–3),
were evaluated in vitro using the MTT assay against eleven cancer
cell lines: HL-60 (human promyelocytic leukemia), OVCAR-8 (ovar-
ian adenocarcinoma), MDA-MB435 (human melanoma), SF295 (hu-
man glioblastoma), HCT-8 (Human colon carcinoma), HCT-116
(human colon carcinoma), PC3 (human prostate carcinoma), DU-
145 (human prostate carcinoma), MCF-7 (human breast adenocar-
cinoma), MX1 (human breast adenocarcinoma) and HS578t (hu-
man breast adenocarcinoma). Doxorubicin was used as the
positive control (Table 3).29 The selectivity of the compounds to-
ward a normal proliferating cell line was investigated using the
MTT assay with human peripheral blood mononucluear cells
(PBMC) after 72 h of drug exposure. To test with normal cells, two
murine fibroblasts cell lines, L929 and V79, were used. As previ-
ously described,30 the compounds were classified according to their
activity as highly active (IC50 <2
(2 M < IC50 < 10 M), or inactive (IC50 >10
In previous studies, we demonstrated that appending a 1,2,3-
lM), moderately active
l
l
lM).
triazole group to nor-b-lapachone can intensify its antitumor31
and trypanocidal32 activities. Molecular hybridization33 was used
as a strategy to obtain new molecules by connecting a quinoidal
moiety and a 1,2,3-triazole ring via click19 chemistry reactions.
Herein, the antitumor activities of 1,4-naphthoquinone-based
1,2,3-triazoles were evaluated and ten compounds were observed
to possess moderate to high activity.
For compounds 7–19, 21 and 24, our approach was not success-
ful and these compounds did not exhibit activity against any of the
cancer cell lines evaluated.
For the quinone-based 1,2,3-triazoles, compounds 1–6 were
considered moderately active with IC values in the range of
2.12–10.0
lM. However, high activity was observed against HL-
60 with compounds 1 (IC50 = 1.37
l
M) and 4 (IC50 = 1.90 M).
l
For the 1,2,3-triazoles, the most significant activity was ob-
served with compounds 1, 4, 21 and 22 against HL-60. They were
highly active, with IC50 values of 1.37, 1.90, 1.19 and 0.80
respectively.
lM,
Recently, our research group has described arylamino-substi-
tuted nor-b-lapachones with significant activity against cancer cell
lines.34 We observed that the presence of a methoxy group in the
arylamino ring intensifies the antitumor activity. Herein, the eval-
uation of the arylamino substituted
a-lapachones 28–35 was de-
scribed. The compounds showed moderate to high activity
against all cancer cell lines evaluated. Interestingly, compound
32, with a methoxy group in the para-position, was found to be
the most active, exhibiting IC50 values in the range of 0.19–
1.76
l
M. The cell line with the most sensitivity toward 32 was
M). Compound 31 has IC50 values < 2.00 lM
HL-60 (IC50 = 0.19
l
and may also represent an important candidate for subsequent
studies.
Compounds 36 and 39 were designed based on the bioisosteric
replacement of the arylamino substituent in a-lapachone as shown
in the Scheme 4. Our objective was to evaluate the influence of the
sulfur atom (chalcogen) on activity. Compound 36 was highly

1616
E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
sure and the reaction mixture was purified on a silica gel column as
O
O
O
O
a gradient mixture of hexane/ethyl acetate or dichloromethane/
ethyl acetate with increasing polarity. Method B: The same proce-
dures described in Method A, but 2 mL DMF, one drop Et3N were
added and the reaction was left under magnetic stirring for
60 min as monitored by TLC.
O
S
O
HN
Pharmacophoric
4.2.1. 2-(4-(4-(((3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)amino)methyl)-1H-1,2,3-triazol-1-yl)butyl)isoindoline-1,3-
dione
Group
Bioisosteric Replacement
Using method B, compound 14 was obtained as an orange so-
Scheme 4. Bioisosteric replacement strategy for 36 and 39.
lid (100 mg, 0.20 mmol, 78%); mp 230–232 °C. IR mmax (cmꢀ1
,
KBr): 3312 (N–H). 1H NMR (400 MHz, DMSO-d6) d: 7.99–7.74
(m, 9H), 7.55 (br s, 1H, NH), 5.02 (d, 2H J = 6.6 Hz), 4.35 (t, 2H,
J = 7.0 Hz), 3.58 (t, 2H, J = 7.1 Hz), 1.83 (qt, 2H, J = 7.0 Hz), 1.57
(qt, 2H, J = 6.6 Hz). 13C NMR (100 MHz, DMSO-d6) d: 179.8,
167.5, 144.6, 134.5, 134.0, 132.4, 131.4, 129.8, 126.2, 122.6,
122.3, 109.2, 48.6, 36.5, 26.8, 24.7. Anal. Calcd for C25H20ClN5O4
0.65 ꢃ H2O, C, 59.86, H, 4.28, N, 13.96. Found. C, 60.24, H, 4.22,
N, 13.59.
effective against the HL-60 cancer cell line (IC50 value = 2.91 lM)
with a high selective index compared with PBMC, L929 and V79.
Compound 39 was highly active against HL-60 and HCT-8.
Despite the small number of compounds, there was a positive
correlation between the electrochemical parameters and cytotox-
icity, allowing the discrimination of compounds 20 (active and
more electrophilic) and 21 and the less cytotoxic compound 10
from the more selective compound 39.
4.2.2. (2R,3R,4S,5R)-2-(Acetoxymethyl)-6-(4-(((3-bromo-1,4-
dioxo-1,4-dihydronaphthalen-2-yl)amino)methyl)-1H-1,2,3-
triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Using method B, compound 20 was obtained as an orange solid
3. Conclusion
In this paper, we synthesized and evaluated the cytotoxicity of
several 1,2,3-triazole-, arylamino- and thio-substituted naphtho-
quinones against cancer and normal cell lines. Of the described
substances, compounds 31 and 32 were considered highly active
(150 mg, 0.23 mmol, 87% yield); mp 152–153 °C. IR mmax (cmꢀ1
,
KBr): 3339 (NH). 1H NMR (400 MHz, acetone-d6) d: 8.23 (s, 1H),
8.07-8.02 (m, 2H), 7.83-7.74 (m, 2H), 7.19 (br s, 1H, NH), 6.22 (d,
1H, J = 9.0 Hz), 5.60 (dd, 1H, J= 9.4 and 9.4 Hz), 5.51 (dd, 1H,
J = 9.0 and 9.0 Hz), 5.25-5.15 (m, 3H), 4.34–4.14 (m, 3H), 2.05
(s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.93 (s, 3H). 13C NMR (100 MHz,
acetone-d6) d: 180.8, 180.2, 170.6, 170.1, 170.0, 169.1, 146.7, 135.4,
133.4, 132.9, 127.5, 127.1, 122.1, 85.8, 75.2, 73.3, 71.2, 68.7, 62.0,
20.5, 20.4, 20.0. Anal. Calcd for C27H27BrN4O11 0.45 ꢃ H2O, C,
48.29, H, 4.19, N, 8.34. Found: C, 48.65, H, 4.59, N, 8.01.
(IC50 <2 lM). Considering the selectivity index, compounds 36
and 39 have been identified as lead compounds for further investi-
gation. As previously shown by Claus Jacob’s group, employing
chalcogens is a productive approach. Our strategy to append aryla-
mino groups to the 1,4-naphthoquinone structure was also suc-
cessful, and several compounds with potent antitumor activity
were presented in this manuscript that will be further investigated.
4.2.3. (2S,3S,4R,5S)-2-(Acetoxymethyl)-6-(4-(((1,4-dioxo-1,4-
dihydronaphthalen-2-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
4. Experimental section
4.1. Chemistry
Using method A, compound 21 was obtained as an orange solid
(150 mg, 0.26 mmol, 62% yield); mp 209–210 °C. IR mmax (cmꢀ1
,
Melting points were obtained on Thomas Hoover and are uncor-
rected. Analytical grade solvents were used. Column chromatogra-
phy was performed on silica gel (SiliaFlash G60 UltraPure—60–
KBr): 3351 (NH). 1H NMR (300 MHz, CDCl3) d: 8.08 (dd, 1H,
J = 7.8 and 0.9 Hz), 8.05 (dd, 1H, J = 7.8 and 0.9 Hz), 7.80 (s, 1H),
7.73 (td, 1H, J = 7.6 and 1.5 Hz), 7.62 (td, 1H, J = 7.6 and 1.5 Hz),
6.35 (br s, 1H, NH), 5.88 (d, 1H, J= 8.5 Hz), 5.79 (s, 1H), 5.41 (t,
1H, J = 4.5 Hz), 5.24 (t, 1H, J = 5.1 Hz), 5.21 (t, 1H, J = 5.1 Hz), 4.52
(d, 1H, J = 5.1 Hz), 4.30 (dd, 1H, J = 12.6 and 4.8 Hz), 4.14 (dd, 1H,
J = 12.6 and 4.8 Hz), 4.00 (dd, 1H, J = 9.9 and 3.3 Hz), 2.07 (s, 3H),
2.05 (s, 3H), 2.01 (s, 3H), 1.86 (s, 3H). 13C NMR (75.5 MHz, CDCl3)
d: 183.0, 181.5, 170.5, 169.8, 169.3, 168.9, 151.4, 134.8, 132.2,
130.4, 126.4, 126.2, 107.6, 85.8, 75.2, 72.4, 70.2, 67.6, 61.4, 38.2,
20.7, 20.5, 20.4, 20.1. Anal. Calcd for C27H28N4O11 C, 55.48, H,
4.83, N, 9.58. Found: C, 55.46, 4.52, N, 9.25.
200 lm, 60 Å). Infrared spectra were recorded on an FTIR Spec-
trometer IR Prestige-21—Shimadzu. 1H and 13C NMR were re-
corded at room temperature using a Bruker AVANCE DRX200,
Varian Mercury 300 and Varian Mercury 400 MHz, in the solvents
indicated, with TMS as internal reference. Chemical shifts (d) are
given in ppm and coupling constants (J) in Hertz. Mass spectra
(electrospray ionization) were obtained using a MicroTOFIc—Bru-
kerDaltonics. Elemental analysis was performed using a CE Instru-
ments (Thermo-Fisher) EA 1110 CHNS-O elemental analyzer. All
the compounds were nominated using the program CS ChemDraw
Ultra version 10.0.
4.2.4. 2-(((1-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-1H-1,2,3-
triazol-4-yl)methyl)amino)naphthalene-1,4-dione
4.2. General procedures to prepare 1,4-naphthoquinone-based
1,2,3-triazoles
Using method A, compound 22 was obtained as an orange solid
(100 mg, 0.24 mmol, 78% yield); mp 228–229 °C. IR mmax (cmꢀ1
,
KBr): 3344 (NH). 1605, 1572 (C@C). 1H NMR (400 MHz, DMSO-
d6) d: 8.65 (s, 1H), 8.13 (dd, 1H, J = 6.0 and 3.2 Hz), 8.06 (dd, 1H,
J = 6.0 and 3.2 Hz), 8.00 (dd, 1H, J = 7.6 and 1.2 Hz), 7.96–7.93 (m,
4H), 7.82 (td, 1H, J = 7.6 and 1.6 Hz), 7.73 (td, 1H, J = 7.6 and
1.6 Hz), 7.48 (s, 1H), 5.87 (s, 1H), 4.64 (d, 2H, J = 6.0 Hz). 13C NMR
(100 MHz, DMSO-d6) d: 183.8, 181.4, 181.3, 178.5, 148.0, 144.0,
140.0, 134.6, 134.5, 134.3, 132.8, 132.1, 131.2, 131.1, 130.1,
Method A: In a flask containing 3 mL acetonitrile, it was added
0.5 mmol of the appropriate azides, followed by 0.6 mmol of the
appropriate alkynes and finally 11 mg (0.06 mmol) of copper(I) io-
dide. The reaction was left under magnetic stirring at 28 °C under
argon atmosphere, for a reaction time ranging from 19 to 24 h.
The end of the reaction was monitored by TLC with dichlorometh-
ane as eluent. The solvent was then removed under reduced pres-

E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
1617
126.5, 126.1, 125.7, 125.6, 125.2, 125.1, 100.6, 36.9. Anal. Calcd for
29.0, 26.4. EI/MS (m/z) [M+H]+: 413.0. Calcd. for [C21H18BrNO3H]+:
C
23H14N4O4, 0.6 ꢃ H2O, C, 65.98; H, 3.64; N, 13.3 Found: C, 65.98;
413.0.
H, 3.48; N, 12.90.
4.2.10. 4-((4-Iodophenyl)amino)-2,2-dimethyl-3,4-dihydro-2H-
benzo[g]chromene-5,10-dione
4.2.5. 2-Chloro-3-(((1-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
1H-1,2,3-triazol-4-yl)methyl)amino)naphthalene-1,4-dione
Using method A, compound 23 was obtained as an orange solid
The compound 30 was obtained as a red solid (229 mg,
0.5 mmol, 50% yield); mp 154–156 °C. IR mmax (cmꢀ1, KBr): 3376
(NH). 1H NMR (200 MHz, CDCl3) d: 8.14–7.99 (m, 2H), 7.77–7.65
(m, 2H), 7.46 (d, 2H, J = 8.8 Hz), 6.50 (d, 2H, J = 8.6 Hz), 4.64 (dd,
1H, J = 5.3 and 3.5 Hz), 2.24 (dd, 1H, J = 14.3 and 3.2 Hz), 1.99
(dd, 1H, J = 14.5 and 5.4 Hz), 1.53 (s, 3H), 1.52 (s, 3H). 13C NMR
(50 MHz, CDCl3) d: 183.8, 180.0, 155.6, 146.5, 138.1, 134.6, 133.4,
132.1, 131.0, 126.6, 126.4, 123.3, 118.6, 116.1, 79.5, 79.3, 43.5,
37.5, 28.8, 24.4. EI/MS (m/z) [M+H]+: 460.0. Calcd for [C21H18INO3-
H]+: 460.0.
(100 mg, 0.23 mmol, 68% yield); mp 219–221 °C. IR mmax (cmꢀ1
,
KBr): 3325 (NH). 1606, 1574 (C@C).1H NMR (400 MHz, DMSO-d6)
d: 8.59 (s, 1H), 8.11–7.94 (m, 7H), 7.83 (t, 1H, J = 7.8 Hz), 7.76 (t,
1H, J = 7.8 Hz), 7.47 (br s, 1H, NH), 5.14 (d, 2H, J = 6.6 Hz). 13C
NMR (100 MHz, DMSO-d6) d: 184.0, 180.1, 178.7, 175.6, 146.4,
140.2, 134.9, 134.5, 132.9, 131.8, 131.2, 130.1, 129.0, 126.6,
125.8. Anal. Calcd for C23H13ClN4O4 1.0 ꢃ H2O, C, 58.87, H, 2.92,
N, 12.48 Found: C, 58.52, H, 2.93, N, 12.19.
4.2.6. 2-Bromo-3-(((1-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
1H-1,2,3-triazol-4-yl)methyl)amino)naphthalene-1,4-dione
Using method A, compound 24 was obtained as an orange solid
4.2.11. 4-((4-Chlorophenyl)amino)-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione
The compound 31 was obtained as a red solid (184 mg,
0.5 mmol, 50% yield); mp 159–161 °C. IR mmax (cmꢀ1, KBr): 3384
(NH). 1H NMR (200 MHz, CDCl3) d: 8.11–7.98 (m, 2H), 7.76–7.64
(m, 2H), 7.15 (d, 2H, J = 8.5 Hz), 6.64 (d, 2H, J = 8.6 Hz), 4.63 (dd,
1H, J = 5.1 and 2.7 Hz), 2.25 (dd, 1H, J = 14.4 and 3.5 Hz), 1.99
(dd, 1H, J = 14.4 and 5.1 Hz), 1.53 (s, 3H), 1.52 (s, 3H). 13C NMR
(50 MHz, CDCl3) d: 183.8, 180.0, 155.6, 145.6, 134.5, 133.4,
132.2, 131.1, 129.3, 126.6, 126.4, 123.3, 118.8, 115.2, 79.3, 43.8,
28.9, 26.4. EI/MS (m/z) [M+H]+ 368.0. Calcd for [C21H18ClNO3H]+
368.0.
(100 mg, 0.20 mmol, 99% yield); mp 168–170 °C. IR mmax (cmꢀ1
,
KBr): 3301 (NH). 1H NMR (400 MHz, DMSO-d6) d: 8.59 (s, 1H),
8.12 (dd, 1H, J = 5.6 and 3.2 Hz), 8.06 (dd, 1H, J = 6.0 and 3.2 Hz),
8.00–7.93 (m, 4H), 7.82 (td, 1H, J = 7.6 and 1.2 Hz) 7.75 (td, 1H,
J = 7.6 and 1.2 Hz), 7.47 (s, 1H), 5.18 (d, 2H, J = 6.8 Hz). 13C NMR
(100 MHz, DMSO-d6) d: 183.8, 179.6, 178.5, 175.2, 147.5, 146.0,
140.0, 134.6, 134.3, 132.6, 131.3, 131.1, 130.0, 126.5, 126.4,
126.1, 125.9, 125.6, 124.8. Anal. Calcd for
C23H13BrN4O4,
1.1 ꢃ H2O, C, 56.46; H, 2.68; N, 11.45 Found: C, 54.58; H, 2.69, H,
10.62.
4.2.12. 4-((4-Methoxyphenyl)amino)-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione
4.2.7. General procedures to synthesis of
arylamino derivatives
a-lapachone
The compound 32 was obtained as a brown solid (137 mg,
0.37 mmol, 37% yield); mp 149–150 °C. IR mmax (cmꢀ1, KBr): 3372
(NH). 1H NMR (200 MHz, CDCl3) d: 8.13–8.04 (m, 2H), 7.77–7.67
(m, 2H), 6.87–6.74 (m, 4H); 4.62–4.55 (m, 1H), 3.77 (s, 3H), 2.24
(dd, 1H, J = 14.2 and 4.0 Hz), 2.00 (dd, 1H, J = 14.1 and 5.6 Hz),
1.56 (s, 3H), 1.49 (s, 3H). 13C NMR (50 MHz, CDCl3) d: 183.8,
179.9, 155.1, 153.1, 141.0, 134.2, 133.1, 132.0, 130.8, 126.3,
119.4, 116.6, 114.8, 79.2, 55.7, 44.7, 37.5, 28.6, 26.4. EI/MS (m/z)
[M+H]+: 364.0. Calcd for [C22H21NO4H]+: 364.0.
Compound 27 (321 mg, 1 mmol) was dissolved in 5 mL of CH2-
Cl2, the respective anilines were added (1.3 mmol) and the mixture
was left under stirring overnight, followed by the addition of 50 mL
of water. The organic phase was extracted with dichloromethane,
washed with HCl 10% (3 ꢃ 50 mL), dried over sodium sulfate, and
filtered. The solvent from the crude product was evaporated under
reduced pressure and it was purified by column chromatography
in silica-gel, using eluents with an increasing polarity gradient
mixture of hexane and ethyl acetate (9/1 to 7/3).
4.2.13. 4-((3-Fluorophenyl)amino)-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione
4.2.8. 2,2-Dimethyl-4-((4-nitrophenyl)amino)-3,4-dihydro-2H-
benzo[g]chromene-5,10-dione
The compound 33 was obtained as a red solid (176 mg,
0.5 mmol, 50% yield); mp 152–154 °C. IR mmax (cmꢀ1, KBr): 3382
(NH). 1H NMR (200 MHz, CDCl3) d: 8.15–7.99 (m, 2H), 7.77–7.64
(m, 2H), 7.19–7.06 (m, 2H), 6.55–6.40 (m, 2H), 4.67 (dd, 1H,
J = 5.5 and 3.5 Hz), 2.27 (dd, 1H, J = 13.8 and 2.2 Hz), 2.02 (dd,
1H, J = 13.8 and 5.0 Hz), 1.54 (s, 3H), 1.53 (s, 3H). 13C NMR
(50 MHz, CDCl3) d: 183.8, 180.0, 172.7 155.6, 148.5 (d,
J = 10.7 Hz), 134.6, 133.4, 132.2, 131.1, 126.5 (d, J = 10 Hz), 118.6,
109.8 (d, J = 2.3 Hz), 104.5 (d, J = 21.8 Hz), 100.5 (d, J = 24.9 Hz),
79.3, 43.6, 37.6, 28.9, 26.4 EI/MS (m/z) [M+H]+: 352.0. Calcd for
[C21H18FNO3H]+: 352.0.
The compound 28 was obtained as a red solid (227 mg,
0.6 mmol, 60% yield); mp 223–225 °C. IR mmax (cmꢀ1, KBr): 3387
(NH), 1337 (N@O). 1H NMR (200 MHz, CDCl3) d: 8.13–7.92 (m,
2H), 7.78–7.59 (m, 2H), 7.23–7.02 (m, 1H), 6.60–6.32 (m, 3H),
4.64 (dd, 1H, J = 5.3 and 3.2 Hz), 2.28 (dd, 1H, J = 14.4 and
3.3 Hz), 1.99 (dd, 1H, J = 14.3 and 5.2 Hz), 1.55 (s, 3H), 1.53 (s,
3H). 13C NMR (50 MHz, CDCl3) d: 183.7, 180.0, 155.6, 148.9,
134.5, 133.4, 132.2, 131.0, 130.7, 130.5; 126.6, 126.3, 118.7,
109.7, 109.6, 100.7, 79.3, 43.4, 37.6, 29.0, 26.3. EI/MS (m/z)
[M+Na]+: 401.1. Calcd for [C21H18N2O5Na]+: 401.1.
4.2.14. 2,2-Dimethyl-4-((3-nitrophenyl)amino)-3,4-dihydro-2H-
benzo[g]chromene-5,10-dione
4.2.9. 4-((4-Bromophenyl)amino)-2,2-dimethyl-3,4-dihydro-2H-
benzo[g]chromene-5,10-dione
The compound 34 was obtained as a orange solid (189 mg,
0.5 mmol, 50% yield); mp 152–154 °C. IR mmax (cmꢀ1, KBr): 3386
(NH). 1H NMR (200 MHz, CDCl3) d: 8.10–7.95 (m, 2H), 7.75–7.58
(m, 2H), 7.09–6.55 (m, 4H), 4.87–4.61 (m, 1H), 2.35–2.17 (m,
1H), 2.14–1.98 (m, 1H), 1.58 (s, 3H), 1.56 (s, 3H). 13C NMR
(50 MHz, CDCl3) d: 183.6, 179.4, 155.5, 149.2, 134.3, 133.3, 131.8,
130.6, 130.5, 129.7, 126.5, 125.9, 125.8, 119.0, 117.8, 112.5, 79.1,
43.0, 37.1, 28.8, 26.0. EI/MS (m/z) [M+H]+: 401.0. Calcd for [C21H18-
N2O5Na]+: 401.0.
The compound 29 was obtained as a red solid (247 mg,
0.6 mmol, 60% yield); mp 174–176 °C. IR mmax (cmꢀ1, KBr): 3386
(NH). 1H NMR (200 MHz, CDCl3) d: 8.22–7.99 (m, 2H), 7.82–7.65
(m, 2H), 7.29 (d, 2H, J = 8.6 Hz), 6.60 (d, 2H, J = 8.5 Hz), 4.64 (dd,
1H, J = 4.9 and 3.4 Hz), 2.25 (dd, 1H, J = 14.4 and 3.6 Hz), 2.00
(dd, 1H, J = 14.7 and 5.5 Hz), 1.54 (s, 3H), 1.53 (s, 3H). 13C NMR
(50 MHz, CDCl3) d: 183.8, 183.0, 155.5, 146.1, 134.6, 133.4, 132.2,
131.0, 126.6, 126.4, 118.8, 115.5, 110.2, 96.6, 79.3, 43.4, 37.6,

1618
E. H. G. da Cruz et al. / Bioorg. Med. Chem. 22 (2014) 1608–1619
4.2.15. 4-((3-Bromophenyl)amino)-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione
yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazo-
lium bromide (MTT) to a blue formazan product as described by
Mosmann.29
The compound 35 was obtained as a red solid (247 mg,
0.6 mmol, 60% yield); mp 182–185 °C. IR mmax (cmꢀ1, KBr): 3410
(NH). 1H NMR (200 MHz, CDCl3) d: 8.10–7.95 (m, 2H); 7.75–7.58
(m, 2H), 7.09–6.55 (m, 4H), 4.17–4.59 (m, 1H), 2.26 (dd, 1H,
J = 14.4 and 2.2 Hz), 1.99 (dd, 1H, J = 14.1 and 5.3 Hz), 1.54 (s,
3H), 1.52 (s, 3H). 13C NMR (50 MHz, CDCl3) d: 183.4, 179.7, 155.3,
148.1, 134.3, 133,2, 131.9, 130.8, 130.5, 126.4, 126.1, 123.2,
121.0, 118.4, 116.0, 112.3, 79.0, 43.1, 37.3, 28.8, 26.1. EI/MS (m/z)
[M+H]+: 413.0. Calcd for [C21H18BrNO3H]+: 413.0.
4.4. Electrochemical studies
Cyclic voltammetry (CV) experiments were performed with a
conventional undivided three electrode cell using an Autolab
PGSTAT-30 potentiostat (Echo Chemie, Utrecht, The Netherlands)
coupled to a microcomputer, interfaced by GPES 4.9 software.
Glassy carbon (GC) (diameter = 3 mm) as the working electrode,
a Pt wire as the counter electrode and the reference electrode an
Ag|AgCl, Clꢀ (saturated) were used. The GC electrode was cleaned
up by polishing with alumina on a polishing felt (BAS polishing
kit). The solvent used in aprotic media studies, DMF from Acros,
was used as received. In CV experiments, the scan rate varied from
10 to 500 mV sꢀ1. All experiments were conducted at room tem-
perature (25 2 °C) and purging an inert gas (Argon). Electrochem-
ical reduction in aprotic media (DMF + TBAP 0.1 mol Lꢀ1) was
performed in the absence of oxygen. Each compound (1 ꢃ 10ꢀ3
mol Lꢀ1) was added to the supporting electrolyte and the solution
was deoxygenated with argon before the measurements by cyclic
voltammetry.
4.2.16. General procedures to the synthesis of
arylamino derivatives
a-lapachone
The respective bromo derivatives (1 mmol) were dissolved in
5 mL of CH2Cl2, and thiophenol was added (1.3 mmol) and the mix-
ture was left under stirring overnight, followed by the addition of
50 mL of water. The organic phase was extracted with dichloro-
methane, dried over sodium sulfate, and filtered. The solvent from
the crude mixture was evaporated under reduced pressure and it
was purified by column chromatography in silica-gel, using eluents
with an increasing polarity gradient mixture of hexane and ethyl
acetate (9/1 to 7/3).
4.2.17. 2,2-Dimethyl-4-(phenylthio)-3,4-dihydro-2H-
benzo[g]chromene-5,10-dione
4.5. X-ray analysis
The compound 36 was obtained as a yellow solid (125 mg,
X-ray data were collected at 150 K using MoKa (0.71073 Å) on
0.5 mmol, 50% yield); mp 182–185 °C. IR mmax (cmꢀ1, KBr): 718
an Agilent–Gemini diffractometer equipped with a CCD area detec-
tor. The CrysAlisPro software package35 was used for data collec-
tion and data reduction. The data were corrected empirically for
absorption using spherical harmonics using the SCALE3 ABSPACK36
scaling algorithm. The structure was solved by direct methods
using SHELXS-9737 and refined by full-matrix least squares on F2
using SHELXL-97.38 All non-hydrogen atoms were successfully re-
fined using anisotropic displacement parameters. Hydrogen atoms
were found in the Fourier difference synthesis and fixed. Crystallo-
graphic data for the structure were deposited in the Cambridge
Crystallographic Data Centre, with number CCDC 964308.
(C–S) cmꢀ1 1H NMR (200 MHz, CDCl3) d: 8.12–8.00 (m, 2H),
.
7.90–7.75 (m, 2H), 7.70–7.62 (m, 2H), 7.46–7.31 (m, 3H), 4.58–
4.50 (m, 1H), 2.10–2.00 (m, 2H), 1.61 (s, 3H), 1.48 (s, 3H). EI/HRMS
(m/z) [M+Na]+ 373.0905. Calcd for [C21H18O3SNa]+ 373.0874.
4.2.18. 2,2-Dimethyl-3-(phenylthio)-2,3-dihydronaphtho[2,3-
b]furan-4,9-dione
The compound 39 was obtained as a yellow solid (150 mg,
0.45 mmol, 45% yield); mp 148–151 °C. IR (KBr): 718 (C–S) cmꢀ1
.
1H NMR (200 MHz, CDCl3) d: 8.15–8.02 (m, 2H), 7.80–7.61 (m,
2H), 7.59–6.51 (m, 2H), 7.35–7.19 (m, 3H), 4.58 (s, 1H), 1.80 (s,
3H), 1.58 (s, 3H). 13C NMR (50 MHz, CDCl3) d: 181.3, 178.2, 158.7,
135.0, 134.5, 133.4, 133.1, 132.7, 131.7, 129.3, 128.0, 126.5,
126.4, 123.6, 94.4, 59.2, 28.5, 24.3. EI/MS (m/z) [M+Na]+: 359.0.
Calcd for [C20H16O3SNa]+: 359.0.
Conflict of interest
Authors declare no conflict of interest.
Acknowledgments
4.3. Cytotoxicity against cancer and normal cell lines
This research was supported by grants from the Conselho Nac-
ional de Desenvolvimento Científico e Tecnológico (CNPq) Project
Universal—MCTI/CNPq n° 14/2012 (480719/2012-8) and Chamada
MCTI/CNPq n° 16/2012, CAPES, Fundação Cearense de Pesquisa e
Cultura (FUNCAP—Edital Pronex), Fundação de Apoio à pesquisa
do Estado de Alagoas (FAPEAL, PRONEX) Fundação de Apoio à pes-
quisa do Estado de Pernambuco (FACEPE, PRONEM). Dr. E.N. da Sil-
va Júnior thanks the Programa Institucional de Auxílio à Pesquisa
de Doutores Recém-Contratados (Edital PRPq—01/2013) and Uni-
versidade Federal de Minas Gerais. The authors would like to thank
Dr. Janaína Versiani by the collaboration in several aspects.
Compounds (0.001–30 lM) were tested for cytotoxic activity
against several human cancer cell lines obtained from National
Cancer Institute, NCI (Bethesda, MD). The L929 cells (mouse fibro-
blast L cells NCTC clone 929) were obtained from American Type
Culture Collection (Manassas, VA), and the Chinese hamster lung
fibroblasts (V79 cells) were kindly provided by Dr. JAP Henriques
(UFRGS). 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 Hist-
opaque-1077 (Sigma Aldrich Co., St. Louis, MO, USA). All cancer cell
lines and PBMC were maintained in RPMI 1640 medium. L929 and
V79 cells were cultivated under standard conditions in MEM with
Earle’s salts. All culture media were supplemented 20% (PBMC) or
10% (cancer, L929 and V79 cells) fetal bovine serum, 2 mM gluta-
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